Abstract

3′-N-(2-Thio-1,3,2-oxathiaphospholane) derivatives of 5′-O-DMT-3′-amino-2′,3′-dideoxy-ribonucleosides (NOTP-N), that bear a 4,4-unsubstituted, 4,4-dimethyl, or 4,4-pentamethylene substituted oxathiaphospholane ring, were synthesized. Within these three series, NOTP-N differed by canonical nucleobases (i.e., AdeBz, CytBz, GuaiBu, or Thy). The monomers were chromatographically separated into P-diastereomers, which were further used to prepare NNPSN′ dinucleotides (3), as well as short P-stereodefined oligo(deoxyribonucleoside N3′→O5′ phosphoramidothioate)s (NPS-) and chimeric NPS/PO- and NPS/PS-oligomers. The condensation reaction for NOTP-N monomers was found to be 5–6 times slower than the analogous OTP derivatives. When the 5′-end nucleoside of a growing oligomer adopts a C3′-endo conformation, a conformational ‘clash’ with the incoming NOTP-N monomer takes place, which is a main factor decreasing the repetitive yield of chain elongation. Although both isomers of NNPSN′ were digested by the HINT1 phosphoramidase enzyme, the isomers hydrolyzed at a faster rate were tentatively assigned the RP absolute configuration. This assignment is supported by X-ray analysis of the protected dinucleotide DMTdGiBuNPSMeTOAc, which is P-stereoequivalent to the hydrolyzed faster P-diastereomer of dGNPST.

Highlights

  • Therapeutic properties of synthetic DNA oligonucleotides (POoligos) targeted against complementary DNA or mRNA molecules have been tested for more than 40 years.[1]

  • Stec on the occasion of his 80th birthday. ‡ Electronic supplementary information (ESI) available: HR MS, 31P NMR, 1H NMR, and 13C NMR spectra of separated P-epimers of 4–6; MS and 31P NMR data for unresolved monomers 4–6; 31P NMR, 1H NMR, 13C NMR spectra, details of crystallographic analysis, and crystal structure of DMTdGiBuNPSMeTOAc obtained from 6Gf; selected data for NPS- and chimeric NPS/PO oligomers

  • Diastereomers, which were used in synthesis of dinucleotides NNPSN0

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Summary

Introduction

Chart 1 B1,B2 1⁄4 Ade, Cyt, Gua or Thy. The asterisks indicate the Pstereogenic centers. 2D NMR ROESY experiments suggested that, contrary to the aforementioned empirical rule, the relative orientation of the sulfur, phosphoryl oxygen and thymidine O50 atoms around the phosphorus atom in the S-methylated triester 3Mf (Chart 3, obtained from the fast-eluting 5Tf) is equivalent to that in phosphorothioate SP-1, which is hydrolyzed by Nuclease P1. Orientations and the absolute con gurations of P atoms in SP-1, and 3Mf are the same This violation of the empirical rule “fast-eluting Dm- and PmOTP-N / RP-NPSN” could not be explained by the C30-endo conformation of the sugar moiety in 30-amino-20,30-dideoxynucleosides, because the C30-endo locked LNA-derived oxathiaphospholane monomers adhere to that rule.[24] In the present work, this unexpected NMR-based assignment was challenged by a successful crystallographic experiment, which showed the rule-obeying RP absolute con guration (Fig. 1) for a non-ionic DMTdGiBuNPSMeTOAc amidodiester (10f, Scheme 1, ESI‡) obtained from the fast-eluting 6Gf (a Pm-derivative) a er Smethylation of the negatively charged amidoester intermediate DMTdGiBuNPSTOAc (9). The released (d) NMPS is further desulfured to form (d)NMP and hydrogen sul de, and this secondary nucleotide product must be taken into account during HPLC-based quanti cation of the products k AMPS-Trp-NH2 was concomitantly obtained from AMPS-Trp-OMe during ammonolysis of the protecting benzoyl groups in the adenosine moiety

15 TNPSdG
Findings
Conclusions
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