P-stereocontrolled synthesis of oligo(nucleoside N3'→O5' phosphoramidothioate)s - opportunities and limitations

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., Ade^Bz, Cyt^Bz, Gua^iBu, or Thy). The monomers were chromatographically separated into P-diastereomers, which were further used to prepare N_NPSN' 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 N_NPSN' were digested by the HINT1 phosphoramidase enzyme, the isomers hydrolyzed at a faster rate were tentatively assigned the R _P absolute configuration. This assignment is supported by X-ray analysis of the protected dinucleotide ^DMTdG^iBu _NPSMeT_OAc, which is P-stereoequivalent to the hydrolyzed faster P-diastereomer of dG_NPST.

Chart 1. B¹,B² = Ade, Cyt, Gua or Thy. The asterisks indicate the P-stereogenic centers.

Scheme 1. A mechanism of the DBU-promoted condensation step in Otp synthesis of N_PSN′ or N_NPSN′ with the use of a mixture of P-epimers of OTP-N (Z = O) or _NOTP-N monomers (Z = NH), respectively. 4, R = H; 5, R = Me; 6, R,R = −(CH₂)₅–. B′ = Ade^Bz, Cyt^Bz, Gua^iBu, or Thy. The codes 6Gf and 6Gs indicate fast- and slow-eluting P-diastereomers of Pm-_NOTP-dG (vide infra).

Chart 2. (D) (R_c)-2-(1-(α-Naphthyl)ethyl)amino)-2-thio-1,3,2-oxathiaphospholane, (E) a 2-thio-1,3,2-oxathiaphospholane derivative of l-tryptophan methyl ester; _NOTP-Trp.

Scheme 2. Effects of silica gel separation of P-diastereomers of 2-thio-1,3,2-oxathiaphospholane monomers 4–6. The descriptors pro-R_P and pro-S_P indicate the absolute configuration of P-atom in the subsequently formed internucleotide phosphoramidothioate linkages. The structure F is given only for comparison with G.

Chart 3. Orientation of the sulfur, phosphoryl oxygen and thymidine O5′ atoms around the phosphorus atoms: Panel A, in phosphorothioate S_P-1, ref. 23; Panel B, 2D NMR ROESY based assignment (here challenged by X-ray analysis of 10f, Scheme 1) in the S-methylated S_P-3Mf, ref. 22 (the phosphoramidothioate diester precursor was obtained from fast-eluting 5Tf). The Arabic numerals 1–4 indicate the priority of substituents around the phosphorus atoms according to the Cahn–Ingold–Prelog rules. Note: to establish the priority of substituents, the formal double bond between the phosphoryl oxygen atom and the phosphorus atom (PO) should be considered a single one.

Fig. 1. Crystal structure of ^DMTdG^iBu_NPSMeT_OAc10f showing partial structure and spatial orientation of substituents around the phosphorus stereogenic center (marked P1). Compound 10 was obtained from the monomer 6Gf (fast-eluting 5′-O-DMT-3′-amino-2′,3′-dideoxy-N6-iBu-guanosine-3′-N-(2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane). The oxygen atom marked O5′A belongs to the thymidine residue.

Scheme 3. The HINT1 catalyzed hydrolysis of adenosine-5′-O-((l-tryptophan amide-Nα-yl)-phosphoramidothioate) (AMPS-Trp-NH₂), followed by desulfuration of the AMPS intermediate leading to the formation of adenosine-5′-O-phosphate.

Fig. 2. Rates of hydrolysis of NPS-dinucleotides 11–17 (data from Table 2). The descriptions fast and slow refer to the relative chromatographic mobility of _NOTP-N precursors. For better visualization the heights of bars depicting the rates of hydrolysis of 16 and 17 have been scaled up by a factor of 10.

Fig. 3. Coupling yields in the consecutive condensation steps, assessed by DMT⁺ assay at 504 nm. Absorptions of the DMT⁺ cation released from the nucleosides attached to the support (blue bars) are taken as 100%.

Fig. 4. Visualization of the lowest energy conformers of 6G (R_PPm-_NOTP-dG and S_PPm-_NOTP-dG). The data were obtained using the Gaussian 16 package. Heteroatom labeling: P-green, O-red, S-yellow, N-navy blue.

Fig. 5. Visualization of the lowest energy conformer of R_P-5T (the C2′-endo atom is marked with an arrow). The data were obtained using the Gaussian 16 package. Heteroatom labeling: P-green, O-red, S-yellow, N-navy blue.

Fig. 6. Predicted by molecular modeling the lowest energy conformations of: S_PPm-OTP-dA, an upper panel; R_P-6A, a middle panel; R_P-5T, a bottom panel. All three compounds are P-stereochemically equivalent (vide supra).

Fig. 7. Analysis of 21f obtained from 5Tf. (Left) An RP HPLC profile (DMT-OFF); (right): an electropherogram (20% PAGE).