Merrifield synthesis

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The Merrifield synthesis is a process developed by the American chemist Robert Bruce Merrifield for the synthesis of peptides from individual amino acids . For this he received the Nobel Prize in Chemistry in 1984 . The process can be automated. The time required to extend the peptide by one amino acid unit is on the order of a few minutes to several hours. In protein biosynthesis , it is seconds.

principle

Schematic presentation of a Merrifield resin with a reactive chlorobenzyl group on the surface of a polymer sphere (left).

The process uses a solid phase ( resin ) made of polystyrene that has CH 2 Cl groups on the surface . This solid phase is called Merrifield resin. The synthesis consists of several introductory steps, propagation steps and a final reaction step. In practice, peptides with a length of about 100 amino acids can be produced.

Introductory response

In the first step, an amino acid protected at the N terminus with the residue R 1 at the C terminus is bound to the surface of the solid phase. In the process, chloride is split off in the course of a nucleophilic substitution . Since the amino acid is now firmly bound to the substrate via an ester group, rinsing is carried out to remove reaction products and excess amino acid.

Immobilization of an N-protected amino acid on the polymeric carrier.

In the next step, the protective group [usually tert- butyloxycarbonyl (BOC), see figure, or fluorenylmethoxycarbonyl (Fmoc)] is split off. The amino acid bound to the polymeric substrate now has a reactive (free) N terminus. It is rinsed again to remove low molecular weight contaminants.

Peptide propagation

An amino acid protected at the N terminus and activated at the C terminus with the residue R 2 is now added. This amino acid activated with dicyclohexylcarbodiimide (DCC) now reacts endergonically with the already bound, forming a peptide bond and formally splitting off water, which is bound as dicyclohexylurea . Another rinse follows. Then the protecting group of the newly added amino acid is removed.

Peptide Propagation: Formation of an immobilized dipeptide ester.

An N -protected amino acid with the residue R 3 can then be added again and the peptide can be extended by a third N -protected amino acid using DCC . The immobilized tripeptide ester is obtained by splitting off the protective group.

Peptide Propagation: Formation of an immobilized tripeptide ester.

Possibly. further N -protected amino acids can be condensed in the same way.

Abort response

With the help of hydrofluoric acid , the ester bond of the amino acid introduced first to the substrate (polymeric carrier) is protonated and cleaved, so that the finished peptide (here a tripeptide ) is released:

Cleavage of the peptide (example: tripeptide) from the polymeric carrier.

history

The development of Merrifield synthesis is closely related to the development of peptide synthesis. The first solid phase synthesis of peptides on solid porous polymers as substrates was carried out in 1966 by Bruce Merrifield and Arnold Marglin with the Merrifield synthesis using the example of insulin. As a result, the yield per reaction cycle rose from 90% to 99.5% and the secondary reaction products and starting materials could be easily removed by washing, whereby longer peptides could also be produced with acceptable yield. A variant of the Merrifield synthesis called SPOT synthesis on cellulose - filter paper as a matrix was described by Ronald Frank in 1992 . It is mainly used for epitope mapping of continuous epitopes of antibodies .

Individual evidence

  1. ^ A b Robert Bruce Merrifield, Solid phase peptide synthesis Journal of the American Chemical Society Volume 85, Issue 14 pp. 2149-2154, doi: 10.1021 / ja00897a025 .
  2. A. Marglin, RB Merrifield: The synthesis of bovine insulin by the solid phase method. In: Journal of the American Chemical Society. Volume 88, Number 21, November 1966, pp. 5051-5052, PMID 5978833 .
  3. Hans-Dieter Jakubke, Hans Jeschkeit: Amino acids, peptides, proteins. Verlag Chemie, Weinheim 1982, ISBN 3-527-25892-2 , pp. 204-222.
  4. a b Information from the Nobel Foundation on the 1984 award ceremony for Robert Bruce Merrifield (English)
  5. Erich Wünsch Synthesis of peptide natural products: Problems of the current state of research Angewandte Chemie Volume 83, Issue 20, pp. 773–782, doi: 10.1002 / anie.19710832002 .
  6. Ronald Frank: Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. In: Tetrahedron. 48, 1992, p. 9217, doi : 10.1016 / S0040-4020 (01) 85612-X .

literature

  • Weng C. Chan, Peter D. White: Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press, Oxford / New York 2000, ISBN 0-19-963724-5 .