Chemical Protein Synthesis by Chemoselective #x03B1;-Ketoacid–Hydroxylamine (KAHA) Ligations with 5-Oxaproline

Farnung, Jakob; Song, Haewon; Bode, Jeffrey W.

doi:10.1007/978-1-0716-1617-8_14

Cited by 3 publications

(3 citation statements)

References 23 publications

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“…Unfortunately, most popular chemical ligation strategies, including a variety of native chemical ligation methods, use reducing agents like tris(2-carboxyethyl)phosphine and thiol additives like 4-mercaptophenylacetic acid or sodium 2-mercaptoethanesulfonate, making these unsuitable for this synthetic approach to VxXXB. [18][19][20] Thus, we investigated enzyme (sortase), 21,22 hydrazone ligation, [23][24][25][26] CuAAC ligation 27,28 and KAHA ligation 16,29,30 approaches, which can form native amide bonds in the absence of reducing agents. Nevertheless, among these four methods, only KAHA ligation was successful in yielding the expected ligated VxXXB NC variants comprising one NTD and one CTD, with an overall yield of ∼10%.…”

Section: Rsc Medicinal Chemistry Research Articlementioning

confidence: 99%

Synthesis of full-length homodimer αD-VxXXB that targets human α7 nicotinic acetylcholine receptors

Abraham

Lewis

2022

RSC Med. Chem.

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Section: Rsc Medicinal Chemistry Research Articlementioning

confidence: 99%

Synthesis of full-length homodimer αD-VxXXB that targets human α7 nicotinic acetylcholine receptors

Abraham

Lewis

2022

RSC Med. Chem.

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“…In order to prepare Ub variants bearing a number of Lys surrogates in a more facile manner, we adapted our previously reported synthesis of Ub using the αketoacid-hydroxylamine (KAHA) ligation. [23] The key starting materials -peptide segments bearing a Cterminal α-ketoacids or N-terminal hydroxylaminescan be prepared, isolated, and stored 'ready-to-ligate', and Ub monomers assembled simply by mixing the appropriate segments as lyophilised powders in DMSO/aq. oxalic acid.…”

Section: Introductionmentioning

confidence: 99%

Atomic Tailoring of Ubiquitin Side Chains Influences E2‐Mediated Ubiquitin Chain Formation

Song,

Mikami,

Majima

et al. 2024

Helvetica Chimica Acta

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Ubiquitin (Ub) is a small, highly conserved protein essential for eukaryotic biology, and is unique in its formation of polyubiquitin chains by conjugation to one of its seven lysine side chains. Here we report that atomic tailoring of Ub side chains – i.e. the insertion, deletion, or replacement of specific atoms – has significant and unexpected consequences on the enzymatic conjugation of Ub oligomers by isopeptide bond formation mediated by E2 conjugating enzymes. These studies employed chemical synthesis and ligation methods to prepare numerous specifically tailored Ub monomers on multi‐milligram scales. While some modifications including N‐terminal acylation and methionine replacement did not affect protein folding or Ub chain formation with Ube2K, other modifications had a pronounced effect of oligomerization with Ubc13/Mms2. We observed that Ala46Hse mutation obliterates the ability of this Ub monomer to accept another Ub at Lys63 in Ubc13‐mediated conjugations. Exhaustive replacement of all seven lysines with shorter surrogates Orn, Dab, or Dap essentially blocks Ub chain formation, and in the case of Dap, precludes proper folding of the Ub protein.

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“…In order to prepare Ub variants bearing a number of Lys surrogates in a more facile manner, we adapted our previously reported synthesis of Ub using the α-ketoacid-hydroxylamine (KAHA) ligation. 22 The key starting materials -peptide segments bearing a C-terminal α-ketoacids or Nterminal hydroxylamines -can be prepared, isolated, and stored 'ready-to-ligate', and Ub monomers assembled simply by mixing the appropriate segments as lyophilised powders in DMSO/aq. oxalic acid.…”

Section: Introductionmentioning

confidence: 99%

Atomic Tailoring of Ubiquitin Side Chains Influences E2-Mediated Ubiquitin Chain Formation

Song

Mikami

Majima

et al. 2023

Preprint

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Ubiquitin (Ub) is a small, highly conserved protein essential for eukaryotic biology, and is unique in its formation of polyubiquitin chains by conjugation to one of its seven lysine side chains. Here we report that atomic tailoring of Ub side chains – i.e. the insertion, deletion, or replacement of specific atoms – has significant and unexpected consequences on the enzymatic conjugation of Ub oligomers by isopeptide bond formation mediated by E2 conjugating enzymes. These studies employed chemical synthesis and ligation methods to prepare numerous specifically tailored Ub monomers on multi-milligram scales. While some modifications including N-terminal acylation and methionine replacement did not affect protein folding or Ub chain formation with Ube2K, other modifications had a pronounced effect of oligomerization with Ubc13/Mms2. We observed that Ala46Hse mutation obliterates the ability of this Ub monomer to accept another Ub at Lys63 in Ubc13-mediated conjugations. Exhaustive replacement of all seven lysines with shorter surrogates Orn, Dab, or Dap essentially blocks Ub chain formation, and in the case of Dap, precludes proper folding of the Ub protein.

show abstract

Chemical Protein Synthesis by Chemoselective #x03B1;-Ketoacid–Hydroxylamine (KAHA) Ligations with 5-Oxaproline

Cited by 3 publications

References 23 publications

Synthesis of full-length homodimer αD-VxXXB that targets human α7 nicotinic acetylcholine receptors

Synthesis of full-length homodimer αD-VxXXB that targets human α7 nicotinic acetylcholine receptors

Atomic Tailoring of Ubiquitin Side Chains Influences E2‐Mediated Ubiquitin Chain Formation

Atomic Tailoring of Ubiquitin Side Chains Influences E2-Mediated Ubiquitin Chain Formation

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