Guide for Resin and Linker Selection in Solid‐Phase Peptide Synthesis

Jason A. Moss1

1 The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 18.7
DOI:  10.1002/0471140864.ps1807s40
Online Posting Date:  June, 2005
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Abstract

For both novice and experienced practitioners of solid‐phase peptide synthesis (SPPS), the vast selection of commercially available linkers and resins has become something of a babel. The purpose of this unit is to clarify the situation, which is best understood by distillation to first principles, through an appreciation of chemical trends and consequences, as well as practical considerations. The most commonly used linkers and resins are presented and described in detail, along with a description of their development and common applications. Key protocols are provided so that the user may prepare appropriate linker‐functionalized resins for the majority of peptide synthesis applications.

Keywords: SPPS; solid‐phase; resin; linker; peptide; synthesis

     
 
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Table of Contents

  • Basic Protocol 1: Functionalization of an Amine‐Derivatized Resin using Preformed Handles
  • Basic Protocol 2: Acylation of Hydroxyl Linkers Via Reaction with a Mixed Anhydride
  • Basic Protocol 3: Qualitative Ninhydrin Test
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Functionalization of an Amine‐Derivatized Resin using Preformed Handles

  Materials
  • 5% (v/v) triethylamine in dimethylformamide (DMF) or 5% (v/v) triethylamine in 1:1 DMF/toluene
  • Linker (free carboxylic acid, Tcp ester, or Pfp ester) to be used in peptide synthesis
  • N,N′‐diisopropylcarbodiimide (DIC; optional)
  • N‐hydroxybenzotriazole (HOBt; optional)
  • Benzotriazol‐l‐yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP; optional)
  • DMF
  • Amine‐derivatized resin to be used in peptide synthesis
  • Glass or polypropylene fritted vessel with Teflon stopcock
  • Rotary shaker
  • Wash bottle

Basic Protocol 2: Acylation of Hydroxyl Linkers Via Reaction with a Mixed Anhydride

  Materials
  • Glass or polypropylene fritted vessel (with Teflon stopcock) containing hydroxyl‐functionalized resin (see protocol 1)
  • Dimethylformamide (DMF)
  • Fmoc‐protected amino acid (to serve as the C‐terminal amino acid in the synthesized peptide)
  • Anhydrous pyridine (packaged under nitrogen in a septum‐equipped bottle; Aldrich or Acros)
  • 2,6‐Dichlorobenzoyl chloride
  • Wash bottle
NOTE: 2,6‐Dichlorobenzoyl chloride is highly reactive. Therefore, it should be purchased in small quantities and exposed to air for as short a period as possible. The use of decomposed 2,6‐dichlorobenzoyl chloride will lead to greatly decreased acylation efficiency and additional undesirable side reactions.

Basic Protocol 3: Qualitative Ninhydrin Test

  Materials
  • Resin to be tested (see protocol 1)
  • Dichloromethane
  • Unreacted aminomethyl‐derivatized resin (with loading capacity similar to that of test resin)
  • 4% (w/v) ninhydrin in ethanol
  • 4:1 (v/v) phenol/ethanol
  • Anhydrous pyridine (packaged under nitrogen in a septum‐equipped bottle; Aldrich or Acros)
  • 3‐ml polypropylene fritted syringe (Applied Separations)
  • Wash bottle
  • Tygon tubing
  • Vacuum source
  • 10 × 75–mm glass culture tubes
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Figures

Videos

Literature Cited

Literature Cited
   Albericio, F. and Barany, G. 1991. Hypersensitive acid‐labile (HAL) tris(alkoxy)benzyl ester anchoring for solid‐phase synthesis of protected peptide segments. Tetrahedron Lett. 32:1015‐1018.
   Albericio, F., Kneib‐Cordonier, N., Biancalana, S., Gera, L., Masada, R.I., Hudson, D., and Barany, G. 1990. Preparation and application of the 5‐(4‐(9‐fluorenylmethyloxycarbonyl)aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid (PAL) handle for the solid‐phase synthesis of C‐terminal peptide amides under mild conditions. J. Org. Chem. 55:3730‐3743.
   Atherton, E. and Sheppard, R.C. 1989. Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Oxford.
   Barlos, K., Chatzi, O., Gatos, D., and Stavropoulos, G. 1991a. 2‐Chlorotrityl chloride resin: Studies on anchoring of Fmoc‐amino acids and peptide cleavage. Int. J. Pept. Protein Res. 37:513‐520.
   Barlos, K., Gatos, D., and Schafer, W. 1991b. Synthesis of prothymosin alpha (pro T‐alpha), a protein consisting of 109 amino acid residues. Angew. Chem. Int. Edit. Engl. 30:590‐593.
   Bayer, E., Goldammer, C., and Zhang, L. 1993. A new acid labile anchor system for the synthesis of fully protected peptide fragments. Chem. Pept. Proteins 5/6(Pt. A):3‐8.
   Bernatowicz, M.S., Daniels, S.B., and Köster, H. 1989. A comparison of acid labile linkage agents for the synthesis of peptide C‐terminal amides. Tetrahedron Lett. 30:4645‐4648.
   Chan, W.C. and White, P.D. 2000. Fmoc Solid Phase Synthesis: A Practical Approach. Oxford University Press, Oxford.
   Clapham, B., Lee, S.H., Koch, G., Zimmermann, J., and Janda, K.D. 2002. The preparation of polymer bound beta‐ketoesters and their conversion into an array of oxazoles. Tetrahedron Lett. 43:5407‐5410.
   Floersheimer, A. and Riniker, B. 1991. Solid‐phase synthesis of peptides with the highly acid‐sensitive HMPB linker. In Peptides 1990: Proceedings of the Twenty‐First European Peptide Symposium (E. Giralt and D. Andreu, eds.) pp. 131‐133. Escom, Leiden, The Netherlands.
   Han, Y., Bontems, S.L., Hegges, P., Munson, M.C., Minor, C.A., Kates, S.A., Albericio, F., and Barany, G. 1996. Preparation and applications of xanthenylamide (XAL) handles for solid‐phase synthesis of C‐terminal peptide amides under particularly mild conditions. J. Org. Chem. 61:6326‐6339.
   Kempe, M. and Barany, G. 1996. CLEAR: A novel family of highly cross‐linked polymeric supports for solid‐phase peptide synthesis. J. Am. Chem. Soc. 118:7083‐7093.
   Matsueda, G.R. and Stewart, J.M. 1981. A p‐methylbenzhydrylamine resin for improved solid‐phase synthesis of peptide amides. Peptides 2:45‐50.
   Meldal, M. 1992. PEGA: A flow‐stable polyethylene glycol‐dimethylacrylamide copolymer for solid‐phase synthesis. Tetrahedron Lett. 33:3077‐3080.
   Mergler, N., Tanner, R., Gosteli, J., and Grogg, P. 1988. Peptide synthesis by a combination of solid‐phase and solution methods I: A new very acid‐labile anchor group for the solid phase synthesis of fully protected fragments. Tetrahedron Lett. 29:4005‐4008.
   Merrifield, R.B. 1963. Solid phase peptide synthesis: I: The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149‐2153.
   Merrifield, R.B. 1964. Solid phase peptide synthesis: II: The synthesis of bradykinin. J. Am. Chem. Soc. 86:304‐305.
   Mitchell, A.R., Erickson, B.W., Ryabtsev, M.N., Hodges, R.S., and Merrifield, R.B. 1976. Tert‐butoxycarbonylaminoacyl‐4‐(oxymethyl)phenylacetamidomethyl‐resin, a more acid‐resistant support for solid‐phase peptide synthesis. J. Am. Chem. Soc. 98:7357‐7362.
   Mitchell, A.R., Kent, S.B., Engelhard, M., and Merrifield, R.B. 1978. A new synthetic route to tert‐butyloxycarbonylaminoacyl‐4‐(oxymethyl)phenylacetamidomethyl‐resin, an improved support for solid‐phase peptide synthesis. J. Org. Chem. 43:2845‐2852.
   Moss, J.A., Dickerson, T.J., and Janda, K.D. 2002. Solid phase peptide synthesis on JandaJel resin. Tetrahedron Lett. 43:37‐40.
   Rink, H. 1987. Solid‐phase synthesis of protected peptide fragments using a trialkoxy‐diphenyl‐methylester resin. Tetrahedron Lett. 28:3787‐3790.
   Sheppard, R.C. and Williams, B.J. 1982. Acid‐labile resin linkage agents for use in solid phase peptide synthesis. Int. J. Pept. Protein Res. 20:451‐454.
   Sieber, P. 1987. A new acid‐labile anchor group for the solid‐phase synthesis of C‐terminal peptide amides by the Fmoc method. Tetrahedron Lett. 28:2107‐2110.
   Wang, S.S. 1973. Para‐alkoxybenzyl alcohol resin and para‐ alkoxybenzyloxycarbonylhydrazide resin for solid‐phase synthesis of protected peptide fragments. J. Am. Chem. Soc. 95:1328‐1333.
   Zalipsky, S., Chang, J.L., Albericio, F., and Barany, G. 1994. Preparation and applications of polyethylene glycol‐polystyrene graft resin supports for solid‐phase peptide synthesis. React. Polym. 22:243‐258.
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