Chemical protein synthesis elucidates key modulation mechanism of the tyrosine-O-sulfation in inducing strengthened inhibitory activity of hirudin

Yang, Ye; Liang, Mingchan; Wang, Rui; He, Chunmao

doi:10.1016/j.cclet.2022.107806

Cited by 7 publications

(7 citation statements)

References 30 publications

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“…Nevertheless, the ligation between 26b and 27 went smoothly to afford the full-length protein 28 (Figures S66-67), albeit a bit slower (i.e., completed within 9 h), which agrees well with the reported method. [33] Interestingly, the HV1(1-27) segment did not undergo C-terminal lactamization (at Lys27) during the ligation, unlike the previous cases for peptides 9 and 16, which suggests that this side-reaction is rather sequence specific or partially suppressed by the formation of thiolactone. It's worth mentioning that, similar to reported observations, [33, 34b] the nP protecting group was removed automatically during the ligation process in the presence of imidazole.…”

Section: Total Synthesis Of the Sulfated Hirudin Variant 1 (Sy 63 -Hv...mentioning

confidence: 75%

“…The neopentyl (nP) protecting group of the sulfate was reported to be stable under typical Fmoc-SPPS and TFA cleavage conditions, [34] hence Fmoc-Tyr(SO 3 nP)-OH was chosen as the monomer. According to initial observations, the HV1(1-27) segment containing four internal Cys residues could form thiolactone almost immediately when the corresponding acyl pyrazole was activated with 3-methylpyrazole, imidazole, or MPAA [33] .…”

Section: Total Synthesis Of the Sulfated Hirudin Variant 1 (Sy 63 -Hv...mentioning

confidence: 99%

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Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis

Liao,

2023

Preprint

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Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS), more efficient ligation methods that circumvent the requirement of peptidyl thioester and thiol additive—which allow the following desulfurization or refolding in one pot—are urgently needed for the synthesis of more complex protein targets and in large quantities. Herein, we discover that the weak acyl donor peptidyl N-acylpyrazole can be activated by azole reagents like 3-methylpyrazole or imidazole to facilitate its ligation directly with an N-terminal cysteine peptide. As it requires no thioester and thiol additive, this ligation strategy can be conveniently combined with metal-free desulfurization (MFD) or oxidative protein folding to allow various one-pot protocols. The utility and generality of the strategy are showcased by the total synthesis of ubiquitin via an N-to-C sequential ligation-MFD strategy, the semi-synthesis of a copper protein azurin, and the efficient assembly of a sulfated hirudin variant and the cyclotide kalata B1, all in a one-pot fashion.

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Section: Total Synthesis Of the Sulfated Hirudin Variant 1 (Sy 63 -Hv...mentioning

confidence: 75%

Section: Total Synthesis Of the Sulfated Hirudin Variant 1 (Sy 63 -Hv...mentioning

confidence: 99%

Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis

Liao,

2023

Preprint

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“…S66 and S67 †), albeit a bit slower (i.e., completed within 9 h), which agrees well with the reported method. 33 Interestingly, the HV1(1-27) segment did not undergo C-terminal lactamization (at Lys27) during the ligation, unlike the previous cases for peptides 9 and 16, which suggests that this side-reaction is rather sequence specic or partially suppressed by the formation of thiolactone. It's worth mentioning that, similar to reported observations, 33,34b the nP protecting group was removed automatically during the ligation process in the presence of imidazole.…”

Section: Semi-synthesis Of Azurin Through a One-pot Expressed Protein...mentioning

confidence: 76%

“…32 CPS has recently evolved as an important tool in accessing sulfated proteins. 32 c ,33 Herein, we document our effort in applying the APCL strategy in the synthesis of the hirudin variant 1 from leech bearing the labile sY modification at Tyr63, sY 63 -HV1. For this purpose, the target protein was disconnected into two segments at Lys27–Cys28, and both were obtained by Fmoc-SPPS.…”

Section: Resultsmentioning

confidence: 99%

Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis

Liao,

2024

Chem. Sci.

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“…[14] The recent study of hirudin variant 1 (HV1) once again underscores the significance of tyrosine sulfation, highlighting its crucial role in enhancing the tight fit of Phe56 within the hydrophobic binding pocket of thrombin. [15] In addition, hirudin variant 1 from the leech H. medicinalis [4,16] and hirudin P6 from the leech H. manillensis [13,17] were the only two hirudin peptides among the hirudin family that have been studied upon their thrombin binding ability in their sulfated form. To further expand our knowledge of this, our aim is to study the different hirudin variants naturally secreted by different leech species by synthesizing chemically and modifying various C-terminus dodecapeptides with tyrosine sulfation and later comparing their thrombin binding and fibrinolytic activities.…”

Section: Introductionmentioning

confidence: 99%

Effect of Sulfotyrosine and Negatively Charged Amino Acid of Leech‐Derived Peptides on Binding and Inhibitory Activity Against Thrombin

Chen,

Shyur,

et al. 2023

ChemBioChem

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Hirudins, natural sulfo(glyco)proteins, are clinical anticoagulants that directly inhibit thrombin, a key coagulation factor. Their potent thrombin inhibition primarily results from antagonistic interactions with both the catalytic and non‐catalytic sites of thrombin. Hirudins often feature sulfate moieties on Tyr residues in their anionic C‐terminus region, enabling strong interactions with thrombin exosite‐I and effectively blocking its engagement with fibrinogen. Although sulfotyrosines have been identified in various hirudin variants, the precise relationship between sulfotyrosine and the number of negatively charged amino acids within the anionic‐rich C‐terminus peptide domain for the binding of thrombin has remained elusive. By using Fmoc‐SPPS, hirudin dodecapeptides homologous to the C‐terminus of hirudin variants from various leech species were successfully synthesized, and the effect of sulfotyrosine and the number of negatively charged amino acids on hirudin‐thrombin interactions was investigated. Our findings did not reveal any synergistic effect between an increasing number of sulfotyrosines or negatively charged amino acids and their inhibitory activity on thrombin or fibrinolysis in the assays, despite a higher binding level toward thrombin in the sulfated dodecapeptide Hnip_Hirudin was observed in SPR analysis.

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Chemical protein synthesis elucidates key modulation mechanism of the tyrosine-O-sulfation in inducing strengthened inhibitory activity of hirudin

Cited by 7 publications

References 30 publications

Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis

Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis

Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis

Effect of Sulfotyrosine and Negatively Charged Amino Acid of Leech‐Derived Peptides on Binding and Inhibitory Activity Against Thrombin

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