4: Solid-supported synthesis of peptides

what is solid support peptide synthesis (SPPS)

the construction of peptides on insoluble functionalised polymer beads or ‘resin’

which side of the amino acid is attached to the resin

C-terminus, the polypeptide chain is built from the N-terminus

how does SPPS work

peptide is immobilised on resin

reagents are in solution

the tap is closed, the reaction happens, the tap is open and the reagents can be washed away after the reaction

product cleaved off after synthesis is complete

resin commonly consists of:

crosslinked polystyrene, an insoluble polymer

how is the first amino acid attached to the resin?

need a functionalised resin that can be covalently linked to carboxylate of amino acid

merrifield resin (chloromethyl polystyrene) is uses, and the amino acid is linked by substitution of the chloride leaving group

conditions for linking amino acid to merrifield resin

amino acid with PG on N-terminus and merrifield resin

base to deprotonate COOH

what is wang resin

resin-CH2-OH

wang resin coupling conditions and product

DIC as coupling reagent

makes ester bond

what is rink amide resin

resin-CH2-NH2

rink amide resin coupling conditions and product

DIC as coupling reagent

makes amide bond

how peptide synthesis on solid support works

1. deprotection, Fmoc or Boc removal

2. coupling, Y-Xaa-OH, coupling agent, base

3. peptide assembly (repeating 1 and 2)

4. cleavage from resin and side chain deprotection

5. purification

additional considerations for SPPS

1. protecting group for N-terminus cannot be boc to avoid releasing peptide during deprotection, so Fmoc

2. coupling reagent must be DIC m(HATU, PyBOP), not DCC for solid phase to avoid an insoluble urea byproduct and precipitates out of solution

3. side chain protecting groups, generally chosen so deprotection occurs with the release of the peptide, so side chain PGs are often not stable to acidic conditions (boc, tBu)

how are peptides released from resin

acidic conditions

cleavage from merrifield resin requires HF (corrosive and toxic acid)

what can be done to avoid using HF for releasing peptides

use other types of resin that contains linkers to allow cleavage under different conditions (usually solution of TFA in an organic solvent)

how is wang resin cleaved

TFA

has +M effect of O

how is sasrin resin cleaved (more acid labile)

dilute TFA = 1%

advantages of SPPS

no work-up or purification of intermediates

can be automated

limitations of SPPS

accumulation of impurities and by-products as no intermediate purification is possible while peptide is bound

purification of final product can be challenging, usually leads to mixtures of products

limited amount of peptide produced