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. 2020 May 29;3(1):67.
doi: 10.1038/s42004-020-0309-y.

N-Terminal selective modification of peptides and proteins using 2-ethynylbenzaldehydes

Jie-Ren Deng #  1 , Nathanael Chun-Him Lai #  1 , Karen Ka-Yan Kung  2 , Bin Yang  1 , Sai-Fung Chung  3 , Alan Siu-Lun Leung  3 , Man-Chung Choi  3 , Yun-Chung Leung  4 , Man-Kin Wong  5
Affiliations

N-Terminal selective modification of peptides and proteins using 2-ethynylbenzaldehydes

Jie-Ren Deng et al. Commun Chem. .

Abstract

Selective modification of the N-terminus of peptides and proteins is a promising strategy for single site modification methods. Here we report N-terminal selective modification of peptides and proteins by using 2-ethynylbenzaldehydes (2-EBA) for the production of well-defined bioconjugates. After reaction screening with a series of 2-EBA, excellent N-terminal selectivity is achieved by the reaction in slightly acidic phosphate-buffered saline using 2-EBA with electron-donating substituents. Selective modification of a library of peptides XSKFR (X = either one of 20 natural amino acids) by 2-ethynyl-4-hydroxy-5-methoxybenzaldehyde (2d) results in good-to-excellent N-terminal selectivity in peptides (up to >99:1). Lysozyme, ribonuclease A and a therapeutic recombinant Bacillus caldovelox arginase mutant (BCArg mutant) are N-terminally modified using alkyne- and fluorescein-linked 2-EBA. Alkyne-linked BCArg mutant is further modified by rhodamine azide via copper(I)-catalyzed [3 + 2] cycloaddition indicating that the reaction has high functional group compatibility. Moreover, the BCArg mutant modified by 2-ethynyl-5-methoxybenzaldehyde (2b) exhibits comparable activity in enzymatic and cytotoxic assays with the unmodified one.

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Conflict of interest statement

The authors declare the following competing interests: M.-K.W. has filed a patent application on the use of 2-ethynylbenzaldehydes for bioconjugation (WO2019219002) and all other authors declare no competing interests.

Figures

Fig. 1
Fig. 1. N-terminus selective modification.
a General mechanism of isoquinolinium formation. b Our design of 2-ethynylbenzaldehydes for N-terminal modification.
Fig. 2
Fig. 2. N-terminal modification of a model peptide.
a N-terminal modification of YTSSSKNVVR 1a with 2-ethynylbenzaldehyde 2a. b Deconvoluted mass spectrum of the reaction mixture. c MS/MS spectrum of N-terminally modified peptide 3a (inset: extracted ion chromatogram of mono-modified peptide). d Model reaction of isoquinolinium salt 3b from l-alanine β-benzylamide 1b and 2a.
Fig. 3
Fig. 3. Substrate scope.
Structures of 2-EBA 2a–t and compounds 4a–c.
Fig. 4
Fig. 4. Effect of pH and substrate substituents.
a Time course of N-terminal modification of YTSSSKNVVR 1a using 2d at pH 6.5–9.0. b Electronic effects of the substituents on 2-EBA towards the N-terminal modification of 1a at pH 6.5–9.0.
Fig. 5
Fig. 5. N-terminal protein modification.
Modification of lysozyme and RNase A with functionalized 2-EBA 2t and 2u.
Fig. 6
Fig. 6. N-terminal protein modification with an alkyne.
Modification of the BCArg mutant with alkyne-functionalized 2-EBA 2t and sequential copper(I)-catalyzed [3 + 2] cycloaddition reaction with rhodamine azide.
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