Mechanistic studies on oxidative condensation of a thymidine 3 ′ - H - phosphonate derivative with 3 ′ - O -acetylthymidine

Oxidation-reduction condensation using triphenylphosphine and 2,2 ′ -dipyridildisulfide was carried out for forming a phosphodiester bond between a mono-anionic thymidine 3 ′ - H - phosphonate derivative and 3 ′ - O -acetylthymidine. This reaction proceeded with simultaneous coupling and oxidation. The detailed NMR analysis of this reaction shows that an S -(2-pyridyl) phosphorothioate diester derivative was formed as an initial intermediate.


Introduction
For internucleotidic bond formation in oligonucleotide synthesis, 1 the H-phosphonate method 2 has been used along with the more popular phosphoramidite method. 3The former requires reagents for dehydration between the H-phosphonate and alcoholic components.For this purpose, a number of condensing agents such as pivaloyl chloride, 1-adamantanecarbonyl chloride, diphenyl phosphorochloridate, Bop-Cl, PyBOP, and BOMP have been developed.2b The initial products of H-phosphonate diester synthesis are oxidized in the last stage to give the phosphodiester derivatives.
Oxidation-reduction condensation, using triphenylphosphine (PPh 3 ) with 2,2′-dipyridyl disulfide (PySSPy) as the condensing agent, was developed in the early years of DNA chemical synthesis by Mukaiyama et al. for peptide and oligonucleotide syntheses. 4In an attempt to develop a new condensing agent for the H-phosphonate method, we found that PPh 3 -PySSPy gave phosphodiester derivatives directly when used as the condensing agent for coupling between 5′-O-dimethoxytritylthymidine 3′-H-phosphonate (1) and 3′-O-acetylthymidine (2).This result was mechanistically interesting, since no reports have appeared about such a condensation with a simultaneous oxidation reaction.In this paper, we report mechanistic studies of this procedure using 31 P NMR analysis.

Results and Discussion
When compound 1 was allowed to react with compound 2 in the presence of 5.0 equiv of PPh 3 and 10.0 equiv of PySSPy in dry pyridine at room temperature for 12 h, DMTrTpToAc 4 was obtained in 88% yield.The structure of this product was characterized by comparison with an authentic sample synthesized from the phosphoramidite approach, as well as by 1 H NMR and high-resolution mass analyses.To clarify the oxidative condensation reaction mechanism, we studied the time-course analysis of the products by 31 P NMR spectroscopy (Figure 2).The peak of Hphosphonate 1 was observed at 2.44 ppm in pyridine-d 5 .After 60 min, this peak disappeared and a new main peak X was observed at 11.20 ppm.Moreover, a minor peak Y appeared at around 21-22 ppm.The peak X slowly decreased, and a peak corresponding to phosphate diester 4 appeared at −0.75 ppm.After 12 h, peak X had disappeared.It is known that the 31 P NMR peaks of H-phosphonate diester and phosphorothioate derivatives are observed at around 10 ppm. 5 To determine the structures responsible for peaks X and Y, we synthesized three compounds that are expected to be formed as intermediates in the present reaction: 5′-O-dimethoxytritylthymidine(3′-5′)3′-O-acetylthymidine H-phosphonate (3), 6 S-(2-pyridyl) 5′-O-dimethoxytritylthymidine(3′-5′)3′-O-acetylthymidine phosphorothioate (5), and S-(2-pyridyl) 5′-O-dimethoxytritylthymidine 3′-phosphorothioate (6).
H-phosphonate 1 was coupled with compound 2 in the presence of Bop-Cl in CH 3 CN.This reaction gave a diastereomeric mixture of dithymidine H-phosphonate derivative 3. 6 After stirring for 30 min, the mixture was extracted with CH 2 Cl 2 /triethylammonium carbonate buffer, and the organic layer was concentrated and dissolved in dry pyridine-d 5 .The 31 P NMR spectrum of the extract showed a set of peaks at 10.30 and 8.71 ppm corresponding to diasteromers 3. When PySSPy was added to the solution, two diastereomeric peaks of the resulting S-pyridyl phosphorothiate derivative 5 appeared after 30 min at 22.47 and 21.93 ppm.Furthermore, the Hphosphonate diester peak of 3 had completely converged spontaneously with the peak of 5 after addition of DBU.These results strongly suggested that the minor peak Y was due to compound 5.Moreover, the 1 H-31 P coupling of peak X was not observed on the proton-coupled 31 P NMR measurement (data not shown).This result suggested that compound X does not have an H-P(O) bond, which should have a large H-P coupling constant. 7To synthesize compound 6 according to the wellestablished procedure, 8 compound 1 was allowed to react with PySSPy in the presence of bis(trimethylsilyl)acetamide (BSA) at room temperature in CD 3 CN for 12 h.The 31 P NMR spectrum of the mixture thus obtained showed a single peak at 10.47 ppm.Based on these results, we concluded that peak X must have been due to compound 6.If this assumption is correct, compound 6 could be obtained under the conditions without adding alcohol 2. To confirm this, compound 1 was dissolved in pyridine-d 5 , and PPh 3 and PySSPy were added.After 60 min, the peak of compound 6 was observed at 11.15 ppm, as expected.Interestingly, another peak at 45.70 ppm appeared: It is likely that this peak, from its chemical shift, was due to phosphorodithioate derivative 7. 9 However, it was difficult to synthesize compound 7 using a different procedure, because it was very unstable and highly reactive.In independent experiments, it was found that compound 4 remained intact when treated with excess PPh 3 and PySSPy in the presence or absence of 2-mercaptopyridine (data not shown).
Compound 6 was previously used as an activated ester to obtain phosphoramidate derivatives. 10However, the reaction of this activated ester with nucleosides proceeded slowly.From these results, it was plausible that compound 5 was derived from the reaction of phosphorodithioate 7 with 2 (Figure 6).Compared with the usual condensing agents such as pivaloyl chloride, diphenyl phosphorochloridate, and BOMP 2b used for the H-phosphonate approach, the condensing agent PPh 3 -PySSPy used for the oxidation-reduction condensation showed its inherent, and entirely different, properties.If this reagent could work as a simple dehydrating reagent between Hphosphonate 1 and alcohol 2, the reaction was expected to proceed initially like the usual Hphosphonate method.Our results did not suggest this course of reaction, and thereby, at the initial stage, the H-phosphonate function was promptly oxidized to the less reactive five-valent species, so that slow condensation with oxidation was observed.
In addition, we could not detect the formation of symmetrical pyrophosphate derivatives, which were exclusively observed when S-phenyl nucleoside phosphorothioate derivatives were activated by condensing agents such as triisopropylbenzenesulfonyl chloride (TPS). 11,12

Conclusions
In summary, it is clear that the coupling reaction between H-phosphonate monoester 1 and alcohol 2 using PPh 3 and PySSPy occurred with simultaneous oxidation to give phosphodiester 4 as the main product.In this reaction, H-phosphonate monoester 1 is initially oxidized by PPh 3 and PySSPy to give S-(2-pyridinyl) phosphorothioate 6.This intermediate compound 6 slowly reacts with alcohol 2 to give phosphodiester 4. Since phosphodiester 4 did not react with PPh 3 and PySSPy, the main route to 5 might be the reaction of 7 with 2. This unique condensing reaction yielding phosphodiester products from alkyl phosphonates and alchohols via a one-step reaction could be used when both components are easily available, and when the final products can be purified without difficulty.

P NMR analysis of formation of 3 and 5.
A solution of 5′-O-dimethoxytritylthymidine 3′-Hphosphonate 1 (213 mg, 0.3 mmol) and 3′-O-acetylthymidine 2 (127 mg, 0.45 mmol) in dry pyridine (3.0 ml) under argon was treated with Bop-Cl (90 mg, 0.33 mmol).After stirring for 30 min at room temperature, the reaction mixture was extracted with CH 2 Cl 2 (20 ml) and 0.1 M triethylammonium carbonate buffer (20 ml × 2).The combined organic layer was dried over anhydrous Na 2 SO 4 and evaporated under reduced pressure.The residue was co-evaporated with toluene and pyridine.The residue was dissolved with pyridine-d 5 (1.5 ml) and the 31 P NMR spectra was measured.PySSPy (660 mg, 3.0 mmol) was added to the solution.The mixture was stirred at room temperature for 30 min, and the 31 P NMR spectra was measured.DBU (90 µl, 0.6 mmol) was added to the mixtures.After 5 min, the 31 P NMR spectra was measured; the results are shown in Figure 3.

31
P NMR analysis of formation of 6 using BSA and PySSPy.Compound 1 (71 mg, 0.1 mmol) was allowed to react with PySSPy (110 mg, 0.5 mmol) in the presence of BSA (122 µl, 0.5 mmol) at room temperature in CD 3 CN (0.5 ml).After 12 h, the 31 P NMR spectra was measured; the results are shown in Figure 4.

Figure 1 .
Figure 1. 31 P NMR spectra of coupling reaction between 1 and 2 in the presence of PPh 3 and PySSPy.

Figure 2 .
Figure 2. 31 P NMR spectrum of the reaction of 1 with 2 in the presence of PPh 3 and PySSPy.

Figure 3 .
Figure 3. 31 P NMR spectrum of the reaction of 1 with 2 in the presence of Bop-Cl.

Figure 4 . 2 +Figure 5 .
Figure 4. 31 P NMR spectrum of the reaction of compound 1 with PySSPy in the presence of bis(trimethylsilyl)acetamide.

Figure 6 .
Figure 6. 31 P NMR spectrum of the reaction of compound 1 with PySSPy in the presence of bis(trimethylsilyl)acetamide.