Use of 2,2′-dithiobis(5-nitropyridine) for the heterodimerization of cysteine containing peptides. Introduction of the 5-nitro-2-pyridinesulfenyl group

Tetrahedron Letters

Volumn 37, Issue 9, 1996, Pages 1347-1350

  Rabanal, Francesc ^a   DeGrado, William F ^a   Dutton, P Leslie ^a  

^a UNIVERSITY OF PENNSYLVANIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; METHODOLOGY; PEPTIDE SYNTHESIS;

EID: 0029872844     PISSN: 00404039     EISSN: None     Source Type: Journal    
DOI: 10.1016/0040-4039(96)00019-6     Document Type: Article

References (23)

1. Abbreviations used for amino acids follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J. Biol. Chem. 1972, 247, 977-983. Single letter code for amino acids used in this article are: C, cysteine; E glutamic acid; F, phenylalanine; G, glycine; H, histidine; K, lysine; L, leucine; R, arginine; S, serine; W, tryptophan. Other abbreviations are: Ac, acetyl; Boc, tert-butyloxycarbonyl; But, terf-butyl; Bzl, benzyl; DIPCDI, N,N′-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; DTNP, 2,2′-dithiobis(5-nitropyridine); Fmoc, 9-fluorenylmethyloxycarbonyl; HOBt, 1-hydroxy-benzotriazole; Nps, 2-nitrophenyl; Npys, 3-nitro-2-pyridinesulfenyl; Pmc, pentamethylchromansulfonyl; pNpys, 5-nitro-2-pyridinesulfenyl; PyrS, 2-pyridinesulfenyl; Trt, trityl or triphenylmethyl;
2. For a recent review in formation of disulfide bonds in synthetic peptides and proteins see: Andreu, D.; Albericio, F.; Sole, N. A.; Munson, M. C.; Ferrer, M.; Barany, G. Methods in Molecular Biology: Peptide Synthesis Protocols, Vol. 35; Pennington, M. W.; Dunn, B. M., Eds; Humana Press Inc.; Totowa, NJ, 1994; pp 91-169.
3. Bernatowicz, M. S.; Matsueda, R.; Matsueda, G. R. Int, J. Peptide Protein Res. 1986, 28, 107-112; Matsueda, R.; Kimura, T. ; Kaiser, E. T. ; Matsueda, G. R. Chem Lett 1978, 6, 737-740; Ruiz-Gayo, M.; Albericio, F.; Pons, M.; Royo, M.; Pedroso, E.; Giralt, E. Tetrahedron Lett. 1988, 29, 3845-3848; Simmonds, R. G.; Tupper, D. E.; Harris, J. R. Int. J. Peptide Protein Res. 1994, 43, 363-366
4. Bernatowicz, M. S.; Matsueda, R.; Matsueda, G. R. Int, J. Peptide Protein Res. 1986, 28, 107-112; Matsueda, R.; Kimura, T. ; Kaiser, E. T. ; Matsueda, G. R. Chem Lett 1978, 6, 737-740; Ruiz-Gayo, M.; Albericio, F.; Pons, M.; Royo, M.; Pedroso, E.; Giralt, E. Tetrahedron Lett. 1988, 29, 3845-3848; Simmonds, R. G.; Tupper, D. E.; Harris, J. R. Int. J. Peptide Protein Res. 1994, 43, 363-366
5. Bernatowicz, M. S.; Matsueda, R.; Matsueda, G. R. Int, J. Peptide Protein Res. 1986, 28, 107-112; Matsueda, R.; Kimura, T. ; Kaiser, E. T. ; Matsueda, G. R. Chem Lett 1978, 6, 737-740; Ruiz-Gayo, M.; Albericio, F.; Pons, M.; Royo, M.; Pedroso, E.; Giralt, E. Tetrahedron Lett. 1988, 29, 3845-3848; Simmonds, R. G.; Tupper, D. E.; Harris, J. R. Int. J. Peptide Protein Res. 1994, 43, 363-366
6. Bernatowicz, M. S.; Matsueda, R.; Matsueda, G. R. Int, J. Peptide Protein Res. 1986, 28, 107-112; Matsueda, R.; Kimura, T. ; Kaiser, E. T. ; Matsueda, G. R. Chem Lett 1978, 6, 737-740; Ruiz-Gayo, M.; Albericio, F.; Pons, M.; Royo, M.; Pedroso, E.; Giralt, E. Tetrahedron Lett. 1988, 29, 3845-3848; Simmonds, R. G.; Tupper, D. E.; Harris, J. R. Int. J. Peptide Protein Res. 1994, 43, 363-366
7. Stewart J. M.; Young, J. D. Solid Phase Peptide Synthesis; W. H. Freeman and Co.; San Francisco, 1969; p. 16.
8. Castell J. V.; Tun-Kyi, A. Helv. Chim. Acta 1979, 62, 2507-2510; Akaji, K.; Fujino,K.; Tatsumi, T. ; Kiso, Y. J. Am. Chem. Soc. 1993, 115, 11384-11392.
9. Castell J. V.; Tun-Kyi, A. Helv. Chim. Acta 1979, 62, 2507-2510; Akaji, K.; Fujino,K.; Tatsumi, T. ; Kiso, Y. J. Am. Chem. Soc. 1993, 115, 11384-11392.
10. 2,2′-dithiobis(5-nitropyridine) is a reagent generally used for the selective detection of thiols. See for instance: Grassetti, D. R.; Murray, D. R. J. Chromatogr. 1969, 41, 121-123. However, during the preparation of this manuscript, a search on the uses of this reagent revealed than it was actually used in one case as a reagent for unsymmetrical formation in the preparation of polymer supports for the synthesis of sulfhydryl containing oligonucleotides: Gupta, K. C.; Sharma, P.; Kumar, P.; Sathyanarayana, S. Nucleic. Acids. Res. 1991, 19, 3019-25.
11. 2,2′-dithiobis(5-nitropyridine) is a reagent generally used for the selective detection of thiols. See for instance: Grassetti, D. R.; Murray, D. R. J. Chromatogr. 1969, 41, 121-123. However, during the preparation of this manuscript, a search on the uses of this reagent revealed than it was actually used in one case as a reagent for unsymmetrical formation in the preparation of polymer supports for the synthesis of sulfhydryl containing oligonucleotides: Gupta, K. C.; Sharma, P.; Kumar, P.; Sathyanarayana, S. Nucleic. Acids. Res. 1991, 19, 3019-25.
12. Ponsati, B.; Ruiz-Gayo, M.; Giralt, E.; Albericio, F.; Andreu, D. J. Am. Chem. Soc. 1990, 112, 5345-5347.
13. The synthesis of this heterodimer is given as a example among the several successfully performed in our laboratory using this procedure. The de novo design of these peptides is discussed extensively in the following references of our groups: Robertson, D. E.; Farid, R. S.; Moser, C.C.; Urbauer, J. F.; Mulholland, S. E.; Pidikiti, R. ; Lear, J. D.; Wand, A. J.; DeGrado, W. F.; Dutton, P. L. Nature 1994, 368, 425-432; Choma, C. T.; Lear, J. D.; Nelson, M. J.; Dutton, P. L.; Robertson, D. E.; DeGrado, W. F. J. Am. Chem. Soc. 1994, 116, 856-865; Rabanal, F.; DeGrado, W. F.; Dutton, P. L. J. Am. Chem. Soc. 1995, in press.
14. The synthesis of this heterodimer is given as a example among the several successfully performed in our laboratory using this procedure. The de novo design of these peptides is discussed extensively in the following references of our groups: Robertson, D. E.; Farid, R. S.; Moser, C.C.; Urbauer, J. F.; Mulholland, S. E.; Pidikiti, R. ; Lear, J. D.; Wand, A. J.; DeGrado, W. F.; Dutton, P. L. Nature 1994, 368, 425-432; Choma, C. T.; Lear, J. D.; Nelson, M. J.; Dutton, P. L.; Robertson, D. E.; DeGrado, W. F. J. Am. Chem. Soc. 1994, 116, 856-865; Rabanal, F.; DeGrado, W. F.; Dutton, P. L. J. Am. Chem. Soc. 1995, in press.
15. The synthesis of this heterodimer is given as a example among the several successfully performed in our laboratory using this procedure. The de novo design of these peptides is discussed extensively in the following references of our groups: Robertson, D. E.; Farid, R. S.; Moser, C.C.; Urbauer, J. F.; Mulholland, S. E.; Pidikiti, R. ; Lear, J. D.; Wand, A. J.; DeGrado, W. F.; Dutton, P. L. Nature 1994, 368, 425-432; Choma, C. T.; Lear, J. D.; Nelson, M. J.; Dutton, P. L.; Robertson, D. E.; DeGrado, W. F. J. Am. Chem. Soc. 1994, 116, 856-865; Rabanal, F.; DeGrado, W. F.; Dutton, P. L. J. Am. Chem. Soc. 1995, in press.
16. The amino acids were coupled as their pentafluorophenyl esters in the presence of HOBt. Protection was as follows: Trt for Cys, Pmc for Arg, Boc for Lys and His, t-But ether for Ser and f-But ester for GIu.
17. Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.; Masada, R. I.; Hudson, D.; Barany, G. J.Org. Chem. 1990, 55, 3730-3743.
18. Both peptides were cleaved with TFA:thioanisole:anisole:ethanedithiol (90:5:3:2) for 2 hours yielding crudes with the corresponding right product as a major component (about 85-90% according to the integrated area of the HPLC traces). Both peptides were purified to homogeneity by preparative reverse phase HPLC in acetonitrile-water-0.1% TFA eluent gradients and characterized by analytical HPLC, UV-spectroscopy (indole absorption) and laser-desorption mass spectrometry.
19. We attempted to prepare des-acetyl H10H24 with an Npys group on its Cys residue by coupling Boc-Cys(Npys)-OH at the last coupling step (DIPCDI mediated coupling, 5 eq., 1 h). However, the use of the PAL handle and the presence of both a tryptophan residue and a Pmc protected arginine in the sequence require good scavenging such as that provide by thiols at the final acidolytic treatment [see for instance: Choi H.; Adrich, J. V. Int. J. Peptide Protein Res. 1994, 42, 58-63; Sieber, P. Tetrahedron Lett. 1987, 28, 6147-6150]. The lability of Npys to thiols prevents their use in the cleavage mixture. Therefore, the thiol-based cleavage mixture was substituted for a silane-based one. The corresponding Boc-Cys(Npys)-H10H24[2-31] was then cleaved with TFA:triisopropylsilane:phenol:water (88:2:5:5) [Sole N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399-5403]. This procedure provided the target peptide in approximately 37% yield in a mixture difficult to purify. The byproducts were not analyzed.
20. We attempted to prepare des-acetyl H10H24 with an Npys group on its Cys residue by coupling Boc-Cys(Npys)-OH at the last coupling step (DIPCDI mediated coupling, 5 eq., 1 h). However, the use of the PAL handle and the presence of both a tryptophan residue and a Pmc protected arginine in the sequence require good scavenging such as that provide by thiols at the final acidolytic treatment [see for instance: Choi H.; Adrich, J. V. Int. J. Peptide Protein Res. 1994, 42, 58-63; Sieber, P. Tetrahedron Lett. 1987, 28, 6147-6150]. The lability of Npys to thiols prevents their use in the cleavage mixture. Therefore, the thiol-based cleavage mixture was substituted for a silane-based one. The corresponding Boc-Cys(Npys)-H10H24[2-31] was then cleaved with TFA:triisopropylsilane:phenol:water (88:2:5:5) [Sole N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399-5403]. This procedure provided the target peptide in approximately 37% yield in a mixture difficult to purify. The byproducts were not analyzed.
21. We attempted to prepare des-acetyl H10H24 with an Npys group on its Cys residue by coupling Boc-Cys(Npys)-OH at the last coupling step (DIPCDI mediated coupling, 5 eq., 1 h). However, the use of the PAL handle and the presence of both a tryptophan residue and a Pmc protected arginine in the sequence require good scavenging such as that provide by thiols at the final acidolytic treatment [see for instance: Choi H.; Adrich, J. V. Int. J. Peptide Protein Res. 1994, 42, 58-63; Sieber, P. Tetrahedron Lett. 1987, 28, 6147-6150]. The lability of Npys to thiols prevents their use in the cleavage mixture. Therefore, the thiol-based cleavage mixture was substituted for a silane-based one. The corresponding Boc-Cys(Npys)-H10H24[2-31] was then cleaved with TFA:triisopropylsilane:phenol:water (88:2:5:5) [Sole N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399-5403]. This procedure provided the target peptide in approximately 37% yield in a mixture difficult to purify. The byproducts were not analyzed.
22. DTNP (Aldrich or Sigma, 3-5 equiv.) was dissolved in the minimum amount of acetic acid:water (3:1, v/v) and the thiol containing peptide (1 equiv.) was added in one portion with vigorous stirring. After the reaction was complete (4-6 h), acetic acid was added to reach a proportion of 9 to 1 and the solvent was eliminated by lyophilization. The solid was extracted with aqueous 0.1% TFA, sonicated, centrifuged, and the supernatant freeze-dried. The pNpys-peptide still contained traces of DTNP and 5-nitro-2-pyridinethiol that were finally fully removed by washing (5×) the lyophilized powder with diethyl ethermethylene chloride (7:3, v/v).
23. To an ammonium acetate buffered solution (1M, pH 3.5-6.5, bubbled with argon) of the Cys(pNpys) containing peptide (1 equiv.), the corresponding free thiol peptide (1 equiv.) was added in one portion either solid or predissolved in aqueous TFA (0.1%, bubbled with argon). The reaction was easily monitored by the intense yellow coloration due to the release of 5-nitro-2-pyridinethiol. HPLC monitoring of the reaction helps in adjusting the actual amounts of peptide added and therefore optimizing yields.