This invention is directed to novel substituted nucleotide bases with a crosslinking arm which accomplish crosslinking between specific sites on adjoining strands of oligonucleotides or oligodeoxynucleotides. The invention is also directed to oligonucleotides comprising at least one of these crosslinking agents and to the use of the resulting novel oligonucleotides for diagnostic and therapeutic purposes. The crosslinking agents of the invention are of the following formula (I'): EQU R.sub.1 --B--(CH.sub.2).sub.q --(Y).sub.r --(CH.sub.2).sub.m --A'(I') wherein, PA1 R.sub.1 is hydrogen, or a sugar moiety or analog thereof optionally substituted at its 3' or its 5' position with a phosphorus derivative attached to the sugar moiety by an oxygen and including groups Q.sub.1 Q.sub.2 and Q.sub.3 or with a reactive precursor thereof suitable for nucleotide bond formation; PA1 Q.sub.1 is hydroxy, phosphate or diphosphate; PA1 Q.sub.2 is .dbd.O or .dbd.S; PA1 Q.sub.3 is CH.sub.2 --R', S--R', O--R', or N--R'R"; PA1 each of R' and R" is independently hydrogen or C.sub.1-6 alkyl; PA1 B is a nucleic acid base or analog thereof that is a component of an oligonucleotide; PA1 Y is a functional linking group; PA1 each of m and q is independently 0 to 8, inclusive; PA1 r is 0 or 1; and PA1 A' is a leaving group. This invention is also directed to novel 3,4-disubstituted and 3,4,-trisubstituted pyrazolo3,4-d!-pyrimidines and to the use of these nucleic acid bases in the preparation of oligonucleotides. The invention includes nucleosides and mono- and oligonucleotides comprising at least one of these pyrazolopyrimidines, and to the use of the resulting novel oligonucleotides for diagnostic purposes.

BACKGROUND OF THE INVENTION 
This invention relates to nucleoside crosslinking agents and to the use of 
these compounds in the preparation of oligonucleotides. It also relates to 
derivatives of pyrazolo3,4-d!pyrimidine which are useful as nucleic acid 
bases for the preparation of oligonucleotides. 
Oligonucleotides are useful as diagnostic probes for the detection of 
"target" DNA or RNA sequences. In the past, such probes were made up of 
sequences of nucleic acid containing purine, pyrimidine or 7-deazapurine 
nucleotide bases (U.S. Pat. No. 4,711,955; Robins et al., J. Can. J. 
Chem., 60:554 (1982); Robins et al., J. Org. Chem., 48:1854 (1983)). The 
method for attaching chemical moieties to these bases has been via an 
acetoxy-mercuration reaction, which introduces covalently bound mercury 
atoms into the 5-position of the pyrimidine ring, the C-8 position of the 
purine ring or the C-7 position of a 7-deazapurine ring (Dale et al., 
Proc. Natl. Acad. Sci. USA, 70:2238 (1973); Dale et al., Biochemistry, 
14:2447 (1975)), or by the reaction of organomercurial compounds with 
olefinic compounds in the presence of palladium catalysts (Ruth et al., J. 
Org. Chem., 43:2870 (1978); Bergstrom et al., J. Am. Chem. Soc., 100:8106 
(1978); Bigge et al., J. Am. Chem. Soc., 102:2033 (1980)). 
The sugar component of oligonucleotide probes has been, until the present, 
composed of nucleic acid containing ribose or deoxyribose or, in one case, 
natural .beta.-arabinose (patent publication EP 227,459). 
A novel class of nucleotide base, the 3,4-disubstituted and 
3,4,6-trisubstituted pyrazolo3,4-d!-pyrimidines, has now been found which 
offers several advantages over the prior art. The de novo chemical 
synthesis of the pyrazolopyrimidine and the resulting nucleotide allows 
for the incorporation of a wide range of functional groups in a variety of 
different positions on the nucleotide base and for the use of different 
sugar moieties. Also, adenine, guanine and hypoxanthine analogs are 
obtained from a single nucleoside precursor. Additionally, the synthesis 
does not require the use of toxic heavy metals or expensive catalysts. 
Similar pyrazolo3,4-d!pyrimidines are known (Kobayashi, Chem. Pharm. 
Bull., 21:941 (1973)); however, the substituents on the group are 
different from those of the present invention and their only use is as 
xanthine oxidase inhibitors The concept of crosslinkable nucleotide probes 
for use in therapeutic and diagnostic applications is related to the 
pioneering work of B. R. Baker, "Design of Active-Site-Directed 
Irreversible Enzyme Inhibitors," Wiley, N.Y., (1967), who used what was 
termed "active-site-directed enzyme inhibitors" in chemotherapeutic 
applications. 
In recent years, the concept of incorporating a crosslink in an 
oligonucleotide has been sporadically discussed in efforts to develop 
superior sequence probes. Knorre and Vlassov, Prog. Nucl. Acid Res. Mol. 
Biol., 32:291 (1985), have discussed sequence-directed cross-linking 
("complementary addressed modification") using an 
N-(2-chloroethyl)-N-methylaniline group attached to either the 3'- or 
5.sup.1 -terminus of oligonucleotides. Summerton and Bartlett, J. Mol. 
Biol., 122:145 (1978) have shown that an 8-atom chain, attached to a 
cytosine residue at its C-4 position and terminating in the highly 
reactive bromomethyl ketone group, can crosslink to the N-7 of guanosine. 
Webb and Matteucci, Nucleic Acids Res., 14:7661 (1986), have prepared 
oligonucleotides containing a 5-methyl-N,N-ethanocytosine base which is 
capable of slow crosslinking with a complementary strand. In a 
conceptually related alkylation via a linker arm within a DNA hybrid, 
Iverson and Dervan, Proc. Natl. Acad. Sci. USA, 85:4615 (1988), have shown 
opposite strand methylation, triggered by BrCN activation of a methylthio 
ether, predominately on a guanine base located two pairs from the base 
bearing the linker. 
Oligonucleotides may be used as chemotherapeutic agents to control the 
expression of gene sequences unique to an invading organism, such as a 
virus, a fungus, a parasite or a bacterium. In nature, some RNA expression 
in bacteria is controlled by "antisense" RNA, which exerts its effect by 
forming RNA:RNA hybrids with complementary target RNAs and modulating or 
inactivating their biological activity. A variety of recent studies using 
plasmid vectors for the introduction of antisense RNAs into eukaryotic 
cells have shown that they effectively inhibit expression of MRNA targets 
in vivo (reviewed in Green, et al., Ann. Rev. Biochem. 55: 569-597 
(1986)). Additionally, a specific mRNA amongst a large number of mRNAs can 
be selectively inactivated for protein synthesis by hybridization with a 
complementary DNA restriction fragment, which binds to the mRNA and 
prevents its translation into protein on ribosomes (Paterson, et al., 
Proc. Natl. Acad. Sci 74: 4370-4374 (1977); Hastie et al., Proc. Natl. 
Acad. Sci. 75: 1217-1221 (1978)). 
In the first demonstration of the concept of using sequence-specific, 
antisense oligonucleotides as regulators of gene expression and as 
chemotherapeutic agents, Zamecnik and Stephenson, Proc. Natl. Acad. Sci. 
USA, 75:280 (1978), showed that a small antisense oligodeoxynucleotide 
probe can inhibit replication of Rous Sarcoma virus in cell culture, and 
that RSV viral RNA translation is inhibited under these conditions 
(Stephenson et al., Proc. Natl. Acad. Sci. USA 75:285 (1978)). Zamecnik et 
al., Proc. Natl. Acad. Sci. USA, 83:4143 (1986), have also shown that 
oligonucleotides complementary to portions of the HIV genome are capable 
of inhibiting protein expression and virus replication in cell culture. 
Inhibition of up to 95% was obtained with oligonucleotide concentrations 
of about 70 .mu.M. Importantly, they showed with labeled phosphate studies 
that the oligonucleotides enter cells intact and are reasonably stable to 
metabolism. 
Uncharged methylphosphonate oligodeoxynucleotides with a sequence 
complementary to the initiation codon regions of rabbit globin mRNA 
inhibited the translation of the mRNA in both cell-free systems and in 
rabbit reticulocytes (Blake et al., Biochemistry 24:6139 (1985)). Another 
uncharged methylphosphonate oligonucleotide analog, an 8-nucleotide 
sequence complementary to the acceptor splice junction of a mRNA of Herpes 
simplex virus, Type 1, can inhibit virus replication in intact Vero cells. 
However, fairly high concentrations (&gt;25 mM) of this nonionic probe were 
required for this inhibition. 
Although the impact of crosslinking oligonucleotides in the 
chemotherapeutic field might be of great significance, their impact in DNA 
probe-based diagnostics is of equally great importance. The ability to 
covalently crosslink probe-target hybrids has the potential to 
dramatically improve background and sensitivity limits in diagnostic 
assays as well as permit novel assay formats. Specific innovations 
(discussed previously by Gamper et al., Nucl. Acids Res., 14, 9943 (1988)) 
include: 
(a) incorporation of a denaturing wash step to remove background; 
(b) use of the crosslink as an additional tier of discrimination; 
(c) crosslinking occurring at or near the melting temperature of the 
expected hybrid to insure exquisite specificity and to substantially 
reduce secondary structure in the target, thereby increasing the 
efficiency of hybrid formation; and 
(d) novel solution hybridization formats as exemplified by the Reverse 
Southern protocol. 
The concept of crosslinking, however, suggests potential problems that must 
be circumvented. For instance, the oligonucleotide containing a 
crosslinking arm might covalently bond to the target sequence so readily 
that mismatching of sequences will occur, possibly resulting in host 
toxicity. On the other hand, the crosslinking reaction must be fast enough 
to occur before correctly matched sequences can dissociate. 
This issue can be addressed by constructing an oligonucleotide that, upon 
hybridization, results in a duplex whose T.sub.m is just above the 
physiological temperature of 37.degree. C. Thus, even a single mismatched 
base will prevent hybrid formation and therefore crosslinkage. The 
optimization can be accomplished by judicious choice of oligonucleotide 
length and base composition, as well as position of the modified base 
within the probe. The probe must be long enough, however, to insure 
specific targeting of a unique site. 
European Patent Application No. 86309090.8 describes the formation of 
chemically modified DNA probes such as 5-substituted uridinyl in which the 
substituent does not crosslink but contains a chemical or physical 
reporter group. WO8707611 describes a process for labeling DNA fragments 
such as by chemically modifying the fragment followed by reaction with a 
fluorescent dye. Yabusaki et al. in U.S. Pat. No. 4,599,303 disclose a 
scheme for covalently crosslinking oligonucleotides such as by formation 
of furocoumarin monoadducts of thymidine which are made to covalently bond 
to other nucleotides upon photoexcitation. EP 0259186 describes adducts of 
macromolecules and biotin which can be used as crosslinking nucleic acid 
hybridization probes. WO8503075 describes crosslinking disulfonic esters 
useful as nucleic acid fragmentation agents. DE3310337 describes the 
covalent crosslinking of single-stranded polynucleotides to such 
macromolecules as proteins with the resulting complex subsequently used as 
a marker in hybridization experiments in the search for complementary 
sequences in foreign polynucleotides. 
A need exists for probe oligonucleotides, consisting of sufficient base 
sequences to identify target sequences with high specificity, that are 
provided with one or more crosslinking arms which readily form covalent 
bonds with specific complementary bases. Such oligonucleotides may be used 
as highly selective probes in hybridization assays. The oligonucleotides 
may also be used as antisensing agents of RNAs, e.g., in chemotherapy. 
SUMMARY OF THE INVENTION 
This invention is directed to crosslinking agents which accomplish 
crosslinking between specific sites on adjoining strands of 
oligonucleotides. The crosslinking reaction observed is of excellent 
specificity. The invention is also directed to oligonucleotides comprising 
at least one of these crosslinking agents and to the use of the resulting 
novel oligonucleotides for diagnostic and therapeutic purposes. 
More particularly, the crosslinking agents of this invention are 
derivatives of nucleotide bases with a crosslinking arm and are of the 
following formula (I'): 
EQU R.sub.1 --B--(CH.sub.2).sub.q --(Y).sub.r --(CH.sub.2).sub.m --A'(I') 
wherein, 
R.sub.1 is hydrogen, or a sugar moiety or analog thereof optionally 
substituted at its 3' or its 5' position with a phosphorus derivative 
attached to the sugar moiety by an oxygen and including groups Q.sub.1 
Q.sub.2 and Q.sub.3, or with a reactive precursor thereof suitable for 
nucleotide bond formation; 
Q.sub.1 is hydroxy, phosphate or diphosphate; 
Q.sub.2 is.dbd.O or .dbd.S; 
Q.sub.3 is CH.sub.2 --R', S--R', O--R', or N--R'R"; 
each of R' and R" is independently hydrogen or c.sub.1-6 alkyl; 
B is a nucleic acid base or analog thereof that is a component of an 
oligonucleotide; 
Y is a functional linking group; 
each of m and q is independently 0 to 8, inclusive; 
r is 0 or 1; and 
A' is a leaving group. 
The invention also provides novel oligonucleotides comprising at least one 
of the above nucleotide base derivatives of formula I'. 
Nucleotides of this invention and oligonucleotides into which the 
nucleotides have been incorporated may be used as probes. Since probe 
hybridization is reversible, albeit slow, it is desirable to ensure that 
each time a probe hybridizes with the correct target sequence, the probe 
is irreversibly attached to that sequence. The covalent crosslinking arm 
of the nucleotide bases of the present invention will permanently modify 
the target strand, or cause depurination. As such, the oligonucleotides of 
this invention are useful in the identification, isolation, localization 
and/or detection of complementary nucleic acid sequences of interest in 
cell-free and cellular systems. Therefore, the invention further provides 
a method for identifying target nucleic acid sequences, which method 
comprises utilizing an oligonucleotide probe comprising at least one of a 
labeled nucleotide base of the present invention. 
This invention also provides novel substituted pyrazolo3,4-d!pyrimidines 
which are useful as a nucleotide base in preparing nucleosides and 
nucleotides, rather than the natural purine or pyrimidine bases or the 
deazapurine analogs.

EXAMPLE 1 
6-(Tritylamino)caproic Acid 
6-Aminocaproic acid (26 g, 0.2 mole) was dissolved in dichloromethane (200 
mL) by the addition of triethylamine (100 mL). Trityl chloride (120 g, 
0.45 mole) was added and the solution stirred for 36 hr. The resulting 
solution was extracted with 1N HCl and the organic layer evaporated to 
dryness. The residue was suspended in 2-propanol/1N NaOH (300 mL/100 mL) 
and refluxed for 3 hr. The solution was evaporated to a thick syrup and 
added to dichloromethane (500 mL). Water was added and acidified. The 
phases were separated, and the organic layer dried over sodium sulfate and 
evaporated to dryness. The residue was suspended in hot 2-propanol, 
cooled, and filtered to give 43.5 g (58%) of 6-(trityl-amino)caproic acid, 
useful as an intermediate compound. 
EXAMPLE 2 
5-(Tritylamino)pentylhydroxymethylenemalononitrile 
To a dichloromethane solution of 6-(tritylamino)-caproic acid (20.0 g, 53 
mmole) and triethylamine (20 mL) in an ice bath was added dropwise over 30 
min isobutyl-chloroformate (8.3 mL, 64 mmole). After the mixture was 
stirred for 2 hr in an ice bath, freshly distilled malononitrile (4.2 g, 
64 mmole) was added all at once. The solution was stirred for 2 hr in an 
ice bath and for 2 hr at RT. The dichloromethane solution was washed with 
ice cold 2N HCl (300 mL) and the biphasic mixture was filtered to remove 
product that precipitated (13.2 g). The phases were separated and the 
organic layer dried and evaporated to a thick syrup. The syrup was covered 
with dichloromethane and on standing deposited fine crystals of product. 
The crystals were filtered and dried to give 6.3 g for a total yield of 
19.5 g (87%) of the product, which is useful as an intermediate. 
EXAMPLE 3 
5-(Tritylamino)pentylmethoxymethylenemalononitrile 
A suspension of the malononitrile of Example 2 (13 g, 31 mmole) in 
ether/dichloromethane (900 mL/100 mL), cooled in an ice bath, was treated 
with a freshly prepared ethereal solution of diazomethane (from 50 mmole 
of Diazald.RTM. (Aldrich Chemical Company)). The solution was stirred for 
6 hr and then neutralized with acetic acid (10 mL). The solution was 
evaporated to dryness and the residue chromatographed on silica gel using 
dichloromethane/acetone (4/1) as the eluent. Fractions containing product 
were pooled and evaporated to a syrup. The syrup was triturated with 
dichloromethane to induce crystallization. The crystals were filtered and 
dried to give 8.3 g (61%) of chromatographically pure product, useful as 
an intermediate compound. 
EXAMPLE 4 
5-Amino-3-(5-tritylamino)pentyl!pyrazole-4-carbonitrile 
To a methanol solution (100 mL) of the product of Example 3 (7.0 g, 16 
mmole) in an ice bath was added hydrazine monohydrate (7.8 mL, 160 mmole) 
dropwise over 15 min. After stirring for 30 min in an ice bath, the 
solution was evaporated to dryness. The residue was suspended in cold 
methanol and filtered to give 7.1 g (100%) of 
5-amino-3-(5-tritylamino)pentyl!pyrazole-4-carbonitrile, useful as an 
intermediate, after drying. An analytical sample was prepared by 
recrystallization from water. 
EXAMPLE 5 
5-Amino-1-(2-deoxy-3,5-di-O-toluoyl-.beta.-D-erythropentofuranosyl)-3-(5-t 
ritylamino)pentyl!pyrazole-4-carbonitrile 
An ice cold solution of the carbonitrile from Example 4 (3.5 g, 8 mmole) 
was treated with sodium hydride and stirred for 30 min at 
0.degree.-4.degree. C. 1-Chloro-1,2-dideoxy-3,5-di-O-toluoylribofuranose 
was added and the solution stirred for 1 hr at 0.degree.-4.degree. C. The 
solution was poured into a saturated solution of sodium bicarbonate and 
extracted with dichloromethane. The organic layer was dried over sodium 
sulfate and evaporated to dryness. The residue was flash chromatographed 
on silica gel using toluene/ethyl acetate (5/1) as eluent. Two major 
products were isolated and identified as the N-1 and N-2 isomers in 57% 
(3.6 g) and 20% (1.2 g) N-1 and N-2 yields, respectively. Approximately 1 
g of a mixture of N-1 and N-2 isomers was also collected. Overall yield of 
glycosylated material was 5.8 g (92%). The N-1 isomer, 
5-amino-1-(2-deoxy-3,5-di-O-toluoyl-.beta.-D-erythropentofuranosyl)-3-(5- 
tritylamino)-pentyl!pyrazole-4-carbonitrile, was used without further 
purification in Example 6. 
EXAMPLE 6 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl)-3-5-(tritylamino)-pentyl!pyrazo 
lo3,4-d!pyrimidin-4-amine 
To a toluene (100 mL) solution of the pyrazole-4-carbonitrile of Example 5 
(3.5 g, 4.4 mmole) was added diethoxymethyl acetate (1.1 mL, 6.7 mmole). 
The solution was kept at 80.degree.-90.degree. C. for 5 hr and then 
evaporated to a syrup. The syrup was dissolved in dichloromethane (10 mL) 
and added to ice cold methanolic ammonia (100 mL) in a glass pressure 
bottle. After two days at RT the contents of the bottle were evaporated to 
dryness. The residue was dissolved in methanol and adjusted to pH 8 with 
freshly prepared sodium methoxide to complete the deprotection. After 
stirring overnight the solution was treated with Dowex.RTM.-50 H+ resin, 
filtered, and evaporated to dryness. The residue was chromatographed on 
silica gel using acetone/hexane (3/2) as eluent to give 2.0 g (77%) of 
analytically pure product. 
EXAMPLE 7 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl)-3-5-(tritylamino)-pentyl!pyrazo 
lo3,4-d!pyrimidin-4-amine 5'-monophosphate 
To an ice cold solution of the pyrazolopyrimidin-4-amine of Example 6 (250 
mg, 0.43 mmole) in trimethyl phosphate (5 mL) was added phosphoryl 
chloride (50 .mu.L) and the solution was kept at 0.degree.-4.degree. C. 
The reaction was monitored by reversed phase HPLC using a linear gradient 
from 0 to 100% acetonitrile in water over 25 min. After stirring for 5 hr, 
an additional aliquot of phosphoryl chloride (25 .mu.L) was added and the 
solution was stirred another 30 min. The solution was poured into 0.1M 
ammonium bicarbonate and kept in the cold overnight. The solution was then 
extracted with ether and the aqueous layer evaporated to dryness. The 
residue was dissolved in water (5 mL) and purified by reversed phase HPLC 
using a 22mm .times.50cm C18 column. The column was equilibrated in water 
and eluted with a gradient of 0 to 100% acetonitrile over 20 min. 
Fractions containing the desired material were pooled and lyophilized to 
give 160 mg (56%) of chromatographically pure nucleotide. 
EXAMPLE 8 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl) -3-(5- 
(6-biotinamido)-hexanamido!pentyl)pyrazolo3,4-d!pyrimidin-4-amine 
5'-monophosphate. 
An ethanol solution (10 mL) of the nucleotide of Example 7, palladium 
hydroxide on carbon (50 mg), and cyclohexadiene (1 mL) was refluxed for 3 
days, filtered, and evaporated to dryness. The residue was washed with 
dichloromethane, dissolved in DMF (1.5 mL) containing triethylamine (100 
mL), and treated with N-hydroxy-succinimidyl biotinylaminocaproate (50 
mg). After stirring overnight an additional amount of 
N-hydroxysuccinimidyl 6-biotinamidocaproate (50 mg) was added and the 
solution was stirred for 18 hr. The reaction mixture was evaporated to 
dryness and chromatographed following the procedure in Example 7. 
Fractions were pooled and lyophilized to give 80 mg of chromatographically 
pure biotinamido-substituted nucleotide. 
EXAMPLE 9 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl)-3-5-(6-biotinamido)-hexanamidop 
entyl!pyrazolo3,4-d!pyrimidin-4-amine 5'-triphosphate. 
The monophosphate of Example 8 (80 mg, ca. 0.1 mmole) was dissolved in DMF 
with the addition of triethylamine (14 .mu.L). Carbonyldiimidazole (81 mg, 
0.5 mmole) was added and the solution stirred at RT for 18 hr. The 
solution was treated with methanol (40 .mu.L), and after stirring for 30 
min tributylammonium pyrophosphate (0.5 g in 0.5 mL DMF) was added. After 
stirring for 24 hr another aliquot of tributylammonium pyrophosphate was 
added and the solution was stirred overnight. The reaction mixture was 
evaporated to dryness and chromatographed following the procedure in 
Example 8. Two products were collected and were each separately treated 
with conc. ammonium hydroxide (1 mL) for 18 hr at 55.degree. C. UV and 
HPLC analysis indicated that both products were identical after ammonia 
treatment and were pooled and lyophilized to give 35.2 mg of nucleoside 
triphosphate. 
EXAMPLE 10 
Nick-Translation Reaction 
The triphosphate of Example 9 was incorporated into pHPV-16 using the nick 
tanslation protocol of Langer et al. (supra). The probe prepared with the 
triphosphate of Example 9 was compared with probe prepared using 
commercially available bio-11-dUTP (Sigma Chemical Co). No significant 
differences could be observed in both a filter hybridization and in in 
situ smears. 
More specifically, the procedure involved the following materials and 
steps: 
Materials: 
DNase (ICN Biomedicals)-4 .mu.g/mL 
DNA polymerase 1 (U.S. Biochemicals)-8 U/mL 
PHPV-16-2.16 mg/mL which is a plasmid containing the genomic sequence of 
human papillomavirus type 16. 
10X-DP-1M Tris,pH7.5(20 mL); 0.5M DTT(80 mL); 1M MgCl.sub.2 (2.8 mL); 
H.sub.2 O (17 mL) 
Nucleotides-Mix A-2 mM each dGTP, dCTP, TTP (Pharmacia) 
Mix U-2 mM each dGTP, dCTP, dATP 
Bio-11-dUTP-1.0 mg/mL (BRL) 
Bio-12-dAPPTP-1.0 mg/mL 
Steps: 
To an ice cold mixture of 10X-DP (4 mL), pHPV-16 (2 mL), nucleotide mix A 
(6 mL), Bio-12-dAPPTP (2 mL), and H.sub.2 O (20 mL) was added DNase (1 mL) 
and DNA polymerase 1 (2.4 mL). The reaction mixture was incubated at 
16.degree. C. for 1 hr. The procedure was repeated using Bio-11-dUTP and 
nucleotide mix U in place of Bio-12-dAPPTP (comprising the triphosphate of 
Example 9) and nucleotide mix A. 
Nucleic acid was isolated by ethanol precipitation and hybridized to 
pHPV-16 slotted onto nitrocellulose. The hybridized biotinylated probe was 
visualized by a streptavidin-alkaline phosphatase conjugate with BCIP/NBT 
substrate. Probe prepared using either biotinylated nucleotide gave 
identical signals. The probes were also tested in an in situ format on 
cervical smears and showed no qualitative differences in signal and 
background. 
EXAMPLE 11 
5-Amino-3-(5-tritylamino)pentyl!pyrazole-4-carboxamide 
Following the procedure of Example 2, except that cyanoacetamide is used 
instead of malononitrile, 
5-(tritylamino)pentylhydroxymethylenecyanoacetamide is prepared from 
6-(tritylamino)caproic acid. This is then treated with diazomethane to 
give the methoxy derivative, following the procedures of Example 3, which 
is then reacted with hydrazine monohydrate, as in Example 4, to give 
5-amino-3- (5-tritylamino)pentyl!pyrazole-4-carboxamide. 
EXAMPLE 12 
4-Hydroxy-6-methylthio-3-(5-tritylamino)pentyl!pyrazolo-3,4-d!pyrimidine. 
The carboxamide from Example 11 is reacted with potassium ethyl xanthate 
and ethanol at an elevated temperature to give the potassium salt of 
4-hydroxypyrazolo3,4-d!pyrimidine-6-thiol. This salt is then reacted with 
iodomethane to give 
4-hydroxy-6-methylthio-3-(5-tritylamino)pentyl!pyrazolo3,4-d!pyrimidine. 
EXAMPLE 13 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl)-4-hydroxy-3-5-(tritylamino)pent 
yl!pyrazolo3,4-d!pyrimidin-6-amine 
Following the procedure of Example 5, the pyrazolopyrimidine of Example 12 
is treated with sodium hydride and reacted with 
l-chloro-1,2-dideoxy-3,5-di-O-toluoylribofuranose. The resulting compound 
is reacted with MCPBA and with methanolic ammonia, and the toluoyl 
protecting groups are removed to give the product. 
EXAMPLE 14 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl)-4-hydroxy-3-5-(6-biotinamido)he 
xanamidopentyl!pyrazolo3,4-d!pyrimidin-6-amine 5'-monophosphate. 
Following the procedure of Example 7, the pyrazolopyrimidine of Example 13 
is reacted with phosphoryl chloride to give the corresponding 
5'-monophosphate. 
Following the procedure of Example 8, the above 5'-monophosphate is reacted 
with palladium/carbon and cyclohexadiene, and the residue is reacted with 
N-hydroxy-succinimidyl biotinylaminocaproate to give 
1-(2-deoxy-.beta.-D-erythropentofuranosyl)-4-hydroxy-3-5-(6-biotinamido)h 
exanamidopentyl!pyrazolo3,4-d!pyrimidin-6-amine 5'-monophosphate. 
EXAMPLE 15 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl)-4-hydroxy-3-5-(6-biotinamido)he 
xanamidopentyl!pyrazolo3,4-d!pyrimidin-6-amine 5'-triphosphate 
Following the procedure of Example 9, the 5'-monophosphate of Example 14 is 
treated with carbonyldiimidazole and then reacted with tributylammonium 
pyrophosphate to give the corresponding 5'-triphosphate. 
EXAMPLE 16 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl)-3-5-(tritylamino)-pentyl!pyrazo 
lo3,4-d!pyrimidine-4-benzoylamine 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl)-3-5-(tritylamino)pentyl!pyrazol 
o3,4-d!pyrimidine-4-amine from Example 6 is reacted with benzoyl chloride 
and pyridine to give 
1-(2-deoxy-3,5-di-O-benzoyl-.beta.-D-erythro-pentofuranosyl)-3-5-(trityla 
mino)pentyl!pyrazolo3,4-d!-pyrimidine-4-dibenzoylamine. This is treated 
with aqueous sodium hydroxide to partially deprotect the compound, giving 
1-(2-deoxy-.beta.-D-erythropentofuranosyl)-3-5-(tritylamino)pentyl!pyrazo 
lo3,4-d!pyrimidine-4-benzoylamine. 
EXAMPLE 17 
1-(2-Deoxy-.beta.-D-erythropentofuranosyl)-3-5-(trifluoroacetamido)pentyl! 
pyrazolo3,4-d!pyrimidine-4-benzoylamine 
Following the procedure of Example 8, the benzoylamine of Example 16 is 
treated with palladium hydroxide on carbon and then with trifluoroacetic 
anhydride to give 
1-(2-deoxy-.beta.-D-erythropentofuranosyl)-3-5-(trifluoroacetamido)pentyl 
!pyrazolo3,4-d!pyrimidine-4-benzoylamine. 
EXAMPLE 18 
1-(2-Deoxy-5-O-dimethoxytrityl-.beta.-D-erythropentofuranosyl)-3-5-(triflu 
oroacetamido)pentyl!pyrazolo3,4-d!pyrimidine-4-benzoylamine 
3'-O-(N,N-diisopropyl)phosphoramidite cyanoethyl ester 
The compound of Example 17 is reacted with dimethoxytrityl chloride and 
pyridine to give the corresponding 5'-dimethoxytrityl compound. This 
compound is then reacted with cyanoethyl chloro-N,N-diisopropyl- 
phosphoramidite (according to the method of Sinha et al., Nucleic Acids 
Res., 12:4539 (1984)) to give the 3'-O-activated nucleoside. 
EXAMPLE 19 
5-(4-Phthalimidobut-1-yn-1-yl)-2'-deoxyuridine 
5-Iodo-2'-deoxyuridine (354 mg, 1 mmol) was dissolved in 10 mL of 
dimethylformamide. Cuprous iodide (76 mg, 0.4 mmol), 
tetrakis(triphenylphosphine)palladium(0) (230 mg, 0.2 mmol), and 
triethylamine (200 mg, 2.0 mmol) were added. 4-Phthalimidobut-1-yne (300 
mg, 1.5 mmol) was added all at once and the reaction kept at 60.degree. C. 
for three hours. The clear yellow reaction was then evaporated and 
methylene chloride was added. Scratching of the flask induced 
crystallization of nearly all of the product which was filtered and 
recrystallized from 95% ethanol to give 335 mg (78%) of title compound as 
fine, feathery needles. 
EXAMPLE 20 
5-(4-Phthalimidobut-1-yl)-2'-deoxyuridine 
1.00 Gram of deoxyridine from Example 19 was dissolved in 95% EtOH and 
about 3 g of neutral Raney nickel was added. After 48 hours, the catalyst 
was removed by cautious filtration and the filtrate was evaporated to a 
solid which was recrystallized from methanol-water to give 960 mg (97%) of 
the title compound. 
EXAMPLE 21 
5-(3-Iodoacetamidopropyl)-2'-deoxyuridine 
5-(3-Trifluoroacetamidoprop-1-yl)-2'-deoxyuridine (0.3 mmol) is treated 
with ammonia and then with N-hydroxy-succinimidyl .alpha.-iodoacetate (0.5 
mmol). The reaction mixture is evaporated to dryness and purified by 
chromatography to give 5-(3-iodoacetamidopropyl)-2'-deoxyuridine. 
EXAMPLE 22 
5-(4-(4-Bromobutyramido)butyl)-2'-deoxyuridine 
Following the procedure of Example 21, 
5-(4-phthalimidobut-1-yl)-2'-deoxyuridine, from Example 20, is treated 
with ammonia and then with N-hydroxysuccinimidyl 4-bromobutyrate to give 
5-(4-(4-bromobutyramido)butyl)-2'-deoxyuridine. 
Preparation of Synthetic Oligonucleotides 
EXAMPLE 23 
Phosphoramidite Preparation and DNA Synthesis 
Nucleosides were 5'-dimethoxytritylated, following known procedures, to 
give around 85% yield, and the 3'-phosphoramidite was made using 
diisopropylamino .beta.-cyanoethylchlorophosphite (as described in 
"Oligonucleotide Synthesis: A Practical Approach", supra) with 
diisopropyl-ethylamine in methylene chloride. The phosphoramidite was made 
into a 0.2N solution in acetonitrile and placed on the automated DNA 
synthesizer. Incorporation of these new and modified phosphoramidites gave 
incorporation similar to ordinary phosphoramidites (97-99% as judged by 
assay of the trityl color released by UV.) 
Oligonucleotides were removed from the DNA synthesizer in tritylated form 
and deblocked using 30% ammonia at 55.degree. C. for 6 hours. Ten .mu.L of 
0.5M sodium bicarbonate was added to prevent acidification during 
concentration. The oligonucleotide was evaporated to dryness under vacuum 
and redissolved in 1.0 mL water. The oligonucleotides were purified by 
HPLC using 15-55% acetonitrile in 0.1N triethylammonium acetate over 20 
minutes. Unsubstituted oligonucleotides came off at 10 minutes; amino 
derivatives took 11-12 minutes. The desired oligonucleotide was collected 
and evaporated to dryness, then it was redissolved in 80% aqueous acetic 
acid for 90 minutes to remove the trityl group. Desalting was accomplished 
with a G25 Sephadex column and appropriate fractions were taken. The 
fractions were concentrated, brought to a specific volume, dilution 
reading taken to ascertain overall yield and an analytical HPLC done to 
assure purity. oligonucleotides were frozen at -20.degree. C. until use. 
Following the above procedures, the nucleoside 
5-(3-trifluoroacetamidoprop-1-yl)-2'-deoxyuridine was converted to the 
5'-O -dimethoxytrityl-3'-(N,N-diisopropyl) -phosphoramidite cyanoethyl 
ester derivative. This was added to a DNA synthesizer and the following 
14-mer oligonucleotide sequence was prepared: 
EQU 3'-CT TCC U.sup.1 TG TAG GTC-5' 
where U.sup.1 is 5-(3-aminoprop-1-yl)-2 '-deoxyuridine (oligo A). 
In the same manner, 5-(4-phthalimidobut-1-yl) -2'-deoxyuridine was 
converted to the 5'-O-dimethoxytrityl-3'-(N,N-diisopropyl)phosphoramidite 
cyanoethyl ester derivative and added to a DNA synthesizer to prepare the 
above 14-mer oligonucleotide sequence where U.sup.1 is 
5-(4-aminobut-1-yl)-2'-deoxyuridine (oligo C). 
A corresponding 14-mer oligonucleotide was also prepared where U.sup.1 is 
the unmodified deoxyuridine. 
EXAMPLE 24 
Derivatization of Oligonucleotides 
In general, to add the crosslinking arm to an aminoalkyloligonucleotide, a 
solution of 10 .mu.g of the aminoalkyloligonucleotide and a 100X molar 
excess of n-hydroxysuccinimide haloacylate such as .alpha.-haloacetate or 
4-halobutyrate in 10 .mu.L of 0.1M borate buffer, pH 8.5, was incubated at 
ambient temperature for 30 min. in the dark. The entire reaction was 
passed over a NAP-10 column equilibrated with and eluted with distilled 
water. Appropriate fractions based on UV absorbance were combined and the 
concentration was determined spectrophotometrically. 
Introduction of the haloacyl moiety was examined by HPLC. A Zorbax.RTM. 
oligonucleotide column (Dupont) eluted with a 20 minute gradient of 60% to 
80% B composed of: A (20% acetonitrile:80% 0.02 N NaH.sub.2 PO.sub.4) and 
B (1.2 N NaCl in 20% acetonitrile:80% 0.02 N NaH.sub.2 PO.sub.4). The 
presence of a reactive .alpha.-haloacyl moiety was indicated by return of 
the retention time of the .alpha.-haloacylamidoalkyl oligonucleotide to 
the corresponding aminoalkyl oligonucleotide after exposure to 1N 
cysteamine. Introduction of cysteamine created equivalent charge patterns 
between the aminoalkyl oligonucleotide and the a-haloacylamido 
oligonucleotide. 
Following the above procedure, the 14-mer oligonucleotide: 
EQU 3'-CT TCC U.sup.1 TG TAG GTC-5' 
where U.sup.1 is 5-(3-aminoprop-1-yl)-2'-deoxyuridine (oligo A, Example 
23), was reacted with n-hydroxysuccinimide .alpha.-iodoacetate to give the 
above 14-mer oligonucleotide where U.sup.1 is 
5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine (oligo B). 
Oligo A and oligo B, as well as the above 14-mer where U.sup.1 is the 
unmodified deoxyuridine were resolved in the Zorbax column, all of 
identical sequence, with the following retention times: unmodified 14-mer, 
9.31 min; aminopropyl 14-mer (oligo A), 7.36 min; and iodoacetamido-propyl 
14-mer (oligo B), 10.09 min. 
In the same manner, the aminopropyl 14-mer (oligo A) was reacted with 
N-hydroxysuccinimide 4-bromobutyrate to give the 14-mer where U.sup.1 is 
5-(3-(4-bromobutyramido)prop-1-yl)-2'-deoxyuridine. 
Also, the aminobutyl 14-mer (oligo C, Example 23) was reacted with either 
N-hydroxysuccinimide .alpha.-iodoacetate or N-hydroxysuccinimide 
4-bromobutyrate to give the 14-mer where U.sup.1 is 
5-(4-iodoacetamidobut-1-yl)-2'-deoxyuridine or 
5-(4-(4-bromobutyramido)but-1-yl)-2'-deoxyuridine, respectively. 
Assays 
EXAMPLE 25 
Assay of Crosslinking Reaction 
The reaction of crosslinking a DNA probe to a target nucleic acid sequence 
contained 1 .mu.g of haloacyl-amidoalkyl probe and 10 ng of .sup.32 
P-labeled cordycepin-tailed target in 200 .mu.L of 0.1M Tris, pH 8.0, and 
0.9M NaCl incubated at 20.degree. or 30.degree. C. Aliquots were removed 
at 24- or 72-hour intervals and diluted in 20 .mu.L of 10 mM cysteamine to 
quench the haloacylamido group. These solutions were stored at RT, and 1 
.mu.L was used for analysis by denaturing polyacrylamide gel 
electrophoresis (PAGE). 
Following the above procedure, two model oligonucleotide sequences were 
utilized to evaluate the crosslinkage potential of the modified probe to 
its complement. The sequences, derived from human papilloma-virus (HPV) or 
human cytomegalovirus (CMV), are shown below: 
##STR9## 
The target for HPV is a 30-mer, and for CMV it is a 24-mer. The 
crosslinking probes were a 14-mer for HPV and two 15-mers for CMV. Each 
probe contained a single modified deoxyuridine designated as U in the 
sequences above. 
Results of the reaction of HPV target with a limiting amount of 
crosslinking probe containing a 5-(3-iodoacetamidopropyl) sidearm are 
shown in FIG. 2. Analysis of the cleavage pattern on a denaturing PAGE gel 
showed the loss of the crosslinked hybrid with the concomitant appearance 
of a discrete low molecular weight band. The intensity of this band was 
dependent upon the extent of crosslinkage in the initial reaction. The 
localization of signal into two discrete bands on the gel strongly argues 
that no non-sequence-directed alkylation of either target or probe strands 
had occurred (including intramolecular probe alkylation). 
Comparison to an authentic 15-mer run in an adjacent lane suggested that 
the major cleaved fragment is a 9-mer. Upon close examination of the 
original autoradiogram, a slower moving band of very weak intensity was 
visible. This pattern would be consistent with major alkylation at G-21 
and minor alkylation at G-20. An examination of a Dreiding model of the 
crosslinkable HPV hybrid shows that the 5-(3-iodoacetamidopropyl) sidearm 
can contact the G-21 residue of the target strand with only minor 
distortion of the helix. 
If alkylation occurs predominately at a guanosine on the target strand 
located two units on the 5' side of the modified-deoxyuridine base pair, 
the CMV sequence should not react. This result was in fact observed. The 
absence of reaction with CMV further supports the specificity of 
crosslinking scheme of the invention. 
EXAMPLE 26 
Time and Temperature Dependence 
Time and temperature dependence studies were carried out with the HPV 
system of Example 25 where U is 
5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine. The target was .sup.32 
P-labeled by cordycepin tailing with terminal transferase (Maniatis et 
al., "Molecular Cloning--A Laboratory Manual", Cold Spring Harbor 
Laboratory, 1982, p. 239) and incubated with excess probe in a pH 8.0 Tris 
buffer at either 20.degree. or 30.degree. C. Aliquots were removed after 
0, 24, or 72 hours incubation, quenched with an equivalent volume of 10 mM 
mercaptoethylamine (which reacts with the iodoacetamide), and stored at RT 
for subsequent analysis by denaturing or non-denaturing PAGE. 
Crosslinkage of the hybrid, which was monitored by denaturing PAGE, was 
evident for the 24 and 72 hour time points at both temperatures (see FIG. 
3). The amount of crosslinked hybrid increased with both temperature and 
time. Approximately 20% of the hybrid was crosslinked after 72 hours 
incubation at 30.degree. C. 
Separate experiments at a range of temperatures indicated that the 
half-life for crosslinking at 37.degree. C. is approximately 2 days, and 
that the reaction is complete after 24 hours at 58.degree. C. This 
time-dependent reaction implies that the iodoacetamido moiety does not 
hydrolyze or react with the buffer. The increased reaction rate at higher 
temperature indicates that the hybrid is maintained, and subsequently the 
rate of alkylation shows the expected increase with temperature. 
EXAMPLE 27 
Site Specificity of Alkylation 
To elucidate the site specificity of alkylation, the crosslinked HPV hybrid 
of Example 25 (where U is 5-(3-iodoacetamidoprop-1-yl)-2'-deoxyuridine) 
was subjected to a 10% piperidine solution at 90.degree. C. for 60 
minutes. As shown by Maxam et al. (Proc. Natl. Acad. Sci. USA, 74: 560 
(1977), this treatment quantitatively cleaves the target strand 3'-to the 
site of alkylation. The resulting data indicated that the alkylation of 
the second guanine above the crosslinker-modified base pair (i.e., the 
guanine above the target base) was the exclusive action observed, 
indicating that the crosslinking reaction in the HPV model system is 
remarkably specific. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 5 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-(3- aminoprop-1-yl)-2'deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-(4- aminobut-1-yl)-2'-deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be the unmodified 
deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-(3- iodoacetamidoprop-1-yl)-2'-deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-(3-(4- bromobutyramido)prop-1-yl)-2'-deoxyuridin 
e" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-(4- iodoacetamidobut-1-yl)-2'-deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-(4-(4- bromobutyramido)but-1-yl)-2'-deoxyuridine" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CTGGATGTUCCTTC14 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
AGACAGCACAGAATTCGAAGGAACATCCAG30 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-3- (alpha-iodoacetamido)-propyl!-2'-deoxyuridin 
e" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-3- (bromobutyramido)-propyl!-2'-deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-4-alpha- iodoacetamido)-butyl!-2'-deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 9 
(D) OTHER INFORMATION: /note= "U may be 
5-4-(4- bromobutyramido)-butyl!-2'-deoxyuridine" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CTGGATGTUCCTTC14 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ACCGTCCTTGACACGATGGACTCC24 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 15 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note= "U may be 
5-3- (alpha-iodoacetamido)-proply!-2'-deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note= "U may be 
5-3-(4- bromobutyramido)-propyl!-2'-deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note= "U may be 
5-4- (alpha-iodoacetamido)-butyl!-2'-deoxyuridine" 
(ix) FEATURE: 
(A) NAME/KEY: modified.sub.-- base 
(B) LOCATION: 6 
(D) OTHER INFORMATION: /note= "U may be 
5-4-(4- bromobutyramido)-butyl!-2'-deoxyuridine" 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
CTCCAUCGTGTCAAG15 
__________________________________________________________________________