Alkynylamino-nucleotides and labeled alkynylamino-nucleotides useful, for example, as chain terminating substrates for DNA sequencing are provided along with several key intermediates and processes for their preparation.

FIELD OF THE INVENTION 
This invention pertains to alkynylaminonucleotides and especially to their 
use in preparing fluorescently-labeled nucleotides as chain-terminating 
substrates for a fluorescence-based DNA sequencing method. 
BACKGROUND OF THE INVENTION 
DNA sequencing is one of the cornerstone analytical techniques of modern 
molecular biology. The development of reliable methods for sequencing has 
led to great advances in the understanding of the organization of genetic 
information and has made possible the manipulation of genetic material 
(i.e. genetic engineering). 
There are currently two general methods for sequencing DNA: the 
Maxam-Gilbert chemical degradation method [A. M. Maxam et al., Meth. in 
Enzym., Vol. 65, 499-559 (1980)] and the Sanger dideoxy chain termination 
method [F. Sanger. et al., Proc. Nat. Acad. Sci. USA, Vol. 74, 5463-5467 
(1977)]. A common feature of these two techniques is the generation of a 
set of DNA fragments which are analyzed by electrophoresis. The techniques 
differ in the methods used to prepare these fragments. 
With the Maxam-Gilbert technique, DNA fragments are prepared through 
base-specific, chemical cleavage of the piece of DNA to be sequenced. The 
piece of DNA to be sequenced is first 5'-end-labeled with .sup.32 P and 
then divided into four portions. Each portion is subjected to a different 
set of chemical treatments designed to cleave DNA at positions adjacent to 
a given base (or bases). The result is that all labeled fragments will 
have the same 5'-terminus as the original piece of DNA and will have 
3'-termini defined by the positions of cleavage. This treatment is done 
under conditions which generate DNA fragments which are of convenient 
lengths for separation by gel electrophoresis. 
With Sanger's technique DNA fragments are produced through partial 
enzymatic copying (i.e. synthesis) of the piece of DNA to be sequenced. In 
the most common version the piece of DNA to be sequenced is inserted, 
using standard techniques, into a "sequencing vector", a large, circular, 
single-stranded piece of DNA such as the bacteriophage M13. This becomes 
the template for the copying process. A short piece of DNA with its 
sequence complementary to a region of the template just upstream from the 
insert is annealed to the template to serve as a primer for the synthesis. 
In the presence of the four natural deoxyribonucleoside triphosphates 
(dNTP's), a DNA polymerase will extend the primer from the 3'-end to 
produce a complementary copy of the template in the region of the insert. 
To produce a complete set of sequencing fragments, four reactions are run 
in parallel, each containing the four dNTP's along with a single 
dideoxyribonucleoside triphosphate (ddNTP) terminator, one for each base. 
(.sup.32 P-Labeled dNTP is added to afford labeled fragments.) If a dNTP 
is incorporated by the polymerase, chain extension can continue. If the 
corresponding ddNTP is selected, the chain is terminated. The ratio of 
ddNTP to dNTP's is adjusted to generate DNA fragments of appropriate 
lengths. Each of the four reaction mixtures will, thus, contain a 
distribution of fragments with the same dideoxynucleoside residue at the 
3'-terminus and a primer-defined 5'-terminus. 
The terms "terminator", "chain terminator" and "chain terminating 
substrate" are used interchangeably throughout to denote a substrate which 
can be incorporated onto the 3'-end of a DNA or RNA chain by an enzyme 
which replicates nucleic acids in a template-directed manner but, once 
incorporated, prevents further chain extension. In contrast, the natural 
deoxynucleotide substrates can be considered to be "chain propagating 
substrates". 
The term "nucleoside" is used throughout to denote a heterocyclic 
base-sugar unit composed of one molecule of pyrimidine or purine (or 
derivatives thereof) and one molecule of a ribose sugar (or derivatives or 
functional equivalents thereof). The term "nucleotide" is used throughout 
to denote either a nucleoside or its phosphorylated derivative. 
In both the Sanger and Mazam-Gilbert methods, base sequence information 
which generally cannot be directly determined by physical methods has been 
converted into chain-length information which can be determined, this 
determination can be accomplished through electrophoretic separation. 
Under denaturing conditions (high temperature, urea present, etc.), short 
DNA fragments migrate as if they were stiff rods. If a gel matrix is 
employed for the electrophoresis, the DNA fragments will be sorted by 
size. The single-base resolution required for sequencing can usually be 
obtained for DNA fragments containing up to several hundred bases. 
To determine a full sequence, the four sets of fragments produced by either 
Maxam-Gilbert or Sanger methodology are subjected to electrophoresis in 
four parallel lanes. This results in the fragments being spatially 
resolved along the length of the gel. The pattern of labeled fragments is 
typically transferred to photosensitive film by autoradiography (i.e. an 
exposure is produced by sandwiching the gel and the film for a period of 
time). The developed film shows a continuum of bands distributed in the 
four lanes, often referred to as a sequencing ladder. The ladder is read 
by visually scanning the film (starting with the short, faster moving 
fragments) and determining the lane in which the next band occurs for each 
step on the ladder. Since each lane is associated with a given base (or 
combination of bases in the Maxam-Gilbert case), the linear progression of 
lane assignments translates directly into base sequence. 
The Sanger and Maxam-Gilbert methods for DNA sequencing are conceptually 
elegant and efficacious but they are operationally difficult and 
time-consuming. Analysis of these techniques shows that many of the 
problems stem from the use of a single radioisotopic reporter. [A reporter 
can be defined as a chemical group which has a physical or chemical 
characteristic which can be readily measured or detected by appropriate 
physical or chemical detector systems or procedures. Ready detectability 
can be provided by such characteristics as color change, luminescence, 
fluorescence, or radioactivity; or it may be provided by the ability of 
the reporter to serve as a ligand recognition site to form specific 
ligand-ligand complexes which contain groups detectable by conventional 
(e.g., colorimetric, spectrophotometric, fluorometric or radioactive) 
detection procedures. The ligand-ligand complexes can be in the form of 
protein-ligand, enzymesubstrate, antibody-antigen, carbohydrate-lectin, 
protein-cofactor, protein-effector, nucleic acid-nucleic acid or nucleic 
acid-ligand complexes.] 
The use of short-lived radioisotopes such as .sup.= P at high specific 
activity is problematic from both a logistical and a health-and-safety 
point of view. The short half-life of .sup.32 P necessitates the 
anticipation of reagent requirements several days in advance and prompt 
use of the reagent. Once .sup.32 P-labeled DNA sequencing fragments have 
been generated, they are prone to self-destruction and must be immediately 
subjected to electrophoretic analysis. The large electrophoresis gels 
required to achieve single base separation lead to large volumes of 
contaminated buffer leading to waste disposal problems. The 
autoradiography required for subsequent visualization of the labeled DNA 
fragments in the gel is a slow process (overnight exposures are common) 
and adds considerable time to the overall operation. Finally, there are 
the possible health risks associated with use of such potent 
radioisotopes. 
The use of only a single reporter to analyze the position of four bases 
lends considerable operational complexity to the overall process. The 
chemical/enzymatic steps must be carried out in separate vessels and 
electrophoretic analysis must be carried out in four parallel lanes. 
Thermally induced distortions in mobility result in skewed images of 
labeled DNA fragments (e.g. the smile effect) which, in turn, lead to 
difficulties in comparing the four lanes. These distortions often limit 
the number of bases that can be read on a single gel. 
The long times required for autoradiographic imaging along with the 
necessity of using four parallel lanes force a "snapshot" mode of 
visualization. Since simultaneous spatial resolution of a large number of 
bands is needed, very large gels must be used. This results in additional 
problems: large gels are difficult to handle and are slow to run, adding 
more time to the overall process. 
Finally there is a problem of manual interpretation. Conversion of a 
sequencing ladder into a base sequence is a time-intensive, error-prone 
process requiring the full attention of a highly skilled scientist. 
Numerous attempts have been made to automate the reading and some 
mechanical aids do exist, but the process of interpreting a sequence gel 
is still painstaking and slow. 
To address these problems, replacement of .sup.32 P/autoradiography with an 
alternative non-radioisotopic reporter/detection system has been 
considered. Such a detection system would have to be exceptionally 
sensitive to achieve a sensitivity comparable to .sup.32 P; each band on a 
sequencing gel contains on the order of 10.sup.-16 mole of DNA. One method 
of detection which is capable of reaching this level of sensitivity is 
fluorescence. DNA fragments could be labeled with one or more fluorescent 
labels (fluorescent dyes). Excitation with an appropriate light source 
would result in a characteristic emission from the label thus identifying 
the band. 
The use of fluorescent labels as opposed to radioisotopic labels, would 
allow easier tailoring of the detection system to this particular 
application. For example, the use of four different fluorescent labels 
distinguishable on the basis of some emission characteristic (e.g. 
spectral distribution, life-time, polarization) would allow linking a 
given label uniquely with the sequencing fragments associated with a given 
base. With this linkage established, the fragments could be combined and 
resolved in a single lane and the base assignment could be made directly 
on the basis of the chosen emission characteristic. 
So far two attempts to develop a fluorescence-based DNA sequencing system 
have been described. The first system, developed at the California 
Institute of Technology, has been disclosed in L. M. Smith, West German 
pat. Appl. #DE 3446635 Al (1984); L. E. Hood et al., West German Pat. 
Appl. #DE 3501306 Al (1985); L. M. Smith et al., Nucleic Acids Research, 
Vol. 13 2399-2412 (1985); and L. M. Smith et al., Nature, Vol. 321, 
674-679 (1986). This system conceptually addresses the problems described 
in the previous section but the specifics of the implementation render 
Smith's approach only partially successful. For example, the large 
wavelength range of the emission maxima of the fluorescently-labeled DNA 
sequencing fragments used in this system make it difficult to excite all 
four dyes efficiently with a single monochromatic source. More 
importantly, the significant differential perturbations in electrophoretic 
mobility arising from dyes with different net charges make it difficult or 
impossible to perform single-lane sequencing with the set of dyes used in 
this system. These difficulties are explicitly pointed out by Smith et al. 
In general, the methodology used to prepare the fluorescence-labeled 
sequencing fragments creates difficulties. For Maxam-Gilbert sequencing, 
5'-labeled oligonucleotides are enzymatically ligated to "sticky ended", 
double-stranded fragments of DNA produced through restriction cleavage 
This limits one to sequencing fragments produced in this fashion. For 
Sanger sequencing, 5'-labeled oligonucleotides are used as primers. Four 
special primers are required. To use a new vector system one has to go 
through the complex process of synthesizing and purifying four new 
dye-labeled primers. The same thing will be true whenever a special primer 
is needed. 
The use of labeled primers is inferior in other respects as well. The 
polymerization reactions must still be carried out in separate vessels. As 
in the Maxam-Gilbert and Sanger sequencing systems, effectively all 
fragments derived from the labeled primer will be fluorescently labeled. 
Thus, the resulting sequencing pattern will retain most of the common 
artifacts (e.g. false or shadow bands, pile-ups) which arise when 
enzymatic chain extension is interrupted by processes other than 
incorporation of a chain terminator. 
In a second approach, W. Ansorge et al., J. Biochem. Biophys. Methods. Vol. 
13, 315-323 (1986), have disclosed a non-radioisotopic DNA sequencing 
technique in which a single 5'-tetramethylrhodamine fluorescent label is 
covalently attached to the 5'-end of a 17-base oligonucleotide primer. 
This primer is enzymatically extended in four vessels through the standard 
dideoxynucleotide sequencing chemistry to produce a series of 
enzymatically copied DNA fragments of varying length. Each of the four 
vessels contains a dideoxynucleotide chain terminator corresponding to one 
of the four DNA bases which allows terminal base assignment from 
conventional electrophoretic separation in four gel lanes. The 
5'-tetramethylrhodamine fluorescent label is excited by an argon ion laser 
beam passing through the width of the entire gel. Although this system has 
the advantage that a fluorescent reporter is used in place of a 
radioactive reporter, all of the disadvantages associated with 
conventional sequencing and with preparing labeled primers still remain. 
Until now, no one has created a DNA sequencing system which combines the 
advantages of fluorescence detection with terminator labeling. If 
appropriate fluorescently-labeled chain terminators could be devised, 
labeled sequencing fragments would be produced only when a labeled chain 
terminator is enzymatically incorporated into a sequencing fragment, 
eliminating many of the artifacts associated with other labeling methods. 
If each of the four chain terminators needed to sequence DNA were 
covalently attached to a different distinguishable fluorescent reporter, 
it should be possible, in principle, to incorporate all four terminators 
during a single primer extension reaction and then to analyze the 
resulting sequencing fragments in a single gel lane. If such 
fluorescently-labeled chain terminators could be devised, these compounds 
would probably also be useful for other types of enzymatic labeling of 
nucleic acids. In particular, analogs of fluorescently-labeled chain 
terminators could be designed to use other, non-fluorescent, reporters or 
to serve as chain-propagating substrates for enzymes which replicate 
nucleic acids in a template-directed manner (e.g., reverse transcriptase, 
RNA polymerase or DNA polymerase). Introducing a reporter into DNA in a 
manner useful for sequencing is one of the most difficult nucleic acid 
labeling problems. Compounds and/or strategies developed for DNA 
sequencing are also likely to be applicable to many other labeling 
problems. 
To be useful as a chain-terminating substrate for fluorescence-based DNA 
sequencing, a substrate must contain a fluorescent label and it must be 
accepted by an enzyme useful for sequencing DNA. Suitable substrate 
candidates are expected to be derivatives or analogs of the 
naturally-occurring nucleotides. Because of the expectation that a 
fluorescent label and a nucleotide will not fit into the active site of a 
replication enzyme at the same time, a well-designed substrate must have 
the fluorescent label separated from the nucleotide by a connecting group 
of sufficient length and appropriate geometry to position the fluorescent 
label away from the active site of the enzyme. The nature of the 
connecting group can vary with both the label and the enzyme used. For 
ease of synthesis and adaptability to variations in label and/or enzyme 
requirements however, it is most convenient to consider the connecting 
group as consisting of a linker which is attached to the nucleotide and to 
the fluorescent label. 
In the design of fluorescently-labeled chain terminators for DNA 
sequencing, the linker must satisfy several requirements: 
1) one must be able to attach the same or a functionally equivalent linker 
to all four bases found in DNA; 
2) the linker must not prevent the labeled nucleotide from being utilized 
effectively as a chain terminating substrate for an enzyme useful for DNA 
sequencing; 
3) the linker (plus optional spacer and label) must perturb the 
electrophoresis of oligonucleotides to which it is attached in a manner 
which is independent of the base to which it is attached; 
4) the attachment of the linker to the base and the spacer or label must be 
stereoselective and regioselective to produce a single well-defined 
nucleotide substrate; and 
5) the linker should preferably contain a primary or secondary amine for 
coupling with the label. 
Although five different types of amine linkers have been disclosed for 
attaching labels to nucleotides and oligonucleotides (see below), none of 
these linkers meet all five of the requirements listed above for use in a 
chain terminating substrate useful in DNA sequencing. 
Bergstrom et al., J. Am. Chem. Soc., Vol. 98, 1587 (1976), disclose a 
method for attaching alkene-amino and acrylate side-chains to nucleosides 
by Pd(II)-catalyzed coupling of 5-mercurio-uridines to olefins. Ruth 
PCT/US84/00279 discloses the use of the above side-chains as linkers for 
the attachment of reporters to non-enzymatically synthesized 
oligonucleotides. Langer et al., Proc. Nat. Acad. Sci. USA, Vol. 78, 6633 
(1981), disclose the use of allylamino linkers for the attachment of 
reporters to nucleotides. The disadvantages of these linkers include the 
difficulty of preparing regioselectively the appropriate mercurial 
nucleotide precursors, the difficulty of separating the mixture of 
products generated by some of these nucleotide/olefin coupling reactions, 
and the potential lability of vinyl substituted nucleosides. Klevan et 
al., WO 86/02929, disclose a method for attaching linkers to the N4 
position of cytidine and the N6 position of adenosine. The disadvantage of 
this method is that there is no analogous site in uridine and guanosine 
for attaching a linker. 
Another potential linker which might satisfy the five requirements listed 
above is an alkynylamino linker, in which one end of the triple bond is 
attached to the nucleoside and the other end of the triple bond is 
attached to a group which contains a primary or secondary amine. To insure 
chemical stability, the amine should not be directly attached to the 
triple bond. Some methods of attaching alkyne groups to nucleosides have 
been disclosed (see below). 
Barr et al., J. Chem. Soc., perkins Trans. I, 1263-1267 (1978), disclose 
the syntheses of 5-ethynyluridine 2'-deoxy-5-ethynyluridine, 
5-ethynylcytosine, 5-ethynylcytidine, 2'-deoxy-5-ethynylcytidine and the 
.alpha.-anomers of the 2'-deoxyribonucleosides. The 
2'-deoxyribonucleosides were prepared by constructing the heterocycles, 
coupling with a functionalized 2-deoxy sugar, separating the anomeric 
mixtures, and removing the protecting groups on the sugars. 
Bergstrom et al., J. Am. Chem. Soc., Vol. 100, 8106 (1978), disclose the 
palladium-catalyzed coupling of alkenes with 5-mercuri or 5-iodo 
derivatives of uracil nucleosides. This method was reported to fail in 
analogous reactions of alkynes with uracil nucleoside derivatives. 
Vincent et al., Tetrahedron Letters, Vol. 22, 945-947 (1981), disclose the 
synthesis of 5-alkynyl-2'-deoxyuridines by the reaction of 
0-3',5'-bis(trimethylsilyl)deoxyuridine with alkynylzinc reagents in the 
presence of palladium or nickel catalysts 
[dichloro-bis(triphenylphosphine)palladium(II). 
dichloro-bis(benzonitrile)palladium(II) or 
dichloro(ethylene-(bis(diphenylphosphine))nickel(II)]. 
Robins et al., J. Org. Chem., Vol. 48, 1854-1862 (1983). disclose a method 
for coupling terminal alkynes, HC.tbd.CR (R.dbd.H, alkyl, phenyl, 
trialkylsilyl, hydroxyalkyl or protected hydroxyalkyl), to 
5-iodo-1-methyluracil and 5-iodouracil nucleosides (protected as their 
p-toluyl esters) in the presence of bis(triphenylphosphine)palladium(II) 
chloride and copper(I) iodide in warm triethylamine. When 
3',5'-di-O-acetyl-5-iodo-2'deoxyuridine was reacted with hexyne, 
4-(p-toluyloxy)butyne, 4-(tetrahydropyranyloxy) or 4-(trityloxy)butyne, 
the major products were the cyclized furano[2,3-d]pyrimidin-2-ones rather 
than the desired alkynyluridines. 
None of the above references discloses a method for attaching an 
alkynylamino linker to nucleosides. The methodology of Bergstrom fails, 
and that of Barr is not directly applicable. The catalysts used by Robins 
et al. and Vincent et al. have the potential to promote numerous 
undesirable side reactions (e.g., cyclization or intermolecular 
nucleophilic addition of the amine to an alkyne) when the alkyne contains 
an amino group. Coupling reactions have been reported only with 
iodonucleosides which contain an electron-deficient uracil base. Since 
Pd-catalyzed coupling reactions generally work best with 
electron-deficient aryl iodides, problems may be anticipated in coupling 
alkynes to any of the other three bases (which are all more electron-rich 
than uracil). 
There remains a need for alkynylamino nucleotides and for methods 
permitting their preparation. 
SUMMARY OF THE INVENTION 
The compounds of this invention are alkynylamino-nucleotides having the 
structure: 
EQU Nuc--C.tbd.C--R.sub.1 --NR.sub.2 R.sub.3 (I), 
wherein R.sub.1 is a substituted or unsubstituted diradical moiety of 1-20 
atoms. R.sub.1 can be straight-chained alkylene, C.sub.1 -C.sub.20, 
optionally containing within the chain double bonds, triple bonds, aryl 
groups or heteroatoms such as N, O or S. The heteroatoms can be part of 
such functional groups as ethers, thioethers, esters, amines or amides. 
Preferably, R.sub.1 is straight-chained alkylene, C.sub.1 -C.sub.10 ; most 
preferably R.sub.1 is --CH.sub.2 --. Substitutents on R.sub.1 can include 
C.sub.1 -C.sub.6 alkyl, aryl, ester, ether, amine, amide or chloro groups; 
R.sub.2 and R.sub.3 are independently H, C.sub.1 -C.sub.4 alkyl, or a 
protecting group such as acyl, alkoxycarbonyl or sulfonyl. Preferably 
R.sub.2 is H, and R.sub.3 is H or trifluoroacetyl; 
Nuc (nucleotide) is R.sub.4 -Het (heterocyclic base: 
##STR1## 
Z is H or NH.sub.2 ; and 
R.sub.4 is a sugar or sugar-like moiety: 
##STR2## 
and wherein R.sub.5 is H, PO.sub.3 H.sub.2, P.sub.2 O.sub.6 H.sub.3, 
P.sub.3 O.sub.9 H.sub.4 or salts thereof, 
and when R.sub.7 .dbd.R.sub.8 .dbd.H, the R.sub.6 .dbd.H, OH, F, N.sub.3 or 
NH.sub.2 ; or when R.sub.7 .dbd.H and R.sub.8 .dbd.OH. then R.sub.6 .dbd.H 
or OH; or when R.sub.7 .dbd.OH and R.sub.8 .dbd.H, then R.sub.6 .dbd.OH. 
The labeled alkynylamino-nucleotides of this invention are structure I 
where R.sub.3 is a reporter (label). 
DETAILED DESCRIPTION OF THE INVENTION 
The strategy used to incorporate reporters in the DNA sequencing fragments 
in a base-specific fashion is a critical feature of any DNA sequencing 
system. The use of the alkynylamino-nucleotides of this invention permit 
the modification of the Sanger methodology most advantageously by 
attaching a reporter (label) to an alkynylamino-nucleotide chain 
terminator. Although the reporter can be chosen from a wide variety of 
readily detectable groups (labels), for convenience the preferred approach 
is illustrated below using fluorescent reporters. 
This approach offers a number of operational advantages. Most importantly, 
terminator labeling firmly links the attached reporter with the 
base-specific termination event. Only DNA sequencing fragments resulting 
from bona fide termination events will carry a reporter. This eliminates 
many of the artifacts observed in conventional sequencing. This approach 
also affords complete flexibility in the choice of sequencing vector since 
no special primers are involved. Automation is facilitated by the fact 
that the reporters are carried by four low molecular-weight reagents which 
can be selectively introduced in a single reaction. 
There are no inherent operational disadvantages; the problems with this 
approach are encountered in the design stage. In general, the enzymes used 
for sequencing DNA are highly substrate selective and there is no reason a 
priori to expect to be able to make a nucleoside triphosphate with a 
covalently attached reporter that is an efficient chain-terminating 
substrate for a sequencing enzyme. It might be thought that attachment of 
a reporter to a substrate would cause sufficiently large changes in the 
steric and electronic character of the substrate to make it unacceptable 
to the enzyme or, even if accepted it would not be incorporated in the DNA 
chain. It has been found, however, that the small size of the alkynylamino 
linker of this invention and the ability to attach the alkynylamino linker 
to the 5-position of the pyrimidine nucleotides and the 7-position of the 
purine nucleotides provide labeled chain-terminating substrates that do 
not interfere excessively with the degree or fidelity of substrate 
incorporation. 
The alkynylamino-nucleotides of this invention will be illustrated through 
the description of fluorescently-labeled alkynylamino nucleotide chain 
terminators. To delineate the structural scope and rationale of 
fluorescently-labeled alkynylaminonucleotides of this invention it is 
useful to break the labeled structure (I) into five components: 
##STR3## 
(i) a triphosphate moiety, R.sub.5 (ii) a "sugar", R.sub.4 
(iii) a heterocyclic base (Het), 
(iv) a linker (--C.tbd.CR.sub.1 NR.sub.2 --), and 
(v) a fluorescent label, R.sub.3. 
(i) Triphosphate Moiety (R.sub.5) 
The triphosphate moiety or a close analog (e.g., .alpha.-thiotriphosphate) 
is an obligate functionality for any enzyme substrate, chain terminating 
or otherwise. This functionality provides much of the binding energy for 
the substrate and is the actual site of the enzyme-substrate reaction. 
##STR4## 
(ii) Sugar (R.sub.4) 
The "sugar" portion corresponds to the 240 -deoxyribofuranose structural 
fragment in the natural enzyme substrates. This portion of the molecule 
contributes to enzyme recognition and is essential for maintaining the 
proper spatial relationship between the triphosphate moiety and the 
heterocyclic base. To be useful for DNA sequencing, when the "sugar" is a 
ribofuranose, the 3'-.alpha.-position must not have a hydroxyl group 
capable of being subsequently used by the enzyme. The hydroxyl group must 
either be absent, replaced by another group or otherwise rendered 
unusable. Such sugars will be referred to as chain-terminating sugars. It 
is known that a number of modified furanose fragments can fulfill this 
requirement, including: 
2'3'-dideoxy-.beta.-D-ribofuranosyl [(a), F. Sanger et al., Proc. Nat. 
Acad. Sci. USA, Vol. 74, 5463-5467 (1977)], 
.beta.-D-arabinofuranosyl, [(b) F. Sanger et al., Proc. Nat. Acad. Sci. 
USA, Vol. 74, 5463-5467 (1977)], 
3'-deoxy-.beta.-D-ribofuranosyl [(c), Klement et al., Gene Analysis 
Technology, Vol. 3, 59-66 (1986)]. 
3'-amino-2',3'-dideoxy-.beta.-D-ribofuranosyl [(d), Z. G. Chidgeavadze et 
al., Nuc. Acids Res., Vol. 12, 1671-1686 (1984)], 
2',3'-dideoxy-3'-fluoro-B-D-ribofuranosyl [(e), Z. G. Chidgeavadze et al., 
FEBS Lett., Vol. 183, 275-278 (1985)], and 
2',3'-dideoxy-2',3'-didehydro-.beta.-D-ribofuranosyl [(f), Atkinson et al., 
Biochem., Vol. 8, 4897-4904 (1969)]. 
##STR5## 
Acyclonucleoside triphosphates, (AcyNTP's), in which the so-called sugar is 
an acyclic group [e.g., 2-oxyethoxymethyl, (g)], can also be used as chain 
terminators in DNA sequencing by the Sanger methodology. The use of 
AcyNTP's as chain-terminating substrates has been demonstrated by carrying 
out conventional Sanger sequencing (.sup.32 P reporter) with the AcyNTP's 
substituting for the ddNTP's. The sequencing ladders produced with 
AcyNTP's were virtually identical to those produced with ddNTP's, except 
that a higher concentration of AcyNTP (approximately 10.times.) was 
required to obtain a similar distribution of DNA fragments. The AcyNTP's 
are effective with both DNA polymerase I (Klenow fragment) and AMV reverse 
transcriptase. The alkynylamino derivatives of AcyNTP's are therefore also 
expected to function as chain-terminating substrates. 
The AcyNTP's have the advantage of being more easily synthesized than the 
ddNTP's. While the synthesis of ddNTP's is not a major problem in 
conventional sequencing, it is significant when structurally complex, 
fluorescently-labeled chain terminators are being prepared. The use of the 
2-oxyethoxymethyl group as a sugar greatly simplifies reagent synthesis 
while maintaining acceptable performance. 
Medicinal research has identified other sugar modifications which can be 
useful for DNA sequencing. For example, 3'-azido-2',3'-dideoxythymidine, 
[AZT; Mitsuya et al., Proc. Nat. Acad. Sci. USA,Vol. 82, 7096-7100 (1985)] 
and 9-[2'-hydroxy-1'-(hydroxymethyl)ethoxymethyl]guanine [DHPG; Aston et 
al., Biochem. Biophys. Res. Comm., Vol. 108, 1716-1721 (1982)] are two 
antiviral agents which are presumed to act by being converted to 
triphosphates which cause chain termination of DNA replication. Nucleoside 
triphosphates with such sugar units can also be useful for DNA sequencing. 
(iii) Heterocyclic Base (Het) 
The heterocyclic base functions as the critical recognition element in 
nucleic acids, acting as a hydrogen-bonding acceptor and donor in a 
particular spatial orientation. The heterocyclic base elements are 
essential for incorporation with the high fidelity necessary for accurate 
sequencing. This structural part is also the site of attachment of the 
linker. 
Preferred heterocyclic bases include: uracil (h), cytosine (i), 
7-deazaadenine (j), 7-deazaguanine (k), and 7-deazahypoxanthine (l). The 
unnatural 7-deazapurines can be employed to attach the linker without 
adding a net charge to the base portion and thereby destabilizing the 
gIycosidic linkage. In addition, other heterocyclic bases which are 
functionally equivalent as hydrogen-bonding donors and acceptors can be 
used, e.g., 8-aza-7-deazapurines and 3,7-dideazaadenine can be used in 
place of 7-deazapurines and 6-azapyrimidines can be used in place of 
pyrimidines. (To simplify the nomenclature, the heterocyclic bases are 
named and numbered throughout as 7-deazapurines.) 
##STR6## 
(iv) Linker 
The linker is an alkynylamino group in which one end of the triple bond is 
attached to an amine through a substituted or unsubstituted diradical 
moiety, R.sub.1, of 1-20 atoms: the other end of the triple bond is 
covalently attached to the heterocyclic base at the 5-position for 
pyrimidines or the 7-position (purine numbering) for the 7-deazapurines. 
The amine nitrogen of the alkynylamino group is attached to a reactive 
functional group (e.g., carbonyl) on the fluorescent label. The linker 
must not significantly interfere with binding to or incorporation by the 
DNA polymerase. The diradical moiety can be straight-chained alkylene, 
C.sub.1 -C.sub.20, optionally containing within the chain double bonds, 
triple bonds, aryl groups or heteroatoms such as N, O or S. The 
heteroatoms can be part of such functional groups as ethers, thioethers 
esters, amines or amides. Substituents on the diradical moiety can include 
C.sub.1 -C.sub.6 alkyl, aryl, ester, ether, amine, amide or chloro groups. 
Preferably, the diradical moiety is straight-chained alkylene, C.sub.1 
-C.sub.10 ; most preferably the diradical is --CH.sub.2 --. 
(v) Fluorescent Label (R.sub.3) 
The fluorescent label provides detectable, emitted radiation following 
excitation by absorption of energy from an appropriate source, such as an 
on ion laser. It is desirable to have unique, distinguishable fluorescent 
reporters for each DNA base encountered in sequencing applications. 
A family of reporters useful for fluorescent labeling in a DNA sequencing 
method based on labeled chain-terminators can be derived from the known 
dye, 9-carboxyethyl-6-hydroxy-3-oxo-3H-xanthene [S. Biggs et al., J. Chem. 
Soc., Vol. 123, 2934-2943 (1923)]. This xanthene family has the general 
structure, 1, 
##STR7## 
where R.sub.9 and R.sub.10 include H, lower alkyl, lower alkoxy, halo, and 
cyano. 
A preferred set of dyes suitable for DNA sequencing is structure 1, a) 
R.sub.9 .dbd.R.sub.10 .dbd.H, abs. 487 nm, emis. 505 nm; b) R.sub.9 
.dbd.H, R.sub.10 .dbd.CH.sub.3, abs. 494 nm, emis. 512 nm; c) R.sub.9 
.dbd.CH.sub.3, R.sub.10 .dbd.H, abs. 501 nm, emis. 519 nm; and d) R.sub.9 
.dbd.R.sub.10 .dbd.CH.sub.3, abs. 508 nm, emis. 526 nm. The instruments 
described by L. M. Smith L. E. Hood et al., L. M. Smith et al. and W. 
Ansorge et al. are capable of detecting sequencing fragments labeled with 
any one of these dyes at concentrations suitable for DNA sequencing, but 
these instruments are not capable or discriminating among the above set of 
four dyes. A method for discriminating the dyes and using this information 
to determine DNA sequences is disclosed in copending application Ser. No. 
07/057,565, hereby incorporated by reference. 
This application discloses a system for sequencing DNA. comprising a means 
for detecting the presence of radiant energy from closely-related yet 
distinguishable reporters, which are covalently attached to compounds 
which function as chain terminating nucleotides in a modified Sanger DNA 
chain elongation method. One distinguishable fluorescent reporter is 
attached to each of four dideoxynucleotide bases represented in Sanger DNA 
sequencing reactions, i.e., dideoxynucleotides of adenine guanine, 
cytosine and thymine. These reporter-labeled chain terminating reagents 
are substituted for unlabeled chain terminators in the traditional Sanger 
method and are combined in reactions with the corresponding 
deoxynucleotides, an appropriate primer, template, and polymerase. The 
resulting mixture contains DNA fragments of varying length that differ 
from each other by one base which terminate on the 3' end with uniquely 
labeled chain terminators corresponding to each of the four DNA bases. 
This new labeling method allows elimination of the customary radioactive 
label contained in one of the deoxynucleotides of the traditional Sanger 
method. 
Detection of these reporter labels can be accomplished with two stationary 
photomultiplier tubes (PMT's) which receive closely-spaced fluorescent 
emissions from laser-stimulated reporters attached to chain terminators on 
DNA fragments. These fragments can be electrophoretically separated in 
space and/or time to move along an axis perpendicular to the sensing area 
of the PMT's. The fluorescent emissions first pass through a dichroic 
filter having both a transmission and reflection characteristic, placed so 
as to direct one characteristic (transmission) to one PMT, and the other 
characteristic (reflection) to the other PMT. In this manner, different 
digital signals are created in each PMT that can be ratioed to produce a 
third signal that is unique to a given fluorescent reporter, even if a 
series of fluorescent reporters have closely spaced emissions. This system 
is capable of detecting reporters which are all efficiently excited by a 
single laser line, such as 488 nm, and which have closely spaced emissions 
whose maxima usually are different from each other by only 5 to 7 nm. 
Therefore, the sequential base assignments in a DNA strand of interest can 
be made on the basis of the unique ratio derived for each of the four 
reporter-labeled chain terminators which correspond to each of the four 
bases in DNA. 
Since these xanthene dyes contain reactive functional groups, the above 
copending application also discloses the design and preparation of 
reagents which are useful for attaching these dyes to the amino group of 
an alkynylamino linker. N-hydroxysuccinimide esters 2 (where R.sub.9 and 
R.sub.10 are as defined structure 1) are preferred examples of such 
reagents. During the preparation of 2, a sarcosine group is added to the 
basic dye structure to minimize side reactions. In the above copending 
application, this optional sarcosine group is referred to as a "spacer". 
Since only the preferred reagents are used herein, the fluorescent part of 
a labeled alkynylamino chain terminator is considered to include a 
sarcosine spacer. After the N-hydroxysuccinimide leaving group has been 
displaced by the amino group of the linker, the fluorescent dye (part 
structure 1) is liberated by treatment with concentrated ammonium 
hydroxide. N-hydroxysuccinimide esters are acylating agents which react 
selectively with highly-nucleophilic amino groups such as the ones present 
in the alkynylamino linkers described above. Control experiments have 
demonstrated that N-hydroxysuccinimide esters similar to 2 react much more 
slowly with the amino groups of the heterocyclic base than with the linker 
amino group. If reaction occurs to a small extent with the heterocyclic 
amino groups, it has been discovered that the resulting amides are 
hydrolyzed by the ammonium hydroxide treatment which liberates the dye. It 
is therefore possible to use esters such as 2 to attach selectively any 
reporter such as a fluorescent dye to the amino group of the linker 
without modifying the nucleotide in an unwanted fashion. 
Fluorescently-labeled alkynylamino-nucleotide chain-terminators of this 
invention function as well in DNA sequencing with AMV reverse 
transcriptase as the corresponding substrates containing allylamino 
linkers which are disclosed in copending application Ser. No. 07/057,565. 
However, the compounds of this invention are easier to synthesize than the 
corresponding substrates containing allylamino linkers because the 
alkynylamino linkers are more easily attached to a preselected position on 
the various bases needed for DNA sequencing. In addition, the alkynylamino 
linkers can be attached to the nucleotides in higher yield. Finally, 
nucleotides containing an alkyne in conjugation with a heterocyclic ring 
are expected to be more stable than corresponding nucleotides containing 
an alkene. Suitable fluorescently-labeled chain terminators derived from 
alkynylamino-nucleotides are shown by structure 3, where Nuc, R.sub.1 
-R.sub.4 and R.sub.6 -R.sub.8 are as defined above, R.sub.5 .dbd.HO.sub.9 
P.sub.3 and provided that when R.sub.8 is H or OH, R.sub.6 must not be OH. 
##STR8## 
Scheme 1 describes methods for preparing the alkynylamino nucleotides of 
this invention where the sugar is a 2,3-dideoxyribofuranosyl group. These 
methods are compatible with all of the sugars of this invention. When 
combined with known methods for modifying the sugars of nucleotides, these 
methods can be used to prepare alkynylamino nucleotides in which the 
2,3-dideoxyribofuranosyl group is replaced by the other sugars of the 
invention. 
##STR9## 
A variety of routes can be used to prepare the first key intermediates, the 
iodonucleosides (4). (In some instances, the corresponding 
bromonucleosides can be used in place of the iodonucleosides.) 
5-Iodo-2',3'-dideoxyuridine can be prepared by treating 
2',3'-dideoxyuridine [Pfitzer et al., J. Org. Chem., Vol. 29, 1508 (1964)] 
with ICl [Robins et al., Can. J. Chem., Vol. 60, 554-557 (1982)]. 
5-Iodo-2',3'-dideoxycytidine can be prepared by converting 
2',3'-dideoxycytidine (Rayco Co.) to the corresponding 5-mercurio 
nucleoside [Bergstrom et al., J. Carbohydrates, Nucleosides and 
Nucleotides, Vol. 4, 257-269 (1977)] and then treating with iodine. 
Although methods for preparing 7-deazaguanosine and 
2'-deoxy-7-deazaguanosine are known, it has been shown [Seela et al., 
Chem. Ber., Vol. III, 2925-2930 (1978)] that electrophilic attack on 
7-deaqzaguanines occurs at both the 7- and 8-positions. However, it has 
now been found that the desired 7-iodo-7-deazapurines can be obtained by 
treatment of 6-methoxy-2-thiomethyl-7-deazapurines with N-iodosuccinimide, 
followed by replacement of the 2-thiomethyl and 6-methoxy substituents as 
shown in Scheme 2 and described in Example 3. The use of N-iodosuccinimide 
for regioselective iodination of a 7-deazapurine ring system is 
unprecedented. 
##STR10## 
7-Iodo-2',3'-dideoxy-7-deazaadenosine can be prepared by deoxygenation of 
tubercidin, followed by mercuration/iodination (Scheme 3). The 
deoxygenation reactions were adapted from procedures disclosed by Moffatt 
et al., [J. Am. Chem. Soc., Vol. 95, 4016-4030 (1972)] and Robins et al., 
[Tetrahedron Lett., Vol. 25, 367-340 (1984)] to give an improved synthesis 
of 2',3'-dideoxy-7-deazaadenosine as shown in Scheme 3 and described in 
Example 4. 
7-Iodo-7-deazaadenosine can be prepared by regioselective 
mercuration/iodination of tubercidin (7-deazaadenosine), as reported by 
Bergstrom et al., J. Carbohydrates, Nucleosides and Nucleotides, Vol. 5, 
285-296 (1978) and Bergstrom et al., J. Org. Chem., Vol. 46, 1424 (1981). 
##STR11## 
Alternative routes to 7-iodo-2',3'-dideoxy-7-deazaadenosine can be used 
which do not use tubercidin, an expensive fermentation product, as a 
starting material. These routes are shown in Schemes 4 and 5 and are 
described in Examples 5 and 6. In one of these routes, the problem of 
regioselectively introducing an iodine in the 7-position was solved by 
another unprecedented iodination. In this case, treatment of 
6-chloro-7-deazapurine 32 with iodine monochloride afforded only the 
7-iodo regioisomer 33. 
##STR12## 
Although a method for coupling terminal alkynes with protected 5-iodouracil 
nucleosides using a Pd(II)/Cu(I) catalyst has been reported by Robins et 
al. [Tetrahedron Lett., Vol. 22, 421-424 (1981)], this method does not 
effect the desired coupling between alkynylamines (e.g., propargylamine) 
and the unprotected 5-iodo-pyrimidine or 7-iodo-purine nucleosides. The 
ability to use alkynylamines in direct coupling was highly desirable to 
provide directly compounds with an amine group for subsequent attachment 
of the fluorescent label. Similarly, a method using unprotected 
nucleosides was sought to provide a more direct route to the desired 
compounds by eliminating an otherwise unnecessary series of 
protection/deprotection reactions. 
Under the conditions described below. alkynylamines were successfully 
coupled to a variety of halonucleosides in excellent Yields using a 
Pd(O)/Cu(I) catalyst. This coupling reaction was also successful when the 
alkynylamine nitrogen was protected by an acyl group such as acetyl and 
trifluoroacetyl, alkoxycarbonyl group such as 9-fluorenylmethyloxycarbonyl 
group, and a sulfonyl group such as p-toluenesulfonyl group. Unexpectedly, 
the number of carbon atoms between the amino group and the triple bond was 
found not to be critical in the procedure described below; 
3-amino-1-propyne (propargylamine), 5-amino-1-pentyne, 
N-(2-propynyl)trifluoroacetamide, 
N-(4-pentynyl)-trifluoroacetamide and N-(11-dodecynyl)-trifluoroacetamide 
were all successfully used in the coupling reaction. 
The broad success of this Pd(O)/Cu(I) catalyzed coupling reaction is 
unexpected in view of the art. For example Bergstrom et al. [J. Am Chem. 
Soc. Vol. 100, 8106 (1978)] noted that alkYnes failed to couple to 
5-mercuri or 5-iodo derivatives of uracil nucleosides using Pd catalysts. 
Also. Robins et al. [J. Org. Chem. Vol. 48, 1854-1862 (1983)] disclosed 
that the Pd(II)/Cu(I) catalyzed reactions of 
3',5'-di-O-acetyl-5-iodo-2'-deoxyuridine frequently produced cyclized 
products. When the process described below is used, the coupling succeeds 
even with alkynylamines (such as 5-amino-1-pentyne) which have the 
potential to cyclize readily. 
Typically, the alkynylamino-nucleosides of this invention can be prepared 
by placing the halonucleoside and Cu(I) in a flask, flushing with Ar to 
remove air, adding dry dimethylformamide, followed by addition of the 
alkynylamine triethylamine and Pd(O). The reaction mixture can be stirred 
for several hours, or until thin layer chromatography (TLC) indicates 
consumption of the halonucleoside. When an unprotected alkynylamine is 
used, the alkynylamino-nucleoside can be isolated by concentrating the 
reaction mixture and chromatographing on silica gel using an eluting 
solvent which contains ammonium hydroxide to neutralize the hydrohalide 
generated in the coupling reaction. When a protected alkynylamine is used, 
methanol/methylene chloride can be added to the reaction mixture, followed 
by the bicarbonate form of a strongly basic anion exchange resin. The 
slurry can be then stirred for about 45 min, filtered, and the resin 
rinsed with additional methanol/methylene chloride. The combined filtrates 
can be concentrated and promptly purified by flash-chromatography on 
silica gel using a methanol-methylene chloride gradient. 
The alkynylamino-nucleotides of this invention are preferably prepared from 
5-iodopyrimidine or 7-iodo-7-deazapurine nucleosides, but the analogous 
bromonucleosides can also be used. [The Pd(O)/Cu(I) catalyzed coupling 
reaction can also be used to introduce alkynylamine groups at other 
positions on the aromatic or heteroaromatic ring, provided only that the 
appropriate halonucleotide is available.] 
Suitable alkynylamines for the Pd(O)/Cu(l) catalyzed coupling reaction are 
terminal alkynes wherein the triple bond is attached to an amine by a 
diradical moiety of 1-20 atoms. The diradical moiety can be straight-chain 
alkylene, (C.sub.1 -C.sub.20 e.g., --C.sub.3 H.sub.6 --), or can contain 
double bonds (e.g., as in --CH.dbd.CHCH.sub.2 --). triple bonds (e.g., as 
in --C.tbd.C--CH.sub.2 --) or aryl groups [e.g., (para --C.sub.6 H.sub.4 
--, or para--CH.sub.2 --C.sub.6 H.sub.3 --]. The diradical can also 
contain heteroatoms such as N, O, or S in the chain as part of ether, 
ester, amine, or amido groups. Suitable substituents on the diradical 
moiety can include C.sub.1 -C.sub.6 alkyl, aryl, ester, ether, amine, 
amide or chloro groups. Preferably, the diradical is a straight-chain 
alkylene (C.sub.1 -C.sub.10); most preferably, the diradical is --CH.sub.2 
--. Suitable substituents on the amine are lower alkyl (C.sub.1 -C.sub.4) 
and protecting groups such as trifluoroacetyl. In general, the amine of 
the alkynylamine can be primary, secondary or tertiary. For use as a 
linker, however the alkynylamine is preferably a primary amine. The amine 
of the alkynylamine is usually protected because amine protection is 
required in the next step. The coupled product is also more readily 
purified when this amine is introduced in protected form. A 
trifluoroacetyl protecting group is preferred because it is easily removed 
after the coupling product is converted to the corresponding 
5'-triphosphate. Generally, a 1.5-3.0 fold excess of alkynylamine 
(relative to iodonucleoside) can be used to insure complete conversion of 
the iodonucleoside to an alkynylamino-nucleoside. 
Suitable solvents for the coupling reaction include polar solvents which 
dissolve the iodo- or bromonucleoside and do not decompose the Pd(O)/Cu(I) 
catalyst system. N,N-Dimethylformamide (DMF), acetonitrile, THF, 
dimethylsulfoxide (DMSO), hexamethylphosphoramide (HMPA), and alcohols can 
all be used; solvents which contain small amounts of water are also 
acceptable. Preferably, the solvent is DMF. Preferably, the concentration 
of the halonucleoside is 0.02-1.0M, most preferably 0.2-0.5M. 
Suitable Pd catalysts are Pd(O) complexes, for example, 
tetrakis(triarylphosphine)Pd(O). Preferably, the Pd(O) catalyst is 
tetrakis(triphenylphosphine)Pd(O). The amount of Pd catalyst used is 
generally 1-25 mol % (based on iodonucleoside), preferably 5-15 mol %. The 
larger amounts of catalyst are used to conduct the reaction on a very 
small scale or to decrease the reaction time for coupling. 
The Cu(I) co-catalyst is preferably a cuprous halide or pseudohalide (such 
as cuprous cyanide), most preferably CuI. 
The mole ratio of Cu(I) co-catalyst to Pd(O) catalyst is more than 1.0 but 
less than 20. When a protected alkynylamine is used, the preferred mole 
ratio of Cu/Pd is 2. When the alkynylamine is unprotected, the unhindered 
basic nitrogen atom diminishes the catalytic activity of the copper. In 
this case, a Cu/Pd ratio of 5 is preferred. In either case, no reaction is 
observed at room temperature when Cu/Pd=1. The reaction rate generally 
increases as the Cu/Pd ratio increases. With protected alkynylamines and 
Cu/Pd ratios greater than 2, however, this increase is accompanied by 
increased side-products as indicated by TLC. 
Triethylamine probably serves as an acid-scavenger in this reaction; other 
strongly basic amines can also be used. An excess of the unprotected 
alkynylamine can also serve as the acid-scavenger, but preferably 
triethylamine is added as well. 
Protected alkynylamino nucleosides can be converted to the corresponding 
5'-triphosphates by treatment with phosphorus oxychloride and then 
tri-n-butylammonium pyrophosphate [Ruth et al., Mol. Pharmacol., 415 
(1981)]. The resulting crude triphosphate can be purified at this stage by 
ion-exchange chromatography by eluting with a volatile buffer such as 
triethylammonium bicarbonate. The desired nucleoside triphosphate is 
well-separated from side products, but lyophilization results in some 
removal of the protecting group on the linker nitrogen when this 
protecting group is a trifluoroacetyl group. Deprotection can be completed 
by treatment with 14% aqueous ammonia and the product can again be 
purified by ion exchange chromatography. Since the nature of the linker 
and its protecting group do not appear to block conversion to a 
triphosphate, this methodology can be used to prepare a wide variety of 
nucleoside mono-, di-, and triphosphates with protected or unprotected 
alkynylamino linkers. 
After the preparation of the alkynylamino-nucleotides of this invention, 
the stage is set for the production of any desired reporter-labeled 
alkynylaminonucleotide of this invention. 
The preferred set of four fluorescently-labeled alkynylamino-nucleotide 
chain-terminators (34-37) shown below is derived from 
alkynylamino-nucleotides of this invention. This set of labeled compounds 
is prepared as described in Example 19 by coupling N-hydroxysuccinimide 
esters 2 with alkynylamino nucleoside triphosphate 6, followed by ammonia 
deprotection. 
##STR13## 
These four fluorescently-labeled chain terminators were used in place of 
the standard dideoxynucleotide chain terminators when sequencing DNA using 
AMV reverse transcriptase according to the procedure of Zagursky et al., 
Gene Analysis Techniques, Vol. 2, 89-94 (1985). The resulting 
fluorescentlylabeled sequencing ladders can be analyzed by an instrument 
designed to detect fluorescent molecules as they migrate during gel 
electrophoresis, preferably by the instrument described in U.S. Pat. No. 
4,833,332, to Robertson et al., issued May 23, 1989. A system of this type 
using two filters is described in the copending Robertson et al. 
application, the contents of which are incorporated herein by reference. 
As described in Robertson et al., a pair of modules are positioned above 
and below a plane in which the reporter exciting light beam scans multiple 
lanes on an electrophoresis gel. Each channel contains reporter-labeled 
DNA fragments. Each detection module comprises a photomultiplier tube 
having a wide entrance area and a separate wavelength selective filter 
positioned between its PMT and the fluorescent species in the gel. These 
filters are interference filters having complementary transmission band 
characteristic which simulate the dichroic filter action. The filters 
permit the PMT's to generate signals that vary in amplitude in different 
senses as a function of the nature of the species. One filter largely 
passes the lower emission wavelengths and rejects the high emission 
wavelengths while the other filter does precisely the reverse. 
Transmission filters may be used with each interference filter to reject 
light from off axis angles greater than a predetermined angle. The 
wavelength filters have roughly complementary transmission vs. wavelength 
characteristics in the emission region of the four dyes, with the 
transition wavelengths occurring near the center of the species radiant 
energy spectra. 
Detection of the fragments can also be carried out by the methods described 
by Smith et al., Hood et al., and Ansorge et al. Simultaneous analysis of 
four bases in a single lane using the information provided by this 
particular set of four fluorescent dyes, however, can only be done by 
means of the signal processing systems described in the above co-pending 
patent application. 
In order to compare the results obtained by fluorescence detection with 
those obtained by standard sequencing techniques, this set of four labeled 
chain terminators has also been used to generate fluorescently-labeled 
sequencing fragments which also contain a .sup.32 P reporter. (The .sup.32 
P reporter can be enzymatically incorporated during primer extension by 
adding labeled dNTP or by 5'-labeling of the primer with a kinase.) The 
resulting doubly-labeled sequencing ladders can be analyzed both by 
autoradiography and by a fluorescent gel reader. These 
fluorescently-labeled sequencing ladders are very similar and functionally 
equivalent to ladders produced by the standard dideoxynucleotide chain 
terminators except that all bands run approximately two bases slower. The 
relative intensity of various bands in these sequencing ladders is 
modified by the addition of a linker and dye but the modified chain 
terminal or does not appear to cause any bands to be missed. Under 
appropriate gel electrophoresis conditions, the linker and dye on these 
chain terminators do not cause any of the bands to migrate anomalously: a 
faster-moving band contains fewer bases than a slower-moving band. The 
spacing between adjacent bands with different dyes varies slightly 
depending on which dyes are next to each other. When using either 
fluorescent or radioactive detection under optimal conditions, these minor 
variations in relative band intensity and position do not interfere with 
the use of these chain terminators to sequence DNA. 
The usefulness of alkynylamino nucleotides for the preparation of labeled 
chain terminating substrates for DNA sequencing is not limited to 
synthesis of only the set of four compounds shown above (34-37). Other 
combinations of sugars, bases and dyes have been assembled by means of an 
alkynylamino linker and these compounds are also useful for sequencing 
DNA. 
In addition to their utility in preparing fluorescently-labeled chain 
terminators, the alkynylamino nucleotides of this invention are generally 
useful for attaching a variety of reporters to nucleotides or 
oligonucleotides. Because the most nucleophilic site in these molecules is 
the amino group introduced with the linker, a reporter containing an 
activated carboxylic acid (e.g., N-hydroxysuccinimide ester), an 
isocyanate, an isothiocyanate, an activated aryl halide (e.g., 
1-fluoro-2,4-dinitrobenzene), or other electrophilic functional groups of 
appropriate reactivity, can be selectively attached to this nitrogen atom. 
The resulting labeled adducts can then be used in other applications to be 
described below. 
Since the heterocyclic base subunit of nucleotides is used in the genetic 
code, the function of many nucleotides is often determined by the nature 
of the sugar subunit. Likewise, the utility of alkynylamino nucleotides 
depends specifically on what type of sugar subunit is present. This 
utility will be diminished if the alkynylamino linker and/or the reporter 
interfere with a needed function of the nucleotide. The alkynylamino 
linker-containing nucleotides of this invention have distinct advantages 
such as: the small steric bulk of the alkynylamino-linker minimizes 
perturbation of the nucleotide; positioning the linker on the 5-position 
of pyrimidine nucleotides and the 7-position of 7-deazapurine nucleotides 
eventually places the linker and reporter in the major groove when the 
nucleotide is incorporated into double-stranded DNA (this will serve to 
minimize interference with hybridization and other processes, which 
require that a double-stranded conformation be possible); and 
alkynylamino-nucleotides with a reporter attached are excellent substrates 
for AMV reverse transcriptase. Because functionally-related enzymes tend 
to interact with their substrates in similar ways, it is likely that these 
nucleotides will also be substrates for other useful enzymes (such as 
other reverse transcriptases, DNA polymerases, and RNA polymerases) which 
perform template-directed nucleotide polymerization. 
The alkynylamino-nucleotides of this invention offer an attractive 
alternative for the chemical (non-enzymatic) synthesis of labeled 
2'-deoxyoligonucleotides. Ruth International Application Number: 
PCT/US84/00279 discloses a method for incorporating a reporter group into 
a defined-sequence single-strand oligonucleotide. The method includes the 
preparation of appropriately protected and activated monomeric nucleotides 
which possess a linker with a protected amino group, use of these 
monomeric nucleotides to synthesize oligonucleotides chemically, followed 
by the selective attachment of a reporter to the linker amino group. The 
small size of the alkynylamino linkers and their location on the 
5-position of pyrimidine nucleotides and the 7-position of 7-deazapurine 
nucleotides are expected to improve the performance of oligonucleotides 
containing them. An appropriately protected and activated monomer, (38), 
similar to one described by Ruth, could be prepared from 
commercially-available 5-iodo-2'-deoxyuridine by the Pd(O)/Cu(I) catalyzed 
attachment of an alkynylamino linker, followed by the selective 
dimethcxytritylation of the 5'-alcohol and finally conversion to a 
3'-phosphoramidite with chloro(diisopropylamino)methoxyphosphine. This 
monomer and other similar alkynylamino-containing monomers are expected to 
be useful oligonucleotide synthesis and reporter attachment according to 
the methods described by Ruth. (The trifluoroacetyl protecting group on 
the linker nitrogen is removed by the basic and/or nucleophilic reagents 
normally used for final deprotection during the chemical synthesis of 
oligonucleotides.) If the reporter is unaffected by the reactions used in 
oligonucleotide synthesis, it could also be attached to the alkynylamino 
linker at an earlier stage. 
##STR14## 
Although chemical synthesis of oligoribonucleotides is currently not as 
efficient or useful as synthesis of 2'-deoxyoligonucleotides, an 
appropriate monomer, [(39), -O-tetrahydropyranyl (OTHP)] could be prepared 
and used to make labeled RNA. 
In yet another application the enzymatic labeling of double-stranded 
nucleic acids can be facilitated through the use of the alkynylamino 
linkers. Langer et al., Proc. Nat. Acad. Sci. USA, 78, 6633 (1981), 
disclosed a nick-translation method for labeling double-stranded DNA with 
biotin reporters. An allylamino linker was used to attach biotin to the 
5-position of 2'-deoxyuridine triphosphate and uridine triphosphate. The 
resulting biotinylated nucleotides are substrates for DNA and RNA 
polymerases. Alternatively, an alkynylamino linker could be used for 
biotin attachment or, in general, for the attachment of other reporters 
such as fluorescent dyes. Adenosine triphosphate analogs (40) and (41) 
with alkynylamino linkers could be prepared more easily than adenosine 
analogs with an allylamino linker. Nucleotide triphosphates analogs of 
(40) and (41) could be used for nick-translation labeling of DNA or RNA by 
the enzymatic procedures of Langer et al. 
##STR15## 
The following Examples illustrate the invention. 
All temperatures are in degrees centigrade. (25.degree. refers to ambient 
or room temperature). All parts and percentages not otherwise indicated 
are by weight, except for mixtures of liquids which are by volume. The 
following abbreviations are employed: DMF--dimethylformamide; 
DMSO--dimethylsulfoxide; NHTFA--trifluoroacetamido-group; 
TEAB--triethylammonium bicarbonate; Tris--tris(hydroxymethyl)aminomethane; 
SF--succinylfluorescein; NMR--nuclear magnetic resonance spectrum; 
IR--infrared spectrum; UV--ultraviolet spectrum or detection; TLC--thin 
layer chromatography on silica gel; HPLC--high pressure liquid 
chromatography; GC--gas chromatography; mp--melting point; mp d--melting 
point with decomposition; bp--boiling point. In reporting NMR data, 
chemical shifts are given in ppm and coupling constants (J) are given in 
Hertz. All melting points are uncorrected. Ion exchange resins were washed 
with appropriate aqueous and organic solvents prior to use. The identity 
of all compounds described herein was established by appropriate 
spectroscopic and analytical techniques. Unless otherwise noted, 
purification by chromatography on silica gel was performed as described by 
Still et al., J. Org. Chem. 43, 2923-2926 (1978).

EXAMPLE 1 
PREATION OF 5-(3-AMINO-1-PROPYNYL)-2',3'-DIDEOXYCYTIDINE 5'-TRIPHOSPHATE 
(42) 
(Compound 42 is an example of structure 6 wherein Het is cytosine (i) and 
R.sub.1 is --CH.sub.2 --. It is the immediate precursor to labeled chain 
terminator 35.) 
A PREATION OF N-PROGYLTRIFLUOROACETAMIDE (43) 
Propargylamine (24.79 g, 0.450 mole; Aldrich, 99%) was added dropwise over 
1 h to methyl trifluoroacetate (69.19 g, 0.540 mole, 1.2 eq, Aldrich) at 
0.degree.. After stirring an additional hour at 0.degree., distillation 
though a 15 cm Vigreux column afforded 62.12 g (91%) of trifluoroacetamide 
43 as a colorless liquid (bp 68.5.degree.-69.5.degree. at 11 torr). This 
material was homogeneous by NMR and GC and was used interchangeably with 
spectroscopically-identical material prepared by acylating propargylamine 
with trifluoroacetic acid anhydride. 
.sup.1 H--NMR (CDCl.sub.3): 6.85 (broad s, 1H, NHTFA), 4.17 (dd, J=5.6 and 
2.5, 2H, CH.sub.2), 2.35 (t, J=2.5, 1H, CH). IR (neat; cm.sup.-1): 3300 
(N--H), 3095 and 2935 (C--H), 2130 (acetylene), 1720 (C.dbd.O), 1550 
(N--H), 1430, 1365, 1160, 1040, 998, 918, 857, 829, 772, and 725. 
B. PREATION OF 5-IODO-2',3'-DIDEOXYCYTIDINE (44) 
A solution of 2',3'-dideoxycytidine (2.11 g, 10 mmol, Raylo) and mercuric 
acetate (3.35 g, 10.5 mmol, Fisher) in 50 mL of methanol was refluxed for 
19 h. The resulting white suspension was diluted with methanol (50 mL) and 
dichloromethane (100 mL). Iodine (3.05 g, 12 mmol) was added and the 
suspension was stirred at 25.degree. until a clear purple solution was 
present After 4 h, the free base form of AG3 X4A resin (20 mL, 38 meq, 
Bio-Rad; a weakly basic polystyrene resin) was added and hydrogen sulfide 
was bubbled into the reaction for 15 min. Complete precipitation of 
mercury(II) was verified by TLC. The reaction was filtered though filter 
aid and the filter aid was washed with 1:1 methanol-dichloromethane. The 
filtrate was evaporated onto silica gel (10 g) and the loaded silica gel 
was placed on top of a 150 g silica gel column. Elution with 5%, 10% and 
20% methanol in dichloromethane afforded 2.79 g (83%) of iodide 44 as a 
colorless crystalline solid. Two recrystallizations from boiling water 
afforded, after vacuum-drying at 50.degree., large, analytically-pure 
prisms (mp: d 178.degree.). 
.sup.1 H--NMR (DMSO-d.sub.6): 8.50 (s, 1H, H.sub.6), 7.73 (broad s, 1H, 
--NH.sub.2 a), 6.53 (broad s, 1H, --NH.sub.2 b), 5.86 (dd, J=6.5 and 2.1, 
1H, H1'), 5.19 (t, 1H, 5'OH), 4.04 (m, 1H, H4'), 3.75 (ddd, J=12.1, 5.2, 
and 2.9, 1H, H5'a), 3.53 (dt, J=12.1 and 3.8, 1H, H5'b), and 2,3-1.7 (m, 
4H, H2' and H3'). Calculated for C.sub.9 H.sub.12 N.sub.3 O.sub.3 I: C 
32.07%, H 3.59%, N12.46%. Found: C 32.05%, H 3.80%, N 12.46%. 
C. A GENERAL PROCEDURE FOR COUPLING AMINOALKYNES TO IODONUCLEOSIDES. 
PREATION OF 5-(3-TRIFLUOROACETAMIDO-1-PROPYNYL)-2',3'-DIDEOXYCYTIDINE 
(45). 
A 50-mL, thee-necked flask was charged with iodocytidine 44 (770 mg, 2.00 
mmol) and cuprous iodide (76.2 mg, 0.400 mmol, 0.20 eq; Aldrich, Gold 
Label). After flushing the flask with argon, dry dimethylformamide (10 mL, 
Aldrich) was added to produce a 0.2M solution of iodocytidine which 
contained suspended cuprous iodide. N-propargyltrifluoroacetamide (0.70 
mL, 6.00 mmol, 3.0 eq) and triethylamine (0.56 mL, 4.00 mmol, 2.0 eq, 
stored over molecular sieves) were added via syringe. 
Tetrakis(triphenylphosphine)palladium(O) (231 mg, 0.20 mmol, 0.10 eq) was 
weighed into a vial in a dry box and added to the reaction mixture. The 
cuprous iodide dissolved, affording a yellow solution which gradually 
darkened over several hours. The reaction was allowed to proceed until TLC 
indicated that the starting material was completely consumed. After 4 h, 
the reaction was diluted with 20 mL of 1:1 methanol-dichloromethane and 
the bicarbonate form of AGI X8 resin (Bio-Rad, 2.0 g, ca. 6 eq; a strongly 
basic, anion exchange, polystyrene resin) was added. After stirring for 
about 15 min, evolution of gas ceased. After 30 min, the reaction mixture 
was filtered and the resin was washed with 1:1 dichloromethanemethanol. 
The combined filtrates were rapidly concentrated with a rotary evaporator. 
(Removal of dimethylformamide required about 10 min at 45.degree. and 2 
torr.) The residue was immediately purified by chomatography on 100 g of 
silica gel using 10%, 15% and 20% methanol in dichloromethane. Removal of 
solvent from the appropriate fractions afforded 651 mg (90%) of 
alkynylamine nucleoside 45 as a pale yellow crystalline foam which was 
homogeneous by TLC and NMR. The product from a similar preparation was 
established to be a hemi-hydrate by elemental analysis. 
.sup.1 H--NMR (DMSO-d.sub.6): 9.96 (broad s, 1H, NHTFA), 8.32 (s, 1H, H6), 
7.76 (broad s, 1H, NH.sub.2 a), 6.78 (broad s, 1H, NH.sub.2 b), 5.88 (dd, 
J=6.5 and 2.5, 1H, H1'), 5.13 (t, J=5.1, 1H, 5'OH) 4.28 (d, J=5.0, 2H, 
--CH.sub.2 --), 4.04 (m, 1H, H4'), 3.73 (ddd, J=12.0, 5.0 and 3.1, 1H, 
H5'a), 3.53 (dt, J=12.1 and 4.0, 1H, H5'b), 2.3-1.7 (m, 4H, H2' and H3'). 
.sup.19 F--NMR (DMSO-d.sub.6): -74.0 (s). UV (MeOH): maxima at 238.5 
(17,100) and 295.5 (9,300). Calculated for C.sub.14 H15N.sub.4 O.sub.4 
F.sub.3.1/2H2 O: C 45.53, H 4.37, N 15.17. Found: C 45.56, H 4.52, N 
15.26. 
D. PREATION OF TRIS(TRI-N-BUTYLAMMONIUM) PYROPHOSPHATE. 
Tetrasodium pyrophosphate decahydrate (4.46 g, 10 mmol) was dissolved in 
the minimum amount of water (about 50 mL) and passed though a column of 
AG50W X8 resin (100-200 mesh, 4.times.10 cm bed; a strongly-acidic, cation 
exchange, polystyrene resin) poured in water. The column was eluted with 
water and the eluent was collected in an ice-cooled flask until pH of the 
eluent approached neutrality. Tri-n-butylamine (Aldrich Gold Label, 7.1 
mL, 30 mmol) was added to the eluent and the two phases were stirred 
vigorously until all of the amine dissolved. The resulting solution was 
lyophilized. The residue was co-evaporated twice with dry pyridine and 
once with dry dimethylformamide. The residue was dissolved in dry 
dimethylforamide (10 mL) and the resulting 1.0M solution was stored (for 
as long as one month) at 0.degree. under argon until used. 
E. A GENERAL PROCEDURE FOR CONVERTING PROTECTED ALKYNYLAMINO NUCLEOSIDES TO 
THE CORRESPONDING 5'-TRIPHOSPHATES AND REMOVING THE TRIFLUOROACETYL 
PROTECTING GROUP. PREATION OF 
5-(3-AMINO-1-PROPYNYL)-2',3'-DIDEOXYCYTIDINE 5'-TRIPHOSPHATE (42) 
Alkynylamino nucleoside 45 (361 mg, 1.00 mmol) was dissolved in trimethyl 
phosphate (2.0 mL, Aldrich Gold Label) while stirring under argon in an 
oven-dried flask. The solution was cooled to -10.degree. and phosphorus 
oxychloride (0.093 mL, 1.00 mmol, Aldrich Gold Label) was added by 
syringe. After stirring the reaction mixture at -10.degree. for 30 min, a 
second aliquot of phosphorus oxychloride (0.093 mL, 1.00 mmol) was added 
and the solution was allowed to warm slowly to 25.degree. while stirring. 
Aliquots from the reaction mixture were quenched with 1N aqueous hydroxide 
and analyzed by HPLC. When conversion to the corresponding nucleotide 
monophosphate was at a maximum (in this case 100 min after the second 
addition of phosphorus oxychloride), the reaction mixture was added 
dropwise to a precooled (-10.degree.) solution of tris(tri-n-butylammonium 
pyrophosphate (6.0 mL of the above 1.0M solution in dry 
dimethylformamide). The solution was allowed to warm slowly to 25.degree. 
while stirring under argon. After 100 min, the reaction solution was added 
slowly to a precooled (0.degree.) solution of triethylamine (1.4 mL) in 
water (20 mL). The solution was stirred with ice-cooling for 15 min and 
then allowed to stand overnight at about 2.degree.. 
The volatiles were removed by vacuum evaporation at 25.degree. and 0.5 
torr. The residue was redissolved in water (75 mL) and applied to a column 
of DEAE-SEPHADEX ion exchanger (A-25-120, 2.6.times.65 cm bed) that had 
been equilibrated with: 1) pH 7.6, 1.0M aqueous TEAB (300 mL), 2) 1.0M 
aqueous potassium bicarbonate (300 mL), and 3) pH 7.6, 0.1M aqueous TEAB 
(300 mL). The column was eluted with a linear gradient of pH 7.6 aqueous 
TEAB from 0.1M (1 L) to 1.0M (1 L). The column was driven at 100 mL/h 
while collecting fractions every 12 min. The elution was monitored by 
absorbance at 270 nm (40 AUFS). The desired material eluted as a 
well-separated, major band near the end of the gradient (Fractions 73-80). 
The product-containing fractions were pooled, concentrated (at below 
30.degree.), and co-evaporated twice with absolute ethanol. The residue 
was taken up in water (20.4 mL) and lyophilized. 
The intermediate product was taken up in water (12.5 mL) and concentrated 
ammonium hydroxide (12.5 mL) was added. After stirring for 3.5 h, the 
solution was stirred under aspirator vacuum for 2 h to remove the excess 
ammonia gas and then lyophilized. The residue was taken up in pH 7.6 0.1M 
aqueous TEAB (10 mL) and applied to a column of DEAE-SEPHADEX ion exchange 
resin (A-25-120, 1.6.times.55 cm bed) that had been prepared as described 
above. The column was eluted while collecting 6 mL fractions with a linear 
gradient of TEAB from 0.1M (280 mL) to 1.0M (280 mL). The product eluted 
as a single major peak. The fractions estimated to contain pure product 
(#39-45) were pooled, concentrated (at below 30.degree.), co-evaporated 
with absolute ethanol (2.times.), and taken up in water (9.8 mL). The 
solution was assayed by Uv absorption and HPLC and then lyophilized. 
A dilute solution of the product showed absorption maxima at 240 and 293.5 
nm in pH 8.2 50 mM aqueous Tris buffer. Assuming an absorption coefficient 
for the product equal to that of the starting material (9,300), the yield 
of product, based on the absorption at 293.5 nm, was 0.32 mmol (32%). HPLC 
(Zorbax SAX, 0.2M pH 6.5 aqueous potassium phosphate, monitoring 270 nm) 
of the final product showed essentially a single peak (&gt;99%). 
.sup.1 H--NMR (D.sub.2 O): 8.57 (s, 1H, H6), 6.03 (dd, J=6.4 and 1.6, 1H, 
H1'), 4.42 (m, 2H, H4'and H5'a), 4.18 (ddd, J=12, 5.5 and 3, 1H, H5'b), 
4.036 (s, 2H, --CH2--), 2.5-1.9 (m, 4H, H2'and H3,), plus counterion 
(triethylammonium) peaks. .sup.31 P--NMR (D.sub.2 O): -9.02 (d, J=20, 1P), 
-9.74 (d, J=20, 1P), -21.37 (t, J=20, 1P). UV (pH 8.2 aq Tris): maxima at 
240 and 293.5 nm. 
EXAMPLE 2 
PREATION OF 5-(3-AMINO-1-PROPYNYL)-2',3'-DIDEOXYURIDINE 5'-TRIPHOSPHATE 
(46) 
(Compound 46 is an example of structure 6 wherein Het is uracil (h) and 
R'is --CH.sub.2 --. It is the immediate precursor to labeled chain 
terminator 34.) 
A. PREATION OF 5-IODO-2',3'-DIDEOXYURIDINE (47) 
Dideoxyuridine (2.122 g, 10.0 mmol) was dissolved in 30 mL of warm methanol 
and, after cooling to 25.degree., iodine monochloride (4.06 g, 25 mmol, 
2.5 eq, Fisher) in methanol (20 mL) was added over 5 min. The dark purple 
reaction mixture was heated in a 50.degree. bath under nitrogen for 20 min 
and then immediately cooled in an ice-water bath. After standing without 
stirring for 165 min, the resulting precipitate was collected by 
filtration and washed with cold methanol (2.times.10 mL). Vacuum-drying 
overnight afforded 2.232 g (66%) of iodide 47 as off-white microcrystals. 
This material was used without further purification in the next reaction, 
but other preparations were purified by chomatography or recrystallization 
from boiling methanol (30 mL/g) to give white needles (mp d 
160.degree.-164.degree.). NMR indicated that the crude precipitate was 
homogeneous, but also that the 5'-hydroxyl proton was very broad due to 
exchange catalyzed by trace impurities. Chomatographed or recrystallized 
materials afforded spectra in which this proton was, as usual, a sharp 
triplet. 
.sup.1 H--NMR (DMSO-d.sub.6): 11.60 (broad s, 1H, H3), 8.57 (s, 1H, H6), 
5.90 (dd, J=2.0 and 6.6, 1H, H1'), 5.2 (broad s, 1H, 5'OH), 4.06 (m, 1H, 
H4'), 3.75 and 3.53 (m, 2H, H5'), 2.26, 2.02 and 1.84 (m, 4H, H2'and H3'). 
B. PREATION OF 5-(3-TRIFLUOROACETAMIDO-1-PROPYNYL)2',3'-DIDEOXYURIDINE 
(48) 
Iodouridine 47 was coupled for 3 h to N-propargyltrifluoroacetamide 
following the general method given in Example 1C. Chomatography with a 
0-5% methanol in dichloromethane gradient afforded material which was 
homogeneous by TLC, but which was difficult to dry. After co-evaporating 
the chomatographed product several times with chloroform and 
vacuum-drying, 536.5 mg of alkynylamino nucleoside 48 was obtained as a 
white foam. This material was homogeneous by TLC and was pure by NMR 
except for a small amount (39 mole%; corrected yield 66%) of chloroform. 
.sup.1 H--NMR (DMSO-d.sub.6): 11.61 (s, 1H, H3), 10.07 (distorted t, 1H, 
NHTFA), 8.35 (s, 1H, H6), 7.26 (s, 0.39H, CHCl.sub.3), 5.89 (dd, J=6.6 and 
3.2, 1H, H1'), 5.15 (t, J=5.2, 1H, 5'OH), 4.22 (broad d, 2H, --CH.sub.2 
N--), 4.04 (apparent hept, J=3.5, 1H, H4'), 3.73 and 3.53 (m, 2H, H5'), 
2.26, 2.03 and 1.84 (m, 4H, H2'and H3'). TLC (95:5 
dichloromethane-methanol, two elutions, UV): Starting iodide 47, R.sub.f 
=0.37; product 48, 0.28; catalysts, 0.95 and 0.80 plus slight streakiness. 
C. PREATION OF 5-(3-AMINO-1-PROPYNYL)-2',3'-DIDEOXYURIDINE 
5'-TRIPHOSPHATE (46). 
Alkynylamino nucleoside 48 (0.30 mmol) was converted to the corresponding 
triphosphate and its trifluoroacetyl group was removed following the 
general procedure given in Example 1E. After addition of the second 
aliquot of phosphorus oxychloride, phosphorylation was allowed to proceed 
for a total of 210 min. Assuming an absorption coefficient for the product 
equal to that of the starting material (13,000), the yield of triphosphate 
46, based on its UV absorption at 291.5 nm, was 18%. 
EXAMPLE 3 
PREATION OF 7-(3-AMINO-1-PROPYNYL)-2',3'-DIDEOXYGUANOSINE 
5'-TRIPHOSPHATE (49) 
(Compound 49 is an example of structure 6 wherein Het is 7-deazaguanine (k) 
and R.sub.1 is --CH.sub.2 --. It is the immediate precursor of labeled 
chain terminator 37.) 
A. PREATION OF 
6-METHOXY-2-METHYLTHIO-9-(3,5-DI-O-p-TOLUOYL-2-DEOXY-.beta.-D-RIBOFURANOSY 
L)-7-DEAZAPURINE (9) 
6-Methoxy-2-methylthio-7-deazapurine (8, 9.2 g, prepared following the 
procedure of F. Seela et al., Chem. Ber., Vol. 111, 2925 (1978)) was 
azeotropically dried by dissolving in 150 mL of dry pyridine and 
evaporating to dryness at 30.degree.-35.degree.. This material was 
suspended in 450 mL of dry acetonitrile at room temperature under nitrogen 
and sodium hydride (2.16 g of a 60% suspension in oil) was added with 
stirring. After 45 min, 
1-chloro-2-deoxy-3,5-di-O-p-toluoyl.alpha.-D-ribofuranose (18.6 g, 
prepared following the procedure of M. Hoffer, Chem. Ber., Vol. 93, 2777 
(1960)) was added in thee equal portions over a 20 min. After stirring the 
reaction mixture for an additional 45 min at room temperature, acetic acid 
(1 mL) and dichloromethane (300 mL) were added. The mixture was suction 
filtered though a pad of filter-aid, and the filtrate was evaporated to 
dryness. The residue was dissolved in benzene and this solution was washed 
with water (2.times. ) and brine (1.times.). After drying the organic 
layer over sodium sulfate and evaporating, the residue was dissolved in 
methanol (400 mL) and allowed to crystallize affording 19.24 g (73.8%) of 
ribosylated product 9 as colorless crystals (mp 106.degree.-107.degree.). 
.sup.1 H--NMR (CDCl.sub.3): 2.42 (s, 3H, toluoyl CH.sub.3), 2.44 (s, 3H, 
toluoyl CH.sub.3), 2.64 (s, 3H, SCH.sub.3), 2.70 and 2.89 (m, 2H, H2'), 
4.08 (s, 3H, OCH.sub.3), 4.56 (m, 1H, H3'), 4.65 (m, 2H, H5'), 5.74 (m, 
1H, H4'), 6.44 (d, J=4, 1H, H7), 6.77 (dd, J=8 and 6, 1H, H1'), 7.05 (d, 
J=4, 1H, H8) and 7.25 and 7.95 (m, 8H, toluoyl H). Recrystallization of a 
sample of the above material from methanol containing a small amount of 
dichloromethane afforded crystals of mp 109.degree.-110.degree.. 
B. PREATION OF 
6-METHOXY-2-METHYLTHIO-9-(2-DEOXY-.beta.-D-RIBOFURANOSYL)-7-DEAZAPURINE 
(10) 
A suspension of ester 9 (19 g) and the hydroxide form of REXYN 201 resin 
(38 g; a strongly basic, anion exchange, polystyrene resin) in 600 mL of 
methanol was refluxed for 1.5 h under nitrogen. The hot suspension was 
suction filtered to remove the resin and the filtrate was evaporated to 
dryness. The solid residue was dissolved in ether (450 mL) and, after 10 
min, the solution was filtered though a pad of filter aid to remove a 
small amount of a colored impurity. The solution was seeded with crystals 
of the desired product obtained from a previous reaction and allowed to 
stand overnight at 25.degree.. Crystalline diol 10 was collected by 
filtration and the mother liquor was concentrated to afford a second crop. 
Each crop was washed thoroughly with ether and dried to afford a total of 
8.43 g (78.0%) of diol 10 as colorless crystals (mp 
129.degree.-130.degree.). 
.sup.1 H--NMR (DMSO-d.sub.6) 2.21 and 2.55 (m, 2H, H2'), 2.56 (s, 3H, 
SCH.sub.3), 3.53 (m, 2H, H5'), 3.82 (m, 1H, H3'), 4.02 (s, 3H, OCH.sub.3), 
4.36 (m, 1H, H4,), 4.90 (t, J=5.5, 1H, 5'OH), 5.30 (d, J=5.5, 1H, 3'H), 
6.48 (d, J=4, 1H, H7), 6.55 (dd, J=8 and 6, 1H, H1'), 7.48 (d, J=1H, 1H, 
H8). Recrystallization of a sample of this material from dichloromethane 
containing a small amount of methanol afforded crystals of mp 
130.degree.-131.degree.. 
C. PREATION OF 
6-METHOXY-2-METHYLTHIO-9-(5-O-TRIPHENYLMETHYL-2-DEOXY-.beta.-D-RIBOFURANOS 
YL)7-DEAZAPURINE (11) 
Diol 10 (7.2 g), was azeotropically dried by dissolving in dry pyridine and 
evaporating the solution to dryness at 35.degree.. The residue was 
dissolved in dry pyridine (100 mL) and triphenylmethyl chloride (8.0 g), 
triethylamine (4.0 mL), and 4-(dimethylamino)pyridine (300 mg) were added. 
After heating the reaction mixture at 65.degree. under nitrogen for 30 
min, a second addition of triphenylmethyl chloride (1.0 g) was made and 
heating was continued for 16.5 h. After cooling, the reaction mixture was 
concentrated and the residue was partitioned between dichloromethane and 
water. The aqueous layer was extracted with dichloromethane and the 
combined organic layers were washed with 0.3N hydrochloric acid, aqueous 
sodium bicarbonate, and brine. After drying over sodium sulfate and 
concentrating, purification of the crude product by chomatography on 
silica gel with 0%, 1%, 1.5% and 2% methanol in dichloromethane afforded 
12.1 g (94.5%) of monotrityl ether 11 as a colorless glass. 
.sup.1 H--NMR (CDCl.sub.3): 2.58 (s, 3H, SCH.sub.3), 2.42 and 2.62 (m, 2H, 
H2'), 3.37 (m, 2H, H5'), 4.04 (m, 1H, H3'), 4.08 (s, 3H, OCH.sub.3), 4.60 
(m, 1H, H4'), 6.40 (d, J=4, 1H, H7), 6.68 (apparent t, J=7, 1H, H1'), 7.00 
(d, J=4, 1H, H8), 7.27 and 7.43 (m, 15H, trityl H). This data was obtained 
from a different batch of 11 prepared as described above. 
D. PREATION OF 
6-METHOXY-2-METHYLTHIO-9-(5-O-TRIPHENYLMETHYL-2,3-DIDEOXY-.beta.-D-RIBOFUR 
ANOSYL)7-DEAZAPURINE (12) 
A solution of trityl ether 11 (12.1 g), 4-dimethylaminopyridine (9.2 g), 
and phenyl chlorothionocarbonate (7.5 mL, Aldrich) in dry dichloromethane 
(220 mL) was stirred at 25.degree. for 2 h under nitrogen. Since TLC 
analysis indicated that the reaction was incomplete, phenyl 
chlorothionocarbonate (4.0 mL) was added and the reaction mixture was 
stirred for an additional 1 h. The solution was diluted with 
dichloromethane (280 mL) and was washed sequentially with 0.5N 
hydrochloric acid (500 mL), 0.5N sodium hydroxide (500 mL), and brine. The 
organic layer was dried over sodium sulfate and evaporated to dryness. 
The resulting crude thionocarbonate was dissolved in dry toluene (350 mL) 
and azoisobisbutyronitrile (350 mg) and tri-n-butyltin hydride (10 mL) 
were added. The resulting solution was heated at 100.degree.-105.degree. 
for 10 min. After cooling, the solution was diluted with a little ether 
and was shaken with 10% aqueous potassium fluoride (350 mL). The two 
layers were filtered though a pad of filter aid (to remove a dark sludge) 
and separated. The organic layer was washed with 0.75N potassium hydroxide 
and brine, dried over sodium sulfate and concentrated. Chomatography of 
the resulting oil on silica gel with 1:1 dichloromethane-ether and then 
with dichloromethane afforded 9.93 g (84.5%) of dideoxynucleoside 12 as a 
colorless solid (mp 122.degree.-124.degree.). 
.sup.1 H--NMR (CDCl.sub.3): 2.10, 2.33, and 2.43 (m, 4H, H2'and H3'), 2.60 
(s, 3H, SCH.sub.3), 3.30 (m, 2H, H5'), 4.08 (s, 3H, OCH.sub.3), 4.29 (m, 
1H, H4'), 6.36 (d, J=3.7, 1H, H7), 6.53 (dd, J=7 and 4, 1H, H1'), 7.09 (d, 
1H, J=3.7, H8), 7.25 and 7.45 (m, 15H, trityl H). 
E. PREATION OF 
7-IODO-6METHOXY-2-METHYLTHIO-9(5-O-TRIPHENYLMETHYL-2,3-DIDEOXY-.beta.-D-RI 
BOFURANOSYL)7-DEAZAPURINE (13) 
N-Iodosuccinimide (10.0 g) was added to a solution of deazapurine 12 (9.9 
g) in dry dimethylformamide (550 mL). After stirring in the dark under 
nitrogen for 16 h, 10% aqueous sodium bicarbonate (2.5 mL) was added and 
the reaction mixture was concentrated in vacuo at 50.degree. to a volume 
of 100 mL. This solution was partitioned between water and ethyl acetate. 
The organic layer was washed with 5% aqueous sodium hydrosulfite and 
brine, dried over sodium sulfate, and concentrated. Chomatography of the 
slightly impure product on silica gel with dichloromethane afforded 11.68 
g (95.6%) of iodide 13 as a colorless glassy solid. 
.sup.1 H--NMR (CDCl.sub.3): 2.06, 2.24, and 2.41 (m, 4H, H2'and H3'), 2 58 
(s, 3H, SCH.sub.3), 3.30 (m, 2H, H5'), 4.10 (s, 3H, OCH.sub.3), 4.29 (m, 
1H, H4'), 6.47 (dd, J=6 and 4, 1H, H1'), 7.19 (s, 1H, H8), 7.30 and 7.46 
(m, 15H, trityl H). This data was obtained from a different batch of 13 
prepared as described above. 
F. PREATION OF 
7-IODO-2-METHYLTHIO-9-(5-O-TRIPHENYLMETHYL-2,3-DIDEOXY-.beta.-D-RIBOFURANO 
SYL)7-DEAZAPURIN-4-ONE (14) 
Sodium thiocresolate was prepared by adding sodium methoxide (1 eq) to a 
solution of thiocresol in methanol and then evaporating to dryness. A 
mixture of methyl ether 13 (4.0 g), sodium thiocresolate (4.0 g), and 
hexamethylphosphoramide (10 mL) in dry toluene (150 mL) was refluxed under 
nitrogen for 4.5 h. After cooling, the mixture was partitioned between 
ethyl acetate and water. The organic layer was washed with water and 
brine, dried over sodium sulfate, and evaporated to dryness. Chomatography 
of the resulting crude product on silica gel with 0% and 2% methanol in 
dichloromethane afforded 3.80 g (97.0%) of deazapurinone 14 as a colorless 
glassy solid. 
.sup.1 H--NMR (CDCl.sub.3): 2.05, 2.25, and 2.42 (m, 4H, H2' and H3'), 2.60 
(s, 3H, SCH.sub.3), 3.30 (m, 2H, H5'), 4.28 (m, 1H, H4'), 6.40 (dd, J=7 
and 4, 1H, H1'), 7.05 (s, 1H, H8), 7.30 and 7.46 (m, 15H, trityl H), 10.00 
(broad s, 1H, H1). 
G. PREATION OF 7-IODO-5'-O-TRIPHENYLMETHYL2',3'-DIDEOXY-7-DEAZAGUANOSINE 
(15) 
Meta-chloroperoxybenzoic acid (1.23 g, 85%, Aldrich) was added to a stirred 
solution of methylthio ether 14 (3.6 g) in dry dichloromethane (150 mL) at 
0.degree. under nitrogen. After 15 minutes, the cooling bath was removed 
and stirring was continued at 25.degree. for 40 min. This solution was 
washed with aqueous sodium bicarbonate and brine and dried over sodium 
sulfate. Methanol (two percent by volume) was added and the resulting 
solution was passed though a short plug of silica gel to remove polar 
impurities. The resulting crude sulfoxide (3.07 g) was dissolved in 
dioxane (40 mL) and placed in a glass-lined bomb. Ammonia (10.0 g) was 
added and the mixture was heated at 100.degree. for 2 h in an autoclave. 
The resulting solution was evaporated to dryness. The residue was 
dissolved in dichloromethane (20 mL) and filtered though a pad of 
filter-aid. Methanol (40 mL) was added to the solution and, on cooling, 
1.57 g of colorless product crystallized. The mother liquor was evaporated 
and purified by medium pressure liquid chomatography on silica gel with 5% 
methanol in dichloromethane to afford an additional 328 mg of product as 
colorless crystals. The total yield of deazaguanosine 15 was 1.90 g 
(55.4%). 
.sup.1 H--NMR (CDCl.sub.3): 2.05, 2.23, and 2,35 (m, 4H, H2' and H3'), 3.29 
(m, 2H, H5'), 4.26 (m, 1H, H4'), 5.90 (broad s, 2H, NH.sub.2), 6.24 (dd, 
J=7 and 4, 1H, H1'), 6.90 (s, 1H, H8), 7.30 and 7.46 (m, 15H, trityl H) 
10.90 (broad s, 1H, H1). Recrystallization of a sample of this material 
from methanol-dichloromethane afforded crystals of mp 
201.degree.-203.degree.. 
H. PREATION OF 2',3'-DIDEOXY-7-IODO-7-DEAZAGUANOSINE (16) 
A solution of trityl ether 15 (1.7 g) in formic acid (12 mL) was stirred at 
room temperature for 10 min. The resulting yellow suspension was then 
quickly evaporated to dryness in vacuo at 30.degree.. Chomatography of the 
residue on silica gel with 5%, 7%, and 10% methanol in dichloromethane 
afforded 940 mg of a colorless solid. Trituration of this solid with ether 
containing a little dichloromethane yielded 838 mg (81.0%) of nucleoside 
16 as colorless crystals. 
.sup.1 H--NMR (DMSO-d.sub.6): 1.95, 2.09, and 2.26 (m, 4H, H2' and H3'), 
3.48 and 3.54 (m, 2H, H5'), 3.98 (m, 1H, H4'), 4.90 (broad t, J=5, 1H, 
5'OH), 6.08 (m, 1H, H1'), 6.32 (broad s, 2H, NH.sub.2), 7.12 (s, 1H, H8), 
10.46 (broad s, 1H, H1). 
I. PREATION OF 
7-(3-TRIFLUOROACETAMIDEO-1-PROPYNYL)2',3'-DIDEOXY-7-DEAZAGUANOSINE (50) 
Iodide 16 (376 mg, 1.00 mmol) was coupled for 2.25 h to 
N-propargyltrifluoroacetamide by the general method given in Example 1C. 
Product and starting material were indistinguishable by TLC, so the 
reaction was monitored by reverse phase HPLC (10 cm ODS, 1 mL/min, 
gradient from 100% water to 100% methanol over 5 min, then 100% methanol, 
with UV detection at 280 nm: starting iodide 16, 5.49 min; product 50, 
5.75 min; intermediate, 6.58 min). The crude product was poorly soluble in 
dichloromethane, so it was concentrated from a dichloromethane-methanol 
solution onto silica gel (5 g) before being loaded onto the chomatography 
column. Elution with 2%, 5%, 7% and 10% methanol in dichloromethane 
afforded 300 mg (78%) of alkynylamino nucleoside 50 as a yellow solid. 
.sup.1 H--NMR (DMSO-d.sub.6): 1.96, 2.08, and 2.28 (m, 4H, H2' and H3'), 
3.47 and 3.55 (m, 2H, H5'), 3.99 (m, 1H, H4'), 4.22 (broad s, 2H, 
--CH.sub.2 --), 4.90 (t, J=5, 1H, 5'OH), 6.09 (dd, J=6 and 4, 1H, H1'), 
6.33 (broad s, 2H, NH.sub.2), 7.30 (s, 1H, H8), 10.05 (broad s, 1H, 
NHTFA), 10.50 (broad s, 1H, H1). .sup.1 H-Decoupled .sup.13 C--NMR 
(DMSO-d.sub.6): 155.5 (q, J=36.5, trifluoroacetyl carbonyl), 157.8, 153.1 
and 149.9 (C2, C4 and C6), 122.6 (C8), 115.9 (q, J=288, CF3), 99.4 and 
97.5 (C7 and C5), 84.2 and 77.4 (acetylenic), 83.2 and 81.0 (Cl' and C4'), 
62.9 (C5'), 29.7 (propargylic), 31.8 and 25.8 (C2' and C3 '). This 
'.sup.13 C--NMR data was obtained from a different batch of 50 prepared as 
described above. 
J. PREATION OF 7-(3-AMINO-1-PROPYNYL)-2',3'DIDEOXY-7-DEAZAGUANOSINE 
5'-TRIPHOSPHATE (49) 
Alkynylamino nucleoside 50 (0.90 mmol) was converted to the corresponding 
5'-triphosphate and the trifluoroacetyl protecting group was subsequently 
removed following the general procedure given in Example 1E. After the 
second addition of phosphorus oxychloride, the reaction was stirred for an 
additional 165 min. Assuming an absorption coefficient for the product 
equal to that of the starting material (11,900), the yield of 
5'-triphosphate 49, based on its absorption at 272.5 mn, was 18%. 
EXAMPLE 4 
PREATION OF 7-(3-AMINO-1-PROPYNYL)-2',3'-DIDEOXY7-DEAZAADENOSINE 
5'-TRIPHOSPHATE (51) 
(Compound 51 is an example of structure 6 wherein Het is 7-deazaadenine (j) 
and R.sub.1 is --CH.sub.2 --. It is the immediate precursor to labeled 
chain terminator 36.) 
A. PREATION OF 2'-ACETOXY-3'-BROMO-5'-(2-ACETOXYISOBUTYRYL)ADENOSINE 
(18) 
2-Acetoxyisobutyryl bromide (19.5 mL, 150 mmol, 5 eq, prepared according to 
the procedure of Russell et al, J. Am. Chem Soc., 95, 4016-4030 (1973)) 
was added over 15 min to a suspension of tubercidin (17, 7-deazaadenosine, 
6.66 g, 25.0 mmol, Sigma) in dry acetonitrile (250 mL, Aldrich). The 
suspended solid dissolved in about 5 min and the reaction was stirred 
under nitrogen for 22 h at 25.degree.. The reaction mixture was added to a 
solution of dipotassium hydrogen phosphate (43.55 g, 300 mmol, 6 eq) in 
water (400 mL). After stirring for 30 min, the solution was extracted with 
ethyl acetate (1.times.400 mL and 2.times.200 mL). The combined organic 
layers were dried over magnesium sulfate and evaporated to dryness to 
afford 14.73 g, (118%) of white foam. This material was greater than 95% 
one slightly broadened spot by TLC (with UV detection), but NMR showed 
that one major and at least one minor product were present. The NMR 
spectrum was consistent with the major product being bromoacetate 18. 
.sup.1 H--NMR (DMSO-d.sub.6) for the major component 18: 8.08 (s, 1H, H2), 
7.34 (d, J=3.7, 1H, H8), 7.12 (broad s, 2H, NH.sub.2), 6.70 (d, J=3.7, 1H, 
H7), 6.32 (d, J=3.8, 1H, H1'), 5.61 (dd, J=2.4 and 3.8, 1H, H2'), 4.89 
(dd, J=2.4 and 4.5, 1H, H3'), 4.43 (m, 1H, H4'), 4.35 (dd, J=12 and 4, 1H, 
H5'a), 4.29 (dd, J=12 and 7, 1H, H5'b), 2.08 (s, 3H, OAc), 2.00 (s, 3H, 
OAc), and 1.49 (s, 6H, 2CH.sub.3). 
B. PREATION OF 2',3'-DIDEOXY-2',3'-DIDEHYDRO7-DEAZAADENOSINE (19) 
Zinc-copper couple was freshly prepared by rapidly (total elapsed time of 
about 10 min) washing zinc dust (20 g, Mallinkrodt) with 1N hydrochloric 
acid (3.times.50 mL), water (2.times.50 mL), 2% cupric sulfate (2.times.50 
mL), water (4.times.50 mL), ethanol (3.times.50 mL) and ether (2.times.50 
mL). During each wash, the zinc dust was stirred in a fritted funnel until 
it was suspended and the wash was removed by suction while minimizing 
exposure of the zinc to air. The couple was vacuum dried for 30 min. The 
above crude bromoacetate (14.63 g) was dissolved in dry dimethylformamide 
(150 mL, Aldrich) and approximately 25 mL of solvent was removed with a 
rotary evaporator (45.degree., at 2 torr). Fresh zinc-copper couple (14.63 
g, about 9 eq) was added and the resulting suspension was stirred under 
nitrogen at 25.degree.. Depending on the quality of the zinc-copper 
couple, this reaction can show an induction period and/or variable rate, 
so the reaction was allowed to proceed until TLC (90:9:1 
dichloromethane-methanol-concentrated ammonium hydroxide: starting 
material R.sub.f =0.45 and products R.sub.f =0.39 and 0.36) indicated the 
starting material had been completely consumed. In this case, the reaction 
was complete in less than 15 min. After 100 min, saturated aqueous sodium 
bicarbonate (75 mL) was added carefully over 10 min to the reaction 
mixture. The reaction mixture was filtered though a filter aid and the 
filter aid was washed with methanol (2.times.50 mL). The combined 
filtrates were evaporated to dryness and the residue was partitioned 
between water (150 mL) and ethyl acetate (150 mL). The aqueous layer was 
extracted with ethyl acetate (2.times.100 mL) and the combined organic 
extracts were dried over magnesium sulfate, concentrated, and vacuum dried 
for 1 h. 
The resulting dark orange semisolid was dissolved in methanol (100 mL) and 
then water (25 mL) and REXYN 201 resin (29 g, 4.3 meq/g, 5 eq, hydroxide 
form) were added. The reaction mixture was refluxed for a total of 210 
min. Monitoring by TLC (85:13:2 dichloromethane-methanol-concentrated 
ammonium hydroxide: intermediate, Rf=0.49; final product 19, 0.24) 
indicated that the reaction had rapidly halted at about 70% conversion, so 
after 165 min, additional resin (29 g) was added. Without cooling, the 
resin was removed by filtration and washed with 1:1 
dichloromethane-methanol (2.times.75 mL). The combined filtrates were 
evaporated to dryness and the resulting purple solid was recrystallized 
from boiling isopropanol (150 mL) to afford 3.778 g, of olefin 19 as a 
off-white needles (mp 205.degree.-206.degree.). A second crop of 0.631 g, 
of product (pale purple needles, mp 202.degree.-203.degree.) was obtained 
by concentrating the mother liquors to 25 mL. Both crops (total 4.409 g, 
76%) were homogeneous by TLC and pure by NMR except for a trace of 
isopropanol. 
.sup.1 H--NMR (DMSO-d.sub.6): 8.07 (s, 1H, H2), 7.15 (d, J=3.6, 1H, H8), 
7.12 (broad s, 1H, H1,), 7.01 (broad s, 2H, NH.sub.2), 6.57 (d, J=3.6, 1H, 
H7), 6.43 and 6.02 (broad d, J=6.0, 1H each, H2'and H3'), 4.95 (t, J=6.5, 
1H, 5'OH), 4.79 (m, 1H, H4'), and 3.52 (m, 2H, H5'). 
C. PREATION OF 2',3'-DIDEOXY-7-DEAZAADENOSINE (20) 
A 450-mL Parr bottle was charged with olefin 19 (3.80 g), ethanol (76 mL), 
10% palladium on carbon (380 mg, Aldrich) and 40 psi of hydrogen. After 
shaking for 4.67 h at 25.degree., 14.5 psi of hydrogen had been absorbed 
and hydrogen uptake had ceased. TLC (two elutions with 85:13:2 
dichloromethane-methanol-concentrated ammonium hydroxide: starting 
material 19, 0.45; product 20, 0.48) showed complete conversion to a 
single UV-active new product. The catalyst was removed by filtration 
though filter aid and washed with ethanol. Removal of solvent from the 
filtrate and vacuum drying overnight afforded 3.98 g, (104%) of 
dideoxynucleoside 20 as a white foam. NMR indicated that the product was 
homogeneous except for the presence of 8 wt % of ethanol (96% corrected 
yield). Similar batches of this material resisted crystallization and 
became extremely hygroscopic upon azeotropic drying with anhydrous 
solvents. Therefore this material was stored under vacuum for about 1 week 
and used when NMR indicated that the material contained 5 wt % of ethanol. 
The lack of crystallinity and spectral characteristics observed for this 
product were in accord with those reported previously by Robins et al., 
Can. J. Chem., Vol. 55, 1259 (1977). 
.sup.1 H--NMR (DMSO-d.sub.6): 8.04 (s, 1H, H2), 7.33 (d, J=3.6, 1H, H8), 
6.97 (broad s, 2H, NH.sub.2), 6.56 (d, J=3.6, 1H, H7), 6.34 (dd, J=5.2 and 
6.4, 1H, H1'), 4.96 (t, J=5.6, 1H, 5'OH), 4.33 (t, J=5.1, 0.43H, ethanol 
OH), 4.04 (m, 1H, H4,), 3.4-3.6 (m, 2.86H, H5' and ethanol CH.sub.2), 
2.33, 2.21 and 2.02 (m, 4H, H2' and H3'), and 1.06 (t, J=7.0, 1.3H, 
ethanol CH.sub.3). 
D. PREATION OF 7-IODO-2',3'-DIDEOXY-7-DEAZAADENOSINE (21) 
A mechanically-stirred solution of 95% pure dideoxynucleoside 20 (2.95 g, 
11.96 mmol), anydrous sodium acetate (4.13 g, 50.3 mmol, 4 eq), and 
mercuric acetate (3.81 g, 11.95 mmol, 1.00 eq, Fisher, 99.9%) in water 
(190 mL) was heated under nitrogen at 65.degree. for 2 h. After cooling 
the resulting white suspension of mercurial to 25.degree., iodine (4.79 g, 
18.9 mmol, 1.6 eq) and ethyl acetate (190 mL) were added. After 1 h, the 
suspended mercurial had been consumed and a clear purple solution 
remained. After 2 h, sodium sulfite (6.35 g) was added and the purple 
color disappeared. After stirring for 30 min, hydrogen sulfide gas was 
gently bubbled into the reaction for 15 min. Mercuric sulfide (a black 
colloid) and iodide 21 (a white powder) precipitated from the reaction. 
Complete precipitation of mercury(II) was assessed by TLC by monitoring 
the disappearance of one of the two major UV-active spots. The reaction 
mixture was filtered though filter aid and separated into two layers. The 
filter aid was washed with boiling ethyl acetate (9.times.100 mL) until 
TLC indicated that no further product was being extracted. Each ethyl 
acetate extract was washed with the aqueous layer. The combined ethyl 
acetate layers were dried over magnesium sulfate and evaporated to 
dryness. The resulting crude solid turned red upon exposure to air. This 
material was dissolved in 3:1 dichloromethanemethanol (100 mL) and the 
free base form of AG3 X4A anion exchange resin (5.0 g, BioRad, 2.9 meq/g 
dry) was added. Hydrogen sulfide was bubbled into the red solution for 10 
min and the red color was discharged. A slight cloudiness was eliminated 
by briefly warming and the solution was rapidly filtered though a 2 cm 
plug (15 g) of silica gel. The silica gel was eluted with additional 3:1 
dichloromethane-methanol (100 mL). Silica gel (50 g) was added to the 
filtrate and hydrogen sulfide was bubbled in for 10 min. The solvent was 
removed from this mixture with a rotary evaporator and the silica gel was 
"dried" by co-evaporating with chloroform (200 mL). This silica gel was 
rapidly loaded onto a silica gel, column (500 g) which had been degassed 
with a stream of nitrogen. Elution under nitrogen with 5% (6 L) and 10% (4 
L) boiling methanol in dichloromethane afforded 2.92 g, (64%) of iodide 21 
as a white powder and 456 mg (7.5%) of less polar 
7,8-diiodo2',3'-dideoxy-7-deazaadenosine. Recrystallization of the major 
product from boiling ethyl acetate (200 mL) afforded 2.626 g, of white 
needles (mp 158.degree.-160.degree.). Concentration of the mother liquors 
to 10 mL afforded a second crop of 0.391 g, of light red needles (mp 
156.degree.-). Both crops were homogeneous according to NMR and TLC and 
together represent a 64% overall yield of iodonucleoside 21 from olefin 
19. .sup.1 H--NMR (DMSO-d.sub.6): 8.09 (s, 1H, H2), 7.67 (s, 1H, H8), 6.65 
(broad s, 2H, NH.sub.2), 6.34 (dd, J=4.4 and 6.8, 1H, H1'), 4.95 (t, 
J=5.5, 1H, 5'OH), 4.04 (apparent hept, J=3.5, 1H, H4'), 3.59 and 3.49 (m, 
2H, H5'), 2.30, 2.28 and 2.00 (m, 4H, H2' and H 3'). 
E. PREATION OF 
7-(3-TRIFLUOROACETAMIDO-1-PROPYNYL)2',3'-DIDEOXY-7-DEAZAADENOSINE (52) 
Iodide 21 (720.3 mg, 2.00 mmol) was coupled for 90 min with 
N-propargyltrifluoroacetamide following the standard procedure given in 
Example 1C. Chomatography with 7% methanol in dichloromethane afforded 
705.8 mg (92%) of coupling product 52 as an off white powder which was 
homogeneous according to NMR and TLC. Recrystallization from boiling ethyl 
acetate (10 mL) afforded 372 mg of white microcrystals (mp 
169.degree.-171.degree.). 
.sup.1 H--NMR (DMSO-d.sub.6): 10.1 (distorted t, 1H, NHTFA), 8.10 (s, 1H, 
H2), 7.78 (s, 1H, H8), 6.0-7.5 (very broad s, 2H, NH.sub.2), 6.34 (dd, 
J=4.5 and 7.0, 1H, H1'), 4.98 (t, J=5, 1H, 5'OH), 4.31 (slightly broadened 
s, H2--CH.sub.2 N--), 4.10 (apparent hept, J=3.5, 1H, H4'), 3.60 and 3.40 
(m, 2H, H5'), 2.37, 2.18 and 2.00 (m, 4H, H2' and H3'). TLC (90:9:1 
dichloromethane-methanol-concentrated ammonium hydroxide; UV): starting 
iodide 21, R.sub.f =0.36; product 52, 0.26). 
F. PREATION OF 7-(3-AMINO-1-PROPYNYL)-2',3'-DIDEOXY7-DEAZAADENOSINE 
5'-TRIPHOSPHATE (51) 
Alkynylamino nucleoside 52 (1.00 'mmol) was converted to the corresponding 
5'-triphosphate and the trifluoroacetyl group was removed following the 
general procedure described in Example 1E. After addition of the second 
aliquot of phosphorus oxychloride, the solution was stirred for 120 min. 
Assuming an absorption coefficient for the product equal to that of the 
starting material (12,700), the yield of triphosphate 51, based on the 
absorption at 279.5 nm, was 40%. 
.sup.1 H--NMR (D.sub.2 O): 7.97 (s, 1H, H2), 7.80 (s, 1H, H8), 6.33 (m, 1H, 
H1'), 4.44 (m, 1H, H4'), 4.27 (m, 1H, H5'a), 4.14 (m, 1H, H5'b), 4.11 
(broad s, 2H, --CH.sub.2 --), 2.6-2.0 (m, 4H, H2' and H3'), plus 
counterion (triethylammonium) peaks. .sup.31 P-NMR (D.sub.2 O): -8.59 
(broad d, J=20, IP), -9.56 (d, J=20, 1P), and -21.38 (m, 1P). UV (pH 8.2 
aq Tris): maxima at 238 and 279.5 nm. 
EXAMPLE 5 
A SECOND PREATION OF 7-IODO-2',3'-DIDEOXY-7-DEAZAADENOSINE (21) 
(Compound 21 is an intermediate prepared and used in Example 4.) 
A. PREATION OF 
6-CHLORO-2-METHYLTHIO-9-(2-DEOXY-.beta.-D-RIBOFURANOSYL)-7-DEAZAPURINE 
(23) 
Methanol (210 mL) and concentrated ammonium hydroxide (210 mL) were added 
to a solution of 
6-chloro-2-methylthio-9-(3,5-di-O-p-toluoyl-2-deoxy-.beta.-D-ribofuranosyl 
)-7-deazapurine (22, 26.8 g, prepared as described by Kazamierczuk et al., 
J. Am. Chem. Soc., Vol. 106, 6379 (1984)) in dichloromethane (210 mL). The 
resulting mixture was stirred at room temperature for 5 d and then 
evaporated to dryness. The residue was dried by co-evaporation with 
ethanol. The crude product was dissolved in dichloromethane and colorless 
crystals precipitated upon standing. The precipitate was collected and 
washed thoroughly with ether to afford 14.5 g, (79.1%) of diol 23 (mp d 
190.degree.-192.degree.). 
.sup.1 H--NMR (DMSO-d.sub.6): 2.26 and 2.55 (m, 2H, H2'), 2.57 (s, 3H, 
SCH.sub.3), 3.54 (m, 2H, H5'), 3.84 (m, 1H, H3'), 4.37 (m, 1H, H4'), 4.95 
(m, 1H, OH), 5.34 (m, 1H, OH), 6.57 (m, 1H, H1'), 6.63 (m, 1H. H7), 7.80 
(m, 1H, H8). This data was obtained from a different batch of diol 23 
prepared as described above. 
B. PREATION OF 
6-CHLORO-2-METHYLTHIO-9-(5-O-TRIPHENYLMETHYL-2-DEOXY-.beta.-D-RIBOFURANOSY 
L)7-DEAZAPURINE (24) 
Diol 23 (14.5 g) was dried by co-evaporation with dry pyridine. 
Triphenylmethyl chloride (16 g), 4-(dimethylamino)pyridine (600 mg), and 
triethylamine (8.0 mL) were added to a solution of the dry diol in dry 
pyridine (200 mL). After stirring the reaction mixture at 65.degree. under 
nitrogen for 6 h, additional triphenylmethyl chloride (2.0 g) and 
triethylamine (1.0 mL) were added and heating was continued for 17 h. 
After cooling, methanol (3 mL) was added and the reaction mixture was 
evaporated to dryness. The residue was partitioned between dichloromethane 
and 0.3N hydrochloric acid. The organic layer was washed with aqueous 
sodium bicarbonate and brine, dried over sodium sulfate, and evaporated to 
dryness. Chomatography of the resulting crude product on silica gel with 
1% and 1.5% methanol in dichloromethane afforded 22.7 g, (88.6%) of 
monotrityl ether 24 as a glassy solid. .sup.1 H--NMR (CDCl.sub.3): 2.48 
and 2.60 (m, 2H, H2'), 2.59 (s, 3H, SCH.sub.3), 3.40 (m, 2H, H5'), 4.08 
(m, 1H, H3'), 4.61 (m, 1H, H4'), 6.43 (m, 1H. H7), 6.68 (m, 1H, H1'), 
7.2-7.5 (m, 16H, trityl H and H8). This data was obtained from a different 
batch of 24 prepared as described above. 
C. PREATION OF 
6-CHLORO-2-METHYLTHIO-9-(5-O-TRIPHENYLMETHYL-2,3-DIDEOXY-.beta.-D-RIBOFURA 
NOSYL)-7-DEAZAPURINE (25) 
4-(Dimethylamino)pyridine (16.5 g) and phenyl chlorothionocarbonate (13.5 
mL) were added to a solution of trityl ether 24 in dry dichloromethane 
(300 mL). After stirring the reaction mixture at room temperature under 
nitrogen for 2.25 h, dichloromethane (200 mL) was added. The solution was 
washed with 0.5N hydrochloric acid (700 mL), 0.5N sodium hydroxide (700 
mL), and brine. The organic layer was dried over sodium sulfate and 
evaporated to dryness. 
The resulting crude thiocarbonate was dissolved in dry toluene (450 mL) and 
the solution was heated to a gentle reflux. Azoisobisbutyronitrile (600 
mg) and tri-n-butyltin hydride (17.7 mL) were added. After stirring at 
reflux under nitrogen for 15 min, additional tri-n-butyltin hydride (2.0 
mL) was added and the reaction mixture was refluxed for another 15 min. 
After cooling, the reaction mixture was diluted with ether (200 mL) and 
washed with 10% aqueous potassium fluoride (500 mL), 0.75N potassium 
hydroxide (500 mL), and brine. After drying over sodium sulfate and 
concentrating, chomatography of the resulting crude product on silica gel 
with 2:1 dichloromethane-ether and dichloromethane afforded 10.1 g, of 
dideoxynucleoside 25. The impure fractions were combined and 
rechomatographed to afford an additional 3.76 g, of pure product These 
products were combined to afford 13.9 g, (63.0%) of 25 as a colorless 
solid (mp 140.degree.-142.5.degree.). 
.sup.1 H--NMR (CDCl.sub.3): 2.11, 2.36, and 2.46 (m, 4H, H2' and H3'), 2.60 
(s, 3H, SCH.sub.3), 3.33 (apparent d, J=4, 2H, H5'), 4.32 (m, 1H, H4'), 
6.39 (d, J=3.7, 1H, H7), 6.52 (dd, J=6.7 and 3.7, 1H, H1,), 7.25 and 7.45 
(m, 15H, trityl H), 7.32 (d, 1H, J=3.7, H8). This data was obtained from a 
different batch of 25 prepared as described above. 
D. PREATION OF 
6-CHLORO-2-METHYLTHIO-9-(2',3'-DIDEOXY-.beta.-D-RIBOFURANOSYL)-7-DEAZAPURI 
NE (26) 
Trifluoroacetic acid (10 mL) was added to a solution of trityl ether 25 
(7.58 g) in 1:1 methanol-dichloromethane (100 mL) and the solution was 
stirred at 25.degree. under nitrogen for 17 h. The reaction mixture was 
partitioned between dichloromethane (500 mL) and aqueous sodium 
bicarbonate, and the aqueous layer was re-extracted with dichloromethane. 
The combined organic layers were dried over sodium sulfate and evaporated 
to dryness. Chomatography of the residue on silica gel with 0% and 5% 
methanol in dichloromethane afforded 4.07 g (97.1%) of nucleoside 26 as a 
thick colorless glass. 
.sup.1 H--NMR (CDCl.sub.3): 2.16, 2.26, and 2.50 (m, 4H, H2' and H3'), 2.63 
(s, 3H, SCH.sub.3), 2.76 (broad s, 1H, OH), 3.67 and 3.93 (m, 2H, H5'), 
4.27 (m, 1H, H4'), 6.38 (dd, J=6.7 and 5.2 Hz, 1H, H1'), 6.51 (d, J=3.7 
Hz, 1H, H7), 7.26 (d, 1H, J=3.7 Hz, H8). This data was obtained from a 
different batch of 26 prepared as described above. 
E. PREATION OF 2',3'-DIDEOXY-2-METHYLTHIO7-DEAZAADENOSINE (27) 
Ammonia (10 g) was distilled into a solution of chloride 26 (1.83 g) in 
methanol (50 mL) in a glass-lined bomb. The solution was heated in an 
autoclave at 100.degree. for 15 h. After cooling, the reaction mixture was 
evaporated to dryness. Purification of the resulting crude product on 
silica gel with 0%, 3% and 5% methanol in dichloromethane afforded 1.27 g, 
(80.4%) of deazaadenosine 27 as a colorless solid (mp 
184.degree.-185.degree.). 
.sup.1 H--NMR (DMSO-d.sub.6): 2.01, 2.21, and 2.39 (m, 4H, H2'and H3'), 
2.45 (s, 3H, SCH.sub.3), 3.50 (m, 2H, H5'), 4.02 (m, 1H, H4'), 4.83 (t, 
J=5.5, 1H, 5'OH), 6.32 (dd, J=7 and 4.5, 1H, H1'), 6.50 (d, J=3.7, 1H, 
H7), 7.07 (broad s, 2H, NH.sub.2), 7.20 (d, 1H, J=3.7, H8) 
F. PREATION OF 2',3'-DIDEOXY-7-DEAZAADENOSINE (20). 
A mixture of 600 mg of 27 and excess Raney Nickel (Aldrich, pre-washed with 
water and methanol) was refluxed under nitrogen until TLC indicated the 
disappearence of the starting material (6 h). The hot solution was 
filtered though filter-aid and the collected Raney nickel was washed well 
with methanol. The combined filtrates were evaporated to afford 424 g 
(84.9%) of 20 as a colorless glassy solid identical to the material 
prepared in Example 4C. 
G. PREATION OF 7-IODO-2',3'-DIDEOXY-7-DEAZAADENOSINE (21) 
Dideoxy-7-deazaadenosine 20 was iodinated following the procedure given in 
Example 4D. 
EXAMPLE 6 
A THIRD PREATION OF 7-IODO-2',3'-DIDEOXY-7-DEAZAADENOSINE (21) 
(Compound 21 is an intermediate prepared and used in Example 4.) 
A. PREATION OF 6-CHLORO-9-(2-DEOXY-.beta.-D-RIBOFURANOSYL)-7-DEAZAPURINE 
(29) 
A solution of concentrated ammonium hydroxide (100 mL) in methanol (175 mL) 
was added to a solution of 
6-chloro-9-(3,5-di-O-p-toluoyl-2-deoxy-.beta.-D-ribofuranosyl)-7-deazapuri 
ne (28, 10.0 g; prepared as described by Z. Kazimierczuk et al., J. Amer. 
Chem. Soc., Vol. 106, 6379 (1984)) in dichloromethane (100 mL). After 
stirring the resulting mixture at 25.degree. for 24 h, additional 
concentrated ammonium hydroxide (50 mL) was added. After stirring for a 
total of 5 d, the reaction mixture was evaporated to dryness and the crude 
product co-evaporated with ethanol. The residue was dissolved in 
dichloromethane and the desired product crystallized. Filtration and 
drying afforded 4.90 g (92%) of nucleoside 29 as colorless crystals (mp 
155.5.degree.-158.5.degree.). 
.sup.1 H--NMR (DMSO-d.sub.6): 2.30 and 2.55 (m, 2H, H2'), 3.58 (m, 2H, 
H5'), 3.85 (m, 1H, H3'), 4.40 (m, 1H, H4'), 4.97 (m, 1H, OH), 5.35 (m, 1H, 
OH), 6.65 (m, 1H, H1,), 6.75 (d, 1H. H7), 8.00 (m, 1H, H8), 8.65 (s, 1H, 
H2). This data was obtained from a different batch of 29 prepared as 
described above. 
B. PREATION OF 6-CHLORO-9-(5-O-TRIPHENYLMETHYL-35 
2-DEOXY-.beta.-D-RIBOFURANOSYL)-7-DEAZAPURINE (30) 
Nucleoside 29 (2.5 g) was dried by co-evaporation with dry pyridine. The 
residue was redissolved in dry pyridine (40 mL) and triphenylmethyl 
chloride (2.5 g), 4-(dimethylamino)pyridine (120 mg), and triethylamine 
(1.6 mL) were added. The reaction mixture was stirred at 65.degree. for 4 
h under nitrogen. Additional triphenylmethyl chloride (1.0 g) and 
triethylamine (0.6 mL) were added and the reaction was stirred at 
75.degree. for 18 h. After cooling, methanol (2 mL) was added and the 
reaction mixture was evaporated to dryness. The residue was partitioned 
between dichloromethane and 0.5N hydrochloric acid. The organic layer was 
washed with aqueous sodium bicarbonate and brine, dried over sodium 
sulfate, and evaporated to dryness. Chromatography on silica gel with 0%, 
1.5% and 3% methanol in dichloromethane afforded 2.26 g, (48%) of trityl 
ether 30 as a glassy solid. 
.sup.1 H--NMR (CDCl.sub.3): 2.46 and 2.65 (m, 2H, H2'), 3.40 (m, 2H, H5'), 
4.10 (m, 1H, H3'), 4.65 (m, 1H, H4'), 6.55 (d, 1H. H7), 6.72 (m, 1H, H1'), 
7.2-7.5 (m, 16H, trityl H and H8), and 8.60 (s, 1H, H2). 
C. PREATION OF 
6-CHLORO-9-(5-O-TRIPHENYLMETHYL2-DEOXY-3-THIONOCARBOPHENOXY-.beta.-D-RIBOF 
URANOSYL)7-DEAZAPURINE (30a) 
4-(Dimethylamino)pyridine (1.35 g) and phenyl chlorothionocarbonate (1.20 
mL) were added to a solution of trityl ether 30 in dry dichloromethane (30 
mL). After stirring the reaction mixture under nitrogen for 2 h at 
25.degree., additional dichloromethane (20 mL) was added and the solution 
was washed with 0.5N hydrochloric acid, 0.5N sodium hydroxide, and brine. 
The organic layer was dried over sodium sulfate and evaporated to dryness. 
Trituration of the residue with dichloromethane-ether afforded 1.53 g, 
(76%) of thiocarbonate 30a as colorless crystals (mp 
186.5.degree.-188.5.degree.). 
.sup.1 H--NMR (CDCl.sub.3): 2.85 and 3.00 (m, 2H, H2'), 3.55 m, 2H, H5'), 
4.50 (m, 1H, H4'), 6.00 (m, 1H, H3'), 6.60 (d, 1H. H7), 6.85 (m, 1H, 
H1'), 7.1-7.5 (m, 20H, trityl and phenyl H), 7.50 (d, 1H, H8), and 8.60 
(s, 1H, H2). 
D. PREATION OF 
6-CHLORO-9-(5-O-TRIPHENYLMETHYL2,3-DIDEOXY-.beta.-D-RIBOFURANOSYL)-7-DEAZA 
PURINE (31) 
A solution of thiocarbonate 30a (1.2 g), azoisobisbutyronitrile (50 mg), 
and tri-n-butyltinhydride (0.60 mL) in dry toluene (50 mL) was heated at 
110.degree. under nitrogen for 15 min. After cooling, the reaction mixture 
was diluted with 50 mL of ether and washed with 10% aqueous potassium 
fluoride (50 mL) and brine. The organic layer was dried over sodium 
sulfate and evaporated to dryness. Chromatography of the resulting crude 
product on silica gel with 0% and 1.5% methanol in dichloromethane 
afforded 0.84 g, (92%) of dideoxynucleoside 31 as a colorless solid (mp 
60.degree.-63.5.degree.). .sup.1 H--NMR (CDCl.sub.3): 2.11, 2.36, and 2.50 
(m, 4H, H2'and H3'), 3.37 (m, 2H, H5'), 4.35 (m, 1H, H4'), 6.50 (d, J=3.7, 
1H, H7), 6.58 (dd, 1H, H1' ), 7.25 and 7.45 (m, 15H, trityl H), 7.55 (d, 
1H, J=3.7, H8), and 8.60 (s, 1H, H2). 
E. PREATION OF 
6-CHLORO-9-(2,3-DIDEOXY-.beta.-D-RIBOFURANOSYL)-7-DEAZAPURINE (31a) 
Trifluoroacetic acid (1.5 was added to a solution of trityl ether 31 (700 
mg) in 1:1 methanoldichloromethane (20 mL). After stirring under nitrogen 
at 25.degree. for 17 h, sodium bicarbonate (1.5 g) was added and the 
mixture was stirred for 30 min. The reaction mixture was filtered and 
evaporated to dryness. Chromatography of the resulting crude product on 
silica gel with 0% and 2% methanol in dichloromethane afforded 300 mg 
(84%) of alcohol 31a as a colorless glass. .sup.1 H--NMR (CDCl.sub.3): 
2.20, 2.40, and 2.65 (m, 4H, H2' and H3'), 3.65 and 4.00 (m, 2H, H5'), 
3.95 (broad s, 1H, OH), 4.35 (m, 1H, H4'), 6.28 (dd, 1H, H1'), 6.62 (d, 
J=4, 1H, H7), 7.40 (d, 1H, J=4, H8), and 8.65 (s, 1H, H2). 
F. PREATION OF 
6-CHLORO-9-(5-ACETOXY-2,3-DIDEOXY-.beta.-D-RIBOFURANOSYL)-7-DEAZAPURINE 
(32) 
Acetic anhydride (2.0 mmol) was added to a solution of alcohol 31a (284 mg) 
in dry pyridine (10 mL). After stirring the solution for 1.25 h at 
25.degree., methanol (10 mL) was added. After stirring an additional 30 
min, the reaction mixture was evaporated to dryness. The residue was 
dissolved in dichloromethane and this solution was washed with 1N 
hydrochloric acid (2.times.) and brine (1.times.). The organic layer was 
dried over sodium sulfate and evaporated to dryness to afford 295 mg (89%) 
of crude acetate 32 as a colorless glass. 
.sup.1 H--NMR (CDCl.sub.3): 2.07 (s, 3H, acetyl), 2.20, 2.45, and 2.55 (m, 
4H, H2'and H3'), 4.25 and 4.35 (m, 2H, H5'), 4.40 (m, 1H, H4'), 6.55 (dd, 
1H, H1'), 6.65 (d, 1H, H7), 7.50 (d, 1H, H8), and 8.60 (s, 1H, H2). 
G. PREATION OF 
6-CHLORO-7-IODO-9-(5-O-ACETYL-2,3-DIDEOXY-.beta.-D-RIBOFURANOSYL)-7-DEAZAP 
URINE (33) 
A solution of iodine monochloride (340 mg; in dichloromethane (about 1 mL) 
was added to a solution of acetate 32 (200 mg) in dry dichloromethane (20 
mL). After stirring at 25.degree. for 3 h, the reaction mixture was 
partitioned between dichloromethane and aqueous sodium hydrosulfite. The 
organic layer was washed with aqueous sodium bicarbonate and brine, dried 
over sodium sulfate and evaporated to dryness. The residue was triturated 
with dichloromethane-ether to afford 135 mg (47%) of colorless crystals 
(mp 132.5.degree.-134.degree.). 
.sup.1 H--NMR (CDCl.sub.3): 2.10, 2.40, and 2.55 (m, 4H, H2' and H3'), 2.17 
(s, 3H, COCH.sub.3), 4.27 and 4.37 (m, 2H, H5'), 4.40 (m, 1H, H4'), 6.55 
(dd, 1H, H1'), 7.72 (s, 1H, H8), and 8.60 (s, 1H, H2). 
H. PREATION OF 7-IODO-2',3'-DIDEOXY-7-DEAZAADENOSINE (21) 
Ammonia (4 g) was added to a solution of 125 mg of chloride 33 in methanol 
(20 mL) in a glass-lined bomb. The bomb was heated in an autoclave at 
100.degree. for 3 h. After cooling, the reaction mixture was evaporated to 
dryness. The residue was dissolved in hot ethyl acetate and the hot 
solution was filtered though a pad of filter aid. After evaporating the 
filtrate to dryness, the residue was triturated with ether to afford 85 mg 
of slightly impure product 21 as colorless crystals. Further purification 
of this material by preparative TLC on a silica gel with 5% methanol in 
dichloromethane afforded 67 mg (63%) iodide 21 as a colorless solid. This 
material was identical to that prepared in Example 4D. 
EXAMPLE 7 
PREATION OF 7-IODO-2',3'-DIDEOXY-7-DEAZAINOSINE (53) 
(Compound 53 is an example of structure 4 wherein Het is 
7-deazahypoxanthine (1)) 
Water (18 mL) was added dropwise to a suspension of deazaadenosine 21 
(720.3 mg, 2.00 mmol) in glacial acetic acid (2.0 mL) under argon to 
produce a clear solution. Solid sodium nitrite (1.38 g, 20.0 mmol, 10 eq) 
was added though a stream of argon in small batches over 10 min. The 
resulting cloudy reaction mixture was mechanically stirred under argon and 
a gummy precipitate gradually formed. After 18 h, the reaction was 
filtered and the precipitate was washed thoroughly with ethyl acetate (100 
mL) and water (about 10 mL). The combined filtrates were partitioned and 
the aqueous layer was extracted with ethyl acetate (2.times.50 mL). The 
combined organic layers were dried over magnesium sulfate and evaporated 
to dryness. According to TLC, both the precipitate and the ethyl acetate 
extracts consisted of product 53 and contaminated with less than 5% of 
unreacted 21. Both batches of product were dissolved in 1:1 
methanol-dichloromethane, combined, and evaporated onto silica gel (7 g). 
The silica gel was co-evaporated with chloroform (50 mL) and placed on a 
silica gel column (50 g). Elution with 8% methanol in dichloromethane 
afforded 558.3 mg (78%) of deazainosine 53 as a pale yellow solid. Two 
crops of white needles were obtained by recrystallizing this material from 
boiling isopropanol. These needles exhibited a melting point with 
decomposition that varied between 200.degree. and 210.degree.. The 
chromatographed and recrystallized products were homogeneous by TLC and 
NMR except for the presence of isopropanol (5 mole %). 
.sup.1 H--NMR (DMSO-d.sub.6): 12.04 (broad s, 1H, H1), 7.93 (s, 1H, H2), 
7.56 (s, 1H, H8), 6.29 (dd, J=4.0 and 6.8, 1H, H1'), 4.94 (t, J=5.0, 1H, 
5'OH), 4.04 (apparent hept, J=3.5, 1H, H4'), 2.36, 2.18 and 1.99 (m, 4H, 
H2' and H3'). 
EXAMPLE 8 
PREATION OF 5-(3-AMINO-1-PROPYNYL)2 -DEOXYCYTIDINE 5'-TRIPHOSPHATE (54) 
(Compound 54 is an example of an alkynylamino nucleotide (I) wherein 
R.sub.1 is --CH.sub.2 --, Het is cytosine (i), R.sub.2, R.sub.3, R.sub.7, 
and R.sub.8 are H, R.sub.6 is OH, and R.sub.5 is P.sub.3 O.sub.9 H.sup.3 
--.) 
A. PREATlON OF 5-IODO-2'-DEOXYCYTIDINE (55) 
A solution of 2,-deoxycytidine monohydrate (1.226 g, 5.00 mmol, Aldrich) 
and mercuric acetate (1.753 g, 5.5 mmol, 1.1 eq, Fisher) in methanol (20 
mL) was refluxed for 14.5 h. The resulting white suspension was diluted 
with methanol (30 mL) and dichloromethane (50 mL) and then iodine (1.522 
g, 6.00 mmol, 1.2 eq) was added. After stirring for 60 min, the resulting 
purple solution had decolorized and unreacted mercurial was still visible 
as a white suspension. After 100 min and 240 min, further additions of 
iodine (0.381 g, 1.5 mmol, 0.3 eq and 0.122 g, 0.50 mmol, 0.1 eq; 
respectively) were made. After a total of 5 h, the reaction was crystal 
clear and purple. AG3 X4A resin in the free base form (5.17 g, 2.9 meq/g, 
3 eq, Bio-Rad) was added and then hydrogen sulfide was bubbled into the 
reaction mixture for 5 min. Complete precipitation of mercury(II) was 
verified by TLC. The reaction was filtered though filter aid and the 
filter aid was washed with 1:1 methanol-dichloromethane. Silica gel (5 g) 
was added to the combined filtrates and the reaction mixture was 
evaporated to dryness. The silica gel was co-evaporated with chloroform 
(50 mL) and placed on a silica gel column (50 g). Elution with 15%, 20% 
and 30% methanol in dichloromethane afforded 1.378 g, (78%) of 
iodocytidine 55 as a white powder. Recrystallization from boiling methanol 
(35 mL) afforded, after vacuum-drying overnight, 0.953 g, of white needles 
(mp 179.degree.-180.degree.). Concentration of the mother liquors to 10 mL 
afforded a second crop of 0.140 g, of pale yellow needles (mp 
172.degree.-174.degree.). With the exception of a trace of methanol, both 
crops (total yield, 62%) were homogeneous according to TLC and NMR. 
.sup.1 H--NMR (DMSO-d.sub.6): 8.28 (s, 1H, H6), 7.8 and 6.6 (broad s, 2H, 
NH.sub.2), 6.08 (t, J=6.3, 1H, H1'), 5.20 (d, J=4, 1H, 3'OH), 4.90 (t, 
J=5, 1H, 5'OH), 4.20 (m, 1H, H4'), 3.77 (distorted q, 1H, H3'), 3.60 and 
3.54 (m, 1H, H5'), 2.12 and 1.98 (m, 1 H, H2'). TLC (75:20:5 
dichloromethane-methanol-concentrated ammonium hydroxide; UV): starting 
material, R.sub.f =0.15; product 55, 0.33; mercury(II), 0.54. 
B. PREATION OF 5-(3-TRIFLUOROACETAMIDO1-PROPYNYL)2'-DEOXYCYTIDINE (56) 
Iodide 55 (353.1 mg, 1.00 mmol) was coupled for 4 h to 
N-propargyltrifluoroacetamide following the general procedure given in 
EXAMPLE 1C. Chomatography of the crude product with a 0-20% methanol in 
dichloromethane gradient afforded 3.84 g, (102%) of white powder after 
vacuum drying overnight. This material was homogeneous by TLC, but 
tenaciously retained solvent. Recrystallization of this powder from 
boiling isopropanol (10 mL) and cooling to -20.degree. afforded 299.6 mg 
(74%) alkynylamino nucleoside 56 as white needles (mp 
168.degree.-170.degree.). NMR showed that the recrystallized product was 
homogeneous and that the crystals contained 0.5 molecules of isopropanol 
per molecule of product 56. 
.sup.1 H--NMR (DMSO-d.sub.6): 9.96 (broad s, 1H, NHTFA), 8.15 (s, 1H, H6), 
7.83 and 6.86 (broad s, 2H, NH.sub.2), 6.10 (t, J=6.5, 1H, H1'), 5.21 (d, 
J=4.5, 1H, 3'OH), 5.06 (t, J=5, 1H, 5'OH), 4.35 (d, J=4, 0.5H, isopropanol 
OH), 4.28 (broad s, 2H, --CH.sub.2 N--), 4.20 (apparent hex, J=3.5, 1H, 
H4'), 3.79 (m, 1.5H, H3' and isopropanol CH), 3.56 (m, 2H, H5'), 2.13 and 
1.97 (m, 1H, H2'), and 1.04 (d, J=6, 3H, isopropanol CH.sub.3). TLC 
(85:13:2 dichloromethane-methanol-concentrated ammonium hydroxide, two 
elutions; UV): starting iodide 55, R.sub.f =0.31; product 56, 0.27. 
C. PREATION OF 5-(3-AMINO-1-PROPYNYL)2'-DEOXYCYTIDINE 5'-TRIPHOSPHATE 
(54) 
Alkynylamino nucleoside 56 (0.275mmol) was converted to the corresponding 
5'-triphosphate and its trifluoroacetyl group was removed following the 
general procedure given in Example 1E. After addition of the second 
aliquot of phosphorus oxychloride, phosphorylation was allowed to proceed 
for 3.5 h. Assuming an absorption coefficient for the product equal to 
that of the starting material (8,780), the yield of triphosphate 54, based 
on its UV absorption at 293 nm, was 17%. 
EXAMPLE 9 
PREATlON OF 5-(3-TRlFLUOROACETAMIDO1-PROPYNYL)-2'-DEOXYURIDINE (57) 
(Compound 57 is an example of an alkynylamino nucleotide (I) wherein 
R.sub.1 is --CH.sub.2 --, R.sub.2 is COCF.sub.3, Het is uracil (h), 
R.sub.3, R.sub.5, R.sub.7 and R.sub.8 are H, and R.sub.6 is OH.) 
5-Iodo-2'-deoxyuridine (7.08 g, 20.0 mmol, Aldrich) was coupled for 4 h to 
N-trifluoroacetylpropargylamine following the general procedure given in 
EXAMPLE 1C except that the reaction was run 2.5 times more concentrated 
than usual. Chromatography of the crude product on silica gel (500 g) with 
10-20% methanol in dichloromethane afforded, 3.50 g, (46%) of alkynylamino 
nucleoside 57 as a tan solid. According to NMR and TLC, this material was 
&gt;95% pure except for the presence of methanol (about 50 mole %) that was 
not removed by vacuum-drying. 
.sup.1 H--NMR (DMSO-d.sub.6): 11.63 (s, 1H, H3), 10.06 (distorted t, 1H, 
NHTFA), 8.19 (s, 1H, H6), 6.10 (apparent t, 1H, H1'), 5.23 (d, J=4, 1H, 
3'OH), 5.07 (t, J=5, 1H, 5'OH), 4.23 (m, 3H, --CH.sub.2 --and H4'), 3.8 
(apparent q, J=4, 1H, H3'), 3.58 (m, 2H, H5'), and 2.12 (m, 2H, H2'). 
EXAMPLE 10 
PREATION OF 5-(5-TRIFLUOROACETAMIDO-1-PENTYNYL)2',3'-DIDEOXYURIDINE (58) 
(Compound 58 is an example of structure 5 wherein 
Het is uracil (h) and R.sub.1 is --(CH.sub.3).sub.3 --.) 
A. PREATION OF 5-TRIFLUOROACETAMIDO-1-PENTYNE (59) 
Sodium hydride (60% dispersion in oil, Alfa) was rendered oil-free by 
thoroughly and rapidly washing with pentane and then vacuum-drying. 
Oil-free sodium hydride (4.40 g, 0.110 mole, 1.1 eq) was added in about 20 
portions over 25 min to a solution of 5-chloropentyne (10.6 mL, 0.100 
mole, 1.0 eq), trifluoroacetamide (14.13 g, 0.125 mole, 1.25 eq), and 
sodium iodide (14.99 g, 0.100 mole, 1.0 eq) in dry dimethylformamide (250 
mL, Aldrich). The reaction mixture was stirred at 25.degree. for 4.5 h and 
at 60.degree. for 21 h. After cooling, the reaction mixture was added to a 
solution of potassium dihydrogen phosphate (43.5 g, 0.250 mole, 2.0 eq) in 
water (500 mL). This solution was extracted with pentane (2.times.500 mL) 
and ether (3.times.500 mL). The combined organic layers were washed with 
water (1.times.100 mL), dried over magnesium sulfate, and concentrated 
with a rotary evaporator. Fractional distillation twice through a 20 cm 
Vigreux column afforded 8.09 g, (45%) of 5-trifluoroacetamido-1-pentyne 
(58) as a colorless, mobile liquid (bp 68.degree..degree.-69.degree. at 13 
torr.) 
.sup.1 H--NMR (CDCl.sub.3): 6.77 (broad s, 1H, NHTFA), 3.53 (q, J=6.7 and 
2.7, 2H, -CH.sub.2 NHTFA), 2.31 (td, J=6.7 and 2.7, 2H, HCCCH.sub.2 --), 
2.04 (t, J=2.7, 1H, HCCCH.sub.2 --), and 1.83 (quintet, J=6.7, 2H, 
--CH2CH2CH2--). 
B. PREATION OF 5-(5-TRIFLUOROACETAMIDO-1-PENTYNYL)2',3'DIDEOXYURIDINE 
(58) 
5-Trifluroacetamido-1-pentyne (59) was coupled for 4 h to 
5-iodo-2',3'-dideoxyuridine (47, prepared as described in Example 2A) 
according to the general procedure described in EXAMPLE 1C. Chromatography 
on silica gel (100 g) with a 0-5% methanol in dichloromethane gradient 
afforded 647.7 mg of alkynylamino nucleoside 58 as a light tan foam. This 
material was homogeneous by TLC and NMR except for the presence of about 
16 mole % of dimethylformamide. Correcting for the presence of 
dimethylformamide, the yield of desired product was 80%. 
.sup.1 H--NMR (DMSO-d.sub.6): 11.52 (s, 1H, H3), 9.47 (distorted t, 1H, 
NHTFA), 5.90 (q, 1H, H1'), 5.12 (t, 1H, 5'OH), 4.04 (m, 1H, H4'), 3.71 and 
3.52 (m, 2H, 5'H), 3.30 (m, 2H, --CH.sub.2 CH.sub.2 CH.sub.2 NHTFA), 2.40 
(t, 2H, --CH.sub.2 CH.sub.2 CH.sub.2 NHTFA), 2.23, 2.01 and 1,85 (m, 4H, 
H2' and H3'), and 1.73 (quintet, 2H, --CH2CH2CH2NHTFA). 
EXAMPLE 11 
PREATION OF 5-(12-TRlFLUOROACETAMIDO-l-DODECYNYL)2',3'-DIDEOXYURIDINE 
(60) 
(Compound 60 is an example of structure 5 wherein Het is uracil (h) and 
R.sub.1 is --(CH.sub.2).sub.10 --.) 
A PREATION OF 11-DODECYN-1-OL (61) 
1-Bromo-10-tetrahydropyranyloxydecane (64.26 g, 0.200 mole, Lancaster, 
"97+%") was added dropwise over 140 min to a precooled suspension of 
lithium acetylide ethylenediamine complex (23.94 g, 0.260 mole, 1.3 eq, 
Aldrich, 90%) in dry dimethylsulfoxide (100 mL) so that the internal 
temperature remained at 5.degree.-10.degree.. After the addition was 
complete, the cooling bath was removed and the reaction mixture was 
stirred for 4.5 h. Water (20 mL) was added dropwise to the reaction 
mixture. After stirring for 10 min, the reaction mixture was poured into 
water (300 mL). This solution was extracted sequentially with pentane 
(2.times.300 mL) and ether (2.times.300 mL). Each organic layer was washed 
individually with water (about 20 mL) and the aqueous washes were combined 
with the main aqueous layer for re-extraction. The combined organic layers 
were dried over magnesium sulfate and evaporated to dryness to afford 
51.38 g (96%) of crude 12-(tetrahydropyranyloxy)-1-dodecyne as an oil. 
A strongly acidic ion exchange resin (AG-50W-X8, 50 g, 5.1 meq/g, Bio-Rad) 
was added to a solution of the above crude product (49.96 g) in a mixture 
of chloroform (260 mL) and methanol (260 mL). The suspension was heated at 
reflux for 4.5 h and then cooled. The reaction mixture was filtered and 
the filtrate was concentrated. Chromatography of the residue on silica gel 
(500 g) with 10%, 20% and 30% ethyl acetate in hexanes afforded 31 g, of 
an oil which was &gt;95% one spot by TLC with detection by phosphomolybdic 
acid. Distillation of this material through a 20 cm Vigreux column 
afforded, after a 0.78 g, forerun, 17.91 g, of 11-dodecyn-1-ol (61) as a 
thick, colorless oil (bp 104-108 at 1.4 torr) which solidified to a white 
solid on standing. This material was 98% one peak by GC. 
.sup.1 H--NMR (CDCl.sub.3) of the chromatographed product before 
distillation: 3.64 (t, 2H, --CH.sub.2 OH), 3.37 and 3.33 (m, about 0.2H, 
impurity), 2.17 (td, 2H, HCCCH.sub.2 --), 1.92 (t, 1H, HCCCH.sub.2 --), 
and 1.2-1.6 (m, 17H, (CH.sub.2).sub.8 and OH). IR (thin film of melt): 
3392 (O--H), 3311, 2930 and 2854 (C--H), 2160 (acetylene), 1466, 1432, 
1394, 1371, 1352, 1328, 1303, 1103, and 1001. 
B. PREATION OF 12-IODO-1-DODECYNE (62) 
Iodine (43.16 g, 170 mmol, 2.0 eq) was added to a suspension of distilled 
alcohol 61 (15.50 g, 85 mmol), imidazole (17.36 g, 255 mmol, 3.0 eq), and 
triphenylphosphine (66.90 g, 255 mmol, 3.0 eq) in dry toluene (425 mL, 
stored over molecular sieves). The reaction mixture was heated at reflux 
with vigorous stirring for 25 min, generating a yellow solution with a 
oily black precipitate. After cooling to 25.degree., saturated aqueous 
sodium bicarbonate (200 mL) and iodine (23.73 g, 93.5 mmol, 1.1 eq) were 
added and the reaction was stirred vigorously for 1 h. Saturated aqueous 
sodium sulfite (40 mL) was added, quenching the purple color. The reaction 
mixture was allowed to separate into two layers and the organic layer was 
washed with brine. The organic layer was dried over magnesium sulfate and 
concentrated. The residue was dissolved in dichloromethane (50 mL) and 
ether (200 mL) was added. After standing for 30 min, the resulting 
precipitate (triphenylphosphine oxide) was removed by filtration and 
washed with ether (100 mL). On further standing, the combined mother 
liquor and ether wash deposited a second crop of crystals which were 
removed as before. The combined mother liquors and ether washes were 
concentrated and dissolved in warm toluene (200 mL). This solution was 
placed on a silica gel column (500 g) and eluted with toluene (3 L) to 
afford 13.55 g, (55%) of iodide 62 as a pale yellow mobile liquid. This 
material was 96% one peak by GC. 
.sup.1 H--NMR (CDCl.sub.3): 3.20 (t, 2H, --CH.sub.2 I), 2.17 (td, 2H, 
HCCCH.sub.2 --), 1.94 (t, 1H, HCCCH.sub.2 --), 1.82, 1.51 and 1.20-1.42 
(m, 16H, (CH.sub.2).sub.8). 
C. PREATION OF 12-TRIFLUOROACETAMIDO-1-DODECYNE (63) 
Sodium hydride (60% dispersion in oil, Alfa) was rendered oil-free by 
rapidly and thoroughly washing with pentane and vacuum-drying. 
Trifluoroacetamide (22.61 g, 200 mmol, 5 eq) was added in about 10 
portions over 50 min to a suspension of oil-free sodium hydride (3.84 g, 
160 mmol, 4 eq) in dry dimethylformamide (90 mL, Aldrich). When it was 
discovered early in this addition that the reaction mixture was getting 
warm, an ice-water bath was added and the rest of the addition was 
performed at an internal temperature of about 10.degree.. The ice-water 
bath was removed and the reaction mixture was stirred until hydrogen 
evolution ceased. After stirring an addition 15 min, a solution of iodide 
63 (11.69 g, 40.0 mmol) in dry dimethylformamide (10 mL) was added 
dropwise over 10 min to the reaction mixture. After stirring for 4 h at 
25.degree., the reaction mixture was rapidly poured into a stirred mixture 
of saturated aqueous ammonium chloride (200 mL), water (200 mL) and 
pentane (200 mL). The reaction vessel was rinsed with a mixture of water 
(50 mL), saturated aqueous ammonium chloride (50 mL) and pentane (200 mL). 
The combined solutions were allowed to separate into two layers and the 
aqueous layer was extracted with pentane (2.times.200 mL). The combined 
organic layers were dried over magnesium sulfate and evaporated to dryness 
to yield 10.42 g, (94%) of trifluoroacetamide 63 as an oil which 
solidified to a waxy solid on standing. Recrystallization of this material 
from boiling hexanes (100 mL) with slow cooling to -20.degree. afforded 
8.145 g (73%) of trifluoroacetamide 63 as pale yellow needles (mp 
46.degree.-47.degree.). 
.sup.1 H--NMR (CDCl.sub.3): 6.27 (broad s, 1H, NHTFA), 3.34 (apparent q, 
2H, --CH.sub.2 NHTFA), 2.18 (td, 2H, HCCCH.sub.2 --), 1.94 (t, 1H, 
HCCCH.sub.2 --), 1.20-1.65 (m, 16H, (CH.sub.2).sub.8). IR (thin film of 
melt): 3312, 3298, 2932 and 2857 (C-H and N--H), 2117 (acetylene), 1706 
(C.dbd.O), 1675 , 1563, 1460, 1448, 1208, 1182, 1166, 722, and 634. 
D. PREATION OF 5-(12-TRIFLUOROACETAMIDO1-DODECYNYL)-2',3'-DIDEOXYURIDINE 
(60) 
Protected alkynylamine 63 was coupled for 24 h to 
5-iodo-2',3'-dideoxyuridine (47, 676.2 mg, 2.00 mmol, prepared as 
described in Example 2A) following the general procedure described in 
EXAMPLE 1C. Chromatography on silica gel (100 g) eluting with a 0-5% 
methanol in dichloromethane gradient afforded a dark red foam. The red 
impurity was removed by chromatography on a reverse phase column (100 g, 
octadecylsilane on 40 micrometer silica gel, Baker) with 40% water in 
methanol. The appropriate fractions were combined, concentrated, and 
co-evaporated twice with absolute ethanol to afford 731 mg of alkynylamino 
nucleoside 60 as a clear oil. This material was homogeneous by TLC and NMR 
except for the presence of residual ethanol (25 mole %, corrected yield 
73%). 
.sup.1 H--NMR (DMSO-d.sub.6): 11.49 (broad s, 1H, H3), 9.38 (distorted t, 
1H, NHTFA), 8.15 (s, 1H, H6), 5.90 (dd 1H, H1'), 5.12 (distorted t, 1H, 
5'OH), 4.35 (t, 0.25H, CH.sub.3 CH.sub.2 OH), 4.03 (m, 1H, H4'), 3.72 and 
3.52 (m, 2H, H5'), 3.43 (m, 0.5H, CH.sub.3 CH.sub.2 OH), 3.16 (quintet, 
2H, --CH.sub.2 NHTFA), 2.34 (t, 2H, propargylic H), 2.16, 2.01, and 1.86 
(m, 4H, H2' and H3'), 1.65-1.15 (m, 16H, (CH.sub.2).sub.8), and 1.06 (t, 
0.75H, CH.sub.3 CH.sub.2 OH). 
EXAMPLE 12 
PREATION OF 5-(5-AMINO-1-PENTYNYL)2',3'-DIDEOXYURIDINE (64) 
(Compound 64 is an example of an alkynylamino nucleotide (I) wherein Het is 
uracil (h), R.sub.1 is (CH.sub.2).sub.3, and R.sub.2, R.sub.3, R.sub.5, 
R.sub.6, R.sub.7 and R.sub.8 are H.) 
A. PREATION OF 5-AMINO-1-PENTYNE (65) 
Ammonia (340 g, 20 mole) was distilled into a bomb which contained 
5-chloropentyne (20.51 g, 0.200 mole) and sodium iodode (7.49 g, 0.050 
mole, 0.25 eq). The bomb was sealed and heated in an autoclave at 
100.degree. for 12 h. The ammonia was allowed to evaporate and the residue 
was stirred with a two phase mixture consisting of sodium hydroxide (40 g, 
1.0 mole, 5 eq), water (100 mL), and ether (100 mL). The resulting mixture 
was filtered and allowed to separate into two layers. The organic layer 
was dried over magnesium sulfate and distilled through a 20 cm Vigreux 
column. Four fractions (12.46 g, bp 95.degree.-127.degree., atmospheric 
pressure) were found by GC to contain significant amounts of product. 
These fractions were combined and carefully distilled through a spinning 
band column to afford 6.55 g, (39%) of 5-amino-1-pentyne (65) as a 
colorless, mobile liquid (bp 125.5.degree.-126 .degree.). This material 
was &gt;99% one peak by GC. 
.sup.1 H--NMR (CDCl.sub.3): 2.81 (t, J=7.5, 2H, --CH.sub.2 NH.sub.2), 2.27 
(td, J=7.5 and 2.5, 2H, HCCCH.sub.2 --), 1.96 (t, J=2.5, 1H, HCCCH.sub.2 
--), 1.66 (quintet, J=7.5, 2H, --CH2CH2CH2--), and 1.07 (broad s, 2H, 
NH.sub.2). 
B A GENERAL PROCEDURE OF COUPLING UNPROTECTED ALKYNYLAMINES TO 
IODONUCLEOSlDES. PREATION OF 5-(5-AMINO-1-PENTYNE)-2',3'-DIDEOXYURIDINE 
(64) 
A dry, 35-mL, round-bottomed flask was charged with 
5-iodo-2',3'-dideoxyuridine (47, 676.2 mg, 2 00 mmol, prepared as 
described in Example 2A) and then flushed with argon. Dry 
dimethylformamide (10 mL, Aldrich), dry triethylamine (0.56 mL, 4.0 mmol, 
2.0 eq, stored over sieves), 5-amino-1-pentyne (0.59 mL, 6.03 mmol, 3.0 
eq), and tetrakis(triphenylphosphine)palladium(O) (231 mg, 0.200 mmol, 0.1 
eq, weighed into a vial in a dry box) were added. The resulting suspension 
was stirred for 45 min, but the palladium catalyst remained at least 
partly undissolved. Cuprous iodide (190.4 mg, 1.00 mmol, 0.5 eq, Aldrich 
Gold Label) was added. After stirring for 15 min, a homogenous blue 
solution had formed and after about 150 min the solution became cloudy. 
After 200 min, TLC showed that all of starting iodide 47 had been 
consumed. After 4 h, the reaction mixture was concentrated with a rotary 
evaporator for about 10 min at 45.degree. and 2 torr. The residue was 
immediately absorbed onto a silica gel column (100 g) and eluted with a 
mixture of dichloromethane, methanol and concentrated ammonium hydroxide 
(400 mL each of 90:9:1, 85:13:2, 75:20:5, 65:30:5 and 50:45:5). The 
fractions containing the major polar product according to TLC were 
combined, co-evaporated twice with ethanol, and vacuum-dried overnight to 
afford 395.9 g, (67%) of alkynylamino nucleoside 64 as a yellow solid. 
This material was homogeneous by TLC and NMR except for the presence of 
ethanol (33 mole %) which was not removed by vacuum-drying. The yield of 
64, corrected for the presence of ethanol, was 64%. 
.sup.1 H--NMR (DMSO-d.sub.6): 8.33 (s, 1H, H6), 5.90 (dd, J=6.6 and 3.0, 
1H, H1'), 4.05 (m, 1H, H4'), 3.73 (dd, J=12.1 and 2.8, 1H, H5'a), 3.53 
(dd, J=12.1 and 3.1, 1H, H5'b), 2.80 (broad s, 2H, --CH.sub.2 H.sub.2), 
2.45 (t, J=7.0, 2H, propargylic H), 2.28, 2.02 and 1.86 (m, 4H, H2' and 
H3'), and 1.70 (quintet, J=7.0 Hz, 2H, --CH.sub.2 CH.sub.2 CH.sub.2 --). 
This NMR data was obtained form a different batch of 64 prepared in a 
manner similar to that described above. The signals for the exchangeable 
hydrogens (H3, 5'OH, and --NH.sub.2) in NMR samples of both materials were 
combined into a single broad (&gt;2 ppm wide) signal which was barely 
resolved from the baseline. 
EXAMPLE 13 
PREATION OF 5-(3-AMINO-1-PROPYNYL)2',3'-DIDEOXYURIDINE (66) 
(Compound 66 is an example of an alkynylamino nucleotide (I) wherein Het is 
uracil (h), R.sub.1 is CH.sub.2, and R.sub.2, R.sub.3, R.sub.5, R.sub.6, 
R.sub.7 and R.sub.8 are H.) 
5-Iodo-2',3'-dideoxyuridine (47, 2.00 mmol) was coupled for 3 h to 
propargylamine (6.00 mmol, Aldrich) according to the procedure described 
in Example 12B except that propargylamine was used in place of 
5-amino1-pentyne. Chromatography as described above returned 794.5 mg of 
impure alkynylamino nucleoside 66 as a yellow solid which afforded a 
single spot when analyzed by TLC. NMR and the mass balance of the reaction 
indicated that this material was contaminated by ethanol and possibly 
inorganic impurities. 
.sup.1 H--NMR (DMSO-d.sub.6): 11.70 (broad s, 1H, H3), 8.40 (s, 1H, H6), 
8.25 (broad s, 2H, NH.sub.2), 5.89 (dd, J=6.6 and 3.0, 1H, H1'), 5.13 (t, 
J=5.0, 1H, 5'OH), 4.07 (m, 1H, H4'), 3.96 (s, 2H, --CH`NH.sub.2), 3.71 and 
3.56 (m, 2H, H5'), 2.30, 2.04 and 1.85 (m, 4H, H2' and H3') and signals 
for ethanol and an unknown impurity. The above NMR data was taken from 
different preparation of 66 performed as above except that 0.2 eq of 
cuprous iodide was used and the reaction did not go to completion. 
EXAMPLE 14 
PREATION OF 1-(2-HYDROXYETHOXYMETHYL)5-(3-AMINO-1-PROPYNYL)CYTOSINE 
TRIPHOSPHATE (67) 
(Compound 67 is an example of an alkynylamino nucleotide (I) wherein 
R.sub.1 is --CH.sub.2 --, R.sub.2 and R.sub.3 are H, Het is cytosine (i), 
R.sub.4 is (g), and R.sub.5 is P.sub.3 O.sub.9 H.sup.3 --.) 
A. PREATION OF 1-(2-HYDROXYETHOXYMETHYL)5-IODOCYTOSINE (68) 
A mixture of 1-(2-hydroxyethoxymethyl)cytosine (1.85 g, 10.0 mmol) and 
mercuric acetate (3.35 g, 10.5 mmol) was refluxed in methanol (50 mL) and 
dichloromethane (100 mL). Iodine (3.05 g, 12.0 mmol) was added and the 
reaction mixture was stirred for 1 h. The free base form of AG3-X4 resin 
(38 meq) was added and the solution bubbled with hydrogen sulfide for 15 
min. The solids were removed by filtration and the filtrate stripped down 
onto silica gel (10 g). The silica was loaded onto a silica gel column 
(4.times.25 cm) and eluted 5%, 10% and 20% methanol in dichloromethane. 
Evaporation followed by vacuum-drying afforded a colorless solid (1.73 g, 
56%). 
Recrystallization from 95% ethanol afforded analytically pure material (mp 
172.degree.). Calculated for C.sub.7 H.sub.10 N.sub.3 O.sub.3 I: C 27.03%, 
H 3.24%, N 13.51%. Found: C 27.08%, H3.41%, N13.51%. UV (methanol): 
maximum at 292.5 (5,300). .sup.1 H--NMR (DMSO-d.sub.6): 3.481 (m, 4H), 
4.659 (t, J=5, 1H), 5.070 (s, 2H, 6.665 (broad s, 1H), 7.869 (broad s, 
1H), and 8.107 (s, 1H), 
B PREATION OF 
1-(2-HYDROXYETHOXYMETHYL)-5-(3-TRIFLUOROACETAMIDO-1-PROPYNYL)CYTOSINE (69) 
Iodide 68 (311 mg, 1.00 mmol) was coupled to N-propargyltrifluoroacetamide 
(43) according to the general procedure described in EXAMPLE 1C. Flash 
chromatography on silica gel (3.times.20 cm) with 5%, 10% and 20% methanol 
in dichlormethane afforded alkynylamino nucleotide 69 as a pale yellow 
foam (77.4 mg, 23%). 
.sup.1 H--NMR(DMSO-d.sub.6): 3.472 (broad s, 4.276 (d, J=5.0, 2H), 4.653 
(broad t, J=4.5, 1H), 5.091 (s, 2H), 6.925 (broad s, 1H), 8.037 (s, 1H), 
and 9.964 (broad s, 1H), 
C. PREATION OF 1-(2-HYDROXYETHOXYMETHYL)5-(3-AMINO-1-PROPYNYL)CYTOSINE 
(67) 
The hydroxyl group of the sugar part of alkynylamino nucleoside 69 (0.167 
mmol) was converted to a triphosphate and the trifluoroacetyl group was 
removed following the general procedure given in Example 1E. After 
addition of the second aliquot of phosphorus oxychloride, phosphorylation 
was allowed to proceed for for 75 min. Assuming an absorption coefficient 
for the product equal to that of the starting material (7,790), the yield 
of triphosphate 67, based on its UV absorption at 291 nm, was 21%. 
EXAMPLE 15 
Preparation of N-Hydroxysuccinimide Ester 2a 
(A preferred reagent for attaching a 505 nm fluorescent dye to an 
alkynylamino-nucleotide wherein R.sub.9 and R.sub.10 are H). 
A. Preparation of 9-(Carboxyethylidene)-3,6-dihydroxy-9H-xanthene (SF-505) 
Resorcinol (33.0 g, 0.300 mol) and succinic anhydride (30.0 g, 0.300 mol) 
were placed in a round bottomed flask and purged with nitrogen. 
Methanesulfonic acid (150 mL) was added and the solution was stirred at 
65.degree. C. for 2 hours under an atmosphere of nitrogen. The reaction 
mixture was added dropwise to rapidly stirred, ice-cooled water (1 L) with 
simultaneous addition of 50% aqueous sodium hydroxide to maintain pH 
2.5+/0.5. The product which appeared as a granular precipitate was 
collected by filtration and rinsed with water (3.times.100 mL) then 
acetone (3.times.100 mL). The product was air-dried then vacuum-dried 
(vacuum oven) at 110.degree. C. for 18 hours to afford a dark red powder 
(37.7 g, 88%). 
An analytical sample was prepared by dissolving 1.0 g, of product in 25 mL 
of hot 0.3N HCl. The precipitate which formed on cooling was removed by 
filtration and discarded. Dilute aqueous sodium hydroxide was added to 
raise the pH to 1.25. The resulting precipitate was collected by 
filtration, rinsed with water, air-dried then vacuum-dried over P.sub.2 
O.sub.5 at 140.degree. C. for 36 hours. 
Anal: Calc. [C(16)H(12)O(5)] C 67.60, H 4.26. 
Found: C 67.37. H 4.34. 0.52% water (K-F). NMR (DMSO-d.sub.6): (mostly 
spirolactone form) .delta. 2.690 (t, J=8.6 hz, 2H): 3.070 (t, J=8.6 hz, 
2H), 6.530 (d, J=1 8 hz, 2H); 6.676 (dd. J=8.7. 1.8 hz, 2H), 7.432 (d, 
J=8.7, 1.8 hz, 2H), 7.432 (d, J=8.7 hz, 2H), and 9.964 (s, 2H), Vis. abs. 
(pH 8.2; 50 mM aq is/HCl): max 486 nm (72,600). 
B. Preparation of 9-(2-Carboxyethyl)-3.6-diacetoxy9-ethoxy-9H-xanthene 
(Ac2EtSF-505) 
SF-505 (29.3 g, 103 mmol] as added to ice-cold acetic anhydride (500 mL) 
followed by pyridine (100 mL). The mixture was stirred in ice for 20 
minutes then added over 20 minutes to rapidly stirred ice-cold water (7 
L). After stirring for an additional 30 minutes, the intermediate product 
was filtered and resuspended in water (4 L) and stirred for another 30 
minutes. The solid was collected by filtration, dissolved in absolute 
ethanol (1 L), and refluxed for 45 minutes. The solution was concentrated 
on a rotary evaporator to 200 mL which resulted in crystallization. The 
product was collected by filtration air-dried then vacuum-dried to afford 
pale-orange microcrystals (21.9 g, 51%). 
Recrystallization from methylene chloride/cyclohexane gave colorless 
microcrystals. M.p.: 142.degree.-143.degree. C. Anal: Calc. 
[C(22)H(22)O(8)] C 6.63.76, H 5.35. Found: C 63.58, H 5.39. NMR 
(DMSO-d.sub.6): .delta. 1.035 (t, J=6.9 hz, 3H), 1.667 (m, 2H), 2.232 (m, 
2H), 2.294 (s, 6H), 2.888 (q, J=6.9 hz, 2H), 7.0-7.1 (m, 4H), and 7.575 
(d, J=9.1 hz, 2H). 
C. Preparation of 
9-(2-(N-Succinimidyloxycarbonyl))ethyl)-3,6-diacetoxy-9-ethoxy-9H-xanthene 
(Ac2EtSF-505-NHS) 
Ac2EtSF-505 (10.4 g, 25.1 mmol) was mixed with methylene chloride (300 mL) 
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (9.70 g. 
50.6 mmol) and N-hydroxysuccinimide (4.32 g, 37.5 mmol) were added. The 
mixture was stirred for one hour and then washed with water (5.times.50 
mL). The combined aqueous layers were back extracted with methylene 
chloride (50 mL) and the pooled organic layers were dried over sodium 
sulfate and stripped down. Trituration with ethanol (75 mL) followed by 
filtration and air-drying afforded the crude product as a light yellow 
solid (c. 10 g). This material was dissolved in methylene chloride (50 mL) 
and cyclohexane (50 mL) was added. One teaspoon of charcoal was added, the 
mixture was filtered, and the product was brought down with an additional 
portion of cyclohexane (100 mL). Collection by filtration. air-drying, and 
vacuum-drying afforded colorless crystals (6.94 g, 54%). 
A second crystallization from ethanol afforded an analytical sample. M.p.: 
162.degree.-3.degree. C. Anal: Calc. [C(26)H(25)N(1)O(10)] C 61.05, H, 
4.93,N 2.74. Found: C 60.78, H 5.01N 2.65. NMR (DMSO-d.sub.6): .delta. 
1.056 (t, J=7.0 hz, 3H), 2.4-2.1 (m, 4H), 2.293 (s, 6H), 2.757 (s, 4H), 
2.922 (q, J=7.0 hz, 2H), 7.069 (m, 4H), and 7.617 (p d, J=9.1 hz, 2H). 
D. Preparation of 
9-(2-(N-methyl-N-(benzyloxycarbonylmethyl)carboxamido)ethyl)-3,6-diacetoxy 
9-ethoxy-9H-xanthene (Ac2EtSF-505-Sar-OBn) 
To a solution of sarcosine benzyl ester* (1.13 g, 6.31 mmol) in methylene 
chloride (50 mL) was added Ac2EtSF-505-NHS (2.58g, 5.05 mmol) and 5% aq 
sodium bicarbonate solution (30 mL). The two-phase mixture was stirred 
rapidly for 20 hours. The layers were separated and the organic layer 
washed with 3.times.15 mL water, dried over sodium sulfate and 
concentrated to 25 mL. The solution was diluted to 150 mL with 
cyclohexane, charcoal-treated, and reduced to 75 mL under a stream of 
nitrogen resulting in the precipitation of the product. The supernatant 
was decanted away and the residue coevaporated with methylene chloride to 
afford a colorless foam (1.70 g, 58%). 
FNT * Sarcosine benzyl ester p-tosylate salt (Adams Chemical Co.) was taken up 
in methylene chloride and washed repeatedly with 5% aqueous sodium 
bicarbonate, then water washed, dried over sodium sulfate, and stripped 
down. 
Extensive vacuum-drying afforded an analytical sample. Anal: Calc. 
[C(32)H(33)N(1)O(9)] C 66.77 H 5.78, N 2.43. Found: C 66.66, H 5.89, N 
2.25. NMR (DMSO-d.sub.6): (Shows 5:2 mixture of amide bond rotamers.) 
.delta. (major and minor) 1.040 and 1.018 (t J=6.7 hz, 3H), 1.789 and 
1.670 (m, 2H), 2.211 (m, 2H), 2.290 and 2.276 (s, 6H), 2.713 and 2.695 (s, 
3H), 2.893 (q, J=6 7 hz, 2H), 3.963 (s, 2H), 5.075 and 5.039 (s, 2H), 
7.044 (m, 4H), 7.324 (m, 5H), and 7.573 and 7.516 (p d, J=9.2 hz, 2H). 
E. Preparation of 
9-(2-(N-Methyl-N-(N'-succinimidyloxycarbonylmethyl)carboxamido)ethyl)-3.6- 
diacetoxy-9-ethoxy-9H-xanthene (Ac2 EtSF-505-Sar-NHS, Structure 2a) 
To a solution of Ac2EtSF-505-Sar-OBn (1.55 g 2.69 mmol) in absolute ethanol 
(60 mL) was added 10% palladium on carbon (0.15 g). The mixture was 
stirred under balloon pressure of hydrogen for 30 minutes. The catalyst 
was removed by filtration and the ethanol stripped off to afford a syrupy 
residue. 
This residue was dissolved in methylene chloride (85 mL) and 
N-hydroxysuccinimide (0.495 g, 4.30 mmol) and 1-(3-dim 
ethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.12 g, 5.84 mmol) 
were added (4.times.25 mL). The solution was concentrated to 25 mL diluted 
to 175 mL with cyclohexane, charcoal treated and reduced in volume to 75 
mL under a stream of nitrogen. The solid product was collected by 
filtration, air-dried, and vacuum-dried to afford a colorless Powder (0.97 
g, 62%). 
Coevaporation with methylene chloride followed by extensive vacuum-drying 
at 40.degree. C. removed traces of cyclohexane and afforded an analytical 
sample as an amorphous solid. Anal: Calc. [C(29)H(30)N(2)O(11)] C 59.79, H 
5.19, N 4.81. Found: C 59.37, H 4.62, N 4.62, 0.93% water (K-F). NMR 
(DMSO.sub.6): (Shows a 4:1 mixture of amide bond rotamers.) .delta. (major 
and minor) 1.034 (t, J=6.9 hz, 3H), 1.827 and 1.935 (m, 2H), 2.223 (m, 
2H), 2.289 (s, 6H), 2.758 (s, 4H), 2.779 and 2.824 (s, 3H), 2.888 (q, 
J=6.8 hz, 2H), 4.333 and 4.473 (s, 2H), 7.043 (m, 4H), and 7.587 (per d, 
J=9.1 hz, 2H). 
EXAMPLE 16 
Preparation of N-Hydroxysuccinimide Ester 2b 
(A preferred reagent for attaching a 512 nm fluorescent dye to an 
alkynylamino-nucleotide wherein R.sub.9 is H and R.sub.10 is CH.sub.3) 
A. Preparation of 4-Methylresorcinol 
2,4-Dihydroxybenzaldehyde (33.97 gm, 0.246 mol) (recrystallized from 
toluene) was dissolved in spectroscopic grade 2-propanol (3 L) in a round 
bottom flask fitted with a gas inlet and a bubbler outlet. 10% Palladium 
on carbon (1.35 gm) was added followed by phosphoric acid (3 mL) and the 
mixture was sparged with nitrogen. The nitrogen flow was switched to 
hydrogen and the mixture was rapidly stirred with ice cooling. After 3 
hours hydrogen uptake was complete and the catalyst was removed by 
filtration. The filtrate was stripped down to 200 mL and 200 mL of ethyl 
acetate was added. The solution was washed with 4.times.200 mL of water 
and the combined water extracts back-extracted with ethyl acetate. These 
organic extracts were water washed and the combined organic layers dried 
over sodium sulfate and stripped down to afford the product as a colorless 
crystalline solid (29.95 gm, 98%). M.p.: 106.degree. C. (Lit. 
106.degree.-107.degree. C. [J. C. Bell W. Bridge, and A. Robertson, J. 
Chem. Soc., 1542-45 (1937)]). NMR (DMSO-d.sub.6): .delta. 1.961 (s, Me). 
6.076 (dd, H-6, J[5, 6]=8 hz, J[2,6]=2 hz), 6.231 (d, H-2), 6.760 (d, H-5) 
8.867 (s, OH), and 9.008 (s, OH). 
B. Preparation of 9-Carboxyethylidene-3,6-dihydroxy2,7-dimethyl- 
9H-xanthene (SF-512) 
4-Methylresorcinol (25.8 g, 0.208 mol) and succinic anhydride (20.8 g, 
0.208 g) were placed in a round bottom flask and the flask was purged with 
nitrogen. Methanesulfonic acid (150 mL) was added and the solution heated 
under nitrogen to 65.degree. C. for 2 hours. The solution was added 
dropwise to 1 L of rapidly stirred, ice-cooled water with the simultaneous 
addition of 50% ag sodium hydroxide to maintain the pH at 2.25+/-0.25. The 
product was collected by centrifugation and washed with water (3.times.) 
and acetone (2.times.). The solid was air-dried, then vacuum-dried at 
110.degree. C. to afford a brick-red powder (24.1 g, 74%). 
Purification was effected by allowing ethyl acetate to slowly diffuse into 
a solution of the product in dimethyl sulfoxide. The precipitate was 
collected by filtration, air-dried, then vacuum-dried. NMR (DMSO-d.sub.6): 
(Shows pure delta form along with one mole each of water and dimethyl 
sulfoxide.) .delta. 2.124 (s, 6H), 3.421 (d, J=7.2 hz 2H), 5.769 (t, J=7.2 
hz, 1H); 6.512 (s, 1H), 6.573 (s, 1H); 7.295 (s, 2H), 9.681 (s, 1H), 9.825 
(s, 1H), and 12.346 (bs, 1H), Vis. abs. (pH 8.2 aq Tris): max 493.5 nm. 
C. Preparation of 
9-Carboxyethyl-3.6-diacetoxy-2,7-dimethyl-9-ethoxy-9H-xanthene 
(Ac2EtSF-512) 
A sample of SF-512 (20.0 g, 64.0 mmol) was added to acetic anhydride (350 
mL) followed by pyridine (80 mL). This was stirred for 1 hour and then 
filtered to remove traces of unreacted dye. The filtrate was poured into 
3.5 L of rapidly stirred water. The solid intermediate was collected by 
filtration, resuspended in 2 L cold water, stirred for 15 minutes, then 
recollected and air-dried to afford the spirolactone intermediate (20.8 
g). This was dissolved in absolute ethanol (600 mL) and refluxed for 45 
minutes. The solution was charcoaltreated and concentrated to 300 mL. The 
product was collected by filtration, rinsed with cold ethanol (2.times.50 
mL), air-dried, and then vacuum-dried to afford colorless microcrystals 
(14.9 g, 53%), M.p.: 143.degree. C. Anal: Calc. [C(24)H(26)O(8)] C 65.15, 
H 5.92. Found: C 65.31, H 5.97. NMR (DMSO-d.sub.6): .delta. 1.027 (t, 
J=6.9 hz, 3H), 1.628 (m, 2H), 2.136 (s, 6H), 2.207 (m, 2H), 2.303 (s, 6H), 
2.884 (q, 6.9 hz, 2H), 6.939 (s, 2H), and 7.417 (s, 2H). 
D. Preparation of 9-(2-(N-Succinimidyloxycarbonyl) 
ethyl)-3,6-diacetoxy-2,7-dimethyl-9-ethoxy-9H-xanthene (Ac2EtSF-512-NHS) 
To a solution of Ac2EtSF-512 (9.42 g, 21.3 mmol) in methylene chloride (175 
mL) was added N-hydroxysuccinimide (3.62 g, 31.5 mmol) followed 
immediately by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride 
(8.05 g, 42.0 mmol). The solution was stirred at room temperature for 2 
hours. The mixture was washed with water (4.times.100 mL) and the aqueous 
washings back-extracted with methylene chloride (2.times.50 mL). The 
combined organic layers were dried over sodium sulfate and stripped down 
to an oil. Absolute ethanol was added and crystallization was induced by 
scratching. The product was collected by filtration air-dried, then 
vacuum-dried to afford pale-orange microcrystals (9.80 g, 85%). 
An analytical sample was prepared by dissolving 1 g, in methylene chloride 
(10 mL) and adding cyclohexane (40 mL). Charcoal treatment followed by 
cooling and scratching induced crystallization affording a colorless 
crystalline solid. M.p.: 159.degree. C. Anal: Calc. [C(28)H(29)N(1)O(10)] 
C 62.33, H 5.42, N 2.60. Found: C 62.06 H 5.71. N 2.39. NMR 
(DMSO-d.sub.6): .delta. 1.053 (t, J=6.9 hz, 3H), 2.149 (s, 6H), 2.304 (s, 
6H), 2.1-2.4 (m, 4H), 2.747 (s, 4H), 2.920 (q, J=6.9 hz, 2H), 6.975 (s, 
2H), and 7.464 (s, 2H). 
E. Preparation of 
9-(2-(N-methyl-N-(benzyloxycarbonylmethyl)carboxamido)ethyl)-3.6-diacetoxy 
2.7-dimethyl-9-ethoxy-9H-xanthene (Ac2EtSF-512-Sar-OBn) 
To a solution of sarcosine benzyl ester (0.72 g, 4.02 mmol) in methylene 
chloride (25 mL) was added Ac2EtSF-512-NHS (1.73 g, 3.21 mmol) and 5% aq 
sodium bicarbonate solution (20 mL). The two-phase mixture was stirred 
rapidly for 20 hours. The layers were separated and the organic layer 
washed with 3.times.15 15 mL water, dried over sodium sulfate, and 
concentrated to 10 mL. The solution was diluted to 60 mL with cyclohexane, 
charcoal-treated, and reduced to 25 mL under a stream of nitrogen 
resulting in the precipitation of the product. The supernatant was 
decanted and the colorless solid vacuum-dried (1.44 g, 74%). 
Recrystallization from methylene chloride/cyclohexane with charcoal 
treatment afforded an analytical sample. M.p.: 150.degree.-2.degree. C. 
Anal: Calc. [C(34)H(37)N(l)O(9)] C 67.65 H 6.18N 2.32. Found: C 67.42 H 
6.08N 2.33. NMR (DMSO-d.sub.6) (Shows 5:2 mixture of amide bond rotamers ) 
.delta. (major and minor) 1.049 and 1.008 (t, J=6.8 hz, 3H), 1.747 and 
1.66 (m, 2H), 2.144 and 2.115 (s, 6H), 2.18 (m, 2H), 2.314 and 2.303 (s, 
6H), 2.694 (s, 3H), 2.907 and 2.884 (q, J=6.8 hz, 2H), 3.961 (s, 2H), 
5.075 and 5.016 (s, 2H), 6.960 and 6.917 (s, 2H), 7.430 and 7.396 (s, 2H), 
and 7.30 (m, 5H). 
F. Preparation of 
9-(2-(N-Methyl-N-(N'-succinimidyloxycarbonylmethyl)carboxamido)ethyl)-3,6- 
diacetoxy-9-ethoxy-2,4,5,7-tetramethyl-9H-xanthene (Ac2EtSF-512-Sar-NHS. 
Structure 2b) 
To a suspension of Ac2EtSF-512-Sar-OBn (0.45 g, 0.745 mol) in absolute 
ethanol (20 mL) was added 10% palladium on carbon (0.05 g). The mixture 
was stirred under balloon pressure of hydrogen for 30 minutes. The 
catalyst was removed by filtration and the ethanol stripped off to afford 
a syrupy residue. 
This residue was dissolved in methylene chloride (25 mL) and 
N-hydroxysuccinimide (0.129 g, 1.12 mmol) and 
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.292 g, 1.52 
mmol) were added. The mixture was stirred for 30 minutes and then washed 
with water (3.times.15 mL). The solution was dried over sodium sulfate, 
concentrated to 10 mL, diluted to 40 mL with cyclohexane charcoal treated 
and reduced in volume to 20 mL under a stream of nitrogen. The supernatant 
was decanted and the residue subjected to a second precipitation from 
methylene chloride to afford a colorless powder (0.27 g, 59%). Anal: Calc. 
[C(31)H(34)N(2)O(11)] C 60.98, H 5.61, N 4.59. Found: C 60.28, H 5.71, N 
4.40, 1.08% water (K-F). NMR (DMSO-d.sub.6); (Shows a 5:1 mixture of 
rotamers about the amide bond.) .delta. (major and minor) 1.043 (t, J=7.0 
hz, 3H), 1.793 and 1.933 (m, 2H), 2.145 and 2.133 (s, 6H), 2.198 (m, 2H), 
2.314 (s, 6H), 2.740 (s, 4H), 2.778 and 2.821 (s, 3H), 2.900 (q, J=7.0 
hz, 2H), 4.334 and 4.469 (s, 2H), 6.960 and 6.925 (s, 2HO, and 7.441 (s, 
2H). 
EXAMPLE 17 
Preparation of N-Hydroxysuccinimide Ester 2c 
(A preferred reagent for attaching a 519 nm fluorescent dye to an 
alkynylamino-nucleotide where-in R.sub.9 is CH.sub.3 and R.sub.10 is H) 
A. Preparation of 
9-(2-Carboxyethylidene)-3,6-dihydroxy-4.5-dimethyl-9H-xanthene (SF-519) 
2-Methylresorcinol (37.2 g, 0.300 mol) and succinic anhydride (30.0 g, 
0.300 mol) were Placed in a round bottomed flask and purged with nitrogen. 
Methanesulfonic acid (150 mL) was added and the solution was stirred at 
65.degree. C. for 4 hours under an atmosphere of nitrogen. The reaction 
mixture was added dropwise to rapidly stirred, ice-cooled water 1 L) with 
simultaneous addition of 50% aqueous sodium hydroxide to maintain pH 
6.0+/-0.5. The finely divided solid was collected by centrifugation and 
rinsed with water (4.times.250 mL), each time resuspending, spinning down, 
and discarding the supernatant. The crude product was suspended in water 
(1 L) and sufficient aqueous sodium hydroxide (50%) was added to raise the 
pH to 10.2. The solution was filtered and the filtrate brought to pH 1.2 
with concentrated HCl. The product was collected by centrifugation and 
rinsed with water (3.times.350 mL) and a cetone (3.times.250 mL) as 
described above. The resulting solid was azeotroped with toluene, 
collected by filtration, and vacuum-dried at 110.degree. C. to afford a 
brick-red powder (24.6 g, 53%). Anal: Calc. [C(18)H(16)O(5)] C 69.22 H 
5.16. Found: C 68.95 H 5.30, 0.80% water (K-F). NMR (DMSO-d.sub.6) (mostly 
delta form): .delta. 2.164 (s, 3H), 2.177 (s, 3H), 3.376 (d, J=7.1 hz, 
2H), 5.749 (t, J=7.2 hz, 1H), 6.642 (d, J=8.8 hz, 1H), 6.672 (d, J=8.8 
hz, 1H), 7.216 (d, J=8.5 hz, 1H), 7.227 (d, J=8.5 hz, 1H), 9.602 (bs, 1H), 
and 9.758 (bs, 1H), Vis. abs. (pH 8.2; 50 mM aq Tris/HCl) max 500 nm 
(69,800). 
B. Preparation of 
9-(2-Carboxyethyl)-3.6-diacetoxy4.5-dimethyl-9-ethoxy-9H-xanthene 
(Ac2EtSF-519) 
SF-519 (15.0 g, 48.0 mmol) was added to acetic anhydride (250 mL) and the 
solid was pulverized. (Sonication is useful to disperse the highly 
insoluble SF-519.) The suspension was ice-cooled, pyridine (50 mL) was 
added, and the mixture stirred for 20 minutes. The solution was filtered 
and added in a slow but steady stream to rapidly stirred ice-cold water (4 
L). After stirring for an additional 20 minutes the intermediate product 
was filtered, resuspended in water (3 L). and stirred for another 25 
minutes. The solid was collected by filtration and air-dried. The dried 
intermediate was dissolved in absolute ethanol (600 mL) and refluxed for 
1 hour. The solution was concentrated on a rotary evaporator to 200 mL 
which resulted in crystallization. The product was collected by 
filtration, air-dried, then vacuum-dried to afford colorless microcrystals 
(12.13 g, 57%). 
An analytical sample was prepared by precipitation from methylene chloride 
solution with cyclohexane. NMR (DMSO-d.sub.6): .delta. 1.033 (t, J=6.9 hz, 
3H), 1.674 (m, 2H), 2.189 (s, 6H), 2.19 (m, 2H), 2.348 (s, 6H) 2.878 (q, 
J=6.9 hz, 2H), 7.006 (d, J=8.6 hz, 2H), and 7.399 (d J=8.6 hz 2H). 
C. Preparation of 
9-(2-(N-Succinimidyloxycarbonyl)ethyl-3,6-diacetoxy-4,5-dimethyl-9-ethoxyl 
9H-xanthene (Ac2EtSF-519-NHS) 
Ac2EtSF-519 (7.80 g, 17.6 mmol) was mixed with methylene chloride (175 mL) 
and N-hydroxysuccinimide (2.75 g, 23.9 mmol) and 
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (7.00 g, 36.5 
mmol) were added. The mixture was stirred for 90 minutes and then washed 
with water (5.times.100 mL). The combined aqueous layers were back 
extracted with methylene chloride (2.times.50 mL) and the pooled organic 
layers were dried over sodium sulfate and stripped down. Trituration with 
ethanol (100 mL) followed by filtration and air-drying afforded the 
product as a light yellow solid (7.45 g, 78%). 
Two recrystallizations from cyclohexane/methylene chloride with charcoal 
treatment afforded an analytical sample. M.p.: 164.degree.-5.degree. C. 
Anal: Calc. [C(28)H(29)N(1)O(10)] C 62.33, H 5.42, N 2.60. Found: C 62.17, 
H 5.47, N 2.48. NMR (DMSO-d.sub.6 : .delta. 1.051 (t, J=7.0 hz, 3H), 
2.4-2.1 (m, 4H), 2.191 (s, 6H), 2.337 (s, 6H), 2.715 (s, 4H), 2.912 (q, 
J=7.0 hz, 2H), 7.015 (d, J=8.6 hz, 2H), and 7.429 (d, J=8.6 hz, 2H). 
D. Preparation of 
9-(2-(N-methyl-N-(benzyloxycarbonylmethyl)carboxamido)ethyl)-3,6-diacetoxy 
-4,5-dimethyl-9-ethoxy-9H-xanthene (Ac2EtSF-519-Sar-OBn) 
To a solution of sarcosine benzyl ester (0.557 g, 3.11 mmol) in methylene 
chloride (19 mL) was added Ac2EtSF-519-NHS (1.30 g, 2.41 mmol) and 5% 
aqueous sodium bicarbonate solution (15 mL). The two-phase mixture was 
stirred rapidly for 18 hours. The layers were separated and the organic 
layer washed with 3.times.10 mL water, dried over sodium sulfate, and 
concentrated to 10 mL. The solution was diluted to 40 mL with cyclohexane, 
charcoal-treated and reduced to 20 mL under a stream of nitrogen resulting 
in the precipitation of the product as a sticky solid. The supernatant was 
decanted away and the residue coevaporated with methylene chloride to 
afford a colorless foam (0.97 g, 67%). 
Extensive vacuum drying afforded an analytical sample. Anal: Calc. 
[C(34)H(37)N(1)O(9)]C 67.65 H 6.18N 2.32. Found: C 67.43 H 6.37N 2.32. NMR 
(DMSO-d.sub.6) (Shows 5:2 mixture of amide bond rotamers.): .delta. (major 
and minor) 1.044 and 1.020 (t=7.0 hz, 3H), 1.824 and 1.714 (m, 2H), 2.17 
(m, 2H), 2.195 and 2.169 (s, 6H), 2.346 and 2.337 (s, 6H), 2.720 and 2.691 
(s, 3H), 2.889 (q, J=7.0 hz, 2H), 3.959 and 3.988 (s, 2H), 5.073 and 5.048 
(s, 2H), 7.000 and 6.954 (d, J=8.6 hz, 2H), and 7.45-7.25 (m, 7H), 
E. Preparation of 
9-(2-(N-Methyl-N-(N'-succinimidyloxycarbonylmethyl)carboxamido)ethyl)-3,6- 
diacetoxy-4,5-dimethyl-9-ethoxy-9H-xanthene (Ac2EtSF-519-Sar-NHS, Structure 
2c) 
To a solution of Ac2EtSF-519-Sar-OBn (1.35 g, 2.24 mmol) in absolute 
ethanol (50 mL) was added 10% Palladium on carbon (0.13 g). The mixture 
was stirred under balloon pressure of hydrogen for 20 minutes. The 
catalyst was removed by filtration and the ethanol stripped off to afford 
a syrupy residue. 
This residue was dissolved in methylene chloride (50 mL) and 
N-hydroxysuccinimide (0.39 g, 3.39 mmol) and 
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.57 g, 8.19 
mmol) were 15 added. The mixture was stirred for 75 minutes and then 
washed with water (4.times.15 mL). The solution was dried over sodium 
sulfate concentrated to 25 mL. diluted to 125 mL with cyclohexane, 
charcoal treated, and reduced in volume to 50 mL under a stream of 
nitrogen. The supernatant was decanted and the remaining oil taken up in 
methylene chloride (5 mL) and added dropwise to rapidly stirred 
cyclohexane (75 mL) to afford a colorless powder (0.587 g, 43%). 
To provide an analytical sample a portion of the product was taken up in 
methylene chloride, dried over molecular sieves, evaporated under a stream 
of nitrogen, and finally dried in a drying pistol at 48.degree. C. over 
phosphorus pentoxide for 20 hours. Anal: Calc. [C(31)H(34)N(2)O(11)]; C 
60.98, H 5.61, N 4.59. Found: C 60.15, H 5.71, N 4.51, water (K-F) 1.51%. 
NMR (DMSO-d.sub.6) (Shows a 4:1 mixture of amide bond rotamers.): .delta. 
(major and minor) 1.039 (t, J=6.9 hz, 3H), 1.841 and 1.945 (m, 2H), 2.19 
(m, 2H), 2.194 (s, 6H), 2.345 (s, 6H), 2.767 and 2.744 (s, 4H), 2.778 and 
2.825 (s, 3H), 2.888 (q, J=6.9 hz, 2H), 4.328 and 4.461 (s, 2H), 7.000 (d 
J=8.6 hz, 2H), and 7.410 (d, J=8.6 hz, 2H). 
EXAMPLE 18 
Preparation of N-Hydroxysuccinimide Ester 2d 
(A preferred reagent for attaching a 526 nm dye to an 
alkynylamino-nucleotide wherein R.sub.9 and R.sub.10 are CH.sub.3) 
A. Preparation of 2,4-Dihydroxy-3-methylbenzaldehyde 
Phosphorus oxychloride (80 mL, 0.86 mol) was added to a stirred mixture of 
N-methylformanilide (102 mL, 0.82 mol) in ether (250 mL). The mixture was 
stirred for 1 hour at room temperature and then cooled in ice. 2-Methyl 
resorcinol (Aldrich, 100 g 0.81 mol) was added and the mixture was allowed 
to warm to room temperature while stirring overnight. The precipitated 
intermediate product was collected by filtration and rinsed with ether 
(3.times.). The intermediate was hydrolyzed by dissolving in a mixture of 
acetone (250 mL) and water (250 mL) and stirring for 30 minutes. Water (2 
L) was added, the mixture was brought to a boil, and then allowed to cool 
and deposit crystalline product. This was recrystallized a second time 
from water (4 L) to afford pure product (70 g, 57%). M.p. 150.degree. C. 
(Lit. 152.degree.-3.degree. C. [W. Baker et al., J. Chem. Soc., 2834-5 
(1949).]. NMR (DMSO-d.sub.6): .delta. 1.973 (s, 3H), 6.551 (d, J=8.5 hz, 
1H), 7.428 (d, J-8.5 hz, 1H), 9.703 (s, 1H), 10.745 (s, 1H), and 11.592 
(s, 1H), 
B. Preparation of 2,4-dimethylresorcinol 
A solution of 2,4-dihydroxy-3-methylbenzaldehyde (30.0 g, 197 mmol) with 
isopropanol (3 L) was ice-cooled in a 5 L 3-neck flask fitted with a 
magnetic stirrer. Phosphoric acid (4 mL) and 10% palladium on carbon were 
added and the solution was sparged with nitrogen, then hydrogen. When 
uptake was judged to be complete (c. 1.5 hour) the solution was again 
sparged with nitrogen and then filtered through Celite.RTM.. The solvent 
was stripped off, the residue taken up in ethyl acetate, and the resulting 
solution washed with water (4.times.100 mL). The water washes were 
back-extracted with ethyl acetate and the combined organic layers dried 
over sodium sulfate and stripped down. Sublimation (95.degree., 0.05 torr) 
afforded a colorless solid (19.6 g, 72%). M.p. 107-8.degree. C. (Lit. 
108-109.degree. C. [W. Baker et al., J. Chem. Soc., 2834-5 (1949).]). NMR 
(DMSO-d.sub.6): .delta. 1.969 (s, 3H), 2.037 (s, 3H), 6.220 (d, J=8.1 hz, 
1H), 6.637 (d, J=8.1 hz, 1H), 7.929 (s, 1H), and 8.785 (s, 1H). 
C. Preparation of 
9-(2-Carboxyethylidene)-3,6-dihydroxy-2,4,5,7-tetramethyl-9H-xanthene 
(SF-526) 
2,4-Dimethylresorcinol (28.4 g, 0.205 mol) and succinic anhydride (20.0 g, 
0.200 mol) were placed in a round bottomed flask and purged with nitrogen. 
Methanesulfonic acid (231 mL) was added and the solution was stirred at 
70.degree. C. for 20 hours under an atmosphere of nitrogen. The reaction 
mixture was added dropwise to a rapidly stirred mixture of aqueous sodium 
hydroxide (95 g, in 150 mL water) and ice (3 L). Sufficient 
methanesulfonic acid was added to bring the final pH from 4.7 to 1.5. The 
resulting solid was collected by centrifugation and washed by suspending, 
spinning down, and decanting from water (5.times.1.2 L). The final 
suspension was collected by filtration, air-dried, then oven-dried at 
110.degree. C. for 6 hours to afford a brick-red solid (30.6 g, 44%). 
A second precipitation from alkaline solution, followed by centrifugation 
and water washes afforded an analytical sample. Anal: Calc. 
[C(16)H(12)O(5)] C 70.57. H 5.92. Found: C 70.39, H 6.00, 0.21% water 
(K-F). NMR (DMSO-d.sub.6) (mostly spirolactone form): .delta. 2.172 (s, 
12H), 2.508 (m, 2H), 3.342 (m, 2H), and 7.604 (s, 2H), Vis. abs. (pH 8.2; 
50 mM ag Tris/HCl): 509 nm (71,300). 
D. Preparation of 
9-(2-Carboxyethyl)-3,6-diacetoxy9-ethoxy-2,4,5,7-tetramethyl-9H-xanthene 
(Ac2EtSF-526) 
SF-526 (25.2 g, 74 mmol) was added to ice-cold acetic anhydride (450 mL) 
followed by pyridine (100 mL) and the mixture was stirred with ice-cooling 
for 150 minutes. The reaction mixture was filtered then added in a slow, 
steady stream to rapidly stirred, ice-cold water (7 L). After stirring for 
an additional 30 minutes, the intermediate product was filtered, washed 
with water, resuspended in water (4 L) and stirred for another 30 minutes. 
The solid was collected by filtration and air-dried to afford the 
spirolactone intermediate (28.9 g). A portion of this intermediate (18.6 
g) was dissolved in absolute ethanol (1 L), and refluxed for 90 minutes. 
The solution was concentrated on a rotary evaporator to 300 mL which 
resulted in crystallization. The product was collected by filtration 
rinsed with ethanol, air-dried, then vacuum-dried to afford colorless 
microcrystals (11.6 g, 52% based on amount of intermediate used). 
Recrystallization from methylene chloride/cyclohexane with charcoal 
treatment gave colorless microcrystals. M.p.: 154-155.degree. C. Two 
evaporations from methylene chloride removed traces of cyclohexane for 
analysis. Anal: Calc. [C(20)H(20)O(5)] C 70.57, H 5.92. Found: C 70.39, H 
6.00, 0.21% water (K-F). NMR (DMSO-d.sub.6) (mostly spirolactone form): 
.delta. 2.172 (s, 12H), 2.508 (m, 2H), 3.342 (m, 2H), and 7.604 (s, 2H), 
Vis. abs. (pH 8.2; 50 mM aq Tris/HCl): 509 nm (71,300). 
E. Preparation of 
9-(2-(N-Succinimidyloxycarbonyl)ethyl)-3,6-diacetoxy-9-ethoxy-2,4,5,7-tetr 
amethyl9H-xanthene (Ac2EtSF-526-NHS) 
Ac2EtSF-526 (4.70 g, 9.99 mmol) was mixed with methylene chloride (75 mL) 
and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.10 g, 
16.2 mmol) and N-hydroxysuccinimide (1.50 g, 13.0 mmol) were added. The 
mixture was stirred for 90 minutes and then washed with water (4.times.50 
mL). The combined aqueous layers were back extracted with methylene 
chloride (50 mL) and the pooled organic layers were dried over sodium 
sulfate and stripped down. Trituration with ethanol (75 mL) followed by 
filtration and air-drying afforded the crude product as a light yellow 
solid (c. 4.7 g). This material was dissolved in methylene chloride (50 
mL) and cyclohexane (50 mL) was added. One teaspoon of charcoal was added, 
the mixture was filtered, and the product was brought down with an 
additional portion of cyclohexane (25 mL). Collection by filtration, 
air-drying, and vacuum-drying afforded colorless crystals (3.14 g, 55%). 
A second precipitation from methylene chloride with cyclohexane afforded an 
analytical sample. Anal: Calc. [C(30)H(33)N(l)O(10)] C 63.48, H 5.86,N 
2.47. Found: C 63.08, H 6.00, N 2.37. NMR (DMSO-d.sub.6)): .delta. 1.058 
(t, J=6.9 hz, 3H), 2.136 (s, 6H), 2.155 (s, 6H), 2.228 (m, 4H), 2.371 (s, 
6H), 2.748 (s, 4H), 2.918 (q, J=6.9 hz, 2H), and 7.300 (s, 2H). 
F. Preparation of 
9-(2-(N-methyl-N-(benzyloxycarbonylmethyl)carboxamido)ethyl)-3,6-diacetoxy 
9-ethoxy-9H-xanthene (Ac2EtSF-505-Sar-OBn) 
To a solution of sarcosine benzyl ester (0.72 g, 4.02 mmol) in methylene 
chloride (40 mL) was added Ac2EtSF-526-NHS (1.82 g, 3.21 mmol) and 5% ag 
sodium bicarbonate solution (30 mL). The two-phase mixture was stirred 
rapidly for 20 hours. The layers were separated and the organic layer 
washed with 4.times.15 mL water, dried over sodium sulfate, and 
concentrated to 15 mL. The solution was diluted to 100 mL with 
cyclohexane, charcoal-treated, and reduced to 50 mL under a stream of 
nitrogen resulting in the precipitation of the product. Filtration 
followed by air-drying afforded a colorless solid (0.96 g, 47%). 
Coevaporation with methylene chloride followed by extensive vacuum drying 
afforded an analytical sample. Anal: Calc. for [C(36)H(41)N(1)O(9)] C 
68.45, H 6.54,N 2.22. Found: C 68.29, H 6.70, .delta. 2.07. NMR 
(DMSO-d.sub.6) (Shows 5:2 mixture of amide bond rotamers.): .delta. (major 
and minor) 1.049 and 1.027 (t, J=6.8 hz, 3H), 1.783 and 1.700 (m,2H), 
2.129 and 2.099 (s, 6H), 2.159 and 2.129 (s, 6H), 2.14 (m, 2H), 2.379 and 
2.371 (s, 6H), 2.699 and 2.690 (s, 3H), 2.873 (q, J=6.8 hz, 2H), 3.958 and 
3.976 (s, 2H), 5.075 and 5.019 (s, 2H), 7.266 and 7.233 (s, 2H), and 
7.25-7.40 (m, 5H). 
G. Preparation of 
9-(2-(N-Methyl-N-(N'-succinimidyloxycarbonylmethyl)carboxamido)ethyl)-3.6- 
1H, H1'), 4.94 (t, J=diacetoxy-9-ethoxy-2,4,5,7-tetramethyl-9H-xanthene 
(Ac2EtSF-526-Sar-NHS, Structure 2d) 
To a solution of Ac2EtSF-526-Sar-OBn (0.96 g, 1.52 mmol) in absolute 
ethanol (40 mL) was added 10% palladium on carbon (0.10 g). The mixture 
was stirred under balloon pressure of hydrogen for 30 minutes. The 
catalyst was removed by filtration and the ethanol stripped off to afford 
a syrupy residue. 
This residue was dissolved in methylene chloride (40 mL) and 
N-hydroxysuccinimide (0.26 g, 2.26 mmol) and 
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.59 g, 3.08 
mmol) were added. The mixture was stirred for 30 minutes and then washed 
with water (4.times.15 mL). The solution was dried over sodium sulfate, 
concentrated to 15 mL, diluted to 100 mL with cyclohexane, charcoal 
treated, and reduced in volume to 50 mL under a stream of nitrogen. The 
product was collected by filtration, air dried, and vacuum dried to afford 
colorless microcrystals (0.573 g, 59%). 
Coevaporation with methylene chloride followed by extensive vacuum drying 
at 40.degree. C. removed traces of cyclohexane and afforded an analytical 
sample as an amorphous solid. NMR (DMSO-d.sub.6) .delta. 1.043 (t, J=6.7 
hz, 3H), 1.82 (m, 2H), 2.130 (s, 6H), 2.157 (s, 6H), 2.15 (m, 2H), 2.378 
(s, 6H), 2.748 (s, 4H), 2.778 (s, 3H), 2.891 (q, J=6.7 hz, H), 4.327 (s, 
2H), and 7.275 (s, 2H). 
EXAMPLE 19 
A GENERAL METHOD FOR COUPLING ALKYNYLAMINONUCLEOTIDES WITH 
N-HYDROXYSUCCINIMIDE ESTERS 2 
PREATION OF FLUORESCENTLY-LABELED CHAIN TERMINATING 
ALKYNYLAMINO-NUCLEOTIDES 34-37 
Alkynylamino-nucleotide triphosphate 49 (10 micromole, from Example 3J) was 
taken up in water (0.050 mL) and diluted With dimethylformamide (0.100 
mL). A solution of N-hydroxysuccinimide ester 2a (12.3 mg, 21 micromole 
2.1 eq. from Example 15E) in dimethylformamide (0.100 mL) was added and 
the mixture was stirred at 50.degree. for 4 hours. Concentrated ammonium 
hydroxide (0.25 mL) was added, the reaction vessel was tightly stoppered, 
and heating at 50.degree. was continued for 25 minutes. The resulting red 
solution was diluted to 10 mL with water and applied to a column of 
DEAE-Sephadex A-25-120 (1.times.19 cm bed) that had been equilibrated with 
1.0 M pH 7.6 aqueous TEAB (50 mL) and then 0.2M pH 7.6 aqueous TEAB (50 
mL). The column was eluted with a linear gradient of pH 7.6 aqueous TEAB 
from 0.4M (150 mL) to 0.7 M (150 mL). The column was driven at 100 mL/h 
collecting fractions every 3 minutes. The eluent was monitored by 
absorbance at 498 nm (40 AUFS). Two lesser by-product bands eluted first 
followed by the stronger product band with baseline resolution. The 
fractions estimated to contain pure product were pooled, stripped down 
(T&lt;30.degree.). co-evaporated three times with absolute ethanol, and taken 
up in water (0.74 mL). The solution was assayed by visible absorption (pH 
8.2 50 mM aqueous Tris buffer) and lyophilized. A dilute solution of the 
product displayed an absorption maximum at 487.5 nm. Assuming an 
absorption coefficient for the product equal to that of the free dye 
(72,600), the yield of labeled alkynylamino-nucleotide 37 was 4.2 
micromole (42%). 
The above procedure produced fluorescently-labeled chain terminator 37 
wherein Het is a 7-deazaguanine (k). Labeled chain terminators 34 (Het is 
uracil (h)), 35, (cytosine (i)), and 36 (7-deazaadenosine (j)) were 
prepared following similar procedures by coupling alkynylaminonucleotide 
triphosphates 46, 42 and 51 with N-hydroxysuccinimides 2d, 2c, and 2b, 
respectively. Other fluorescently-labeled nucleotide triphosphates were 
also prepared by the sam methods.