Selective N-terminal functionalization of native peptides and proteins

Diao, Chen; Disotuar, Maria M.; Xiong, Xiaochun; Wang, Yuanxiang; Chou, Danny Hung‐Chieh

doi:10.1039/c6sc04744k

Cited by 126 publications

(116 citation statements)

References 52 publications

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“…It is therefore likely that N‐terminal modification will not affect the inherent function of wild‐type proteins or enzymes in most cases . Since an N‐terminal α‐amino group has a lower p K a value than the ϵ‐amino group of the Lys side chain, a reagent targeting the N‐terminal amino acid is expected to be particularly effective for modification of this site . For example, Francis and co‐workers, and other researchers have reported a site‐specific and one‐step N‐terminal α‐amino group modification Scheme using 2‐pyridinecarbaldehyde derivatives …”

Section: Figurementioning

confidence: 99%

Triazolecarbaldehyde Reagents for One‐Step N‐Terminal Protein Modification

et al. 2020

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Site-specific modification of peptides and proteins is ak ey aspect of protein engineering.W ed eveloped am ethod for modification of the Nterminus of proteins using 1H-1,2,3-triazole-4-carbaldehyde( TA4C) derivatives, which can be prepared in one step. The N-terminal specific labelingo fb ioactive peptides and proteins with the TA4C derivatives proceeds under mild reactionc onditions in excellent conversion (angiotensinI: 92 %, ribonuclease A: 90 %). This method enables site-specific conjugation of various functional molecules such as fluorophores,b iotin, and polyethylene glycol attached to the triazole ring to the Nterminus. Furthermore, af unctional molecule modified with ap rimary amine moiety can be directly converted into aT A4C derivativethrough aDimroth rearrangement reaction with 1-(4-nitrophenyl)-1H-1,2,3-triazole-4-carbaldehyde. This methodc an be used to obtain N-terminal-modifiedp roteins via only two steps:1 )convenient preparation of aT A4C derivativew ith af unctional group and 2) modification of the Nterminus of the protein with the TA4C derivative.

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Section: Figurementioning

confidence: 99%

Triazolecarbaldehyde Reagents for One‐Step N‐Terminal Protein Modification

et al. 2020

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“…The methodology is compatible with peptides produced by any method where the linker can be semisynthetically conjugated to a selective amine, preferably the Nterminus 24 . The linker can be further optimized to enhance the biophysical properties of synthetic intermediates.…”

Section: Discussionmentioning

confidence: 93%

Synthesis of disulfide-rich heterodimeric peptides through an auxiliary N, N-crosslink

et al. 2018

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Insulins, relaxins, and other insulin-like peptides present a longstanding synthetic challenge due to their unique cysteine-rich heterodimeric structure. While their three disulfide signature is conserved within the insulin superfamily, sequences of the constituent chains exhibit considerable diversity. As a result, methods which rely on sequence-specific strategies fail to provide universal access to these important molecules. Biomimetic methods utilizing native and chemical linkers to tether the A-chain N-terminus to the B-chain Cterminus, entail complicated installation, and require a unique proteolytic site, or a two-step chemical release. Here we present a strategy employing a linkage of the A-and B-chains Ntermini offering unrestricted access to these targets. The approach utilizes a symmetrical linker which is released in a single chemical step. The simplicity, efficiency, and scope of the method are demonstrated in the synthesis of insulin, relaxin, a 4-disulfide insulin analog, two penicillamine-substituted insulins, and a prandial insulin lispro.

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“…In the present study, the change of a pair of lysine-mutated scFvs in scFv dimer showed different affinity and cytotoxicity, suggesting that the side chain of lysine residue was dominantly reacted; however, there is no critical evidence that only the γ-amino group was reacted. Recently, several specific reactions for N-terminus have been reported [38,39]. Utilization of the reaction for the capping of N-terminus would ensure the homogeneous formation of scF dimers.…”

Section: Discussionmentioning

confidence: 99%

Chemically Crosslinked Bispecific Antibodies for Cancer Therapy: Breaking from the Structural Restrictions of the Genetic Fusion Approach

Ueda

Umetsu

Nakanishi

et al. 2020

IJMS

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Antibodies are composed of structurally and functionally independent domains that can be used as building blocks to construct different types of chimeric protein-format molecules. However, the generally used genetic fusion and chemical approaches restrict the types of structures that can be formed and do not give an ideal degree of homogeneity. In this study, we combined mutation techniques with chemical conjugation to construct a variety of homogeneous bivalent and bispecific antibodies. First, building modules without lysine residues—which can be chemical conjugation sites—were generated by means of genetic mutation. Specific mutated residues in the lysine-free modules were then re-mutated to lysine residues. Chemical conjugation at the recovered lysine sites enabled the construction of homogeneous bivalent and bispecific antibodies from block modules that could not have been so arranged by genetic fusion approaches. Molecular evolution and bioinformatics techniques assisted in finding viable alternatives to the lysine residues that did not deactivate the block modules. Multiple candidates for re-mutation positions offer a wide variety of possible steric arrangements of block modules, and appropriate linkages between block modules can generate highly bioactive bispecific antibodies. Here, we propose the effectiveness of the lysine-free block module design for site-specific chemical conjugation to form a variety of types of homogeneous chimeric protein-format molecule with a finely tuned structure and function.

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Selective N-terminal functionalization of native peptides and proteins

Abstract: A highly site-selective modification of peptides/proteins with aldehydes or carbohydrates under mild conditions was achieved.

Cited by 126 publications

References 52 publications

Triazolecarbaldehyde Reagents for One‐Step N‐Terminal Protein Modification

Triazolecarbaldehyde Reagents for One‐Step N‐Terminal Protein Modification

Synthesis of disulfide-rich heterodimeric peptides through an auxiliary N, N-crosslink

Chemically Crosslinked Bispecific Antibodies for Cancer Therapy: Breaking from the Structural Restrictions of the Genetic Fusion Approach

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