Synthesis of 3'-thioamido-modified 3'-deoxythymidine-5'-triphosphates and their use as chain terminators in Sanger-DNA sequencing.

Article abstract of DOI:10.1081/NCN-100002450

The thioamide derivatives 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2-methyl-1-thioxo- propyl)amino]thymidine 1 and 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-((6-([(9H-(fluo-ren-9- ylmethoxy)carbonyl]-amino)-1-thioxohexyl)amino) thymidine 2 were synthesized

Full text of DOI:10.1081/NCN-100002450

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SYNTHESIS OF 3′-THIOAMIDO-MODIFIED 3′-
DEOXYTHYMIDINE-5′-TRIPHOSPHATES AND THEIR USE
AS CHAIN TERMINATORS IN SANGER-DNA SEQUENCING
K. Schwarzer a , C. Wojczewski a & J. W. Engels b
a Johann Wolfgang Goethe-Universität , Frankfurt am Main, Germany
b Johann Wolfgang Goethe-Universität , Frankfurt am Main, Germany
Published online: 07 Feb 2007.
To cite this article: K. Schwarzer , C. Wojczewski & J. W. Engels (2001) SYNTHESIS OF 3′-THIOAMIDO-MODIFIED 3′-
DEOXYTHYMIDINE-5′-TRIPHOSPHATES AND THEIR USE AS CHAIN TERMINATORS IN SANGER-DNA SEQUENCING, Nucleosides,
Nucleotides and Nucleic Acids, 20:4-7, 879-882, DOI: 10.1081/NCN-100002450
To link to this article: http://dx.doi.org/10.1081/NCN-100002450
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS, 20(4–7), 879–882 (2001)
SYNTHESIS OF 3ꢀ-THIOAMIDO-MODIFIED
3ꢀ-DEOXYTHYMIDINE-5ꢀ-TRIPHOSPHATES
AND THEIR USE AS CHAIN TERMINATORS
IN SANGER-DNA SEQUENCING
K. Schwarzer, C. Wojczewski, and J. W. Engels∗
Johann Wolfgang Goethe-Universita¨t, Frankfurt am Main, Germany
ABSTRACT
The thioamide derivatives 3ꢀ-deoxy-5ꢀ-O-(4,4ꢀ-dimethoxytrityl)-3ꢀ-[(2-methyl-
1-thioxo-propyl)amino]thymidine 1 and 3ꢀ-deoxy-5ꢀ-O-(4,4ꢀ-dimethoxytrityl)
-3ꢀ- {{6-{[(9H-(fluo-ren-9-ylmethoxy)carbonyl]-amino}-1-thioxohexyl}amino}
thymidine 2 were synthesized by regioselective thionation of their correspond-
ing amides 7 and 8 with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphet-
ane-2,4-disulfide (Lawesson’s reagent). The thioamides were converted into
the corresponding 5ꢀ-triphosphates 3 and 4. Compound 3 was chosen for DNA
sequencing experiments and 4 was further labelled with fluorescein.
INTRODUCTION
Dye-terminator sequencing is characterized by an enzymatic incorporation
of chain-terminating, fluorescent nucleotides. These terminators are attached to
fluorescent dyes at their base moiety. Apart from this labeling strategy, there are
other potential positions within nucleosides that may be used for labeling. We have
shown that 3ꢀ-amino- and amido-modified nucleoside-5ꢀ-triphosphates are excellent
terminators (1,2). However, the ability of DNA polymerases to hydrolyze ester- and
amide-bonds at the 3ꢀ-position limits the use of these 3ꢀ-dye-terminators for DNA
sequencing since the label is lost during the incorporation process (3). Therefore, we
∗Corresponding author.
879
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C
Copyright 2001 by Marcel Dekker, Inc.
www.dekker.com

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SCHWARZER, WOJCZEWSKI, AND ENGELS
decided to alter the link between dye and sugar moiety to find a bond that is stable
against enzymatic degradation. In this context, we describe an efficient synthesis of
3ꢀ-thioamido-modified thymidines. We synthesized the thioamide derivative 1 as a
model compound for the investigation of the thionation reaction with Lawesson’s
reagent. In a second synthetic route we optimized the thionation reaction. The
addition of exact amounts of pyridine to the reaction mixture proved to be essential
for an efficient transformation. The corresponding 5ꢀ-triphosphate 4 was further
labeled with fluorescein and 3 was chosen for DNA sequencing experiments.
RESULTS AND DISCUSSION
A broad spectrum of reactions exists for the synthesis of thioamides, only
the conversion of amides into thioamides seemed to be suitable for nucleoside
chemistry. Therefore, we synthesised the readily accessible amide 7 as a model
compound for the investigation of the thionation reaction (Fig. 1). Direct action of
hydrogen sulfide or phosphorus pentasulfide on 7/8 would result in an undesired
exchange of the two carbonyl oxygen atoms at C(2) and C(4) of the base moiety.
A more sophisticated thionation reagent is Lawesson’s reagent, which converts
carbonyl groups into thiocarbonylgroups in high yield (4–6). Therefore, 7 was
reacted with Lawesson’s reagent for 30 minutes at RT.
No by-products were observed under these reaction conditions, but the yield
of 1 was rather poor (22%). 33% of the reactant was recovered unchanged. The
synthesis of 2 under the same conditions resulted in even lower yields caused mainly
by 5ꢀ-hydroxy deprotection. Therefore we tested basic solvents as scavengers for
the phosphoric acids.
Thionation of 8 in pure anh. pyridine instead of anh. THF did not at all
indicate any formation of 2 even when the amount of Lawesson’s reagent was
drastically increased. Thus, the reaction of 8 with Lawesson’s reagent was accom-
plished in mixtures of anh. THF/pyridine. We observed that lowering the amount of
Figure 1. Synthesis of the thioamide derivatives 1 and 2.

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SYNTHESIS OF 3ꢀ-THIOAMIDO-MODIFIED THYMIDINES
881
pyridine gradually resulted in an increased yield. Finally, the addition of 1 equiv.
of pyridine and 4 equiv. of Lawesson’s reagent in anh. THF as solvent yielded 2 in
50%; almost 50% of the intact educt was recovered. 1 and 2 were deprotected in
80% aq. AcOH solution at room temperature. The syntheses of the triphosphates
3 and 4 were achieved according to the procedure of Ludwig and Eckstein (7). The
aminofunction of 4 was deprotected with 20% piperidine in pyridine and DMF at
room temperature and succeedingly coupled with fluoresceinisothiocyanate (FITC)
in aq. 0.1M NaHCO3 (pH = 9.3) and DMF at room temperature for 24 hours.
Both thioamides 1/2 were clearly identified by mass spectrometry and NMR spec-
troscopy. The electrospray-ionization MS (ESI-MS) showed formation of only one
product with an increased weight of 16 Da compared with the educt, representing
the exchange of an O-atom against a S-atom. The 13C-NMR spectrum revealed
the identity of the carbonyl group involved. The 13C-chemical shifts of the carbon
atoms C(2) and C(4) of the base moiety remained unchanged, whereas the carbonyl
signal at the 3ꢀ-terminal moved downfield as expected.
Thethioamides1and2wereconvertedintothecorresponding5ꢀ-triphosphates
3 and 4. Compound 4 was further labelled with fluorescein and 3 was investigated
in DNA sequencing experiments (8). No band pattern was detected with Sequenase
or with Thermosequenase. With Taq DNA polymerase, we obtained a band pat-
tern which did not correlate with the standard ddTTP sequence. It was, however,
identical with the band pattern obtained with a 3ꢀ-thioether 3ꢀ-deoxy-thymidine-5ꢀ-
triphosphate. Although 3 can obviously not be defined as specific terminator for the
enzymatic DNA synthesis under the chosen conditions, the result can not be called
nonselective, since two structurally different nucleotides were incorporated at ex-
actly the same positions within the DNA. This excludes a random process. Variation
of the terminator concentration only resulted in a different fragment length distri-
bution. The reason for this could be a conformationally driven selection process.
CONCLUSION
We demonstrated that 3ꢀ-thioamido modified nucleosides can efficiently be
synthesised from 3ꢀ-amido tethered nucleosides by a regioselective thionation strat-
egy applying Lawesson’s reagent. Addition of 1 equiv. of pyridine to the reaction
mixture was necessary to prevent degradation of the nucleoside and ensure good
yields at the same time. Thioamide 3 was accepted as a substrate by the Taq DNA
polymerase. However, specific incorporation did not occur. Rather the band pattern
showed a distinct correlation with a thioether tethered nucleoside triphosphate.
ACKNOWLEDGMENTS
We would like to thank the Bundesministerium fu¨r Bildung und Forschung
(BEO0311088), the Verband der Chemischen Industrie and the Deutsche Forsch-
ungsgemeinschaft (Graduiertenkolleg) for financial support as well as Roche

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SCHWARZER, WOJCZEWSKI, AND ENGELS
Diagnostics for cooperation and financial support. DNA sequencing experiments
were done by Dr. S. Klingel and Dr. G. Sagner at Roche Diagnostics, Penzberg,
Germany.
REFERENCES
1. Herrlein, M. K.; Konrad, R. E.; Engels, J. W.; Holletz, T.; Cech, D., Helv. Chim. Acta
1994, 77, 586.
2. Wojczewski, C.; Faulstich, K.; Engels, J. W., Nucleosides Nucleotides 1997, 16, 751.
3. Canard, B.; Cardona, B.; Sarfati, R. S., Proc. Natl. Acad. Sci. USA 1995, 92, 10859.
4. Yde, B.; Yousif, N. M.; Pedersen, U.; Thomsen, I.; Lawesson, S.-O., Tetrahedron 1984,
40, 2047.
5. Cava, M. P.; Levinson, M. I., Tetrahedron 1985, 41, 5061.
6. Cherkasov, R. A.; Kutyrev, G. A.; Pudovik, A. N., Tetrahedron 1985, 41, 2567.
7. Ludwig, J.; Eckstein, F., J. Org. Chem. 1989, 54, 631.
8. Wojczewski, C.; Schwarzer, K.; Engels, J. W., Helv. Chim. Acta 2000, 83, 1268.

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