Side reactions in solid-phase peptide synthesis and their applications

Side reactions in peptide synthesis indicate steps needing improvement as well as opportunities for structural diversification in combinatorial design. Among the side reactions observed in this study, transesterification of Boc-Glu(OBzl) occurred in TMAH-catalyzed resin attachment, leading to Boc-DKKREE(OMe) in solid-phase synthesis of Boc-DKKREE. Acetylation of Boc-Arg(NO₂)-resin occurred during resin capping with Ac₂O/Et₃N, leading to GPR(Ac) in GPR synthesis. His- and Lys-modification occurred during GHRPLDKKREE cleavage from resin by Pd(OAc)₂-catalyzed hydrogenation in DMF. To verify these side reactions, model experiments were performed, which indicated rapid transesterification of Boc-Glu(OBz1) in methyl. isopropyl, or tert-butyl alcohol into the corresponding ester by TMAH, but not by Cs. This TMAH ability was used to devise a convenient procedure for peptide cleavage. TLC studies of acetylation showed that both Boc-Arg(NO₂) and Boc-Arg(Tos) were stable to Ac-Im treatment, but were modified by Ac₂O/Et₃N. Since transfer hydrogenation of Boc-His(Bzl) and Boc-Lys(Z) in HCOOH or ammonium formate did not generate the formylated side-products of catalytic hydrogenation, DMF and not its decomposed product, HCOOH, appeared involved in side-chain modification. Elimination of the side reactions, by using Cs-derived Boc-Glu(OBzl)-resin for peptide synthesis and catalytic hydrogenation in NMP-HOPr for peptide cleavage. increased the GHRPLDKKREE yield by 1/3. On the other hand, the side reactions provided modified peptides, whose bioassays revealed different importance of the modified side-chains. © Munksgaard 1996.     

Convergent solid-phase peptide synthesis

The solid-phase synthesis, photolytic detachment from the solid support and purification in solution, of a fully-protected octapeptide containing a methionine residue (protected as the sulphoxide) is described. Protection of methionine in this manner avoids problems associated with the oxidation of this residue during the photolysis. The peptide has been purified by medium pressure liquid chromatography using solvent mixtures containing a high proportion of dimethylformamide in order to avoid precipitation of the peptide on the column.     

Multi-component methodologies in solid-phase organic synthesis

Solid-phase organic synthesis, particularly when used in conjunction with combinatorial techniques, is emerging as a revolutionary technology in chemistry. Multi-component reaction systems are particularly valued because several elements of diversity can be introduced in a single transformation thereby expanding the diversity of compound libraries. A variety of multi-component reactions have been successfully adapted for solid-phase technology as described in this review.     

Spectrophotometric monitoring in continuous-flow Boc-based solid-phase peptide synthesis

Abstract: It has been demonstrated that SPPS with Boc amino group protection can be monitored spectrophotometrically if it is performed in a continuous-flow reactor of variable volume. It is shown that this approach provides useful and adequate evidence on the beginning/end-point of most steps of the SPPS cycle. At the deprotection step the spectrophotometric monitoring enables real-time recording of the initial and final moments of the process. When synthesizing a ‘difficult’ polyalanine sequence, we were able to monitor variation in the deprotection dynamics associated with the aggregation phenomena. The time actually necessary for the Boc protecting group removal appeared to be significantly smaller than that usually preset in the available Boc-SPPS protocols.     

Incomplete Fmoc deprotection in solid-phase synthesis of peptides

During solid-phase peptide synthesis of homo-oligopeptides containing leucine or alanine using the Fmoc strategy, we have observed ineffective N-α-deprotection with piperidine in a sequence-dependent manner. Incomplete deprotection was found to be associated with subsequent slow or incomplete amino acid coupling. Optimization of the deprotection step was carried out by varying the experimental conditions e.g. deprotection time, temperature, solvents and addition of chaotropes. Coupling and deprotection steps have been investigated using color monitoring, as well as FAB MS and HPLC for product analysis. The phenomena of difficult coupling and deprotection steps in the investigated systems have been demonstrated to have the same physical chemical origins, p-sheet formation. © Munksgaard 1994.     

Side reactions in peptide synthesis

During the hydrogenolytic removal of the benzyl ester from the pentapeptide Boc-Asp(OBzl)-Ala-Phe-Ile-Gly-OEt, either with hydrogen gas or ammonium formate in the presence of palladium-on-charcoal as catalyst, variable amounts (5–30%) of an unexpected by-product were always obtained. The compound was eventually identified as Boc-Asc-Ala-Phe-Ile-Gly-OEt (Asc = aminosuccinyl). The application of field desorption mass spectrometry as a diagnostic tool is reported. The study of this side reaction, carried out on the pentapeptide and two other dipeptide models, showed that: 1) palladium-on-charcoal may induce Asc formation; 2) the contemporary presence of the catalyst and a base (even in trace amounts) greatly increases the by-product formation; 3) the side reaction is sequence and solvent dependent; 4) the Asc formation is completely prevented by adding a few equivalents of acetic or formic acid. Some mechanistic considerations are also reported.     

Multicomponent synthesis and anticonvulsant activity of monocyclic 2,6-diketopiperazine derivatives

In this study, a series of diastereomerically pure monocyclic 2,6-diketopiperazine (2,6-DKP) derivatives were synthesized. The key synthetic step involved a multicomponent Ugi five-center, four-component reaction which was used to generate the convertible tert-butylamidoesters with both good yields and high diastereoselectivity toward the desired bioactive (S,S) absolute configuration. In subsequent steps, selective tertbutyl cleavage by use of BF₃·CH₃COOH and base-induced intramolecular cyclocondensation gave the final 2,6-DKP derivatives. The relative stereochemistry of the target molecules was confirmed by ¹H NMR experiments. The compounds obtained were submitted to in vivo screening in animal models of epilepsy. Some of them displayed good activity in maximal electroshock seizure and 6 Hz tests.     

Aggregation of resin-bound peptides during solid-phase peptide synthesis

Nonrandom incomplete aminoacylation of a pendent peptide chain on an insoluble polymeric support during solid-phase peptide synthesis is sequence-dependent and is caused by aggregation of peptide chains, manifested by a decreased swelling capacity. The volume of the swollen peptidyl-resin after each coupling during the syntheses of 87 sequence unrelated peptides was measured, and for each amino acid an aggregation parameter, 〈P_a〉, was derived that reflects the propensity of the swollen volume of peptidyl-resin to decrease during peptide synthesis. These aggregation parameters were used to predict potentially difficult sequences.     

A novel oxidation-labile linker for solid-phase peptide synthesis

The use of an oxidation-labile phenylhydrazide group as a linker for solid phase peptide synthesis (SPPS) is discussed.