Device and method for photoactivation

A device comprising a light source, a sample holder and a temperature control chamber. The sample holder supports the vessels in a fixed relationship relative to the light source. The temperature control chamber surrounds the sample holder and vessels, so that a temperature control fluid may be passed around the sample vessels effectively maintaining the temperature of the sample vessels within a desired temperature range.

FIELD OF THE INVENTION 
The present invention relates to a device and method for photoactivating 
new and known compounds. 
BACKGROUND 
Nucleic acid technology has made possible the manipulation, amplification, 
selection and characterization of a potentially very large number of 
eukaryotic, prokaryotic and viral genes. Most importantly, application of 
nucleic acid techniques allows for the isolation of any nucleic acid 
sequence within a complex genome, the modification of the sequence, and 
the introduction of the sequence into diverse species. 
With the prospect of advertently or inadvertently releasing nucleic acid 
sequences into nature that are either: a) modified but present in their 
normal host species, or b) normal but present in a foreign host species, 
there is some concern that nucleic acid techniques pose a risk to human 
health. Regulatory approaches to this risk have focused on physical or 
biological containment of organisms that contain foreign or modified 
nucleic acid sequences. National Institutes of Health, Federal Register 
41:27902 (1976); National Institutes of Health, Federal Register 43:60108 
(1978). Such approaches are bolstered by studies that assess the impact of 
different laboratory protocols and various types of human error and 
equipment failures on the incidence and extent of uncontained organisms. 
E. Fisher and D. R. Lincoln, Recomb. DNA Tech. Bull. 7:1 (1984). 
With this effort directed at nucleic acids in organisms, little attention 
has been paid to the problem of naked nucleic acid, i.e., nucleic acid 
that is free from a host organism. Depending on the particular 
circumstances, naked nucleic acid can be an infectious or transforming 
agent. R. W. Old and S. B. Primrose, Principles of Gene Manipulation, pp. 
167-168 (Univ. of Cal. Press, 2d Edition 1981). Furthermore, naked nucleic 
acid can interfere with other laboratory reactions because of carry-over. 
Carry-over 
Carry-over is broadly defined here as the accidental introduction of 
nucleic acid into a reaction mixture. Of course, the types of accidental 
introductions are numerous. Nucleic acids can be introduced during a spill 
or because of poor laboratory technique (e.g., using the same reaction 
vessel or the same pipette twice). Of more concern, however, is the 
introduction of nucleic acids that occurs even during normal laboratory 
procedures, including inadvertent transfer from contaminated gloves. As 
with modified organisms, one of the most troubling source of this type of 
accident is aerosolization. 
Aerosols are suspensions of fine liquid or solid particles, as in a mist. 
Aerosols can occur by disturbing a solution (e.g., aerosols are created 
during a spill), but they can also occur simply by disturbing the small 
amount of material on a container surface (e.g., the residue on the inner 
surface of a cap of a plastic tube is frequently aerosolized at the moment 
the tube is opened). Because of the latter, any container having highly 
concentrated amounts of nucleic acid is a potential source of nucleic acid 
carry-over. 
It should be pointed out that the question of whether there is carry-over 
is only significant to the extent that such carry-over interferes with a 
subsequent reaction. In general, any laboratory reaction that is directed 
at detecting and/or amplifying a nucleic acid sequence of interest among 
vastly larger amounts of nucleic acid is susceptible to interference by 
nucleic acid carry-over. 
Amplification Techniques 
The circumstances in the modern laboratory where both: a) containers having 
highly concentrated amounts of nucleic acid are present, and b) reactions 
directed at amplifying nucleic acid sequences are performed, are 
relatively common. The screening of genomic DNA for single copy genes is 
perhaps the best example of a procedure involving both concentrated 
nucleic acid and amplification. There are a number of alternative methods 
for nucleic acid amplification, including: 1) the replication of 
recombinant phage through lytic growth, 2) amplification of recombinant 
RNA hybridization probes, and 3) the Polymerase Chain Reaction. 
1. Recombinant Vectors 
Most cloning vectors are DNA viruses or bacterial plasmids with genomic 
sizes from 2 to approximately 50 kilobases (kb). The amplification of 
genomic DNA into a viral or plasmid library usually involves i) the 
isolation and preparation of viral or plasmid DNA, ii) the ligation of 
digested genomic DNA into the vector DNA, iii) the packaging of the viral 
DNA, iv) the infection of a permissive host (alternatively, the 
transformation of the host), and v) the amplification of the genomic DNA 
through propagation of virus or plasmid. At this point, the recombinant 
viruses or plasmids carrying the target sequence may be identified. T. 
Maniatis et al, Molecular Cloning, pp. 23-24 (Cold Spring Harbor 
Laboratory 1982). Identification of the recombinant viruses or plasmids 
carrying the target sequence is often carried out by nucleic acid 
hybridization using plasmid-derived probes. 
Bacterial viruses (bacteriophage) can infect a host bacterium, replicate, 
mature, and cause lysis of the bacterial cell. Bacteriophage DNA can, in 
this manner, be replicated many fold, creating a large quantity of nucleic 
acid. 
Plasmids are extrachromosomal elements found naturally in a variety of 
bacteria. Like bacteriophages, they are double-stranded and can 
incorporate foreign DNA for replication in bacteria. In this manner, large 
amounts of probes can be made. 
The use of plasmid-derived probes for the screening of phage libraries in 
hybridization reactions avoids the problem of hybridization of vector DNA 
(e.g., phage-phage, plasmid-plasmid). In the construction of a viral 
library, it is therefore essential that no plasmid DNA carry-over into the 
phage-genomic DNA mixture. If, for example, 10 picograms of clonable 
plasmid DNA were to carry-over into a viral-genomic mixture containing 1 
microgram of genomic DNA (0.001% carry-over by weight), every 11 clones 
assessed to contain the target sequence would, on average, represent 10 
false positives (i.e., plasmid-plasmid hybridization) and only 1 true 
positive (probe-target hybridization), assuming a frequency of 1 target 
insert in 1.times.10.sup.6 inserts. 
2. Recombinant RNA Probes 
P. M. Lizardi et al., Biotechnology 6:1197 (1988), describe recombinant RNA 
molecules that function both as hybridization probes and as templates for 
exponential amplification by Q.beta. replicase. Each recombinant consists 
of a specific sequence (i.e., an "internal probe") within the sequence of 
MDV-1 RNA. MDV-1 RNA is a natural template for the replicase. D. L. Kacian 
et al., Proc. Nat. Acad. Sci USA 69:3038 (1972). The recombinant can 
hybridize to a target sequence that is complementary to the internal probe 
and that is present in a mixture of nucleic acid. Various isolation 
techniques (e.g., washing) can then be employed to separate the hybridized 
recombinant/target complex from a) unbound recombinant and b) nucleic 
acids that are non-complementary to the internal probe. B. C. F. Chu et 
al., Nucleic Acids Res. 14:5591 (1986). See also Biotechnology 7:609 
(1989). Following isolation of the complex, Q.beta. replicase is added. In 
minutes a one-billion fold amplification of the recombinant (i.e., 
"recombinant RNA probe amplification") occurs, indicating that specific 
hybridization has taken place with a target sequence. 
While a promising technique, recombinant RNA probe amplification works so 
well that carry-over is of particular concern. As little as one molecule 
of template RNA can, in principle, initiate replication. Thus, the 
carry-over of a single molecule of the amplified recombinant RNA probe 
into a new reaction vessel can cause RNA to be synthesized in an amount 
that is so large it can, itself, be a source of further carry-over. 
3. Polymerase Chain Reaction 
K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and 4,683,202, describe a 
method for increasing the concentration of a segment of a target sequence 
in a mixture of genomic DNA without cloning or purification. This process 
for amplifying the target sequence consists of introducing a large excess 
of two oligonucleotide primers to the DNA mixture containing the desired 
target sequence, followed by a precise sequence of thermal cycling in the 
presence of a DNA polymerase. The two primers are complementary to their 
respective strands of the double stranded target sequence. To effect 
amplification, the mixture is denatured and the primers then anneal to 
their complementary sequences within the target molecule. Following 
annealing, the primers are extended with a polymerase so as to form a new 
pair of complementary strands. The steps of denaturation, primer 
annealing, and polymerase extension can be repeated many times (ie., 
denaturation, annealing and extension constitute one "cycle;" there can be 
numerous "cycles") to obtain a high concentration of an amplified segment 
of the desired target sequence. The length of the amplified segment of the 
desired target sequence is determined by the relative positions of the 
primers with respect to each other, and therefore, this length is a 
controllable parameter. By virtue of the repeating aspect of the process, 
the method is referred to by the inventors as the "Polymerase Chain 
Reaction" (hereinafter PCR). Because the desired amplified segments of the 
target sequence become the predominant sequences (in terms of 
concentration) in the mixture, they are said to be "PCR amplified." 
With PCR, it is possible to amplify a single copy of a specific target 
sequence in genomic DNA to a level detectable by several different 
methodologies (e.g., hybridization with a labelled probe; incorporation of 
biotinylated primers followed by avidin-enzyme conjugate detection; or 
incorporation of .sup.32 p labelled deoxynucleotide triphosphates, e.g., 
dCTP or DATP, into the amplified segment). In addition to genomic DNA, any 
oligonucleotide sequence can be amplified with the appropriate set of 
primer molecules. In particular, the amplified segments created by the PCR 
process itself are, themselves, efficient templates for subsequent PCR 
amplifications. 
The PCR amplification process is known to reach a plateau concentration of 
specific target sequences of approximately 10.sup.-8 M. A typical reaction 
volume is 100 .mu.l, which corresponds to a yield of 6.times.10.sup.11 
double stranded product molecules. At this concentration, as little as one 
femtoliter (10.sup.-9 microliter) of the amplified PCR reaction mixture 
contains enough product molecules to generate a detectable signal in a 
subsequent 30 cycle PCR amplification. If product molecules from a 
previous PCR are carried over into a new PCR amplification, it can result 
in a false positive signal during the detection step for the new PCR 
reaction. 
Handling of the reaction mixture after PCR amplification can result in 
carry-over such that subsequent PCR amplifications contain sufficient 
previous product molecules to result in a false positive signal. S. Kwok 
and R. Higuchi, Nature 339, 286 (1989). PCR Technology, H. A. Erlich (ed.) 
(Stockton Press 1989). This can occur either through aerosols or through 
direct introduction, as described above for other types of carry-over. 
Control Of Carry-Over 
At present, there are three approaches for the control of carry-over. These 
can be broadly defined as: 1) containment, 2) elimination, and/or 3) 
prevention. With the containment approach, amplification is performed in a 
closed system. Usually, this means a designated part of the laboratory 
that is closed off from all other space. Of course, the designated area 
must be appropriately configured for the particular amplification assay. 
In the case of replication of recombinant phage through lytic growth, the 
area must allow for the amplification of the genomic DNA through 
propagation of virus or plasmid. The area must also provide all the 
requisite equipment and reagents for amplification and subsequent 
detection of the amplified segment of the target sequence. 
The problem with containment is that it is very inconvenient. In order for 
the containment area to be configured to provide conditions appropriate 
for all the steps of amplification, the laboratory must commit a separate 
set of equipment. This duplicate set of equipment, furthermore, is also 
subject to carry-over. Over time it can be rendered unusable. 
The elimination approach is used when carry-over has already occurred. New 
stocks of enzymes, buffers, and other reagents are prepared along with a 
complete and thorough cleaning of the laboratory area where amplification 
is performed. All surfaces are scrubbed and all disposable supplies 
replaced. Suspect laboratory equipment is either discarded or removed from 
the area. 
The elimination approach is also unsatisfactory. First, it does not 
entirely render the area free of carry-over. Indeed, the cleaning process 
can, itself, generate aerosols. Second, the level of thoroughness needed 
in the cleaning requires too much time. Finally, it is not practical to 
constantly be discarding or removing laboratory equipment. 
One preventative approach to dealing with plasmid carry-over in phage 
libraries is the purification of the probe. Purifying the probe so that it 
is essentially free of plasmid DNA can reduce the incidence of 
plasmid-plasmid hybridization. 
There are a number of problems with this approach. First, while reducing 
the incidence of plasmid-plasmid hybridization, this method leaves the 
carry-over in the library. Second, purification is never 100%; the method 
can only reduce, not eliminate, the problem. This carry-over is an 
inherent problem with all cloning vectors including not only bacterial 
viruses and plasmids, but also animal and plant viruses and plasmids as 
well as the more recent technologies such as yeast chromosomal vectors. 
There is at present one preventative approach to dealing with 
recombinant-RNA probe carry-over. This involves base treatment to destroy 
RNA carry-over. This approach will not harm DNA target. However, it is 
obviously inadequate as a treatment for RNA target. 
The only prevention method for PCR carry-over that has been considered up 
to now involves the use of nested primers. While originally applied to PCR 
to improve specificity, the nested primer technique can also be applied to 
PCR as a means of reducing the problem of carry-over. Nested primers are 
primers that anneal to the target sequence in an area that is inside the 
annealing boundaries of the two primers used to start PCR. K. B. Mullis et 
al., Cold Spring Harbor Symposia, Vol. LI, pp. 263-273 (1986). When 
applied to the carry-over problem, nested primers are used that have 
non-overlapping sequences with the starting primers. Because the nested 
primers anneal to the target inside the annealing boundaries of the 
starting primers, the predominant PCR-amplified product of the starting 
primers is necessarily a longer sequence than that defined by the 
annealing boundaries of the nested primers. The PCR amplified product of 
the nested primers is an amplified segment of the target sequence that 
cannot, therefore, anneal with the starting primers. If this PCR-amplified 
product of the nested primers is the nucleic acid carried over into a 
subsequent PCR amplification, the use of the starting primers will not 
amplify this carry-over. 
There are at least two problems with the nested primer solution to 
carry-over in PCR reactions. First, the carry-over is neither removed, nor 
inactivated (inactivation is defined as rendering nucleic acid 
unamplifiable in PCR). Second, the amplified product of the nested primers 
will be amplified if the same nested primers are used in a subsequent PCR. 
Of course, another solution to carry-over in subsequent PCR amplifications 
is to use different primers altogether. This is not, however, a practical 
solution. First, making new primers for every new PCR amplification would 
be extremely time consuming and costly. Second, PCR amplification with 
each primer pair must be individually optimized. Third, for a target 
sequence of a given length, there is a limit to the number of 
non-overlapping primers that can be constructed. 
The present invention offers the first definitive method for controlling 
carry-over. These methods involve the use of compounds, including 
psoralens and isopsoralens. 
Psoralens 
Psoralens are tricyclic compounds formed by the linear fusion of a furan 
ring with a coumarin. Psoralens can intercalate between the base pairs of 
double-stranded nucleic acids, forming covalent adducts to pyrimidine 
bases upon absorption of longwave ultraviolet light. G. D. Cimino et al., 
Ann. Rev. Biochem. 54:1151 (1985). Hearst et al., Quart. Rev. Biophys. 
17:1 (1984). If there is a second pyrimidine adjacent to a 
psoralen-pyrimidine monoadduct and on the opposite strand, absorption of a 
second photon can lead to formation of a diadduct which functions as an 
interstrand crosslink. S. T. Isaacs et al., Biochemistry 16:1058 (1977). 
S. T. Isaacs et al., Trends in Photobiology (Plenum) pp. 279-294 (1982). 
J. Tessman et al., Biochem. 24:1669 (1985). Hearst et al., U.S. Pat. No. 
4,124,589 (1978). Hearst et al., U.S. Pat. No. 4,169,204 (1980). Hearst et 
al., U.S. Pat. No. 4,196,281 (1980). 
Isopsoralens 
Isopsoralens, like psoralens, are tricyclic compounds formed by the fusion 
of a furan ring with a coumarin. See Baccichetti et al., U.S. Pat. No. 
4,312,883. F. Bordin et al., Experientia 35:1567 (1979). F. Dall'Acqua et 
al., Medeline Biologie Envir. 9:303 (1981). S. Caffieri et al., Medecine 
Biologie Envir. 11:386 (1983). F. Dall'Acqua et al., Photochem Photobio. 
37:373 (1983). G. Guiotto et al., Eur. J. Med. Chem-Chim. Ther. 16:489 
(1981). F. Dall'Acqua et al., J. Med. Chem. 24:178 (1984). Unlike 
psoralens, the rings of isopsoralen are not linearly annulated. While able 
to intercalate between the base pairs of double-stranded nucleic acids and 
form covalent adducts to nucleic acid bases upon absorption of longwave 
ultraviolet light, isopsoralens, due to their angular geometry, normally 
cannot form crosslinks with DNA. See generally, G. D. Cimino et al., Ann. 
Rev. Biochem. 54:1151 (1985). Objects and advantages of the present 
invention will be apparent from the following description when read in 
connection with the accompanying figures. 
SUMMARY OF THE INVENTION 
The present invention relates to a device and method for photoactivating 
new and known compounds. The present invention further contemplates 
devices for binding new and known compounds to nucleic acid. 
In general, the present invention relates to a photoactivation device for 
treating photoreactive compounds, comprising: a) means for providing 
appropriate wavelengths of electromagnetic radiation to cause activation 
lengths of electromagnetic radiation to cause activation of at least one 
photoreactive compound; b) means for supporting a plurality of sample 
vessels in a fixed relationship with the radiation providing means during 
activation; and c) means for maintaining the temperature of the sample 
vessels within a desired temperature range during activation. In one 
embodiment, the photoactivation device further comprises means for 
controlling the radiation providing means. In one embodiment, the 
controlling means comprises a timer. 
In a preferred embodiment, the photoactivation device further comprises 
means for containing the radiation providing means, such that a user is 
shielded from said wavelengths of electromagnetic radiation. The radiation 
containing means, in one embodiment, comprises an opaque housing 
surrounding the radiation providing means. 
In a preferred embodiment, the temperature maintaining means comprises a 
chamber positioned interior to the housing and in a fixed relationship to 
the radiation providing means, and the sample vessel supporting means 
comprises intrusions in the chamber. In another preferred embodiment, the 
chamber has exterior and interior walls, the interior walls of said 
chamber form a trough, and the sample vessel supporting means comprises a 
sample rack detachably coupled to the housing above the trough. 
Alternative sample covers are contemplated to be dimensioned to overlay 
the sample rack. 
It is preferred that the chamber has inlet and outlet ports so that 
temperature control liquid may enter and exit. 
In another embodiment, a photoactivation device for treating photoreactive 
compounds, comprises: a) means for providing electromagnetic radiation, 
having a wavelength cutoff at 300 nanometers, to cause activation of at 
least one photoreactive compound; b) means for supporting a plurality of 
sample vessels in a fixed relationship with the radiation providing means 
during activation; and c) means for maintaining the temperature of the 
sample vessels within a desired temperature range during activation. 
In still another embodiment, the photoactivation device for treating 
photoreactive compounds, comprises: 1) a fluorescent source of ultraviolet 
radiation having wavelengths capable of causing activation of at least one 
photoreactive compound; b) means for supporting a plurality of sample 
vessels in a fixed relationship with the fluorescent radiation source 
during activation; and c) means for maintaining the temperature of the 
sample vessels within a desired temperature range during activation. 
In still another embodiment, the photoactivation device for treating 
photoreactive compounds, comprises: a) a fluorescent source of ultraviolet 
radiation having wavelengths capable of causing activation of at least one 
photoreactive compound; b) means for supporting a plurality of sample 
vessels positioned with respect to the fluorescent source, so that, when 
measured for the wavelengths between 300 and 400 nanometers, an intensity 
flux grater than 15 mW cm.sup.-2 is provided to the sample vessels; and c) 
means for maintaining the temperature of the sample vessels within a 
desired temperature range during activation. 
In still another embodiment, the photoactivation device for treating 
photoreactive compounds comprises: a) means for continuously flowing 
sample liquid containing photoreactive compound; and b) means for 
providing appropriate wavelengths of electromagnetic radiation in a fixed 
relationship with said continuous flowing means to cause activation of at 
least one photoreactive compound. In one embodiment, the continuous flow 
photoactivation device fruther comprises means for maintaining the 
temperature of the continuously flowing sample liquid within a desired 
temperature range during activation. In another embodiment, the continuous 
flow photoactivation fruther comprises means for containing the radiation 
providing means, such that a user is shielded from wavelengths of 
electromagnetic radiation. The radiation containing means, in one 
embodiment, comprises an opaque housing surrounding the radiation 
providing means. In one embodiment, the continuous flowing means comprises 
a chamber interior to the housing and positioned in a fixed relationship 
to the radiation providing means. The continuous flow photoactivation 
device chamber, in one embodiment, has inlet and outlet ports so that 
sample liquid may enter and exit. 
The present invention also contemplates a method for photoactivating 
photoreactive compounds, comprising: a) supporting a plurality of sample 
vessels, containing one or more photoreactive compounds, in a fixed 
relationship with a fluorescent source of electromagnetic radiation; b) 
irradiating the sample vessels simultaneously with electromagnetic 
radiation to cause activation of at least one photoreactive compound; and 
c) maintaining the temperature of sample vessels within a desired 
temperature range during activation.

DESCRIPTION OF THE INVENTION 
The present invention relates to: i) new methods of synthesis of known 
compounds, ii) new compounds and methods of synthesis of new compounds, 
iii) methods for binding new and known compounds to nucleic acid, and iv) 
methods for using new and known compounds to inhibit template-dependent 
enzymatic synthesis of nucleic acid. 
The description of the invention is divided into: I) Compound Synthesis, 
II) Photoactivation Devices and Methods, III) Binding of Compounds to 
Nucleic Acid, IV) Capture of Nucleic Acid, V) Inhibiting 
Template-Dependent Enzymatic Synthesis, and VI) Sterilization. 
I. COMPOUND SYNTHESIS 
"Activation compounds" defines a family of compounds that undergo chemical 
change in response to triggering stimuli. Triggering stimuli include, but 
are not limited to, thermal stimuli, chemical stimuli and electromagnetic 
stimuli. "Photoreactive, activation compounds" (or simply "photoreactive 
compounds"), defines a genus of compounds in the activation compound 
family that undergo chemical change in response to electromagnetic 
radiation (Table 1). 
TABLE 1 
Photoreactive Compounds 
Actinomycins 
Anthracyclinones 
Anthramycin 
Benzodipyrones 
Fluorenes And Fluorenones 
Furocoumarins 
Mitomycin 
Monostral Fast Blue 
Norphillin A 
Organic Dyes 
Phenanthridines 
Phenazathionium Salts 
Phenazines 
Phenothiazines 
Phenylazides 
Polycyclic Hydrocarbons 
Quinolines 
Thiaxanthenones 
One species of photoreactive compounds described herein is commonly 
referred to as the furocoumarins. The furocoumarins belong to two main 
categories: 1) psoralens 7H-furo(3,2-g)-(1)-benzopyran-7-one, or 
.delta.-lactone of 6-hydroxy-5-benzofuranacrylic acid!, which are linear: 
##STR1## 
and in which the two oxygen residues appended to the central aromatic 
moiety have a 1,3 orientation, and further in which the furan ring moiety 
is linked to the 6 position of the two ring coumarin system, and 2) the 
isopsoralens 2H-furo(2,3-h)-(1)-benzopyran-2-one, or .delta.-lactone of 
4-hydroxy-5-benzofuranacrylic acid!, which are angular: 
##STR2## 
in which the two oxygen residues appended to the central aromatic moiety 
have a 1,3 orientation, and further in which the furan ring moiety is 
linked to the 8 position of the two ring coumarin system. Psoralen 
derivatives are derived from substitution of the linear furocoumarin at 
the 3, 4, 5, 8, 4', or 5' positions, while isopsoralen derivatives are 
derived from substitution of the angular furocoumarin at the 3, 4, 5, 6, 
4', or 5 positions. 
Tables 2 and 3 set forth the nomenclature used for the furocoumarin 
derivatives discussed herein. FIGS. 1 and 2 set forth the overall scheme 
for the synthesis of the furocoumarin derivatives of the present 
invention. 
The present invention contemplates labelled and unlabelled furocoumarin 
derivatives. FIGS. 1 and 2 set forth how each furocoumarin derivative may 
be labelled. Where both an unlabelled and radiolabelled version of a 
compound may be synthesized by methods of the present invention, the 
radiolabel is indicated in parentheses. 
If labelled, the compound will have at least one label attached or 
integrated into its structure. Labels are generally intended to facilitate 
i) the detection of the inhibiting compounds, as well as ii) the detection 
of molecules bound to the inhibiting compounds (e.g., nucleic acid). 
Labels are chosen from the group consisting of enzymes, fluorophores, 
high-affinity conjugates, chemiphores and radioactive atoms 
TABLE 2 
______________________________________ 
Furocoumarin Derivatives (MIP Series) 
# Compound Abbrev. 
______________________________________ 
1 7-Hydroxy-5-methylcoumarin 
H5MC 
2 7-(2,2-diethoxyethyloxy)-5-methylcoumarin 
DEMC 
3 5-Methylisopsoralen MIP 
4 4',5'-H.sub.2 !-4',5'-dihydro-5-methyl-isopsoralen 
DHMIP 
5 5-Halomethylisopsoralen XMIP 
5-Bromomethylisopsoralen 
BMIP 
5-Chloromethylisopsoralen 
CMIP 
6 5-Hydroxymethylisopsoralen 
HMIP 
7 5-Formylisopsoralen FIP 
8 5-Iodomethylisopsoralen IMIP 
9 5-Hexamethylenetetraminomethylisopsoralen 
HMTAMIP 
10 5-Aminomethylisopsoralen 
AMIP 
11 5-N-(N,N'-Dimethyl-1,6-hexanediamine)- 
DMHMIP 
methyl-isopsoralen 
12a 5-N-N,N'-Dimethyl-(6-biotinamido!- 
BIOMIP 
hexanoate)-1,6-hexanediamine!)- 
methyl-isopsoralen 
12b 5-N-N,N'-dimethyl-N'-(2-{biotinamido}- 
DITHIOMIP 
ethyl-1,3-dithiopropionate)-1,6- 
hexanediamine!-methyl-isopsoralen 
12c 5-N-N,N'-dimethyl-N'-(carboxyfluorescein 
FLUORMIP 
ester)-1,6-hexanediamine)- 
methyl-isopsoralen 
______________________________________ 
TABLE 3 
______________________________________ 
Furocoumarin Derivatives (DMIP Series) 
13 7-Hydroxy-4-methylcoumarin 
H4MC 
14 7-(.beta.-haloallyloxy)-4-methylcoumarin 
XAMC 
7-(.beta.-chloroallyloxy)-4-methylcoumarin 
CAMC 
15 7-Allyloxy-6-(.beta.-haloallyl)-4-methylcoumarin 
RXAMC 
7-Butyroxy-6-(.beta.-chloroallyl)-4- 
BCAMC 
methylcoumarin 
16 4,5'-Dimethylisopsoralen DMIP 
17 4',5'-.sup.3 H.sub.2 !-4',5'-dihydro-4,5'- 
DHDMIP 
dimethylisopsoralen 
18 4'-Halomethyl-4,5'-dimethylisopsoralen 
XMDMIP 
4'-Chloromethyl-4,5'-dimethylisopsoralen 
CMDMIP 
4'-Bromomethyl-4,5'-dimethylisopsoralen 
BMDMIP 
19 4'-Hydroxymethyl-4,5'-dimethylisopsoralen 
HMDMIP 
20 4'-Formyl-4,5'-dimethylisopsoralen 
FDMIP 
21 4'-Phthalimidomethyl-4,5'- 
PHIMDMIP 
dimethylisopsoralen 
22 4'-Aminomethyl-4,5'-dimethylisopsoralen 
AMDMIP 
23 4'-Iodomethyl-4,5'-dimethylisopsoralen 
IMDMIP 24 
4'-N-(N,N'-dimethyl-1,6-hexanediamine)- 
HDAMDMIP 
methyl-4,5'-dimethylisopsoralen 
25a 4'-N-N,N'-dimethyl-N'-(6-{biotinamido}- 
BIODMIP 
hexanoate)-1,6-hexanediamine!-methyl-4,5'- 
dimethylisopsoralen 
25b 4'-N-N,N'-dimethyl-N'-(2-{biotinamido}- 
DITHIODMIP 
ethyl-1,3-dithiopropionate)-1,6- 
hexanediamine!-methyl-4,5'- 
dimethylisopsoralen 
25c 4'-N-N,N'-dimethyl-N'-(6-carboxyfluorescein 
FLUORDMIP 
ester)-1,6-hexanediamine)-methyl- 
4,5'-dimethylisopsoralen 
______________________________________ 
("radiolabels"). While others may be used, 1) enzymes contemplated include 
alkaline phosphatase, .beta.-galactosidase and glucose oxidase, 2) an 
affinity conjugate system contemplated is the biotin-avidin system, 3) 
fluorescein is contemplated as a fluorophore, 4) lumninol is contemplated 
as chemiphore, and 5) the preferred radiolabels contemplated by the 
present invention include .sup.3 H and .sup.14 C. 
It is not intended that the present invention be limited by the nature of 
the label used. The present invention contemplates single labelling (e.g., 
a radiolabel, a fluorophore, etc.) and double labelling (e.g., two 
radiolabels, a radiolabel and a fluorphore, etc.). 
While not limited to any particular label, a preferred label of the present 
invention for facilitating the detection of compounds is tritium (.sup.3 
H). A preferred label of the present invention for facilitating the 
detection of molecules bound to the compounds is biotin. While FIGS. 1 and 
2 have been drafted to show these preferred labels (as well as some other 
labels), it is not intended thereby to limit the present invention. 
As shown in FIGS. 1 and 2, the synthesis pathway for the compounds of the 
present invention involves starting with: 
##STR3## 
where R is either --CH.sub.3 or --H, respectively. 
Where R is --CH.sub.3 (FIG. 1; Table 2), the starting compound is 
5-methylresorcinol. Where R is --H (FIG. 2; Table 3), the starting 
compound is resorcinol. Accordingly, the description of the compound 
synthesis methods of the present invention proceeds in two parts. 
A. T ONE: R EQUALS --CH.sub.3 
Where R is --CH.sub.3 (FIG. 1; Table 2), the synthesis proceeds via one of 
two new synthesis methods to MIP (Compound #3), a known compound. One of 
these novel synthesis methods for MfP proceeds via new compound DEMC 
(Compound #2). 
After MIP is formed, the synthesis can continue on to create i) new 
compounds XMIP (Compound #5), HMIP (Compound #6), FMIP (Compound #7), IMIP 
(Compound #8), HMTAMIP (Compound #9), AMIP (Compound #10), DMHMIP 
(Compound #11), BIOMIP (Compound #12a), DITHIOMIP (Compound 12b), and/or 
FLUORMIP (Compound 12c), or ii) radiolabelled compounds. (In FIG. 1, 
radiolabels are indicated in parentheses where the compound can be 
synthesised unlabelled as well as labelled.) In addition to the tritiated 
compounds indicated in FIG. 1, the analogous .sup.14 C derivatives may be 
prepared (i.e., .sup.14 C labelled 5-methylresorcinol). 
All methods for synthesizing new compounds AMIP, BIOMIP, DITHIOMIP and 
FLUORMIP proceed via new compound intermediate XMIP. XMIP is defined as 
either CMIP or BMIP. Some methods for synthesizing AMIP, BIOMIP, DITHIOMIP 
and FLUORMIP proceed from XMIP through new compound IMIP (Compound #8). 
The synthesis methods of the present invention where R equals --CH.sub.3 
begins with novel synthesis methods for MIP. 
1) MIP Synthesis 
The invention contemplates novel approaches to the synthesis of MIP and/or 
labelled MIP prior to the synthesis of novel MIP derivatives. 
Two new methods are provided for the synthesis of MIP (FIG. 1). The first 
step of the first method for MIP synthesis involves a reaction of 
5-methylresorcinol with malic acid to yield H5MC (Compound #1). The second 
step of the first method involves a reaction of H5MC with a 
haloacetaldehyde diethylacetal to yield the diethoxyethylether of H5MC, 
DEMC (Compound #2). The haloacetaldehyde diethylacetal can be chloro-, 
iodo- or bromo-acetaldehyde diethylacetal. In the third step of the first 
method, DEMC is treated to close the ring to yield the two isomers, 
5-methylpsoralen and MIP, which are separated to isolate pure MIP 
(Compound #3). 
The first step of the second method for MIP synthesis is identical to the 
first step of the first method. The second step of the second method, 
however, involves the synthesis of MIP directly from H5MC (i.e., compound 
#2 to #3) via a haloethylene carbonate. 
1') Radiolabelled MIP Synthesis 
Methods are provided for the synthesis of radiolabelled MIP (FIG. 1). These 
methods build on the two methods of MIP synthesis with additional known 
steps: 1) catalytic hydrogenation of the 4',5' (furan-side) double bond 
using tritium gas to provide the tritiated compound, DHMIP, followed by 2) 
catalytic reoxidation of this bond with a hydrogen donor to yield the 
tritiated MIP (.sup.3 H-MIP). S. Isaacs et al., Nat. Cancer Inst. 
Monograph No. 66 (1985). Alternatively, the reaction can be continued 
until catalytic hydrogenation of the 3,4 (pyrone-side) double bond 
resulting in the formation of the 3,4,4',5'-tetrahydro-.sup.3 H.sub.4 
!-5-methylisopsoralen (THMIP). THMIP has the advantage of allowing for 
compounds of higher specific activity and the disadvantage of poor yield 
relative to DHMIP (for convenience only, DHMIP is shown in FIG. 1). 
The present invention contemplates that the catalyst is selected from the 
group consisting of palladium on charcoal, palladium on barium sulfate, 
Adams catalyst (NH.sub.4).sub.2 PtCl.sub.6 !, PtO.sub.2, rhodium, 
ruthenium, copper chromite and Raney nickel. The present invention 
contemplates that the hydrogen donor in the reoxidation step is selected 
from the group consisting of diphenylether and cyclohexene. 
2) XMIP Synthesis 
As noted above, all methods for synthesizing new compounds AMIP, BIOMIP, 
DITHIOMIP and FLUORMIP proceed via new compound XMIP as an intermediate. 
XMIP is defined as a halomethylisopsoralen selected from the group 
consisting of bromomethylisopsoralen (BMIP) and chloromethylisopsoralen 
(CMIP). 
The synthesis method of XMIP of the present invention is a free radical 
halogenation of MIP with an N-halosuccinimide and a peroxide initiator. 
The preferred N-halosuccinimide is N-bromosuccinimide but the present 
invention contemplates the use of N-chlorosuccinimide as well. 
2') Radiolabelled XMIP Synthesis 
The present invention contemplates radiolabelled XMIP. Methods are provided 
for the synthesis of radiolabelled XMIP (FIG. 1). In one embodiment, the 
method builds on the methods of synthesizing radiolabelled MIP (e.g., #3 
to #4*, #4* to #3*, and #3* to #5*, where * indicates a radiolabelled 
compound). In another embodiment, the methods proceed via new compounds 
HMIP and FMIP (e.g., #5 to #6, #6 to #7, #7 to #6*, and #6* to #5*, where 
* indicates a radiolabelled compound). Combining the radiolabelling steps 
for MIP with the HMIP/FMIP radiolabelling method provides for 
double-radiolabelling of XMIP (e.g., #3 to #4*, #4* to #3*, and #3* to 
#5*, #5* to #6*, #6* to #7*, #7* to #6**, and #6** to #5**, where ** 
indicates a double-radiolabelled compound). 
3) AMIP Synthesis 
As shown in FIG. 1, four alternative synthesis methods are provided when 
proceeding via new compound intermediate XMJP as starting material to new 
compound AMIP. One method proceeds in four steps from XMJP to new compound 
AMIP via new compound intermediate HMIP (i.e., compound #5 to #6, #6 to 
#5, #5 to #9, and #9 to #10). Another method proceeds in five steps from 
XMIP to new compound AMIP via new compound intermediates HMIP and IMIP 
(i.e., #5 to #6, #6 to #5, #5 to #8, #8 to #9, and #9 to #10) Still 
another proceeds in two steps from XMIP to new compound AMIP (i.e., 
compound #5 to #9, and #9 to #10). Still another proceeds in three steps 
from XMIP to new compound AMIP via new compound IMIP (i.e., #5 to #8, #8 
to #9, #9 to #10). 
Methods one and two allow the synthesis to be interrupted at new compound 
intermediate HMIP, which is stable and can be stored indefinitely without 
decomposition. The third method (the preferred method) proceeds in two 
steps from XMIP to new compound AMIP. While this third method offers the 
most direct route to new compound AMIP, it is inappropriate if stopping 
the synthesis sequence prior to completion is anticipated. This follows 
from the hydrolytic instability of XMIP, which must be maintained in a 
strictly inert environment to prevent hydrolytic decomposition. Again, 
XMIP is selected from the group bromomethylisopsoralen (BMIP) and 
chloromethylisopsoralen (CMIP) (instability increases in the order 
BMIP&gt;CMIP). 
In the four step method and the five step method, XMIP may be the same 
halomethylisopsoralen or may be a different isopsoralen. (In general, the 
reactivity of XMIP will increase as X is changed from chloro to bromo; a 
significant advantage of higher reactivity is correspondingly shorter 
reaction times for conversions such as XMIP.fwdarw.HMIP.) 
In the fourth method for synthesizing AMIP, the synthesis proceeds via 
IMIP. In this regard, it is known that benzyl iodides are more reactive 
than the corresponding bromides or chlorides, which follows from the 
relative ability of each halide to act as a leaving group in an S.sub.N 2 
(second order, nucleophilic displacement) reaction. Accordingly, to take 
advantage of the resulting high reactivity and corresponding short 
reaction times provided by the benzyl iodide analog, the new compound IMIP 
is prepared in the method of the present invention via the Finkelstein 
reaction. 
When combined with the two methods for producing MIP, the present invention 
provides eight methods for synthesizing AMIP from 5-methylresorcinol: 
I #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6to #5, #5 to #9, and #9 to #10. 
II #1 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #9, and #9 to #10. 
III #1 to #2, #2 to #3, #3 to #5, #5 to #9, and #9 to #10. 
IV #1 to #3, #3 to #5, #5 to #9, and #9 to #10 
V #1 to #2, #2 to #3, #3 to #5, #5 to #8, #8 to #9, and #9 to #10. 
VI #1 to #3, #3 to #5, #5 to #8, #8 to #9, and #9 to #10. 
VII #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #8, #8 to #9, 
and #9 to #10. 
VIII #1 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #8, #8 to #9, and #9 to 
#10. 
3') Radiolabelled AMIP Synthesis 
FIG. 1 also shows two methods provided for proceeding via new compound 
intermediate XMIP as starting material to new compound, radiolabelled 
AMIP. Both of the methods proceeding via new compound intermediate XMIP as 
starting material to new compound, radiolabelled AMIP, proceed via HMIP 
and new compound FMIP. One method is a six step method (i.e., compound #5 
to #6, #6 to #7, #7 to #6*, #6* to #5*, #5* to #9*, and #9* to #10*); the 
other is a seven step method (i.e., compound #5 to #6, #6 to #7, #7 to 
#6*, #6* to #5*, #5* to #8*, #8* to #9*, and #9* to #10*). 
When combined with the two methods for producing MIP, the present invention 
provides two additional methods (for a total of four methods) for 
synthesizing radiolabelled AMIP from 5-methylresorcinol; when combined 
with the two methods for producing radiolabelled MIP, the present 
invention provides eight additional methods for producing radiolabelled 
AMIP for a total of twelve methods: 
I #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to 
#5*, #5* to #9*, and #9* to #10*. 
II #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to #5*, #5* 
to #9*, and #9* to #10*. 
III #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* 
to #5*, #5* to #8*, #8* to #9*, and #9* to #10*. 
IV #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to #5*, #5* 
to #8*, #8* to #9*, and #9* to #10*. 
V #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #9*, and 
#9* to #10*. 
VI #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #9*, and #9* to 
#10*. 
VII #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #8*, #8* 
to #9*, and #9* to #10*. 
VIII #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #8*, #8* to #9*, 
and #9* to #10*. 
IX #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to 
#5*, #5* to #9*, and #9* to #10*. 
X #1 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to #5*, #5* to 
#9*, and #9* to #10*. 
XI #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to 
#5*, #5* to #8*, #8* to #9*, and #9* to #10*. 
XII #1 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to #5*, #5* to 
#8*, #8* to #9*, and #9* to #10*. 
where * indicates a labelled compound. Methods V, VI, IX and X are 
preferred. 
The present invention also contemplates double-labelling. In one 
embodiment, the double-labelling method of the present invention involves 
the combination of the labelling steps for MIP (Compound #3 to Compound 
#4) and the labelling steps for AMIP. Where the label is a radiolabel, 
this provides, among other advantages, the advantage of increasing the 
specific activity of the compounds of the present invention. The present 
invention contemplates the following double-radiolabelling methods (where 
** indicates a double-labelled compound): 
I #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, to #5* to #6*, #6* 
to #7*, #7* to #6**, #6** to #5**, #5** to #9**, and #9** to #10**. 
II #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to #7*, #7* 
to #6**, #6** to #5**, #5** to #9**, and #9** to #10**. 
III #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, to #5* to #6*, 
#6* to #7*, #7* to #6**, #6** to #5**, #5** to #8**, #8** to #9**, and 
#9** to #10**. 
IV #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to #7*, #7* 
to #6**, #6** to #5**, #5** to #8**, #8** to #9**, and #9** to #10**. 
Methods I and II are preferred. 
4) BIOMIP, DITHIOMIP and FLUORMIP Synthesis 
The BIO-, DITHIO- and FLUOR- derivatives of MIP of the present invention 
(compounds #12a, 12b, and 12c, respectively) can each be generally 
described as a three part compound consisting of the following three 
units: 
MIP--SER--LABEL 
The spacer contemplated by the present invention has the general formula 
R.sub.1 HN--(CH.sub.2).sub.n --NHR.sub.2. In general, R.sub.1 =--H, 
--CH.sub.3, --C.sub.2 H.sub.5, --C.sub.3 H.sub.7 or --C.sub.4 H.sub.9, 
R.sub.2 =--H or --CH .sub.3, --C.sub.2 H.sub.5, --C.sub.3 H.sub.7 or 
--C.sub.4 H.sub.9, and n is between 6 and 16, inclusive. It is 
contemplated that, where the BIOMIP compound is bound to another molecule 
(e.g., nucleic acid), sufficient length is provided for the biotin moiety 
to span the distance between the site of attachment to another molecule 
and the avidin binding site when n.ltoreq.6. Since the biotin binding site 
is reported to be 9 .ANG. below the surface of the avidin molecule Green 
et al., Biochem. J. 125:781 (1971)!, shorter spacers see e.g., J. P. 
Albarella et al., Nucleic Acids Res. 17:4293 (1983)! may hinder the 
formation of the biotin-avidin complex. Adequate chain length helps reduce 
steric hinderance associated with the avidin-biotin interaction, and 
accordingly, the stability of the avidin-biotin complex should increase 
when the appropriate chain length is employed. 
Chemical (synthetic) considerations come into play when considering the 
preferred spacer for the BIO-, DITHIO- and FLUOR- derivatives of MIP. 
While for spacer R.sub.1 HN--(CH.sub.2).sub.n --NHR.sub.2, R.sub.1 can be 
--H, --CH.sub.3, --C.sub.2 H.sub.5, --C.sub.3 H.sub.7 or --C.sub.4 
H.sub.9, and R.sub.2 can be --H, --CH.sub.3, .sub.--c 2H.sub.5, C.sub.3 
H.sub.7 or C.sub.4 H.sub.9, most preferably R.sub.1 and R.sub.2 are both 
--CH.sub.3, .sub.--c 2H.sub.5, --C.sub.3 H.sub.7 or C.sub.4 H.sub.9. While 
not limited to any particular theory, in the reaction to prepare DMHMIP 
from XMIP (or IMIP) where R.sub.1 and R.sub.2 are both --CH.sub.3, the 
spacer nitrogens can react at either nitrogen with only one or two 
equivalents of XMIP (or IMIP). 
So that the desired mono-N-substituted product (i.e., DMHMIP) is favored, 
the present invention contemplates that a high ratio of spacer to XMIP (or 
IMIP) is employed in the reaction. Nonetheless, even where 1) R.sub.1 and 
R.sub.2 are both --CH.sub.3, --C.sub.2 H.sub.5, --C.sub.3 H.sub.7 or 
--C.sub.4 H.sub.9, and 2) a high ratio of spacer to XMIP (or IMIP) is 
employed, the present invention contemplates side products from the 
reaction of more than one XMIP (or IMIP) with the spacer. These side 
products include one di-N,N-substituted product (i.e., two isopsoralens at 
the same nitrogen on the spacer), one di-N,N'- substituted product (i e., 
two isopsoralens at each of the spacer nitrogens), one 
tri-N,N,N'-substituted product and one tetra-N, N, N', N'-substituted 
product. 
As noted, the present invention does contemplate the case where R.sub.1 and 
R.sub.2 are --H. While this spacer can be used, the number of possible 
multi-substituted spacer side products is increased, making subsequent 
purification of the desired mono-N-substituted product (i.e., DMHMIP) more 
difficult. 
The label on the BIO-, DITHIO- and FLUOR- derivatives of MIP of the present 
invention is comprised of two elements: 1) the reporter moiety, and 2) the 
linking arm which binds the reporter moiety to the spacer. Two types of 
reporter moieties are shown in FIG. 1. The first, biotin, is an indirect 
reporter moiety, as it functions to bind avidin, which in turn is attached 
to the signal generating system (e.g., BluGENE; BRL). The second, 
fluorescein, is a direct reporter moiety which provides a highly 
fluorescent signal upon excitation with appropriate wavelengths of light. 
Both biotin and fluorescein are appended to the spacer via an amide bond, 
with zero to seven bridging atoms making up the linking arm between the 
spacer amido carbonyl and the reporter moiety. In some cases (e.g., 
DITHIOMIP), the linking arm may contain a disulfide linkage, which is 
useful for subsequent cleavage of the reporter moiety from the 
isopsoralen. 
The reaction to form the amide bond between the spacer nitrogen and the 
label carbonyl uses an activated ester, preferably the 
N-hydroxy-succinimide ester. Other active esters, however, are 
contemplated, such as the imidazolides (from N, N'-carbonyldiimidizoles) 
and the sulfosuccinimidyl esters. 
a) BIOMIP Synthesis 
As shown in FIG. 1, the present invention contemplates four alternative 
synthesis methods for proceeding via new compound intermediate XMIP to new 
compound BIOMIP (Compound #12a). Two of the four methods proceed via HMIP; 
one proceeds from HMIP via IMIP (i.e., #5 to #6, #6 to #5, #5 to #8, #8 to 
#11, and #11 to #12a) and one proceeds from HMIP via DMHMIP (i.e., #5 to 
#6, #6 to #5, #5 to #11, and #11 to #12a). The other two methods proceed 
directly from XMIP (i.e., without HMIP); one proceeds from XMIP via IMIP 
(i.e., #5 to #8, #8 to #11, and #11 to #12a) and one proceeds from XMIP 
via DMHMIP (i.e., #5 to #11, and #11 to #12a). The latter is preferred. 
As discussed above for the synthesis of AMIP, the routes via HMIP offer the 
advantage of allowing for interruptions in the synthesis (often necessary 
in a production facility) because of the stability of HMIP. The XMIP route 
is more direct, however, and should be used where continued synthesis is 
possible. 
When combined with the two methods for synthesizing MIP, the present 
invention provides eight methods for synthesizing BIOMIP (methods III and 
IV are preferred): 
I #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #11, and #11 to 
#12a. 
II #1 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #11, and #11 to #12a. 
III #1 to #2, #2 to #3, #3 to #5, #5 to #11, and #11 to #12a. 
IV #1 to #3, #3 to #5, #5 to #11, and #11 to #12a. 
V #1 to #2, #2 to #3, #3 to #5, #5 to #8, #8 to #11, and #11 to #12a. 
VI #1 to #3, #3 to #5, #5 to #8, #8 to #11, and #11 to #12a. 
VII #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #8, #8 to #11, 
and #11 to #12a. 
VIII #1 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #8, #8 to #11, and #11 
to #12a. 
a') Radiolabelled BIOMIP Synthesis 
The present invention further contemplates synthesis methods for proceeding 
via new compound intermediate XMIP to radiolabelled BIOMIP. Both methods 
involve synthesis of HMIP and labelled HMIP. One proceeds via IMIP (i.e., 
#5 to #6, #6 to #7, #7 to #6*, #6* to #5*, #5* to #8*, #8* to #11*, and 
#11* to #12a*) and one proceeds directly via DMHMIP (i.e., #5 to #6, #6 to 
#7, #7 to #6*, #6* to #5*, #5* to #11*, and #11 * to #12a*). 
When combined with the two methods to produce MIP and two methods to 
produce labelled MIP, the present invention provides twelve methods for 
synthesizing radiolabelled BIOMIP (methods V, VI, IX and X are preferred): 
I #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to 
#5*, #5* to #11*, and #11* to #12a*. 
II #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to #5*, #5* 
to #11*, and #11* to #12a*. 
III #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* 
to #5*, #5* to #8*, #8* to #11*, and #11* to #12a*. 
IV #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to #5*, #5* 
to #8*, #8* to #11*, and #11* to #12a*. 
V #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #11*, and 
#11* to #12a*. 
VI #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #11*, and #11* to 
#12a*. 
VII #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #8*, #8* 
to #11*, and #11* to #12a*. 
VIII #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #8*, #8* to #11*, 
and #11* to #12a*. 
IX #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to 
#5*, #5* to #11*, and #11* to #12a*. 
X #1 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to #5*, #5* to 
#11*, and #11* to #12a*. 
XI #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to 
#5*, #5* to #8*, #8* to #11*, and #11* to #12a*. 
XII #1 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to #5*, #5* to 
#8*, #8* to #11*, and #11* to #12a*. 
These twelve methods of radiolabelling BIOMIP offer one approach to 
doublelabelling (the compound has both .sup.3 H and biotin). As with 
labelled AMIP, the present invention also contemplates 
double-radiolabelling of BIOMIP (in this case, however, to create a 
triple-labelled compound). The double-radiolabelling method combines the 
radiolabelling steps for MIP with the radiolabelling steps for BIOMIP. 
b) DITHIOMIP Synthesis 
As shown in FIG. 1, the present invention contemplates four alternative 
synthesis methods for proceeding via new compound intermediate XMIP to new 
compound DITHIOMIP (Compound #12b). As with the synthesis for BIOMIP, two 
of the four methods proceed via HMIP; one proceeds from HMIP via IMIP 
(i.e., #5 to #6, #6 to #5, #5 to #8, #8 to #11, and #11 to #12b) and one 
proceeds from HMIP via DMHMIP (i.e., #5 to #6, #6 to #5, #5 to #11, and 
#11 to #12b). The other two methods proceed directly from XMIP (i.e., 
without HMIP); one proceeds from XMIP via IMIP (i.e., #5 to #8, #8 to #11, 
and #11 to #12b) and one (the preferred) proceeds from XMIP via DMHMIP 
(i.e., #5 to #11, and #11 to #12b). The HMIP route advantages discussed 
above must again be balanced with the more direct routes. 
When combined with the two methods for synthesizing MIP, the present 
invention provides eight methods for synthesizing DITHIOMIP (methods III 
and IV are preferred): 
I #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #11, and #11 to 
#12b. 
II #1 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #11, and #11 to #12b. 
III #1 to #2, #2 to #3, #3 to #5, #5 to #11, and #11 to #12b. 
IV #1 to #3, #3 to #5, #5 to #11, and #11 to #12b. 
V #1 to #2, #2 to #3, #3 to #5, #5 to #8, #8 to #11, and #11 to #12b. 
VI #1 to #3, #3 to #5, #5 to #8, #8 to #11, and #11 to #12b. 
VII #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #8, #8 to #11, 
and #11 to #12b. 
VIII #1 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #8, #8 to #11, and #11 
to #12b. 
b') Radiolabelled DITHIOMIP Synthesis 
The present invention further contemplates synthesis methods for proceeding 
via new compound intermediate XMIP to radiolabelled DITHIOMIP. Both 
methods involve synthesis of HMIP and labelled HMIP. One proceeds via IMIP 
(ie., #5 to #6, #6 to #7, #7 to #6*, #6* to #5*, #5* to #8*, #8* to #11*, 
and #11* to #12b*) and one proceeds directly via DMHMIP (i.e., #5 to #6, 
#6 to #7, #7 to #6*, #6* to #5*, #5 to #11*, and #11* to #12b*). 
When combined with the two methods to produce MIP and two methods to 
produce labelled MIP, the present invention provides twelve methods for 
synthesizing radiolabelled DITHIOMIP (methods V, VI, IX and X are 
preferred): 
I #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to 
#5*, #5* to #11*, and #11* to #12b*. 
II #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to #5*, #5* 
to #11*, and #11* to #12a*. 
III #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* 
to #5*, #5* to #8*, #8* to #11*, and #11* to #12b*. 
IV #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to #5*, #5* 
to #8*, #8* to #11*, and #11* to #12b*. 
V #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #11*, and 
#11* to #12b*. 
VI #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #11*, and #11* to 
#12*b. 
VII #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #8*, #8* 
to #11*, and #11* to #12b*. 
VIII #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #8* to #11*, and #11* to 
#12b*. 
IX #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to 
#5*, #5* to #11*, and #11* to #12b*. 
X #1 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to #5*, #5* to 
#11*, and #11* to #12b*. 
XI #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to 
#5*, #5* to #8*, #8* to #11*, and #11* to #12b*. 
XII #1 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to #5*, #5* to 
#8*, #8* to #11*, and #11* to #12b*. 
These twelve methods of radiolabelling DITHIOMIP offer one approach to 
double-labelling (the compound has both .sup.3 H and cleavable biotin). As 
with labelled BIOMIP, the present invention also contemplates 
double-radiolabelling of DITHIOMIP (creating a triple-labelled compound). 
The double-radiolabelling method combines the radiolabelling steps for MIP 
with the radiolabelling steps for DITHIOMIP. 
c) FLUORMIP Synthesis 
As shown in FIG. 1, the present invention contemplates four alternative 
synthesis methods for proceeding via new compound intermediate XMIP to new 
compound FLUORMIP (Compound #12c). As with both BIOMIP and DITHIOMIP, two 
of the four methods proceed via HMIP; the other two methods proceed 
directly from XMIP. When combined with the two methods for synthesizing 
MIP, the present invention provides eight methods for synthesizing 
FLUORMIP (methods III and IV are preferred): 
I #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #11, and #11 to 
#12c. 
II #1 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #11, and #11 to #12c. 
III #1 to #2, #2 to #3, #3 to #5, #5 to #11, and #11 to #12c. 
IV #1 to #3, #3 to #5, #5 to #11, and #11 to #12c. 
V #1 to #2, #2 to #3, #3 to #5, #5 to #8, #8 to #11, and #11 to #12c. 
VI #1 to #3, #3 to #5, #5 to #8, #8 to #11, and #11 to #12c. 
VII #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #8, #8 to #11, 
and #11 to #12c. 
VIII #1 to #3, #3 to #5, #5 to #6, #6 to #5, #5 to #8, #8 to #11, and #11 
to #12c. 
c') Radiolabelled FLUORMIP Synthesis 
The present invention further contemplates synthesis methods for proceeding 
via new compound intermediate XMIP to radiolabelled FLUORMIP. As with 
BIOMIP and DITHIOMIP, both methods involve synthesis of HMIP and labelled 
HMIP. 
When combined with the two methods to produce MIP and two methods to 
produce labelled MIP, the present invention provides twelve methods for 
synthesizing radiolabelled FLUORMIP (methods V, VI, IX and X are 
preferred): 
I #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to 
#5*, #5* to #11*, and #11* to #12c*. 
II #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, 6* to #5*, #5* 
to #11* to #12c*. 
III #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, 5* to #6*, #6* 
to #5*, #5to #8*, #8*to #11*, and #11 to #12c*. 
IV #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #6*, #6* to #5*, #5* 
to #8*, #8* to #11*, and #11* to #12c*. 
V #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #11*, and 
#11* to #12c*. 
VI #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #11*, and #11* to 
#12c*. 
VII #1 to #2, #2 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #8*. #8* 
to #11*, and #11* to #12c. 
VIII #1 to #3, #3 to #4*, #4* to #3*, #3* to #5*, #5* to #8*, #8* to #11*, 
and #11* to #12c*. 
IX #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to 
#5*, #5* to 11*, and #11* to #12c*. 
X #1 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to #5*, #5* to 
#11*, and #11* to #12c*. 
XI #1 to #2, #2 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to 
#5*, #5* to #8*, #8* to #11*, and #11* to #12c*. 
XII #1 to #3, #3 to #5, #5 to #6, #6 to #7*, #7* to #6*, #6* to #5*, #5* to 
#8*, #8* to #11*, and #11* to #12c*. 
These twelve methods of radiolabelling FLUORMIP offer one approach to 
double-labelling (the compound has both .sup.3 H and fluorescein). The 
present invention also contemplates double-radiolabelling of FLUORMIP to 
create a triple-labelled compound. The double-radiolabelling method 
combines the radiolabelling steps for MIP with the radiolabelling steps 
for FLUORMIP. 
One important advantage of the synthesis methods of the present invention 
for new compounds AMIP, BIOMIP, DITHIOMIP, and FLUORMIP (and new compound 
intermediates) as well as the above-named radiolabelled compounds, is that 
these synthesis methods avoid the use of toxic compounds. As discussed 
below, preparation of some isopsoralen derivatives requires the use of 
chloromethylmethyl ether. This compound is highly volatile, extremely 
toxic and a well known carcinogen (OSHA regulated carcinogen CFR Title 29, 
Part 1910.1006; L. Bretherick, Hazards in the Chemical Laboratory, (Royal 
Society, London 1981) (p. 247). Its use requires special equipment and 
precautions to avoid exposure of the worker or release to the environment. 
The synthesis methods for providing MIP derivatives of the present 
invention do not require this hazardous compound. 
Other advantages of the synthesis methods of the present invention for the 
MIP derivatives are i) ease of synthesis (fewer steps) and ii) superior 
overall yield. In this regard, the preparation of the new compounds first 
requires the synthesis of MIP, which has been previously reported by 
Baccichetti et al. U.S. Pat. No. 4,312,883; Eur. J. Med. Chem. 16:489 
(1981). The methods of the present invention differ from the methods 
reported by Baccichetti in that Baccichetti's two methods include a four 
step procedure: 
5-methyiresorcinol.fwdarw.H5MC.fwdarw.7-allyloxy-5-methylcoumarin 
.fwdarw.8-allyl-7-hydroxy-5-methyl-coumarin.fwdarw.MIP and a seven step 
procedure: 
5-methylresorcinol.fwdarw.H5MC.fwdarw.7-allyloxy-5-methylcoumarin.fwdarw.8- 
allyl-7-hydroxy-5-methylcoumarin.fwdarw.7-acetoxy-8-allyl-5-methylcoumarin. 
fwdarw.7-acetoxy-8-(2',3'-epoxypropyl)-5-methylcoumarin 
.fwdarw.8-(7-acetoxy-5-methyl)-coumarinylacetaldehyde.fwdarw.MIP. 
The overall yields of these two methods of Baccichetti et al. are 
approximately 3.8% and 2.8%, respectively. 
By contrast, the present invention provides a two and a three step method 
for MIP synthesis (see FIG. 1). The overall yields of these methods of the 
present invention are approximately 8.4% and 7.1%. Thus, the methods of 
the present invention for MIP synthesis involve fewer steps and a better 
overall yield. 
B. T TWO: R EQUALS--H 
Where R is --H (FIG. 2; Table 3), the present invention contemplates a 
novel synthesis method for DMIP (Compound #16), a known compound; the 
method proceeds via new compounds XAMC (Compound #14) and RXAMC (Compound 
#15). From DMIP, the synthesis builds on the novel synthesis to yield 
known compound AMDMIP (Compound #22) or proceeds to new compounds HDMADMIP 
(Compound #24), BIODMIP (Compound #25a), DITHIODMIP (Compound #24b), 
FLUORDMIP (Compound #24c). New methods for radiolabelling compounds are 
also shown. In addition to the tritiated compounds indicated in FIG. 2, 
the analogous .sup.14 C derivatives may be prepared from labelled 
5-methylresorcinol. 
1) DMIP Synthesis 
The present invention provides a new synthesis method for DMIP. The 
approach utilizes a Claisen rearrangement to build the furan ring. This 
approach has heretofore only been used for synthesizing psoralens. See D. 
R. Bender et al., J. Org. Chem. 44:2176 (1979); D. R. Bender et al., U.S. 
Pat. No. 4,398,031. 
Baccichetti et al. have reported the synthesis of DMIP from 
7-hydroxy-4-methylcoumarin in five steps. These workers elected to build 
the 5'-methylfiran moiety via a five step conversion: 1) o-alkylation with 
allyl bromide, 2) Claisen rearrangement to provide the mixed isomers 
(6-allyl and 8-allyl), 3) acetylation of the phenolic hydroxide, 4) 
bromination of the allylic bond, and 5) alkaline ring closure to provide 
DMIP. They report an overall yield for the five steps of 7.7%. Eur. J. 
Med. Chem. (1981). 
The synthesis procedure described in the present invention, by contrast, 
requires fewer steps and provides a better yield of DMIP. DMIP is prepared 
in three steps: 1) o-alkylation with a 2,3-dihalo-alkene, 2) Claisen 
rearrangement to provide the two allylic isomers (6-allyl and 8-allyl), 
and 3) ring closure to provide DMIP. The overall three step yield is 26%. 
The method of the new synthesis improves the prior procedure as follows. 
First, an alkyl anhydride is used during the Claisen rearrangement, which 
provides the esterified phenol (instead of esterifying as a separate 
step). Esterification concomitant with rearrangement enhances the yield of 
the rearranged product due to protection of the phenolate from subsequent 
undesired high temperature oxidation. While acetic or proprionic 
anhydrides may be used, the higher boiling butyric anhydride is preferred 
because it allows the reaction temperature to remain closer to the boiling 
point of the solvent (diisopropylbenzene). Second, the present invention 
uses a 2,3-dihaloalkene instead of the allyl moiety, which obviates the 
requirement for subsequent bromination prior to the ring closure step. 
Like the o-allyl moiety, the o-(2-halo)alkene undergoes Claisen 
rearrangement primarily to the 8 position of the coumarin, but distinct 
from the allylic moiety, the rearranged haloalkene is in fact a masked 
ketone. Under acidic conditions, conversion of the haloalkene to the 
ketone occurs along with simultaneous acid catalyzed cleavage of the 
alkylester. The resulting phenolic ketone subsequently undergoes 
conversion to the ring-closed compound. A third advantage of the new 
synthesis is that alkaline conditions are avoided in all steps, which 
eliminates loss of product due to hydrolysis of the coumarin lactone to 
the cis cinnimate, which undergoes subsequent (irreversible) isomerization 
to the thermodynamically more favored trans isomer. 
1') Radiolabelled DMIP 
The present invention also contemplates labelled DMIP. A two step method is 
provided: 1) mixing DMIP with a catalyst, acetic acid and tritium gas to 
yield the tritiated compound DHDMIfP, and 2) mixing DHDMIP with a catalyst 
and diphenyl ether to yield tritiated DMIP (.sup.3 H-DMIP). 
The present invention contemplates that the catalyst is selected from the 
group consisting of palladium on charcoal, palladium on barium sulfate, 
Adams catalyst (NH.sub.4).sub.2 PtCl.sub.6 !, PtO.sub.2, rhodium, 
ruthenium, copper chromite and Raney nickel. 
2) AMDMIP Synthesis 
The present invention contemplates a new approach to the synthesis of known 
compound AMDMIP and new compound .sup.3 H-AMDMIP. The approach builds on 
the novel synthesis method described above for DMIP. AMDMIP is thereafter 
made in one of two ways: i) with a halomethylation step, or ii) without a 
halo-methylation step. 
a) AMDMIP via Halomethylation 
In one method of the present invention for synthesizing AMDMIP, DMIP is 
made by the novel synthesis method described above; AMDMIP is then made by 
derivatizing DMIP to provide a halomethyl derivative followed by 
hydrazinolysis of the corresponding phthalimidomethyl derivative (prepared 
by the Gabriel synthesis) with hydrazine hydrate according to the method 
described by F. Dall'Acqua et al., J. Med. Chem 24:178 (1981). 
Because of the novel synthesis method of the present invention for DMIP, 
the approach of the present invention has the advantage over other methods 
of synthesizing AMDMIP. For example, the procedure reported by Baccichetti 
et al. (U.S. Pat. No. 4,312,883) for the synthesis of AMDMIP relies on a 
method of DMIP synthesis that, as discussed above, is less efficient. 
As shown in FIG. 2, the present invention contemplates a number of 
variations using the halomethylation step. After XMDMIP (Compound #18) is 
synthesized, the synthesis can proceed via HMDMIP (Compound #19) in two 
ways: 
XMDMIP.fwdarw.HMDMIP.fwdarw.XMDMIP.fwdarw.PHIMDMIP.fwdarw.AMDMIP 
or 
XMDMIP.fwdarw.HMDMIP.fwdarw.XMDMP.fwdarw.IMDMIP.fwdarw.PHIMDMIP.fwdarw.AMDM 
IP. 
On the other hand, the present invention also contemplates two ways of 
proceeding to AMDMIP without HMDMIP: 
XMDMIP.fwdarw.PHIMDMIP.fwdarw.AMDMIP 
or 
XMDMIP.fwdarw.IMDMIP.fwdarw.PHIMDMIP.fwdarw.AMDMIP. 
As discussed with respect to the hydroxy derivative of MIP and HMIP, the 
hydroxy derivative of DMIP and HMDMIP, is stable and can be stored. This 
offers the convenience of interrupting the synthesis scheme. The 
non-HMDMIP routes, however, are more direct. They are, therefore, 
preferred where interruptions in the synthesis scheme are not anticipated. 
a') Radiolabelled AMDMIP via Halomethylation 
The halomethylation route for the synthesis of AMDMIP can further be used 
to synthesize labelled AMDMIP. In one approach of the present invention, 
radiolabelled AMDMIP is synthesized via new compound FDMIP (Compound #20). 
The present invention contemplates two methods that are variations of this 
approach: 
XMDMIP.fwdarw.HMDMIP.fwdarw.FMDMIP.fwdarw.*HMDMIP.fwdarw.*XMDMIP.fwdarw.*PH 
IMDMIP.fwdarw.*AMDMIP 
and 
XMDMIP.fwdarw.HMDMIP.fwdarw.FMDMIP.fwdarw.*HMDMIP.fwdarw.*XMDMIP.fwdarw.*IM 
DMIP.fwdarw.*PHIMDMIP.fwdarw.*AMDMIP 
where (*) indicates a radiolabelled compound. 
Together with the novel radiolabelling method of the present invention for 
DMIP, the present invention provides the following four (single) 
radiolabelling methods for AMDMJP (methods I and III are preferred): 
I #13 to #14, #14 to #15, #15 to #16, #16 to #17*, #17* to #16*, #16* to 
#18*, #18* to #21*, #21* to #22*. 
II #13 to #14, #14 to #15, #15 to #16, #16 to #17*, #17* to #16*, #16* to 
#18*, #18* to #23*, #23* to #21*, #21* to #22*. 
III #13 to #14, #14 to #15, #15 to #16, #16 to #18, #18 to #19, #19 to #20, 
#20 to #19*, #19* to #18*, #18* to #21*, #21* to #22*. 
IV #13 to #14, #14 to #15, #15 to #16, #16 to #18, #18 to #19, #19 to #20, 
#20 to #19*, #19* to #18*, #18* to #23*, #23* to #21*, #21* to #22*. 
The present invention also contemplates double-labelling, including 
double-radiolabelling. FIG. 2 shows two ways for synthesizing 
double-radiolabelled AMDMIP. 
b) AMDMIP without Halomethylation 
While the halomethylation route described above, combined with the novel 
method of the present invention for the synthesis of the AMDMIP precursor, 
DMIP, provides a novel and useful method for the synthesis of AMDMIP, 
halomethylation can require toxic compounds. For example, 
chloromethylation requires the use of chloromethylmethyl ether. This 
compound, as discussed earlier, is highly volatile, extremely toxic and a 
well known carcinogen (OSHA regulated carcinogen CFR Title 29, Part 
1910.1006). Its use requires special equipment and precautions to avoid 
exposure of the worker or release to the environment. To avoid the the 
danger and inconvenience of using chloromethylmethyl ether, the present 
invention provides a novel method for the synthesis of AMDMIP without 
halomethylation. 
The present invention contemplates conversion of DMIP to PHIMDMIP by direct 
phthalimidomethylation of the 4' furan position with a nitrogen donor. The 
present invention cotemplates that the nitrogen doner may be selected from 
the group consisting of N-hydroxymethyl phthalimide and derivatives 
thereof. 
In converting DMIP directly to PHIMDMIP, the present invention adapts and 
modifies a procedure that has heretofore only been used for psoralens. N. 
D. Heindel et al., J. Hetero. Chem. 22:73 (1985). The present invention 
contemplates that this adapted and modified procedure is suitable for 
isopsoralens which a) do contain a methyl group at the 4 position, and b) 
do not contain hydroxy, amino or other like substituents which result in 
poly-substitution. 
This approach is an improvement over the reported procedures for PHIMDMIP 
synthesis in that 1) no carcinogen is used, 2) the method requires one 
step instead of two, and 3) the method provides product (PHIMDMIP) in 
higher yield. From PHIMDMIP, the method proceeds to AMDMIP as described 
above. 
b') Radiolabelled AMDMIP without Halomethylation 
The route for the synthesis of AMDMIP without halomethylation can further 
be used to synthesize labelled AMDNHP via radiolabelled DMIP: 
*DMIP.fwdarw.*PHIMDMIP.fwdarw.*AMDMIP 
where (*) indicates a radiolabelled compound. 
With the novel radiolabelling method of the present invention for DMIP, the 
present invention provides the following single radiolabelling method for 
AMDMIP without halomethylation: 
I #13 to #14, #14 to #15, #15 to #16, #16 to #17*, #17* to #16*, #16* to 
#21*, #21* to #22*. 
3) BIODMIP, DITHIODMIP And FLUORDMIP Synthesis 
The BIO-, DITHIO- and FLUOR- derivatives of DMIP of the present invention 
(Compounds #25a, 25b, and 25c, respectively) can each be generally 
described as a three-part compound consisting of the following three 
units: 
DMIP--SER--LABEL 
The spacer contemplated by the present invention has the general formula 
R.sub.1 HN--(CH.sub.2).sub.n --NHR.sub.2. In general, R.sub.1 =--H, 
--CH.sub.3, --C.sub.2 H.sub.5, --C.sub.3 H.sub.7 or --C.sub.4 H.sub.9, 
R.sub.2 =--H --CH.sub.3, --C.sub.2 H.sub.5, --C.sub.3 H.sub.7 or --C.sub.4 
H.sub.9, and n is between 6 and 16, inclusive. It is contemplated that, 
where the BIODMIP compound is bound to another molecule (e.g., nucleic 
acid), sufficient length is provided for the biotin moiety to span the 
distance between the site of attachment to another molecule and the avidin 
binding site when n &gt;6. As noted earlier, shorter spacers see e.g., J. P. 
Albarella et al., 17:4293 (1989)! may hinder the formation of the 
biotin-avidin complex. Adequate chain length helps reduce steric 
hinderance associated with the avidin-biotin interaction, and accordingly, 
the stability of the avidin-biotin complex should increase when the 
appropriate chain length is employed. 
Chemical (synthetic) considerations come into play when considering the 
preferred spacer for the BIO-, DITHIO- and FLUOR- derivatives of DMIP. 
While for spacer R.sub.1 HN --(CH.sub.2).sub.n --NHR.sub.2, R.sub.1 can be 
--H, --CH.sub.3, --C.sub.2 H.sub.3, --C.sub.2 H.sub.5, --C.sub.3 H.sub.6 
or --C.sub.4 H.sub.7, and R.sub.2 can be --H, --CH.sub.3, --C.sub.2 
H.sub.5, --C.sub.3 H.sub.7 or --C.sub.4 H.sub.9, most preferably R.sub.1 
and R.sub.2 are both --CH.sub.3, --C.sub.2 H.sub.5, --C.sub.3 H.sub.7 or 
--C.sub.4 H.sub.9. While not limited to any particular theory, in the 
reaction to prepare HDAMDMIP from XMDMIP of (IMDMIP) where R.sub.1 and 
R.sub.2 are both --CH.sub.3, --C.sub.2 H.sub.5, --C.sub.3 H.sub.7 or 
--C.sub.4 H.sub.9, the spacer nitrogens can react at either nitrogen with 
only one or two equivalents of XMDMIP (or IMDMIP). 
So that the desired mono-N-substituted product (i.e., HDAMDMIP) is favored, 
the present invention contemplates that a high ratio of spacer to XMDMIP 
(or IMDMIP) is employed in the reaction. Nonetheless, even where 1) 
R.sub.1 and R.sub.2 are both --CH.sub.3, --C.sub.2 H.sub.5, --C.sub.3 
H.sub.7 or --C.sub.4 H.sub.9 and 2) a high ratio of spacer to XMDMIP (or 
IMDMIP) is employed, the present invention contemplates side products from 
the reaction of more than one XMDMIP (or IMDMIP) with the spacer. These 
side products include one di-N,N-substituted product (i.e., two 
isopsoralens at the same nitrogen on the spacer), one di-N,N'-substituted 
product (i.e., two isopsoralens at each of the spacer nitrogens), one 
tri-N,N,N'-substituted product and one tetra-N, N, N', N'-substituted 
product. 
As noted, the present invention does contemplate the case where R.sub.1 and 
R.sub.2 are --H. While this spacer can be used, the number of possible 
multi-substituted spacer side products is increased, making subsequent 
purification of the desired mono-N-substituted product (i.e., HDAMDMIP) 
more difficult. 
The label on the BIO-, DITHIO- and FLUOR- derivatives of DMIP of the 
present invention is comprised of two elements: 1) the reporter moiety, 
and 2) the linking arm which binds the reporter moiety to the spacer. Two 
types of reporter moieties are shown in FIG. 2: i) biotin and ii) 
fluorescein. Both biotin and fluorescein are appended to the spacer via an 
amide bond, with zero to seven bridging atoms making up the linking arm 
between the spacer amido carbonyl and the reporter moiety. In some cases 
(e.g., DITHIODMIP), the linking arm may contain a disulfide linkage, which 
is useful for subsequent cleavage of the reporter moiety from the 
isopsoralen. 
The reaction to form the amide bond between the spacer nitrogen and the 
label carbonyl uses an activated ester, preferrably the 
N-hydroxy-succinimide ester. Other active esters, however, are 
contemplated, such as the imidazolides (from N, N'-carbonyldiimidizoles) 
and the sulfosuccinimidyl esters. 
a) BIODMIP Synthesis 
As shown in FIG. 2, the present invention contemplates four alternative 
synthesis methods for proceeding via new compound intermediate XMDMIP to 
new compound BIODMIP (Compound #25a). Two of the four methods proceed via 
HMDMIP: 
XMDMIP.fwdarw.HMDMIP.fwdarw.XMDMIP.fwdarw.HDAMDMIP.fwdarw.BIODMIP 
or 
XMDMIP.fwdarw.HMDMIP.fwdarw.XMDMIP.fwdarw.IMDMIP.fwdarw.HDAMDMIP.fwdarw.BIO 
DMIP 
The other two methods (the first of which is preferred) proceed directly 
from XMDMIP: 
XMDMIP.fwdarw.HDAMDMIP.fwdarw.BIODMIP 
or 
XMDMIP.fwdarw.IMDMIP.fwdarw.HDAMDMIP.fwdarw.BIODMIP 
a') Radiolabelled BIODMIP Synthesis 
The present invention also provides methods for radiolabelling BIODMIP. Two 
methods are provided for synthesizing radiolabelled BIODMIP from DMIP and 
two methods are provided for synthesizing (single) radiolabelled BIODMIP 
from radiolabelled DMIP, for a total of four (single) radiolabelling 
methods: 
I #16 to #18, #18 to #19, #19 to #20, #20 to #19*, #19* to #18*, #18* to 
#24*, #24* to #25a*. 
II #16 to #18, #18 to #19, #19 to #20, #20 to #19*, #19* to #18*, #18* to 
#23*, #23* to #24*, #24* to #25a*. 
III #16 to #17*, #17* to #16*, #16* to #18*, #18* to #24*, #24* to #25a*. 
IV #16 to #17*, #17* to #16*, #16* to #18*, #18* to #23*, #23* to #24*, 
#24* to #25a*. 
where * indicates a labelled compound. Methods I and III are preferred. 
The present invention also contemplates double-radiolabelling of BIODMIP. 
FIG. 2 shows two methods of double-radiolabelling BIODMIP. In one 
embodiment, the double-radiolabelling method of the present invention 
involves the combination of the radiolabelling steps for DMIP (Compound 
#16 to Compound #17*) and the radiolabelling steps for BIODMIP (above). As 
noted, this provides, among other advantages, the advantage of increasing 
the specific activity of the compounds of the present invention. The 
present invention contemplates the following double-radiolabelling methods 
(where ** indicates a double-labelled compound): 
I #16 to #17*, #17* to #18*, #18* to #19*, #19* to #20*, #20* to 19**, 
#19** to #18**, #18** to #24**, #24** to 25a**. 
II #16 to #17*, #17* to #18*, #18* to #19*, #19* to #20*, #20* to 19**, 
#19** to #18**, #18** to #23**, #23** to #24**, #24** to 25a**. 
b) DIOTHIODMIP Synthesis 
As shown in FIG. 2, the present invention contemplates four alternative 
synthesis methods for proceeding via new compound intermediate XMDMIP to 
new compound DITHIODMIP (Compound #25b). Two of the four methods proceed 
via HMDMIP: 
XMDMIP.fwdarw.HMDMIP.fwdarw.XMDMIP.fwdarw.HDAMDMIP.fwdarw.DITHIODMIP 
or 
XMDMIP.fwdarw.HMDMIP.fwdarw.XMDMIP.fwdarw.IMDMIP.fwdarw.HDAMDMIP.fwdarw.DIT 
HIODMIP 
The other two methods (the first of which is preferred) proceed directly 
from XMDMIP: 
XMDMIP.fwdarw.HDAMDMIP.fwdarw.DITHIODMIP 
or 
XMDMIP.fwdarw.IMDMIP.fwdarw.HDAMDMIP.fwdarw.DITHIODMIP 
b') Radiolabelled DITHIODMIP 
The present invention also provides methods for radiolabelling DITHIODMIP. 
Two methods are provided for synthesizing radiolabelled DITHIODMIP from 
DMIP and two methods are provided for synthesizing (single) radiolabelled 
DITHIODMIP from radiolabelled DMIP, for a total of four (single) 
radiolabelling methods: 
I #16 to #18, #18 to #19, #19 to #20, #20 to #19*, #19* to #18*, #18* to 
#24*, #24* to #25b*. 
II #16 to #18, #18 to #19, #19 to #20, #20 to #19*, #19* to #18*, #18* to 
#23*, #23* to #24*, #24* to #25b*. 
III #16 to #17*, #17* to #16*, #16* to #18*, #18* to #24*, #24* to #25b*. 
IV #16 to #17*, #17* to #16*, #16* to #18*, #18* to #23*, #23* to #24*, 
#24* to #25b*. 
where * indicates a labelled compound. Methods I and III are preferred. 
The present invention also contemplates double-radiolabelling of 
DITHIODMIP. FIG. 2 shows two methods of double-radiolabelling DITHIODMIP. 
In one embodiment, the double-radiolabelling method of the present 
invention involves the combination of the radiolabelling steps for DMIP 
(Compound #16 to Compound #17*) and the (single) radiolabelling steps for 
DITHIODMIP (above). As noted, this provides, among other advantages, the 
advantage of increasing the specific activity of the compounds of the 
present invention. The present invention contemplates the following 
double-radiolabelling methods (where ** indicates a double-labelled 
compound): 
I #16 to #17*, #17* to #18*, #18* to #19*, #19* to #20*, #20* to 19**, 
#19** to #18**, #18** to #24**, #24** to 25b**. 
II #16 to #17*, #17* to #18*, #18* to #19*, #19* to #20*, #20* to 19**, 
#19** to #18**, #18** to #23**, #23** to #24**, #24** to 25b**. 
c) FLUORDMIP Synthesis 
As shown in FIG. 2, the present invention contemplates four alternative 
synthesis methods for proceeding via new compound intermediate XMDMIP to 
new compound FLUORDMIP (Compound #25c). Two of the four methods proceed 
via HMDMIP: 
XMDMIP.fwdarw.HMDMIP.fwdarw.XMDMIP.fwdarw.HDAMDMIP.fwdarw.FLUORDMIP 
or 
XMDMIP.fwdarw.IMDMIP.fwdarw.XMDMIP.fwdarw.IMDMIP.fwdarw.HDAMDMIP.fwdarw.FLU 
ORDMIP 
The other two methods (the first of which is preferred) proceed directly 
from XMDMIP: 
XMDMIP.fwdarw.HDAMDMIP.fwdarw.FLUORDMIP 
or 
XMDMIP.fwdarw.IMDMIP.fwdarw.HDAMDMIP.fwdarw.FLUORDMIP 
c') Radiolabelled FLIJORDMIP Synthesis 
The present invention also provides methods for radiolabelling FLUORDMIfP. 
Two methods are provided for synthesizing radiolabelled FLUORDMIP from 
DMIlP and two methods are provided for synthesizing (single) radiolabelled 
FLUORDMIP from radiolabelled DNIP, for a total of four (single) 
radiolabelling methods: 
I #16 to #18, #18 to #19, #19 to #20, #20 to #19*, #19* to #18*, #18* to 
#24*, #24* to #25c*. 
II #16 to #18, #18 to #19, #19 to #20, #20 to #19*, #19* to #18*, #18* to 
#23*, #23* to #24*, #24* to #25c*. 
III #16 to #17*, #17* to #16*, #16* to #18*, #18* to #24*, #24* to #25c*. 
IV #16 to #17*, #17* to #16*, #16* to #18*, #18* to #23*, #23* to #24*, 
#24* to #25c*. 
where * indicates a labelled compound. Methods I and III are preferred. 
The present invention also contemplates double-radiolabelling of 
FLUORDMIfP. FIG. 2 shows two methods of double-radiolabelling FLUORDMIfP. 
In one embodiment, the double-radiolabelling method of the present 
invention involves the combination of the radiolabelling steps for DMIP 
(Compound #16 to Compound #17*) and the radiolabelling steps for 
FLUORDMIfP (above). The present invention contemplates the following 
double-radiolabelling methods (where ** indicates a double-labelled 
compound): 
I #16 to #17*, #17* to #18*, #18* to #19*, #19* to #20*, #20* to 19**, 
#19** to #18**, #18** to #24**, #24** to 25c**. 
II #16 to #17*, #17* to #18*, #18* to #19*, #19* to #20*, #20* to 19**, 
#19** to #18**, #18** to #23**, #23** to #24**, #24** to 25c**. 
II. PHOTOACTIVATION DEVICES AND METHODS 
The present invention contemplates devices and methods for photoactivation 
and specifically, for activation of photoreactive compounds. The present 
invention contemplates devices having an inexpensive source of 
electromagnetic radiation that is integrated into a unit. In general, the 
present invention contemplates a photoactivation device for treating 
photoreactive compounds, comprising: a) means for providing appropriate 
wavelengths of electromagnetic radiation to cause activation of at least 
one photoreactive compound; b) means for supporting a plurality of sample 
vessels in a fixed relationship with the radiation providing means during 
activation; and c) means for maintaining the temperature of the sample 
vessels within a desired temperature range during activation. The present 
invention also contemplates methods for photoactivating, comprising: a) 
supporting a plurality of sample vessels, containing one or more 
photoreactive compounds, in a fixed relationship with a fluorescent source 
of electromagnetic radiation; b) irradiating the plurality of sample 
vessels simultaneously with said electromagnetic radiation to cause 
activation of at least one photoreactive compound; and c) maintaining the 
temperature of the sample vessels within a desired temperature range 
during activation. 
It is intended that the devices of the present invention serve to replace 
the specialized instruments of photochemists investigating basic 
photochemistry of a photoactivator in vitro. These specialized instruments 
have expensive, high energy light sources such as high pressure arc lamps 
or medium pressure mercury lamps. In addition, each has its own peculiar 
sample holders with varying geometries relative to the lamp source and 
with varying filter devices (eg., glass cut-off filters or liquid 
solutions that transmit only a specific region of the electromagnetic 
spectrum or ultraviolet spectrum). This lack of standardization makes it 
difficult to compare data between different labs since there are both 
intensity variations in each of the different irradiation apparatuses and 
differences in the spectral energy distribution. Furthermore, the 
specialized irradiation devices that are available usually lack inherent 
safety. 
The major features of one embodiment of the device of the present invention 
involve: A) an inexpensive source of ultraviolet radiation in a fixed 
relationship with the means for supporting the sample vessels, B) rapid 
photoactivation, C) large sample processing, D) temperature control of the 
irradiated samples, and E) inherent safety. 
A. Electromagnetic Radiation Source 
A preferred photoactivation device of the present invention has an 
inexpensive source of ultraviolet radiation in a fixed relationship with 
the means for supporting the sample vessels. Ultraviolet radiation is a 
form of energy that occupies a portion of the electromagnetic radiation 
spectrum (the electromagnetic radiation spectrum ranges from cosmic rays 
to radio waves). Ultraviolet radiation can come from many natural and 
artificial sources. Depending on the source of ultraviolet radiation, it 
may be accompanied by other (non-ultraviolet) types of electromagnetic 
radiation (e.g., visible light). 
Particular types of ultraviolet radiation are herein described in terms of 
wavelength. Wavelength is herein described in terms of nanometers ("nm"; 
10.sup.-9 meters). For purposes herein, ultraviolet radiation extends from 
approximately 180 nm to 400 nm. When a radiation source does not emit 
radiation below a particular wavelength (e.g., 300 nm), it is said to have 
a "cutoff" at that wavelength (e.g., "a wavelength cutoff at 300 
nanometers"). 
When ultraviolet radiation is herein described in terms of irradiance, it 
is expressed in terms of intensity flux (milliwatts per square centimeter 
or "mW cm.sup.-2 "). "Output" is herein defined to encompass both the 
emission of radiation (yes or no; on or off) as well as the level of 
irradiance. 
A preferred source of ultraviolet radiation is a fluorescent source. 
Fluorescence is a special case of luminescence. Luminescence involves the 
absorption of electromagnetic radiation by a substance and the conversion 
of the energy into radiation of a different wavelength. With fluorescence, 
the substance that is excited by the electromagnetic radiation returns to 
its ground state by emitting a quantum of electromagnetic radiation. While 
fluorescent sources have heretofore been thought to be of too low 
intensity to be useful for photoactivation, in one embodiment the present 
invention employs fluorescent sources to achieve results thus far 
achievable on only expensive equipment. 
As used here, fixed relationship is defmed as comprising a fixed distance 
and geometry between the sample and the light source during the sample 
irradiation. Distance relates to the distance between the source and the 
sample as it is supported. It is known that light intensity from a point 
source is inversely related to the square of the distance from the point 
source. Thus, small changes in the distance from the source can have a 
drastic impact on intensity. Since changes in intensity can impact 
photoactivation results, changes in distance are avoided in the devices of 
the present invention. This provides reproducibility and repeatability. 
Geometry relates to the positioning of the light source. For example, it 
can be imagined that light sources could be placed around the sample 
holder in many ways (on the sides, on the bottom, in a circle, etc.). The 
geometry used in a preferred embodiment of the present invention allows 
for uniform light exposure of appropriate intensity for rapid 
photoactivation. The geometry of a preferred device of the present 
invention involves multiple sources of linear lamps as opposed to single 
point sources. In addition, there are several reflective surfaces and 
several absorptive surfaces. Because of this complicated geometry, changes 
in the location or number of the lamps relative to the position of the 
samples to be irradiated are to be avoided in that such changes will 
result in intensity changes. 
B. Rapid Photoactivation 
The light source of the preferred embodiment of the present invention 
allows for rapid photoactiv-ation. The intensity characteristics of the 
irradiation device have been selected to be convenient with the 
anticipation that many sets of multiple samples may need to be processed. 
With this anticipation, a fifteen minute exposure time is a practical 
goal. 
A fifteen minute exposure, in addition to its convenience, provides for 
reproducible results. In this regard, it should be noted that the binding 
levels of photoactive compounds to polynucleotides increases with 
increasing exposure to activating light. A plateau of binding density is 
ultimately achieved. This plateau results from competing photochemical 
reactions. Most photoreactive compounds which undergo addition reactions 
to the base moieties of nucleic acid also undergo photodecomposition 
reactions when free in solution. For a given intensity flux 
(watts/cm.sup.2) the relative rates of these competing reactions will 
determine when, in the course of a time course of an irradiation process, 
the plateau level will be achieved. For reproducible binding, it is 
desirable to have irradiation protocols that result in plateau levels of 
binding. Plateau levels of binding will avoid minor intensity differences 
that can arise from small differences in sample position (i.e., while the 
means for supporting the sample vessels can be in a fixed relationship 
with the source of irradiation, each sample in a large number of samples 
cannot occupy precisely the same point in space relative to the source). 
When plateau binding is used, identical reaction mixtures in different 
positions will show the same level of binding. 
In designing the devices of the present invention, relative position of the 
elements of the preferred device have been optimized to allow for plateau 
binding in fifteen minutes of irradiation time through Eppendorf tubes for 
most photoreactive compounds thus far tested. The present invention 
contemplates for a preferred device: a) a fluorescent source of 
ultraviolet radiation, and b) a means for supporting a plurality of sample 
vessels, positioned with respect to the fluorescent source, so that, when 
measured for the wavelengths between 300 and 400 nanometers, an intensity 
flux greater than 15 mW cm.sup.-2 is provided to the sample vessels. 
Similarly, in the preferred method of the present invention, the following 
steps are contemplated: a) providing a fluorescent source of ultraviolet 
radiation, b) supporting a plurality of sample vessels with respect to the 
fluorescent source of ultraviolet radiation, so that, when measured for 
the wavelengths between 300 and 400 nanometers, an intensity flux greater 
than 15 mW cm.sub.-2 is provided simultaneouusly to the plurality of 
sample vessels, and c) simultaneously irradiating the plurality of sample 
vessels. 
C. Processing Of Large Numbers Of Samples 
As noted, another important feature of the photoactivation devices of the 
present invention is that they provide for the processing of large numbers 
of samples. In this regard, one element of the devices of the present 
invention is a means for supporting a plurality of sample vessels. In the 
preferred embodiment of the present invention, the supporting means 
comprises a sample rack detachably coupled to the housing of the device. 
The sample rack provides a means for positioning the plurality of sample 
vessels. The positioning means has been designed to be useful in 
combination with commonly used laboratory sample vessels. Commonly used 
laboratory sample vessels include, but are not limited to, test tubes, 
flasks, and small volume (0.5-1.5 ml) plastic tubes (such as Eppendorf 
tubes). By accepting commonly used laboratory sample vessels, the sample 
rack of the preferred embodiment of the present invention allows for 
convenient processing of large numbers of samples. 
The detachable aspect of the sample rack in the preferred embodiment also 
provides for interchange-ability of the supporting means. Sample racks 
having different features suited to different size sample vessels and/or 
different size photoactivation jobs can be interchanged freely. 
The embodiments of the device of the present invention also provide for the 
processing of a large liquid sample. In the preferred embodiment of the 
device of the present invention, a trough is provided for holding 
temperature control liquid (see next section). In an alternative 
embodiment, it is contemplated that the trough serve as a built-in 
container for liquid that is to be irradiated. In such a case, the device 
of the present invention provides a flow-through trough, having inlet and 
outlet ports for liquid. It is contemplated that the flow-through trough 
serve as a container for continuous liquid flow during irradiation. 
Temperature control of this flow-through system can still be achieved by 
use of an external temperature control means (e.g., a temperature 
controlled reservoir). 
D. Temperature Control 
As noted, one of the important features of the photoactivation devices of 
the present invention is temperature control. Temperature control is 
important because the temperature of the sample in the sample vessel at 
the time of exposure to light can dramatically impact the results. For 
example, conditions that promote secondary structure in nucleic acids also 
enhance the affinity constants of many psoralen derivatives for nucleic 
acids. Hyde and Hearst, Biochemistry, 17, 1251 (1978). These conditions 
are a mix of both solvent composition and temperature. With single 
stranded 5S ribosomal RNA, irradiation at low temperatures enhances the 
covalent addition of HMT to 5S rRNA by two-fold at 4.degree. C. compared 
to 20.degree. C. Thompson et al., J. Mol. Biol. 147:417 (1981). Even 
further temperature induced enhancements of psoralen binding have been 
reported with synthetic polynucleotides. Thompson et al., Biochemistry 
21:1363 (1982). 
Temperature control is also an important factor for hybridization assays 
that detect allele specific nucleic acid targets. Allelic variants of a 
specific target nucleic acid may differ by a single base. Sickle cell 
anemia is an example of a human genetic disease that results from the 
change of a single base (A to T) in the gene for the human .beta. globin 
molecule. The specific hybridization of a single oligonucleotide probe to 
one of two allelic variants that differ by only a single base requires 
very precise temperature control. Wood et al., Proc. Nat. Acad. Sci. 
82:1585 (1985). The irradiation of psoralen monoadducted oligonucleotide 
probes under hybridization equilibrium conditions results in the covalent 
attachment of these probes to their targets. Allele specific 
discrimination of a single base change is possible with these 
crosslinkable probes. However, discrimination is sharply dependant upon 
temperature. A 2.degree. C. change during the irradiation procedure will 
have dramatic effect on the level of discrimination that is observed. 
E. Inherent Safety 
Ultraviolet radiation can cause severe burns. Depending on the nature of 
the exposure, it may also be carcinogenic. The light source of a preferred 
embodiment of the present invention is shielded from the user. This is in 
contrast to the commercial hand-held ultraviolet sources as well as the 
large, high intensity sources. In a preferred embodiment, the irradiation 
source is contained within a housing made of material that obstructs the 
transmission of radiant energy (i.e., an opaque housing). As noted above, 
sample vessels are placed in the sample rack which is detachably coupled 
to the housing above the rack. As a final precaution, a sample overlay is 
provided that extends over and covers the sample vessels. This sample 
overlay provides two functions. First, it helps to maintain the position 
of the sample vessels when liquid is in the trough. Second, and more 
importantly, it closes off the only opening of the housing and, thereby, 
seals the device. The sealed device allows no irradiation to pass to the 
user. This allows for inherent safety for the user. 
III. BINDING OF COMPOUNDS TO NUCLEIC ACID 
The present invention contemplates binding new and known compounds to 
nucleic acid, including (but not limited to) a) nucleic acid target 
sequences, probes, and primers, as well as b) nucleic acid used as 
template, and c) amplified nucleic acid. Target sequences are regions of 
nucleic acid having one or more segments of known base sequence. Target 
sequences are "targets" in the sense that they are sought to be detected 
(i.e., sorted out from other nucleic acid). Detection is frequently 
performed by hybridization with probes. Probes are nucleic acids having a 
base sequence that is partially or completely complementary with all or a 
portion of a target sequence. 
Some molecular biological techniques use template and primers. Template is 
defined simply as nucleic acid that is substrate for enzymatic synthesis. 
Frequently, it is nucleic acid suspected of containing target sequence(s). 
Primers act to control the point of initiation of synthesis of target 
sequences when they are present in the template. Other molecular 
biological techniques use template and replicating probes. 
The present invention contemplates that the binding to all these forms of 
nucleic acid (as well as others) can be non-covalent binding and/or 
covalent binding. The present invention contemplates specific embodiments 
of binding including, but not limited to dark binding and photobinding. 
A. Dark Binding 
One embodiment of the binding of the present invention involves dark 
binding. "Dark Binding" is defined as binding to nucleic acid that occurs 
in the absence of photoactivating wavelengths of electromagnetic 
radiation. Dark binding can be covalent or non-covalent. "Dark Binding 
Compounds" are defmed as compounds that are capable of dark binding. In 
one embodiment, the dark binding of the present invention involves the 
steps of: a) providing a dark binding compound; and b) mixing the dark 
binding compound with nucleic acid in the absence of photoactivation 
wavelengths of light, where the dark binding compound is selected from the 
group consisting of DEMC (Compound #2), XMIP (Compound #5), FIMIP 
(Compound #6), FMIP (Compound #7), IMIP (Compound #8), HMTAMIP (Compound 
#9), AMIP (Compound #10), DMHMIP (Compound #11), BIOMIP (Compound #12a), 
DITHIOMIP (Compound 12b), FLUORMIP (Compound 12c), XAMC (Compound #14) 
RXAMC (Compound #15), BMDMIP (Compound #18, where X=Br), FDMIP (Compound 
#20), IMDMIP (Compound #23), HDMADMIP (Compound #24), BIODMIP (Compound 
#25a), DITHIODMIP (Compound #25b), FLUORDMIP (Compound #25c), and their 
radiolabelled derivatives. 
The present invention further contemplates the product of dark binding, 
ie., a dark binding compound:nucleic acid complex, where the dark binding 
compound is selected from the group consisting of DEMC (Compound #2), XMIP 
(Compound #5), HMIP (Compound #6), FMJP (Compound #7), IMIP (Compound #8), 
HMTAMIP (Compound #9), AMIP (Compound #10), DMHMIP (Compound #11), BIOMIP 
(Compound #12a), DITHIOMIP (Compound 12b), FLUORMIP (Compound 12c), XAMC 
(Compound #14) RXAMC (Compound #15), BMDMIP (Compound #18, where X=Br), 
FDMIP (Compound #20), IMDMIP (Compound #23), HDMADMIP (Compound #24), 
BIODMIP (Compound #25a), DITHIODMIP (Compound #25b), FLUORDMIP (Compound 
#25c), and their radiolabelled derivatives. 
The present invention also contemplates dark binding of photoproduct. 
"Photoproduct" is defined as a product of the reaction of a compound and 
activating wavelengths of electromagnetic radiation that, once formed, is 
later capable of binding to nucleic acid in the absence of electromagnetic 
radiation. 
In considering photoproduct binding, it should be noted that previous work 
towards the modification of nucleic acids with furocoumarins has 
historically proceeded by a method having the temporal steps: 1) providing 
a specific furocoumarin derivative, 2) providing a particular nucleic acid 
or nucleic acid sequence, and 3) mixing the furocoumarin with the nucleic 
acid in the presence of activating wavelengths of electromagnetic 
radiation. Depending on the details of the particular reaction, including 
the particular furocoumarin derivative, radiation source irradiation time, 
buffer, temperature and other factors used for the procedure, a given 
level of covalent modification, with almost exclusively cyclobutyl type 
2+2 photocycloaddition products, resulted. 
In one embodiment, the present invention contemplates a radical departure 
from this historical approach to photobinding. In one embodiment of the 
method of the present invention, the temporal sequence is the following: 
1) providing one or more flrocoumarin derivative(s), 2) exposing the 
furocoumarin derivative(s) to activating wavelengths of electromagnetic 
radiation, 3) providing a particular nucleic acid sample or nucleic acid 
sequence, and 4) mixing the irradiated furocoumarin derivative(s) with the 
nucleic acid sequence in the absence of activating wavelengths of 
electromagnetic radiation. In this embodiment, the furocoumarin derivative 
is irradiated prior to mixing with nucleic acid. The experimental 
investigation of this novel temporal sequence has established the 
existence of furocoumarin photoproduct. Application of the novel temporal 
sequence has useful applications but was neither predicted nor expected 
from the chemical or biochemical literature concerning furocoumarins. 
"Photoproduct" is best understood by considering the possible reactions of 
photoreactive compound when exposed to activating wavelengths of 
electromagnetic radiation. While not limited to any precise mechanism, it 
is believed that the reaction of photoreactive compound in its ground 
state ("C") with activating wavelengths of electromagnetic radiation 
creates a short-lived excited species ("C*"): 
C.fwdarw.C* 
What happens next is largely a function of what potential reactants are 
available to the excited species. Since it is short-lived, a reaction of 
this species with nucleic acid ("NA") is believed to only be possible if 
nucleic acid is present at the time the excited species is generated. 
Thus, the reaction must, in operational terms, be in the presence of 
activating wavelengths of electromagnetic radiation, ie., it is 
"photobinding"; it is not dark binding. The reaction can be depicted as 
follows: 
C*+NA.fwdarw.NA:C 
The product of this reaction is hereinafter referred to as "Photoaddition 
Product" and is to be distinguished from "Photoproduct." 
With this reaction described, one can now consider the situation where 
nucleic acid is not available for binding at the time the compound is 
exposed to activating wavelengths of electromagnetic radiation. Since the 
excited species is short-lived and has no nucleic acid to react with, the 
excited species may simply return to its ground state: 
C*.fwdarw.C 
On the other hand, the excited species may react with itself (i.e., a 
ground state or excited species) to create a ground state complex ("C:C"). 
The product of these selfreactions where two compounds react is referred 
to as "photodimer" or simply "dimer." The self-reactions, however, are not 
limited to two compounds; a variety of multimers may be formed (trimers, 
etc.). 
The excited species is not limited to reacting with itself. It may react 
with its environment, such as elements of the solvent ("E") (e.g., ions, 
gases, etc.) to produce other products: 
C*+E.fwdarw.E:C Furthermore, it may simply internally rearrange 
("isomerize") to a ground state derivative (""): 
C*.fwdarw. 
Finally, the excited species may undergo other reactions than described 
here. 
The present invention and the understanding of "photoproduct" does not 
depend on which one (if any) of these reactions actually occurs. 
"Photoproduct"--whatever its nature--is deemed to exist if, following the 
reaction of a compound and activating wavelengths of electromagnetic 
radiation, there is a resultant product formed that is later capable of 
binding to nucleic acid in the absence of electromagnetic radiation, i.e., 
capable of dark binding (whether non-covalent dark binding or covalent 
dark binding). 
It is important to note that, while the defmition of "photoproduct" demands 
that, once formed by exposure to electromagnetic radiation, the product be 
"capable" of binding to nucleic acid in the absence of electromagnetic 
radiation, it is not necessary that the product bind only in the dark. 
Photoproduct may bind under the condition where there is exposure to 
electromagnetic radiation; it simply does not require the condition for 
binding. Such a definition allows for both "photobinding" and 
"photoproduct binding" to nucleic acid to occur at the same time. Such a 
definition also allows a single compound to be "photoproduct" and 
"photobinding compound." 
In one embodiment, the present invention contemplates dark binding of both 
psoralen photoproduct and isopsoralen photoproduct. With psoralens such as 
4' hydroxymethyl-4,5',8-trimethylpsoralen (HMT), the present invention 
contemplates there are a number of resultant products produced when the 
HMT is exposed to activating wavelngths of electromagnetic radiation. The 
present invention contemplates that a number of resultant products are 
similarly produced when isopsoralens such as AMIP and AMDMIP are exposed 
to activating wavelengths of electromagnetic radiation (particularly when 
irradiated with the CE-III device). The major resultant products of HMT 
are two cyclobutyl photodimers. In one of the dimers, the two pyrone rings 
are linked in a cis-syn configuration, while in the other dimer, the 
linkage occurs between the flran end of one molecule and the pyrone end of 
the other, again with cis-syn configuration. A third resultant product of 
HMT is a monomeric HMT photoisomer. In this isomer, the central ring 
oxygens assume a 1, 4 instead of the normal 1, 3 orientation. While the 
two photodimers would not be expected to have an intercalating activity 
due to geometrical considerations, the photoisomer remains planar, and 
accordingly, it is contemplated that it has a positive intercalative 
association with double stranded nucleic acid. Analogously, it is 
contemplated that some of the resultant products of AMIP and AMDMIP also 
have a positive intercalative association with nucleic acid. While not 
limited to any particular theory, non-covalent dark binding is anticipated 
where monomeric isomers are formed, and particularly, where the positively 
charge aminomethyl moiety is retained in the structure. 
B. Photobinding 
One approach of the present invention to binding activation compounds to 
nucleic acid is photobinding. Photobinding, as noted above, is defined as 
the binding of photobinding compounds in the presence of photoactivating 
wavelengths of light. Photobinding compounds are compounds that bind to 
nucleic acid in the presence of photoactivating wavelengths of light. The 
present invention contemplates a number of methods of photobinding, 
including 1) photobinding with photobinding compounds of the present 
invention, 2) high photobinding with new and known compounds, and 3) 
photobinding to label nucleic acids. 
1) Photobinding With New Compounds 
One embodiment of the method of the present invention for photobinding 
involves the steps: a) providing a photobinding compound; and b) mixing 
the photobinding compound with nucleic acid in the presence of 
photoactivation wavelengths of electromagnetic radiation, where the 
photobinding compound is selected from the group consisting of DEMC 
(Compound #2), XMIP (Compound #5), HMIP (Compound #6), FMIP (Compound #7), 
IMIP (Compound #8), HMTAMIP (Compound #9), AMIP (Compound #10), DMHMIP 
(Compound #11), BIOMIP (Compound #12a), DITHIOMIP (Compound 12b), FLUORMIP 
(Compound 12c), XAMC (Compound #14) RXAMC (Compound #15), BMDMIP (Compound 
#18, where X=Br), FDMIP (Compound #20), IMDMIP (Compound #23), HDMADMIP 
(Compound #24), BIODMIP (Compound #25a), DITHIODMIP (Compound #25b), 
FLUORDMIP (Compound #25c), and their radiolabelled derivatives. 
In another embodiment, the steps of the method comprise: a) providing a 
photobinding compound; b) providing one or more nucleic acid target 
sequences; and c) mixing the photobinding compound with the nucleic acid 
target sequences in the presence of photoactivation wavelengths of 
electromagnetic radiation. Again, in one embodiment, the photobinding 
compound is selected from the group consisting of DEMC (Compound #2), XMIP 
(Compound #5), HMIP (Compound #6), FMIP (Compound #7), IMIP (Compound #8), 
HMTAMIP (Compound #9), AMIP (Compound #10), DMHMIP (Compound #11), BIOMIP 
(Compound #12a), DITHIOMIP (Compound 12b), FLUORMIP (Compound 12c), XAMC 
(Compound #14) RXAMC (Compound #15), BMDMIP (Compound #18, where X=Br), 
FDMIP (Compound #20), IMDMIP (Compound #23), HDMADMIP (Compound #24), 
BIODMIP (Compound #25a), DITHIODMIP (Compound #25b), FLUORDMIP (Compound 
#25c), and their radiolabelled derivatives. 
The present invention further contemplates the product of photobinding, 
i.e., a photobinding compound: nucleic acid complex. In one embodiment, 
the photobinding compound of the complex is selected from the group 
consisting of DEMC (Compound #2), XMIP (Compound #5), HMIP (Compound #6), 
FMIP (Compound #7), IMIP (Compound #8), HMTAMIP (Compound #9), AMIP 
(Compound #10), DMHMIP (Compound #11), BIOMIP (Compound #12a), DITHIOMIP 
(Compound 12b), FLUORMIP (Compound 12c), XAMC (Compound #14) RXAMC 
(Compound #15), BMDMIP (Compound #18, where X=Br), FDMIP (Compound #20), 
IMDMIP (Compound #23), HDMADMIP (Compound #24), BIODMIP (Compound #25a), 
DITHIODMIP (Compound #25b), FLUORDMIP (Compound #25c), and their 
radiolabelled derivatives. 
The invention further contemplates a method for modifying nucleic acid, 
comprising the steps: a) providing photobinding compound and nucleic acid; 
and b) photobinding the photobinding compound to the nucleic acid, so that 
a compound:nucleic acid complex is formed, wherein the photobinding 
compound is selected from the group consisting of DEMC (Compound #2), XMIP 
(Compound #5), HMIP (Compound #6), FMIP (Compound #7), IMIP (Compound #8), 
HMTAMIP (Compound #9), AMIP (Compound #10), DMHMIP (Compound #11), BIOMIP 
(Compound #12a), DITHIOMIP (Compound 12b), FLUORMIP (Compound 12c), XAMC 
(Compound #14) RXAMC (Compound #15), BMDMIP (Compound #18, where X=Br), 
FDMIP (Compound #20), IMDMIP (Compound #23), HDMADMIP (Compound #24), 
BIODMIP (Compound #25a), DITHIODMIP (Compound #25b), FLUORDMIP (Compound 
#25c), and their radiolabelled derivatives. 
A preferred embodiment of the method of the present invention for 
photobinding involves the steps: a) providing a photobinding compound; and 
b) mixing the photobinding compound with nucleic acid in the presence of 
photoactivation wavelengths of electromagnetic radiation, where the 
photobinding compound is selected from the group consisting of AMIP 
(Compound #10), BIOMIP (Compound #12a), DITHIOMIP (Compound 12b), FLUORMIP 
(Compound 12c), BIODMIP (Compound #25a), DITHIODMIP (Compound #25b), 
FLUORDMIP (Compound #25c), and their radiolabelled derivatives. 
In another preferred embodiment, the steps of the method comprise: a) 
providing a photobinding compound; b) providing one or more nucleic acid 
target sequences; and c) mixing the photobinding compound with the nucleic 
acid target sequences in the presence of photoactivation wavelengths of 
electromagnetic radiation, where the photobinding compound is selected 
from the group consisting of AMIP (Compound #10), BIOMIP (Compound #12a), 
DITHIOMIP (Compound 12b), FLUORMIP (Compound 12c), BIODMIP (Compound 
#25a), DITHIODMIP (Compound #25b), FLUORDMIP (Compound #25c), and their 
radiolabelled derivatives. 
In still another preferred embodiment, the present invention contemplates a 
photobinding compound:nucleic acid complex, where the photobinding 
compound of the complex is selected from the group consisting of AMIP 
(Compound #10), BIOMIP (Compound #12a), DITHIOMIP (Compound 12b), FLUORMIP 
(Compound 12c), BIODMIP (Compound #25a), DITHIODMIP (Compound #25b), 
FLUORDMIP (Compound #25c), and their radiolabelled derivatives. 
In still an additional preferred embodiment, the invention contemplates a 
method for modifying nucleic acid, comprising the steps: a) providing 
photobinding compound and nucleic acid; and b) photobinding the 
photobinding compound to the nucleic acid, so that a compound:nucleic acid 
complex is formed, wherein the photobinding compound is selected from the 
group consisting of AMIP (Compound #10), BIOMIP (Compound #12a), DITHIOMIP 
(Compound 12b), FLUORMIP (Compound 12c), BIODMIP (Compound #25a), 
DITHIODMIP (Compound #25b), FLUORDMIP (Compound #25c), and their 
radiolabelled derivatives. 
2) High Photobinding 
The present invention provides isopsoralens with high photobinding affinity 
and conditions for using isopsoralens to allow for high photobinding. High 
photobinding is defmed here as photobinding to nucleic acid that results 
in significantly higher levels of addition than reported for the known 
compound AMDMIP. 
Baccichetti et al. and Dall' Acqua et al. previously reported the nucleic 
acid binding characterization of AMDMIP. Baccichetti et al., U.S. Pat. No. 
4,312,883; Dall' Acqua et al., J. Med. Chem. 24:178 (1981). these workers 
reported that, while AMDMIP has a high dark binding affinity for DNA, 
photobinding with AMDMIP results in low levels of addition to nucleic 
acid. In fact, AMDMJP was found to photobind to DNA less than the parent 
compound, DMIP. (No RNA binding data for AMDMIP was provided.) 
The present invention provides photobinding methods for i) known 
isopsoralens, and ii) new isopsoralens. With respect to methods for known 
isopsoralens, the present invention provides methods for photobinding of 
AMDMIP that allow for photobinding of AMDMIP to DNA at a level greater 
than 1 photobound AMDMIP per 15 base pairs, and to RNA at a level greater 
than 1 photobound AMDMIP per 20 RNA bases. With respect to photobinding 
methods for new isopsoralens, the present invention provides photobinding 
methods for new compound AMIP that allow for photobinding at a level 
greater than 1 photobound AMIP per 15 base pairs of DNA and a level 
greater than 1 photobound AMIP per 20 bases of RNA. 
While not limited to any particular theory, the photobinding methods of the 
present invention take into consideration two concepts as they relate to 
photobinding capacity: a) nucleic acid base pair/compound ratio, and b) 
isopsoralen structure. 
a) Nucleic Acid Base Pair/Compound Ratio 
Dall' Acqua et al. compared AMDMIP photobinding with the photobinding of 
the parent compound, DMIP. The parent compound (as well as other 
compounds) was tested at concentrations at or near its solubility limit 
(i.e., DMIP was tested at 10.1 .mu.g/ml; DMIP's maximum aqueous 
solubility, as reported by Dall' Acqua et al., is 8 .mu.g/ml). For 
comparative purposes, AMDMIP was tested in this concentration range as 
well (specifically, at 13.1 .mu.g/ml). Given this concentration range, 
both DMIP and AMDMIP photobinding was determined at a DNA base 
pair:isopsoralen ratio of 24.3 to 1 (1.14.times.10.sup.-3 M DNA: 
4.7.times.10.sup.-5 M isopsoralen). With this ratio, photobinding of 
AMDMIP resulted in 1 photobound AMDMIP per 151 DNA base pairs, which was 
lower photobinding than that observed for the parent compound, DMIP. Dall' 
Acqua et al. J. Med. Chem. 24:178 (1981). Thus, where concentrations of 
DMIP and AMDMIP were approximately equal, DMIP was reported as the better 
photobinder. 
The photobinding methods of the present invention take into consideration 
the nucleic acid base pair/compound ratio. The photobinding methods of the 
present invention involve carrying out the photobinding step under 
conditions where the isopsoralen concentration is increased relative to 
the concentration of nucleic acid base pairs. Importantly, increasing the 
isopsoralen concentration takes advantage of the solubility of the 
isopsoralen; with isopsoralens which have high aqueous solubility, higher 
concentrations are possible to obtain. 
By increasing the concentration of AMDMIP relative to nucleic acid, the 
present invention takes into consideration the much better solubility of 
AMDMIP as compared with the parent compound, DMIP. While DMIP is reported 
to be a better intrinsic photobinder than AMDMIP, the level of addition to 
nucleic acid is governed by the photobinding compound concentration 
(relative to nucleic acid) which is (with the concentration of nucleic 
acid constant) governed by solubility of the photobinding compound. Thus, 
while DMIP is a better intrinsic photobinder, the poor solubility of DMIP 
results in a relatively low level of addition (adducts per base pair) to 
nucleic acid. 
By taking advantage of the higher solubility of AMDMIP, higher 
concentrations of AMDMIP can be used, thus providing a higher ratio of 
photobinding compound to nucleic acid base pairs prior to irradiation. 
While AMDMIP is reported to be a less efficient photobinder relative to 
DMIP, the present invention contemplates increasing the concentration of 
AMDMIP so that a high level of photoaddition to nucleic acid is achieved, 
ie., high photobinding. 
The nucleic acid base pair/compound ratio of the photobinding methods of 
the present invention is preferably less than 3:1. With this ratio, the 
methods of the present invention allow for photobinding of AMDMIP to DNA 
at a level greater than 1 photobound AMDMIP per 15 base pairs, and to RNA 
at a level greater than of 1 photobound AMDMIP per 20 RNA bases. 
b) Isopsoralen Structure 
While the present invention takes into consideration the nucleic acid base 
pair/compound ratio, the methods of the present invention further consider 
isopsoralen structure. Inspection of the molecular structure of AMDMIP 
(FIG. 2) shows two methyl groups at the 4 and 5' carbons and the 
aminomethyl moiety at the 4' carbon. It is known that methylation improves 
the ability of a psoralen or isopsoralen to photobind to nucleic acid. It 
has been reported that the trialkyl isopsoralens, particularly trimethyl, 
are better DNA photobinding ligands than the corresponding dialkyl 
compounds, and analogously, the dialkyl compounds are better photobinders 
than the monoalkyl analogs. Guiotto et al., J. Med. Chem. 27:959 (1984). 
In particular, methyl groups at the 4, 4', 5, 5' and 6 positions of the 
isopsoralen system increase photobinding activity. 
Psoralens which contain aminoalkyl moieties at the 4', 5, 5', or 8 
positions are charged and show enhanced dark binding and enhanced 
photoreactivity relative to the uncharged analogs. In particular, the 4' 
and 5' aminoalkyl-trimethylpsoralens show enhanced photobinding to nucleic 
acid. S. Isaacs et al., Biochemistry 16:1058 (1977). I. Willis and J. M. 
Menter, Nat. Cancer Inst. Monograph 66 (1985). 
While not limited to any particular theory, the methods of the present 
invention take into consideration these structural relationships which 
suggest that the position of the aminomethyl moiety in AMDMIP may not be 
optimum for high photobinding activity. The 4'-aminomethyl moiety, through 
association with external phosphate groups, could be skewing the 
intercalated complex such that the critical alignment between the 4', 5' 
and/or 3, 4 double bonds and the 5, 6 pyrimidine double bond is 
disfavored, resulting in a significant reduction in the quantum efficiency 
of 2+2 photocycloaddition to form the cyclobutane ring. 
The present invention provides new compounds where the aminomethyl moiety 
is at the 5 position (e.g., AMIP). It was hoped that such compounds of the 
present invention might promote a different and potentially more favorable 
double bond geometry, resulting in higher photobinding than AMDMIP 
provides. While it was not predictable that this new geometry could 
overcome the disadvantage of a compound having no additional methyl groups 
present on the ring system, the new compounds of the present invention 
display high photobinding. In this regard, while the known compound AMDMIP 
has the highest K.sub.a (DNA association constant) of all reported 
isopsoralens, and new compound AMIP of the present invention has a K.sub.a 
which is only 28% as strong, AMIP can provide 43% of the modification 
density provided by AMDMIP. Since i) solubility of the two compounds is 
essentially the same, and ii) testing was performed (photobinding) at the 
same concentration, it appears that placing the aminomethyl moiety at the 
5 position rather than at the 4' position enhances photobinding, relative 
to dark binding; AMIP is the better photobinding per isopsoralen molecule, 
even though AMDMIP give the highest number of isopsoralens bound. 
3) Labelling Nucleic Acid 
As noted, one utility of the compounds is their ability to bind to nucleic 
acids (RNA and DNA). Furthermore, because the compounds bind nucleic 
acids, they also bind nucleic acid target sequences. While target 
sequences are normally present in a mixture of nucleic acids, they may be 
purified to homogeneity and reacted with photoreactive compounds of the 
present invention. 
While unlabelled compounds bind to nucleic acids, labelled compounds are 
particularly useful for assessing the level of binding to nucleic acids 
because, as noted above, labels facilitate detection of the compound as 
well as the detection of molecules bound to the compound, such as nucleic 
acids and nucleic acid target sequences. In this manner it is also easier 
to separate unbound from bound reactants (e.g., unbound isopsoralen from 
bound isopsoralen). Furthermore, when there is binding, the separation and 
isolation of bound reactants allows for a yield of substantially pure 
bound product. 
The present invention contemplates binding of the above-named compounds to 
all types of nucleic acids under a wide variety of conditions and thereby 
labelling nucleic acids. Of course, the degree of binding will vary 
according to the particular compound, the particular nucleic acid, and the 
conditions used. The particular advantage of using isopsoralens such as 
those of the present invention is that labelling can be carried out 
without interfering with subsequent hybridization. 
The present invention contemplates labelling of nucleic acid and nucleic 
acid target sequences by 1) labelled compounds synthesized by methods of 
the present invention and 2) labelled compounds of the present invention 
synthesized by methods of the present invention. 
In one embodiment, the present invention contemplates using BIOMIP and 
BIODMIP to provide an appended biotin on nucleic acid target, and then 
using that labelled nucleic acid in a subsequent detection step. For 
example, the present invention contemplates mixing BIOMIP or BIODMIP with 
the total DNA extracted from a blood sample suspected of harboring a 
virus. Irradiation of the mixture causes the BIOMIP or BIODMIP to 
photobind to the nucleic acid. The present invention contemplates that the 
next step of the method involves use of nucleic acid probes specific 
(i.e., complementary) for the viral nucleic acid sequences. These probes 
are added to the BIOMIP(or BIODMIP)-treated nucleic acid. It is 
contemplated that the probes are introduced on a solid support such as 
polystyrene beads. After hybridization and washing, the solid support 
(containing the probe/target-biotin complex) is treated with a signal 
development system (e.g., an avidin-HRP complex) for detection of binding. 
In a strictly analogous manner, the new compounds FLUORMIP and FLUORDMIP 
may be used to provide labelled nucleic acid. With these compounds, the 
nucleic acid can be detected by fluorescence techniques. 
The photochemical labelling methods of the present invention have 
advantages over other nucleic acid labelling techniques. First, as 
mentioned above, labelling with isopsoralens does not interfere with 
subsequent hybridization. Second, photochemical labelling allows for 
labelling of an entire nucleic acid target sequence. This is in contrast 
to enzymatic labelling methods such as the BIO-UTP system. P. R. Langer et 
al., Proc. Nat. Acad. Sci. U.S.A 78:6633 (1981). Furthermore, enzymatic 
labelling (e.g., nick translating) usually results in labelled products 
that are only about 200 base pairs in length. Finally, labelling nucleic 
acid with isopsoralens offers the additional advantages of repeatability 
and low cost. 
It is not intended that the labelling methods of the present invention be 
limited by the nature of the nucleic acid. In one embodiment, the present 
invention contemplates that nucleic acid is selected from the group human 
genomic DNA and human RNA. In another embodiment, the present invention 
contemplates that the nucleic acid sequences are selected from the group 
consisting of sequences of viral DNA and sequences of viral RNA. In still 
a further embodiment, the present invention contemplates that the nucleic 
acid is selected from the group consisting of viral, bacterial, flngal, 
mycoplasma and protozoan nucleic acid. 
IV. CAPTURE OF NUCLEIC ACIDS 
The present invention contemplates that the compounds of the present 
invention be used to label and capture nucleic acid and nucleic acid 
sequences. In one embodiment, probe DNA is reacted with a cleavable 
biotin-isopsoralen, such as DITHIOMIP and DITHIODMIP, to yield 
biotinylated (probe) DNA. This biotinisopsoralen/nucleic acid complex is 
then hybridized to target DNA in a mixture of DNA. After hybridization, 
the biotin-probe-target complex is passed through an avidin-agarose 
column. The avidin-biotin-probe-target complex is retained on the column, 
while non-hybridized (non-target) DNA is washed through. Following the 
wash, the probe-target DNA hybrid is recovered by cleaving the biotin from 
the isopsoralen via reduction of the disulfide bond of DITHIOMIP or 
DITHIODMIP. Reduction is readily accomplished with reagents such as 
dithiothreitol or sodium borohydride. 
The present invention also contemplates capture with non-cleavable 
biotinisopsoralen such as BIOMIP and BIODMIP. In one embodiment, the probe 
DNA is reacted with BIOMIP or BIODMIP. The subsequent hybridization, 
capture and wash steps are the same as described for the cleavable 
compound. However, following the wash, the present invention contemplates 
that either the entire probe-target complex is removed from the avidin 
column by the addition of a reagent that breaks up the biotinavidin 
interaction (e.g., 8M guanidinium chloride), or alternatively, the 
captured target sequence is specifically released from the probe-target 
complex by denaturation of the hybridized nucleic acid. This latter 
procedure leaves the probe bound to the column and washes the target off 
the column. In one embodiment, this step is accomplished by elution with a 
solution that provides denaturing conditions within the column matrix 
(e.g., 60% formamide). 
V. INHIBITING TEMPLATE-DEPENDENT ENZYMATIC SYNTHESIS 
Enzymatic synthesis that involves nucleic acid, either solely as a template 
(e.g., translation involves the use of nucleic acid as a template to make 
polypeptides) or as both a template and a product (replication and 
transcription use nucleic acid as a template to produce nucleic acid) is 
hereinafter referred to as "template-dependent enzymatic synthesis." 
In the case of replication, nucleic acid polymerases replicate a nucleic 
acid molecule ("template") to yield a complementary ("daughter") nucleic 
acid molecule. For example, DNA polymerase I, isolated from E. Coli, 
catalyzes the addition of deoxyribonucleoside triphosphates to the 3' end 
of a short segment of DNA ("primer") hybridized to a template strand to 
yield a daughter of the template, starting from a mixture of precursor 
nucleotides (dATP, dGTP, dCTP, and dTTP). This 5' to 3' template-dependent 
enzymatic synthesis is also called "primer extension." The reaction will 
not take place in the absence of template. The reaction can be measured if 
one or more of the precursor nucleotides are labelled (usually they are 
radiolabelled with .sup.32 P). 
There are a nunber of known methods of DNA modification that block 
template-dependent enzymatic synthesis. For example E. coli polymerase I 
terminates copying single stranded DNA one nucleotide before encountering 
DNA lesions such as pyrimidine dimers induced by ultraviolet light, P. D. 
Moore et aL, Proc. Natl. Acad. Sci. 78:110 (1981), carcingen adducts, P. 
D. Moore et al., Proc. Natl. Acad. Sci. 79:7166 (1982), and 
proflavin-mediated guanine residue photooxidation, J. G. Piette and P. D. 
Moore, Photochem. Photobiol. 35:705 (1982). 
C. M. Ou et al., Biochemistry 17:1047 (1978) investigated whether DNA 
replication by DNA polymerase I from E. coli. B could be inhibited by 
covalently bound 8-methoxypsoralen (8-MOP) or by 5,7-dimethoxycoumarin 
(DMC). 8-MOP is a psoralen and was used to crosslink the DNA. DMC is a 
coumarin derivative that lacks the flryl carbon-carbon double bond 
necessary for photoaddition to pyrimidine bases of DNA; DMC cannot form 
crosslinks. It was found that the crosslinked DNA (8-MOP-modified) lost 
97% of its template activity for the enzyme used. The DMC-modified 
(uncrosslinked) DNA lost only 50% of its template activity. It was 
proposed that the crosslinking of DNA was responsible for the difference 
in inhibition of template activity. 
J. G. Piette and J. E. Hearst, Proc. Natl. Acad. Sci. 80:5540 (1983) 
reported that E. Coli polymerase I, when nick-translating a 
double-stranded template, is not inhibited by covalently bound psoralen 
4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT)! monoadducts or 
isopsoralen (5-methylisopsoralen) monoadducts. The enzyme is, however, 
effectively blocked by psoralen crosslinks. 
J. G. Piette and J. E. Hearst, Int. J. Radiat. Biol. 48:381 (1985) later 
reported that E.Coli polymerase I, when carrying out template-dependent 
enzymatic synthesis on a single-stranded template (single-stranded 
bacteriophage DNA), was inhibited by HMT monoadducts. It was concluded 
that DNA structure (single-stranded versus double-stranded) must account 
for the different results. 
G. Ericson and P. Wollenzien, Analytical Biochem. 174:215 (1988) examined 
psoralen crosslinks on RNA and their ability to block reverse 
transcriptase. They reported that a psoralen crosslink is an absolute stop 
for avian myeloblastosis virus reverse transcriptase. Psoralen monoaddcuts 
showed little inhibition of the enzyme. 
These experiments showed that blocking of enzymatic synthesis of nucleic 
acids could be accomplished with psoralen crosslinks and, in some cases, 
inhibition could be achieved with psoralens monoadducts. Importantly, the 
one attempt to block enzymatic synthesis with an isopsoralen showed no 
inhibition. 
The present invention provides the surprising result that 
template-dependent enzymatic synthesis of nucleic acid can be effectively 
inhibited with one or more "inhibition agents" wherein the inhibition 
agents are compounds selected from the group consisting of isopsoralens 
and photoproduct. As noted earlier, isopsoralens cannot form crosslinks. 
("Photoproduct" has been extensively defined and discussed above.) 
The present invention contemplates inhibiting template-dependent enzymatic 
synthesis by A) Site-Specific Covalent Addition, B) Random Covalent 
Addition, and C) Photoproduct Addition, and reveals D) Compound/Enzyme 
Specificity. 
A. Site-Specific Addition 
The present invention contemplates inhibiting of template-dependent 
elongation by site-specific binding of new and known isopsoralens to 
nucleic acid. In one embodiment, the method of the present invention for 
the construction of specifically placed isopsoralen adducts begins with 
two short oligonucleotides that are complementary to each other, but that 
differ in length. These oligonucleotides, along with an isopsoralen are 
placed together under conditions where the oligonucleotides are base 
paired as a double stranded molecule. This non-covalent complex is 
irradiated to cause addition of the isopsoralen (320-400 nm) or psoralen 
(&gt;380 nm) to the oligonucleotides. Following irradiation, monoadducted 
oligonucleotides are isolated by HPLC or denaturing polyacrylamide gel 
electrophoresis (PAGE). Because of the differential length of the original 
short oligonucleotides, monoadducted oligonucleotides specific to each 
strand are isolated. The present invention contemplates further that the 
short monoadducted oligonucleotides may be appended to longer 
oligonucleotides through the use of a ligation reaction and a 
complementary splint molecule (the longer, ligated molecules are purified 
by PAGE). 
Such specifically constructed monoadducted oligonucleotides are useful to 
determine the differential site-specificity of photoreactive compounds. 
The present invention contemplates the use of different compounds for 
different site-specifities. For example, AMIP has a different 
site-specificity from AMDMIP. 
B. Random Addition 
The present invention also contemplates randomly adding isopsoralens to 
produce covalent complexes of isopsoralen and nucleic acid. By random it 
is not meant that the particular isopsoralen will not display preferential 
placement. By random it is meant that the level of addition (one, two or 
three adducts, etc.) is not limited to one adduct per strand; the compound 
has access to a larger number of sites. The present invention further 
contemplates mixing isopsoralens to create a "cocktail" for random 
addition. Randomly added cocktails can be used where multiple adducts per 
strand are desired and where preferential placement is sought. The present 
invention contemplates that consideration be given to the nature of the 
nucleic acid (A:T rich, A:T poor, etc.) in selecting both single mixtures 
and cocktails for random addition. 
C. Photoproduct Addition 
Previous work towards the blocking of replication of nucleic acids with 
furocoumarins has historically proceeded by a method having the temporal 
steps: 1) providing a specific psoralen derivative, 2) providing a 
particular nucleic acid or nucleic acid target sequence(s), 3) mixing the 
psoralen with the nucleic acid in the presence of activating wavelengths 
of electromagnetic radiation. In one embodiment, the present invention 
contemplates a radical departure from this historical approach to 
blocking. In one embodiment of the method of the present invention, the 
temporal sequence is the following: 1) providing furocoumarin 
derivative(s), 2) exposing the furocoumarin derivative(s) to activating 
wavelengths of electromagnetic radiation, 3) providing a particular 
nucleic acid or nucleic acid target sequence(s), 4) mixing the irradiated 
furocoumarin derivative(s) with the nucleic acid. In this embodiment, the 
furocoumarin is irradiated prior to mixing with nucleic acid. The 
experimental investigation of this novel temporal sequence has established 
that furocoumarin photoproduct exists and that photoproduct can inhibit 
template-dependent enzymatic synthesis, e.g., primer extension. 
In one embodiment, the present invention contemplates using AMDMIP 
photoproduct and AMIP photoproduct ("a photoproduct cocktail") to inhibit 
polymerase activity. While not limited to any particular molecular 
mechanism for inhibition, it is contemplated that inhibition is 
specifically due to the interaction of photoproduct with nucleic acid. In 
one embodiment, the method of the present invention comprises: a) 
providing pre-irradiated AMIP and AMDMIP; b) providing one or more nucleic 
acid target sequences; and c) adding the pre-irradiated AMIP and AMDMIP to 
the one or more nucleic acid sequences, so that the one or more sequences 
cannot be extended by polymerase. Again, while not limited to any 
particular molecular mechanism, it is contemplated that photoproduct is 
formed which undergoes subsequent thermal addition to the nucleic acid. It 
is believed that the photoproduct:nucleic acid complex cannot serve as a 
template for polymerase. 
Advantages of photoproduct inhibiting methods of the present invention 
include the ability to pre-form the inhibition agent in the absence of 
target. The photoproduct can then be provided at the appropriate point in 
the process (i.e., when a polymerase inhibiting moiety is required to be 
added to the nucleic acid or nucleic acid sequence). This pre-irradiation 
is contemplated particularly where thermally sensitive reagents are used 
for inhibition. For example, compounds which are thermally sensitive would 
not be suitable for some types of template-dependent enzymatic synthesis. 
Such compounds would lose their utility due to thermal decomposition prior 
to photoactivation. With the novel temporal sequence of the method of 
photoproduct inhibition of the present invention, the need for thermal 
stability is obviated since photoproduct can be pre-formed and added at 
the conclusion of thermal cycling. 
D. Compound/Enzyme Specificity 
The present invention provides results that suggest there is some 
compound/enzyme specificity, e.g., some isopsoralens inhibit particular 
polymerases better than other isopsoralens. For example, MIP and AMIP 
adducts will inhibit primer extension by Taq polymerase, T4 polymerase and 
reverse transcriptase. MIP and AMIP adducts, however, do not show the same 
level of inhibition of primer extension by E. Coli polymerase or Klenow 
fragment. By contrast, AMDMJP adducts show the same level of inhibition of 
primer extension by all of these enzymes. 
VI. STERILIZATION 
The present invention contemplates a method of sterilization that is useful 
for, among other uses, solving the carry-over problem associated with 
amplification of nucleic acid. The overall approach of the method involves 
rendering nucleic acid after amplification substantially unamplifiable 
(hence "Post-Amplification Sterilization"), before a carry-over event can 
occur. 
Post-amplification sterilization is designed to control carry-over. It is 
desirable to concurrently run reagent controls to assure that carry-over 
is absent in the first place. 
It was noted earlier that target sequences are "targets" in the sense that 
they are sought to be sorted out from other nucleic acid. Amplification 
techniques have been designed primarily for this sorting out. 
"Amplification" is a special case of replication involving template 
specificity. It is to be contrasted with non-specific template replication 
(i.e., replication that is template-dependent but not dependent on a 
specific template). Template specificity is here distinguished from 
fidelity of replication (i.e., synthesis of the proper polynucleotide 
sequence) and nucleotide (ribo- or deoxyribo-) specificity. 
Template specificity is achieved in most amplification techniques by the 
choice of enzyme. Amplification enzymes are enzymes that, under conditions 
they are used, will process only specific sequences of nucleic acid in a 
heterogenous mixture of nucleic acid. For example, in the case of Q.beta. 
replicase, MDV-1 RNA is the specific template for the replicase. D. L. 
Kacian et al., Proc. Nat. Acad. Sci USA 69:3038 (1972). Other nucleic acid 
will not be replicated by this amplification enzyme. Similarly, in the 
case of T7 RNA polymerase, this amplification enzyme has a stringent 
specificity for its own promoters. M. Chamberlin et al., Nature 228:227 
(1970). In the case of T4 DNA ligase, the enzyme will not ligate the two 
oligonucleotides where there is a mismatch between the oligonucleotide 
substrate and the template at the ligation junction. D. Y. Wu and R. B. 
Wallace, Genomics 4:560 (1989). Finally, Taq polymerase, by virtue of its 
ability to function at high temperature, is found to display high 
specificity for the sequences bounded and thus defined by the primers; the 
high temperature results in thermodynamic conditions that favor primer 
hybridization with the target sequences and not hybridization with 
nontarget sequences. PCR Technology, H. A. Erlich (ed.) (Stockton Press 
1989). 
Enzymes such as E. coli DNA polymerase I and Klenow are not specific 
enzymes. Indeed, within their range of activity (temperature, pH, etc.), 
they are promiscuous; they will elongate any nucleic acid having short 
double-stranded segments exposing a 3' hydroxyl residue and a protruding 
5' template. This, of course, is not to say that these enzymes cannot be 
used in an amplification protocol. For example, these enzymes can be used 
with homogeneous nucleic acid to produce specific target. 
It is not intended that the sterilization method of the present invention 
be limited by the nature of the particular amplification system producing 
the nucleic acid to be sterilized. Some amplification techniques take the 
approach of amplifying and then detecting target; others detect target and 
then amplify probe. Regardless of the approach, amplified nucleic acid can 
carry-over into a new reaction and be subsequently amplified. The present 
invention contemplates sterilizing this amplified nucleic acid before it 
can carry-over. 
A. Sterilization In General 
Something is "sterilized" when it is rendered incapable of replication. 
While the term "sterilization" has typically been applied only in the 
context of living organisms, it is here meant to be applied to in vitro 
amplification protocols of polynucleotides where a template polynucleotide 
functions in the nature of a germination seed for its further propagation. 
Sterilization "sensitivity" is an operationally defined term. It is defmed 
only in the context of a "sterilization method" and the particular 
detection method that is used to measure templates (or organisms). 
Sterilization sensitivity is the number of germination seeds (e.g., viable 
bacterial cells or polynucleotide templates) that result in a measurable 
signal in some sterilization method and defined detection assay. 
To appreciate that a "sterilization method" may or may not achieve 
"sterilization," it is useful to consider a specific example. A bacterial 
culture is said to be sterilized if an aliquot of the culture, when 
transferred to a fresh culture plate and permitted to grow, is 
undetectable after a certain time period. The time period and the growth 
conditions (e.g., temperature) define an "amplification factor." This 
amplification factor along with the limitations of the detection method 
(e.g., visual inspection of the culture plate for the appearance of a 
bacterial colony) define the sensitivity of the sterilization method. A 
minimal number of viable bacteria must be applied to the plate for a 
signal to be detectable. With the optimum detection method, this minimal 
number is 1 bacterial cell. With a suboptimal detection method, the 
minimal number of bacterial cells applied so that a signal is observed may 
be much greater than 1. The detection method determines a "threshold" 
below which the "sterilization method" appears to be completely effective 
(and above which "sterilization" is, in fact, only partially effective). 
This interplay between the amplification factor of an assay and the 
threshold that the detection method defines, can be illustrated. Referring 
now to Table 4, bacterial cells are applied to a plate under two different 
sets of conditions: in one case, the growth conditions and time are such 
that an overall amplification of 10.sup.4 has occurred; in the other case, 
the growth conditions and time are such that an overall amplification of 
10.sup.8 has occurred. The detection method is arbitarily chosen to be 
visual inspection. The detectable signal will be proportional to the 
number of bacterial cells actually present after amplification. For 
calculation purposes, the detection threshold is taken to be 10.sup.6 
cells; if fewer than 10.sup.6 cells are present after amplification, no 
cell colonies are visually detectable and the sterilization method will 
appear effective. Given the amplification factor of 10.sup.4 and a 
detection threshold of 10.sup.6, the sterilization sensitivity limit would 
be 100 bacterial cells; if less than 100 viable bacterial cells were 
present in the original aliquot of the bacterial culture after the 
sterilization method is performed, the culture would still appear to be 
sterilized. Alternatively, if the time and growth conditions permitted an 
amplification of 10.sup.8, then the sterilization sensitivity limit 
(assuming the same detection threshhold) would be 1 bacterial cell. 
TABLE 4 
______________________________________ 
Amplification 
# Of Viable Bacterial Cells Applied To A Plate 
Factor 1 10 100 1000 
______________________________________ 
10.sup.4 10.sup.4 
10.sup.5 
10.sup.6 
10.sup.7 
# of Bacterial cells 
after Amplification 
- - + ++ Detection (+/-) 
10.sup.8 10.sup.8 
10.sup.9 
10.sup.10 
10.sup.11 
# of Bacterial cells 
after Amplification 
++ +++ +++ ++++ Detection (+/-) 
______________________________________ 
Under the latter conditions, the sterilization method must be suffiently 
stringent that all bacterial cells are, in fact, incapable of replication 
for sterilization to appear complete (i.e., the sterilization method would 
need to cause sterilization, not just substantial sterilization). 
B. Sterilization Of Potential Carry-Over 
The same considerations of detection threshold and amplification factor are 
present when determining the sensitivity limit of a sterilization method 
for nucleic acid. Again, by "sterilization" it is meant that the nucleic 
acid is rendered incapable of replication, and specifically, 
unamplifiable. 
The post-amplification sterilization method of the present invention 
renders nucleic acid substantially unamplifiable. In one embodiment, the 
post-amplification sterilization method renders amplified nucleic acid 
unamplifiable but detectable. In still another embodiment, the 
post-amplification sterilization method of the present invention 
contemplates that the number of carry-over molecules of amplifiable 
nucleic acid that has occurred is small enough that, in a subsequent 
amplification, any amplified product reflects the presence of true target 
in the sample. In a preferred embodiment, the post-amplification 
sterilization method of the present invention renders amplified segments 
of a target sequence substantially unamplifiable but detectable prior to a 
carry-over event. 
It is not intended that the post-amplification sterilization method of the 
present invention be limited by the nature of the nucleic acid; it is 
contemplated that the postamplification sterilization method render all 
forms of nucleic acid (whether DNA, mRNA, etc.) substantially 
unamplifiable. 
"Template" encompasses both the situation where the nucleic acid contains 
one or more segments of one or more target sequences, and the situation 
where the nucleic acid contains no target sequence (and, therefore, no 
segments of target sequences). "Template" also encompasses both the 
situation where the nucleic acid contains one or more replicatable probes, 
and the situation where the nucleic acid contains no replicatable probes. 
Where template is used for amplification and amplification is carried out, 
there is "amplification product." Just as "template" encompasses the 
situation where no target or probe is present, "amplification product" 
encompasses the situation where no amplified target or probe is present. 
The present invention provides "sterilizing compounds" and methods for 
using "sterilizing compounds." "Sterilizing compounds" are defmed such 
that, when used to treat nucleic acid according to the sterilization 
method of the present invention, the nucleic acid is rendered 
substantially unamplifiable, i.e., substantially sterilized. The preferred 
sterilizing compounds of the present invention are activation compounds. 
While it is not intended that the present invention be limited to any 
theory by which nucleic acid is rendered substantially unamplifiable by 
the methods and compounds, it is expected that sterilization occurs by 
either 1) modification of nucleic acid, or 2) inhibition of the 
amplification enzyme itself. Again, while not limited to any mechanism, it 
is expected that, if modification of nucleic acid occurs with sterilizing 
compounds, it probably occurs because the compounds react with amplified 
nucleic acid to create sufficient adducts per base (i.e., sufficient 
"modification density") such that statistically all strands are prevented 
from either 1) subsequent use of the denatured nucleic acid in single 
stranded form as template for amplification or 2) dissociation of the 
double stranded form of the nucleic acid into single strands, thereby 
preventing it from acting as a template for subsequent amplification. On 
the other hand, it is expected that, if direct inhibition of the 
amplification enzyme occurs, it probably occurs because the sterilizing 
compound acts via 1) hydrophobic and hydrophylic interactions, or 2) 
steric hindrance. 
In the case of sterilizing compounds modifying nucleic acid, it is 
preferred that interaction of the nucleic acid (whether DNA, mRNA, etc.) 
with the sterilizing compound causes the amplification enzyme to 
differentiate between actual target sequences and carry-over nucleic acid, 
such that, should amplified nucleic acid be carried over into a subsequent 
amplification, it will not be amplified. 
C. Selective Sterilization 
It is further contemplated that the sterilization method of the present is 
useful in conjunction with amplification, without regard to the carry-over 
problem. In one embodiment, it is contemplated that sterilization is 
performed in a selective manner so that with respect to a mixture of 
nucleic acids, nucleic acid desired to be rendered unamplifiable is 
rendered substantially unamplifiable, but nucleic acid desired to remain 
amplifiable (hereinafter "sheltered nucleic acid") remains amplifiable. 
The present invention contemplates three general approaches to this 
selective sterilization (and consequent selective amplification). 
First, the present invention contemplates taking advantage of the 
site-specificity of activation compounds, and in particular, photoreactive 
activation compounds. In this approach, an activation compound is selected 
that has a known site-specificity (or site preference) for nucleic acid 
(e.g., TpA site-specificity). It is preferred that the site-specificity is 
selected with the knowledge of the sequence of the sheltered nucleic acid. 
In this manner, a site-specificity can be chosen where the activation 
compound will bind at non-sterilizing modification densities to the 
sheltered nucleic acid (or not bind at all) but will bind at sterilizing 
modification densities to the remaining nucleic acid. 
Second, the present invention contemplates taking advantage of the unique 
secondary and tertiary structural requirements of some activation 
compounds for binding with nucleic acid. In this case, the sheltered 
nucleic acid must have different secondary or tertiary structure than the 
remaining nucleic acid. An activation compound requiring secondary or 
tertiary structure that is lacking in the sheltered nucleic acid is 
selected and added to the nucleic acid mixture. The activation compound 
binds at non-sterilizing modification densities with the sheltered nucleic 
acid and sterilizing modification densities with the remaining nucleic 
acid. 
Finally, the present invention contemplates multiple sterilizations in a 
multiple amplification protocol. Multiple amplification systems have been 
suggested where a first amplification is carried out by a first 
polymerase, followed by a second amplification with a second polymerase. 
For example, the first amplification can be used to introduce promotor 
sites for the enzyme of the second amplification. Mullis et al, Cold 
Springs Harbor Symposia, Vol. L1, p. 263 (1986). G. J. Murakawa et al., 
DNA 7:287 (1988). In the sterilization approach to these multiple 
amplification systems, the present invention takes advantage of the unique 
polymerase specificities of activation compounds, and in particular 
photoreactive activation compounds. A first activation compound is 
selected that, when bound to nucleic acid, will inhibit amplification by 
the first polymerase in the first amplification, but that will not inhibit 
the second polymerase in the second amplification. Post-amplification 
sterilization is carried out after the first amplification with this first 
activation compound. Amplified nucleic acid treated in this manner will 
not be amplified by the first polymerase but can be amplified by the 
second polymerase. Post-amplification sterilization can later be performed 
after the second amplification with a second activation compound that, 
when bound to nucleic acid, will inhibit amplification with the second 
polymerase. 
D. Selecting Activation Compounds for Sterilization 
As noted above, the preferred sterilizing compounds of the present 
invention are activation compounds. FIG. 3 outlines the methods by which 
activation compounds can be screened for use as sterilizing compounds. 
Four "Sterilization Modes" are shown along with the temporal points where 
potential reactants of each Mode are added to the amplification system 
(the amplification system is contemplated to encompass all amplification 
methods, e.g., target-amplifying or probe-amplifying). 
The Sterilization Modes consist of the following temporal steps: 
Mode I: Add activation compound then amplify sample, followed by activation 
("triggering") of the activation compound; 
Mode II: Amplify sample then add activation compound, followed by 
activation ("triggering") of the activation compound; 
Mode III: Add pre-activated ("triggered") activation compound then amplify 
sample; 
Mode IV: Amplify sample then add pre-activated ("triggered") activation 
compound. 
In the general case, an activation compound is "triggered" to an active 
form. This form provides the sterilizing activity to the system. The type 
of triggering required depends on the properties of the sterilizing 
compound. For example, thermally reactive compounds are triggered by 
providing the correct temperature while photoreactive compounds are 
triggered by providing the appropriate activating wavelengths of 
electromagnetic radiation. Thoughtful consideration of FIG. 3 allows any 
activation compound to be analyzed as a potential sterilizing compound and 
defines its appropriate Mode of application (if any). 
A new compound ("X") can be evaluated as a potential sterilizing compound. 
X is initially evaluated in Step A of Mode I. In Step A, X is added to the 
sample during the sample preparation step prior to amplification. The 
amplification process is performed and the yield of the amplified product 
compared to an identical sample amplified without X. If the amplification 
yield is similar in both samples, the sterilization activity of X is 
evaluated in Step B of Mode I. In Step B, the appropriate "trigger" is 
pulled to activate X after amplification has occurred. For example, if X 
is a thermal reagent, the appropriate temperature is provided to generate 
the activated form of the compound (X*= generically activated X). The 
sterilization effect of X* on the amplified products is then determined by 
reamplification of the amplified products after treatment. If an 
acceptable level of sterilization is realized, a separate evaluation is 
performed to determine the effect of the modification provided by X* on 
subsequent detection of the modified target molecules. In this manner, 
both the effectiveness of X as a Mode I sterilization reagent and the 
compatibility of the modified amplified target with subsequent detection 
formats is evaluated. 
Alternatively, X may inhibit the amplification process in Mode I, Step A. 
In this event, X cannot be effectively used in Mode I; X is thereafter 
evaluated as a Mode II sterilization reagent. In Mode II, the temporal 
order of amplification, compound addition and triggering are changed 
relative to Mode I. X is added following amplification in Mode II, thereby 
avoiding the amplification inhibition detected in Mode I. In this fashion, 
the sterilization effect of X* on the amplified products can be determined 
independent of the negative effect of X on amplification. Evaluation of 
the Mode II sterilization activity is done in the same fashion as for Mode 
I, Step B. 
The two additional methods which use X for sterilization are Modes III and 
IV. In both these Modes, X is triggered to provide X* prior to addition to 
the sample. X* is then added to the system either before (Mode III) or 
after (Mode IV) amplification. 
In Mode III, X* may be provided then added to the sample prior to 
amplification. In the case where X is a photoreactive compound, X* is the 
resultant product of the exposure of photoreactive compound to activating 
wavelengths of electromagnetic radiation. If amplification is inhibited 
with this resultant product, it may reasonably be suspected that exposure 
of X to activating wavelengths of electromagnetic radiation results in 
photoproduct. 
In Mode IV, X* is provided then added to the system following 
amplification, thereby avoiding any issue of compatablity with the 
amplification process. X*, whether a thermally activated or 
photoactivated, when provided and used according to Mode IV, can provide 
effective sterilization via more than one mechanism. X* may react with 
amplified target, non-nucleic acid components of the system, or both. 
Table 5 summarizes the above. 
TABLE 5 
______________________________________ 
Evaluation Of Potential Sterilization Reagents 
Mode/Step 
Result* Interpretation/Next Step 
______________________________________ 
I/A +ampl Compound is compatible with 
amplification/Evaluate in Mode I, Step B 
I/A -ampl Compound is incompatible with 
amplification/Evaluate in Mode II, Steps A and B 
I/A + B 
+ster Compound is a useful sterilization reagent in 
Mode I/Evaluate detection 
I/A + B 
-ster Compound is ineffective as a sterilization reagent 
in Mode I/Evaluate in Modes II, III and IV 
II +ster Compound is useful for sterilization in Mode II/ 
Evaluate detection 
II -ster Compound is ineffective as a sterilization reagent 
in Mode II/Evaluate in Modes III and IV 
III -ampl Compound may be useful in Mode IV. 
III +ampl Compound is compatible with amplification but 
not useful for sterilization by definition. 
IV +ster Compound is a useful sterilization reagent in 
Mode IV/Evaluate detection 
IV -ster Compound is an ineffective as a sterilization 
reagent in Mode IV. 
______________________________________ 
*+/-ampl = amplification inhibited/amplification not inhibited 
+/-ster = sterilization effective/sterilization ineffective 
As noted in FIG. 3, the choice of the appropriate activation compound for 
post-amplification sterilization also depends in part on the detection 
method employed. If the detection procedure involves a hybridization step 
with the amplified nucleic acid sequences, it is desired that the 
amplified sequences be both available and hybridizable, i.e., they should 
not be irreversibly double stranded. If the detection procedure need not 
involve hybridization (e.g., incorporation of labelled nucleic acid 
precursors or the use of biotinylated primers, which are subsequently 
detected), the amplified sequence can normally remain double stranded. 
With the preferred method of sterilization, it is desired that the 
modification caused by the inactivation procedure not interfere with 
subsequent detection steps. In the case of post-amplification 
modifications to amplified target sequences, it is preferred that there be 
no impact on hybridization to or detection of the amplified segment of the 
target molecule. 
Environmental factors are important considerations--particularly during 
sample preparation. The preferred compound will not require special 
handling due to toxicity or sensitivity to the normal laboratory/clinical 
environment, including the normal incandescent or fluorescent lighting 
found in such environments. Compounds which are toxic to the user and/or 
sensitive to room light will require a special environment for use. 
Special environments make the assay inherently more cumbersome and complex 
and correspondingly more subject to error. The supporting instrumentation 
for such assays likewise becomes more complicated. 
Because it is desired that amplified nucleic acids not be exposed to the 
environment until they are sterilized, a preferred embodiment of the 
present invention contemplates the use of photoreactive compounds for 
sterilization. As noted earlier, "photoreactive compounds" are defined as 
compounds that undergo chemical change in response to appropriate 
wavelengths of electromagnetic radiation. Photoreactive compounds possess 
the advantage of allowing inactivation without opening the reaction vessel 
(when appropriate reaction vessels are used). Furthermore, because it is 
desired that the modification of the amplified nucleic acid not interfere 
with subsequent steps, the present invention contemplates the use of 
photoreactive compounds that do not interfere with detection. 
In the preferred embodiment, the invention contemplates amplifying and 
sterilizing in a closed system, i.e., the amplified nucleic acid is not 
exposed to the environment until modified. In one embodiment, the present 
invention contemplates having the photoreactive compound present in the 
reaction mixture during amplification. In this manner, the reaction vessel 
need not be opened to introduce the sterilizing compound. 
The use of photoreactive compounds in closed containers requires that 
sufficient light of appropriate wavelength(s) be passed through the 
vessel. Thus, a light instrument must be used in conjunction with the 
present invention to irradiate the sample. As noted above ("II. 
PHOTOACTIVATION DEVICES"), instruments with these features are 
contemplated by the present invention. 
In general, the sterilization method of the present invention is a method 
for treating nucleic acid comprising: a) providing in any order; i) 
nucleic acid, ii) amplification reagents, iii) one or more amplification 
enzymes, iv) one or more sterilizing compounds, and v) means for 
containing a reaction, as reaction components; b) adding to said reaction 
containing means, in any order, said nucleic acid and said amplification 
reagents, to make a reaction mixture; and c) adding to said reaction 
mixture, without specifying temporal order, i) said one or more 
amplification enzymes, and ii) said one or more sterilizing compounds. 
In a preferred embodiment, sterilization comprises the sequential steps of: 
a) providing, in any order, i) one or more photoreactive compounds, ii) 
nucleic acid, iii) amplification reagents, iv) one or more amplification 
enzymes, and v) means for containing a reaction; b) adding to the reaction 
containing means, in any order, one or more photoreactive compounds, 
nucleic acid, and amplification reagents, to make a reaction mixture; 
adding said one or more amplification enzymes to said reaction mixture; 
and d) treating said mixture with appropriate wavelengths of 
electromagnetic radiation so that said photoreactive compounds are 
photoactivated. 
"Amplification reagents" are defined as those reagents (primers, 
deoxyribonucleoside triphosphates, etc.) needed for amplification except 
for nucleic acid and the amplification enzyme. In one embodiment, the 
means for containing is a reaction vessel (test tube, microwell, etc.). 
In another embodiment, sterilization comprises the sequential steps of: a) 
providing, in any order, i) one or more photoreactive compounds, ii) 
nucleic acid, iii) amplification reagents, iv) one or more amplification 
enzymes, and v) means for containing a reaction; b) adding to said 
reaction containing means, said nucleic acid and said amplification 
reagents, followed by one or more amplification enzymes, to make a 
reaction mixture; c) adding said one or more photoreactive compounds to 
said reaction mixture; d) treating said mixture with appropriate 
wavelengths of electromagnetic radiation so that said photoreactive 
compounds are photoactivated. 
While the various embodiments illustrate that the mixing of photoreactive 
compound(s), amplification reagents, nucleic acid, and amplification 
enzyme(s) can be in any order, it is preferred that photoreactive 
compound(s) be added prior to initiation of amplification (note: the 
adding of amplification enzyme(s) to the reaction mixture containing 
amplification reagents and nucleic acid will initiate amplification). The 
method of the present invention may have the additional step of detecting 
amplified nucleic acid. 
In one embodiment, the photoreactive compound is selected from the group 
consisting of psoralens and isopsoralens. The preferred photoreactive 
compounds are isopsoralens. In one embodiment, a cocktail of isopsoralens 
is used. In another embodiment, the isopsoralen(s) is selected from the 
group consisting of 5-methylisopsoralen, 5-bromomethylisopsoralen, 
5-chloromethylisopsoralen, 5-hydroxymethylisopsoralen, 
5-formylisopsoralen, 5-iodomethylisopsoralen, 
5-hexamethylenetetraminomethylisopsoralen, 5-aminomethylisopsoralen, 
5-N-(N,N'-dimethyl-1,6-hexanediamine)-methylisopsoralen, 
5-N-N,N'-dimethyl-(6-biotinamido!-hexanoate)-1,6-hexane-diamine!)-methyl 
isopsoralen, 
5-N-N,N'-dimethyl-N'-(2-{biotinamido}-ethyl-1,3-dithiopropionate)-1,6-hex 
anediamine!-methylisopsoralen, 5-N-N,N'-dimethyl-N'-(carboxy-fluorescein 
ester)-1,6-hexanediamine)-methylisopsoralen, and their radiolabelled 
derivatives. In still another embodiment, the isopsoralen is selected from 
the group consisting of 4,5'-dimethylisopsoralen, 
4'-chloromethyl-4,5'-dimethylisopsoralen, 
4'-bromomethyl-4,5'-dimethylisopsoralen, 
4'-hydroxymethyl-4,5'-dimethylisopsoralen, 
4'-formyl-4,5'-dimethylisopsoralen, 
4'-phthalimidomethyl-4,5'-dimethylisopsoralen, 
4'-aminomethyl-4,5'-dimethylisopsoralen, 4'-iodomethyl-4,5' 
dimethylisopsoralen, 
4'-N-(N,N'-dimethyl-1,6-hexanediamine)-methyl-4,5'-dimethylisopsoralen, 
4'-N-N,N'-dimethyl-N'-(6-{biotinamido}-hexanoate)-1,6-hexanediamine!-meth 
yl-4,5'-dimethylisopsoralen, 
4'-N-N,N'-dimethyl-N'-(2-{biotinamido}-ethyl-1,3-dithiopropionate)-1,6-he 
xanediamine!-methyl-4,5'-dimethylisopsoralen, 
4'-N-N,N'-dimethyl-N'-(6-carboxyfluorescein 
ester)-1,6-hexanediamine)-methyl-4,5'-dimethylisopsoralen, and their 
radiolabelled derivatives. 
While the preferred compound for controlling carry-over according to the 
methods of the present invention is an isopsoralen, the present invention 
contemplates sterilization with psoralens as well. In one embodiment, the 
linear furocoumarin 4'-aminomethyl-4,5', 8-trimethylpsoralen (AMP) is used 
as a post-amplification sterilization reagent. 
The present invention contemplates using photoproduct for sterilization. In 
this embodiment, sterilization comprises the sequential steps: a) 
providing, in any order, i) photoproduct, ii) nucleic acid, iii) 
amplification reagents, iv) one or more amplification enzymes, and v) a 
means for containing a reaction; b) adding to said reaction containing 
means, in any order, said nucleic acid and said amplification reagents, to 
make a reaction mixture; c) adding said one or more amplification enzymes 
to said reaction mixture; and d) adding said photoproduct to said reaction 
mixture. 
In this embodiment, photoproduct is created prior to amplification but 
introduced after amplification. In another embodiment, photoproduct is 
made after and introduced after amplification. Photoproduct is made, 
therefore, at any time prior to mixing with the amplified nucleic acid. 
However, in both embodiments, photoproduct is not present in the reaction 
mixture during amplification. 
Photoproduct can be both provided and added manually or by an automated 
system. For example, it is contemplated that photoproduct be made in and 
introduced from a compartment in a reaction vessel. The compartment is 
separated from the remaining vessel by a barrier (e.g., a membrane) that 
is removable in a controlled manner. In the removable barrier approach, 
photoproduct is made by exposing the entire reaction vessel with the 
compartment containing photoreactive compound(s)! to the appropriate 
wavelength(s) of electromagnetic radiation. When photoproduct is to be 
added, the barrier is then removed and the newly formed photoproduct added 
to the amplified product. 
In another embodiment, photoproduct is made separately in a first reaction 
vessel and then injected into a second reaction vessel containing nucleic 
acid without opening the second reaction vessel. Injection is by a small 
needle; the needle can be permanently fixed in the side of the vessel if 
desired. 
In still another embodiment, photoproduct is made in a first reaction 
vessel and pipetted into a second reaction vessel containing nucleic acid. 
While other arrangements are possible, it is preferred that the pipetting 
be performed in an automated manner in the housing of a machine large 
enough to contain the first reaction vessel and the second reaction 
vessel. The housing serves to contain any carry-over while the reaction 
vessels are open. 
The sterilization method utilizing photoproduct may comprise additional 
steps such as a detection step. In one embodiment, the detection step 
involves detection of amplified target(s). In another embodiment, the 
detection step involves detection of amplified probe(s). 
E. Polymerase Chain Reaction 
In one embodiment, the present invention contemplates controlling the 
carryover associated with PCR. This embodiment is broadly referred to as 
"Post-Amplification Sterilization of Target from PCR" or "PAST PCR." For 
purposes here, a "target sequence" is further defined as the region of 
nucleic acid bounded by the primers used for PCR. A "segment" is defined 
as a region of nucleic acid within the target sequence. As noted above, 
the present invention contemplates sterilization whether or not the sample 
prepared for amplification contains a segment of a target sequence that 
can be amplified. Whether or not there is a segment of a target sequence 
that can be amplified, there is "PCR product." For definitional purposes, 
"PCR product" refers to the resultant mixture of compounds after two or 
more cycles of the PCR steps of denaturation, annealing and extension are 
complete. "PCR product" encompasses both the case where there has been 
amplification of one or more segments of one or more target sequences, and 
the case where there has been no amplification. 
A general nucleic acid screening protocol involving PCR amplification is 
schematically illustrated in FIG. 4. The steps are broadly characterized 
as 1) sample preparation, 2) amplification, and 3) detection. The lower 
time lines of FIG. 4 schematically illustrate the temporal sequence for a) 
the addition of sterilizing compound(s), and b) the activation of 
sterilizing compound(s) according to a preferred embodiment of the method 
of the present invention. 
Amplification cycling requires "PCR reagents." "PCR reagents" are here 
defmed as all reagents necessary to carry out amplification except 
polymerase and template. PCR reagents normally include nucleic acid 
precursors (dCTP, dTTP, etc.) and primers in buffer. See K. B. Mullis et 
al., U.S. Pat. Nos. 4,683,195 and 4,683,202, both of which are hereby 
incorporated by reference. 
PCR is a polynucleotide amplification protocol. The amplification factor 
that is observed is related to the number (n) of cycles of PCR that have 
occurred and the efficiency of replication at each cycle (E), which in 
turn is a function of the priming and extension efficiencies during each 
cycle. Amplification has been observed to follow the form E.sup.n, until 
high concentrations of PCR product are made. At these high concentrations 
(approximately 10.sup.-8 M/l) the efficiency of replication falls off 
drastically. This is probably due to the displacement of the short 
oligonucleotide primers by the longer complementary strands of PCR 
product. At concentrations in excess of 10.sup.-8 M, the kinetics of the 
two complementary PCR amplified product strands of finding each other 
during the priming reactions become sufficiently fast that they will occur 
before or concomitantly with the extension step of the PCR procedure. This 
ultimately leads to a reduced priming efficiency, and therefore, a reduced 
cycle efficiency. Continued cycles of PCR lead to declining increases of 
PCR product molecules. PCR product eventually reaches a plateau 
concentration. 
Table 6 illustrates the relationship of PCR cycle number to the number of 
PCR product strands that are made as a function of a wide range of 
starting target molecules (initial copy number). The efficiency of 
amplification was taken to be 1.85 per cycle, a value measured while using 
methods of the present invention for the HIV system with the SK38/SK39 
primers. Included in Table 6 is the PCR product concentration that is 
observed if the PCR reaction were to take place in 100 .mu.l volume. The 
gray area indicates conditions that give rise to PCR products that are in 
excess of 10.sup.-8 M/l. In these gray regions, PCR product would be 
expected to be approaching the plateau stage. Also shown in Table 6 is a 
signal (CPM) for a hybridization assay that is used to detect the presence 
of PCR product. This signal is calculated on the basis of having a 10% 
hybridization efficiency of a .sup.32 P labelled probe (3000 Ci/mM) to a 
20 .mu.l aliquot of the 100 .mu.l PCR reaction mix. 
While not limited to any particular theory, the present invention 
contemplates that, when an adduct is present on a PCR target sequence 
within the segment of the target sequence bounded by the primer sequences, 
the extension step of the PCR process will result in a truncated, 
complementary strand that is incapable of being replicated in subsequent 
cycles of the PCR process. As discussed above ("V. INHIBITING 
TEMPLATE-DEPENDENT ENZYMATIC SYNTHESIS") isopsoralens attached to a DNA 
polymer represent a stop for Taq polymerase extension reactions. In one 
embodiment, the present invention contemplates that such isopsoralens are 
effective in rendering a fraction of the starting target molecules 
incapable of amplification by the PCR process with Taq polymerase. 
Importantly, the sterilization protocol will be incomplete if some of the 
target molecules escape modification by the photochemical modification 
process. This process is, by its nature, a statistical process. This 
process can be characterized by measuring an average number (a) of adducts 
per DNA strand. Not all of the strands will have a adducts per strand. If 
the addition reaction is governed by Poisson statistics, the fraction of 
molecules that contain n modifications in a large population of molecules 
that have an average of a modifications is given by f.sub.a (n) (see Table 
7). A fraction of molecules, f.sub.a (0), will contain no modifications 
and are therefore considered non-sterilized. Table 7 evaluates 
TABLE 6 
__________________________________________________________________________ 
Amplication 
Initial Copy Number 
Cycle 
Factor (1.85).sup.n 
1 10 10.sup.2 
10.sup.3 
10.sup.4 
10.sup.5 
3 .times. 10.sup.5 
__________________________________________________________________________ 
* 
20 2.20 .times. 10.sup.5 
2.2 .times. 10.sup.5 
2.2 .times. 10.sup.6 
2.2 .times. 10.sup.7 
2.2 .times. 10.sup.8 
2.2 .times. 10.sup.9 
2.2 .times. 10.sup.10 
6.6 .times. 10.sup.10 
PCR Product 
Molecules 
3.6 .times. 10.sup.-15 
3.6 .times. 10.sup.-14 
3.6 .times. 10.sup.-13 
3.6 .times. 10.sup.-12 
3.6 .times. 10.sup.-11 
3.6 .times. 10.sup.-10 
10 .times. 10.sup.-9 
0 0 2.4 24 240 2,400 7,100 
25 4.78 .times. 10.sup.6 
4.8 .times. 10.sup.6 
4.8 .times. 10.sup.7 
4.8 .times. 10.sup.8 
4.8 .times. 10.sup.9 
4.8 .times. 10.sup.10 
4.8 .times. 10.sup.11 
1.4 .times. 10.sup.12 
PCR Product 
(M/I) 
7.9 .times. 10.sup.-14 
7.9 .times. 10.sup.-13 
7.9 .times. 10.sup.-12 
7.9 .times. 10.sup.-11 
7.9 .times. 10.sup.-10 
7.9 .times. 10.sup.-9 
2.4 .times. 10.sup.- * 
0 5 52 520 5,200 5.2 .times. 10.sup.4 
1.5 .times. 10.sup.5 
30 1.03 .times. 10.sup.8 
1.0 .times. 10.sup.8 
1.0 .times. 10.sup.9 
1.0 .times. 10.sup.10 
1.0 .times. 10.sup.11 
1.0 .times. 10.sup.12 
CPM's 
1.7 .times. 10.sup.-12 
1.7 .times. 10.sup.-11 
1.7 .times. 10.sup.-10 
1.7 .times. 10.sup.-9 
1.7 .times. 10.sup.-8 
11 110 1,100 1.1 .times. 10.sup.4 
1.1 .times. 10.sup.5 
35 2.24 .times. 10.sup.9 
2.2 .times. 10.sup.9 
2.2 .times. 10.sup.10 
2.2 .times. 10.sup.11 
2.2 .times. 10.sup.12 
3.6 .times. 10.sup.-11 
3.6 .times. 10.sup.-10 
3.6 .times. 10.sup.-9 
3.6 .times. 10.sup.-8 
240 2,400 2.4 .times. 10.sup.4 
2.4 .times. 10.sup.5 
40 4.86 .times. 10.sup.10 
4.9 .times. 10.sup.10 
4.9 .times. 10.sup.11 
4.9 .times. 10.sup.12 
8.0 .times. 10.sup.-10 
8.0 .times. 10.sup.-9 
8.0 .times. 10.sup.-8 
5,200 5.2 .times. 10.sup.4 
5.2 .times. 10.sup.5 
__________________________________________________________________________ 
*1 ug genomic DNA 
the non-sterilized fraction of DNA strands that are expected if an average 
of a modifications per strand exists. 
TABLE 7 
______________________________________ 
Poisson Statistics Applied To Sterilization 
f.sub.a (n) = .sub.a n.sub.e - a! / n| 
N = 10.sup.6, f.sub.g (0) = e.sup.-a 
a f.sub.a (0) 
Nf.sub.a (0) 
3 0.050 5.0 .times. 10.sup.4 
4 0.018 1.8 .times. 10.sup.4 
5 0.007 6.7 .times. 10.sup.3 
6 0.0025 2.5 .times. 10.sup.3 
7 0.0009 9.1 .times. 10.sup.2 
8 0.0003 3.3 .times. 10.sup.2 
9 0.00012 1.2 .times. 10.sup.2 
10 0.000045 45.0 
11 0.000017 17.0 
12 0.0000061 6.1 
13 0.0000023 2.2 
14 0.00000083 
.8 
15 0.00000030 
.3 
16 0.00000011 
.1 
17 0.00000004 
0.04 
______________________________________ 
a = Average number of adducts per strand. 
f.sub.a (0) = Fraction of strands with zero adducts when the average 
number of adducts per strand is a. 
Nf.sub.a (0) = The number of nonsterilized molecules, calculated for a 
total of 10.sup.6 molecules (N = 10.sup.6). 
Although the fraction of molecules with no modifications is small for all 
values of a, the expected number of non-sterilized molecules is large when 
sterilization is applied to a large number of molecules (N). For example, 
if carry-over consisted of 10.sup.6 product strands, Table 7 shows that 
2.5.times.10.sup.3 non-sterilized target molecules are expected if there 
is an average of 6 effective adducts per strand of PCR product. Effective 
adducts are those adducts that occur in the segment of a target molecule 
that is bounded by the primer sequences. For the HIV system this 
corresponds to 6 adducts in the 56 base long segment between the primer 
sequences on the 115-mer target molecule. This level of effective adducts 
corresponds to an average strand modification density of 1 adduct per 9.3 
bases. 
Ideally, one would like to be able to sterilize a PCR reaction mixture such 
that a major spill of the reaction would not lead to a carry-over problem. 
A 100 .mu.l PCR sample mixture with PCR product at a plateau concentration 
of 1.times.10.sup.-8 M contains 6.times.10.sup.11 complementary PCR 
product strands. Sterilization of this sample to a level where the 
expected number of non-sterilizing target molecules is less than one 
requires that f.sub.a (0) * 6.times.10.sup.11 be less than one (here 
"sterilized target molecule" means a target molecule that contains at 
least one adduct). By extending the data in Table 7 and assuming a 
reaction volume of 100 .mu.l, the statistical view of sterilization 
predicts that 28 adducts per strand of PCR product is sufficient to 
achieve this level of sterilization. If the number of target strands made 
by the PCR procedure is increased or reduced, the average number of 
adducts per strand required to achieve this level of sterilization will 
change accordingly. For the HIV 115-mer system that has reached plateau 
concentrations of product (6.times.10.sup.11 molecules), this level of 
sterilization occurs (28 average effective adducts per strand) when the 
average modification density is increased to 1 adduct per 2 bases. 
Alterations of the modification density can be expected through the use of 
different photoreactive compounds, or the use of the same photoreactive 
compound at different concentrations. In particular, the modification 
density is expected to increase through the use of the same photochemical 
agent at higher concentrations, and attaching the photochemical agent by 
exposure to actinic light from a device whose optical properties enhance 
covalent binding. 
For a fixed modification density there is another method of improving the 
sterilization sensitivity limit. The important statistical parameter for 
sterilization 
TABLE 8 
______________________________________ 
Expected Number Of Non-Sterilized PCR 
Molecules As A Function Of PCR Product Length 
Length of PCR 
*Average effective 
Non-Sterilized Molecules per 
Product Adducts/Strand 
6 .times. 10.sup.11 Starting Molecules 
______________________________________ 
CASE A: (1 adduct per 5 bases) 
100 10 2.7 .times. 10.sup.7 
150 20 1.2 .times. 10.sup.3 
200 30 &lt;1 
250 40 &lt;&lt;1 
300 50 &lt;&lt;1 
CASE B: (1 adduct per 9 bases) 
100 5.5 2.4 .times. 10.sup.9 
150 11.1 9.1 .times. 10.sup.6 
200 16.6 3.7 .times. 10.sup.4 
250 22.2 137 
300 27.7 &lt;1 
______________________________________ 
*Assumes that to be effective, the adducts must be in the segment of the 
PCR product that is bounded by the primers. For calculation purposes, the 
primer lengths were taken to be 25 bases each. 
sensitivity is the average number of adducts per PCR strand. By choosing 
PCR primers judiciously, the length of the PCR products can be varied, and 
therefore, the average number of adducts per strand can be varied. Table 8 
illustrates this effect for two different modification densities. In Case 
A, a modification density of 1 adduct per 5 bases is assumed. Under these 
conditions a PCR product oligonucleotide 200 bases in length should have 
approximately 30 effective adducts per strand. At this level of 
modification, less than one PCR product molecule in a 100 .mu.l PCR 
reaction tube would be expected to have no adducts per strand, and 
therefore, essentially all of the molecules in the reaction tube would be 
expected to be sterilized. Case B in Table 8 considers the situation in 
which the modification density is reduced to 1 adduct per 9 bases. Under 
these conditions the same level of sterilization requires that the PCR 
product be at least 300 bases in length for a sufficient number of 
effective adducts to be present on each strand. 
EXPERIMENTAL 
The following examples serve to illustrate certain preferred embodiments 
and aspects of the present invention and are not to be construed as 
limiting the scope thereof. 
In the experimental disclosure which follows, the following abbreviations 
apply: eq (equivalents); M (Molar); pLM (micromolar); N (Normal); mol 
(moles); mmol (millimoles); .mu.mol (micromoles); nmol (nanomoles); gm 
(grams); mg (milligrams); .mu.g (micrograms); L (liters); ml 
(milliliters); .mu.l (microliters); cm (centimeters); mm (millimeters); 
.mu.m (micrometers); nm (nanometers); .degree.C.(degrees Centigrade); Ci 
(Curies); mp (melting point); m/e (ion mass); MW(molecular weight); OD 
(optical density); EDTA (ethylenediamine-tetracetic acid); 1.times.TE 
(buffer: 10 mM Tris/1 mM EDTA, pH 7.5); 1.times.Taq (buffer: 50 mM KCl, 
2.5 mM MgCl.sub.2, 10 nM Tris, pH 8.5, 200 .mu.g/ml gelatin); C/M 
(chloroform/methanol); C/E/T (chloroform/ethanol/triethylamine); C/B/A/F 
(chloroform/n-butanol/acetone/formic acid); DMF (N.N-dimethylformnamide); 
PAGE (polyacralamide gel electrophoresis); UV (ultraviolet); V (volts); 
W(watts); mA (milliamps); bp (base pair); CPM (counts per minute); DPM 
(disintegrations per minute); TLC (Thin Laser Chromatography); HPLC (High 
Pressure Liquid Chromatography); FABMS (Fast Atom Bombardment Mass 
Spectrometry-spectra obtained on a Kratos MS50 instrument-Kratos 
Analytical. Manchester, England ); EIMS (Electron Impact Mass 
Spectrometry-spectra obtained on an AEI MS-12 Mass Spectrometer-Associated 
Electric Industries,. Manchester England); NMR (Nuclear Magnetic 
Resonance; spectra obtained at room temperature on either a 200 MHz or 250 
MHz Fourier Transform Spectrometer); Aldrich (Aldrich Chemical Co., 
Milwaukee, Wis.); Baker (J. T. Baker, Jackson, Tenn.); Beckman (Beckman 
Instruments, San Ramon, Calif.); BRL (Bethesda Research Laboratories, 
Gaithersburg, Md.); Cyro (Cyro Industries, Wood Cliff Lake, N.J.); DNEN 
(Dupont-New England Nuclear, Wilmington, Del. 19805); Gelman (Gelman 
Sciences, Ann Arbor, Miss.); Eastman (Eastman Kodak, Rochester, N.Y.); 
Eastman TLC Plates (#13181 TLC plates with fluorescent indicator, 
Eastman); EM (EM Science. Cherry Hill. N.J.); Lawrence (Lawrence Berkeley 
Laboratory, Berkeley, Calif.); Mallinckrodt (Mallinckrodt. St. Louis, 
Mo.); Pierce (Pierce Chemical Co., Rockford, Ill.); Polycast (Polycast 
Technology Corp., Stamford, Conn.); Rohm and Haas (Rohm and Hass Co., Los 
Angeles Calif.): Sigma (Sigma Chemical Co., St. Louis, Mo.); Spectrum 
(Spectrum Medical Industries, Los Angeles, Calif.). 
To better characterize the devices of the present invention, a customized 
light instrument (hereinafter referred to as "the PTI device") was 
constructed from commercially available parts (at a cost of approximately 
$10.000.00) to serve as a control. The device is a modified version of a 
described device. G. D. Cimino et al., Biochemistry 25, 3013 (1986). Some 
machining was necessary to retrofit some of the commercial parts and to 
make specialized adapters and holders. 
A 500 watt Hg/Xe arc lamp (Model A5000, Photon Technology International) 
positioned at the focal point of an elliptical mirror in a commercial lamp 
housing provides the light for the PTI device. The output from the lamp 
housing passes into an adaptor tube which provides physical support for 
additional optical accessories and prevents harmful stray UV radiation 
from emanating into the lab. A mirror deflects the optical beam in the 
adaptor tube so that it passes through the other optical components Two 
water-cooled, liquid filters are used. These filters have been selected to 
provide wavelengths of electromagnetic radiation that are appropriate for 
furocoumarin photochemistry. (Other photoreactive compounds may have 
wavelength requirements which are quite different from the furocoumarins.) 
The first filter is fitted with suprasil windows, filled with H.sub.2 O, 
and is used to filter out infrared radiation (IR). Exclusion of IR is 
required to prevent undesired heating of the sample chamber during 
irradiation, since addition of furocoumarins to nucleic acid is reduced at 
elevated temperatures. The second liquid filter provides a Window of 
320-400 nm light for use with furocoumarin photochemical reactions. This 
particular wavelength window (320-400 nm) excludes both shorter and longer 
wavelengths which are inappropriate for furocoumarin photochemistry. For 
example. furocoumarin:nucleic acid complexes undergo photochemical 
reversal at wavelengths below 313 nm. Exclusion of these wavelengths is 
necessary for irreversible photobinding of the furocoumarins to occur. 
This filter (9 cm in length) is fitted with 0.6 cm pyrex windows and 
filled with an aqueous solution of 0.85% cobaltous nitrate, 2% sodium 
chloride. An optical diffuser between the first filter and the second 
filter provides even illumination over the entire width of the light beam. 
This diffuser consists of a ground suprasil plate (0.6 cm) fitted into a 
lens holder. 
Light exiting from the first filter passes through an iris so that beam 
intensity can be controlled. Two lenses focus the beam within the sample 
holder by first passing the beam through a shutter system, then through 
the exit of the adapter tube and finally across a second mirror. The 
shutter system consists of a rotary solenoid attached to a metal blade 
which passes between the exit hole of the adaptor tube and a similar hole 
in a second aluminum plate. This second plate resides adjacent to the exit 
port of the adaptor tube and also serves as a mount for the solenoid. The 
sample holder is composed of rectangular brass and can be irradiated 
either through the side or from the top. It has been machined with 
passages for the flow of liquids. Thermoregulation of a sample is achieved 
by connecting this holder to a thermoregulated circulating water bath. The 
sample holder also contains passages that allow the flow of gases over the 
surfaces of the sample vessels (i.e., cuvette faces, etc.) to prevent 
condensation of water on these surfaces while irradiating at low 
temperatures. The orifice for the sample vessel in the sample holder is 1 
inch by 1 inch by 2.5 inches. A brass adaptor, with slots for the passage 
of light, permits standard cuvettes to be used, as well as 13 mm test 
tubes and Eppendorf tubes. The base of the sample holder is hollow so that 
a bar magnet attached to a small motor can be inserted beneath the sample 
vessel and function as a magnetic stirrer. Alternatively, the holder can 
be placed on top of a laboratory stir plate to achieve stirring 
capabilities. With this irradiation device, the light beam is 
approximately 0.8 cm diameter at the focal point and it has an intensity 
of 340 mW/cm.sup.2, as measured with a Model J-221 UV meter (UV Products, 
San Gabriel, Calif.). 
The PTI device allows for comparisons of the performance characteristics of 
the devices of the present invention against the performance 
characteristics of the more expensive PTI device. The performance 
characteristics examined in some of the examples below include: A) Thermal 
Stability, B) Spectral Output, C) Irradiation Intensity, D) Irradiation 
Uniformity, and E) Photoactivation Efficiency. 
Unless otherwise noted, all sample solutions prepared for irradiation were 
contained in Eppendorf tubes and irradiated through the sides of the tubes 
(CE-I, CE-II and CE-III) or through the top of the tubes (PTI). Eppendorf 
tubes have a transmittance of only 8 to 15% for wavelengths in the range 
of 300 nm to 400 nm (data not shown). Therefore, approximately 90% of the 
actinic light is lost by the use of these sample vessels. Although 
Eppendorf tubes are the most convenient sample vessels for biochemical and 
molecular biological procedures, other types of irradiation vessels having 
better transmission characteristics are contemplated (e.g., quartz, pyrex, 
polycarbonate etc.) 
Concentrations for photoproduct are given in terms of the amount of 
unirradiated starting material, where subsequent irradiation is performed 
in the absence of nucleic acid. For example, if 50 .mu.g/ml of 
unirradiated starting material is subsequently irradiated in the absence 
of DNA, the concentration of resulting photoproduct is given as 50 
.mu.g/ml. 
The starting compound (unirradiated compound) for photoproduct is sometimes 
indicated when photoproduct is referred to. For example, the photoproduct 
produced the following irradiation of AMDMIP is referred to as AMDMIP 
photoproduct. 
Where polyacrylamide gel electrophoresis (PAGE) is used denaturing (7 or 8M 
urea) polyacrylamide gels (28 cm.times.35 cm.times.0.4 mm) were poured and 
pre-electrophoresed for 30 to 60 minutes at 2000 Volts, 50 Watts, 25 
milliamps. 12% gels were used for oligonucleotides between 40 and 200 base 
pairs in length 8% gels were used for longer sequences. Depending on the 
length of the DNA to be analized, samples were loaded in either 8M urea, 
containing 0.025% tracking dyes (bromphenol blue and xylene cyanol), or in 
80% formamide, 10% glycerol, 0.025% tracking dyes, then electrophoresed 
for 2-4 hours at 2000 Volts, 50 Watts, 25 milliamps. Following PAGE 
individual bands were, in most cases, visualized by autoradiography. 
Autoradiography involved exposure overnight at -70.degree.. to Kodak XAR-5 
films with an intensifying screen. In some cases, the visualized bands 
were cut from the gel and collected for scintillation counting. 
Scintillation counting involved the use of a scintillation fluid and a 
commercial scintillation counter. (Searle Analytic 92, Model #000006893). 
Generally, PCR was carried out using 175-200 .mu.M dNTPs 
(deoxyribonucleoside 5'-triphosphates) and 0.5 to 1.0 .mu.M primers. 5 
Units/100 .mu.l of Taq polymerase was used. PCR reactions were overlaid 
with 30-100 .mu.l light mineral oil. A typical PCR cycle for HIV 
amplification using a Perkin-Elmer Cetus DNA Thermal Cycler (Part No. 
N8010150) was: denaturation at 93.degree. C. for 30 seconds: annealing at 
55.degree. C. for 30 seconds: and extension at 72.degree. C. for 1 minute. 
PCR cycles were normally carried out in this manner for 30 cycles followed 
by 7 minutes at 72.degree. C. 
In many cases. PCR was carried out on an HIV system. This system provides a 
115-mer product designated HRI 46: 
5'-ATAATCCACCTATCCCAGTAGGAGAAATTTATAAAAGATGGA 
TAATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC CAGCATTCTGGACATAAGACAGACCAAA-3 
' 
and its complement. designated HRI 47: 
3'-TATTAGGTGGATAGGGTCATCCTCTTTAAATATTTCTACCTA 
TTAGGACCCTAATTTATTTTATCATTCTTACATATCGGGATGGT 
CGTAAGACCTGTATTCTGTTCCTGGTTT-5' 
These sequences were used by C. Y. Ou et al., Science 239:295 (1988). 
In many of the examples below, compounds are referred to by their 
abbreviation (see Tables 2 and 3) and/or number (see FIGS. 1 and 2). For 
example, "(MIP,3)" indicates the compound is 5-Methylisopsoralen (Table 2) 
and compound 3 in FIG. 1. 
EXAMPLE 1 
Synthesis Of 5-Methylisopsoralen (MIP,3): Method 1 (three steps) 
Step 1: 5-methylresorcinol monohydrate (284 gm. 2.0 mol: Aldrich) was 
thoroughly mixed with malic acid (280 gm. 2.10 mol: Aldrich) and then 
placed in a reaction flask containing sulfuric acid (600 ml) and a trace 
of sodium bisulfite (1.0 gm: Aldrich). The reaction mixture was heated to 
90.degree. C. while being mechanically stirred until evolution of carbon 
dioxide subsided (about 5 hours). The resulting reddish-orange solution 
was poured into sufficient ice (with vigorous stirring) make up 1 liter. 
The receiving flask was maintained at 0.degree.C. by external cooling 
until the addition of the reaction mixture was complete. The resulting 
light-orange product that precipitated was collected by suction filtration 
then washed thoroughly with water. The crude product was air dried on the 
filter, then recrystallized twice from tetramethylene glycol (1800 ml) to 
give pure 7-hydroxy-5-methylcoumarin (H5MC,1) as product (220 gm. 62% 
yield: mp 252.degree.-255.degree. C.). 
Step 2: H5MC (177 gm. 1.0 mol), bromoacetaldehyde diethylacetal (207 gm, 
1.05 mol. Aldrich), potassium carbonate (100 gm) and freshly distilled 
dimethylformamide (125 ml) were mixed in a 3 neck reaction flask fitted 
with a mechanical stirrer and argon line. The reaction was heated and 
stirred at 100.degree. C. for 43 hours after which all the starting 
material had been converted to a high Rf TLC spot (Eastman TLC Plates: 
developed with CIM 98:2: detection with 260 nm ultraviolet light). 
Unreacted acetal and solvent were removed by distillation under reduced 
pressure. Water (1500 ml) was added to the residue followed by extraction 
with chloroform (1000 ml). The chloroform was then repeatedly washed with 
1N sodium hydroxide until colorless. Evaporation of the solvent gave the 
product, the diethoxyethyl ether of 7-hydroxy-5-methylcoumarin (DEMC,2) as 
an oil (217 gm; 82.4% yield). 
Step 3: Glacial acetic acid (310 ml) and zinc chloride (98 gm, 0.72 mol) 
were placed in a flask fitted with an internal thermometer then heated to 
between 100.degree. and 114.degree.. DEMC (50 gm, 0.17 mol) was added to 
the hot solution and stirred vigorously at temperature for 17 minutes. The 
hot solution was then poured onto a mixture of ice (1000 ml) and 
CHCl.sub.3 (500 ml) with vigorous stirring, which was continued until all 
the ice had dissolved. The CHCl.sub.3 layer was then separated (emulsion) 
followed by repeated washing with water (500 ml portions). Washing was 
continued until the pH of the water was neutral. Finally, the CHCl.sub.3 
layer was washed with saturated NaCl (500 ml), dried (MgSO.sub.4), and the 
solvent removed by distillation. The crude dark product (7.7 gm) (mixture 
of 5-methylisopsoralen and f-methylpsoralen) was dissolved in a small 
volume of chloroform, washed with 1N NaOH (to remove phenols), washed with 
water then brine, then chromatographed on a flash column (EM silica gel, 
200-400 mesh), eluting with ethyl acetate/hexanes 70:30 to give the pure 
product, 5-methylisopsoralen (MIP) (4.7 gm. 13.8%: mp 
189.5.degree.-191.5.degree. C.). NMR (CDC13) d 2.59 (3 H. s), 6.37 (1 
H.d), 7.05 (1 H. d), 7.23 (1 H, s), 7.59 (1 H. d), 7.96 (1 H, d). 
EXAMPLE 2 
Synthesis Of MIP: Method 2 (two steps) 
Step 1: H5MC was made from 5-methylresorcinol hydrate as described in Step 
1 of Method 1 (Example 1). 
Step 2: H5MC (1.76 gm, 10 mmol) and chloroethylene carbonate (6.13 gm, 50 
mmol, Aldrich) were heated between 150.degree.-165.degree. for 1.5 hours. 
Following this period the dark reaction mixture was poured onto ice. This 
was extracted with chloroform, the chloroform washed with base (0.5N 
NaOH), water and then dried (Na.sub.2 SO.sub.4). Removal of the solvent 
under reduced pressure gave a brownish syrup (0.7 gm), from which pure 
product, MIP, was isolated by flash chromatography (EM silica gel, 200-400 
mesh), eluted with C/M 98:2. The yield of MIP by this second method was 
270 mg (13.5%). 
EXAMPLE 3 
Radiolabelled MIP Synthesis 
Step 1: MIP (58 mg; 0.29 mmol), 10% palladium on charcoal (29 mg. Aldrich), 
and glacial acetic acid (7.0 ml) were placed in a small round bottom 
flask, attached to a vacuum line, frozen with liquid nitrogen, and then 
the reaction vessel was evacuated. Carrier free tritium gas (Lawrence: 60 
Ci/mmol) was added to slightly below 1 atmosphere, and the round bottomed 
flask was warmed briefly in a 60.degree. C. water bath to redissolve the 
MIP. The heterogeneous mixture was stirred at room temperature for 1 hour 
after which approximately .31 mmol tritium gas had been consumed. The 
mixture was frozen. the tritium gas evacuated. methanol (10 ml) added. and 
the slurry centrifuged to remove the catalyst. The supernatant was 
decanted, frozen and then lyophilized. Following lyophilization, TLC 
(chloroform) of the residue revealed unreacted starting material, a low Rf 
blue fluorescent spot corresponding to 4',5'-.sup.3 H.sub.2 
!-4'5'-dihydro-5-MIP and a high Rf nonfluorescent spot corresponding to 
3,4,4',5'-.sup.3 H.sub.4 !-3,4,4',5' tetrahydro-5-MIP. 4', 5'-.sup.3 
H.sub.2 !-4'5'-dihydro-5-MIP was isolated by column chromatography on a 
0.5.times.8-inch silica column (60-200 mesh. Baker) eluted with CH.sub.2 
Cl.sub.2. The recovery was 30-40 mg. This compound was used in Step 2 
directly for the preparation of labelled MIP. 
Step 2: 4',5'-.sup.3 H.sub.2 !-4'5'-dihydro-5-MIP of Step 1 (30-40 mg), 
10% palladium on charcoal (32 mg, Aldrich and diphenylether (5.0 ml, 
Aldrich) were placed in a small round bottom flask with attached argon 
line then refluxed for 28 hours. Following this period, TLC (CH.sub.2 
Cl.sub.2) indicated that most of the starting material had been converted 
to 4',5'-.sup.3 H.sub.2 !-5-MIP (as determined by co-chromatography with 
authentic MIP). The product was purified by chromatography on 2 silica 
columns (60-200 mesh, Baker, eluted with CH.sub.2 Cl.sub.2). The fractions 
containing the purified product were combined, the solvent evaporated 
under reduced pressure, and the residue dissolved in absolute ethanol. The 
specific activity of the compound was established by measuring the optical 
density of the stock solution to determine its concentration, then 
counting appropriate aliquots of the stock. In this manner, the specific 
activity of tritiated 4',5'-.sup.3 H.sub.22 !-5-MIP ("tritiated MIP") 
product was determined to be 7.4 Ci/mmol. 
The radiochemical purity was determined by HPLC. Approximately 10.sup.6 CPM 
of tritiated MIP was mixed with 10 .mu.g unlabelled MIP in 50 .mu.l of 
ethanol (100%). The sample was injected on a C18 octadecasilyl reverse 
phase chromatography column (Beckman) and eluted with a water/methanol 
gradient as follows: 0-10 minutes. 100% H.sub.2 O; 10-70 minutes. 100%s 
H.sub.2 O-100% CH.sub.3 OH: 70-80 minutes. 100% CH.sub.3 OH. Eighty 1.0 ml 
fractions were collected and 40 .mu.l of each fraction counted. Greater 
than 99% of the radioactivity co-chromatographed with the peak 
corresponding to tritiated MIP. 
EXAMPLE 4 
Halomethylisopsoralen Synthesis 
This example involves the synthesis of a halome- thylisopsoralen, in this 
case 5-bromomethylisopsoralen (BMIP,5) from MIP. MIP (1.80 gm. 9 mmol) was 
dissolved CCl.sub.4 (193 ml) at reflux. N-bromosuccinimide (1.65 gm, 9 
mmol, Aldrich) and dibenzoylperoxid (0.22 gm, 0.9 mmol, Aldrich) were 
added to the boiling solution and the mixture refluxed for four hours 
while being monitored by TLC (Eastman TLC Plates: developed with C/M 98:2; 
detection with 260 nm ultraviolet light). Following this period, the 
boiling mixture was filtered hot and the filtrate set aside to cool then 
held at 0.degree. C. for 24 hrs. The resulting crystals (light yellow 
needles) were collected by filtration, dissolved in CHCl.sub.3 (140 ml) 
then washed with water (140 ml.times.4). The CHCl.sub.3 solution was dried 
(anhydrous MgSO.sub.4) then concentrated by rotary evaporation under 
reduced pressure to provide the product, BMIP, as yellow crystals (1.75 
gm, 68.7%, m p. 201.degree.-204.degree. C. with decomposition). NMR 
(CDCl.sub.3)d 8.06 (d. H-4), 7.64 (d. H-5'), 7.41 (s. H-6).7.05 (m, H-4'), 
6.43 (d, H-3), 4.72 (s. CH.sub.2 Br). 
EXAMPLE 5 
Synthesis Of 5-Aminomethylisopsoralen (AMIP, 10): Method 1 (four steps) 
Step 1: The first step of the first method of the present invention for 
synthesizing AMIP from XMIP involves the synthesis of 
5-Hydroxymethylisopsoralen (HMIP,6). XMIP is chosen to be BMIP for 
purposes of this step. 
BMIP (0.2 gms, 0.71 mmol) was refluxed in distilled water (20 ml) while 
being monitored by TLC (Eastman TLC Plates: developed with C/M 98:2, 
detection with 260 nm ultraviolet light). After 3 hours, no starting 
material remained and a new low Rf spot had appeared. Upon cooling of the 
reaction mixture, the product, HMIP, precipitated as very light yellow 
needles and was collected by suction filtration (0.15 gms, 96.8% m.p 
184.degree.-187.degree. C.). 
Step 2: The second step of the first method of the present invention for 
synthesis of AMIP involves synthesis of XMIP from HMIP, XMIP is chosen to 
be 5-chloromethylisopsoralen (CMIP,5) for purposes of this step. 
HMIP (858 mg, 4.0 mmol; obtained from combining numerous runs of Step 1) is 
dissolved in freshly distilled chloroform (60 ml, dried over 4.ANG. 
molecular sieves). Thionyl chloride (1.49 gm, 12.6 mmol, Aldrich) is added 
followed by stirring at room temperature. Additional portions of thionyl 
chloride are added after 1 hour (990 mg. 8.4 mmole) and 16 hr (990 mg, 8.4 
mmol ). After a total of 25 hr., no starting material remains (TLC, 
CH.sub.2 Cl.sub.2) and a new high Rf spot appears. The new spot 
co-chromatographs on TLC with authentic CMIP using several different 
solvent systems (CH.sub.2 Cl.sub.2 ; CHCl.sub.3 ; EtOAc:Hexanes 1:3). 
Step 3: The third step of the first method of the present invention for 
synthesizing AMIP involves the synthesis of 
5-hexamethylenetetraminomethylisopsoralen (HMTAMIP,9) from XMIP (in this 
case, CMIP). The product of Step 2, CMIP, is brought up in 30 ml sieve 
dried chloroform and hexamethylenetetramine (680 mg, 4.9 mmol) is added. 
The mixture is stirred at 55.degree. for 43 hours, after which an 
additional portion of hexamethylenetetramine (680 mg, 4.9 mmol) is added. 
The mixture continues to be stirred at 55.degree. C. for another 48 hours, 
after which HCl (0.1N. 60 ml) and chloroform (30 ml) are added. The 
chloroform is then removed and the aqueous phase washed three more times 
with chloroform (30 ml). The aqueous phase is evaporated under reduced 
pressure to yield the solid product, HMTAMIP. The product is characterized 
by TLC, co-chromatographing on TLC with authentic HMTAMIP in several 
different solvent systems (C/M 98:2; C/M 95:5; C/M 90:10). 
Step 4: The next step involves the synthesis of AMIP from HMTAMIP. The 
solid of Step 3was suspended in 12 ml ethanol:concentrated HCl (3:1) at 
room temperature for 72 hours and then concentrated in vacuo. The residue 
was added to dilute NaOH, extracted with CHCl.sub.3 and washed with water. 
The CHCl.sub.3 extract was then further extracted with HCl (0.1N). The 
aqueous phase was then separated, adjusted to pH 12-13 with NaOH, and 
extracted with CHCl.sub.3. The CHCl.sub.3 extract was washed with water, 
dried (MgSO.sub.4) and the solvent removed under reduced pressure to 
provide the product as the free base, which was converted to the 
hydrochloride salt with HCl gas (310 mg. 31% yield, based on HMIP). Mass 
spectrum m/e (relative intensity) 215 (M+,100%). 
EXAMPLE 6 
Synthesis Of AMIP: Method 2 (five steps) 
Step 1: The first step of the second method, synthesis of HMIP from XMIP, 
is identical to the first step of the first method, since XMIP has been 
chosen to be BMIP in both cases. The first step proceeds, therefore, 
according to Step 1 of EXAMPLE 5. 
Step 2: The second step of the second method of the present invention for 
synthesis of AMIP, synthesis of XMIP from HMIP, is the same as the second 
step of the first method since XMIP has been chosen to be CMIP in both 
examples. Thus, the second step proceeds as in Step 2 of EXAMPLE 5. 
Step 3: The third step of the second method of the present invention for 
synthesizing AMIP involves the synthesis of 5-iodomethylisopsoralen 
(IMIP,8) from XMIP (in this case CMIP). 
CMIP (577 mg: 2.25 mmol). sodium iodide (1.77 gm, 11.53 mmol; Baker; dried 
overnight at 120.degree. C.) and acetone (25 ml. Mallinckrodt) are 
refluxed for 48 hours. Following this period, the reaction mixture is 
filtered to remove the inorganic salts (mixed NaCl and NaI), the filtrate 
evaporated under reduced pressure, the residual filtrate evaporated under 
reduced pressure, the residual crude product dissolved in chloroform, 
loaded on a 1/2".times.20" silica gel column (60-200 mesh, Baker), and 
eluted with the same solvent. The fractions containing the product, IMIP, 
are identified by TLC, combined, and the solvent removed under reduced 
pressure (489 mg; 66.7%). 
Step 4: The next step involves the synthesis of HMTAMIP from IMIP. IMIP 
(489 mg; 1.5 mmol) and hexamethylenetramine (360 mg; 2.6 mmol) are 
refluxed in dry CHCl.sub.3 until all the starting IMIP is consumed, as 
shown by TLC. The resulting precipitate is collected by suction 
filtration, suspended in dilute acid (0.1N HCl), washed several times with 
an equivalent volume of CHCl.sub.3, then recovered from the aqueous phase 
by evaporation. The product, HMTAMIP, is characterized by comparative TLC 
in several different solvent systems (C/M 98:2; C/M 95:5; C/M 90:10)1 
Step 5: The final step of this second method involves the synthesis of AMIP 
from HMTAMIP. This was carried out in the manner described in Step 4 of 
EXAMPLE 5. 
EXAMPLE 7 
Synthesis Of AMIP: Method 3 (two steps) 
In the two step method, XMIP may again be CMIP or BMIP. For this example, 
the two step method proceeds according to the following scheme: 
BMIP.fwdarw.HMTAMIP.fwdarw.AMIP 
Step 1: The first step of the second method of the present invention for 
synthesizing AMIP involves the synthesis of HMTAMIP from XMIP (in this 
case, BMIP). 
BMIP (540 mg. 2.5 mmol) and hexamethylene-tetramine (610 mg, 4.4 mmol) were 
refluxed in dry CHCl.sub.3 for 40 hours. The resulting precipitate was 
collected by suction filtration and directly for Step 2. 
Step 2: The next step involves the synthesis of AMIP from HMTAMIP. The 
solid of Step 1 was suspended in 12 ml ethanol:concentrated HCl (3:1) at 
room temperature for 72 hours and then concentrated in vacuo. The step 
then proceeds as in Step 4 of Example 5. 
EXAMPLE 8 
Radiolabelled AMIP Synthesis 
As noted above, the present invention provides twelve methods for producing 
radiolabelled AMIP from MIP. Where the method relies on tritiated MIP, the 
method proceeds initially according to the steps of EXAMPLE 3 to make 
tritiated MIP, and then continues according to the steps of EXAMPLE 7. In 
this example, however, tritiated MIP is not used. For this example, the 
method proceeds according to the following scheme (nine steps): 
5-methylresorcinol.fwdarw.H5MC.fwdarw.MIP.fwdarw.XMIP.fwdarw.H- 
.fwdarw.NIP.fwdarw.FIP.fwdarw.*HMIP.fwdarw.*XMIP.fwdarw.*HMTAMIP.fwdarw.*AM 
IP 
where * indicates a labelled compound. 
Steps 1-2: MIP was synthesized via H5MC from 5-methylresorcinol according 
to the two step method described in EXAMPLE 2, above. 
Step 3: For this example, the first XMIP was chosen to be BMIP (later, XMIP 
is radiolabelled CMIP; see reactions of Step 7 below). BMIP was 
synthesized from MIP according to the method described in EXAMPLE 4 above. 
Step 4: HMIP was synthesized from BMIP according to the method described in 
Step 1 of method 1 of EXAMPLE 5. 
Step 5: New compound 5-Formylisopsoralen (FIP,7) was synthesized from HMIP. 
3,5-Dimethylpyrazole (180 mg: 1.9 mmol; Aldrich) was added to a suspension 
of chromium trioxide (Aldrich: 190 mg: 1.9 mmol) in methylene chloride (6 
ml) and the mixture stirred for 15 minutes under argon at room 
temperature. HMIP from Step 4 (150 mg: 0.69 mmol) was added in one portion 
and the reaction mixture stirred at room temperature for 2.5 hours, after 
which TLC (CHCl.sub.3) indicated the reaction was complete. the solvent 
was removed under reduced pressure and the residue dissolved in a small 
volume of CHCl.sub.3, loaded on a silica gel column (Baker; 60-200 mesh) 
and then eluted with CHCl.sub.3. The fractions containing product were 
combined and the solvent removed to provide the aldehyde (120 mg; 81% 
yield). Further purification was accomplished by re-crystallization from 
95% EtOH giving yellow needles. 
Step 6: Tritiated HMIP was synthesized from FIP. FIP (71 mg: 0.35 mmol) 
from Step 5 and .sup.3 H.sub.4 -sodium borohydride (DNEN; 1.98 mg; 0.0476 
mmol) were mixed in 95% ethanol (10 ml) then stirred at room temperature 
for one hour. After this period, TLC showed all the formyl compound had 
been reduced to 5-(hydroxy-.sup.3 H!-methyl)isopsoralen (".sup.3 
H-HMIP"). The solvent was removed under reduced pressure, methanol added 
(10 ml) and then evaporated. This was repeated a total of four times. The 
residual solid was then dissolved in 3 ml C/M (99:1) and loaded on a 1 
cm.times.30 cm silica gel column (Baker: 60-200 mesh) then eluted with the 
same solvent mix. The produce fractions were combined and the solvent 
evaporated to provide the labelled alcohol, .sup.3 H-HMIP (yield not 
determined). 
Step 7: The .sup.3 H-HMIP of Step 6 was used directly for conversion to the 
chloromethyl derivative. 5-(chloro.sup.3 H!-methyl)isopsoralen (".sup.3 
H-CMIP"). .sup.3 H-HMIP from Step 6 was dissolved in 10 ml chloroform 
(dried over 4 .ANG. molecular sieves), then thionyl chloride (248 mg; 2.1 
mmol: Aldrich) was added. The reaction mix was stirred under argon at room 
temperature. Another 165 mg (1.4 mmol) portion of thionyl chloride was 
added after one hour. This was left stirring for another 16 hours at which 
point a third portion of thionyl chloride (165 mg; 1.4 mmol) was added. 
The reaction was evaporated under reduced pressure after another five 
hours to give the product, .sup.3 H-CMIP (yield not determined). 
Step 8: The product of Step 7 was brought up in 5 ml chloroform (sieve 
dried and hexamethylene tetramine (85 mg; 0.61 mmol; Aldrich) was added. 
This was stirred at 550 for 43 hours, at which point another portion of 
hexamethylene tetramine (95 mg; 0.68 mmol) was added. Heating was 
continued for another 48 hours, after which HCl (0.1N; 10 ml) chloroform 
(5 ml) were added. The chloroform was removed and the aqueous phase washed 
three more times with chloroform (5 ml). The aqueous phase was then 
evaporated under reduced pressure and the solid product, 
5-(Hexamethyl-tetramino-.sup.3 H!-methyl)isopsoralen (".sup.3 
H-HMTAMIP"), was brought up in 12 ml ethanol: concentrated HCl (3:1) for 
the next reaction. 
Step 9: .sup.3 H-HMTAMIP from Step 8 was stirred at room temperature in the 
ethanol:HCl mixture. An additional 2 ml of concentrated HCl was added and 
the mixture stirred at 40.degree.. for 15 hours. Following this period, 
the pH was adjusted to 7 with NaOH and the solution evaporated under 
reduced pressure. The solid solution evaporated under reduced pressure. 
the solid was brought up in 10 ml of 0.1M NaOH and extracted three times 
with chloroform (5 ml). The chloroform washes were combined and washed 
twice with water (10 ml). The chloroform was then extracted with HCl 
(0.1N; 10 ml) and the acidic aqueous phase then was washed three times 
with chloroform (5 ml). The aqueous phase was evaporated under reduced 
pressure and the solid dissolved in ethanol (10 ml). Aliquots of this 
solution were removed and counted. While the concentration of the stock 
was determined by UV absorption. The product, 5-(Aminomethyl-.sup.3 
H!-methyl)isopsoralen (".sup.3 H-AMIP"), was determined to be 347 .mu.g/ml 
and the specific activity 3.1.times.10.sup.5 CPM/.mu.g (117 Ci/mol). Over 
all recovery was 1.6 mCi, 3.47 mg, 0.014 mmol (72% yield based on .sup.3 
H.sub.4 -NaBH.sub.4). 
EXAMPLE 9 
BIOMIP Synthesis 
The BIOMIP synthesis method for the following example proceeds according to 
the scheme: 
XMIP.fwdarw.HMIP.fwdarw.XMIP.fwdarw.IMIP.fwdarw.DMHNIP.fwdarw.BI-OMIP 
Again, XMIP can be either CMIP or BMIP. For the example, XMIP is CMIP and 
BMIP, respectively. 
Step 1: MIP is reacted to form XMIP. In this step, XMIP is CMIP. MIP (1.80 
gm, 9 mmol) is dissolved in CCl.sub.4 at reflux. N-chlorosuccinimide (1.20 
gm, 9 the mixture boiled until no starting material remains (as determined 
by TLC). Following this period, the boiling mixture is filtered (hot) and 
the filtrate set aside at 0.degree.. The resulting precipitate is 
collected by suction filtration, dissolved in CHCl.sub.3 washed with 
water, dried (anhydrous MgSO.sub.4) and concentrated by rotary evaporation 
under reduced pressure to provide the product, CMIP. 
Step 2: HMIP is then synthesized from XMIP (in this case, CMIP). CMIP (233 
mg. 1.0 mmol) is refluxed in distilled water (50 ml) while being monitored 
by TLC (Eastman TLC Plates; developed with chloroform, detection with 260 
nm ultraviolet light). After 2 hours, no starting material remains and a 
new low Rf spot appears. Upon cooling of the reaction mixture, the 
product, HMIP, precipitates as white needles and is collected by suction 
filtration. 
Step 3: XMIP is then synthesized from HMIP. In this case, XMIP is BMIP. 
HMIP (270 mg; 1.25 mmol) is dissolved in freshly distilled chloroform (20 
ml, dried over 4 .ANG. molecular sieves). Thionyl bromide (384 mg; 3.0 
mmol; Aldrich) is added followed by stirring at room temperature. 
Additional portions of thionyl bromide are added after 1.5 hours (126 mg, 
1.0 mmol) and 3.0 hours (126 mg; 1.0 mmol). After a total of 6.5 hours, no 
starting material remains (Eastman TLC Plates; CH.sub.2 Cl.sub.2) and a 
new high Rf spot appears. The solvent is removed under reduced pressure 
and the residue dissolved in a small volume of CH.sub.2 Cl.sub.2, loaded 
on a 1/2".times.20" silica gel column (Baker, 60-200 mesh), and eluted 
with the same solvent. The fractions containing the product are identified 
by TLC, combined, and the solvent removed under reduced pressure. 
Step 4: IMIP is then synthesized from BMIP via the Finkelstein reaction. In 
a small round-bottomed flask fitted with a reflux condenser and argon 
line, a mixture of BMIP (279 mg; 1.0 mmol), sodium iodide (767 mg; 5.15 
mmol; Baker; dried overnight at 120.degree. C.) and methyl ethyl ketone 
(10 ml, Mallinckrodt) are refluxed for 48 hours. Following this period, 
the reaction mixture is filtered to remove the inorganic salts (mixed NaI 
and NaBr), the filtrate evaporated under reduced pressure, the residual 
crude product dissolved in chloroform, loaded on a 1/2".times.20" silica 
gel column (60-200 mesh, Baker), and eluted with the same solvent. The 
fractions containing the product, IMIP, are identified by TLC, combined, 
and the solvent removed under reduced pressure. 
Step 6: 5-N-(N,N'-dimethyl-1,6-hexanediamine (4.1gm; 28.5 mmol; Aldrich) 
are refluxed in dry toluene (45 ml) under argon while being monitored by 
TLC. After a short period no starting material remains and a new, low Rf 
spot appears. The solvent is removed under reduced pressure and the solid 
residue dissolved in HCl (1.0N; 60 ml), washed with chloroform (3.times.25 
ml), the acidic aqueous phase made basic with 1.0N NaOH (pH 12), the basic 
aqueous phase extracted with chloroform (3.times.50 ml), the chloroform 
extract washed with water (2.times.40 ml), saturated sodium chloride 
(1.times.40 ml) and finally dried (MgSO.sub.4). The solvent is removed 
under reduced pressure, the residue dissolved in a small volume of C/E/T 
(9:1:0.25), loaded on a 0.5".times.12" silica gel column (60-200 mesh, 
Baker) and eluted with C/E/T. The fractions containing the pure product, 
DMHMIP, are identified by TLC, combined and evaporated to provide the 
product. 
Step 7: 
5-N-N.N'-Dimethyl-(6-biotinamido!-hexanoate)-1,6-hexanediamine!)-methyli 
sopsoralen (BIOMIP,12a) was made from DMHMIP. DMHMP (630 mg; 1.8 mmol), 
biotin-amidocaproate N-hydroxysuccinimide ester (Pierce; 100 mg; 0.22 
mol), and DMF (2.7 ml, freshly distilled onto 4.ANG. sieves) were placed 
in a 10 ml round bottomed flask with attached argon line. The reaction was 
magnetically stirred at room temperature while being monitored by TLC 
(C/E/T; 9:1:0.25; the product ran as a high Rf spot relative to starting 
material). After the reaction was complete, the solvent was removed under 
vacuum and the residue dissolved in a small volume of CH.sub.2 Cl.sub.2 
:CH.sub.3 OH (10:1), loaded on a silica gel column (60-200 mesh; 
0.5".times.20"), and eluted with the same solvent. The product was 
isolated, the elution solvent removed, and the free amine dissolved in 10 
ml ethanol. HCl gas was bubbled through the solution, followed by argon. 
The ethanol was removed to give the product, BIOMIP (HCl salt) (190 mg; 
14.7% yield; FABMS m/e 682 (MH +, 25%)). 
EXAMPLE 10 
BIOMIP Synthesis 
The synthesis method for the following example proceeds according to the 
scheme: 
XMIP.fwdarw.DMHMIP.fwdarw.BIOMIP 
In this example, XMP is BMIP. 
Step 1: BMIP (400 mg; 1.43 mmol), freshly distilled 
N,N'-dimethyl-1,6-hexanediamine (3.1 gm; 21.5 mmol; Aldrich) was refluxed 
in dry toluene (45 ml) under argon while being monitored by TLC. After 1.5 
hours, no starting material remained and a new, low Rf spot appeared. The 
solvent was removed under reduced pressure and the solid residue dissolved 
in 1.0N HCl (60 ml), washed with chloroform (3.times.25 ml), the acidic 
aqueous phase made basic with 1.0N NaOH (pH 12), the basic aqueous phase 
extracted with chloroform (3.times.50 ml), the chloroform extract washed 
with water (2.times.40 ml), saturated sodium chloride (1.times.40 ml) and 
finally dried (MgSO.sub.4). The solvent was removed under reduced 
pressure, the residue dissolved in a small volume of C/E/T (9:1:0.25), 
loaded on a 0.5".times.12" silica gel column (Baker, 60-200 mesh) then 
eluted with C/E/T. The fractions containing the pure product were 
identified by TLC, combined and evaporated to provide the product, DMHMIP, 
as a viscous oil which solidified upon standing (300 mg; 61% yield). 
Step 2: BIOMIP (HCl salt) was then synthesized from DMHMIP as in Step 7 of 
Example 9. 
EXAMPLE 11 
Tritiated BIOMIP Synthesis 
The present invention also provides methods for synthesizing labelled 
BIOMIP. One method involves radiolabelling BIOMIP according to the 
following scheme: 
.sup.3 H-XMIP.fwdarw..sup.3 H-DMHMIP.fwdarw..sup.3 H-BIOMIP 
In this example, .sup.3 H-XMIP is .sup.3 H-CMIP. 
Step 1: .sup.3 H-CMIP from Step 7 of Example 8 (24 mg, 0.1 mmol) is 
refluxed in dry toluene (5 ml) under argon while being monitored by TLC. 
After several hours, no starting material remains and a new, low Rf spot 
appears. The solvent is removed under reduced pressure and the solid 
residue dissolved in HCl (1.0N), extracted with chloroform, the acidic 
aqueous phase separated and made basic with naOH (1.0N), the basic aqueous 
phase is extracted with chloroform, the chloroform extract washed with 
water, saturated sodium chloride then dried (MgSO.sub.4). The solvent is 
removed under reduced pressure, the residue dissolved in a small volume of 
C/E/T 9:1:0.25, loaded on a 0.5".times.4" silica gel column (60-200 mesh; 
Baker) and eluted with C/E/T. The fractions containing the pure product 
are identified by TLC, combined and evaporated to provide the product. The 
product is further characterized by UV and TLC (co-chromatography with 
authentic material in several different solvent systems). The 
5-N-(N,N'-dimethyl-1,6-hexanediamine)-.sup.3 H!-methylisopsoralen (.sup.3 
H-DMHMIP) so prepared is used directly in Step 2 for the preparation of 
.sup.3 H!-BIOMIP (and other compounds). 
Step 2: .sup.3 H-DMHMIP (17.5 mg; 0.05 mmol), biotinamidocaproate 
N-hydroxysuccinimide ester (25 mg; 0.055 mmole; Pierce), and DMF (1.8 ml, 
freshly distilled onto 4A sieves) are placed in a 5 ml round bottomed 
flask with attached argon line. The reaction is magnetically stirred at 
room temperature while being monitored by TLC (C/E/T; 9:1:0.25; the 
product runs as a high Rf spot relative to starting material). After the 
reaction is complete, the solvent is removed under vacuum and the residue 
dissolved in a small volume of CH.sub.2 Cl.sub.2 :CH.sub.3 OH 10:1, loaded 
on a silica gel column (60-200 mesh, 0.5".times.5"), and eluted with the 
same solvent. The product is isolated as the free base, the elution 
solvent removed, then dissolved in absolute ethanol. HCl gas is bubbled 
through the solution, followed by argon. The ethanol is removed to give 
5-N-N, N'-dimethyl-N'-{6-biotinamido}-hexanoate)-1, 
6-hexanediamine!-.sup.3 H!-methylisopsoralen (.sup.3 H-BIOMIP) as the 
monohydrochloride salt. 
EXAMPLE 12 
DITHIOMIP Synthesis 
The present invention provides methods for synthesizing DITHIOMIP. For this 
example, the synthesis proceeds according to the following scheme: 
XMIP.fwdarw.DMHMIP.fwdarw.DITHIOMIP 
where XMIP is BMIP. 
Step 1: DMHMIP is synthesized from BMIP according to the method described 
in Step 1 of EXAMPLE 10. 
Step 2: DMHMIP from Step 1 (41 mg; 1.20 mmol, 1.0 eq) and 
sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (102 mg; 
0.168 mmol; 1.40 eq) is dissolved in freshly distilled DMF (3.0 ml) in a 
small, dry round bottomed flask with attached argon line then stirred at 
room temperature for 5 hours. After this period, the DMF is removed under 
reduced pressure and the residue suspended in NaOH (1.0N), extracted with 
chloroform/isopropanol 3:1, and the organic extract washed with water 
(10.times.2) then dried (MgSO.sub.4), filtered and evaporated under 
reduced pressure to five the crude product. This is dissolved in ethanol 
and HCl gas is bubbled through the solution. The ethanol is removed to 
give the product, 
5-N-N,N'-dimethyl-N'-(2-(biotinamido}-ethyl-1,3-dithiopropionate)-1,6-hex 
anediamine!-methyl-isopsoralen (DITHIOMIP, 12b) (HCl salt). This is 
dissolved in ethanol and further characterized by sample treatment with 
sodium borohydride (Aldrich) or mercaptoethanol (Aldrich). The product of 
these reactions is compared to untreated material to verify cleavage 
occurred as expected. 
EXAMPLE 13 
.sup.3 H-DITHIOMIP Synthesis 
The present invention provides methods for synthesizing .sup.3 H-DITHIOMIP. 
For this example, the synthesis proceeds according to the following 
scheme: 
.sup.3 H-XMIP.fwdarw..sup.3 H-DMHMIP.fwdarw..sup.3 H-DITIEOMIP 
In this example, .sup.3 H-XMIP is .sup.3 H-CMIP. 
Step 1: .sup.3 H-DMHMIP is prepared as described in Step 1 of Example 11. 
Step 2: .sup.3 H-DMHMIP (24 mg, 0.07 mmol, 1.0 eq and 
sulfosuccinimidyl-2-(biotinamido)-ethyl-1-1,3-dithiopropionate (60 mg; 
0.10 mmol, 1.4 eq) is dissolved in freshly distilled DMF (1.5 ml) in a 
small, dry round bottomed flask with attached argon line then stirred at 
room temperature for 5 hr. After this period, the DMF is removed under 
reduced pressure and the residue suspended in 0.1N naOH (5 ml), extracted 
with chlorofonm/isopropanol 3:1 (3.times.5 ml), and the organic extract 
washed with water (5.times.2) then dried (MgSO.sub.4), filtered and 
striped to give the product, .sup.3 H-DITHIOMIP. DITHIOMIP. The product is 
then characterized by ultraviolet absorption (comparison with authentic 
material) and TLC (co-chromatography with authentic material in several 
different solvent systems). Radiochemical purity is determined by HPLC. 
Approximately 10.sup.6 CPM of tritiated .sup.3 H-DITHOMIP is mixed with 10 
.mu.g of unlabeled DITHIOMIP in 50 .mu.l ethanol (100%). The sample is 
injected on a C18 octadecylsilyl reverse phase chromatography column 
(Beckman) and eluted with an acetonitrile/ammonium acetate (0.1M, pH 7) 
gradient, as follows: 0-10 minutes, 100% ammonium acetate: 10-70 minutes, 
100% ammonium acetate.fwdarw.100% acetonitrile; 70-80 minutes, 100% 
acetonitrile. Eighty 1.0 ml fractions are collected and 40 .mu.l of each 
fraction is then counted. The product is further characterized by 
dissolving a sample in ethanol and treating with either sodium borohydride 
(Aldrich) or mercaptoethanol (Aldrich). The product of these reactions is 
compared to untreated material to verify cleavage occurs as expected. 
EXAMPLE 14 
FLUORMIP Synthesis 
The present invention also contemplates methods for synthesizing FLUORMIP. 
For this example, the synthesis proceeds according to the following 
scheme: 
XMIP.fwdarw.DMHMIP.fwdarw.FLUORMIP 
where XMIP is BMIP. 
Step 1: DMHMIP is synthesized from BMIP according to the method described 
in Step 1 of EXAMPLE 10. 
Step 2: DMHMIP from Step 1 (13.7 mg, 0.04 mmol, 1.0 eq) in DMF 
(Mallinckrodt; 1.0 ml distilled onto 4.ANG. sieves), and 
6-carboxyfluorescein-N-hydroxysuccinimide succinimid ester Pierce; 20.7 
mg, 0.044, 1.1 eq) in DMF (1.0 ml) are mixed in a 5 ml round bottomed 
flask with attached argon line. The reaction mix is stirred several hours 
at room temperature. After this period, the majority of the DMHMIP is 
consumed as indicated by TLC (C/B/A/F; 4:1:1:1). The solvent is removed 
with gentle heating (&lt;50.degree.) under reduced pressure. The residue is 
dissolved in HCl (0.1N) and extracted with chloroform/isopropanol 3:1. The 
organic extract is then reduced in volume, loaded onto a glass backed 
preparative silica gel TLC plate (20 cm.times.20 cm .times.2 mm; Baker), 
and eluted with C/B/A/F. The major low Rf band is scraped from the plate, 
eluted with C/M (90:10), the silica removed by filtration and the solvent 
evaporated under reduced pressure. The product is dissolved in a small 
volume of ethanol and HCl gas bubbled through the solution. The ethanol is 
then evaporated to provide 
5-N-N,N'-dimethyl-N'-(carboxyfluoresceinester)-1,6-hexanediamine)-methyli 
sopsoralen (FLUORMIP, 12c) as the monohydrochloride salt. 
EXAMPLE 15 
.sup.3 H-FLUORMIP Synthesis 
The present invention also contemplates methods for synthesizing .sup.3 
H-FLUORMIP. For this example, the synthesis proceeds according to the 
following scheme: 
.sup.3 H-DMHMIP.fwdarw..sup.3 H-FLUORMIP 
.sup.3 DMHMIP from Step 1 of Example 11 (2.5 mg; 0.007 mmol; 117 Ci/mmol; 
1.0 eq) in DMF (Mallinckrodt; 1.0 ml distilled onto 4A sieves) and 
6-carboxyfluorescein-N-hydroxy-succinimide hydroxy-succinimide ester (3.7 
mg; 0.008 mmol; 1.1 eq; Pierce) in DMF (1.0 ml) are mixed in a 5 ml round 
bottomed flask with attached argon line. The reaction mix is stirred 
several hours at room temperature. After this period, the majority of the 
.sup.3 H-DMHMIP is consumed as indicated by TLC (C/B/A/F; 4:1:1:1). The 
DMF is removed with gentle (&lt;50.degree.) under reduced preasure. The crude 
product is dissolved HCL (0.1N; 3 ml) and extracted with 
chloroform/isopropanol 3:1 (3 ml.times.3). The organic extract is then 
reduced to 2 ml, loaded onto a glass backed preparative silica gel TLC 
plate (20 cm.times.20 cm.times.2 mm; Baker), and eluted with C/B/A/F. The 
major low Rf band is scraped from the plate and eluted with methanol. The 
silica is removed by filtration and the solvent evaporated under reduced 
pressure. The product is dissolved in a small volume of ethanol and HCl 
gas bubbled through the solution. The ethanol is then evaporated to 
provide .sup.3 H-FLUORMIP as the monohydrochloride salt. The product is 
characterized by ultraviolet absorption (comparison with authentic 
material) and TLC (co-chromatography with authentic material in several 
different solvent systems). The specific activity is determined by 
determining the optical density of an ethanolic stock of the compound and 
scintillation counting of aliquots of this solution (117 Ci/mmol). 
Radiochemical purity is determined by HPLC. Approximately 10.sup.6 CPM of 
.sup.3 H-FLUORMIP is mixed with 10 .mu.g of unlabelled FLUORMIP in 50 
.mu.l ethanol (100%). The sample is injected on a C18 octadecasilyl 
reverse phase chromatography colurn (Beckman) and eluted with 
acetonitrile/ammonium acetate (0.1M; pH 7) gradient, as follows: 0-10 
minutes, 100% ammonium acetate; 10-70 minutes, 100% ammonium 
acetateo.fwdarw.100% acetonitrile; 70-80 minutes, 100% acetonitrile. 
Eighty 1.0 ml fractions are collected and 40 .mu.l of each fraction is 
then counted. 
EXAMPLE 16 
DMIP Synthesis 
This example describes the method of the present invention for synthesizing 
DMIP. From resorcinol, the method proceeds in four steps according to the 
following scheme: 
Resorcinol.fwdarw.H4MC.fwdarw.CAMC.fwdarw.BCAMC.fwdarw.DMIP 
Step 1: Resorcinol (110 gm; 1.0 mol. Aldrich) is mixed with 
ethylacetoacetate (130 gm, 1.0 mol, Aldrich) and placed in a dropping 
funnel. This mixture is added dropwise to a chilled (10.degree. C.) 
solution of sulfuric acid (1000 ml) in a three necked flask fitted with a 
mechanical stirrer and internal thermometer. The rate of addition is such 
that the internal temperature does not exceed 10.degree. C. The solution 
is stirred for 12 hours then slowly poured onto 2 kg of ice and 3 liters 
of water. Following vigorous stirring, the precipitate is collected by 
suction filtration, washed with water, dissolved in 5% aqueous NaOH (1500 
ml), then reprecipitated by addition of dilute sulfuric acid (650 ml) with 
vigorous stirring. The product, 7-Hydroxy-4-methylcoumarin (H4MC,13), is 
collected by filtration, washed with water, then allowed to air dry (145 
gm; 41% yield; mp 185.degree. C.). 
Step 2: H4mc (145 gm; 0.82 mol; Aldrich) was treated with 
2,3-dichloro-1-propene (107.8 gm; 0.97 mol; Aldrich) in DMF (1178 ml; 
Mallickrodt)/toluene (931 ml; Mallickrodt) in the presence of potassium 
carbonate (154 gm; 1.12 mol; Baker) and a catalytic amount of potassium 
iodide (7.1 gm; 0.05 mmol; Baker). The mixture was heated to 95.degree. C. 
with stirring for 12 hour, after which TLC indicated no starting material 
remained. The solvent was removed under reduced pressure and the residual 
paste extracted with hot chloroform. The chloroform was washed with water, 
saturated NaCl, dried (MgSO.sub.4) then the solvent removed under reduced 
pressure. The residual solid was dissolved in absolute ethanol at reflux 
(1200 ml) and set aside. The resulting crystals were collected by suction 
filtration, washed with cold ethanol and vacuum dried to provide 
7-(.beta.-chloroallyloxy)-4-methylcoumarin (CAMC,14) (143.5 gm, 70% 
yield). NMR (CDCl.sub.3) d 7.5 (1H, d), 6.8-6.9 (2H,m), 6.2 (1H,d), 
5.5-5.6 (2H,M), 4.2 (2H, m), 2.4 (H,M). 
Step 3: Rearrangement of CAMC was accomplished in high yield by refluxing 
the allyl ether from Step 2 (143.5 gm; 0.55 mole) in a mixture of 
p-diisopropylbenzene (Aldrich, 1000 ml) and butyric anhydride (96 ml, 92.8 
gm; 0.59 mol, Aldrich) under argon for 18 hours. The cooled reaction 
mixture was diluted with chloroform, washed with water and then saturated 
sodium bicarbonate, dried (MgSO.sub.4) then evapaorated under pressure. 
Folloeing recrystallization from ethanol, 79.9 grams of mixed 6 and 
8-(.beta.-chloroallyl)-7-butyroxy-4-methylcoumarin (BCAMC,15) were 
obtained. HPLC analysis (Beckman; C18-ODS reverse phase column, isocratic 
elution with 60% CH.sub.3 OH/40%H.sub.2 O) showed the mixture to contain 
85.7% of the desired 8-substituted isomer, of which a sample was purified 
by column chromatography (60-200 mesh silica gel, elution with CH.sub.2 
Cl.sub.2). The structures of the two isomers were confined by NMR. NMR 
(CDCl.sub.3) d 7.56 (1H,d), 7.11 (1H,d), 6.26 (1 h,d), 5.05-5.19 (2H, m, 
3.84 (2H, m), 2.55-2.62 (2H, m), 2.43 (3H, d), 1.78-1.82 (2H, m), 
1.01-1.09 (3H,t). 
Step 4: Ring closure was achieved by treatment of 20 grams of the mixed 
isomers with 70% sulfuric acid at 5.degree. C., precipitating the product 
by addition of the reaction mixture to a 50:50 mixture of water and ice 
(3000 ml). Following extraction with chloroform, 12.5 grams (94%) of the 
mixed isomeric products 4.5' dimethylisopsoralen (DMIP, 16) and 4.5' 
dimethylpsoralen were obtained. Purification of the desired 4.5' 
dimethylisopsoralen was accomplished by repeated recrystallization from 
ethanol (to give approximately 85% yield of pure DMIP). 
EXAMPLE 17 
Radiolabelled DMIP Synthesis 
Step 1: DMIP (21.4 mg, 0.1 mmol), 10% palladium on charcoal (15 mg, 
Aldrich) and glacial acetic acid (Mallinkrodt, 2 ml) are placed in a 25 ml 
round bottom glass and stirred with tritium gas (Lawrence; 150 Ci) until 
no more tritium is absorbed. The catalyst is removed by centrifugation, 
followed by evaporation of the supernatant under vacuum. The residual 
solid is dissolved in methylene chloride (1 ml), loaded on a 1/2" by 5" 
silica gel column (60-200 mesh, Baker), and eluted with methylene 
chloride. Column fractions containing the non-fluorescent 
3,4,4',5'-.sup.3 H.sub.4 !-tetrahydro-4,5'-dimethylisopsoralen methylene 
chloride. Column fractions containing the non-fluorescent 
3,4,4',5'-.sup.3 H.sub.4 !tetrahydro-4, 5-dimethylisopsoralen (.sup.3 
H-THDMIP,17) are identified by TLC, combined, and the solvent removed 
under reduced pressure. This material is further characterized by 
comparison with authentic unlabelled compound (UV; cochromatography on TLC 
in CHCl.sub.3 ; C/M98:2). 2.45 Ci of material was recovered, corresponding 
to a preliminary specific activity of 24.5 Ci/mmol. This material was then 
used in Step 2 for the preparation of tritiated DMIP. 
Step 2: .sup.3 H-THDMIP, prepared as described above, is placed in a 25 ml 
round bottomed flask along with diphenyl ether (5 ml) and 10% palladium on 
charcoal (30 mg; Aldrich). A nitrogen bubbler is attached and the mixture 
refluxed for 24-36 hours. After cooling to room temperature, absolute 
ethanol is added (5 ml) and the catalyst removed by centrifugation. The 
supernatant is partially evaporated, loaded on a 1/2 " by 5" silica gel 
column (Baker, 60-200 mesh), and eluted with methylene chloride. The 
fractions containing the product are combined, the solvent volume reduced 
and the chromatography repeated on a 1/2".times.10" column as above. 
Column fractions containing the product are combined and the solvent 
removed. The (3,4'-.sup.3 H.sub.4 !)-4,5'-dimethylisopsoralen .sup.3 
H-DMIP) so obtained is stored in ethanol to inhibit radiolysis. 
The specific activity of the .sup.3 H-DMIP is established by measuring the 
optical density of the stock solution to determine its concentration, then 
counting appropriate aliquots of the stock. 
The radiochemical purity is determined by HPLC. Approximately 10.sup.6 CPM 
of .sup.3 H-DMIP is mixed with 10 .mu.g unlabelled DMIP in .mu.l of 
ethanol (100%). The sample is injected on a C18 octadecasilyl reverse 
phase chromatography column (Beckman) and eluted with a water/methanol 
gradient as follows: 0-10 minutes, 100% H.sub.2 O; 10-70 minutes, 100% 
H.sub.2 O.fwdarw.100% CH.sub.3 OH; 70-80 minutes, 100% CH.sub.3 OH. Eight 
1.0 ml fractions are collected and 40 .mu.l of each fraction counted. 
Greater than 99% of the radioactivity cochromatographs with the optical 
peak corresponding to .sup.3 H-DMIP. 
EXAMPLE 18 
CMDMIP Synthesis 
4'-Chloromethyl-4,5'-dimethylisopsoralen (CMDMIP,18) was prepared from DMIP 
as follows: DMIP (11.9 gm; 55.6 mmol) was dissolved in acetic acid (600 
mmol) added. The homogeneous solution remained at room temperature for 16 
hours, after which a second portion of chloromethyl methylether (46 ml; 
48.7 gm; 600 mmol) was added. The solution was left at room temperature 
another 53 hours after which crystals began to form. The reaction flask 
was cooled at 0.degree. C. for 78 hours, resulting in the formation of a 
large mass of white precipitate, which was collected by suction filtration 
then dried on the filter. The yield was 10.9 gm (74.7%). The NMR spectra 
of the product, CMDMIP, was consistent with that described by Dall'Acqua 
et al., J. Med. Chem 24, 178 (1981). 
EXAMPLE 19 
Radiolabelled CMDMIP Synthesis 
The present invention also contemplates labelled CMDMIP. The present 
example describes one method of the present invention involving four 
steps: 
CMDMIP.fwdarw.HMDMlP.fwdarw.FDMIP.fwdarw..sup.3 H-HMDMIP.fwdarw..sup.3 
H-CMDMIP 
Step 1: 4'-hydroxymethyl-4,5'-dimethylisopsoralen (HMDMIP,19) was prepared 
from CMDMIP. CMDMIP (1.0 gm; 3.8 mmol) was placed in placed in a 250 ml 
round bottomed flask and refluxed with water for four hours. TLC (C/M 
95:5) showed that all the starting material had been converted to a single 
low Rf spot after this time. The reaction mixture was cooled to 0.degree. 
C. for 2 hours and then the product collected by suction filtration. 
Step 2: New compound 4'-Formyl-4.5'-dimethylisopsoralen (FDMIP,20) was 
prepared from HMDMIP by a novel synthesis method of the present invention. 
3,5-Dimethylpyrazole (830 mg. 8.7 mmol, Aldrich) was added to suspension 
of chromium trioxide (874 mg; 8.8 mmol) in methylene chloride (25 ml) and 
the mixture stirred for 30 min under argon at room temperature. HMDMIP 
from Step 1 (800 mg; 3.3 mmol) was added in one portion and the reaction 
mixture stirred at room temperature for 2.5 hours. TLC showed the reaction 
was over after 3 hours (CHCl.sub.3). The solvent was removed under reduced 
pressure and the residue dissolved in a small volume of CHCl.sub.3 loaded 
on a silica gel column (60-200 mesh. Baker) then eluted with CH.sub.2 
Cl.sub.2. The fractions containing product were determined by TLC, 
combined and the solvent removed to provide FDMIP (647 mg. 81%). Further 
purification was accomplished by recrystallization from 95% EtOH giving 
yellow needles. 
Step 3: 4'(hydroxy-.sup.3 H!-methyl)-4,5'-dimethylisopsoralen (.sup.3 
H-HMDMIP) was prepared from FDMIP. The FDMIP of Step 2 (18 mg; 0.0743 
mmol) and sodium-.sup.3 H.sub.4 !-borohydride (DNEN, 1.8 mg. 0.0476 mmol; 
60 Ci/mmol) were stirred in 95% ethanol (8 ml) at room temperature for 5 
hours. After this period, TLC showed the FDMIP (Rf 0.7) had been 
completely reduced to .sup.3 H-HMDMIP (Rf 0.15). The solvent was removed 
by lyophilization and the residual solid dissolved in C/M (99:1, 1 ml), 
loaded on a 1 cm.times.30 cm chromatography column (60-200 mesh silica 
gel; Baker) and eluted with C/M (99:1). The fractions which contained 
.sup.3 H-HMDMIP were identified by TLC, combined and evaporated. The 
product was used directly in Step 4 for the preparation of 4'-(.sup.3 
H!-Chloromethyl-4,5'-dimethylisopsoralen (.sup.3 H-CMDMIP). 
Step 4: .sup.3 H-CMDMIP was prepared from .sup.3 H-HMDMIP with thoinyl 
chloride. (Alternatively, 4'-(.sup.3 
H!-Bromomethyl)-4.5'-dimethylisopsoralen (.sup.3 H-BMDMIP) can be prepared 
from .sup.3 H-HMDMIP using thionyl bromide. .sup.3 H-BMDMIP is preferred 
due to its higher reactivity in S.sub.N 2 displacement reactions, such as 
BMDMIP.fwdarw.HDAMDMIP and BMDMIP.fwdarw.PHIMDMIP; accordingly, thionyl 
bromide is the reagent of choice to provide XMDMIP where X=Br). .sup.3 
H-HMDMIP, prepared as described in Step 3, was placed in a small round 
bottomed flask and dissolved in freshly distilled chloroform (5 ml, dried 
over 4.ANG. molecular sieves). Thionylchloride (41 mg; 0.35 mmol; Aldrich) 
was added and the yellow solution stirred for one hour. TLC (CH.sub.2 
Cl.sub.2) indicated all the starting alcohol had been converted to the 
product following this period. The solvent was removed under reduced 
pressure, benzene added (5 ml) and then evaporated under reduced pressure 
(twice). The white solid residue was used directly for the preparation of 
additional labelled compounds. 
EXAMPLE 20 
AMDMIP Synthesis 
This example describes the synthesis of AMDMIP from DMIP according to the 
following scheme: 
DMIP.fwdarw.PHIMDMIP.fwdarw.AMDMIP 
Step 1: DMIP (51 gm, 0.41 mole) is dissolved with heat in CH.sub.2 Cl.sub.2 
then cooled to room temperature. N-Hydroxymethylphtalimide (59.6 gm; 0.34 
mol) is added and the mixture cooled to 8.degree. C. A mixture of CF.sub.3 
SO.sub.3 H (19.8 ml; 33.6 gm; 0.22 mol) and CF.sub.3 COOH (280 ml; 189 gm; 
1.66 mol) is added from a dropping funnel over a period of 40-50 min, 
during which the temperature of the reaction mix is maintained between 
8.degree. C.-12.degree. C. by external cooling. Following the addition, 
the reaction flask is brought to room temperature then refluxed until all 
the starting material is consumed (TLC; CH.sub.2 Cl.sub.2). After the 
reaction is complete, one of two procedures is employed for work-up. In 
the first, the solvent is removed under reduced pressure, the residual 
yellow solid dissolved in chloroform, the chloroform washed with water, 
0.3M NaOH, water, then dried (MgSO.sub.4). The product is isolated by 
column chromatography (60-200 mesh silica gel, Baker). Alternatively, the 
reaction mixture is reduced to half volume, then the crude product is 
precipitated by the addition of methanol, followed by filtration. The 
precipitate is washed with methanol then recrystallized from 
ethanol:chloroform (1:1), providing 
4'-phthalimido-4.5'-dimethylisopsoralen (PHIMDMIP, 21). 
Step 2: PHIMDMIP (40 gm; 0.11 mol) is dissolved in 95% ethanol (1800 ml) 
followed by the addition of hydrazine hydrate (15 ml; 85% in water; 
Aldrich). The solution is heated (60.degree. C.) with stirring for 17 
hours after which additional hydrazine (15 ml) is added. After another 4 
hours, TLC (C/M 98.2) indicates all the starting material is consumed. The 
solvent is evaporated under reduced pressure and the residual solid 
dissolved in a mixture of chloroform (500 ml) and 0.1M NaOH (500 ml). The 
chloroform is separated and the basic aqueous phase extracted twice more 
with chloroform (250 ml). The combined chloroform extract is washed twice 
with water (500 ml), then back-extracted with 0.1M HCl (3.times.250 ml) to 
form the protonated amine. The acidic aqueous phase is then made basic 
with NaOH (1.0N) and extracted three times with chloroform. The chloroform 
extracts are combined, washed with water, dried (MgSO.sub.4), then 
evaporated under reduced pressure and the residual solid dissolved in 1000 
ml ethanol. HCl gas is passed through the chilled ethanol solution to 
provide the hydrochloride slat of 4'-aminomethyl14.5'-dimethylisopsoralen 
(AMDMIP,22), which is filtered, washed with ethanol, then dried under 
vacuum (23 gm; 75% yield). All analytical data (NMR, elemental analysis) 
is checked to be consistent with published results. 
EXAMPLE 21 
Radiolabelled AMDMIP Synthesis 
The present invention also contemplates labelled PHIMDMIP and labelled 
AMDMIP. One method of the present invention involves the scheme: 
.sup.3 H-CMDMIP.fwdarw..sup.3 H-PHIMDMIP.fwdarw..sup.3 H-AMDMIP 
Step 1: .sup.3 H-CMDMIP, prepared as described above, was dissolved in 2 ml 
freshly distilled DMF in the presence of 4.ANG. molecular sieves. 
Potassium phthalimide (43 mg, 0.23 mmol) was added and the mixture heated 
to 40.degree. C. and stirred for 42 hours. Following this period, the 
solvent was removed and the residual solid dissolved in chloroform: 
methanol 98:2 (1 ml), loaded on a 1 cm.times.20 cm silica gel column 
(60-200 mesh) then eluted with C/M 98:2. The fractions containing product 
were identified by TLC (CH.sub.2 Cl.sub.2), combined and evaporated under 
reduced pressure. The solid residue, 4'-(phthalimido-.sup.3 
H!-methyl)-4.5'-dimethylisopsoralen (.sup.3 H-PHIMDMIP), was used directly 
for the preparations of tritiated AMDMIP. 
Step 2: .sup.3 H-PHIMIDMIP, prepared as described above, was dissolved in 
95% ethanol (3 ml). Hydrazine hydrate (5 mg. 0.1 mmol) was added and the 
solution heated (60.degree. C.) and stirred for 17 hr after which 
additional hydrazine (0.04 mmol) was added. After another 4 hr. TLC 
indicated all the starting material had been consumed. The solvent was 
evaporated under reduced pressure and the residual solid disolved in a 
mixture of chloroform (5 ml) and NaOH (0.1N; 5 ml). The chloroform was 
separated and the basic aqueous phase extracted twice more with chloroform 
(5 ml). The combined chloroform extracts were washed twice with water (10 
ml) then backextracted with HCl (0.1N; 10 ml) to form the protonated 
amine. The chloroform was removed and the acidic aqueous phase washed 
three more times with chloroform (5 ml). The aqueous phase was then 
evaporated under reduced pressure and the residual solid dissolved in 
ethanol (10 ml). An appropriate dilution of the stock solution was made, 
counted, and the concentration determined by optical density. Overall 
recovery of the product was 4.57 mg (16.8% based on .sup.3 H-CMDMIP). The 
specific activity was 2.2.times.10.sup.5 CPM/.mu.g (93 mCi/mmol). 
EXAMPLE 22 
BIODMIP Synthesis 
The example describes one method of the present invention for the synthesis 
of BIODMIP according to the following scheme: 
CMDMIP.fwdarw.HDAMDMIP.fwdarw.BIODMIP.fwdarw. 
Step 1: CMDMIP (250 mg. 0.9 mmole, 1.0 eq), N,N'-dimethyl-1.6-hexanediamine 
(1.83 gm: 12.7 mmol: 14.0 eq: aldrich) and toluene (28 ml, freshly 
distilled onto 4.ANG. sieves) were placed in a 50 ml round bottomed flask 
with attached reflux condensor and argon line. The reaction mix was 
brought to reflux with a heating mantle while being magnetically stirred. 
TLC after 1 hour (benzene/methanol 1:1) found approximately 80% of the 
starting material converted to a single low Rf spot. The total reflux time 
was 17 hours after which essentially no starting material remained. The 
toluene was removed under reduced pressure on the rotovap and the residual 
yellowish oil dissolved in a small volume of chloroform. This solution was 
loaded on a small (0.5".times.5") chromatography column (60-200 mesh 
silica gel; Baker) then eluted with 95% ethanol:concentrated NH.sub.4 OH 
(4:1), collecting 15 1-ml fractions. Fractions containing product were 
identified by TLC, combined, the solvent removed under reduced pressure, 
and the residue reloaded on a second silica column (0.5".times.20") and 
re-eluted with C/E/T/(9:1:0.25). This solvent system effected separation 
of the product from unreacted N,N'dimethyl-1,6-hexanediamine. This 
separation was confirmed by development of the TLC place with iodine or 
ninhydrin (0.5% in ethanol) following elution. Fractions containing 
purified product were combined and the solvent removed under reduced 
pressure and the residual oil placed under high vacuum to constant weight. 
The yield of 
4'-N-(N,N'-dimethyl-1,6-hexanediaminemethyl-4,5'-dimethylisopsoralen 
(HDAMDMIP, 24) was approximately 50%. Mass spectrum m/e (relative 
abundance) 370, (M+, 1.09); absorption spectra maxima (nm): 252, 303. 
Step 2: HDAMDMIP (18.4 mg, 0.5 mmole, 1.0 eq), biotinamidocaproate 
N-hydroxysuccinimide ester (45.5 mg; 0.10 mmol; 2.0 eq; Pierce), and DMF 
(1.5 ml, freshly distilled onto 4.ANG. sieves) were placed in a 10 ml 
round bottomed flask with attached argon line. The reaction was 
magnetically stirred at room temperature. TLC after 1 hour (C/E/T 
9:1:0.25) showed a high Rf product spot. After 5 hr reaction time, another 
20 mg of biotin starting material was added and the reaction was continued 
for another hours. The solvent was removed under vacuum and the residue 
dissolved in a total of 10 ml chloroform: isopropanol 3:1 and transferred 
to a separatory funnnel. HCl (0.1N; 10 ml) was added and the layers mixed 
thoroughly then allowed to separate. The organic phase was removed and the 
aqueous phase adjusted to pH 13 then extracted again with chloroform : 
isopropanol 3:1. The organic extracts were combined and the solvent 
removed under reduced pressure. The residual solid (39.4 mg) was dissolved 
in C/E/T, loaded onto a silica gel column (60-200 mesh, 0.5".times.20"), 
and eluted with the same solvent. The product was isolated (as the free 
amine), the elution solvent removed, and the free amine dissolved in 
ethanol (10 ml). HCl gas was bubbled through the solution, followed by 
argon. The ethanol was removed to give 31.7 mg of 
4'-N-N,N'-dimethyl-N'-(6-{biotinamido}-hexanoate)-1,6-hexanediamine!-meth 
yl-4, 5'-dimethylisopsoralen (BIODMIP, 25a) (HCl salt) (85% yield). FABMS 
m/e (relative abundance) 710 (MH+, 60%). 
EXAMPLE 23 
Radiolabelled BIODMIP Synthesis 
This example describes the synthesis of radiolabelled HDAMDMIP and BIODMIP 
from radiolabelled CMDMIP according to the following scheme: 
.sup.3 H-CMDMIP.fwdarw..sup.3 H-HDAMDMIP.fwdarw..sup.3 H-BIODMIP 
Step 1: .sup.3 H-CMDMIP (70 mg; 0.27 mmol), prepared as described above, 
and N,N'-dimethyl-1,6-hexanediamine (500 mg, 3.5 mmole)and freshly 
distilled toluene (8 ml) were mixed in a 50 ml round bottomed flask. The 
solution was stirred overnight. TLC (chloroform/benzene/acetone/formic 
acid 4:1:1:1) showed that product had formed and essentially all the 
starting material had been consumed. The toluene was removed under reduced 
pressure and the residual solid was dissolved in 1-2 ml of C/E/T 
(9:1:0.25). This was loaded onto a silica gel column (0.5".times.2", 
60-200 mesh, Baker) and eluted with the same solvent. Fractions containing 
the product (not separated from the hexanediamine) were combined and the 
solvent removed under reduced pressure. The solid was dissolved in 1-2 ml 
in the same solvent, loaded onto a larger column (0.5"-20") and eluted 
with the same solvent. The presence of the hexanediamine compound on TLC 
was detected by using 1% ninhydrin in ethanol (the solution was sprayed 
onto the TLC plates and the plates heated at 70.degree.-80.degree.). The 
column fractions containing pure 
4'-N-(N,N'-dimethyl-1,6-hexanediamine)-.sup.3 H!-methyl-4, 
5'-dimethylisopsoralen .sup.3 H-HDAMDMIP)were thus identified, combined 
and the solvent removed under reduced pressure, providing 53.7 mg (58% 
yield). 
Step 2: .sup.3 H-HDAMDMIP (2mg; 0.005 mmol; 1 eq) and biotinamidocaproate 
N-hydroxysuccinimide hydroxysuccinimide ester (6.2 mg; 0.014 mmol; 2.8 eq) 
and DMF (1.0 ml, over sieves) were mixed in a 10 ml round bottomed flask. 
The reaction and work-up procedures were identical to the preparation of 
the unlabelled compound. Recovered 1.8 mg product (47% yield) at a 
specific activity of 2.6.times.10.sup.7 CPM/.mu.mol (3.5.times.10.sup.4 
CPM/.mu.g). 
EXAMPLE 24 
DITHIODMIP Synthesis 
This example describes one method of the present invention for the 
synthesis of DITHIODMIP according to the following scheme: 
HDAMDMIP.fwdarw.DITHIODMIP 
HDAMDMIP (30 mg: 0.80 mmol; 1.0 eq) and 
sulfosulfosucinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (68 mg; 
0.112 mmol; 1.40 eq) were dissolved in freshly distilled DMF (1.5 ml) in a 
small, dry round bottom flask with attached argon line then stirred at 
room temperature for 3 hr. After this period, the DMF was removed under 
reduced pressure and the residue suspended in 0.1N NaOH (7 ml), extracted 
with chloroform/isopropanol 3:1 (20 ml.times.2), and the organic extract 
washed with 0.5N HCL (20 ml.times.2), then dried (MgSO.sub.4), filtered 
and striped to give the crude product as a yellow oil (5.4 mg). The acidic 
extract from the organic wash was made basic by the addition of 1.0N Na OH 
(30 ml) then extracted with chloroform (25 ml.times.2). The chloroform was 
dried (Na.sub.2 SO.sub.4), filtered and stripped to give additional 
product (24 mg). To further characterize the product, 
4'-N-N,N!-dimethyl-N'-(2{biotinamido}-ethyl-1,3-dithiopropiate)-1,6-hexan 
ediamine-methyl-4,5'-dimethylisopsoralen (DITHIODMIP, 25b) (free base), 
small aliquots of the stock (ethanolic solution) were treated with sodium 
borohydride (Aldrich) or mercaptoethanol (Aldrich), then the product of 
these reactions compared to untreated material. In both cases the product 
was cleaved as expected. 
EXAMPLE 25 
Radiolabelled DITHIODMIP Synthesis 
This example describes one embodiment of the method of the present 
invention for the synthesis of .sup.3 H-DITHIODMIP according to the 
following scheme: 
.sup.3 H-CMDMIP.fwdarw..sup.3 H-IMDMIP.fwdarw..sup.3 
H-HDAMDMIP-.fwdarw..sup.3 H-DITHIODMIP 
Step 1: .sup.3 H-CMDMIP (20 mg; 0.75 mmol) prepared as described in Step 4 
of acetone (Mallinckrodt are refluxed for 48 hours. Following this period, 
the reaction mixture is filtered to remove the resulting NaCl, the 
filtrate evaporated under reduced pressure, and the residual crude product 
dissolved in chloroform, loaded on a 1/4.times.5"silica gel column (60-200 
mesh; Baker) then eluted with chloroform. The fractions containing the 
product, 4'-(iodo-.sup.3 H!-methyl)-4,5'-dimethyl-isopsoralen (.sup.3 
HIMDMIP,23), are identified by TLC, combined and solvent removed under 
reduced pressure. The product is used directly for Step 2. 
Step 2: Using the .sup.3 H-IMDMIP prepared in Step 1, .sup.3 H-HDAMDMIP is 
prepared. The synthesis and work-up are identical to the procedures 
described in Step 1 of Example 23 except .sup.3 H-CMDMIP is replaced by 
.sup.3 H-HDAMDMIP is further purified by column chromatography as 
described then used directly for the preparation of .sup.3 H-DITHIODMIP. 
Step 3: Using the .sup.3 H-HDAMDMIP prepared in Step 3, .sup.3 H-DITHIODMIP 
is prepared. .sup.3 H-HDAMDMIP (5 mg; 0.013 mmol; 1.0 eq) and 
sulfosuccinimidyl-2-(biotinamido)-ethyl-1, 3-dithiopropionate (11 mg; 
0.019 mmol; 1.5 eq) is dissolved in freshly distilled DMF (1.5 ml) in a 
small, dry round bottomed flask with attached argon line then stirred at 
room temperature for 3 hours. After this period, the DMF is removed under 
reduced pressure suspended in NaOH (0.1N; 3 ml), extracted with 
chloroform/isopropanol 3:1 (7 ml.times.2), water (5 ml.times.2) then dried 
(MgSO.sub.4), filtered and stripped to provide the crude product as a 
yellow oil. The product is characterized by ultraviolet absorption 
(comparison with authentic material) and TLC (cochromatography with 
authentic material) and TLC (cochromatography with authentic material in 
several different solvent systems). Radiochemical purity is determined by 
HPLC. Approximately 10.sup.6 CPM of tritiated .sup.3 H-DITHIODMIP is mixed 
with 10 .mu.g of unlabelled DITHIODMIP in 50 .mu.l ethanol (100%). The 
sample is injected on a C18 octadecylsilyl reverse phase chromatography 
column (Beckman) and eluted with an acetonitrile/ammonium acetate (0.1M; 
pH 7) gradient, as follows: 0-10 minutes, 100% ammonium acetate; 10-70 
minutes, 100% ammonium acetate.fwdarw.100% acetonitrile; 70-80 minutes, 
100% acetonitrile. Eight 1.0 ml fractions are collected and 40 .mu.l of 
each fraction is then counted. The product is further characterized by 
dissolving a sample in ethanol and treating it with either sodium 
borohydride (Aldrich) or mercaptoethanol (Aldrich). The product of these 
reactions is compared to untreated material to verify that cleavage occurs 
as expected. 
EXAMPLE 26 
FLUORDMIP Synthesis 
This example describes one method of the present invention for the 
synthesis of FLUORDMIP according to the following scheme: 
CMDMIP-HDAMDMIP-FLUORDMIP 
Step 1: HDAMDMIP was prepared from CMDMIP as described in Step 1 of Example 
22. 
Step 2: HDAMDMIP from Step 1 (10 mg; 0.027 mmol; 1.0 eq) in DMF (1.0 ml, 
distilled onto 4.ANG. sieves; Mallinckrodt) and 
6-carboxyfluorescein-N-hydroxysuccinimide ester (15.1 mg; 0.032 mmol; 1.1 
eq; Pierce) in DMF (1.0 ml, distilled onto 4.ANG. sieves; Mallinckrodt) 
were mixed in a 5 ml round bottomed flask with attached argon line. The 
reaction mix was stirred for several hours at room temperature, after 
which the majority of the starting material had been consumed as indicated 
by TLC (C/B/A/F--4:1;1:1). The solvent was removed with gentle heating 
(&lt;50.degree. C.) under reduced pressure. The residue was dissolved in HCl 
(5 ml; 0.1N) then extracted with chloroform/isopropyl alcohol (3:1). The 
organic extract was then reduced in volume, loaded onto a glass backed 
preparative silica gel TCL place (20 cm.times.20 cm.times.2 mm; Baker) 
then eluted with C/B/A/F. The major low Rf band was scraped off, eluted 
with chloroform/isopropanol (3:1), the silica removed by filtration and 
the solvent evaporated under reduced pressure. The product was dissolved 
in a small volume of ethanol and HCl gas bubbled through the solution. The 
ethanol was then evaporated to provide 
4'-N'N,N'-dimethyl-N'-(6-carboxyfluorescein 
ester)-1,6-hexanediamine)-methyl-4,5'-dimethylisopsoralen (FLUORDMIP, 25c) 
as the monohydrochloride salt (FABMS) (free base) m/e 729, M+: absorption 
spectrum relative maximum (nm) 447. 
EXAMPLE 27 
Radiolabelled FLUORDMIP Synthesis 
This example describes one embodiment of the method of the present 
invention for the synthesis of radiolabelled FLUORDMIP according to the 
following scheme: 
FDMIP.fwdarw..sup.3 H-HMDMIP.fwdarw..sup.3 H-CMDMIP.fwdarw..sup.3 
H-HDAMDMIP.fwdarw..sup.3 H-FLUORDMIP 
Step 1: .sup.3 H-HMDMIP was prepared from FDMIP as described in Step 3 of 
Example 19. 
Step 2: .sup.3 H-CMDMIP was prepared from .sup.3 H-HMDMIP as described in 
Step 4 of Example 19. 
Step 3: .sup.3 H-HDAMDMIP was prepared from .sup.3 H-CMDMIP as described in 
Step 1 of Example 23. 
Step 4: .sup.3 H-FLUORDMIP was prepared from .sup.3 H-HDAMDMIP. .sup.3 
H-HDAMDMIP (3.1 mg; 0.0084 mmol; 1.0 eq) in DMF (1.7 ml, distilled over 
4.ANG. sieves) and 6-carboxyfluorescein-N-hydroxysuccinimide (4.0 mg; 
0.0084 mmol; 1.0 eq) in DMF (1.0 ml) were mixed in a 5 ml round bottomed 
flask with attached argon line. This mixture was stirred overnight. The 
work-up was similar to the preparation in Step 2 of Example 26, except the 
preparative TLC plate was eluted with C/B/A/F (5:1:1:1; this solvent gave 
a slightly higher Rf and better separation of the product). Approximately 
2.1 mg product was obtained (a 42% yield) with a specific activity of 93 
mCi/mmol. 
EXAMPLE 28 
Solubility Of Photoreactive Compounds 
The solubilities of AMIP, AMDMIP, DMIP, MIP and isopsoralen (IP) were 
determined experimentally according to the scheme set forth in FIG. 5. To 
perform the solubility measurement, it was first necessary to establish 
known optical densities for a 1 .mu.g/ml solution of each compound. These 
were determined by preparing either ethanolic or aqueous stocks of each 
compound at known concentrations in 1.times.TE, then measuring the optical 
density of each solution. From this information, the absorption of a 1 
.mu.g/ml solution was computed. Alternatively, the extinction coefficient 
may be thus determined and used in the same fashion. In this manner, the 
following absorption data was collected: 
______________________________________ 
Compound Wavelength (nm) 
O.D. 1 .mu.g/ml (1 .times. TE) 
Emax 
______________________________________ 
AMIP 249 0.087 2.19 .times. 10.sup.4 
AMDMIP 249.5 0.082 2.29 .times. 10.sup.4 
DMIP 250.5 0.102 2.18 .times. 10.sup.4 
MIP 249 0.092 1.97 .times. 10.sup.4 
IP 247 0.105 1.95 .times. 10.sup.4 
______________________________________ 
To determine the solubility of each compound an excess (2-25 mg) of each 
(except for AMDMIP; see below) was placed in 1.times.TE (0.37-2 ml) then 
heated (50.degree.-70.degree. C.) for several hours. After this, each 
mixture was stirred for several hours at room temperature in the presence 
of undissolved solid (this procedure assumed that supersaturation did not 
occur). Following this step, undissolved compound was removed by 
filtration using a 0.2.mu. nylon-66 syringe filter (Arco LC13; Gelman). 
The concentration of the remaining soluble compound was then determined by 
measuring the optical density of the filtrate and computing the solubility 
from the known optical density of a 1 .mu.g/ml solution of that compound. 
The solubility of the compounds was thus detemined to be as follows: 
______________________________________ 
Compound Solubility in 1 .times. TE (.mu.g/ml) 
______________________________________ 
AMIP 21,000 
AMDMIP &gt;22,700* 
DMIP nd** 
MIP nd 
IP 2 
______________________________________ 
*The solubility of AMDMIP waws a minimum of 22,700 .mu.g/ml as the initia 
mix of the compound was entirely soluble in the volume of 1 .times. TE 
used. 
**nd = not detectable (solubility was less than 1 .mu.g/ml). 
EXAMPLE 29 
Dark Binding Of Photoreactive Compounds 
Equilibrium Dialysis was used to determine the association ("dark binding") 
constants of AMIP, AMDMIP and MIP with calf thymus DNA. The tritium 
labelled isopsoralens were used for the experiment. The method was a 
modification of that of Isaacs et al., Biochemistry 16, 1058-1066 (1977). 
Spectrapor 2 dialysis tubing (Spectrum was pretreated by boiling in 
saturated sodium bicarbonate then rinsed thoroughly with double distilled 
water. Calf thymus DNA was prepared at a concentration of 50 .mu.g/ml in 
1.times.TE. The samples were prepared by placing 1.0 ml of the DNA 
solution inside the bag and using enough isopsoralen to provide an 
isopsoralen:DNA base pair ratio of 1:15. 
TABLE 9 
______________________________________ 
Volume of 
Volume of 1 .times. 
Mass of Location of 
DNA Stock in 
TE Stock in 
Sample Drug Drug Sialysis Bag 
Dialysis Bag 
______________________________________ 
MIP in 1.27 .mu.g 
inside bag 1 ml 10 ml 
HMT in 1.43 .mu.g 
" " " 
AMIP in 
1.30 .mu.g 
" " " 
AMDMIP 1.01 .mu.g 
" " " 
in 
MIP out 
1.27 .mu.g 
outside bag 
1 ml 10 ml 
HMT out 
1.43 .mu.g 
" " " 
AMIP out 
1.30 .mu.g 
" " " 
AMDMIP 1.01 .mu.g 
" " " 
out 
______________________________________ 
The samples were prepared with the tritiated isopsoralen ("Drug") placed 
either inside or outside of the dialysis bag (each sample) was placed in a 
scintillation vial containing a small magnetic stir bar). The samples were 
stirred at 4.degree. C. for five days in the dark. After this period the 
bags were removed, opened, and the radioactivity inside and outside the 
bag determined by scintillation counting. The concentration of the DNA was 
determined by measuring optical density at 260 nm. The dissociation 
constant (K.sub..alpha.) was calculated from this information (Table 9). 
Preparing the samples in duplicate with the isopsoralen both inside and 
outside the dialysis bag provided a check that the system had come to 
equilibrium at the end of the dialysis period (the amount of isopsoralen 
inside and outside the bag at equilibrium should be, and was, the same in 
both cases). 
The association constant, K.sub..alpha., is defined here as 
##EQU1## 
where P=free drug in solution, DNA=DNA binding site for the drug, and 
P:DNA=drug associated with the DNA. The following summarizes the 
K.sub..alpha. values determined for the compounds: 
______________________________________ 
Sample K.sub..alpha. 
______________________________________ 
AMIP in 2.96 .times. 10.sup.4 
AMIP out 2.21 .times. 10.sup.4 
AMDMIP in 7.66 .times. 10.sup.4 
AMDMIP out 1.07 .times. 10.sup.4 
MIP in 9.08 .times. 10.sup.4 
MIP out 2.07 .times. 10.sup.4 
______________________________________ 
EXAMPLE 30 
Photoactivation Device 
One embodiment of the photoactivation device of the present invention is 
designated "CE-I." CE-I is an irradiation device having the following 
features: (1) an inexpensive source of electromagnetic radiation, (2) 
temperature control of the sample, (3) a multisample holder, (4) a 
multiple sample irradiation format, and (5) a compact design that requires 
minimal bench space. 
FIG. 6 is a perspective view of CE-I, integrating the above-named features. 
The figure shows the bottom platform of a housing (100) with the rest of 
the housing removed (not shown), having six bulbs (101-106) connectable to 
a power source (not shown) arranged around a chamber (107) having a 
plurality of intrusions (108) for supporting a plurality of sample vessels 
(109). The bulbs serve as a source of electromagnetic radiation and, in 
one embodiment, ultraviolet radiation. While not limited to the particular 
bulb type, the embodiment is configured to accept an industry standard, 
F8T5BL hot cathode dual bipin lamp. 
The chamber (107), in addition to holding sample vessels (109), holds 
temperature control liquid (not shown), thereby serving as a means for 
controlling the temperature of the sample vessels (109). 
FIG. 7 is a cross-sectional view of CE-I along the lines of a--a of FIG. 6. 
FIG. 7 shows the arrangement of the sources (101-106) around the chamber 
(107). FIG. 7 also shows the chamber (107) punctuated with sample holder 
intrusions (108) with dimensions designed to accommodate the sample 
vessels (109). 
It is not intended that the present invention be limited by the nature of 
the material used to form the chamber (107). In one embodiment, it is made 
of glass. In another embodiment, it is made of plastic. In a preferred 
embodiment, it is made of UV transmitting acrylic selected from the group 
of commercial acrylics consisting of ACRYLIC-VT (Polycast), PLEXIGLAS 
11-UVT (Rohm & Haas), PLEXIGLAS GUVT (Rohm & Haas) and ACRYLITE OP-4 
(Cyro). 
Similarly, it is not intended that the present invention be limited by the 
nature of the method used to form the chamber (107). In one embodiment, it 
is molded as one piece. In another embodiment, it is modeled as separate 
pieces and then assembled. 
FIG. 6 shows that the temperature control liquid is introduced via a liquid 
inlet port (111) and removed via a liquid outlet port (112). It is 
preferred that the liquid inlet port (111) and the liquid outlet port 
(112) connect via tubes (113, 114) to a liquid source (not shown). It is 
further preferred that the liquid source allow for recirculation of the 
liquid. To improve temperature control, static temperature control liquid 
(not shown) may be placed in the intrusions (108). 
It is not intended that the present invention be limited to any particular 
temperature control liquid. One inexpensive temperature control liquid 
contemplated by the invention is water. 
FIG. 8 shows the CE-I embodiment with seven intrusions (108) placed within 
the boundary defined by the inlet (111) and outlet (112) ports. While the 
number of intrusions (108) and their placement may be selected to suit the 
convenience of the user, some configurations may impact irradiation 
efficiency. 
FIGS. 6 and 7 also show an array of reflectors (115, 116, 117). It is 
preferred that the reflectors are made from UV reflecting metal. 
While not limited to any particular dimensions (the drawings are not drawn 
to scale), it is preferred that the intrusions (108) be approximately 4 cm 
deep, that the intrusions be spaced approximately 3 cm apart, and the 
distance from the top surface of the housing to the opening of the 
intrusions be approximately 6.5 cm. In such an arrangement, the lower 
bulbs (103, 104) are preferably 2.3 cm apart when measured from their 
centers (their centers are preferably 1.2 cm above the housing when 
measured from the surface of the reflector 117). This allows the lower 
bulbs (103, 104) to be less than 1.5 cm in distance from the bottom of the 
reaction vessel (109). 
The other bulbs can be viewed as two more sets (101, 102 and 105, 106) (for 
a total of three, two bulb sets in all). Within a set, it is preferred 
that the bulbs are 2.3 cm apart when measuring from their centers (their 
centers are preferably 1.2 cm away from the surface of the reflectors 115, 
116). It is preferred that the reflectors 115 and 116 are approximately 11 
cm apart when measuring from their sides. 
It is preferred that the relationship of the chamber (107) length ("CL") to 
the bulb (101-106) length ("BL") and to the reflector (115,116,117) length 
("RL") is as follows: 
RL&gt;BL&gt;CL 
Preferred lengths are RL=29.5 cm, BL=between 26 cm and 29 cm, and 
CL=approximately 25 cm. 
EXAMPLE 31 
Photoactivation Device 
One embodiment of the photoactivation device of the present invention is 
designated "CE-II." CE-II is an irradiation device created to serve as a 
convenient photoactivation device having the following features: (1) an 
inexpensive source of electromagnetic radiation, (2) temperature control 
of the sample, (3) a multisample holder, (4) a multiple sample irradiation 
format, (5) a housing that shields the user from stray electromagnetic 
radiation, and (6) a compact design that requires minimal bench space. 
FIG. 9 is a perspective view of CE-II, integrating the above-named 
features, showing a housing (200) containing six bulbs (201-206) 
connectable to a power source (not shown) arranged around a detachable 
chamber (207). FIG. 10 shows that the chamber (207) has a plurality of 
intrusions (208) for supporting a plurality of sample vessels (not shown). 
The bulbs serve as a source of electromagnetic radiation and, in one 
embodiment, ultraviolet radiation. While not limited to the particular 
bulb type, the embodiment is configured to accept an industry standard, 
F8T5BL hot cathode dual bipin lamp. 
The housing (200) also serves as a mount for several electronic components. 
A main power switch (209) controls the input current from the AC power 
source (not shown). For convenience, this power switch (209) is wired to a 
count down timer (210) which in turn is wired in parallel to an hour meter 
(211) and to the coils (not shown) of the source of electromagnetic 
radiation. The count down timer (210) permits a user to preset the 
irradiation time to a desired level of sample exposure. The hour meter 
(211) maintains a record of the total number of radiation hours that are 
provided by the source of electromagnetic radiation. This feature permits 
the bulbs (201-206) to be monitored and changed before their output 
diminishes below a minimum level necessary for rapid photoactivation. 
The chamber (207), in addition to holding sample vessels, holds temperature 
control liquid (not shown), thereby serving as a means for controlling the 
temperature of the sample vessels. 
It is not intended that the present invention be limited by the nature of 
the material used to form the chamber (207). In one embodiment, it is made 
of glass. In another embodiment, it is made of plastic. In a preferred 
embodiment, it is made of UV transmitting acrylic selected from the group 
of commercial acrylics consisting of ACRYLIC-UVT (Polycast), PLEXIGLAS 
11-UVT (Rohm & Haas), PLEXIGLAS GUVT (Rohm & Haas) and ACRYLITE OP-4 
(Cyro). 
Similarly, it is not intended that the present invention be limited by the 
nature of the method used to form the chamber (207). In one embodiment, it 
is modeled as one piece. In another embodiment, it is molded in separate 
pieces and then assembled. 
FIG. 9 shows that the temperature control liquid is introduced via tubes 
(212, 212) from a liquid source (not shown) and is circulated via liquid 
inlet and outlet ports (not shown). It is not intended that the present 
invention be limited to any particular temperature control liquid. One 
inexpensive temperature control liquid contemplated by the invention is 
water. 
FIG. 10 shows the positioning of reflectors (214,215). It is preferred that 
the reflectors be made from UV reflecting metal. 
FIG. 9 shows CE-II with twenty intrusions (208) placed within the boundary 
defined by tubes (212, 213). While the number of intrusions (208) and 
their placement may be selected to suit the convenience of the user, there 
can be a significant impact on irradiation efficiency. In this embodiment, 
the intrusions (208) were aligned in two rows with the intrusion (208) of 
one row lined up opposite the intrusion (208) of the other row. 
Performance data obtained subsequent to the design indicated that this 
arrangement of intrusions (208) may cause the electromagnetic radiation to 
be partially blocked, ie., the intrusion (208) of one row is blocking 
electromagnetic radiation coming from one side of the device so that the 
intrusion (208) opposite from it in the other row receives less 
electromagnetic radiation. 
FIG. 10 shows the "V-shape" geometry of the bulb (201-206) placement of 
this embodiment in relation to the chamber (207). The V-shape geometry is, 
in large part, dictated by the dimension demands placed on the chamber 
(207) by virtue of the double row arrangement of the intrusions (208). The 
distance from the center of one intrusion (208) in one row and to the 
center of the opposite intrusion (208) in the other row is greater than 
5.5 cm. This causes the upper level bulbs (201, 206) to be placed almost 
1.5 cm apart (measured center to center), the middle level bulbs (202, 
205) to be placed almost 11.5 cm apart (measured center to center), and 
the lower level bulbs (203, 204) to be placed almost 4.0 cm apart 
(measured center to center). 
It is preferred that the relationship of the chamber (207) length ("CL") to 
the bulb (201-206) length ("BL") and to the reflector (214,215) length 
("RL") is as follows: 
RL&gt;BL&gt;CL 
In this embodiment, CL=approximately 37 cm. 
EXAMPLE 32 
Photoactivation Device 
A preferred embodiment of the photoactivation device of the present 
invention is designated "CE-III." CE-III is an irradiation device created 
to optimize rapid photoactivation having the following features: (1) an 
inexpensive source of electromagnetic radiation, (2) temperature control 
of the sample, (3) control of irradiation time, (4) a multisample holder, 
(5) a multiple sample irradiation format, (6) a housing that shields the 
user from stray electromagnetic radiation, and (7) a compact design that 
requires minimal bench space. 
FIGS. 11, 12, and 13 are view of CE-III, integrating the above-named 
features, showing a housing (300) containing eight bulbs (301-308) 
connectable to a power source (not shown) arranged around a detachable 
chamber (309), having interior (310) and exterior walls (311). The 
interior walls (310) form a trough (312). FIGS. 11 and 13 show one 
embodiment of an interchangeable, detachable sample rack (313A). The 
sample (313A) is detachably coupled to the housing (300) above the trough 
(312). Sample vessels (315) fit in the sample rack (313A) and are thereby 
aligned in the trough (312). A sample overlay (314) extends over and 
covers the interchangeable sample rack (313A) sealing the unit and 
shielding the user from electromagnetic radiation when the device is in 
operation. 
FIG. 14 shows an alternative embodiment of an interchangeable, detachable 
sample rack (313B). Note that in this embodiment, the placement of sample 
vessels (315) in two rows is staggered to avoid blocking electromagnetic 
radiation (compare with CE-II, above). 
FIG. 12 shows a unitary reflector (316) extending around all the bulbs 
(301-308) of the device. It is preferred that the reflector (316) is made 
of UV reflecting material. 
The chamber (309) holds circulating temperature control liquid (317) 
between the interior (310) and exterior walls (311), thereby serving as a 
means for controlling the temperature of the sample vessels (315). FIG. 13 
shows that the circulating temperature control liquid (317) is introduced 
from a liquid source (not shown) via liquid inlet port (318) and removed 
via a liquid outlet port (319), allowing for recirculation of the liquid. 
To improve temperature control, static temperature control liquid (not 
shown) may be placed in the trough. It is not intended that the present 
invention be limited to any particular temperature control liquid. One 
inexpensive temperature control liquid contemplated by the invention is 
water. 
It is not intended that the present invention be limited by the nature of 
the material used to form the chamber (309). In one embodiment, it is made 
of glass. In another embodiment, it is made of plastic. In a preferred 
embodiment, it is made of UV transmitting acrylic selected from the group 
of commercial acrylics consisting of ACRYLIC-UVT (Polycast), PLEXIGLAS 
11-UVT (Rohm & Haas), PLEXIGLAS G-UVT (Rolum & Haas) and ACRYLITE OP-4 
(Cyro). 
Similarly, it is not intended that the present invention be limited by the 
nature of the method used to form the chamber (309). In one embodiment, it 
is molded as one piece. In another embodiment, it is molded in separate 
pieces and then assembled. 
The housing (300) also serves as a mount for several electronic components. 
A main power switch (320) controls the input current from the AC power 
source (not shown). For convenience, this power switch is wired to a timer 
activation switch (321); in the "ON" position, the timer activation switch 
(321) provides power to a count down timer (322). The count down timer 
(322) in turn controls the current to an hour meter (323) and to the coils 
(not shown) of the source electromagnetic radiation. The count down timer 
(322) permits a user to preset the irradiation time to a desired level of 
sample exposure. The hour meter (323) maintains a record of the total 
number of radiation hours that are provided by the source of 
electromagnetic radiation. This feature permits the bulbs (301-308) to be 
monitored and changed before the output diminishes below a level necessary 
to achieve rapid photoactivation. In the "OFF" position, timer activation 
switch (321) is wired such that it bypasses the count down timer (322) and 
provides continual power to the hour meter (323) and the coils (not shown) 
of the source of electromagnetic radiation. 
FIG. 12 shows the "U-shape" geometry of the bulb (301-308) placement of 
this embodiment in relation to the chamber (309). (Compare with the 
V-shape geometry of CE-II). The U-shape geometry is, in large part, 
allowed by the smaller dimensions of the chamber (207) by virtue of the 
trough (312) design. 
While not limited by the particular dimensions, the width of the trough 
(312), when measured by the length of the bottom exterior wall (311) is 6 
cm. The upper level bulbs (301, 308) are placed less than 9 cm apart 
(measured center to center). 
Again, it is preferred that the chamber (309) length ("CL"), the bulb 
(301-308) length ("BL") and the reflector (317) length ("RL") follow the 
relationship: RL&gt;BL&gt;CL. 
EXAMPLE 33 
Photoactivation Device: Temperature Control 
Temperature changes can have a drastic impact on photoactivation chemistry. 
It is desired that the devices of the present invention provide 
temperature control to limit the possibility of uncontrolled changes on 
photoactivation results. 
FIG. 15 illustrates the problem of lack of temperature control for the 
devices of the present invention. CE-I, CE-III and the PTI device were 
allowed to irradiate 1.5 ml Eppendorf tubes without using the means for 
controlling the temperature of the sample vessels of the present 
invention. The temperature of the control liquid was measured over time. 
measurements were conducted with type T thermocouple immersed into a 0.5 
ml Eppendorf tube containing 100 .mu.l of dH.sub.2 O. The Eppendorf tube 
was irradiated in each device. For irradiations with the PTI device, the 
tube was irradiated from the top down with the cap closed. Temperature was 
monitored on an Omega Temperature Controller, Model 148 (Omega 
Engineering, Inc., Stamford, CT). 
The results (FIG. 15A) show that the temperature of the sample vessel 
rapidly increases in temperature without temperature control. By contrast, 
irradiations with temperature control (FIG. 1B) show a constant 
temperature. 
EXAMPLE 34 
Photoactivation Device: Energy Output 
This example investigates energy output as it relates to optimum 
photobinding kinetics. FIG. 16 shows the relative energy output of the 
devices of the present invention. The PTI device has a tremendously strong 
intensity relative to CE-III. From the relative intensity output it would 
appear that the fluorescent source of ultraviolet irradiation is not of 
sufficient flux for rapid photoactivation. At the very least, a dramatic 
difference in kinetics of photobinding was expected for the two machines. 
The impact of this difference on binding was investigated as shown in FIG. 
17. .sup.3 H-HMT was used to measure binding to calf thymus DNA. .sup.3 
H-HMT was mixed with the DNA and irradiated. The product was then 
extracted with chloroform to separate the unbound .sup.3 H-HMT. The 
nucleic acid was then precipitated and solubilized. Bound HMT was 
determined by scintillation counting along with measuring the optical 
density of the DNA solution. 
The results are shown in FIG. 18. Surprisingly, the kinetics of the CE-III 
device are essentially the same as the costly PTI device. Plateau binding 
for the CE-III and PTI machines was reached in less than five minutes. 
Interestingly, CE-II did not reach plateau binding under the conditions of 
the experiment. (Plateau binding might be reached with the CE-II device in 
one of two ways: (1) additional radiation time, or (2) use of a sample 
vessel with better UV transmission properties such as a polycarbonate 
tube). 
EXAMPLE 35 
Photoactivation Device: Sample Position 
The impact of the small differences in position of samples within the 
photoactivation device was investigated. FIG. 19 shows the intensity of 
the light of CE-III at the surface of the trough (FIG. 12, element 312) 
according to sample position. Samples from the center position and the end 
position of the sample rack (FIG. 11, element 313A) were examined for 
photobinding in the manner outlined in FIG. 17. The results are shown in 
FIG. 20. It is clear that some difference in photoaddition kinetics exists 
when the irradiation time is below two minutes. This illustrates the 
importance of plateau binding to nullify such small positional differences 
(contrast FIG. 20 with FIG. 18). 
EXAMPLE 36 
Photoactivation Device: Photoproduct 
In the first part of this example, the various embodiments of the 
photoactivation device of the present invention were investigated for 
their ability to create photoproduct. In the second part, photoproduct is 
shown to bind to nucleic acid. 
FIG. 21 shows schematically the manner in which photoproduct generation was 
investigated. FIG. 22 shows the production of photoproduct over time on 
the CE-III device for known compound AMDMIP. While the AMDMIP standard 
(unirradiated compound) shows a single peak on HPLC (FIG. 22A), AMDMIP 
photoproduct peaks increase and the AMDMIP peak diminishes from two 
minutes (FIG. 22B), five minutes (FIG. 22C) and fifteen minutes (FIG. 
22D). 
FIG. 23 shows production of photoproduct according to the photoactivation 
device used for known compound AMDMIP. Again the AMDMIP standard is a 
single peak on HPLC (FIG. 23A). By contrast, the fifteen minute 
irradiation with CE-III shows increase in photoproduct peaks and a 
decrease in the AMDMIP peak (FIG. 23, compare A to B and note scale 
change). Irradiation for the same time period, however, on the PTI device 
shows very little reduction in the AMDMIP peak and very little generation 
of photoproduct peaks (FIG. 23, compare A to C and note scale change). 
Clearly, the CE-III device generates more AMDMIP photoproduct than does 
the PTI device. 
FIG. 24 shows production of photoproduct according to the photoactivation 
device used for novel compound AMIP. The AMIP standard is primarily a 
single peak on HPLC (FIG. 24A). By contrast, the fifteen minute 
irradiation with CE-I devices shows the appearance of photoproduct peaks 
and a decrease in the AMIP peak (FIG. 24, compare A to B and note scale 
change). Similarly, the fifteen minute irradiation with CE-III device 
shows the appearance of photoproduct peaks and a decrease in the AMIP peak 
(FIG. 24, compare A to C and not scale change). Interestingly, irradiation 
of AMIP for the same time period with the PTI device shows approximately 
the same amount of photoproduct formations as with the CE-I and CE-III 
devices (FIG. 24, compare D with B and C). 
The nucleic acid binding properties of photoproduct were investigated with 
calf thymus DNA. 200 .mu.g/ml of .sup.3 H-AMDMIP (1.times.10.sup.5 CPM/ml) 
in Taq buffer was irradiated at room temperature with either the CE-III 
device or the PTI device. This irradiation was performed for 15 minutes in 
the absence of nucleic acid. Following the irradiation, 200 .mu.l of the 
irradiated solution was mixed with 200 .mu.l of 100 .mu.g/ml DNA solution 
in Taq buffer. Several identical samples were prepared in this manner. One 
set of samples was allowed to react with the DNA for 2 hours at room 
temperature. The other set of samples was subjected to 30 cycles of 
heating and cooling by placing the samples in a thermal cycler 
Perkin-Elmer Cetus DNA Thermal Cycler (Part No. N8010150); each cycle 
involved 93.degree. C. for 30 seconds, 55.degree. C. for 30 seconds and 
72.degree. C. for 1 minute!. When the DNA reactions were complete, all 
sample were analyzed for DNA-associated tritium counts (CPM) by following 
the flow chart outlined in FIG. 17. 
The DNA-associated counts are given in terms of adducts per 100 base pairs 
in Table 10. For calculation purposes, these adducts are reported as if 
they represented monomers of AMDMIP. Regardless of the accuracy of this 
approximation, the process of irradiating .sup.3 H-AMDMIP clearly results 
in a tritiated product which associates (i.e., binds) with DNA in the 
absence of subsequent activating wavelengths of electromagnetic radiation. 
The extent of the association is shown in Table 10 to depend upon the 
reaction conditions. 
Binding is influenced by the photoactivation device used. When .sup.3 
H-AMDMIP irradiated with the CE-III device is compared with .sup.3 
H-AMDMIP irradiated with the PTI device, a significantly greater amount of 
associated counts is observed. This can be viewed as consistent with the 
observation (FIG. 23) that more photoproduct is made with CE-III than with 
the PTI device after the same exposure time. Interestingly, thermal 
cycling results in a five fold higher association than a 2 hour reaction 
at room temperature (regardless of the device used). 
TABLE 10 
______________________________________ 
Binding of Photoproduct to Nucleic Acid 
Dark Reaction 
Adducts per 1000 
Device Irradiation 
Treatment Base Pairs 
______________________________________ 
None None Room Temp 1.1 
None None Thermally Cycled 
0.9 
CE-II 15 mins. Room Temp 4.4 
CE-III 15 mins. Thermally Cycled 
24.3 
PTI 15 mins. Room Temp 1.7 
PTI 15 mins. Thermally Cycled 
5.7 
______________________________________ 
As seen by the results with no irradiation, the isolation procedure that 
was applied to the DNA samples to remove unbound reactants removes most of 
the noncovalently associated reactants. The increase in counts seen when 
irradiated AMDMIP reacts with DNA, relative to those counts seen when 
unirradiated AMDMIP is reacted, is, therefore presumed to be due to 
covalent association of photoproduct with DNA. 
That is not say, however, that the counts must be due to covalent 
interactions. It is possible that photoproduct has a very high 
non-covalent association with nucleic acid. This association may be high 
enough that photoproduct is non-covalently associated with DNA even after 
the rigorous work up. 
EXAMPLE 37 
Nucleic Acid Binding 
It will be desirable in some situations to have precise control of the 
binding levels of a photoactive compound to nucleic acids. As shown 
earlier in FIG. 18, binding levels are a function of the irradiation time 
providing that the irradiation time is sufficient to achieve the plateau 
level, a constant level of binding can be achieved. In addition to light 
exposure, the concentration of the photoactive compound also affects the 
ultimate binding levels. As discussed in the introduction, photoactive 
compounds such as psoralen and isopsoralen undergo competing reactions 
during exposure to actinic light. They will undergo photo decomposition at 
the same time as they add to polynucleotides. Although the structural 
properties of a particular photoactive compound determine the relative 
rates of photo decomposition to photoaddition reactions, the initial 
concentration of the compounds does affect the plateau level of binding. 
This example investigates the binding levels as a function of 
concentration. Following the procedure outlined in FIG. 17, .sup.3 
H-AMDMIP was used at different concentrations to measure binding to calf 
thymus DNA. The results are as shown in FIG. 25. Clearly, the 
concentration of AMDMIP affects the binding levels achieved with calf 
thymus DNA. providing irradiations are of sufficient duration to achieve 
plateau levels, the concentration dependence can be used to precisely 
control addition reactions to a desired level of photobinding. 
EXAMPLE 38 
Nucleic Acid Binding 
FIG. 17 can again be referred to as a flow chart schematically showing the 
manner in which covalent binding was measured for the compounds 
synthesized by the methods of the present invention. .sup.3 H-AMIP 
(3.1.times.10.sup.5 cpm/.mu.g), .sup.3 H-AMDMIP (2.2.times.10.sup.5 
cpm/.mu.g), and .sup.3 H-AMIP (2.1.times.10.sup.5 cpm/.mu.g) were added to 
300 .mu.l of calf thymus DNA (Sigma) in 1.times.TE buffer. All of the 
isopsoralen compounds were added at a nominal concentration of 100 
.mu.g/ml. However, actual concentrations obtained empirically (counted 
after sitting overnight at room temperature) were: .sup.3 H-AMIP (105 
.mu.g/ml), .sup.3 H-AMDMIP (115 .mu.g/ml), and .sup.3 H-MIP (&lt;10 
.mu.g/ml). 
To assess binding, three samples were prepared for each compound; two were 
irradiated (15 minutes at 25.degree. C. with the CE-I device) while one 
was unirradiated as a control. Following irradiation, the samples were 
extracted four times with CHCl.sub.3 then precipitated twice. The final 
pellet was brought up in 1 ml of 1.times.TE buffer and resuspended by 
shaking at 40.degree. C. overnight. 50 .mu.l of each sample was then 
diluted to 0.5 mls with H.sub.2 O. The concentration of DNA was determined 
by UV absortion (A.sub.260) and the amount of covalently bound isopsoralen 
("adduct") was determined by scintillation counting (3.times.100 .mu.l 
aliquots). From these numbers, the following binding ratios (adducts:DNA 
base pair) were determined: 
______________________________________ 
.sup.3 H-Compound 
Ratio 
______________________________________ 
AMIP 1:16.7 
AMDMIP 1:7.2 
MIP 1:44.2 
______________________________________ 
From these ratios it is clear that AMDMIP has the highest binding and MIP 
has the lowest binding. It is not clear, however, that MIP's low binding 
ratio is due to low affinity with DNA. Indeed, from the fact that 
empirically determined concentration of MIP was &lt;10 .mu.g/ml as opposed to 
100 .mu.g/ml for AMDMIP, it would appear that the low ratio is due 
primarily to low solubility of MIP. 
On the other hand, the fact that the concentrations of AMIP and AMDMIP were 
approximately the same would suggest that the difference in ratios 
represents a difference in affinity. 
EXAMPLE 39 
Nucleic Acid Binding 
.sup.3 H-AMIP (0.04 .mu.mol ) or .sup.3 H-AMDMIP (0.035 .mu.mol) were added 
to 5 .mu.g (0.1 nmol) each of HRI 46 and HRI 47 (complementary 115-mers) 
in a total volume of 100 .mu.l of either standardirradiation buffer (0.1M 
NaCl, 10 mM Tris pH 7, and 1 mM EDTA) or 1.times.Taq buffer (50 mM KCl, 50 
mM Tris pH 8.5, 2.5 mM MgCl.sub.2, 200 .mu.g/ml gelatin). To assess 
binding, three samples were prepared for each compound; one was irradiated 
for 15 minutes at 25.degree. C. with the CE-I photoactivation device; one 
was irradiated for 15 minutes at 25.degree. C. with the PTI 
photoactivation device; one was unirradiated as a control. All the samples 
were then extracted four times with 100 .mu.l CHCl.sub.3 brought to 0.2M 
NaCl and 5 mM MgCl.sub.2, and precipitated with 250 .mu.l ethanol at 
-20.degree. C. The pellets were dried, resuspended and reprecipitated. The 
final pellets were brought up in 0.5 ml H.sub.2 O. The concentration of 
DNA was determined by UV absorption (A.sub.260) and the amount of adduct 
was determined by scintillation counting (4.times.100 .mu.l aliquots). 
Binding was calculated using molecular weights (AMIP*HCl MW=251.5; 
AMDMIP*HCl MW=279.5; 115-mer complex MW=74800) and concentrations (30 
.mu.g/ml oligo per 1 OD at 260 nm). From these numbers, the following 
binding ratios (adducts:DNA base pair) were determined: 
______________________________________ 
.sup.3 H-Compound 
Device Buffer Ratio 
______________________________________ 
AMIP CE-I NaCl 1:9.3 
AMIP PTI NaCl 1:14.2 
AMIP CE-I Taq 1:7.6 
AMIP PTI Taq 1:12.5 
AMDMIP CE-I NaCl 1:2.7 
AMDMIP PTI NaCl 1:3.9 
AMDMIP CE-I Taq 1:2.5 
AMDMIP PTI Taq 1:4.2 
______________________________________ 
From these ratios it is clear that AMDMIP again has the highest binding. 
Interestingly, the CE-I device shows better ratios (regardless of the 
compound used) than the PTI device. Most importantly, the use of discrete 
viral sequences of DNA results in better binding than calf thymus genomic 
DNA. 
This latter point is probably due to the A:T rich nature of the sequences 
used. HRI 46 and 47 have almost 60% A:T sequences. Since isopsoralens are 
thought to intercalate preferentially at these sites, an A:T rich nucleic 
acid such as used here should show increased binding with these 
photoreactive compounds. 
This increased binding is due to two factors: (1) using a higher 
isopsoralen concentration relative to nucleic acids and (2) the 
above-mentioned A:T rich nature of the sequences used. It is expected that 
increasing the concentration of photoreactive compound (relative to 
concentration of nucleic acid) will increase binding up to a point. A 
higher relative concentration of AMIP to DNA was used here as compared to 
the relative concentration in Example 38, and a corresponding increase in 
covalent binding was realized (1:9:0.3 versus 1:16.7). 
EXAMPLE 40 
Nucleic Acid Binding 
BIODMIP (100 .mu.g/ml) was irradiated for 30 minutes at room temperature 
with the PTI device in 500 .mu.l of 1.times.TE buffer with 2 .mu.g of 
Hind-III restriction fragments of lambda DNA. After the first irradiation, 
the mixture was transferred to a new tube containing 50 .mu.g additional 
BIODMIP and this was incubated at 37.degree. C. for one hour to dissolve 
the compound. This was followed by a second 30 minute irradiation at room 
temperature with the PTI device. It was observed that, under these 
conditions BIODMIP added to the DNA at a ratio of 1 covalently bound 
BIODMIP per 20 base pairs. 
EXAMPLE 41 
Nucleic Acid Binding 
This example describes photobinding of .sup.3 H-AMIP, .sup.3 H-AMDMIP and 
BIOMIP to RNA. A tRNA stock was prepared at a concentration of 765 
.mu.g/ml in 1.times.TE/10 mM NaCl. Appropriate amounts of each compound 
were prepared in separate reaction vessels such that upon addition of 200 
.mu.l of the tRNA solution (153 .mu.g) the final concentration of each 
compound was 100 .mu.g/ml. .sup.3 H-AMDMIP and .sup.3 H-AMIP were used in 
the experiment: the BIOMIP used was unlabelled. Each reaction vessel was 
then irradiated for 30 minutes at 25.degree. C. in the CE-I11device. 
Identical unirradiated reaction mixtures were used as controls for each 
compound. Following irradiation, each mixture was extracted with 
CHCl.sub.3 (4.times.200 .mu.l) then precipitated (.times.2). The pellets 
were resuspended in 1.times.TE and the binding levels determined as 
follows. For the two labelled compounds (.sup.3 AMDMIP and .sup.3 H-AMIP), 
the nucleic acid concentration was determined optically and radioactivity 
measured by scintillation counting of aliquots of the stock solutions. 
Binding of the compounds was then calculated to be as follows: 
______________________________________ 
Sample Adducts:RNA Bases 
______________________________________ 
AMIP Control 1:51000 
AMIP + hv 1:18 
AMDMIP Control 1:15000 
AMDMIP + hv 1:18 
______________________________________ 
To determine if binding of BIOMIP to tRNA occurred, it was necessary to use 
a nonradioactive format which detected the biotin moiety on the compound. 
A commercial kit ("BluGene"; BRL) was used for this purpose. The kit 
instructions were followed for detection of the both the control DNA 
(supplied with the kit) and BIOMIP treated tRNA. Samples containing 20, 
10, 5, 2 or 0 pg of control DNA and between 1 .mu.g and 10 .mu.g of BIOMIP 
treated tRNA (+/-light) were spotted onto a dry nitrocellulose membrane 
then fixed by baking under vacuum at 80.degree. C. for 2 hours. The blot 
was then developed as specified with the following results. All of the 
control DNA samples gave the expected color patter, with even the 2 pg 
sample visible on the blot. A clear increase in signal was seen for the 
irradiated samples (1 .mu.g&lt;5 .mu.g&lt;10 .mu.g) while no signal was evident 
from the unirradiated controls. This demonstrated that BIOMIP had 
covalently bound to the tRNA. 
EXAMPLE 42 
Template-Dependent Enzimatic Synthesis 
The sequences that are presented in FIG. 26 describe several 
oligonucleotides that are used in the synthesis of either a normal 71-mer 
or the identical 71-mer containing a site-specifically placed psoralen or 
isopsoralen monoadduct. The 71-mer is a sub-sequence of the Human 
Immunodeficiency Virus (HIV) sequence. (An HIV DNA system is described in 
a co-pending application Ser. No. 231,440.) SK-39 is a primer 
oligonucleotides that is complementary to the 3' end of the 71-mers. This 
primer oligonucleotide can be extended on the 71-mer template to make a 
complementary strand of the 71-mer. 
FIG. 27 shows the manner in which the monoadducted template was derived. 
Preparation of 71-mers which contain site-specific monoadducts involves 
(1) preparation of different 15-mer monoadducts from the same unmodified 
15-mer (HRI-42), and (2) ligation of the different 15-mer monoadducts to 
the same 56-mer "extender oligonucleotide" (HRI 102) using a 25-mer 
oligonucleotide (HRI-45) as a splint. The arrows in FIG. 27 indicate the 
direction of synthesis, while the monoadduct is indicated by a short line 
that is perpendicular to the oligomer. While each of the 71-mer 
monoadducts contains the adduct at a base position that is greater than 56 
bases from the 5' end, the precise position of the monoadducts is not 
meant to be indicated. 
To prepare the 15-mer adducts, the 15-mer was incubated with a 
complementary 10-mer along with psoralen or isopsoralen under 
hybridization conditions. The mixture was then irradiated to provide the 
monoadducted 15-mer. While the invention is not dependent on knowing the 
precise mechanism of coupling, it has generally been believed that the 
10-mer directs the isopsoralen to a single TpA site within the 
double-stranded helix formed by the 10-mer/5-mer hybrid. After isolation 
of an HPLC peak believed to contain the 15-mer with a single psoralen or 
isopsoralen monoadduct, these 15-mer monoadducts were ligated to a 56-mer 
extender in order to provide the monoadducted 71-mers for use as 
polymerase templates. The ligation reaction therefore utilized three 
oligonucleotides: the particular psoralen or isopsoralen monoadducted 
15-mer, the 56-mer extender, and the 25-mer splint. The ligation complex 
was hybridized together then ligated. The ligated product was then 
isolated as a single band by denaturing PAGE. 
To provide highly purified 71-mers which contain a single monoadduct, it 
was necessary to provide highly purified 15-mer monoadduct prior to the 
ligation step. This was accomplished by repurification of the HPLC 
purified monoadducted 15-mers by PAGE. In this way, essentially all the 
non-monoadducted 15-mer was removed prior to ligation. Separation of 
15-mer monoadduct from unmodified 15-mer was readily accomplished by PAGE 
(while the same technique is not effective for separation of the 
corresponding unmodified 71-mer and monoadducted 71-mer sequences). In 
this manner, exceedingly pure monoadducted 71-mers were produced for the 
primer extension reactions: monoadducted 71-mers (as well as unmodified 
71-mers) are used in template-dependent enzymatic extension experiments in 
examples 43-47 below. 
EXAMPLE 43 
Template-Dependent Enzimatic Synthesis 
In this experiment, monoadducted 71-mers (as well as unmodified 71-mers) 
were used in template-dependent enzymatic extension. AMIP, AMDMIP, and MIP 
71-mer monoadducts were made as in Example 42. FIG. 28 shows the manner in 
which extension is achieved. Note that the 3' end of the 71-mer (HRI 55) 
is complementary to the primer (SK-39) (see also FIG. 26). Each of the 
extension experiments was run at 37.degree. C. for 0, 5 or 15 minutes. 
Each of the reactions was initiated by providing the templates and 
deoxyribonucleoside 5'-triphosphates (dATP, dGTP, dCTP, and dTTP are 
collectively abbreviated as dNTPs), and by adding the particular 
polymerase last to start the reaction. For detection, the primer extension 
reaction utilized 5.sup.'32 -P-labelled primer. The reactions were stopped 
by adding EDTA. Analysis was by denaturing PAGE followed by 
autoradiography. 
Reaction conditions for each of the different polymerases were as follows: 
1. E. coli DNA Polymerase: 50 mM Tris Buffer (pH 7.5); 10 mM MgCl.sub.2 ;1 
mM DTT; 50 .mu.g/ml bovine serum albumin (BSA); 100 .mu.M dNTPs; 3 units 
of polymerase (for 25 .mu.l volume); 1.times.10.sup.-8 M primer; 
1.times.10.sup.-9 M 71-mer; 
2. Klenow Polymerase: (same as E. coli except add 3 units of Klenow instead 
of E. coli polymerase); 
3. T4 Polymerase: 50 mM Tris Buffer (pH 8.0); 5 mM MgCl.sub.2 ; 5 mM DTT; 
50 mM KCl; 50 .mu.g/ml BSA; 5 units T4 polymerase; 
4. Reverse Transcriptase: 50 mM Tris Buffer (pH 8.0); 5 mM MgCl.sub.2 ; 5 
Reverse Transcriptase. 
The results with MIP, AMIP, and AMDMIP are shown in FIGS. 29, 30 and 31 
respectively. Both MIP and AMIP adducts appear to be complete stops for T4 
polymerase and reverse transcriptase, but not complete stops for E. coli 
DNA polymerase and Klenow. AMDMIP adducts appear to be a complete stop for 
all of the polymerases tested. 
EXAMPLE 44 
Template-Dependent Enzymatic Synthesis 
In this experiment, monoadducted 71-mers (as well as unmodified 71-mers) 
were used in template-dependent enzymatic extension. AMIP, AMDMIP, and MIP 
71-mer monoadducts were made as in Example 42. Extension was carried out 
as in Example 43 except that in this experiment, Taq I DNA polymerase, a 
thermostable DNA polymerase isolated from Thermus aquaticus (Stratagene, 
Inc., La Jolla, Calif.) was assessed for its ability to read past 
isopsoralens. 
The reaction mixture was in 80 .mu.l total volume and comprised 1.times.Taq 
buffer, 200 .mu.M each of dNTPs, 0.05 units of Taq per .mu.l of reaction 
volume; 1.times.10.sup.-9 M in 71-mer MA; 1.times.10.sup.-8 M in .sup.32 
P-labelled SK-39. Each reaction was set up with everything except Taq 
polymerase. The samples were initially heated to 95.degree. C. for 5 
minutes, then incubated at 55.degree. C. for 3 minutes. The extension 
reaction was initiated by addition of Taq polymerase. At the indicated 
time points (0.5, 1.0, 5.0 minutes), 20 .mu.l of the reaction mix was 
removed to a tube containing 1 .mu.l of 0.5M EDTA to stop the reaction. 
All products were analyzed on a 20% polyacrylamide, 7M urea gel followed 
by autoradiography. 
The results are shown in FIG. 32. The lanes with the control template (HRI 
55) show the position of the full length 71-mer extension product. Each 
time point was measured in duplicate. The extension reaction appears to be 
complete at the earliest time point (30 sec). The lanes with the targets 
containing the isopsoralens (MIP, AMIP, and AMDMIP) indicate stops at 
positions that are shorter than the full length 71-mer product. 
Unexpectedly, each adduct results in a stop at a different position within 
the sequence of the 71-mer. The MIP stop appears to be at about the 
position of the TpA sequence in the 10-mer that was used to create the 
monoadduct. AMIP has multiple stops. AMDMIP has a different stop 
altogether. The longest extension product with AMIP and the AMDMIP stop 
indicate that the isopsoralens are probably located on the initial 15-mer 
outside the region of the 10-mer/15-mer interaction. This shows that the 
isopsoralens don't necessarily follow the rules reported for psoralen 
derivatives (i.e., that an intercalation site is required, and further 
that a TpA site is preferred). 
EXAMPLE 45 
Template-Dependent Enzymatic Synthesis 
This experiment investigated whether blocking of Taq polymerase by 
monoadducts is complete or whether monoadducts can be bypassed by the 
enzyme. In order to view the process of bypass synthesis, it is necessary 
to cycle the extension reaction of the primer with the 71-mer templates. 
Cycling in this case consists of mixing the primer with the 71-mer 
template, adding Taq polymerase and appropriate reagents, extending 
heating to induce strand separation, reannealing of the primer to the 
71-mer template, extending again, and subsequent strand separation. This 
process can be repeated as necessary. During this process, only the 
complement of the 71-mer template is being synthesized. It is accumulating 
in a linear fashion with the number of thermal cycles (in contrast to PCR 
where both strands are being synthesized and accumulate geometrically). 
The templates and reaction conditions were as in Example 44 above except 
that three samples were used for each monoadduct and each sample was 
tested under different conditions: 
Samples (1): one sample for each monoadduct was used to carry out extension 
at 55.degree. C. as in Example 43; 
Samples (2): one sample for each monoadduct was used to carry out extension 
by the following series of steps: denaturing at 95.degree. C.; incubating 
for 30 seconds at 55.degree. C.; adding Taq polymerase for 3 minutes at 
55.degree. C.; cooling the reaction for 1 minute at 7.degree. C.; stopping 
the reaction with EDTA for one minute at 95.degree. C.; 
Samples (3): one sample for each monoadduct was used to carry out extension 
by repeating the following series of steps nine times: denaturing at 
95.degree. C.; incubating for 30 seconds at 55.degree. C.; adding Taq 
polymerase for 3 minutes at 55.degree. C.; cooling the reaction for 1 
minute at 7.degree. C. At the end, the reaction was stopped with EDTA for 
one minute at 95.degree. C. 
The results are shown in FIG. 33. At either 55.degree. C. or one cycle of 
extension, there appears to be no read through (i.e., Taq polymerase is 
completely blocked). After nine cycles there is evidence of full length 
extension product. It is not clear, however, if these results indicated 
actual read through or just extension or nonmonoadducted 71-mer 
contaminant. 
EXAMPLE 46 
Template-Dependent Enzymatic Synthesis 
It was investigated whether blocking of Taq polymerase is complete or 
whether blocking can be overcome. While the results described in Example 
45 suggested bypass occurred (since full length extension products were 
produced after 10 cycles using isopsoralen monoadducted 71-mer template), 
it was not clear if the results were due to actual bypass of the 
monoadduct or to extension of non-monoadducted 71-mer that was present as 
a contaminant. Non-monoadducted 71-mer could have been present due to 
incomplete separation of 15-mer monoadduct from unmodified 15-mer prior to 
ligation during the preparation of the 71-mer (see Example 42). 
To investigate this question further, a new 5MIP monoadducted 71-mer by 
prepared by repurification of the HPLC purified 5-MIP monoadducted 15-mer 
by PAGE. In this way, more of the non-monoadducted 15-mer was removed 
prior to ligation. The purified 71-mer was then used as template in an 
extension experiment as described in Example 45. After 10 cycles, there 
was still evidence of extension product (FIG. 34). Excision of the band 
and counting found this full length extension product constituted 2.3% of 
the total extension products (i.e. 97.7% of the extension products were 
truncated). 
EXAMPLE 47 
Template-Dependent Enzimatic Synthesis 
From Examples 43, 44 and 45, it is clear that some isopsoralens may be used 
for polymerase blocking. It is important to note that isopsoralens form 
monoadducts with double stranded nucleic acid but do not form crosslinks 
because of their angular structure. Because of this, these 
isopsoralen-modified single strands remain detectable by hybridization 
procedures. On the other hand, it may be useful under some circumstances 
to block replication with psoralens. This experiment examines the ability 
of HMT to block Taq polymerase on an HIV template. 
Monoadducted 71-mer was constructed as described, but for HMT. Extension 
was carried out and analyzed as before on PAGE. The results for Taq 
polymerase are shown in FIG. 35. Clearly, HMT monoadducts stop Taq 
polymerase. Full length 71-mer is not made and a shorter strand 
corresponding to the position of the HMT adduct was made. 
EXAMPLE 48 
Template-Dependent Enzimatic Synthesis 
In this experiment, the ability of AMIP, AMDMIP and MIP to block 
replication of 71-mer is investigated by randomly adding each compound to 
the 71-mer. Of course, addition may only be random in the sense that one 
or more adducts may be formed with any one strand of nucleic acid. The 
actual placement of the isopsoralen may be governed by preferential 
binding at particular sites (e.g., A:T). In addition to blockage by random 
adducts, photoproducts of the various isopsoralens may be providing an 
inhibitory effect. No attempt to separate the effects of photoproduct from 
the effects of covalent adducts on the 71-mer template was made in this 
series of experiments. 
In these experiments, the 71-mer template (10.sup.-9 M) was primed with 
.sup.32 P-SK-39 (10.sup.-8 M) and extended with Taq polymerase in a total 
volume of 20 .mu.l. The dNTP concentration was 200 .mu.M. In the control 
samples, the 71-mer, the primer, and the dNTPs were mixed in Taq buffer in 
a volume of 18 .mu.l. These samples were initially denatured at 95.degree. 
C. for 5 minutes, followed by equilibration at 55.degree. C. for 3 
minutes. Taq enzyme was then added (0.5 units) and the extension reaction 
was carried out for 5 minutes at 55.degree. C. The reaction was stopped by 
making the solution 10 mM in EDTA. In another set of samples, the 71-mer 
in 10 .mu.l was mixed with either AMIP (200 .mu.g/ml), AMDMIP (200 
.mu.g/ml), or MIP (60 .mu.g/ml). Half of these samples were irradiated for 
15 minutes on the CE-I device at 25.degree. C. The other half were kept in 
the dark as controls. These samples were then mixed with the primer and 
dNTPs as before, and were subjected to the same thermal profile and 
extension reactions as the samples were described earlier. The results 
were examined by PAGE. The extended product bands were identified by 
autoradiography, excised and counted (data not shown). 
The unirradiated controls that contained the isopsoralens resulted in full 
length extension products; no truncated products were observed. 
Quantitation of these extension products showed they were equivalent to 
the amount of extension products seen in the samples that did not contain 
isopsoralens, which corresponded to about 8% of the primer being extended. 
The irradiated samples that contained either AMIP or AMDMIP were not 
extended at all. The irradiated sample that contained MIP resulted in some 
full length extension product and minor truncated product. The amount of 
full length product with irradiated MIP was half of that observed with the 
control samples. 
EXAMPLE 49 
Template-Dependent Enzimatic Synthesis 
In this experiment, the ability of two different Phenylazide derivatives 
(see Table 1), photobiotin (Vector Labs) and monoazide ethidium chloride, 
to block replication of 71-mer was investigated by randomly adding each 
compound to the 71-mer. Again, addition may only be random in the sense 
that one or more adducts may be formed with any one stand of nucleic acid. 
The actual placement of these compounds may be governed by preferential 
binding at particular sites (e.g., A:T). In addition to blockage by random 
adducts, there may be inhibition by photoproducts. As in Example 48, no 
attempt was made to separate the impact of photoproducts from that of 
covalent binding of photoreactive compound on the 71-mer. 
The two compounds, photobiotin and monoazide ethidium chloride, have 
different spectral characteristics. To activate these compounds, two 
different wavelength regions were selected using a single light source 
(General Electric Sunlamp, Model RSM, 275 watt). The light source was 
positioned 10 cm above uncapped Eppendorf tubes which contained samples to 
be irradiated. The samples were kept on ice during irradiation. A pyrex 
dish was placed between the lamp and the samples. 
For samples containing photobiotin, 2.5 cm of water was added to the pyrex 
dish to help remove some of the infrared radiation. The samples were 
irradiated for 15 minutes. 
For samples containing monoazide ethidium chloride, wavelengths less than 
400 nm were filtered out by using 2.5 cm of an aqueous solution of 2.9M 
NaNO.sub.2. Removal of short wavelengths (i.e., wavelengths shorter than 
400 nm) is necessary for the use of monoazide ethidium chloride. 
Irradiation of this compound with shorter wavelengths results in 
conversion to non-active forms (data not shown). Wavelengths below 400 nm 
are therefore undesirable for use with this compound. 
In this experiment, the 71-mer template (2.times.10.sup.-9 M) in 10 .mu.l 
was mixed with either no photoreactive compound, photobiotin 
(6.times.10.sup.-6 M), or monoazide ethidium (1.4.times.10.sup.-3 M). Half 
of each of these samples were irradiated on ice for 15 minutes with 
wavelengths appropriate for each specific photoreactive compound. The 
other half of the samples were kept in the dark as controls. The samples 
containing no photoreactive compound were exposed with the water filter in 
place. .sup.32 P-SK-39 primer (1.times.10.sup.-8 M), dNTPs (200 .mu.M), 
and additional buffer were added to yield a volume of 18 .mu.l. The 
samples were then denatured at 95.degree. C. for 5 minutes, and then 
equilibrated at 55.degree. C. for 3 minutes. Taq polymerase was then added 
and extension was allowed to proceed for 5 minutes at 55.degree. C. The 
reactions were stopped by bringing the samples 10 mM in EDTA. The products 
were examined by PAGE (data not shown). The controls containing no 
photoreactive compound, no photoreactive compound plus light, and 
photobiotin (Dark control) all showed similar amounts of fill length 
extension product. No truncated products were observed with these samples. 
The dark control with monoazide ethidium chloride resulted in inhibition 
of extension. Photobiotin, by contrast, showed inhibition only after 
irradiation. 
EXAMPLE 50 
Template-Dependent Enzymatic Synthesis 
This example demonstrates that AMDMIP photoproduct inhibits primer 
extension. This example also demonstrates that the inhibitory effect of 
photoproduct is not sufficient to account for all the inhibition seen when 
a 71-mer target is irradiated in the presence of AMDMIP and subsequently 
extended. 
71-mer was made up in Taq buffer. Samples were prepared as follows: (1) 
dark control samples were not subjected to activating light; (2) 10 .mu.l 
samples of the 71-mer (2.times.10.sup.-9 M) were irradiated in the 
presence of 200 .mu.g/ml AMDMIP, and (3) 10 .mu.l samples of a 200 
.mu.g/ml solution of AMDMIP were irradiated in the absence of the 71-mer 
target. All irradiations were carried out with the CE-I device at 
25.degree. C. After preparing the above samples, unirradiated AMDMIP was 
added to one set of the dark control samples. Another set of dark controls 
received irradiated AMDMIP. .sup.32 P-SK39 primer and dNTPs were then 
added to all samples and the volume was adjusted to 18 .mu.l. These 
samples were denatured at 95.degree. C. for 5 minutes, followed by 
equilibration at 55.degree. C. for 3 minutes. Taq polymerase was then 
added and the extension reaction was carried out for 5 minutes. The 
reaction was stopped by bringing the samples to 10 mM EDTA. The final 
concentrations of all reagents during the extension reactions were: 
______________________________________ 
Buffer 1 .times. Taq 
Taq Polymerase 0.5 units 
dNTPs 200 .mu.M 
.sup.32 P-SK-39 Primer 
1 .times. 10.sup.-8 M 
71-mer 1 .times. 10.sup.-9 M 
AMDMIP 100 .mu.g/ml 
______________________________________ 
The results were analyzed by PAGE (data not shown). A visual inspection of 
the autoradiograph showed that the dark controls yielded full length 
extension product. 71-mer that was irradiated in the presence of AMDMIP 
resulted in no extension products at all. The sample that contained AMDMIP 
photoproduct and unirradiated 71-mer resulted in full length extension 
product, but at about 10% of the level seen in the dark control samples. 
The observation that some extension product was made in the presence of 
photoproduct but not with directly irradiated 71-mer indicates that the 
effects of photoproduct and covalent addition of AMDMIP to a template 
oligonucleotide may be synergistic. 
EXAMPLE 51 
Post-Amplification Sterilization 
FIG. 36 describes a series of oligonucleotides that can be used with the 
PCR amplification technique. Two primers are described (SK-38 and SK-39) 
that are complementary to a segment of the HIV genome. Each primer is 
complementary to sequences at the 5' ends of one of each of two strands of 
a 115 base pair long segment of the HIV genome. In addition, FIG. 36 
describes a crosslinkable probe molecule which is capable of hybridizing 
and crosslinking to one strand of the 115-mer PCR product. Repeated 
thermal cycling of SK-38 and SK-39 in the presence of polymerase, 
appropriate reagents, and a target polynucleotide containing at least the 
115-mer segment bounded by SK-38/SK-39, will result in the synthesis of 
both strands of the 115-mer. Therefore, PCR amplification will occur, with 
both strands of the 115-mer accumulating geometrically. This is in 
contrast to the oligonucleotides described in FIG. 26. Only one primer 
(SK-39) is described in FIG. 26 which is capable of hybridizing to the 3' 
end of the 71-mer target oligonucleotide (HRI-55). The lack of a second 
primer in the system of oligonucleotides described by FIG. 26 prevents 
this system from being amplified geometrically. Repeated thermal cycling 
of HRI-55 and SK-39 in the presence of Taq polymerase and appropriate 
reagents will result in the linear accumulation of the complement of 
HRI-55. 
The system described in FIG. 36 for the HIV DNA system was used for PCR 
sterilization. In FIG. 36, the arrows indicate the polymerase extension 
direction for the primers. The HMT monoadduct on SK-19-MA is shown by 
(.rect-ver-solid.). A block denotes a natural, conserved 5-TpA-3' 
crosslinking site in the DNA sequence of HIV. 
PCR amplification requires numerous cycles of denaturation replication. 
Because denaturation is most conveniently accomplished by heat, the 
polymerase is ideally thermostable. See K. B. Mullis et al., U.S. Pat. 
Nos. 4,683,195 and 4,683,195 (incorporated by reference). Taq I DNA 
polymerase, a thermostable DNA polymerase isolated from Thermus aquaticus 
(Cetus Corp., Emeryville, Calif.) was used for all amplifications. 
Unless otherwise noted, the PCR amplification procedure follows the broad 
temporal steps of FIG. 4: (1) template preparation, (2) amplification 
cycling, and (3) detection. 
AMDMIP photoproduct was made by irradiating AMDMIP (using the CE-III 
device) in 1.times.Taq buffer (50 mM KCI, 2.5 mM MgCl.sub.2, 10 mM Tris, 
pH 8.5, 200 .mu.g/ml gelatin) in a separate vessel for 15 minutes at room 
temperature (RT). Aliquots of AMDMIP photoproduct and the unirradiated 
compound were added (by pipetting) at 50, 100, 200 and 300 .mu.g/ml to 1 
.mu.l aliquots of a 10.sup.6 dilution of PCR product copies. PCR product 
was provided by (a) preparing template, (b) providing template, (c) 
providing PCR reagents, (d) providing polymerase, (3) mixing PCR reagents, 
template, and polymerase to initiate PCR, (f) cycling to synthesize PCR 
product. 
Step (a): Template Preparation. The templates were derived from actual 
patient samples. Ficoll-Hypaque separated peripheral blood mononuclear 
cells (PBMCs) are prepared from individuals enrolled in a longitudinal 
AIDS study. Approximately 1.times.10.sup.6 cells are added to 10 ml of 
RPMI containing 10% fetal calf serum. The cells are pelleted by 
centrifugation at 200.times.g for 5 minutes and washed twice with 10 ml 
PBS. The cell pellet is resuspended in a solution of 50 mM KCl, 10 mM TRis 
HCl (pH 8.3), 2.5 mM MgCl.sub.2 and subsequently lysed by the addition of 
0.5% Tween 20 and 0.5% NP40. Samples are digested with 60 .mu.g/ml 
proteinase K (Sigma) for 1 hour at 60.degree. C. Inactivation of the 
proteinase K is achieved by heating the sample at 95.degree. for 10 
minutes. 
Step (b): Providing Template. For the reaction, 10 .mu.l of template 
(equivalent to approximately 3.times.10.sup.4 cells) is placed in a 
reaction vessel (0.5 ml Eppendorf tube) for later amplification. 
Steps (c), (d), (e) and (f). PCR reagents were provided, mixed to a final 
volume of 20 .mu.l and cycled as described above. 
To evaluate the efficacy of photoproduct as a sterilization reagent, a 
subsequent polymerase chain reaction was carried out for 30 cycles in the 
presence of .alpha..sup.-32 P-dCTP. PCR reaction products were then 
visually examined by running them on a 12% acrylamide/8M urea gel followed 
by autoradiography. The results are shown in FIG. 37. The irradiated 
compound (i.e., photoproduct) shows complete sterilization (i.e., no PCR 
product is evident) at concentrations above 50 .mu.g/ml (Lanes 4, 6 and 
8); photoproduct shows partial sterilization (i.e., some PCR product is 
evident) at 50 .mu.g/ml (Lane 2). The unirradiated compound (i.e., the 
control) shows no sterilization (Lanes 1, 3, 5 and 7). 
With the concentration spectrum for photoproduct sterilization of PCR 
broadly defined, an additional experiment was performed to more 
specifically pinpoint the cutoff for photoproduct sterilization of PCR. 
Again, AMDMIP photoproduct was made by separately irradiating AMDMIP (with 
the CE-III device) in 1.times.Taq buffer for 15 minutes at RT. This time, 
however, aliquots of AMDMIP photoproduct were added to PCR product to give 
a concentration range of between 0.25 and 50 .mu.g/ml. 
Again, to evaluate the efficacy of this method of sterilization, a 
subsequent polymerase chain reaction was carried out for 30 cycles in the 
presence of .alpha..sup.-32 P-dCTP. PCR product was then quantitated by 
running gels (see above), cutting the bands (detected by autoradiography) 
and counting the bands on a commercial scintillation counter. The results 
are plotted in FIG. 38. 
FIG. 38 shows that as little as 5 .mu.g/ml of photoproduct can result in as 
much as a 50% reduction in the amount of PCR product (as measured by DPM). 
On the other hand, very little reduction in PCR product is seen at 0.25 
.mu.g/ml; with concentrations of photoproduct below 0.25 .mu.g/ml, 
photoproduct sterilization of PCR is insignificant. 
EXAMPLE 52 
Post-Amplification Sterilization 
It may be desired that photoproduct sterilization of PCR be avoided. Other 
methods of sterilization--methods that are preferred over photoproduct 
sterilization--may be employed without interference of photoproduct 
sterilization by either (1) working with photoreactive compound 
concentrations that are below that where photoproduct sterilization can 
occur, and (2) by selecting conditions where less photoproduct is 
generated. 
In this experiment, conditions were selected where less photoproduct was 
generated. These conditions involve irradiation of AMDMIP with the PTI 
light source. More intact AMDMIP remains after irradiation with the PTI 
light source (in contrast with irradiation of ADMIP with the CE-III 
device; see FIG. 23). 
AMDMIP in a 1.times.Taq buffer was irradiated in the PTI source for 15 
minutes at RT. As a control, AMDMIP in 1.times.Taq buffer was irradiated 
with the CE-III device for 15 minutes at RT. In both cases, photoproduct 
was added to 1 .mu.l aliquots of a 10.sup.6 dilution of a PCR product 
mixture to give a final concentration of 100 .mu.l ml photoproduct. As a 
control, unirradiated AMDMIP was added to similar PCR product mixtures at 
the same concentration as the photoproduct. PCR product was provided as 
described earlier. 
To evaluate the efficacy of this sterilization method, a new PCR reaction 
was carried out for 30 cycles in the presence of .alpha..sup.-32 P-dCTP. 
PCR product was examined on gels as in Example 51 (FIG. 37 ) and the gels 
were subjected to autoradiography. The results are shown in FIG. 39. 
In both cases where unirradiated AMDMIP was used (FIG. 39, Lanes 1 and 3) 
PCR product is clearly evident. Where AMDMIP irradiated in the CE-III 
device is used, extensive sterilization is observed. By contrast, where 
AMDMIP irradiated in the PTI device is used, little sterilization is 
observed. AMDMIP irradiated with the PTI light source shows the same 
results as unirradiated ANDMIP (control), suggesting that photoproduct is 
at a concentration below which its effects are seen by the PCR assay. 
The dramatic difference in results between the two light sources may be due 
to the fact that the CE-III source has a shorter wavelength (300 nm 
cutoff) relative to the PTI source (320 nm cutoff). The absorption 
spectrum of AMDMIP suggests it would be more reactive with the broader 
light window provided by the CE-III source. 
EXAMPLE 53 
Post-Amplification Sterilization 
To systematically evaluate the effect of the presence of increasing 
concentrations of isopsoralen on PCR, AMDMIP (0, 100, 200, 400 .mu.g/ml) 
were added to 10.sup.7 copies of HIV 115-mer as template. PCR was then 
carried out for 30 cycles in the presence .alpha..sup.-32 P-dCTP. The PCR 
product was run on denaturing polyacrylamide gel and autoradiographed. The 
bands were thereafter cut and counted. The results were as follows: 
______________________________________ 
AMDMIP! Counts (CPM) 
______________________________________ 
0 42200 
100 77200 
200 77500 
400 69400 
______________________________________ 
The results show that unirradiated isopsoralen does not cause PCR 
sterilization. Indeed, with this particular isopsoralen, AMDMIP, there is 
enhancement of PCR product. Importantly, even high concentrations (400 
.mu.g/ml) of AMDMIP show no appreciable impact on amplification. 
EXAMPLE 54 
Post-Amplification Sterilization 
In this embodiment, the method of sterilizing comprises: (1) providing PCR 
reagents, (2) providing template. (3) providing polymerase, (4) mixing PCR 
reagents, template, and polymerase to initiate PCR, (5) cycling to provide 
PCR product, (6) providing isopsoralen, (7) adding isopsoralen to PCR 
product, and (8) irradiating PCR product. 
Note that while the isopsoralen could be introduced to the mixture at any 
time prior to irradiation (e.g., at the time the template is added in 
order to avoid opening the reaction vessel again prior to irradiating), in 
this embodiment, the isopsoralen is added after amplification. 
To demonstrate the effectiveness of the method of the present invention, 
the sterilized carryover must be shown to be unamplifiable. For the 
purposes of this determination, the steps of the following experiment 
include (see FIG. 40): (a) preparing template,(b) providing template, (c) 
providing PCR reagents, (d) providing polymerase, (e) remixing PCR 
reagents, template, and polymerase to initiate PCR, (f) cycling to provide 
PCR product, (g) carrying over of PCR product into new tubes, (h) 
providing isopsoralen, (i) adding isopsoralen to the carryover, (j) 
irradiating, (k) addition of new PCR reagents, and (1) subsequent PCR. 
Steps (a), (b), (c), (d), (e), and (f). PCR product was provided as 
described earlier. 
Step (g): Carrying over of PCR product. Aliquots of PCR product were added 
into new reaction tubes at 10.sup.6 copies per tube. 
Step (h): Providing Isopsoralen. AMDMIP was synthesized as described and 
diluted. 
Step (i): Addition of Isopsoralen. AMDMIP (100 .mu.g/ml, approximately 
10.sup.-4 M) was added to reaction vessels containing carryover. 
Step (j): Irradiation. Irradiation was performed on the PTI device to avoid 
photoproduct. Three of the new reaction tubes were irradiated while the 
others were left unirradiated as controls. Irradiation was for 15 minutes 
at RT as above. 
Step (k): PCR Reagents and Taq Polymerase Were Added at Appropriate 
Concentrations. The final volume was increase two fold such that AMDMIP 
photoproducts were at 50 .mu.g/ml. 
Step (1): Subsequent PCR. Amplification was performed in closed reaction 
vessels using primer pair SK-38/SK-39 for 20, 25 or 30 cycles, using the 
temperature profile for cycling described above in the presence of 
.alpha..sup.-32 P-dCTP. 
The results were evaluated by gel electrophoresis and autoradiography (FIG. 
41). To the right of the gel lanes, the bands corresponding to starting 
material and product are indicated. As expected, the control reactions 
that have no carry-over (Lanes 1, 5, and 9) show no amplified product. On 
the other hand, the control reactions that contain carryover produced in 
the first amplification without AMDMIP (Lanes 2, 6, and 10) show 
amplification. The control reactions that contain carryover produced in 
the first amplification with AMDMIP, but that were not light-treated 
(Lanes, 3, 7, and 11), also show amplification. Importantly, the reactions 
that received carryover, AMDMIP and irradiation (Lanes 4, 8 and 12) show 
isopsoralen sterilization of PCR to a degree that is cycle dependent. With 
twenty cycles, sterilization appears to be complete (i.e., the 
twenty-cycle amplification does not provide detectable product). With 
twenty-five cycles, PCR product is visible (Lane 8), albeit reduced 
relative to controls (Lanes 6 and 7). Finally, with thirty cycles, no 
significant sterilization is observed; PCR product (Lane 12) is 
approximately the same relative to controls (Lanes 10 and 11). 
On the basis of visual examination of the bands, AMDMIP, when 
photoactivated in the presence of carryover, appears to be very effective 
at 20 cycles. However, at thirty cycles, sterilization appears to be 
overwhelmed. This illustrates the interplay of amplification factor and 
sterilization sensitivity. 
FIG. 41 can be interpreted in terms of Table 6. At 20 cycles of PCR, 
sterilization appeared to be completely effective (Lane 4 compared to Lane 
3). If 100 CPM is taken to be the threshold for seeing a band on the 
autoradiograph, then Table 6 shows that the sterilization protocol of this 
example with AMDMIP left less than 10.sup.4 target molecules that were 
capable of being replicated by the PCR procedure. At 25 cycles of PCR, a 
very measurable band was observed (Lane 8), suggesting that at least 
10.sup.3 target molecules retained replicating capabilities. At 30 cycles 
of PCR it is difficult to distinguish the control signal from the signal 
obtained with the sterilized sample (Lane 11 compared with Lane 12). This 
is consistent with both the control sample and the AMDMIP treated sample 
reaching the plateau region of the PCR amplification process. 
EXAMPLE 55 
Post-Amplification Sterilization 
To demonstrate the effectiveness of the method of the present invention, 
the sterilized carryover is again shown to be unamplifiable. In this 
example, however, isopsoralen is introduced prior to amplification. 
For the purposes of this determination, the steps of the following 
experiment include (see FIG. 4): (a) preparing template, (b) providing 
template, (c) providing isopsoralen, (d) providing PCR reagents, (e) 
providing polymerase, (f) mixing isopsoralen, PCR reagents, template, and 
polymerase to initiate PCR, (g) cycling to provide PCR product, (h) 
irradiating, (i) carrying over of PCR product into new tubes at specific 
copy numbers, and (j ) amplifying in a subsequent PCR. 
Step (a): Template Preparation. HIV 115-mer was used as template. 
Step (b): Providing Template. For the reaction, template (equivalent to 
approximately 10.sup.7 copies) in buffer was provided for the reaction 
vessel. 
Step (c): Providing Isopsoralen. AMDMIP (400 .mu.g/ml) was provided as the 
isopsoralen. This is a higher concentration of AMDMIP than used in the 
previous example. 
Steps (d), (e), (f) and (g). PCR reagents and polymerase were provided, 
mixed and cycled as described above. This time, however, isopsoralen is 
part of the preamplification mixture. 
Step (h): Irradiation. Irradiation was performed on the CE-III device for 
15 minutes at 25.degree. C. 
Step (i): Carrying Over of PCR Product. Aliquots of PCR product were added 
into six new reaction tubes--two tubes for each dilution. Dilutions to 
yield 10.sup.7, 10.sup.5 and 10.sup.3 copies were made. (Note that the 
dilutions were made from a concentration of approximately 10.sup.101 
copies/.mu.l; thus, the dilutions produce a concentration of photoproduct 
that is far too low to consider photoproduct sterilization.) 
Step (j): Subsequent PCR. New PCR reagents and polymerase were provided and 
mixed. Amplification was performed in closed reaction vessels using primer 
pair 
SK-38/SK-39 for 30 cycles, using the temperature profile for cycling 
described above in the presence of .alpha..sup.-32 P-dCTP. 
The results were evaluated by gel electrophoresis and autoradiography (FIG. 
43). The control reactions that contained carryover produced in the first 
amplification with AMDMIP, but that were not light-treated (Lanes 1, 3, 
and 5) show amplification. Importantly, the reactions that received 
carryover from the first PCR after irradiation in the presence of AMDMIP 
(Lanes 2, 4, and 6) show complete, post-amplification sterilization. 
EXAMPLE 56 
Photobiotin and monoazide ethidium chloride were previously tested for 
their ability to block template-dependent enzymatic synthesis (see Example 
49). The effect of photoprodut (if any) was not investigated at that time. 
In this experiment, photobiotin and monoazide ethidium chloride were tested 
as PCR sterilization reagents. The temporal steps were performed to 
examine photoproduct effects (if any). Solutions of photobiotin and 
monoazide ethidium chloride were made up in 1.times.Taq buffer. 
Concentrations of photobiotin ranged from 7.times.10.sup.-4 M to 
7.times.10.sup.-10 M; concentrations of the monoazide ethidium chloride 
ranged from 3.times.10.sup.-6 M to 3.times.10.sup.-10 M. The high-end of 
these concentration series was based on earlier experiments that showed 
that higher concentrations of these compounds shut down PCR by dark 
binding. Each compound solution was divided into two parts: One part was 
irradiated under a GE sunlamp through a pyrex filter (300 nm cutoff); the 
other half was irradiated under a GE sunlamp through a 2.9M NaNO.sub.2 
liquid filter (400 nm cutoff). Irradiations were carried out on ice for 15 
minutes. After irradiation, aliquots of each tube were carried over into 
tubes containing PCR reagents and target (HIV 115-mer); PCR was then 
carried out for 30 cycles in the presence of .alpha..sup.-32 P-dCTP. After 
amplification aliquots were analyzed on 12% acrylamide/8M urea gels. 
The results obtained show that monoazide ethidium chloride, when tested in 
this mode, does not inhibit PCR; 115-mer amplified at the high 
concentration point. By contrast, when used in this mode, photobiotin shut 
down PCR at the highest concentration used (7.times.10.sup.-4 M) (115-mer 
amplified at all lower concentrations). 
Given the results, it is believed that blocking of primer extension seen 
earlier (Example 49) with the monoazide ethidium chloride was probably due 
to photobinding and not photoprodut binding. The results seen here with 
photobiotin, however, suggest that the previous blocking was probably due 
to photobiotin photoproduct. 
EXAMPLE 57 
Post-Amplification Sterilization 
A PCR sample is prepared for amplification with the following changes. 
Instead of AMDMIP, AMT is added prior to amplification at a concentration 
of 100 .mu.g/ml. Instead of primer pair SK-38/SK-39, the biotinylated 
analogs, in which biotin has been appended to the 5' end of one or both 
primers via an intervening tetraethyleglycol bridge (ester linkage to the 
biotin), are used. Following 30 cycles of PCR, the reaction vessel is 
exposed to 300-400 nm light on the CE-III device. Following irradiation, 
the PCR reaction vessel is opened and the PCR product removed. Free primer 
is then removed by spinning the PCR reaction mix through a Centricon 100 
(Amicon Division, W R Grace & Co., Danvers, Mass.). The Centricon filters 
consist of a semipermeable membrane which permits the passage of short 
oligonucleotides, but not long oligonucleotides. PCR product is 
differentially retained in the retentate. Several washes are required 
(these membranes are conveniently mounted in a disposable plastic tube 
that is spun in a centrifuge for 5 minutes at 2000.times.g). After the 
final wash, the retentate is immobilized on a nylon membrane or a 
nitrocellulose membrane by filtration. The filter is then baked under 
vacuum for 2 hours at 80.degree. C. After immobilization, the PCR product 
is detected with a commercially available biotin detection system 
(Blu-Gene Detection System; Catalog #8179 SA; BRL). 
Alternatively, detection may be realized by the incorporation of 
.alpha..sup.-32 P-deoxyribonucleoside triphosphates during the PCR 
amplification step. The steps here are the same as the first method except 
for the detection step. Instead of immobilization following the 
irradiation step, a portion of the sterilized sample is loaded on an 8M 
urea (denaturing) 11% polyacrylamide gel and electrophoresed for 2-3 hours 
at 50 watts (25 mA/2000 V) until the marker dye bromphenol blue just runs 
off the gel. The crosslinked double stranded PCR product is then 
visualized by autoradiography (XAR-5 X-ray film; Kodak); a typical 
exposure time is 12-16 hours. 
To show that AMT treated product is sterilized, aliquots of AMT treated PCR 
reaction mix containing 10.sup.4 to 10.sup.10 copies of PCR reagents are 
added and the samples reamplified for 30 cycles, and .alpha..sup.-32 
P-dCTP is present during the amplification. Following PCR, the reamplified 
samples are analyzed by denaturing PAGE as described above. In all cases, 
the crosslinked (AMT treated) PCR product does not reamplify. 
In a third method, a solution of AMT (100 .mu.g/ml) is prepared and 
irradiated for 15 minutes on the CE-III device. This solution is then 
added to a PCR reaction tube which contains target DNA for amplification 
along with all the reagents necessary for PCR. The mix is then amplified 
for 30 cycles in the presence of .alpha..sup.-32 P-dCTP, then analyzed by 
PAGE, as above. No reamplification was observed, hence the AMT 
photoproduct is in itself an effective inhibitor of PCR. The mechanism of 
photoproduct inhibition is not understood at this time, but is clearly 
concentration dependent. 
EXAMPLE 58 
Post-Amplification Sterilization 
As discussed generally for sterilization, it is expected that the 
sensitivity of sterilization will depend upon both the modification 
density and the length of the PCR target sequences. In this experiment, 
the effect of modification density and target length on sterilization 
sensitivity were examined by sterilizing two different length PCR products 
with either AMIP or AMDmIP. Each isopsoralen was used two different 
concentrations for the sterilization procedure to produce differing 
modification densities on PCR targets. 
The two PCR targets used in these experiments were a 115-mer (SK-38/Sk-39 
HIV system) and a 500-mer. The 500-mer target is obtained from a PCR 
amplification of a lambda plasmid with primers PCR 01/02. This system is 
provided by Cetus/Perkin-Elmer as a control in their commercial kits of 
PCR reagents (Catalogue No. N801-0055). For both of these systems, 
equivalent copy numbers of each target were prepared in the following 
manner: An initial 30 cycle PCR reaction was carried out for each system 
with the appropriate primers and targets. Aliquots (approximately 10.sup.5 
-10.sup.6 target copies) of each of these reactions were transferred to a 
second set of PCR reactions. These second sets of PCR amplifications were 
carried out in the presence of .beta..sup.-32 P-dCTP, again for 30 cycles. 
Aliquots of these reactions were removed and counted by liquid 
scintillation counting. With these numbers, the specific activity of the 
.beta..sup.-32 P-dCTP, and the sequence of each of the PCR product 
oligonucleotides (115-mer and 500-mer), the concentrations of each of the 
two PCR product oligonucleotides in the second set of PCR reaction tubes 
was determined. Both the 115-mer and the 500-mer concentrations were then 
adjusted to exactly 1.times.10.sup.-8 M by the addition of additional Taq 
buffer. These stocks of equivalent copy number of PCR product were then 
used for further investigation. Each of the stock solutions then was 
subdivided into four reaction tubes. The reaction tubes were adjusted to 
contain the following: Tube 1, 100 .mu.g/ml AMIP; Tube 2, 400 .mu.g/ml 
AMIP; Tube 3, 100 .mu.g/ml AMDMIP; and Tube 4, 400 .mu.g/ml AMDMIP. Each 
of these samples were again split into two portions, one part being 
irradiated for 15 minutes at room temperature with the CE-III device and 
the other part kept in the dark. Serial dilutions of the irradiated and 
the unirradiated targets were then carried out on these samples for 30 
cycles in the presence of .alpha..sup.-32 P-dCTP. Aliquots of these 
samples were analyzed on denaturing polyacrylamide gels. The PCR product 
bands were visualized by autoradiography, cut, and counted in a liquid 
scintillation counter. 
The sterilization effect of 100 .mu.g/ml AMIP on the 115-mer PCR product is 
illustrated in FIG. 44A. A 10.sup.8 fold dilution of the irradiated PCR 
carryover product, corresponding to 600 carryover molecules, resulted in a 
diminished PCR signal compared to its unirradiated control after 30 cycles 
of amplification. At 10.sup.6 fold or less dilutions of the PCR carryover 
products, both the irradiated and the unirradiated samples yield similar 
signals. Apparently at 100 .mu.g/ml, AMIP has an insufficient modification 
density on the 115-mer to effectively sterilize more than about 10,000 
molecules of carryover. When the concentration of AMIP was increased to 
400 .mu.g/ml, sterilization sensitivity was improved with the 115-mer 
target. FIG. 44B shows that a carryover of 600,000 molecules of 
unirradiated PCR product results in a signal which is consistent with the 
concentration of the PCR product being in the plateau region of PCR 
amplification. The equivalent amount of irradiated PCR carryover product 
does not produce a measurable PCR signal at all. 100 fold more of the 
irradiated PCR carryover product does start to overwhelm this 
sterilization protocol, indicating that the sterilization sensitivity 
limit with 400 .mu.g/ml AMIP and the 115-mer PCR product is about 10.sup.6 
carryover molecules. 
The effect of PCR product length is illustrated by comparing FIG. 44C to 
FIG. 44A. At 100 .mu.g/ml of AMIP, the 500-mer PCR product is clearly more 
effectively sterilized than the 115-mer PCR product. At 400 .mu.g/ml AMIP, 
the irradiated 500-mer was not amplified at all for any of the dilution 
series up 6.times.10.sup.9 molecules of carryover (data not shown). Larger 
amounts of carryover were not tested. The unirradiated 500-mer with 400 
.mu.g/ml AMIP yielded signals comparable to those in FIG. 44C. 
FIG. 44D shows that AMDMIP at 100 is .mu.g/ml better sterilization agent 
with the 115-mer PCR product that is AMIP at a similar concentration 
(compare with FIG. 44A). When AMDMIP was used at 400 .mu.g/ml, the 
irradiated carryover series yielded no signal at all up to 
6.times.10.sup.9 molecules of carryover with the 115-mer PCR product. The 
unirradiated controls yield normal levels of PCR signals from the 
carryover molecules. When AMDMIP was used with the 500-mer PCR product, 
both 100 .mu.g/ml and 400 .mu.g/ml concentrations resulted in no signal in 
the irradiated dilution series. AMDMIP at 100 .mu.g/ml has a high enough 
modification density on the 500-mer target that there appears to be no 
non-sterilized 500-mers in 6.times.10.sup.9 carryover molecules with the 
.alpha..sup.-32 P assay for PCR product. 
EXAMPLE 59 
It is believed that isopsoralens form monoadducts with double stranded 
nucleic acid but do not form crosslinks because of their angular 
structure. Because of this, these isopsoralen-modified single strands 
should remain detectable by hybridization procedures. The following 
experiments demonstrate the particular usefulness of isopsoralens by 
virtue of their compatibility with two different hybridization formats: 
(1) Oligonucleotide Hybridization (OH) and (2) Crosslinkable 
Oligonucleotide Probe Analysis (COP) (FIG. 45). The experiments show that 
AMDMIP sterilized target molecules remain detectable by both OH ad COP 
procedure. The presence of AMDMIP on the target 115-mer appears not to 
inhibit probe hybridization and likewise, does not reduce the 
crosslinkable of these sterilized target molecules, when assayed and 
examine visually on gels. 
Preparation Of Samples 
The HIV 115-mer system with primers SK-38/SK-39 was used for the experiment 
(see FIG. 36, "HIV Oligonucleotide System"). Two types of starting 
template were used: previously amplified 115-mer or genomic DNA, isolated 
from an individual known to be infected with HIV (MACS sample). For 
previously amplified 115-mer between 10.sup.5 to 10.sup.6 copies were used 
for starting template. For the genomic (MACS) sample, approximately 1 
.mu.g (3.times.10.sup.5 copies) of genomic DNA were used. Two PCR samples 
were prepared for each type of template then amplification was carried out 
for either 20 or 30 cycles. One of the two PCR samples contained AMDMIP at 
200 .mu.g/ml while the other was AMDMIP free. Following amplification, the 
AMDMIP containing samples were divided and half were irradiated. Analysis 
(OH or COP) was then done on the three sample from each set (AMDMIP free, 
AMDMIP unirradiated, and AMDMIP irradiated) for each cycle number. The 
AMDMIP free samples served as control, the AMDMIP unirradiated samples 
addressed the effect of non-covalently bound AMDMIP on detection, and 
AMDMIP irradiated samples addressed the effect of covalently bound AMDMIP 
on detection. 
COP and OH assays were performed as follows: 10 .mu.l of the PCR reaction 
mixture was added to 3.3 .mu.l of "probe mix" 5'-labelled SK-19 (normal 
or monoadducted) at 10.sup.-8 M containing EDTA and an appropriate salt 
mixture!, overlaid with 30-40.mu. of light mineral oil, then heated to 
95.degree.-100.degree. C. for 5 minutes. For COP, the hybridization 
mixture was placed in the PTI device at 56.degree. C. and irradiated for 
15 minutes. Following this, loading dyes (containing urea or formamide) 
were added, the sample heated to 95.degree.-100.degree. C. for 5 minutes, 
quick chilled on wet ice, then loaded on a denaturing PAGE gel followed by 
electrophoresis under denaturing conditions. For OH reactions, the 
hybridization mixture was incubated at 56.degree. C. for 30 minutes, 
loading dyes added, and aliquots loaded directly into a native PAGE gel 
followed by electrophoresis under native conditions. 
1. COP Results: The results with COP are shown in FIG. 46. Samples 1-6 
contain previously amplified 115-mer that was reamplified either 20 
(Samples 1, 3, and 5) or 30 (Samples 2, 4, and 6) cycles then detected by 
COP. Samples 1 and 2 are controls (without AMDMIP); Samples 3 and 4 are 
(with AMDMIP and with light) sterilized samples; Samples 5 and 6 are (with 
AMDmIP but without light) The bands corresponding to amplified 115-mer 
crosslinked to labelled 41-mer probe (SK-19 MA) are the upper bands in 
FIG. 46. Samples 11-16 are the same series except the MACS samples was 
used template. 
Inspection of FIG. 46 shows that only the samples amplified 30 times 
generated significant PCR product (band excision allowed the 20 cycle 
samples to quantitated; see below). The visual intensity of all of the 
hybrid bands in the 30 cycles series appear to be quite similar. For the 
30 cycle series, comparison of the (no compound) control amplification 
(Lanes 2/12) with the test amplification with AMDmIP and light (Lanes 
4/14)! with the (no light) control amplification (Lanes 6/16) shows little 
difference in band intensity. Better quantitation was obtained by excising 
the bands and counting which provided the following numbers: 
______________________________________ 
Sample CPM (%) Sample CPM (%) 
______________________________________ 
1 3104 (100) 11 878 (100) 
3 1944 (62) 13 460 (52) 
5 4480 (144) 15 464 (53) 
2 43948 (100) 12 53304 (100) 
4 36452 (83) 14 41777 (78) 
6 39596 (90) 16 35176 (66) 
______________________________________ 
The trends in both the 20 and 30 cycle series were similar. Comparison of 
the (with AMDMIP and light) samples (3, 4, 13, 14) with the corresponding 
(without AMDMIP and without light) samples (1, 2, 11, 12) shows the 
hybridization signal is reduced between 52 and 83%. Comparison with the 
(with AMDMIP but without light) controls also show a reduction in 
hybridization signal (except Sample 5). It is expected that these (no 
light) samples are samples which contained ANDMIP but were not sterilized 
prior to the COP analysis. However, light is added during the COP 
procedure, and since AMDMIP is present, photoaddition occurs during COP. 
2. OH Results: The results with OH are shown in FIG. 47. This experiment 
used the same amplified samples described above; it was identical to the 
COP experiment except that detection was by OH. Samples 1-6 again contain 
previously amplified 115-mer that was reamplified either 20 (samples 1, 3, 
5) or 30 (samples 2, 4, 6) cycles followed by OH analysis. Samples 1 and 2 
are (without compounds and without light) controls; samples 3 and 4 are 
(with AMDMIP and with light) samples; samples 5 and 6 are (with AMDMIP but 
without light) controls. The bands corresponding to amplified 115-mer 
hybridized to labelled 41-mer probe SK-19 are the upper bands in FIG. 47. 
Lanes 11-16 are the same series but the MACS sample was used as the 
template. The sample in the middle is a negative (reagent) control, while 
"M1" and "M2" are probe alone (as marker). 
Visual inspection of FIG. 47 shows that only the samples amplified 30 times 
provided significant PCR product. In the 30 cycle series, the band 
intensities all appear to be similar. Comparison of the control 
amplification (Lanes 2/12) with the test (with AMDMIP and light; Lanes 
4/14) and the control (with AMDMIP but without light; Lanes 6/16) shows 
similar intensity. It was not possible to obtain reliable counts from the 
bands in this gel (native gels do not tolerate the band excision process), 
so more quantitative comparisons were not made. Relying on the visual 
signal of the gels one can conclude that there may be no impact of the 
photoreactive compound (whether covalently or non-covalently bound to 
nucleic acid) on subsequent detection of the amplified target. Assuming 
the quantitation of the OH (if it could be done) shows the same trend as 
the COP data, there may be a difference of up factor of 2 in hybridization 
signal caused by the presence of the photoreactive compound. 
EXAMPLE 60 
Post-Amplification Sterilization 
Four samples containing 1 .mu.g of Molt-4 human genomic DNA target were 
prepared for PCR with primers KM 29 (5'-GGTTGGCCAATCTACTCCCAGG) and HRI-12 
(5'-GGCAGTAACGGCAGACTACT). These primers give a 174 bp product within the 
human beta globin gene. All four samples contained AMDMIP at 100 .mu.g/ml, 
and were irradiated for 0, 5, 10 or 15 minutes prior to PCR amplification. 
A duplicate set of control samples were also prepared which did not 
contain AMDMIP. Amplifications were carried out in 1.times.Taq buffer (50 
mM KCl, 10 mM Tris pH 8.5, 2.5 mM MgCl.sub.2, 200 .mu.g/ml gelatin), 175 
.mu.M each dNTP, 20 .mu.M primer with 2.5 units of Taq polymerase and 100 
.mu.g/ml AMDMIP present during amplification. PCR was carried out for 30 
cycles; one cycle=30 sec at 94.degree. C. (denaturing), 30 sec at 
55.degree. C. (primer annealing), and 60 sec at 72.degree. (extension). 
.alpha..sup.-32 P-dCTP was used as an internal label. After amplification, 
the samples were analyzed for product by PAGE followed by autoradiography. 
As shown in FIG. 48, irradiation in the presence of AMDMIP at all time 
points (Lanes 5-8 are, respectively, 0, 5, 10 and 15 minutes) resulted in 
no amplification of the sterilized genomic target. Irradiation in the 
absence of AMDMIP at all time points (Lanes 1-4 are, respectively, 0, 5, 
10 and 15 minutes) resulted in amplification of the genomic target. 
Note that the concentration of AMDMIP was selected to be 100 .mu.g/ml. This 
concentration of unirradiated AMDMIP was determined to have no significant 
impact on amplification (data not shown). Even when the sterilizing 
compound is the same, it is desirable to determine appropriate 
concentrations for each particular amplification system (in this example, 
Globin) and not rely on concentrations determined for other systems (e.g., 
HIV). In general, results with the present invention indicate that the 
longer the length of the amplification product, the lower the 
concentration of inactivated compound needed to inhibit amplification 
(data not shown). 
EXAMPLE 61 
Post-Amplification Sterilization 
This example investigated the concentrations at which non-psoralen 
compounds inhibited PCR in the absence of light. The compounds tested were 
the following: (1) ethidium bromide (a Phenanthridine, see Table 1), (2) 
xylene cylanol (an Organic Dye, see Table 1), (3) bromphenol blue (an 
Organic Dye, see Table 1), (4) coumarin, and (5) methylene blue (a 
Phenazathionium Salt, see Table 1). 
The first dark control was run with compounds 1-3. The results are shown in 
FIG. 49. All the compounds showed some inhibition of PCR at the higher 
concentrations used. 
A separate experiment examined PCR inhibition with coumarin and methylene 
blue in the absence of light. The following concentrations of methylene 
blue were tried: 4.3.times.10.sup.-2, 4.3.times.10.sup.-3, 
4.3.times.10.sup.-4, 4.3.times.10.sup.-5 M. Concentrations of coumarin 
tried included: 7.times.10.sup.-3, 7.times.10.sup.-4, 7.times.10.sup.-5, 
7.times.10.sup.-6 M. Compound was added to PCR tube containing 
.alpha.-=P-dCTP and target (1 .mu.l 10.sup.65 .times.dil.; PCR'd MACS 1555 
per assay-point). PCR was carried out for 30.times.cycles. Samples were 
loaded onto a 12%/8M urea gel. The results showed that methylene blue 
inhibited PCR at concentrations above 4.3.times.10.sup.-5 M. Coumarin did 
not inhibit PCR at any of the concentrations tested. 
EXAMPLE 62 
As discussed in Example 60, it cannot be assumed that the particular 
sterilizing compound concentration compatible with one PCR system will be 
compatible with another. The impact of a given concentration of 
sterilizing compound on PCR amplification efficiency must be determined on 
a system by system basis. For example, the HIV 115-mer system is 
compatible with concentrations of AMDMIP up to 400 .mu.g/ml, and therefore 
this concentration of AMDmIP may be used for sterilization. However, this 
concentration of AMDMIP may not be compatible with other PCR systems. 
Indeed, the amplification efficiency of some PCR systems may be 
compromised by high concentrations of sterilizing compounds. 
High concentrations of sterilizing compounds may function to stabilize PCR 
product (particularly long PCR products or PCR products which are 
exceptionally GC rich) such that less of the double stranded product will 
denature in each cycle. This reduced availability of single stranded 
product for subsequent priming and extension would reduced the product 
yield in each PCR cycle. This reduced efficiency over many PCR cycles 
would result in drastic reduction in the yield of PCR product. 
One method to overcome stabilization of PCR product is to modify the PCR 
conditions such that the melting temperature of the PCR product is 
lowered. In so doing, more of the double stranded PCR product is denatured 
in each cycle thereby providing more single stranded target for subsequent 
priming and extension. The net result of the modified PCR conditions is a 
higher yield of PCR product. 
One modification of PCR conditions which provides more denatured (single 
stranded) PCR product is to raise the pre-set denaturation temperature 
above 95.degree. C. for each PCR cycle. This modification has the 
disadvantage of concomitant inactivation of Taq at temperatures above 
95.degree. C. Another modification is adding a co-solvent to the PCR 
buffer which allows denaturation of the PCR product to occur at a lower 
temperature. One such co-solvent is dimethyl sulfoxide (DMSO). Under some 
conditions, DMSO has been shown to facilitate PCR. PCR Technology, H. A., 
Erlich (ed.) (Stockton Press 1989). 
In this example, the effect of DMSO on PCR amplification in the presence of 
high concentrations of sterilizing compound (AMDMIP) was investigated. 
Samples were prepared for PCR which contained 1 g of human placental DNA 
either with or without (unirradiated AMDMIP (200 .mu.g/ml ). The samples 
were amplified 30 cycles, under standard PCR conditions in the presence of 
0%, 1%, 5% or 10% DMSO. The reaction mix contained .alpha..sup.-32 P-dCTP. 
Following amplification, the samples were analyzed by PAGE (data not 
shown). 
The results indicated that while (unirradiated) AMDMIP inhibits 
amplification at a concentration of 200 .mu.g/ml to the point where there 
is virtually no detectable amplification product, addition of DMSO as a 
PCR co-solvent allowed amplification to proceed in the presence of AMDMIP. 
Excision and counting of the product bands provided the following results 
for the AMDMIP-containing samples (% DMSO/CPM): 0%/2500; 1%/2800; 
5%/86,000; 10%/102,000). 
Comparison of the control (no AMDMIP) PCR samples as a function of % DMSO 
showed a regular decrease in amplification yield. Excision and counting of 
the product bands (reported as the average of the duplicate samples) 
confirmed the visual observation that increasing concentrations of DMSO 
showed increased inhibition of PCR (% DMSO/CPM; 0%/139,000; 1%/137,000; 
5%/94,000; 10%/76,000).