Semisynthesis of Ubiquitin and SUMO-Rhodamine 110-Glycine through Aminolysis of Boc-Protected Thioester Counterparts

Fan, Jian; Ye, Yinshan; Chu, Guo‐Chao; Zhang, Zhongping; Fu, Yao; Li, Yiming; Shi, Jing

doi:10.1021/acs.joc.9b01529

Cited by 6 publications

(6 citation statements)

References 50 publications

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“…18 Synthesis of SUMO fluorogenic substrates was previously reported using a fully Boc-protected protein in DMSO and with HPLC purification of intermediates. 34 Our results indicate that fluorogenic SUMO reagents can also be directly obtained in pure form from SUMO C-terminal thioesters in aqueous reaction conditions in analogy to Ubiquitin-RhoG. 27 Moreover, they demonstrate that their native state can be retained during purification by ion exchange 35 instead of the previously employed reversed-phase chromatography, which circumvents organic solvents and refolding.…”

Section: Native Synthesis and Purification Of Fluorogenic Andmentioning

confidence: 79%

Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases

Zhao,

O’Dea,

Wendrich

et al. 2023

J. Am. Chem. Soc.

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Post-translational modifications with ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are regulated by isopeptidases termed deubiquitinases (DUBs) and Ubl proteases. Here, we describe a mild chemical method for the preparation of fluorescence polarization substrates for these enzymes that is based on the activation of C-terminal Ub/Ubl hydrazides to acyl azides and their subsequent functionalization to isopeptides. The procedure is complemented by native purification routes and thus circumvents the previous need for desulfurization and refolding. Its broad applicability was demonstrated by the generation of fully cleavable substrates for Ub, SUMO1, SUMO2, NEDD8, ISG15, and Fubi. We employed these reagents for the investigation of substrate specificities of human UCHL3, USPL1, USP2, USP7, USP16, USP18, and USP36. Pronounced selectivity of USPL1 for SUMO2/3 over SUMO1 was observed, which we rationalize with crystal structures and biochemical assays, revealing a SUMO paralogue specificity mechanism distinct from SENP family deSUMOylases. Moreover, we investigated the recently identified Fubi proteases USP16 and USP36 and found both to act as bona fide deFubiylases, harboring catalytic activity against isopeptide-linked Fubi. Surprisingly, we also noticed the activity of both enzymes toward ISG15, previously not identified in chemoproteomics, which makes USP16 and USP36 the first human DUBs with specific isopeptidase activity toward three distinct modifiers. The methods described here for the preparation of isopeptide-linked, fully folded substrates will aid in the characterization of further DUBs/Ubl proteases. More broadly, our findings highlight possible limitations associated with fluorogenic substrates and Ubl activity-based probes and stress the importance of isopeptide-containing reagents for validating isopeptidase activities and quantifying substrate specificities.

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Section: Native Synthesis and Purification Of Fluorogenic Andmentioning

confidence: 79%

Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases

Zhao,

O’Dea,

Wendrich

et al. 2023

J. Am. Chem. Soc.

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“…Note that in all these cases, the mono-and di-functionalized derivatives were isolated mainly in "closed" lactone. Recently, Jing Shi and coworkers [21] have successfully performed the condensation reaction of Rho110 and Boc glycine in DMF using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate (HATU) in the presence of N,N-diisopropylethylamine (DIPEA). We have tried similar conditions, involving the initial treatment of L 1 (4 equiv.)…”

Section: Synthesismentioning

confidence: 99%

Functionalization of Rhodamine Platforms with 3-Hydroxy-4-pyridinone Chelating Units and Its Fluorescence Behavior towards Fe(III)

et al. 2022

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Functionalization of xanthene fluorophores with specific receptor units is an important topic of research aiming for the development of new analytical tools for biological sciences, clinical diagnosis, food and environmental monitoring. Herein, we report a new dihydrorosamine containing two active amino groups, which was functionalized with 3-benzyloxy-1-(3′-carboxypropyl)-2-methyl-4-pyridinone through an amide coupling strategy. Benzylated mono- and di-functionalized dihydrorosamine derivatives (H in position 9 of the xanthene) were obtained, but with modest reaction yields, requiring long and laborious purification procedures. Looking for a more efficient approach, rhodamine 110 was selected to react with the carboxypropyl pyridinone, enabling the isolation of the corresponding mono- and di-functionalized derivatives in amounts that depend on the excess of pyridinone added to the reaction. The structure of all compounds was established by 1H and 13C NMR, MS (ESI) and their absorption and emission properties were evaluated in dichloromethane. The fluorescence behavior of the debenzylated mono-rhodamine 110 derivative in the presence of Fe(III) was studied, making it an interesting fluorogenic dye for future optical sensing applications.

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“…[α]D (20.0 °C, c = 1.0 in CHCl3) = -3.21°; 80% ee, from (R)-L2. According to the reported literature [20] , 115 was obtained from 114 as white solid (112.3 mg, 93%, 90:10 dr).…”

Section: Hplc Analysismentioning

confidence: 99%

“…According to the reported literature [20] , 122 was obtained from 121 as colorless oil (78.6 mg, 80% yield) [α]D (20.0 °C, c = 1.0 in CHCl3) = -14.59°; 92% ee, from (S,S)-L1.…”

Section: Hplc Analysismentioning

confidence: 99%

Catalytic Asymmetric Reductive Alkylation of Enamines to Chiral Aliphatic Amines

Wang¹,

Li²,

Nie³

et al. 2020

Preprint

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<p>Herein, we report a mild and general nickel-catalysed asymmetric reductive alkylation to effectively convert enamines—a class of important yet underexploited feedstock chemicals—into drug-like α-branched chiral amines and derivatives. This reaction involves the regio- and stereoselective hydrometallation of an enamine to generate a catalytic amount of enantioenriched alkylnickel intermediate, followed by C–C bond formation via alkyl electrophiles.</p>

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Semisynthesis of Ubiquitin and SUMO-Rhodamine 110-Glycine through Aminolysis of Boc-Protected Thioester Counterparts

Cited by 6 publications

References 50 publications

Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases

Native Semisynthesis of Isopeptide-Linked Substrates for Specificity Analysis of Deubiquitinases and Ubl Proteases

Functionalization of Rhodamine Platforms with 3-Hydroxy-4-pyridinone Chelating Units and Its Fluorescence Behavior towards Fe(III)

Catalytic Asymmetric Reductive Alkylation of Enamines to Chiral Aliphatic Amines

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