Synthesis of Peptide−Protein Conjugates Using N-Succinimidyl Carbamate Chemistry

Abstract

Peptide-protein conjugates are useful tools in different fields of research as, for instance, the development of vaccines and drugs or for studying biological mechanisms, to cite only few applications. N-Succinimidyl carbamate (NSC) chemistry has been scarcely used in this area. We show that unprotected peptides, featuring one lysine residue within their sequences, can be converted in good yield into NSC derivatives by reaction with disuccinimidylcarbonate (DSC). No hydrolysis of the NSC group was observed during RP-HPLC purification, lyophilization, or storage. NSC peptides reacted efficiently within minutes with lysozyme used as model protein. To illustrate usefulness of the method consisting of the synthesis of a peptide-protein conjugate of biological interest, a NSC peptide derived from a peptide substrate for tyrosylprotein sulfotransferase (TS) was synthesized and ligated to receptor-binding nontoxic B-subunit of Shiga toxin (STxB). Immunofluorescence studies showed the intracellular delivery of the TS-STxB conjugate and its ability to circulate to the Golgi as the native STxB protein. Moreover, we demonstrate that the TS label could be sulfated by tyrosylprotein sulfotransferases present in the Golgi. Thus, NSC chemistry permitted rapid synthesis of a peptide-protein conjugate worthwhile for studying the transport of proteins from the plasma membrane to the Golgi. The second part of this article describes a more general method for synthesizing peptide-protein conjugates without any limitation of the peptide sequence. The conjugates were assembled by combining NSC chemistry and alpha-oxo semicarbazone ligation. To this end, a glyoxylyl NSC peptide was synthesized and reacted with lysozyme. The glyoxylyl groups on the protein were then reacted with a semicarbazide peptide to produce the target peptide-protein conjugate. Both reactions, namely, urea bond formation and alpha-oxo semicarbazone ligation, were carried at pH 8.0 using a one-pot procedure.

α-Oxo Aldehyde or Glyoxylyl Group Chemistry in Peptide Bioconjugation

“…One reason for this can be the lack of methods giving access to semicarbazide peptides such as 95 , i.e., peptides featuring a hydrazinocarbonyl group on their N-terminus (Scheme ). It was shown recently that a hydrazinocarbonyl group can be easily attached to the N-terminus of peptides after the Fmoc-SPPS elongation step by using tert -butylcarbazate derivative 94 . , Semicarbazide peptide 95 survived the TFA cleavage and deprotection step but hydrolyzed rapidly during analytical or preparative RP-HPLC at pH 2. In contrast, the use of an eluent system buffered at pH 6.5 permitted prevention of semicarbazide bond hydrolysis and isolation of semicarbazide peptides 95 in good yield.…”

“…Another interesting observation is the capacity of the semicarbazide group of peptide 95 to react with an α-oxo aldehyde moiety at pH 8 (Table , entry 6). This property was exploited for designing a one-pot procedure permitting the synthesis of peptide–protein conjugates based on a carbonylation-semicarbazone bond forming reaction sequence (Scheme ) . For this, bifunctional peptide A 97 featuring an α-oxo aldehyde group and an N -succinimidylcarbonyl group was reacted at pH 8.0 with surface-exposed amino groups of a protein to form stable urea bonds.…”

SIB-DOTA: A trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies

“…To overcome this, we also plan to replace the N -hydroxysuccinimide ester with hydrolytically more stable N -succinimidyl carbamate moiety. 45 …”

A universal and ready-to-use heterotrifunctional cross-linking reagent for facile synthetic access to sophisticated bioconjugates

“…Interestingly, such a one pot synthesis has been recently reported by Mhidia et al for the preparation of peptideprotein conjugates by means of combining two reactions namely amine acylation with N-succinimidyl carbamate (NSC chemistry) and a-oxo semicarbazone ligation. 43…”