Epimerisation in Peptide Synthesis

Abstract

Epimerisation is basically a chemical conversion that includes the transformation of an epimer into another epimer or its chiral partner. Epimerisation of amino acid is a side reaction that sometimes happens during peptide synthesis. It became the most avoided reaction because the process affects the overall conformation of the molecule, eventually even altering the bioactivity of the peptide. Epimerised products have a high similarity of physical characteristics, thus making it difficult for them to be purified. In regards to amino acids, epimerisation is very important in keeping the chirality of the assembled amino acids unchanged during the peptide synthesis and obtaining the desirable product without any problematic purification. In this review, we report several factors that induce epimerisation during peptide synthesis, including how to characterise and affect the bioactivities. To avoid undesirable epimerisation, we also describe several methods of suppressing the process.

Keywords: cyclisation; epimerisation; peptide synthesis; side reaction; solid-phase peptide synthesis.

Figure 1

Structure of (a) doxorubicin and (b) epirubicin (Paul Launchbury and Habboubi, 1993).

Figure 2

Epimerisation of N-acetylglucosamine (GlcNAc) into N-acetylmannosamine (NANA) catalysed by a renin-binding protein [5].

Figure 3

Proposed epimerisation of amino acid by AdoMet radical proteusin epimerases [6].

Figure 4

Mechanism of epimerisation/racemisation through oxazolone intermediate [13].

Figure 5

Various types of coupling reagents.

Figure 6

Reactive O-Acylisourea ester increases racemisation.

Figure 7

DIC/DMAP coupling agent mechanism.

Figure 8

Structure of 1-ethyl-3-[3-(dimethylaminopropy1) carbodiimide] (EDC).

Figure 9

Epimerisation mechanism using carbodiimide coupling agent.

Figure 10

BOP coupling reagent.

Figure 11

Structures of (a) phenylglycine and (b) methionine.

Figure 12

Proton abstraction of methionine by base.

Figure 13

Structure of NMe-IB-01212.

Figure 14

Structure of coibamide A.

Figure 15

Potential racemisation pathways of Fmoc-(L)-phenylglycine during solid-phase peptide synthesis.

Figure 16

Epimerisation study in glycopeptide synthesis.

Figure 17

Comparison of the geometries at amide (a), 2-substituted 1,3-thiazine (b), 1,3-thiazoline (c), and 1,3-thiazole (d).

Figure 18

Possible intermolecular H-bond-assisted enamination/racemisation mechanism in non-Pro*-Thi containing analogues.

Figure 19

Mechanism of racemisation through alpha hydrogen attack.

Figure 20

Racemisation process in BOP activation with the effect of base [45].

Figure 21

Base-induced cleavage of Fmoc and subsequent quenching of dibenzofulvene by a nucleophile.

Figure 22

Epimerisation via the aspartimide mechanism in the deprotection step.

Figure 23

Structure of dipeptide Phg-Ala and Phg-Val.

Figure 24

Phenylglycine-containing peptide.

Figure 25

Crystal structures of LO4 butanol solvate and LO2 methanol solvate, with color-coded atoms sulphur—yellow, oxygen—red, nitrogen—blue, carbon—black, a carbon Met/MetO2—green; solvent clouds: butanol—brown, water—red, methanol—cyan [44].

Figure 26

A representation of the APED model that is based on the Primary Pump Scenario and displays the antagonism between two chemical cycles that potentially induces dynamically controlled states such that D- or L-amino acids can prevail as a result of the reproduction of chirality through dipeptide epimerisation.

Figure 27

(a) Lawesson’s reagent, xylene, 130 °C, 30 min, 33%, (b) PhCH₂Br, aq. NaOH/CH₂Cl₂, sonication, r.t., 30 min, 94%, (c) AgNO₃, ^tBuOH/water 9:1, r.t., 2.5 h, 35%, (d) aq.HCl/^tBuOH, 55 °C, 20 min, quant, (e) HgCl₂/CaCO₃, AcCN, r.t., 2 h, 45%, (f) TFA, ^tBuOH/water, 15 h, r.t., 35%.

Figure 28

Changing the configuration of methionine from L to D using KOH base catalyst.

Figure 29

Epimerisation product in cyclisation step nocardiotide A.

Figure 30

Mechanism of coupling reaction facilitated by coupling reagent HATU/HOAt.

Figure 31

The difference in structure between OxymaPure and Oxyma-B.

Figure 32

Structure of ynamides (MSMsA and MYTsA).

Figure 33

Epimerisation mechanism in histidine amino acid residue.

Figure 34

Structures of protected histidine.

Figure 35

Comparison study of the epimerisation rate at C-Terminal Cys of protected peptide acid on the resin.

Figure 36

Copper(II)-mediated and classical peptide arylester synthesis and subsequent elongation.

Figure 37

Epimerisation-free mechanism of 3 hydrosilane-mediated type coupling.

Figure 38

General steps for generation of peptide thioesters via Boc-based SPPS utilising special thioester linkers.

Figure 39

Native Chemical Ligation of peptide thioester Ac-AVGPPGVACOSR 3 h with N-terminal cysteine peptide CRFAS-NH₂. HPLC/MS traces of peptide thioester 3 h and ligated product. SR ¼ S-(CH₂)₂-COOC₂H₅.

Figure 40

Ligation of peptide thioester Ac-GNSARKGRSNTFID-COSR 3Q with N-terminal cysteine peptide CPTGPRPNEPMWITY-NH₂. MS of the ligated product. SR ¼ S-(CH₂)₂-COOC₂H₅.

Figure 40

Ligation of peptide thioester Ac-GNSARKGRSNTFID-COSR 3Q with N-terminal cysteine peptide CPTGPRPNEPMWITY-NH₂. MS of the ligated product. SR ¼ S-(CH₂)₂-COOC₂H₅.

Figure 41

General route for the conversion of peptide hydrazide to peptide acid.

Figure 42

Analytical HPLC traces (210 nm) of (A) crude C-AhPDF 1.1b acid (1) using trityl(2-Cl) chloride resin and (B) crude C-AhPDF 1.1b hydrazide (2) using hydrazine-trityl(2-Cl) resin; * denotes the epimer of hydrazide 2.

Figure 43

HPLC spectra in epimerisation study of microwave-assisted peptide synthesis. (A). Process flow diagram. Amino acid, activating agent, and DIEA are merged together using three HPLC pumps. (B). HPLC spectra of L-Cys and D-Cys residue. (C) Flow rate vs. %D dastereomer diagram of Cys residue. (D). HPLC spectra of L-His and D-His residue (E). Flow rate vs. %D dastereomer diagram of His residue.