Modification of N-Terminal α-Amino Groups of Peptides and Proteins Using Ketenes

Abstract

A method of highly selective N-terminal modification of proteins as well as peptides by an isolated ketene was developed. Modification of a library of unprotected peptides XSKFR (X varies over 20 natural amino acids) by an alkyne-functionalized ketene (1) at room temperature at pH 6.3 resulted in excellent N-terminal selectivity (modified α-amino group/modified ε-amino group = >99:1) for 13 out of the 20 peptides and moderate-to-high N-terminal selectivity (4:1 to 48:1) for 6 of the 7 remaining peptides. Using an alkyne-functionalized N-hydroxysuccinimide (NHS) ester (2) instead of 1, the modification of peptides XSKFR gave internal lysine-modified peptides for 5 out of the 20 peptides and moderate-to-low N-terminal selectivity (5:1 to 1:4) for 13 out of the 20 peptides. Proteins including insulin, lysozyme, RNaseA, and a therapeutic protein BCArg were selectively N-terminally modified at room temperature using ketene 1, in contrast to the formation of significant or major amounts of di-, tri-, or tetra-modified proteins in the modification by NHS ester 2. The 1-modified proteins were further functionalized by a dansyl azide compound through click chemistry without the need for prior treatment.

Diazo ester insertion in NH bonds of amino acid derivatives and insulin catalyzed by water-soluble iron and ruthenium porphyrin complexes (FeTSPPCl) as application of carbenoid transfer in aqueous media

“…Because the difference of pK a values of the N-terminal group (∼7. [6][7][8] and the NH 2 group in the side chain of lysine residue (10.0-10.2), this reaction (81% yield) can also be optimized to maximize the selectivity for the N-terminal insertion by the reaction being performed under weakly basic conditions (pH 9). Actually cysteine ethyl ester was also tested to detect any selectively.…”

A Facile Method for Preferential Modification of the N-Terminal Amino Group of Peptides Using Triazine-Based Coupling Reagents

“…Interestingly, the pH values of the reaction media did not a ect the selectivity between the N-terminus and the Lys side chains (Nterminus : Lys=100 : 4-6). is tendency was also observed for the ketene-mediated reactions, 5) in which formation of the di-modi ed peptides increases with increasing conversion rates, but without a ecting the selectivity. is implies that, under the conditions that lead to high conversion rates, the reaction occurs rst at the N-terminal amino group of most of the peptides, followed by the modi cation at the Lys side chains to generate the di-modi ed peptides.…”

“…However, even under judiciously controlled pH conditions, the selectivity is o en unsatisfactory, accompanying a considerable level of modi cation at the Lys side chains. 5) As an alternative approach, utilization of transamination reaction was developed, 6) in which the N-terminal amino acid residue is speci cally converted into α-ketoacid using aldehyde reagents for the subsequent introduction of various components having a hydroxylamine moiety. While this method is demonstrated to be e ective in terms of selectivity, it is incompatible with peptides having the particular amino acid residues, such as Ser, r, Cys, and Trp, at the N-terminus because of their side reactions with aldehydes, limiting the range of applications.…”

“…7) Likewise, Chan et al also reported a method for selective N-terminal modi cation using ketene-containing compounds. 5) Although above-cited methods allow highly selective modi cations of the peptide N-terminus regardless of the amino acid structure, they are disadvantageous in that they require laborious and time-consuming preparation of reagents containing a 2-pyridinecarboxyaldehyde or ketene moiety. Moreover, since ketene is highly reactive, care must be taken to avoid degradation of the reagents during storage.…”

A peptide ligase and the ribosome cooperate to synthesize the peptide pheganomycin

“…Furthermore, PGM1 may be useful for modifying the N-terminus of proteins of interest, since there are many reports demonstrating such trials by chemical and enzyme methods for a diverse range of aims [30][31][32][33] .…”