Preliminary Communication
Full Access
N, N-diisopropyl-bis(4-chlorobenzyl) phosphoramidite: A versatile phosphitylating agent for the phosphorylation of hydroxy amino acids and preparation of protected phosphopeptides
H. B. A. de Bont,
J. H. van Boom,
R. M. J. Liskamp,
H. B. A. de Bont
Department of Organic Chemistry, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands
Search for more papers by this authorJ. H. van Boom
Department of Organic Chemistry, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands
Search for more papers by this authorR. M. J. Liskamp
Department of Organic Chemistry, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands
Search for more papers by this authorH. B. A. de Bont,
J. H. van Boom,
R. M. J. Liskamp,
H. B. A. de Bont
Department of Organic Chemistry, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands
Search for more papers by this authorJ. H. van Boom
Department of Organic Chemistry, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands
Search for more papers by this authorR. M. J. Liskamp
Department of Organic Chemistry, Gorlaeus Laboratories, University of Leiden, PO Box 9502, 2300 RA Leiden, The Netherlands
Search for more papers by this authorAbstract
N, N-diisopropyl-bis(4-chlorobenzyl) phosphoramidite is an easily accessible phosphitylating agent, which readily phosphorylates Boc-Ser-OMe, Boc-Thr-OMe and Boc-Tyr-OMe in good to high yields after t-BuOOH or MCPBA oxidation. The Boc-group can be removed without affecting the phosphate 4-chlorobenzyl groups and vice versa. Extension of the Boc-deprotected phosphoamino acids with a subsequent amino acid yields the corresponding protected phosphopeptides
References and Notes
- 1 E. G. Krebs, and J. A. Beavo, Ann. Rev. Biochem. 48, 923 (1979). P. Cohen, Nature 296, 613 (1982).
- 2 Evidence that protein phosphorylation is a regulatory element in carcinogenesis and associated with certain diseases: M. P. Kamps, S. S. Taylor and B. M. Sefton, Nature 310, 589 (1984); C. R. Lin, W. S. Chen, W. Kruiger, L. S. Stolarsky, W. Weber, R. M. Evans, I. M. Verma, G. N. Hill, and M. G. Rosenfeld, Science 224, 843 (1984); L. Gierasch, Adv. Prot. Chem. 37, 1 (1985).
- 3 H. B. A. de Bont, G. H. Veeneman, J. H. van Boom, and R. M. J. Liskamp, Recl. Trav. Chim. Pays-Bas, 106, 641 (1987).
- 4 H. B. A. de Bont, R. M. J. Liskamp, C. A. O'Brian, C. Erkelens, G. H. Veeneman and J. H. van Boom, Int. J. Peptide Protein Res. 33, 115 (1989).
- 5 A. H. van Oijen, C. Erkelens, J. H. van Boom and R. M. J. Liskamp, J. Am. Chem. Soc. (1989), in press.
- 6 C. M. Dreef-Tromp, C. Erkelens, G. A. van der Marel and J. H. van Boom, J. Am. Chem. Soc. 111, 6470 (1989).
- 7 T. B. Johnson and J. K. Coward, J. Org. Chem 52, 1771 (1987)
- 8 P. F. Alewood, J. W. Perich, and R. B. Johns, Synth. Comm. 12, 821 (1982).
- 9 Synth. Comm., Aust. J. Chem. 37, 429 (1984).
- 10 Synth. Comm., Tetrahedron Lett. 25, 987 (1984).
- 11 Synth. Comm., Tetrahedron Lett. 27, 1373 (1986).
- 12 J. W. Perich and R. B. Johns, J. Org. Chem. 53, 4103 (1988)
- 13 J. Org. Chem. J. Chem. Soc. Chem. Comm. 1988, 664.
- 14 A. Beld, C. A. A. Claesen, E. S. Roersma, W. J. M. Schippers, L. M. Keizer, and G. I. Tesser, Recl. Trav. Chim. Pays-Bas, 103, 196 (1984).
- 15 D. H. Schlesinger, A. Buku, H. R. Wyssbrod, and D. I. Hay, Int. J. Peptide Protein Res. 30, 257 (1987).
- 16 J. W. Perich, P. F. Alewood, and R. B. Johns, Synthesis, 1986, 572.
- 17 W. Bannwarth and A. Trzeciak, Helv. Chim. Acta, 70, 175 (1987).
- 18 J. W. Perich, and R. B. Johns, J. Org. Chem. 54, 1750 (1989).
- 19 J. Org. Chem., Tetrahedron Lett. 29, 2369 (1988).
- 20 Conditions of the removal of the Fmoc group will probably lead to β-elimination (Z.A. Shabarova, in “Progress in Nucleic Acids Research and Molecular Biology”, J. N. Davidson, and W. E. Cohn, eds., Academic Press, London, 1970, vol. 10. p. 145)
- 21 N,N-diisopropyl-dichlorophosphoramidite was prepared according to Houben & Weyl;, Methoden Der Organischen Chemie, Georg Thieme Verlag, Stuttgart vol. 12/2, p. 96 (1964). N,N-diisopropyl-bis(4-chlorobenzyl)phosphoramidite was synthesized by addition of 4-chlorobenzylalcohol (8.55 g, 60 mnol) and Et3N (9.2 mL, 66 mmol) in dry CH2Cl2 (50 mL) to a solution of N,N-diisopropyl-dichlorophosphoramidite (6.06 g, 30 mmol) in dry CH2Cl2 (50 mL). The work-up was carried out as described in ref. 17. Purification was carried out by short column chromatography, using 2% Et3N in pet. ether 40-60 as eluent.
- 22 Phosphorylated hydroxy amino acids 2; general procedure After coevaporation of the amino acid derivative 1 (2 mmol) with dioxane (2 × 15 mL), the reaction flask is charged with Argon and N,N-diisopropylamino-bis(4-chlorobenzyl)phosphor-amidite (1.03 g, 2.5 mmol) is introduced, followed by addition of a solution (0.5 M, 6 mL) of 1H-tetrazole in acetonitrile to the solid reaction components. The reaction mixture is stirred until dissolution and another 1-2 h, necessary for completion of the reaction as was monitored by TLC (eluent MeOH/CH2Cl2, 5/95, by vol.). After cooling the reaction mixture to 0°C, 80% t-BuOOH (0.62 mL, 5 mmol; preparation of 1a and 1b) or 55% MCPBA (1.57 g, 5 mmol; preparation of 1b and 1c) is added and stirring is continued at RT for 1 h (1a and 1b) or 2 h (1b and 1c). Subsequently, excess oxidizing agent is destroyed with 10% aq. Na2S03 (10 mL) and the mixture is transferred to a separatory funnel. Butanon (70 mL) is added and the organic layer is washed with 10% aq. Na2S03 (10 mL), sat. aq. NaHC03 (10 mL, only in the preparation of 1c), H2O (10 mL), brine (10 mL) and dried (Na2SO4). Evaporation of the solvent affords the crude phosphorylated hydroxy amino acid derivatives 2a-c, which are purified by crystallization (2a, from MeOH/H20) or column chromatography (2b and 2c, eluent pet. ether 40-60/ether 3/2 to 1/4, by vol.).
- 23 Compounts 2-5 were characterized by 13C and 31P NMR ( 200 MHz, Solvent CDCI3 for 2 and 3, CD3OD for 4 and 5, shifts in ppm, relative to TMS or 85% H3PO4, In parentheses: Jp-c in Hz): 2a: 13C: 28.1, 52.6, 53.9(7.3), 67.5(4.4), 68.6(5.9), 128.7, 129.2, 133.8(7.3), 134.5, 169.4; 31P: -0.63; 2b: 13C: 18.3, 28.0, 52.3, 57.7(7.3), 63.4(4.4), 75.6(5.9), 80.1, 128.6, 129.0, 133.9(7.3), 134.3, 155.5, 170.0; 31P: -1.39; 2c: 13C:28.1, 37.5, 52.1, 54.2, 69.1(5.9), 79.9, 119.9(4.4), 128.7, 129.3, 130.5, 133.1, 133.7(5.9), 134.5, 149.1(7.3), 155.4, 171.9; 31P: -5.80; 3a: 13C: 28.7, 52.9, 55.0(5.9), 65.7, 80.7, 157.9, 172.7; 31P: 2.55; 3b: 19.4, 28.7, 50.3, 60.3(5.9), 72.3(4.4), 80.7, 158.2, 173.1; 31P: 1.63; 3c: 13C: 28.6, 37.8, 52.6, 56.6, 80.6, 121.3(4.4), 130.8, 132.4, 153.7(5.9), 174.2; 31P: -2.12; 4a:13C: 18.3, 28.2, 50.1, 52.6(7.3), 67.0(4.4), 68.8(4.4), 79.9, 128.8, 129.3, 133.8(7.3), 134.6, 168.9, 172.8; 31P: -0.21; 4b: 13C: 17.9, 18.1, 28.1, 49.9, 52.4, 56.0(7.3), 68.4(4.4), 75.5(5.9), 79.8, 128.5, 129.1(2.9), 133.9(7.3), 134.3, 155.2, 169.3, 173.1; 31P: -1.39; 4c: 13C: 18.0, 28.1, 37.0, 52.1, 53.0, 69.0(4.4), 80.0, 119.8(4.4), 128.6, 129.2, 130.4, 133.0, 133.6(5.9),134.4, 149.2(7.3), 155.2, 171.3, 172.3; 31P: -5.83; 5a: 13C: 18.7, 28.7, 51.5 52.9, 54.7(7.3), 65.5(4.4), 80.6, 157.5, 171.6, 175.8; 31P: 2.67; 5b: 13C: 18.5, 19.3, 28.7, 51.5, 53.0, 59.0, 72.1, 80.6, 157.3, 172.0, 176.2; 31P: 1.89; 5c: 13C 18.4, 28.7, 37.6, 52.7, 55.0, 80.6, 121.3(2.9), 130.8, 131.9, 153.8(5.9), 157.3, 173.2, 175.8; 31P: -2.05; 24. If the hydrogenation is carried out in a unbuffered reaction mixture, the deprotected phosphate causes cleavage of the Boc-group.
- 25 M. Meldal and J. W. Kindtier Acta Chim. Scand. B40, 235 (1986); M. Meldal Acta Chim. Scand., B40, 242 (1986); M. Meldal, Acta Chim. Scand., B40, 250 (1986).
- 26 e.g. the preparation of myo-inositol phosphates see e.g.: K-L Yu, and B. Fraser-Reid, Tetrahedron Lett. 29, 979 (1988).
