One-pot enzymatic preparative synthesis of 6-thio-2′-deoxyguanosine

An improved biotechnological method for producing 6-thio-2′-deoxyguanosine, a modified nucleoside exhibiting a uniquely broad spectrum of antitumor activity, has been developed. The one-pot preparative method involves incubation of 6-thioguanine and 2′-deoxythymidine at 45 °C in phosphate buffer in the presence of recombinant Escherichia coli thymidine phosphorylase and purine nucleoside phosphorylase. This method is distinct from the known analogue method in that 2′-deoxythymidine and 6-thioguanine are added to the reaction mixture in amounts that far exceed their water solubility. Utilizing the disclosed method of producing 6-thio-2′-deoxyguanosine increases yield of the end product based on the starting 2′-deoxythymidine from 14.6 to 33.5 % compared to the known prototype method and raises volumetric yield of the end product from 4.13 to 75.8 g/l of the reaction mixture. This significantly improves the efficiency of using a unit volume of the bioreactor. The proposed technology is high-tech and can be successfully applied under largescale production conditions.

1. Jordheim L. P., Durantel D., Zoulim F., Dumontet C. Advances in the development of nucleoside and nucleotide analogues for cancer and viral diseases. Nature Reviews. Drug Discovery, 2013, vol. 12, no. 6, pp. 447–464. https://doi.org/10.1038/nrd4010

2. Van Giesen K. J. D., Thompson M. J., Meng Q., Lovelock S. L. Biocatalytic synthesis of antiviral nucleosides, cyclic dinucleotides, and oligonucleotide therapies. JACS Au, 2023, vol. 3, no. 1, pp. 13−24. https://doi.org/10.1021/jacsau.2c00481

3. De Clercq E. Selected milestones in antiviral drug development. Viruses, 2024, vol. 16, no. 2, art. 169. https://doi.org/10.3390/v16020169

4. Sussmuth R. D., Kulike-Koczula M., Gao P., Kosol S. Fighting antimicrobial resistance: innovative drugs in antibacterial research. Angewandte Chemie International Edition, 2025, vol. 64, no. 10, art. e202414325. https://doi.org/10.1002/anie.202414325

5. Lewkowicz E. S., Iribarren A. M. Whole cell biocatalysts for the preparation of nucleosides and their derivatives. Current Pharmaceutical Design, 2017, vol. 23, no. 45, pp. 6851–6878. https://doi.org/10.2174/1381612823666171011101133

6. Romero E. O., Saucedo A. T., Hernandez-Melendez J. R., Yang D., Chakrabarty S., Narayan A. R. Enabling broader adoption of biocatalysis in organic chemistry. JACS Au, 2023, vol. 3, no. 8, pp. 2073–2085. https://doi.org/10.1021/jacsau.3c00263

7. Verma S., Paliwal S. Recent developments and applications of biocatalytic and chemoenzymatic synthesis for the generation of diverse classes of drugs. Current Pharmaceutical Biotechnology, 2024, vol. 25, no. 4, pp. 448–467. https://doi.org/10.2174/0113892010238984231019085154

8. Del Arco J., Fernandez-Lucas J. Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives. Applied Microbiology and Biotechnology, 2018, vol. 102, no. 18, pp. 7805–7820. https://doi.org/10.1007/s00253-018-9242-8

9. Yu W., Wei X., Wu Y., Jiang C., Tu Y., He B. Microbial biosynthesis of nucleos(t)ide analogs: applications, and engineering optimization. Critical Reviews in Microbiology, 2024, vol. 51, no. 5, pp. 879–897. https://doi.org/10.1080/1040841x.2024.2435668

10. Mender I., Gryaznov S., Dikmen Z. G., Wright W. E., Shay J. W. Induction of Telomere Dysfunction Mediated by the Telomerase Substrate Precursor 6-Thio-2′-Deoxyguanosine. Cancer Discovery, 2015, vol. 5, no. 1, pp. 82–95. https://doi.org/10.1158/2159-8290.cd-14-0609

11. Yu Sh., Wei S., Savani M., Lin X., Du K., Mender I., Siteni S. [et al.]. A Modified Nucleoside 6-Thio-2′-Deoxyguanosine Exhibits Antitumor Activity in Gliomas. Clinical Cancer Research, 2021, vol. 27, no. 24, pp. 6800−6814. https://doi.org/10.1158/1078-0432.ccr-21-0374

12. Eglenen-Polat B., Kowash R. R., Huang H. C., Siteni S., Zhu M., Chen K., Bender. M. E. [et al.]. A telomere-targeting drug depletes cancer initiating cells and promotes anti-tumor immunity in small cell lung cancer. Nature Communications, 2024, vol. 15, no. 1, art. 672. https://doi.org/10.1038/s41467-024-44861-8

13. Hanna N. B., Ramasamy K., Robins R. K., Revankar G. R. A convenient synthesis of 2′-deoxy-6-thioguanosine, araguanine, ara-6-thioguanine and certain related purine nucleosides by the stereospecific sodium salt glycosylation procedure. Journal of Heterocyclic Chemistry, 1988, vol. 25, no. 6, pp. 1899–1903. https://doi.org/10.1002/jhet.5570250653

14. Xu Y. Z., Zheng Q., Swann P. F. Simple synthesis of 4-thiothymidine, 4-thiouridine and 6-thio-2′-deoxyguanosine. Tetrahedron Letters, 1991, vol. 32, no. 24, pp. 2817−2820. https://doi.org/10.1016/0040-4039(91)85095-m

15. Onizuka K., Taniguchi Y., Sasaki S. A new odorless procedure for the synthesis of 2′-deoxy-6-thioguanosine and its incorporation into oligodeoxynucleotides. Nucleosides, Nucleotides and Nucleic Acids, 2009, vol. 28, no. 8, pp. 752–760. https://doi.org/10.1080/15257770903155576

16. Bieber A. L., Sartorelli A. C. Enzymatic synthesis and some biochemical effects of 2′-deoxy-6-thioguanosine. Nature, 1964, vol. 201, art. 624. https://doi.org/10.1038/201624a0

17. Beresnev A., Kvasyuk E., Kvach S., Zinchenko A. Attack on telomeres, or an Approach to cancer therapy using 6-thio-2′-deoxyguanosine. Nauka i innovatsii [Science and Innovation], 2016, no. 9, pp. 58−61 (in Russian).

18. Horn U., Strittmatter W., Krebber A., Knüpfer U., Kujau M., Wenderoth R., Müller K., Matzku S., Plückthun A., Riesenberg D. High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions. Applied Microbiology and Biotechnology, 1996, vol. 46, pp. 524−532. https://doi.org/10.1007/s002530050855