2-(Phenylthio)ethyl as a Novel Two-Stage Base Protecting Group for Thymidine Analogues

Abstract

2-(Phenylthio)ethyl is here proposed for temporary masking the thymine residue in the synthesis of sugar modified thymidine derivatives. This protection has been devised as a ‘two-stage’ system. The 2-(phenylthio)ethyl residue can be easily and regiospecifically inserted at the N3-position of the pyrimidine by a Mitsunobu reaction with 2-(phenylthio)ethanol and is perfectly stable also to strongly basic conditions. This allowed us to selectively achieve O-alkylation of the ribose moieties in satisfactory yields, avoiding undesired base alkylations. After oxidation of the thioether to sulfone, the thymine protecting group can be totally removed, by a β-elimination mechanism, upon the same basic treatment required for the final deprotection and detachment of oligonucleotides from the support in solid-phase synthesis protocols.

protecting groups - Mitsunobu reactions - thymidine - oligonucleotides - solid-phase synthesis

References and Notes

1 For a recent review, see for example: Reese CB. Org. Biomol. Chem.  2005,  3:  3851 

2 For a recent review, see for example: Natarajan V. Jeong KS. Byeang K. Curr. Med. Chem.  2003,  10:  1973 

3 For a recent review, see for example: Stawinski J. Kraszewski A. Acc. Chem. Res.  2002,  35:  952 

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4b Reese CB. Skone PA. J. Chem. Soc., Perkin Trans. 1  1984,  1263 

5 Reese CB. Song Q. Rao MV. Beckett I. Nucleosides Nucleotides  1998,  17:  451 

6a Kozai S. Maruyama T. Kimura T. Yamamoto I. Chem. Pharm. Bull.  2001,  49:  1185 

6b Robles R. Rodrìguez C. Izquierdo I. Plaza MT. Mota A. Cienfuegos LA. Tetrahedron: Asymmetry  2000,  11:  3069 

6c Luzzio FA. Menes ME. J. Org. Chem.  1994,  59:  7267 

6d Dobson N. McDowell DG. French DJ. Brown LJ. Mellor JM. Brown T. Chem. Commun.  2003,  1234 

7 D’Onofrio J. de Champdoré M. De Napoli L. Montesarchio D. Di Fabio G. Bioconjugate Chem.  2005,  16:  1299 

8 Hotoda H. Koizumi M. Koga R. Kaneko M. Momota K. Ohmine T. Furukawa H. Agatsuma T. Nishigaki T. Sone J. Tsutsumi S. Kosaka T. Abe K. Kimura S. Shimada K. J. Med. Chem.  1998,  41:  3655 

9 Hotoda H. Momota K. Furukawa H. Nakamura T. Kaneko M. Nucleosides Nucleotides  1994,  13:  1375 

10 Jaksa S. Kralj B. Pannecouque C. Balzarini J. De Clercq E. Kobe J. Nucleosides, Nucleotides Nucleic Acids  2004,  23:  77 

11 Schelhaas M. Waldmann H. Angew. Chem., Int. Ed. Engl.  1996,  35:  2056 

12a Kocienski PJ. In Protecting Groups   3rd ed.:  Thieme; Stuttgart: 2003. 

12b Greene TW. Wuts PGM. In Protective Groups in Organic Synthesis   3rd ed.:  Wiley; New York: 1999. 

13

Selected Data for Compound 2.
¹H NMR (400 MHz, CDCl₃): δ = 7.61-6.82 (13 H, complex signals, arom. protons), 6.38 (1 H, dd, H-1′, J = 6.2, 6.8 Hz), 4.49 (1 H, m, H-3′), 4.01-3.96 (3 H, overlapped signals, CH₂N and H-4′), 3.79 (6 H, s, OCH₃), 3.45 (1 H, dd, H-5′_a, J = 4.0, 12.0 Hz), 3.25 (1 H, dd, H-5′_b, J = 4.0, 12.0 Hz), 2.33-2.17 (2 H, complex signal, H₂-2′), 1.54 (3 H, s, CH₃ T), 0.97 (2 H, m, CH₂Si), 0.83 {9 H, s, [(CH₃)₃C]Si of TBDMS group}, 0.07 [9 H, s, (CH₃)₃Si], -0.02 and -0.04 [3 H each, s, Si(CH₃)₂ of TBDMS group]. ¹³C NMR (100 MHz, CDCl₃): δ = 163.22 (C-4), 150.57 (C-2), 135.40 (C-6), 158.61, 144.28, 133.19, 129.95, 126.98, 113.15 (arom. carbons), 110.18 (C-5), 86.81 (quat. C of DMT group), 86.56 (C-1′), 85.35 (C-4′), 71.97 (C-3′), 62.82 (C-5′), 55.14 (OCH₃), 41.53 (C-2′), 37.96 [(CH₃)₃SiCH₂ CH₂N], 25.59 {[(CH₃)₃C]Si of TBDMS group}, 17.81 [(CH₃)₃ C of TBDMS group], 15.87 [(CH₃)₃SiCH₂CH₂N], 12.54 (CH₃ T), -1.89 [(CH₃)₃SiCH₂CH₂N], -4.98 [Si(CH₃)₂ of TBDMS group]. MS: m/z calcd for C₄₂H₅₈N₂O₇Si₂: 758.38. ESI-MS found: m/z = 759.61 [M + H⁺]; m/z = 781.53 [M + Na⁺]; m/z = 797.50 [M + K⁺].

14

Typical Procedure for the Insertion of 2-(Trimethyl-silyl)ethyl Group: Synthesis of 2
The amount of 1.0 g (1.52 mmol) of 5′-O-(4,4′-dimethoxy-triphenylmethyl)-3′-O-(tert-butyldimethylsilyl)thymidine was dissolved in 10 mL of benzene at 0 °C and treated with 160 µL (1.14 mmol) of 2-(trimethylsilyl)ethanol and 470 µL (1.90 mmol) of tributylphosphine. After 10 min the reaction mixture was taken to r.t. and treated with 480 mg (1.90 mmol) of 1,1′-(azodicarbonyl)dipiperidine (ADDP).
After 18 h, the crude was taken to dryness, redissolved in 100 mL of EtOAc and washed twice with H₂O. The organic layer was concentrated under reduced pressure and purified by silica gel chromatography [eluent system: 2% MeOH in CHCl₃-pyridine (1:0.05)], affording 745 mg (0.98 mmol, 65%) of pure 2.

15a de Champdoré M. De Napoli L. Di Fabio G. Messere A. Montesarchio D. Piccialli G. Chem. Commun.  2001,  2598 

15b De Napoli L. Di Fabio G. D’Onofrio J. Montesarchio D. Synlett  2004,  1975 

16 Smrt J. Collect. Czech. Chem. Commun.  1974,  39:  972 

17

Selected Data for Compound 3.
¹H NMR (400 MHz, CDCl₃): δ = 7.47-6.85 (18 H, complex signals, arom. protons), 6.48 (1 H, dd, H-1′, J = 5.6, 9.6 Hz), 4.27 (1 H, d, H-3′, J _{3 ′,2 ′b} = 12.0 Hz), 4.22 (2 H, m, CH₂N), 4.01 (1 H, br s, H-4′), 3.82 (6 H, s, OCH₃), 3.68 (1 H, dd, H-5′_a, J = 4.0, 12.0 Hz), 3.29 (1 H, dd, H-5′_b, J = 4.0, 12.0 Hz), 3.19 (2 H, t, CH₂S, J = 8.0, 8.0 Hz), 1.87 (3 H, s, CH₃ T), 1.76 (1 H, dd, H-2′_a, J = 4.0, 12.0 Hz), 1.57 (1 H, complex signal, H-2′_b), 0.83 {9 H, s, [(CH₃)₃C]Si}, -0.02 and -0.08 [3 H each, s, Si(CH₃)₂]. ¹³C NMR (100 MHz, CDCl₃): δ = 162.83 (C-4), 150.37 (C-2), 135.86 (C-6), 158.29, 150.37, 144.70, 135.95, 135.28, 133.27, 129.87, 129.75, 128.51, 127.99, 127.86, 127.51, 126.63, 125.35, 112.83 (arom. carbons), 109.62 (C-5), 86.81 (C of DMT group), 86.16 (C-1′), 85.12 (C-4′), 74.55 (C-3′), 63.12 (C-5′), 54.80 (OCH₃), 40.59 (CH₂N), 39.55 (C-2′), 29.22 (CH₂S), 25.34 {[(CH₃)₃C]Si}, 17.77 [(CH₃)₃ C], 12.68 (CH₃ T), -5.86 and -6.18 [Si(CH₃)₂]. MS: m/z calcd for C₄₅H₅₄N₂O₇SSi: 794.34. ESI-MS (positive ions) found: m/z 833.75 [M + K⁺].

18

Typical Procedure for the Insertion of 2-(Phenylthio)-ethyl Group: Synthesis of 3 The amount of 900 mg (1.37 mmol) of 5′-O-tert-butyl-dimethylsilyl-3′-O-(4,4′-dimethoxytriphenylmethyl)thy-midine was dissolved in 10 mL of benzene at 0 °C and treated with 38 µL (1.02 mmol) of 2-(phenylthio)ethanol and 422 µL (1.71 mmol) of tributylphosphine. After 10 min the reaction mixture was taken to r.t., treated with 431 mg (1.71 mmol) of ADDP and left at r.t. for 18 h. The crude was then taken to dryness, redissolved in 100 mL of EtOAc and washed twice with H₂O. The organic layer was concentrated under reduced pressure and purified by silica gel chromatography [eluent system: 2% acetone in CHCl₃-pyridine (1:0.05)], affording 980 mg (1.23 mmol, 90%) of pure 3.

19

Selected Data for Compound 5.
³¹P NMR (161.98 MHz, CDCl₃): 149.3. MS: calcd for C₄₈H₅₇N₄O₁₀PS: 912.35. ESI-MS (positive ions) found: m/z = 935.89 [M + Na⁺]; m/z = 952.90 [M + K⁺].

20

Removal of the 2-(phenylsulfonyl)ethyl group by aq NH₃ at 55 °C after an overnight treatment was only partial, requiring longer reaction times to go to completion (ca.
72 h). On the other hand, a 0.1 M NaOH solution ensured complete thymine deprotection after an overnight treatment at 50 °C, i.e. conditions usually adopted also for oligo-nucleotide deprotection and detachment from the solid support.

21

Data for ^{5 ′DBB} TGGGAG ^{³ ′OH}
MS: m/z calcd for C₈₁H₉₂N₂₇O₃₆P₅: 2173.49; MALDI-TOF (negative ions) found: 2171.76 [M - H]^-.