HETEROCYCLES

Communication | Regular issue | Vol. 91, No. 6, 2015, pp. 1164-1169
Received, 9th April, 2015, Accepted, 12th May, 2015, Published online, 26th May, 2015.
DOI: 10.3987/COM-15-13223

■ A New Strategy for Synthesis of the Dinucleotide pdCpA: A Convenient Method for the Deprotection of Cyanoethyl, TBDMS, and Benzoyl Groups in One Step at High Pressure

Masanori Kataoka,* Chiharu Fukui, Akiko Mimoto, Hideaki Kuge, Koichi Honke, and Hiyoshizo Kotsuki*

Laboratory of Natural Products Chemistry, Faculty of Science, Kochi University, 2-5-1, Akebono-cho, Kochi 780-8520, Japan

Abstract

A new improved method for the preparative-scale synthesis of the dinucleotide pdCpA was developed. This method takes advantage of the complete deprotection of cyanoethyl, TBDMS, and benzoyl groups in one step under high-pressure conditions in the final stage of the synthetic sequence.

The incorporation of unnatural amino acids into proteins at predetermined sites has been extensively used to investigate protein structures and functions,1 for the design of glycoprotein substrates that can participate in several important biological processes,2 and for the discovery of new drug candidates.3 In general, such cell-free protein syntheses commence with the chemical aminoacylation of pdCpA (1)4 and the subsequent condensation with an abbreviated suppressor tRNA under catalysis with T4 RNA ligase (Scheme 1).1d
Despite this enormous utility in molecular engineering science, there is still a strong demand for improved product yields and greater purity.5 Some of the major problems that remain to be overcome in this regard include the difficulty of obtaining sufficient amounts of the starting pdCpA (1) and the need for special techniques for its purification as well as sample regulation.
In our extensive efforts to develop a new synthetic method for a variety of nucleotides,6 it became necessary to prepare large quantities of the dinucleotide pdCpA (1). Here we describe a new and convenient method for the synthesis of this key compound using a high-pressure-promoted deprotection strategy.

Our synthetic procedure is shown in Scheme 2. Condensation of bis-TBDMS- and N-Bz-protected adenosine (2, 1.8 g)7 with 1.2 eq of N-Bz-protected 2’-deoxycytidinephosphoramidite (3) in the presence of 1.5 eq of benzimidazolium triflate8 and MS 4A in dichloromethane followed by oxidation with 0.53 eq of 2-butanone peroxide9 at 25 °C for 0.5 h gave, after careful acidification by exposure to trichloroacetic acid at ambient temperature, phosphotriester 4 (2.4 g) in 75% overall yield. Next, 4 was converted to the

corresponding 5’-phosphorylated compound 5 in 90% yield by conventional phosphorylation (1.9 g): i.e., treatment with 1.5 eq of bis(2-cyanoethyl)-N,N-diisopropylphosphoramidite in the presence of 2.0 eq of benzimidazolium triflate8 followed by oxidation with 0.53 eq of 2-butanone peroxide9 at 25 °C for 0.5 h. Finally, deprotection of all of the cyanoethyl, TBDMS, and benzoyl groups was first attempted under normal conditions using ammonium hydroxide.5c In this case, however, the TBDMS group remained undeprotected and subsequent desilylation was required to complete this synthetic sequence.
To shorten this stepwise methodology, we anticipated that the application of high pressure would be useful, since it is known that deprotection / hydrolysis of this type can be effectively accelerated under high-pressure conditions.10 Accordingly, when a mixture of 5 and excess aq ammonium hydroxide in a mixed solvent system composed of H2O-MeOH-dioxane (9 : 7 : 1) was reacted at 0.8 GPa and 25 °C for 14 h, the desired ammonium salt of pdCpA (1, R = H) was obtained in 60% yield (Method A).11, 12 To our delight, simultaneous desilylation of double TBDMS groups could be achieved even under such fluoride-free conditions.13 Furthermore, the superiority of this technique was exemplified by the use of tetra-n-butylammonium hydroxide as a base,14 and the corresponding tetra-n-butylammonium salt of pdCpA (6, R = n-Bu) was obtained in almost quantitative yield (Method B).11, 12 This substance was sufficiently pure and could be used directly in further experiments to introduce unnatural amino acids to a ribose core. Notably, the latter improved strategy does not require a tedious counter cation exchange process,15 and may be very useful for further elaboration in nucleotide chemistry.
In summary, we have developed a new convenient method for synthesis of the dinucleotide pdCpA (1) on a preparative scale. This method takes advantage of the simultaneous complete deprotection of cyanoethyl, TBDMS, and benzoyl groups under high-pressure conditions in the final stage. Further studies on protein engineering using the dinucleotide pdCpA (1) are now in progress in our laboratory.
ACKNOWLEDGEMENTS
We are grateful to Dr. K. Onodera and Mrs. M. Yuhi of Kochi University for MALDI-TOF/MS and HPLC measurements. This work was supported by Adaptable and Seamless Technology Transfer Program through target-driven R&D, JST (Japan Science and Technology Agency) and by the Program to Disseminate Tenure Tracking System of the MEXT (Japan).

References

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11. All high-pressure reactions were performed in a piston-cylinder type apparatus (Hikari Koatsu HR-15-B3).
12. Method A: To a solution of the protected pdCpA 5 (57 mg, 46 μmol) in MeOH (750 μL) and 1,4-dioxane (110 μL) was added 28% aq NH4OH (900 μL), and the mixture was transferred to a Teflon reaction vessel and kept at 0.8 GPa and 25 °C for 14 h. After concentration, the crude product was subjected to reverse-phase short-column chromatography on 30 μm ODS (30 g) eluted with a mixture of double-distilled H2O and MeCN (100 : 0 → 95 : 5) to provide the ammonium salt of pdCpA 1 as an amorphous solid (19 mg, 60% yield); Method B: To a solution of the protected pdCpA 5 (370 mg, 300 μmol) in MeOH (3.7 mL), 1,4-dioxane (540 μL), and double-distilled H2O (1.8 mL) was added 20% aq n-Bu4NOH (2.5 mL, 1.8 mmol), and the mixture was transferred to a Teflon reaction vessel and kept at 0.8 GPa and 25 °C for 14 h. After dilution by the addition of double-distilled H2O, the mixture was subjected to gel filtration on Sephadex G-10 (480 mL, eluted with double-distilled H2O / MeOH = 80 : 20) to give the tetra-n-butylammonium salt of pdCpA 1 (480 mg, 97% yield estimated by OD260) as an amorphous solid; 1 (as its tetra-n-butylammonium salt): FTIR (KBr) ν 1653, 1604, 1576, 1488, 1378 cm–1; UV (MeOH) λmax 260 nm (ε 23,800); 1H NMR (500 MHz, CD3OD) δ 8.62 (1H, s), 8.17 (1H, s), 8.10 (1H, d, J = 7.5 Hz), 6.41 (1H, dd, J = 5.4, 8.3 Hz), 6.11 (1H, d, J = 6.3 Hz), 5.94 (1H, d, J = 7.5 Hz), 4.98–4.92 (1H, m), 4.72 (1H, dd, J = 6.0, 6.9 Hz), 4.59–4.54 (1H, m), 4.32–4.28 (1H, m), 4.26–4.22 (1H, m), 4.13–3.97 (4H, m), 3.62 (1H, t, J = 6.9 Hz), 3.26–3.14 (24H, m), 2.65–2.56 (1H, m), 2.40 (1H, t, J = 7.2 Hz), 2.28–2.19 (1H, m), 1.69–1.59 (24H, m, TBA), 1.40 (24H, sextet, J = 7.5 Hz), 1.01 (36H, t, J = 7.5 Hz); 31P NMR (202.5 MHz, CD3OD) δ 4.28, –0.46; MALDI-TOF/MS Calcd for C19H27N8O13P2 [M + H]+ m/z 637.12, found m/z 637.52.
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14. At the end of the reaction, the mixture showed a pH of ca. 8.0 as a result of neutralization by the deprotection of cyanoethyl and benzoyl groups.
15. (a) J. A. Ellman, D. Mendel, S. Anthony-Cahill, C. J. Noren, and P. G. Schultz, Methods Enzymol., 1991, 202, 301; CrossRef (b) M. J. Damha and K. K. Ogilvie, Methods Mol. Biol., 1993, 20, 81