Combinatorial Chemistry Advanced Medicinal Chemistry (Pharm 5219): Section A Md. Saifuzzaman Assoc. Professor saifuzzaman17@yahoo.com Ref.: An Introduction to Medicinal Chemistry, 3rd ed. 2005, G.L.Patrick, Oxford University press

The Solid support Earliest form of resin (used by Merrifield) – polystyrene beads – styrene cross-linked with 1% divinylbenzene. Derivatized with a chloromethyl group (anchor/linker) – amino acids can be coupled via an ester group. This ester group is stable to reaction conditions but cleaved at end of synthesis using acids (e.g., HF).

The Solid support Disadvantages of polystyrene beads Growing peptide chain is hydrophobic, so not solvated and folds itself & forms internal H bonds thus, hinders access of further amino acids to growing chain

The Solid support Therefore, more polar solid phages – developed e.g., Sheppard’s polyamide. For non-peptides, Tentagel resin 80% polyethylene glycol grafted to cross-linked polystyrene Provides ether or tetrahydrofuran like environment.

Solid support Beads should be capable of swelling in solvent Most reactions in solid phase synthesis take place in interior of beads rather than surface. Each bead - a polymer and swelling involves unfolding of the polymer chains so that solvent and reagents can move between the chains into the core of polymer.

Solid support Besides bead, pins – designed to maximize surface area, maximize amount of compounds linked to solid support. Functionalized glass surfaces – suitable for oligoneucleotide synthesis.

Anchor / Linker A molecular unit covalently attached to polymer chain making up solid support Contains a reactive functional group with which starting material can react and attach to the resin Resulting link – stable to reaction conditions but cleavable to release final product Most linkers – in interior of polymer beads, so swelling is important.

Anchor / Linker Choice of linkers depends on….. Functional group present on starting material Functional group to be present on final product upon release

Anchor / Linker Resins of different linkers have different names Wang resin – has a linker suitable for attachment and release of carboxylic acids Rink resin – for attachment of carboxylic acids and release of carboxamides Dihydropyran-derivatized resin – suitable for attachment and release of alcohols

Anchor / Linker Wang resin – Used in peptide synthesis where N-protected amino acid – linked to resin by means of ester link. Ester link – remains stable to coupling and deprotection steps in synthesis and cleaved using trifluoroacetic acid (TFA) to release final product.

Anchor / Linker Rink resin – Attach starting material (with carboxylic acid) via amide link When reaction is complete, treatment with TFA releases final product with primary amide group.

Anchor / Linker Dihydropyran-derivatized resin– Attach primary & secondary alcohols in presence of pyridinium 4-toluenesulfonate (PPts) in dichloromethane. Upon completion of reaction, cleavage using TFA.

Combinatorial synthesis with a Dihydropyran-derivatized resin

Protecting groups Important functional groups (not involved in reaction) – should be protected Selection is extremely important Should be stable in reaction condition but capable of being removed under mild conditions after synthesis

Boc/benzyl protection strategy N-terminus of each amino acid – protected by a tert- butyloxycarbonyl (Boc) group After adding a.a. with growing peptide chain, Boc group is removed with TFA to free up amino group Next protected a.a. = coupled on to the chain Bond connecting new peptide to linker – stable to TFA, so remains unaffected by synthesis; but susceptible to strong acid and after synthesis, HF – used to release peptide

Boc/benzyl protection strategy An amide that is less stable than the protein amide is formed and then removed The tert-butoxycarbonyl amide (BOC) protecting group is introduced with di-tert-butyl dicarbonate Removed by brief treatment with trifluoroacetic acid 21

Boc/benzyl protection strategy Functional groups on a.a. residues – to be protected during synthesis (that protecting group has to be stable to TFA) Benzyl-type groups – stable to TFA but susceptible to HF

Boc/benzyl protection strategy So, HF both releases final peptide & deprotects residues Disadvantage of using HF: nasty chemical dissolves glass so use Teflon equipment too harsh condition – decomposition of peptide serious health risk (HF on skin!!!!)

Fmoc/t-Bu strategy 9-fluorenylmethoxycarbonyl (Fmoc) group – for terminal amino group – removed using mild base (e.g., piperidine) Functional groups on a.a. residues – to be protected with t- butyl group – can be removed by TFA Orthogonal strategy – base for removing Fmoc & acid for t- butyl

Fmoc/t-Bu strategy Link to resin – susceptible to TFA, no need to use HF Both peptide cleavage & functional group deprotection simultaneously