Disulfide Bond Formation in Peptides

Lin Chen1, Ioana Annis2, George Barany3

1 AxCell Biosciences Corporation, Newtown, 2 Union Carbide Corporation, Bound Brook, 3 University of Minnesota, Minneapolis
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 18.6
DOI:  10.1002/0471140864.ps1806s23
Online Posting Date:  May, 2001
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Abstract

The formation of disulfide bridges is often a crucial final stage in peptide synthesis. There is compelling evidence that the disulfide pattern can be critical in the folding and structural stabilization of many natural peptide and protein sequences, while the artificial introduction of disulfide bridges into natural or designed peptides may often improve biological activities/specificities and stabilities. This unit provides a highly selective, albeit state‐of‐the‐art, menu of procedures that can be performed to establish intramolecular or intermolecular disulfide bridges in targets of varying complexities.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Air Oxidation
  • Alternate Protocol 1: On‐Resin Air Oxidation
  • Alternate Protocol 2: Charcoal/Air‐Mediated Intramolecular Disulfide Formation
  • Basic Protocol 2: Intramolecular Disulfide Formation by Potassium Ferricyanide Oxidation
  • Alternate Protocol 3: Intermolecular or Intramolecular Disulfide Formation by Potassium Ferricyanide Oxidation
  • Alternate Protocol 4: On‐Resin Disulfide Formation by Potassium Ferricyanide Oxidation
  • Basic Protocol 3: Oxidation Under Slightly Acidic pH Conditions by DMSO
  • Alternate Protocol 5: Oxidation Under Slightly Basic pH Conditions by DMSO
  • Basic Protocol 4: Oxidation by Redox Buffers
  • Basic Protocol 5: Oxidation Mediated by Solid‐Phase Ellman's Reagent
  • Basic Protocol 6: Simultaneous Deprotection/Oxidation with Iodine
  • Alternate Protocol 6: Simultaneous On‐Resin Deprotection/Oxidation of S‐ACM with Iodine
  • Basic Protocol 7: Simultaneous Deprotection/Oxidation of S‐ACM with Tl(III)
  • Alternate Protocol 7: On‐Resin Simultaneous Deprotection/Oxidation of S‐ACM with Tl(III)
  • Basic Protocol 8: Alkyltrichlorosilane‐Sulfoxide Oxidation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Air Oxidation

  Materials
  • Poly(thiol) peptide, previously purified (see unit 8.7 for purification techniques)
  • Buffer, selected from among:
  • 0.1 to 0.2 M Tris⋅Cl, pH 7.7 to 8.7 ( appendix 2E)
  •  Tris⋅acetate, pH 7.7 to 8.7
  • 0.01 M phosphate buffers, pH 7 to 8 ( appendix 2E)
  •  0.01 M ammonium bicarbonate, pH 8
  • Air or oxygen
  • Additional reagents and equipment for dialysis (unit 4.4 and appendix 3B), HPLC (unit 8.7), and gel‐filtration (unit 8.3) or ion‐exchange (unit 8.2) chromatography

Alternate Protocol 1: On‐Resin Air Oxidation

  Materials
  • Protected peptide‐resin, prepared by optimized linear SPPS chain assembly
  • Deprotection reagents and solvents
  • Appropriate wash solvents
  • 0.02 to 0.175 M triethylamine in N‐methylpyrrolidone (NMP)
  • Air or oxygen
  • Appropriate peptide cleavage cocktail (unit 18.5), and materials for workup
  • 2‐ml plastic syringe fitted with a polypropylene frit (use larger syringe for larger amounts of resin)
  • Additional reagents and equipment for HPLC (unit 8.7) and gel‐filtration (unit 8.3) or ion‐exchange (unit 8.2) chromatography

Alternate Protocol 2: Charcoal/Air‐Mediated Intramolecular Disulfide Formation

  Materials
  • Bis(thiol) peptide, previously purified
  • 5% (v/v) aqueous NH 4OH
  • Granulated charcoal
  • Ellman's reagent (unit 18.3)
  • Additional reagents and equipment for assaying free sulfhydryls with Ellman's reagent (unit 18.3)

Basic Protocol 2: Intramolecular Disulfide Formation by Potassium Ferricyanide Oxidation

  Materials
  • Poly(thiol) peptide, previously purified
  • 0.01 M phosphate buffer, pH ∼7
  • 0.01 M aqueous K 3Fe(CN) 6 solution
  • 10% (v/v) aqueous NH 4OH
  • 50% (v/v) aqueous acetic acid
  • Celite resin (Aldrich)
  • AG‐3 anion‐exchange resin
  • Additional reagents and equipment for HPLC (unit 8.7) and gel‐filtration (unit 8.3) or ion‐exchange (unit 8.2) chromatography

Alternate Protocol 3: Intermolecular or Intramolecular Disulfide Formation by Potassium Ferricyanide Oxidation

  Materials
  • Protected peptide‐resin, prepared by optimized linear SPPS chain assembly
  • Dimethylformamide (DMF)
  • 0.1 to 0.5 M K 3Fe(CN) 6 solution
  • Dichloromethane (CH 2Cl 2)
  • Appropriate (thiol‐free) peptide cleavage cocktail (unit 18.5), and materials for workup
  • 2‐ml plastic syringe fitted with a polypropylene frit (use larger syringe for larger amounts of resin)
  • Additional reagents and equipment for HPLC (unit 8.7) and gel‐filtration (unit 8.3) or ion‐exchange (unit 8.2) chromatography

Alternate Protocol 4: On‐Resin Disulfide Formation by Potassium Ferricyanide Oxidation

  Materials
  • Poly(thiol) peptide, previously purified
  • 5% (v/v) aqueous acetic acid
  • Ammonium carbonate, (NH 4) 2CO 3
  • Dimethylsulfoxide (DMSO)
  • 0.05% (v/v) trifluoroacetic acid (TFA)/5% (v/v) aqueous acetonitrile
  • Additional reagents and equipment for HPLC (unit 8.7)

Basic Protocol 3: Oxidation Under Slightly Acidic pH Conditions by DMSO

  Materials
  • Poly(thiol) peptide, previously purified
  • 0.01 M phosphate buffer, pH 7.5
  • Dimethylsulfoxide (DMSO)
  • Additional reagents and equipment for HPLC (unit 8.7)

Alternate Protocol 5: Oxidation Under Slightly Basic pH Conditions by DMSO

  Materials
  • Poly(thiol) peptide, previously purified
  • recipeGlutathione redox buffer (see recipe)
  • EDTA
  • Sephadex G‐10 or G‐25 column
  • Additional reagents and equipment for dialysis (unit 4.4 and appendix 3B), HPLC (unit 8.7), and gel‐filtration (unit 8.3) or ion‐exchange (unit 8.2) chromatography

Basic Protocol 4: Oxidation by Redox Buffers

  Materials
  • Poly(thiol) peptide, previously purified
  • 2:1:1 (v/v/v) suitable buffer (pH 2.7‐7.0)/acetonitrile/CH 3OH
  • Solid‐phase Ellman's reagent (Annis et al., )
  • Dichloromethane (CH 2Cl 2)
  • Dimethylformamide (DMF)
  • 50‐ml plastic syringe fitted with a polypropylene frit (use larger syringe for larger amounts of resin)
  • Septum or plastic lock cap
  • Additional reagents and equipment for HPLC (unit 8.7)

Basic Protocol 5: Oxidation Mediated by Solid‐Phase Ellman's Reagent

  Materials
  • S‐protected peptide (protected by S‐Acm, S‐Xan, S‐Tmob, or S‐Trt), previously purified
  • 80% (v/v) acetic acid
  • Iodine
  • Carbon tetrachloride (CCl 4)
  • Additional reagents and equipment for HPLC (unit 8.7) and gel‐filtration (unit 8.3) or ion‐exchange (unit 8.2) chromatography

Basic Protocol 6: Simultaneous Deprotection/Oxidation with Iodine

  Materials
  • S‐protected peptide resin (protected by S‐Acm, S‐Xan, S‐Tmob, or S‐Trt, prepared by optimized linear SPPS chain assembly)
  • Dimethylformamide (DMF)
  • Iodine
  • Suitable peptide cleavage cocktail
  • Additional reagents and equipment for for dialysis (unit 4.4 and appendix 3B), HPLC (unit 8.7), and gel‐filtration (unit 8.3) or ion‐exchange (unit 8.2) chromatography

Alternate Protocol 6: Simultaneous On‐Resin Deprotection/Oxidation of S‐ACM with Iodine

  Materials
  • S‐protected peptide (protected by S‐Acm, S‐Xan, S‐Tmob, or S‐Trt), previously purified
  • 19:1 (v/v) trifluoroacetic acid (TFA)/anisole
  • Thallium (III) trifluoroacetate [Tl(tfa) 3]
  • Ethyl ether
  • 10 to 20‐ml screw‐cap centrifuge tube
  • Additional reagents and equipment for HPLC (unit 8.7)

Basic Protocol 7: Simultaneous Deprotection/Oxidation of S‐ACM with Tl(III)

  Materials
  • S‐protected peptide‐resin (protected by S‐Acm, S‐Tmob, prepared by optimized linear SPPS chain assembly)
  • Thallium (III) trifluoroacetate (Tl(tfa) 3)
  • Dimethylformamide (DMF)
  • Anisole
  • Dichloromethane (CH 2Cl 2)
  • Suitable (thiol‐free) peptide cleavage cocktail (unit 18.5)
  • 2‐ml plastic syringe fitted with a polypropylene frit
  • Additional reagents and equipment for for dialysis (unit 4.4 and appendix 3B), HPLC (unit 8.7), and gel‐filtration (unit 8.3) or ion‐exchange (unit 8.2) chromatography

Alternate Protocol 7: On‐Resin Simultaneous Deprotection/Oxidation of S‐ACM with Tl(III)

  Materials
  • S‐protected peptide (protected by S‐Acm, StBu, S‐Mob, or S‐Meb), previously purified
  • Trifluoroacetic acid (TFA)
  • Diphenylsulfoxide (Ph(SO)Ph)
  • Trichloromethylsilane (CH 3SiCl 3)
  • Anisole
  • Ammonium fluoride (NH 4F)
  • Diethyl ether
  • Sephadex G‐15 column
  • 4 N aqueous acetic acid
  • Additional reagents and equipment for HPLC (unit 8.7) and gel‐filtration (unit 8.3) or ion‐exchange (unit 8.2) chromatography
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Figures

Videos

Literature Cited

   Albericio, F., Hammer, R.P., García‐Echeverría, C., Molins, M.A., Chang, J.L., Munson, M.C., Pons, M., Giralt, E., and Barany, G. 1991. Cyclization of disulfide‐containing peptides in solid‐phase synthesis. Int. J. Peptide Protein Res. 37:402‐413.
   Annis, I., Chen, L., and Barany, G. 1998. Novel solid‐phase reagents for facile formation of intramolecular disulfide bridges in peptides under mild conditions. J. Am. Chem. Soc. 120:7226‐7238.
   Eritja, R., Ziehler‐Martin, J.P., Walker, P.A., Lee, T.D., Legesse, K., Albericio, F., and Kaplan, B.E. 1987. On the use of S‐t‐butylsulphenyl group for protection of cysteine in solid‐phase peptide synthesis using Fmoc‐amino acids. Tetrahedron 43:2675‐2680.
   Hargittai, B. and Barany, G. 1999. Controlled syntheses of natural and disulfide‐mispaired regioisomers of α‐conotoxin SI. J. Peptide Res. 54:468‐479.
   Munson, M.C. and Barany, G. 1993. Synthesis of α‐conotoxin SI, a bicyclic tridecapeptide amide with two disulfide bridges: Illustration of novel protection schemes and oxidation strategies. J. Am. Chem. Soc. 115:10203‐10210.
   Munson, M.C., Lebl, M., Slaninov´, J., and Barany, G. 1993. Solid‐phase synthesis and biological activity of the parallel dimer of deamino‐oxytocin. Peptide Res. 6:155‐159.
   Tam, J.P. and Shen, Z.‐Y. 1992. Efficient approach to synthesis of two‐chain asymmetric cysteine analogs of receptor‐binding region of transforming growth factor‐α. Int. J. Peptide Protein Res. 39:464‐471.
   Tam, J.P., Wu, C.‐R., Liu, W., and Zhang, J.‐W. 1991. Disulfide bond formation in peptides by dimethyl sulfoxide. Scope and applications. J. Am. Chem. Soc. 113:6657‐6662.
   Volkmer‐Engert, R., Landgraf, C., and Schneider‐Mergener, J. 1998. Charcoal surface‐assisted catalysis of intramolecular disulfide bond formation in peptides. J. Peptide Res. 51:365‐369.
Key References
   Albericio, F., Annis, I., Royo, M., and Barany, G. 2000. Preparation and handling of peptides containing methionine and cysteine. In Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W.C. Chan and P.D. White, eds.) pp.77‐114. Oxford University Press, Oxford.
  Relatively recent coverage of cysteine protection and disulfide formation, in the context of a 14‐chapter monograph on Fmoc solid‐phase methodology.
   Andreu, D., Albericio, F., Solé, N.A., Munson, M.C., Ferrer, M., and Barany, G. 1994. Formation of disulfide bonds in synthetic peptides and proteins. In Methods in Molecular Biology, Vol. 35: Peptide Synthesis Protocols (M.W. Pennington and B.M. Dunn, eds.) pp.91‐169. Humana Press, Totowa, N.J.
  State‐of‐the‐art review at the time that it was written. Some experimental information and 358 literature citations.
   Annis, I., Hargittai, B., and Barany, G. 1997. Disulfide bond formation in peptides. Methods Enzymol. 289:198‐221.
  Another book chapter, more recent but less comprehensive than the Andreu et al. reference above. The parent volume, edited by G.B. Fields, is a highly useful 32‐chapter compendium of methods of solid‐phase peptide synthesis.
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