Novel solid-phase reagents for facile formation of intramolecular disulfide bridges in peptides under mild conditions

Journal of the American Chemical Society

Volumn 120, Issue 29, 1998, Pages 7226-7238

  Annis, Ioana ^a   Chen, Lin ^a   Barany, George ^a  

^a University of Minnesota   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

APAMIN; CONOTOXIN; DISULFIDE; OXYTOCIN; PEPTIDE; SOMATOSTATIN;

ARTICLE; COVALENT BOND; DERIVATIZATION; DIMERIZATION; OLIGOMERIZATION; OXIDATION; PROTEIN STRUCTURE;

EID: 0032578173     PISSN: 00027863     EISSN: None     Source Type: Journal    
DOI: 10.1021/ja981111p     Document Type: Article

References (81)

1. Abbreviations and conventions are defined at first use in the text and are summarized in full in the Supporting Information. Abbreviations used repeatedly for this work include: CPG, controlled pore glass; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); El, 5-thio-2-nitrobenzoyl; PAC, p-alkoxybenzyl ester (Peptide Acid Linker); PAL, 5-[[(4-amino)methyl]-3,5-dimethoxyphenoxy]valeric acid handle (Peptide Amide Linker); PEG-PS, poly(ethylene glycol)-polystyrene (graft resin support); TNB, 5-thio-2-nitrobenzoic acid; and Xan, 9H-xanthen-9-yl.
2. Portions of this work were reported in preliminary form: (a) Barany, G.; Chen, L. In Innovation and Perspectives in Solid-Phase Synthesis & Combinatorial Libraries, 1996, Collected Papers, Fourth International Symposium, Edinburgh, Scotland, September 12-16, 1995; Epton, R., Ed.; Mayflower Scientific: Kingswinford, U.K., 1997; pp 181-186. (b) Annis, I.; Barany, G. In Peptides - Chemistry, Structure and Biology: Proceedings of the Fifteenth American Peptide Symposium, Nashville, TN, June 14-19, 1997; Tam, J. P., Kaumaya, P. T. P., Eds.; Kluwer: Dordercht, The Netherlands, 1998, in press. (c) Annis, I.; Chen, L.; Barany, G. In Innovation and Perspectives in Solid-Phase Synthesis & Combinatorial Libraries, 1998, Collected Papers, Fifth International Symposium, London, U.K., September 2-6, 1997; Epton, R., Ed.; Mayflower Scientific Ltd.: Kingswinford, U.K., 1998, in press.
3. Portions of this work were reported in preliminary form: (a) Barany, G.; Chen, L. In Innovation and Perspectives in Solid-Phase Synthesis & Combinatorial Libraries, 1996, Collected Papers, Fourth International Symposium, Edinburgh, Scotland, September 12-16, 1995; Epton, R., Ed.; Mayflower Scientific: Kingswinford, U.K., 1997; pp 181-186. (b) Annis, I.; Barany, G. In Peptides - Chemistry, Structure and Biology: Proceedings of the Fifteenth American Peptide Symposium, Nashville, TN, June 14-19, 1997; Tam, J. P., Kaumaya, P. T. P., Eds.; Kluwer: Dordercht, The Netherlands, 1998, in press. (c) Annis, I.; Chen, L.; Barany, G. In Innovation and Perspectives in Solid-Phase Synthesis & Combinatorial Libraries, 1998, Collected Papers, Fifth International Symposium, London, U.K., September 2-6, 1997; Epton, R., Ed.; Mayflower Scientific Ltd.: Kingswinford, U.K., 1998, in press.
4. Portions of this work were reported in preliminary form: (a) Barany, G.; Chen, L. In Innovation and Perspectives in Solid-Phase Synthesis & Combinatorial Libraries, 1996, Collected Papers, Fourth International Symposium, Edinburgh, Scotland, September 12-16, 1995; Epton, R., Ed.; Mayflower Scientific: Kingswinford, U.K., 1997; pp 181-186. (b) Annis, I.; Barany, G. In Peptides - Chemistry, Structure and Biology: Proceedings of the Fifteenth American Peptide Symposium, Nashville, TN, June 14-19, 1997; Tam, J. P., Kaumaya, P. T. P., Eds.; Kluwer: Dordercht, The Netherlands, 1998, in press. (c) Annis, I.; Chen, L.; Barany, G. In Innovation and Perspectives in Solid-Phase Synthesis & Combinatorial Libraries, 1998, Collected Papers, Fifth International Symposium, London, U.K., September 2-6, 1997; Epton, R., Ed.; Mayflower Scientific Ltd.: Kingswinford, U.K., 1998, in press.
5. For reviews, see: (a) Richardson, J. S. Adv. Prot. Chem. 1981, 34, 167-339. (b) Thornton, J. M. J. Mol. Biol. 1981, 151, 261-287. (c) Creighton, T. E. BioEssays 1988, 8, 57-63. (d) Raines, R. T. Nat. Struct. Biol. 1997, 4, 424-427.
6. For reviews, see: (a) Richardson, J. S. Adv. Prot. Chem. 1981, 34, 167-339. (b) Thornton, J. M. J. Mol. Biol. 1981, 151, 261-287. (c) Creighton, T. E. BioEssays 1988, 8, 57-63. (d) Raines, R. T. Nat. Struct. Biol. 1997, 4, 424-427.
7. For reviews, see: (a) Richardson, J. S. Adv. Prot. Chem. 1981, 34, 167-339. (b) Thornton, J. M. J. Mol. Biol. 1981, 151, 261-287. (c) Creighton, T. E. BioEssays 1988, 8, 57-63. (d) Raines, R. T. Nat. Struct. Biol. 1997, 4, 424-427.
8. For reviews, see: (a) Richardson, J. S. Adv. Prot. Chem. 1981, 34, 167-339. (b) Thornton, J. M. J. Mol. Biol. 1981, 151, 261-287. (c) Creighton, T. E. BioEssays 1988, 8, 57-63. (d) Raines, R. T. Nat. Struct. Biol. 1997, 4, 424-427.
9. For reviews, see: (a) König, W.; Geiger, R. In Perspectives in Peptide Chemistry; Eberle, A., Geiger, R., Wieland, T., Eds.; S. Karger: Basel, 1981; pp 31-44. (b) Büllesbach, E. E. Kontakte (Darmstadt) 1992, 1, 21-29. (c) Andreu, D.; Albericio, F.; Solé, N. A.; Munson, M. C.; Ferrer, M.; Barany, G. In Methods in Molecular Biology, Vol. 35, Peptide Synthesis Protocols; Pennington, M. W., Dunn, B. M., Eds; Humana: Totowa, NJ, 1994; pp 91-169. (d) Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-Böhner, S.; Siedler, F. Biopolymers 1996, 40, 207-234. (e) Annis, I.; Hargittai, B.; Barany, G. Methods Enzymol. 1997, 289, 198-221.
10. For reviews, see: (a) König, W.; Geiger, R. In Perspectives in Peptide Chemistry; Eberle, A., Geiger, R., Wieland, T., Eds.; S. Karger: Basel, 1981; pp 31-44. (b) Büllesbach, E. E. Kontakte (Darmstadt) 1992, 1, 21-29. (c) Andreu, D.; Albericio, F.; Solé, N. A.; Munson, M. C.; Ferrer, M.; Barany, G. In Methods in Molecular Biology, Vol. 35, Peptide Synthesis Protocols; Pennington, M. W., Dunn, B. M., Eds; Humana: Totowa, NJ, 1994; pp 91-169. (d) Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-Böhner, S.; Siedler, F. Biopolymers 1996, 40, 207-234. (e) Annis, I.; Hargittai, B.; Barany, G. Methods Enzymol. 1997, 289, 198-221.
11. For reviews, see: (a) König, W.; Geiger, R. In Perspectives in Peptide Chemistry; Eberle, A., Geiger, R., Wieland, T., Eds.; S. Karger: Basel, 1981; pp 31-44. (b) Büllesbach, E. E. Kontakte (Darmstadt) 1992, 1, 21-29. (c) Andreu, D.; Albericio, F.; Solé, N. A.; Munson, M. C.; Ferrer, M.; Barany, G. In Methods in Molecular Biology, Vol. 35, Peptide Synthesis Protocols; Pennington, M. W., Dunn, B. M., Eds; Humana: Totowa, NJ, 1994; pp 91-169. (d) Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-Böhner, S.; Siedler, F. Biopolymers 1996, 40, 207-234. (e) Annis, I.; Hargittai, B.; Barany, G. Methods Enzymol. 1997, 289, 198-221.
12. For reviews, see: (a) König, W.; Geiger, R. In Perspectives in Peptide Chemistry; Eberle, A., Geiger, R., Wieland, T., Eds.; S. Karger: Basel, 1981; pp 31-44. (b) Büllesbach, E. E. Kontakte (Darmstadt) 1992, 1, 21-29. (c) Andreu, D.; Albericio, F.; Solé, N. A.; Munson, M. C.; Ferrer, M.; Barany, G. In Methods in Molecular Biology, Vol. 35, Peptide Synthesis Protocols; Pennington, M. W., Dunn, B. M., Eds; Humana: Totowa, NJ, 1994; pp 91-169. (d) Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-Böhner, S.; Siedler, F. Biopolymers 1996, 40, 207-234. (e) Annis, I.; Hargittai, B.; Barany, G. Methods Enzymol. 1997, 289, 198-221.
13. For reviews, see: (a) König, W.; Geiger, R. In Perspectives in Peptide Chemistry; Eberle, A., Geiger, R., Wieland, T., Eds.; S. Karger: Basel, 1981; pp 31-44. (b) Büllesbach, E. E. Kontakte (Darmstadt) 1992, 1, 21-29. (c) Andreu, D.; Albericio, F.; Solé, N. A.; Munson, M. C.; Ferrer, M.; Barany, G. In Methods in Molecular Biology, Vol. 35, Peptide Synthesis Protocols; Pennington, M. W., Dunn, B. M., Eds; Humana: Totowa, NJ, 1994; pp 91-169. (d) Moroder, L.; Besse, D.; Musiol, H.-J.; Rudolph-Böhner, S.; Siedler, F. Biopolymers 1996, 40, 207-234. (e) Annis, I.; Hargittai, B.; Barany, G. Methods Enzymol. 1997, 289, 198-221.
14. For monographs and reviews, see: (a) Biochemical Aspects of Reactions on Solid Supports; Stark, G. R., Ed.; Academic: New York, 1971. (b) Polymer-Supported Reactions in Organic Synthesis; Hodge, P., Sherrigton, D. C., Eds.; John Wiley & Sons: New York, 1980. (c) Merrifield, R. B. Science 1986, 232, 341-347. (d) Früchtel, J. S.; Jung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17-42. (e) Barany, G.; Kempe, M. In A Practical Guide to Combinatorial Chemistry; Czarnik, A. W., DeWitt, S. H., Eds.; American Chemical Society: Washington, DC, 1997; pp 51-97.
15. For monographs and reviews, see: (a) Biochemical Aspects of Reactions on Solid Supports; Stark, G. R., Ed.; Academic: New York, 1971. (b) Polymer-Supported Reactions in Organic Synthesis; Hodge, P., Sherrigton, D. C., Eds.; John Wiley & Sons: New York, 1980. (c) Merrifield, R. B. Science 1986, 232, 341-347. (d) Früchtel, J. S.; Jung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17-42. (e) Barany, G.; Kempe, M. In A Practical Guide to Combinatorial Chemistry; Czarnik, A. W., DeWitt, S. H., Eds.; American Chemical Society: Washington, DC, 1997; pp 51-97.
16. For monographs and reviews, see: (a) Biochemical Aspects of Reactions on Solid Supports; Stark, G. R., Ed.; Academic: New York, 1971. (b) Polymer-Supported Reactions in Organic Synthesis; Hodge, P., Sherrigton, D. C., Eds.; John Wiley & Sons: New York, 1980. (c) Merrifield, R. B. Science 1986, 232, 341-347. (d) Früchtel, J. S.; Jung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17-42. (e) Barany, G.; Kempe, M. In A Practical Guide to Combinatorial Chemistry; Czarnik, A. W., DeWitt, S. H., Eds.; American Chemical Society: Washington, DC, 1997; pp 51-97.
17. For monographs and reviews, see: (a) Biochemical Aspects of Reactions on Solid Supports; Stark, G. R., Ed.; Academic: New York, 1971. (b) Polymer-Supported Reactions in Organic Synthesis; Hodge, P., Sherrigton, D. C., Eds.; John Wiley & Sons: New York, 1980. (c) Merrifield, R. B. Science 1986, 232, 341-347. (d) Früchtel, J. S.; Jung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17-42. (e) Barany, G.; Kempe, M. In A Practical Guide to Combinatorial Chemistry; Czarnik, A. W., DeWitt, S. H., Eds.; American Chemical Society: Washington, DC, 1997; pp 51-97.
18. For monographs and reviews, see: (a) Biochemical Aspects of Reactions on Solid Supports; Stark, G. R., Ed.; Academic: New York, 1971. (b) Polymer-Supported Reactions in Organic Synthesis; Hodge, P., Sherrigton, D. C., Eds.; John Wiley & Sons: New York, 1980. (c) Merrifield, R. B. Science 1986, 232, 341-347. (d) Früchtel, J. S.; Jung, G. Angew. Chem., Int. Ed. Engl. 1996, 35, 17-42. (e) Barany, G.; Kempe, M. In A Practical Guide to Combinatorial Chemistry; Czarnik, A. W., DeWitt, S. H., Eds.; American Chemical Society: Washington, DC, 1997; pp 51-97.
19. Initial efforts in this regard are reported in refs 2a-c and Chen, L.; Barany, G. Lett. Pept. Sci. 1996, 3, 283-292.
20. We are aware of two additional, possibly related, solid-phase approaches to facilitate disulfide formation. In one approach, poly(thiol) snake toxin substrates were passed over a commercially available agarose glutathione-2-pyridyl disulfide support, and the oxidized products were eluted by gradients of reduced and oxidized glutathione at pH 8.7. See: (a) Smith, D. C.; Hider, R. C. Biophys. Chem. 1988, 31, 21-28. In the second approach, a commercially available methacrylate resin, derivatized in an unspecified manner, gave a support termed Ekathiox. This was incubated with solutions of dihydrooxytocin and dihydrocalcitonin in dilute acetic acid and said to promote rapid, high-yield transformations to the corresponding intramolecular disulfides. See: (b) Clark, B. R.; Pai, M. Presented at the 14th American Peptide Symposium, Columbus, OH, June 18-23, 1995; Poster 90. (c) Clark, B. R.; Pai, M. Chem. Abstr. (Selects Plus: Amino Acids, Peptides and Proteins) 1996, 14, 23, 125: 12365k.
21. We are aware of two additional, possibly related, solid-phase approaches to facilitate disulfide formation. In one approach, poly(thiol) snake toxin substrates were passed over a commercially available agarose glutathione-2-pyridyl disulfide support, and the oxidized products were eluted by gradients of reduced and oxidized glutathione at pH 8.7. See: (a) Smith, D. C.; Hider, R. C. Biophys. Chem. 1988, 31, 21-28. In the second approach, a commercially available methacrylate resin, derivatized in an unspecified manner, gave a support termed Ekathiox. This was incubated with solutions of dihydrooxytocin and dihydrocalcitonin in dilute acetic acid and said to promote rapid, high-yield transformations to the corresponding intramolecular disulfides. See: (b) Clark, B. R.; Pai, M. Presented at the 14th American Peptide Symposium, Columbus, OH, June 18-23, 1995; Poster 90. (c) Clark, B. R.; Pai, M. Chem. Abstr. (Selects Plus: Amino Acids, Peptides and Proteins) 1996, 14, 23, 125: 12365k.
22. We are aware of two additional, possibly related, solid-phase approaches to facilitate disulfide formation. In one approach, poly(thiol) snake toxin substrates were passed over a commercially available agarose glutathione-2-pyridyl disulfide support, and the oxidized products were eluted by gradients of reduced and oxidized glutathione at pH 8.7. See: (a) Smith, D. C.; Hider, R. C. Biophys. Chem. 1988, 31, 21-28. In the second approach, a commercially available methacrylate resin, derivatized in an unspecified manner, gave a support termed Ekathiox. This was incubated with solutions of dihydrooxytocin and dihydrocalcitonin in dilute acetic acid and said to promote rapid, high-yield transformations to the corresponding intramolecular disulfides. See: (b) Clark, B. R.; Pai, M. Presented at the 14th American Peptide Symposium, Columbus, OH, June 18-23, 1995; Poster 90. (c) Clark, B. R.; Pai, M. Chem. Abstr. (Selects Plus: Amino Acids, Peptides and Proteins) 1996, 14, 23, 125: 12365k.
23. Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70-77.
24. (a) Habeeb, A. F. S. A. Methods Enzymol. 1972, 25, 457-464.
25. (b) Riddles, P. W.; Blakeley, R. L.; Zerner, B. Methods Enzymol. 1983, 91, 49-61.
26. This term, which implies the kinetic basis for minimizing intersite reactions, was introduced along with experimental documentation for a benzyne system by (a) Mazur, S.; Jayalekshmy, P. J. Am. Chem. Soc. 1979, 101, 677-683. See also: (b) Barany, G.; Merrifield, R. B. In The Peptides - Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic: New York, 1979; Vol. 2, pp 1-284, especially pp 27-29.
27. This term, which implies the kinetic basis for minimizing intersite reactions, was introduced along with experimental documentation for a benzyne system by (a) Mazur, S.; Jayalekshmy, P. J. Am. Chem. Soc. 1979, 101, 677-683. See also: (b) Barany, G.; Merrifield, R. B. In The Peptides - Analysis, Synthesis, Biology; Gross, E., Meienhofer, J., Eds.; Academic: New York, 1979; Vol. 2, pp 1-284, especially pp 27-29.
28. The approach taken in the present study is reciprocal to other work in which the peptide substrate for disulfide formation is attached to the support, as reviewed in refs 4c and 4e and also covered in (a) Albericio, F.; Hammer, R. P.; García-Echeverría, C.; Molins, M. A.; Chang, J. L.; Munson, M. C.; Pons, M.; Giralt, E.; Barany, G. Int. J. Pept. Protein Res. 1991, 37, 402-413. (b) Munson, M. C.; Barany, G. J. Am. Chem. Soc. 1993, 115, 10203-10216, and references therein. Due to pseudodilution, intramolecular cyclization is favored, but intermolecular processes do compete. Also, when intramolecular pathways are not available to the resin-bound peptide substrates, conditions can often be found under which intermolecular disulfide formation occurs in reasonably high yield, as shown in (c) Bhargava, K. K.; Sarin, V. K.; Le Trang, N.; Cerami, A.; Merrifield, R. B. J. Am. Chem. Soc. 1983, 105, 3247-3251. (d) Munson, M. C.; Lebl, M.; Slaninovà, J.; Barany, G. Pept. Res. 1993, 6, 155-159.
29. The approach taken in the present study is reciprocal to other work in which the peptide substrate for disulfide formation is attached to the support, as reviewed in refs 4c and 4e and also covered in (a) Albericio, F.; Hammer, R. P.; García-Echeverría, C.; Molins, M. A.; Chang, J. L.; Munson, M. C.; Pons, M.; Giralt, E.; Barany, G. Int. J. Pept. Protein Res. 1991, 37, 402-413. (b) Munson, M. C.; Barany, G. J. Am. Chem. Soc. 1993, 115, 10203-10216, and references therein. Due to pseudodilution, intramolecular cyclization is favored, but intermolecular processes do compete. Also, when intramolecular pathways are not available to the resin-bound peptide substrates, conditions can often be found under which intermolecular disulfide formation occurs in reasonably high yield, as shown in (c) Bhargava, K. K.; Sarin, V. K.; Le Trang, N.; Cerami, A.; Merrifield, R. B. J. Am. Chem. Soc. 1983, 105, 3247-3251. (d) Munson, M. C.; Lebl, M.; Slaninovà, J.; Barany, G. Pept. Res. 1993, 6, 155-159.
30. The approach taken in the present study is reciprocal to other work in which the peptide substrate for disulfide formation is attached to the support, as reviewed in refs 4c and 4e and also covered in (a) Albericio, F.; Hammer, R. P.; García-Echeverría, C.; Molins, M. A.; Chang, J. L.; Munson, M. C.; Pons, M.; Giralt, E.; Barany, G. Int. J. Pept. Protein Res. 1991, 37, 402-413. (b) Munson, M. C.; Barany, G. J. Am. Chem. Soc. 1993, 115, 10203-10216, and references therein. Due to pseudodilution, intramolecular cyclization is favored, but intermolecular processes do compete. Also, when intramolecular pathways are not available to the resin-bound peptide substrates, conditions can often be found under which intermolecular disulfide formation occurs in reasonably high yield, as shown in (c) Bhargava, K. K.; Sarin, V. K.; Le Trang, N.; Cerami, A.; Merrifield, R. B. J. Am. Chem. Soc. 1983, 105, 3247-3251. (d) Munson, M. C.; Lebl, M.; Slaninovà, J.; Barany, G. Pept. Res. 1993, 6, 155-159.
31. The approach taken in the present study is reciprocal to other work in which the peptide substrate for disulfide formation is attached to the support, as reviewed in refs 4c and 4e and also covered in (a) Albericio, F.; Hammer, R. P.; García-Echeverría, C.; Molins, M. A.; Chang, J. L.; Munson, M. C.; Pons, M.; Giralt, E.; Barany, G. Int. J. Pept. Protein Res. 1991, 37, 402-413. (b) Munson, M. C.; Barany, G. J. Am. Chem. Soc. 1993, 115, 10203-10216, and references therein. Due to pseudodilution, intramolecular cyclization is favored, but intermolecular processes do compete. Also, when intramolecular pathways are not available to the resin-bound peptide substrates, conditions can often be found under which intermolecular disulfide formation occurs in reasonably high yield, as shown in (c) Bhargava, K. K.; Sarin, V. K.; Le Trang, N.; Cerami, A.; Merrifield, R. B. J. Am. Chem. Soc. 1983, 105, 3247-3251. (d) Munson, M. C.; Lebl, M.; Slaninovà, J.; Barany, G. Pept. Res. 1993, 6, 155-159.
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42. The first explicit application of DTNB to intramolecular disulfide formation was reported in ref 11a, although in hindsight, several aspects of the experiment were suboptimal and not all of the observed oxidation can be attributed to DTNB. Thus, a linear oxytocin sequence assembled by solid-phase peptide synthesis was selectively deblocked, and the resin-bound peptide dithiol was treated with DTNB (0.5 equiv) in "buffered" DMF, pH 7.5, for 1 h. Cleavage from the support revealed monomeric oxytocin in 65% absolute yield, along with a major byproduct, detected by HPLC, which was believed to contain TNB on the basis of UV absorbance at 220, 280, and 340 nm.
43. A preparative solution experiment used DTNB (10 equiv) to carry out disulfide cyclization at pH 6.8 on a peptide (2 mM) that was labile and sluggishly reactive under standard alkaline pH air-oxidation conditions. The desired product was isolated in 36% yield, and an extra HPLC peak was observed and postulated to be the peptide with both Cys thiol groups linked to TNB groups through disulfide bonds. See: Engebretsen, M.; Agner, E.; Sandosham, J.; Fischer, P. M. J. Peptide Res. 1997, 49, 341-346.
44. Our own preliminary studies (see Experimental Section) showed that solution oxidations with DTNB in the oxytocin, somatostatin, and conotoxin families provide, at best, 50-85% monomeric intramolecular disulfide, and identified significant experimental difficulties that limit the practical usefulness of the method.
45. (a) Barany, G.; Albericio, F.; Solé, N. A.; Griffin, G. W.; Kates, S. A.; Hudson, D. In Peptides 1992. Proceedings of the Twenty-Second European Peptide Symposium; Schneider, C. H., Eberle, A. N., Eds.; ESCOM Science: Leiden, 1993; pp 267-268.
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53. Several methods for activating Sephadex have been reported previously, but they give maximal loadings of 0.1-60 μmol/g. See: (a) Axén, R.; Porath, J.; Ernback, S. Nature 1967, 214, 1302-1304. (b) Parikh, I.; March, S.; Cuatrecasas. Methods Enzymol. 1975, 34, 77-102. (c) Brocklehurst, K.; Carlsson, J.; Kierstan, M. P. J.; Crook, E. M. Methods Enzymol. 1975, 34, 531-547. As this work was being readied for publication, a method for converting the commercially available HiTrap Sepharose derivative to a form with 0.2 mmol/g of amino functionalization was reported; see: (d) Tegge, W.; Frank, R. J. Peptide Res. 1997, 49, 355-362.
54. Several methods for activating Sephadex have been reported previously, but they give maximal loadings of 0.1-60 μmol/g. See: (a) Axén, R.; Porath, J.; Ernback, S. Nature 1967, 214, 1302-1304. (b) Parikh, I.; March, S.; Cuatrecasas. Methods Enzymol. 1975, 34, 77-102. (c) Brocklehurst, K.; Carlsson, J.; Kierstan, M. P. J.; Crook, E. M. Methods Enzymol. 1975, 34, 531-547. As this work was being readied for publication, a method for converting the commercially available HiTrap Sepharose derivative to a form with 0.2 mmol/g of amino functionalization was reported; see: (d) Tegge, W.; Frank, R. J. Peptide Res. 1997, 49, 355-362.
55. Several methods for activating Sephadex have been reported previously, but they give maximal loadings of 0.1-60 μmol/g. See: (a) Axén, R.; Porath, J.; Ernback, S. Nature 1967, 214, 1302-1304. (b) Parikh, I.; March, S.; Cuatrecasas. Methods Enzymol. 1975, 34, 77-102. (c) Brocklehurst, K.; Carlsson, J.; Kierstan, M. P. J.; Crook, E. M. Methods Enzymol. 1975, 34, 531-547. As this work was being readied for publication, a method for converting the commercially available HiTrap Sepharose derivative to a form with 0.2 mmol/g of amino functionalization was reported; see: (d) Tegge, W.; Frank, R. J. Peptide Res. 1997, 49, 355-362.
56. Several methods for activating Sephadex have been reported previously, but they give maximal loadings of 0.1-60 μmol/g. See: (a) Axén, R.; Porath, J.; Ernback, S. Nature 1967, 214, 1302-1304. (b) Parikh, I.; March, S.; Cuatrecasas. Methods Enzymol. 1975, 34, 77-102. (c) Brocklehurst, K.; Carlsson, J.; Kierstan, M. P. J.; Crook, E. M. Methods Enzymol. 1975, 34, 531-547. As this work was being readied for publication, a method for converting the commercially available HiTrap Sepharose derivative to a form with 0.2 mmol/g of amino functionalization was reported; see: (d) Tegge, W.; Frank, R. J. Peptide Res. 1997, 49, 355-362.
57. This compound has been prepared previously by a similar method: Beylin, V. G.; Goel, O. P. Org. Prep. Proced. Int. 1987, 19, 78-80.
58. Han, Y.; Barany, G. J. Org. Chem. 1997, 62, 3841-3848.
59. Munson, M. C.; García-Echeverría, C.; Albericio, F.; Barany, G. J. Org. Chem. 1992, 57, 3013-3018.
60. (a) Hiskey, R. G.; Mizoguchi, T.; Igeta, H. J. Org. Chem. 1966, 31, 1188-1192.
61. (b) Photaki, I.; Taylor-Papadimitriou, J.; Sakarellos, C.; Mazarakis, P.; Zervas, L. J. Chem. Soc., Chem. Commun. 1970, 2683-2687.
62. It is instructive to compare our approach to the work of Fridkin and collaborators, whose goal was to prepare solid-phase aromatic thiols. Both symmetrical and unsymmetrical carboxyl-containing disulfides were coupled, following which borohydride reduction gave the desired products. Information about the ratio of single and dual attachments at the disulfide stage was not reported. See: (a) Stern, M.; Warshawsky, A.; Fridkin, M. Int. J. Pept. Protein Res. 1981, 17, 531-538. (b) Stern, M.; Fridkin, M.; Warshawsky, A. J. Polym. Sci., Polym. Chem. Ed. 1982, 20, 1469-1487.
63. It is instructive to compare our approach to the work of Fridkin and collaborators, whose goal was to prepare solid-phase aromatic thiols. Both symmetrical and unsymmetrical carboxyl-containing disulfides were coupled, following which borohydride reduction gave the desired products. Information about the ratio of single and dual attachments at the disulfide stage was not reported. See: (a) Stern, M.; Warshawsky, A.; Fridkin, M. Int. J. Pept. Protein Res. 1981, 17, 531-538. (b) Stern, M.; Fridkin, M.; Warshawsky, A. J. Polym. Sci., Polym. Chem. Ed. 1982, 20, 1469-1487.
64. It is known (ref 23) that pairwise oxidation of S-Xan-protected thiols with iodine gives disulfides directly, raising the possibility to save a step in the chemistry of Scheme 4, right side. However, this conversion was ambiguous for ® = PEG-PS due to problems already discussed with iodine, and for ® = Sephadex G-15, the reaction was slow and difficult to carry out to completion. The residual (unoxidized) S-Xan-protected sites on the support led to a new problem at the next stage, when such supports were used in experiments to oxidize peptide substrates with two (or for that matter, one) thiol function(s). Thus, under the semiaqueous reaction conditions, the S-Xan group apparently transferred from the resin-bound aromatic thiol to the aliphatic peptide thiol.
65. (a) Albericio, F.; Barany, G. Int. J. Pept. Protein Res. 1987, 30, 206-216.
66. (b) Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera, L.; Masada, R. I.; Hudson, D.; Barany, G. J. Org. Chem. 1990, 55, 3730-3743.
67. Solé, N. A.; Barany, G. J. Org. Chem. 1992, 57, 5399-5403.
68. For reviews, see: (a) Barany, G.; Kneib-Cordonier, N.; Mullen, D. G. Int. J. Pept. Protein Res. 1987, 30, 705-739. (b) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214. (c) Fields, G. B.; Tian, Z.; Barany, G. In Synthetic Peptides: A User's Guide; Grant, G. A., Ed.; W. H. Freeman: New York, 1992; pp 77-183.
69. For reviews, see: (a) Barany, G.; Kneib-Cordonier, N.; Mullen, D. G. Int. J. Pept. Protein Res. 1987, 30, 705-739. (b) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214. (c) Fields, G. B.; Tian, Z.; Barany, G. In Synthetic Peptides: A User's Guide; Grant, G. A., Ed.; W. H. Freeman: New York, 1992; pp 77-183.
70. For reviews, see: (a) Barany, G.; Kneib-Cordonier, N.; Mullen, D. G. Int. J. Pept. Protein Res. 1987, 30, 705-739. (b) Fields, G. B.; Noble, R. L. Int. J. Pept. Protein Res. 1990, 35, 161-214. (c) Fields, G. B.; Tian, Z.; Barany, G. In Synthetic Peptides: A User's Guide; Grant, G. A., Ed.; W. H. Freeman: New York, 1992; pp 77-183.
71. The reduced linear precursors were 64-92% pure (<5% disulfide) upon cleavage from the support by appropriate TFA-scavanger mixtures and workup. For much of our work, these materials were used in oxidation reactions directly, that is, without any preliminary purification. Portions of both reduced and oxidized substrates were purified to homogeneity, quantified by amino acid analysis, and used to determine accurate HPLC response factors.
72. (a) du Vigneaud, V.; Ressler, C.; Swan, J. M.; Roberts, C. W.; Katsoyannis, P. G.; Gordon, S. J. Am. Chem. Soc. 1953, 75, 4879-4880.
73. (b) Live, D. H.; Agosta, W. C.; Cowburn, D. J. Org. Chem. 1977, 42, 3556-3561.
74. (c) Chen, L.; Zoulíková, I.; Slaninová, J.; Barany, G. J. Med. Chem. 1997, 40, 864-876, and references therein.
75. Rivier, J.; Kaiser, R.; Galyean, R. Biopolymers 1978, 17, 1927-1938, and references therein.
76. To the best of our knowledge, this is the first successful synthesis of apamin by an Fmoc strategy. For stepwise synthesis or segment condensation with Boc chemistry, see: (a) Van Rietschoten, J.; Granier, C.; Rochat, H.; Lissitzky, S.; Miranda, F. Eur. J. Biochem. 1975, 56, 35-40. (c) Sandberg, B. E. B.; Ragnarsson, U. Int. J. Pept. Protein Res. 1978, 11, 238-245. (d) Albericio, F.; Granier, C.; Labbé-Juillié, C.; Seagar, M.; Couraud, F.; Van Rietschoten, J. Tetrahedron 1984, 40, 4313-4326.
77. To the best of our knowledge, this is the first successful synthesis of apamin by an Fmoc strategy. For stepwise synthesis or segment condensation with Boc chemistry, see: (a) Van Rietschoten, J.; Granier, C.; Rochat, H.; Lissitzky, S.; Miranda, F. Eur. J. Biochem. 1975, 56, 35-40. (c) Sandberg, B. E. B.; Ragnarsson, U. Int. J. Pept. Protein Res. 1978, 11, 238-245. (d) Albericio, F.; Granier, C.; Labbé-Juillié, C.; Seagar, M.; Couraud, F.; Van Rietschoten, J. Tetrahedron 1984, 40, 4313-4326.
78. To the best of our knowledge, this is the first successful synthesis of apamin by an Fmoc strategy. For stepwise synthesis or segment condensation with Boc chemistry, see: (a) Van Rietschoten, J.; Granier, C.; Rochat, H.; Lissitzky, S.; Miranda, F. Eur. J. Biochem. 1975, 56, 35-40. (c) Sandberg, B. E. B.; Ragnarsson, U. Int. J. Pept. Protein Res. 1978, 11, 238-245. (d) Albericio, F.; Granier, C.; Labbé-Juillié, C.; Seagar, M.; Couraud, F.; Van Rietschoten, J. Tetrahedron 1984, 40, 4313-4326.
79. Chen, L.; Bauerová, H.; Slaninová, J.; Barany, G. Pept. Res. 1996, 9, 114-121.
80. In the case of conotoxin, authentic standards of unambiguously synthesized mispaired regioisomers were available; see: Hargittai, B.; Barany, G. In Peptides - Chemistry, Structure and Biology: Proceedings of the Fifteenth American Peptide Symposium; Tam, J. P., Kaumaya, P. T. P., Eds.; Kluwer: Dordercht, The Netherlands, 1998, in press.
81. The DMSO oxidation procedure was developed by Tam, J. P.; Wu, C.-R.; Liu, W.; Zhang, J.-W. J. Am. Chem. Soc. 1991, 113, 6657-6662. For applications to the conotoxin system, see ref 11b and Experimental Section of this paper.