Fast and Stable N‐Terminal Cysteine Modification through Thiazolidino Boronate Mediated Acyl Transfer

Li, Kaicheng; Wang, Wenjian; Gao, Jianmin

doi:10.1002/ange.202000837

Cited by 16 publications

(9 citation statements)

References 28 publications

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“…Since the Michael adduct (3a)a nd the transcyclizationp roduct (4)p ossess the same exact mass, we also synthesized the compounds (together with 3b)a nd characterized them in detail via 1 Ha nd 13 CNMR spectroscopy to confirmt heir chemical identity.C ompound 3a was generated from 1a and 2 in MeOH to keep the free amino group protonated and avoid the ring closing reaction. In contrast, 4 was synthesized from the same educts, however in 100 mm PB solutiona tp H7.4.…”

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confidence: 99%

“…On the other hand, ac ross peak in the heteronuclear multiple bond correlation (HMBC) spectrum of the proton located at N2 (8.07 ppm) and C6 (186.07 ppm), in line with the newly formed bond. The presence of diastereomers in case of 3a was indicated by two 13 Cs ignals for each carbon atom. Notably, the retention time of both synthesized compoundsi nt he HPLC perfectlyf itted to the co-incubation experimentsi nF igure 3, confirming the peak assignment.…”

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confidence: 99%

“…The respective transcyclization can be achieved simplyb ya ne xtended incubation time in buffered solution.Int he last years alot of work was put into the discovery of SH conjugation moieties other than maleimides, which also increase the stability against retro-Michael reactions. For example exo-cyclic maleimides, [10] sulfones, [11] carbonylacrylic reagents [12] or 2-formylphenylboronic acids [13] (Figure S3). Fact is, however,t hat most of the thiol-coupling reagents commercially available are still common (alkyl) maleimides:f or example, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is one of the most popularl inkersf or antibody coupling, N-(g-maleimidobutyryloxy)succinimide ester (GMBS) or similard erivativesa re used for peptide coupling and dibenzoazacyclooctyne-maleimide (Mal-DBCO) for click chemistry or crosslinking reagentsw ith two or more maleimide moieties.…”

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confidence: 99%

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Improving the Stability of Maleimide–Thiol Conjugation for Drug Targeting

Lahnsteiner

Kastner

Mayr

et al. 2020

Chemistry A European J

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Maleimides are essential compounds for drug conjugation reactions via thiols to antibodies, peptides and other targeting units. However,o ne main drawback is the occurrence of thiol exchange reactions with, for example, glutathione resulting in loss of the targeting ability.A new strategyt oo vercome such retro-Michael exchange processes of maleimide-thiol conjugates by stabilization of the thiosuccinimide via at ranscyclization reactioni s presented. This reaction enablest he straightforward synthesis of stable maleimide-thiol adducts essential in drugconjugation applications.

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Improving the Stability of Maleimide–Thiol Conjugation for Drug Targeting

Lahnsteiner

Kastner

Mayr

et al. 2020

Chemistry A European J

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“…In conclusion, two-fold symmetric tridentate linchpins that contain aldehyde and two thiol-reactive groups enable a robust one-pot bicyclization of peptides SXnCXmC. Such libraries can be used to discover pro- 78 Finally, the lower symmetry of the TSL-style linkers allows their diversification with any chemotype of C2-symmetry. 71 It offers a significant expansion of the bicyclization repertoire beyond traditional architectures produced from three-fold symmetric cross-linkers.…”

Section: Resultsmentioning

confidence: 92%

Genetically-Encoded Discovery of Proteolytically Stable Bicyclic Inhibitors of Morphogen NODAL

Wong¹,

Mukherjee²,

Bilyk³

et al. 2021

Preprint

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In this manuscript, we developed a Two-fold Symmetric Linchpin (TSL) that converts readily available phage display peptides libraries made of 20 common amino acids to genetically-encoded libraries of bicyclic peptides displayed on phage. TSL combines an aldehyde-reactive group and two thiol-reactive groups; it bridges two side chains of cysteine [C] with an N-terminal aldehyde group derived from the N-terminal serine [S], yielding a novel bicyclic topology that lacks a free N-terminus. Phage display libraries of SX1CX2X3X4X5X6X7C sequences, where Xi is any amino acids but Cys, were converted to a library of bicyclic TSL-[S]X1[C]X2X3X4X5X6X7[C] peptides in 45 ± 15% yield. Using this library and protein morphogen NODAL as a target, we discovered bicyclic macrocycles that specifically antagonize NODAL-induced signaling in cancer cells. At a 10 µM concentration, two discovered bicyclic peptides completely suppressed NODAL-induced phosphorylation of SMAD2 in P19 embryonic carcinoma. The TSL-[S]Y[C]KRAHKN[C] bicycle inhibited NODAL-induced proliferation of NODAL-Tky-nu ovarian carcinoma cells with apparent IC50 1 µM. The same bicycle at 10 µM concentration did not affect the growth of the control Tky-nu cells. TSL-bicycles remained stable over the course of the 72 hour-long assays in a serum-rich cell-culture medium. We further observed general stability in mouse serum and in a mixture of proteases (PronaseTM) for 33 diverse bicyclic macrocycles of different ring sizes, amino acid sequences, and cross-linker geometries. TSL-constrained peptides expand the previously reported repertoire of phage display bicyclic architectures formed by cross-linking Cys side chains. We anticipate that it will aid the discovery of proteolytically stable bicyclic inhibitors for a variety of protein targets.

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“…As well as mono-sulfone PEG, alternatives to maleimide-PEG as the conjugate starting material include exocyclic maleimides ( Kalia et al, 2016 ), and carbonylacrylic reagents ( Bernardim et al, 2016 ). Also 2-formylphenylboronic acids ( Li et al, 2020 ) provide stable alternatives compared to maleimide-PEG.…”

Section: Discussionmentioning

confidence: 99%

Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit

et al. 2021

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In order to use a Hemoglobin Based Oxygen Carrier as an oxygen therapeutic or blood substitute, it is necessary to increase the size of the hemoglobin molecule to prevent rapid renal clearance. A common method uses maleimide PEGylation of sulfhydryls created by the reaction of 2-iminothiolane at surface lysines. However, this creates highly heterogenous mixtures of molecules. We recently engineered a hemoglobin with a single novel, reactive cysteine residue on the surface of the alpha subunit creating a single PEGylation site (βCys93Ala/αAla19Cys). This enabled homogenous PEGylation by maleimide-PEG with >80% efficiency and no discernible effect on protein function. However, maleimide-PEG adducts are subject to deconjugation via retro-Michael reactions and cross-conjugation to endogenous thiol species in vivo. We therefore compared our maleimide-PEG adduct with one created using a mono-sulfone-PEG less susceptible to deconjugation. Mono-sulfone-PEG underwent reaction at αAla19Cys hemoglobin with > 80% efficiency, although some side reactions were observed at higher PEG:hemoglobin ratios; the adduct bound oxygen with similar affinity and cooperativity as wild type hemoglobin. When directly compared to maleimide-PEG, the mono-sulfone-PEG adduct was significantly more stable when incubated at 37°C for seven days in the presence of 1 mM reduced glutathione. Hemoglobin treated with mono-sulfone-PEG retained > 90% of its conjugation, whereas for maleimide-PEG < 70% of the maleimide-PEG conjugate remained intact. Although maleimide-PEGylation is certainly stable enough for acute therapeutic use as an oxygen therapeutic, for pharmaceuticals intended for longer vascular retention (weeks-months), reagents such as mono-sulfone-PEG may be more appropriate.

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Fast and Stable N‐Terminal Cysteine Modification through Thiazolidino Boronate Mediated Acyl Transfer

Cited by 16 publications

References 28 publications

Improving the Stability of Maleimide–Thiol Conjugation for Drug Targeting

Improving the Stability of Maleimide–Thiol Conjugation for Drug Targeting

Genetically-Encoded Discovery of Proteolytically Stable Bicyclic Inhibitors of Morphogen NODAL

Stability of Maleimide-PEG and Mono-Sulfone-PEG Conjugation to a Novel Engineered Cysteine in the Human Hemoglobin Alpha Subunit

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