Peptide and Protein Cysteine Modification by Virtue of Highly Chemo-, Regio- and Stereoselective Hydrosulfuration of Ynamide

doi:10.21203/rs.3.rs-1982339/v1

Download PDF

Article

Peptide and Protein Cysteine Modification by Virtue of Highly Chemo-, Regio- and Stereoselective Hydrosulfuration of Ynamide

https://doi.org/10.21203/rs.3.rs-1982339/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

Chemoselective modification of peptides and proteins has wide applications in chemical biology and pharmaceutical development. A highly efficient chemo-, regio- and stereoselective hydrosulfuration of ynamide was developed and identified as an efficient strategy for peptides and proteins Cys modification. It proceeded efficiently in a slightly basic aqueous conditions (pH 8) to provide exclusively the Z-isomer of the corresponding conjugates with superior stability. All the reactive peptide side chain functional groups such as amino, carboxyl, primary amide, and hydroxyl groups, as well as the unprotected imidazole and indole NH are compatible. This method displayed a broad substrate scope including linear and cyclic peptides, proteins and antibody. The potential application of this method in peptide and protein chemical biology was further exemplified by Cys-bioconjugation with ynamides containing functional molecules including small molecular drugs, fluorescent and affinity tags. In addition, this strategy was also compatible with click chemistry (performed in one-pot), which remarkably extended its application. Furthermore, the chemoselective biotinylation of ubiquitin(G47C) variant with a biotinylated ynamide, as well as the regioselective modification of Cys14 and Cys38 in bovine pancreatic trypsin inhibitor (BPTI), Cys34 of BSA and the antibody (Trastuzumab), could be accomplished readily without perturbation of the other disulfide bonds. This method offered a novel and robust platform for peptides and proteins Cys modification and opened new horizons for the production of peptide/protein/antibody-drug conjugates.

Protein functions are greatly diversified and adjusted by various post-translational modifications (PTMs) which are intricately mediated by enzymes.^[1] However, it is still challenging to produce proteins with precise PTMs using biological techniques.^[2] To address such issue, chemists have developed a variety of chemical tools to mimic the function of some protein PTMs.^[3] Consequently, chemical modifications of peptides and proteins have made enormous impact for the developments of biotherapeutics^[4] as well as for basic biological studies.^[5] Despite the progress in the chemoselective bioconjugation of previously unexplored amino acid side chains, including serine (Ser),^[6] selenocysteine (Sec),^[7] histidine (His),^[8] methionine (Met),^[9] selenomethionine (Sem),^[10] tyrosine (Tyr)^[11] and tryptophan (Trp),^[12] nevertheless lysine (Lys)^[13] and cysteine (Cys)^[14] are still the preferred residues for chemical protein modification due to their unique nucleophilicity.^[15] While Lys is highly common in protein (make up ~6% of human proteins sequences^[16]), many of which are solvent exposed, Cys is one of the least frequent amino acids in proteins (~ 1.7%),^[17] especially those in the reduced form on the protein surfaces, which are readily accessible for chemical modifications. Various Cys-specific modifications have been developed, including Michael addition,^[18] hypervalent iodine-mediated reactions,^[19] metal-mediated reactions,^[20] nucleophilic substitution^[21] and radical transformations.^[22] Among these reactions, Michael addition is a widely used strategy to conjugate small molecules containing α,β-unsaturated systems, such as maleimides,^[23] to the proteins of interest both in vivo and in vitro.^[24] Although the rapid kinetics of these reactions has attracted considerable attention,^[25] two major drawbacks including the low chemoselectivity (see the Supporting Information (SI) for detailed chemoselectivity experiments, Figure S55-S65) and poor stability of the conjugated products in the presence of external thiols (like glutathione) plagued their applications in chemical biology (Figure 1a).^[26] Although recent innovative progresses including Loh’s allenamide (Figure 1b)^{[14b, 27]} and Hackenberger’s phosphorus containing electron-deficient alkynes^[28] mitigated the situation in some extent, challenges in the stereo- and regioselectivities, and the stability of the conjugated products,^[29] which are important for drug discovery,^[30] still remain (Figure 1c).^{[24b, 26a, 31]}

We recently have disclosed that ynamides could be used as a class of novel general coupling reagents for facilitating amide^[32] and ester^[33] bond formation by taking advantage of the α-addition of carboxylic acid to ynamides. During our study, we found that the nucleophilic α-addition of ynamides could be reversed to a β-addition by employing basic reaction conditions.^[34] Herein, we developed a highly chemo-, regio- and stereoselective hydrosulfuration of ynamide by virtue of the rare β-addition chemistry and successfully developed a novel and robust Cys-specific peptide and protein modification strategy on the basis of this unique reaction (Figure 1d).

Discovery of β-Hydrosulfuration of Ynamide. During our study of peptide and protein synthesis and modification,^{[7a, 32a]} we became interested in developing novel reactions which proceeded efficiently under mild reaction conditions and in a click manner. Our previous study led to the discovery of β-hydroamidation^[34a] and β-hydrophosphorylation^[34b] of ynamides via a very rare base-promoted β-addition. The simple and mild reaction conditions, excellent chemo-, regio- and stereoselectivities of base-promoted ynamide β-addition encouraged us to expand such chemistry to hydrosulfuration by employing thiols as the nucleophile. If successful, we might provide an efficient approach to construct S-[(Z)-2-aminovinyl]-D-cysteine (AviCys) subunit which is a unique motif widely found in lanthipeptide natural products and plays a crucial role in their potent antimicrobial or anticancer activity^[35]. Obviously, this method would also provide a novel and promising Cys modification strategy, which might address the notorious issues of current strategies.

Our study was initiated by employing t-butyl thiol (1a) and N-methylynetoluenesulfonamide (MYTsA, 2a) as model substrates. To our delight, the base promoted β-addition reaction proceeded smoothly to afford the target product (Z)-N-(2-(tert-butylthio) vinyl)-N,4-dimethyl-benzenesulfonamide 3a in 86% yield with excellent regio- and stereoselectivity (Z:E > 99:1) (Table 1, entry 1). Different solvent, base as well as the loading of base and thiol were screened to optimize the reaction conditions (Table 1). The best results were obtained when the reaction proceeded with 1.2 equiv of 1a and 1.5 equiv of Cs₂CO₃ in i-propanol at 37 °C (Table 1, entry 10).

With the optimized reaction conditions in hand, we explored the substrate scope of both ynamides and thiols. As shown in Table 2, both thiols (1a, 1c, 1e) and thiophenols (1b, 1d, 1f) were tolerated with terminal ynamide (2a-2f) to give the corresponding (Z)-products in good to excellent yields. The hydrosulfuration of ynamides with higher steric hindrance, including internal ynamides (2g-2i), also proceeded smoothly, albeit required prolonged reaction time (2d) and higher temperature (2g-2i). As expected, stereoselective modifications of protected single Cys (1g-1j) or Cys-containing short peptides (1j) were implemented with different ynamides (2a, 2e, 2j). Intriguingly, dipeptide containing AviCys^[35] analogous (3kk) could also be constructed easily. Notably, only Z-isomer of the conjugated products were detected in all cases (see the Supporting Information for details), illustrating an excellent stereoselectivity (Z:E > 99:1).

A plausible reaction mechanism was proposed to rationalize the excellent outcome of the β-hydrosulfuration of ynamides (Figure 2). Theoretically, there are two polarized resonance structures for ynamides due to the direct attachment of the electron-donating nitrogen atom to the triple bond and the electron-withdrawing group (EWG) on the nitrogen atom,^[30] both of which could react with nucleophiles to deliver the corresponding α- and β-addition products, respectively. Interestingly, the acid promoted α-addition of ynamide could be completely excluded under the basic reaction conditions,^[32b] thus guaranteeing excellent chemo- and regioselectivities. The base-promoted hydrosulfuration reaction proceeded via a nucleophilic addition of the thiolate anion, which forms readily under basic reaction conditions, to the β-site of ynamide.^[36] The exclusive formation of Z-isomer might be attributed to the fact that a favorable intermediate 1 with low free energy would be formed via the trans-addition (path a) and the following protonation afforded the Z-product (Figure 2). In contrast, unfavorable charge repulsion between the S lone-pair electrons and the negative charge of the formed carbanion at C_a in intermediate 2 led to higher free energy and inhibited the formation of E-isomer of the product via cis-addition (path b, Figure 2), which is in good agreement with the DFT calculations carried out by Hentz et al.^[34c] for the β-addition of endolate ions to ynamides.

Rational Design of Ynamide for Cys-Specific Modification of Peptides and Proteins. The robustness of the hydrosulfuration of ynamide made it a promising strategy for peptide and protein Cys modification. Previously, Yorimitsu^[37] and Castle^[38] studied the radical version of hydrosulfuration of ynamides by employing internal and terminal ynamides, respectively. However, the harsh radical reaction conditions and complex mechanism made it difficult to control the chemo- and stereoselectivity and rendered the radical hydrosulfuration not suitable for peptide and protein Cys modification (see the SI for details). On the contrary, our base promoted ynamide hydrosulfuration proceeded smoothly under simple and mild reaction conditions with excellent chemo-, regio- and stereoselectivities. In addition, our preliminary study suggested that this hydrosulfuration could also be performed in aqueous media to modify Cys residues in peptides and proteins. To this end, a model dipeptide Boc-Ala-Cys-OEt (4a, 1 mM) and MYTsA (2a, 2 equiv) were mixed in MeCN/phosphate buffer (PB) (1:1, pH 8). Delightfully, the conjugation reaction proceeded smoothly to give the target conjugate 5a in 85% yield with excellent stereoselectivity (Z/E > 99:1) in the absence of any other reagent, which verified the feasibility for peptide Cys modification in buffer media. However, the sluggish kinetics (reaction time of ~24 h) suggested that a rational design of the ynamide reagent was necessary to fulfill its application for protein modification (Table 3). Next, a broad range of ynamides were synthesized and evaluated, and the representative results were summarized in Table 3. Ynamide containing a more hydrophobic p-^tBu (2l) on the phenyl group was inert under the optimized reaction conditions, presumably owning to its poor solubility in aqueous media. To address this issue, R² substituent (methyl group in 2a) was replaced by a 2-hydroxyethyl group (2m) with a hope of improving the solubility. Indeed, the reaction of 2m proceeded smoothly in phosphate buffer with 30% MeCN as a co-solvent and afforded the target product 5am in 89% isolated yield. Clearly, the EWG on the nitrogen atom of ynamide plays a crucial role for controlling the reactivity of ynamide, and a stronger EWG would be beneficial to increase its activity for acting as an acceptor for nucleophiles (Figure 2). Thus, the p-toluenesulfonyl (Ts) was replaced by a stronger electron-withdrawing and more hydrophilic trifluoromethylsulfonyl group (triflyl, Tf) to offer a novel ynamide with a more electron-deficient C-C triple bond (2n). Using this ynamide, the conjugation reaction was completed within 20 min in phosphate buffer with only 5% MeCN as a co-solvent, giving a single product in 95% isolated yield, which was identified as the Z isomer by NMR and HPLC (Z:E > 99:1, see zoom in ¹H NMR spectra in Table 3 and further details in SI). Similarly, ynamides (2o) and (2p) with different functional groups showed comparable efficiency, which further implied the potential diversification of ynamides via functionalization of R².

Optimization of the Reaction Conditions. With the optimal ynamide (2n) in hand, we tested its reactivity under different reaction conditions (Table 4). Varying pH conditions (entries 1−4, Figure S10) suggested that the reaction was sensitive to pH, and was sluggish under neutral conditions (pH 6-7.5), but highly efficient at slight basic reaction condition (pH 8), which was consistent with our expectation. Screening for the loading of the ynamide 2n (entries 4−6, Figure S11) suggested that the conjugation reaction could be completed within 5 min with only 5.0 equiv (entry 6, Figure S11). Varying the concentration of the reagents (entries 8−11, Figure S12) revealed that a comparable efficiency could be retained when it was performed at as low as 10 µM of peptide 4a (entry 11, Figure S12). Furthermore, when the reaction was carried out in the presence of different types of radical scavengers, no negative effect on the reaction efficacy was observed and thus excluding a radical mechanism for this transformation (see the SI for detailed experiments, Scheme S5-S6, Figure S66).

Chemoselectivity Study. With the established reaction conditions in hand, we moved forward to investigate the chemoselectivity of this strategy in the presence of other unprotected amino acid side chains (Table 5a). When Asp and Glu containing tripeptides (4b and 4c) were treated with 2n under the standard reaction conditions, no reaction could be detected even after extended reaction times (5 h), indicating that the normal α-addition of carboxylic acids to ynamides was completely inhibited.^[32b] This was attributed to the fact that basic reaction condition deprotonated carboxyl group and thus prohibited the protonation initiated α-addition of carboxylic acid to ynamide^[32b]. Furthermore, considering that the potential β-hydroamidation of ynamides with indole^[34c] and amides^[34a] in the presence of strong base,tripeptides containing histidine (His, 4d), lysine (Lys, 4e), arginine (Arg, 4f) and tryptophan (Trp, 4g) were investigated. We were delighted to find that these side chains were all kept intact under our mild reaction conditions. Nucleophilic serine (Ser, 4h) and tyrosine (Tyr, 4i) were also compatible under our optimized reaction conditions.

Next, the chemoselective modification of Cys was performed in the presence of all possible reactive side chains (4j and 4l). Notably, the reactions of 4j and 4l were completed within 20 min and 2 h, respectively, providing a single conjugated product in excellent yields, and the modification site was confirmed to be the Cys residue by trypsin digestion of 5ln (Figure S25). As expected, no modification could be detected when Cys was substituted with Met (4k). All the chemoselectivity study toward the side chain functional groups in peptides demonstrated that the base promoted β-addition of ynamide was orthogonal to the thiol group of Cys.

The Scope of Peptides in the Reaction. The robustness of this methodology was further evaluated with complex peptide substrates (4m-4q, Table 5b and 5c). Reduced glutathione (GSH, 4m) could be modified by ynamide 2n in excellent yield under the standard reaction conditions. Further, while oxidized oxytocin was inert, the reduced oxytocin (4n), containing a terminal Cys and an internal Cys, could be bis-functionalized in excellent yield within 20 min, indicating that the position of Cys in the sequence did not affect the efficiency of the modification.

Moreover, a potent αVβ3 integrin-binding cyclic peptide^[21e] (4o) was employed in this transformation to provide the desired Cys-specific modified product in 76% yield. Finally, two peptides with highly positively and negatively charged sequences, (4p) and (4q), respectively, were selected as key examples to evaluate the influence of electrostatic effect on this transformation. Both Cys-specific modifications of 4p and 4q proceeded smoothly under the standard conditions, illustrating that highly charged peptides did not have negative effect on the reaction.

Peptide Modification by Ynamide-Based Biologically Relevant Molecules. To demonstrate the applicability of this strategy in peptide and protein chemical biology, different biologically relevant functional moieties, including fluorescent tag (coumarin, 2q), complex drug molecule (indomethacin, 2r), affinity tag (biotin, 2s) and alkyne handle for bioconjugation (2t), were introduced to ynamides (see SI for details) and evaluated by employing the Cys containing peptides (Table 6). The Cys-selective modification of protected peptide (4a), cyclic peptide (4o) and unprotected peptide (4r) with a variety of important ynamide-based functional molecules (2q-2t) proceeded smoothly in buffer with 10%-50% MeCN as the co-solvent (for solubility reasons) to afford the desired products in excellent yields (Table 6).

One-Pot Dual Functionalization of Cys with Click Chemistry. Considering the great importance of click reaction^[39], the compatibility of the Cys-specific modification with click reaction was studied. As shown in Figure 3, the Cys of peptide 4l was first modified efficiently by ynamide 2t to give the Z-conjugated product 5lt containing an additional C-C triple bond (Figure 3). To our delight, 5lt took part in the proceeding click reaction with biotin-PEG₃-azide (2u) readily to anchor a biotin tag to the peptide in a one-pot manner furnishing the target dual functionalized product (5lu) in 90% yield (Figure 3). The excellent efficiency of such one-pot, two-step dual functionalization unambiguously illustrated the potential application and flexibility of this strategy to anchor a biotin tag or other useful functional groups to a peptide of interest.

Stability Study of the Peptide Conjugates. The stability of the conjugated products is utmost important for developing a powerful peptide and protein modification strategy. To examine the stability of the ynamide-modified peptides under different conditions, we incubated product 5an (1 mM) in four different buffer solutions at pH 2, 8, 10 and 13. The modified peptides were stable under these conditions and kept intact after 10 h (Figure 4, Figure S50). Furthermore, the ynamide modified peptide, 5an (10 µM), did not undergo β-syn elimination, which was commonly encountered in other methods when treated with large excess of H₂O₂ (at pH 4, 8 and 10 at 37 °C, Figure 4), even after extended time (24 h, Figure S52). Further, 5an remained intact after 10 h of incubation in bovine serum (Figure 4, Figure S53). Moreover, the cleavage of 5am in the presence of external thiol to regenerate the unmodified 1a, which occurred in other methods^{[14a, 26a]} was not detected in our case (2-mercaptoethanol, Figure S51). The remarkable stability of the modified products further supports the potential application of this chemical approach in peptide and protein conjugations.

Modification of Cys-Containing Proteins and Antibody. Encouraged by the excellent results on peptides, we went further to investigate the application of ynamide hydrosulfuration for protein Cys modification. Biotinylated ynamide (2s) was readily prepared (see SI) and used as a model substrate for protein modification. A ubiquitin(G47C) variant (6, 250 µmol, prepared by chemical protein synthesis, see the SI for details)^[40] was treated with 5 equiv of 2s at pH 8 and 37 °C, to afford the biotin-labeled ubiquitin(G47C*) (7) in 43% isolated yield after 12 h (Figure 5a, and Figure S44 in the SI). In contrast, no modification of ubiquitin(G47A) variant^[7a] was observed under the same reaction conditions even after 18 h, confirming the Cys selectivity. The efficient chemo-, regio- and stereoselective biotinylation of ubiquitin(G47C) variant (6) unambiguously illustrated the potential application of this strategy in the construction of protein conjugates. Furthermore, the modification of bovine pancreatic trypsin inhibitor (BPTI), a 58-residue Cys-rich protein with three disulfide bonds, was also examined.^{[40d, 41]} First, BPTI (8, 250 µmol) was treated with 1.5 equiv of tris(2-carboxyethyl)phosphine (TCEP), which selectively reduced the partly solvent exposed Cys14-Cys38 disulfide bond,^[41d] leaving the other two disulfide bonds intact, followed by the addition of 10 equiv of 2n/2s, separately. After 12 h, the corresponding bis-modified proteins (9a and 9b) were isolated in 21% and 29% yields, respectively (Figure 5b). Although 8% oxidation of the reduced Cys residues to form disulfide bonds (dimer of 6 and native 8) were detected in both cases, no perturbation on other disulfides of BPTI was observed. BSA is another disulfide-rich protein, with 17 disulfide bonds and one free Cys residue (C34). When BSA was treated with ynamide 2s, only the monoadduct was observed as judged by MALDI-TOF (Figure 5c). The compatibility of disulfide bond in this transformation implied the potential applications of this protocol in complex biological systems. For example, Her2-targeting IgG therapeutic, the antibody tratsuzumab^[42] (12) was chosen as a model to probe the feasibility of using ynamide for antibody-drug conjugates (ADC) production. The conjugation was conducted using a typical reduction-addition procedure^[28a]. 40 mins of reduction with 10 equiv of TCEP at 37 ˚C was performed to unlock the solvent-exposed interchain disulfide bridges. The reduced antibody was then incubated with prototypical ynamide 2n at 37 ˚C for 20 h, while the heterogeneous N-glycan was removed to simplify the analysis. Labelling was confirmed according to LC-MS analysis (Figure 5d). A labeling degree of 3.7 per antibody could be observed, including one ynamide (2n, +217.5 Da, LC₁) labeling in light chain, as well as one ynamide (+215.2 Da, HC₁), two ynamides (+433.5 Da, HC₂) and three ynamides (+651.5 Da, HC₃) labeling in the heavy chain (Figure 5d). In a control reaction without TCEP reduction, no antibody labeling was observed (Figure S54), showing the high chemo-selectivity of ynamides towards Cys residues. Considering I. the high chemo-, regio-, and stereoselectivity of the reaction and II. the great stability of the formed C-C bond, ynamides hold great potential in ADC-based therapeutics.

In summary, we have developed a novel chemo-, regio- and stereoselective base promoted ynamide β-hydrosulfuration, and successfully developed a peptide and protein Cys modification strategy based on such unique chemistry. The three flexible sites on ynamides, including the alkynyl moiety and two substituents on the nitrogen atom, offered great chemical space for optimization and diversification. The strong electron-withdrawing triflyl group played a crucial role, which guaranteed the stability and improves the β-addition reactivity of ynamide towards Cys. In addition, the other substituent on the nitrogen atom offers a handle for functional expansion with various functional moeities. This method exhibited excellent chemoselectivity for thiol group in the presence of other peptide side chains including carboxylate, free amino, primary amide, and hydroxyl groups, as well as NH of imidazole and indole. The ruboustness of this method in chemical biology was exemplified by applications in chemical modifications of peptides (both linear and cyclic peptides) and proteins (a Cys-containing ubiquitin variant, partly reduced BPTI, BSA and antibody). Furthermore, ynamide-modified peptide conjugates displayed remarkable stability under rigorous conditions, including acidic, basic, and oxidative conditions, as well as competition with external thiols, which is an important advantage over maleimides and other related strategies. This study provides a useful chemical tool for peptide and protein precise modification for future drug discovery and development. Further exploration of the manipulation of peptides and proteins in vivo and in vitro with ynamides is underway in our laboratories.

ASSOCIATED CONTENT

The Supporting Information is available free of charge on the ACS Publications website at DOI:

Materials and methods, experimental procedures, characterization, HPLC, and LC-MS data, as well as HRMS,¹H and ¹³C NMR spectra (PDF).

AUTHOR INFORMATION

Corresponding Authors

Junfeng Zhao -- Key Laboratory of Molecular Target & Clinical Pharmacology and the NMPA & State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences & The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436, Guangdong, P. R. China; orcid.org/0000-0003-4843-4871; [email protected]

Norman Metanis -- Institute of Chemistry, The Center for Nanoscience and Nanotechnology, Casali Center for Applied Chemistry, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel; [email protected]

Author Contributions

†C. W., Z. Z., R. G. and Z. D. contributed equally.

Authors

Changliu Wang -- Key Laboratory of Molecular Target & Clinical Pharmacology and the NMPA & State Key Laboratory of Respiratory Disease, School of Pharmaceutical Sciences & The Fifth Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436, Guangdong, P. R. China; National Research Center for Carbohydrate Synthesis, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, Jiangxi, P. R. China

Zhenguang Zhao -- Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel

Reem Ghadir -- Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel

Zhe Ding -- National Research Center for Carbohydrate Synthesis, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, Jiangxi, P. R. China

Yuan Cao -- Department of Process Development, BeiGene Guangzhou Biologics Manufacturing Co., Ltd., Guangzhou 510700, Guangdong, P. R. China.

Yuqing Li -- National Research Center for Carbohydrate Synthesis, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, Jiangxi, P. R. China

Frank Qiu -- Department of Process Development, BeiGene Guangzhou Biologics Manufacturing Co., Ltd., Guangzhou 510700, China.

Yujie Zhang -- Department of Process Development, BeiGene Guangzhou Biologics Manufacturing Co., Ltd., Guangzhou 510700, China.

Yongli Zhao -- National Research Center for Carbohydrate Synthesis, College of Chemistry and Chemical Engineering, Jiangxi Normal University, Nanchang 330022, Jiangxi, P. R. China

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

This work was supported by the National Natural Science Foundation of China (91853114, 21778025, 22067008) and the Natural Science Foundation of Jiangxi Province (20202ACBL203004). N.M. acknowledges financial support from the Israel Science Foundation (783/18). Z.Z. is grateful for a CSC fellowship, R.G. acknowledge the support of the Neubauer PhD fellowship.

[1] Walsh, C. T., Garneau-Tsodikova, S. & Gatto Jr., G. J. Protein Posttranslational Modifications: The Chemistry of Proteome Diversifications. Angew. Chem. Int. Ed. 44, 7342-7372 (2005).

[2] a) Kent, S. B. Total chemical synthesis of proteins. Chem. Soc. Rev. 38, 338-351 (2009); b) Kent, S. Chemical protein synthesis: Inventing synthetic methods to decipher how proteins work. Bioorg. Med. Chem. 25, 4926-4937 (2017).

[3] a) Hoyt, E. A., Cal, P. M. S. D., Oliveira, B. L. & Bernardes, G. J. L. Contemporary approaches to site-selective protein modification. Nat. Rev. Chem. 3, 147-171 (2019); b) Wright, T. H. et al. Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity. Science 354, agg1465 (2016).

[4] a) Hsieh-Wilson, L. C. & Griffin, M. E. Improving Biologic Drugs via Total Chemical Synthesis. Science 342, 1332-1333 (2013); b) Liu, Y. et al. Development and application of novel electrophilic warheads in target identification and drug discovery. Biochem. Pharmacol. 190, 114636 (2021).

[5] a) Tamura, T. & Hamachi, I. Chemistry for Covalent Modification of Endogenous/Native Proteins: From Test Tubes to Complex Biological Systems. J. Am. Chem. Soc. 141, 2782-2799 (2019); b) Shiraiwa, K., Cheng, R., Nonaka, H., Tamura, T. & Hamachi, I. Chemical Tools for Endogenous Protein Labeling and Profiling. Cell Chem. Biol. 27, 970-985 (2020).

[6] Vantourout, J. C. et al. Serine-Selective Bioconjugation. J. Am. Chem. Soc. 142, 17236-17242 (2020).

[7] a) Zhao, Z., Shimon, D. & Metanis, N. Chemoselective Copper-Mediated Modification of Selenocysteines in Peptides and Proteins. J. Am. Chem. Soc. 143, 12817-12824 (2021); b) Cohen, D. T., Zhang, C., Pentelute, B. L. & Buchwald, S. L. An Umpolung Approach for the Chemoselective Arylation of Selenocysteine in Unprotected Peptides. J. Am. Chem. Soc. 137, 9784-9787 (2015); c) Cohen, D. T. et al. A chemoselective strategy for late-stage functionalization of complex small molecules with polypeptides and proteins. Nat. Chem. 11, 78-85 (2019).

[8] Jia, S., He, D. & Chang, C. J. Bioinspired Thiophosphorodichloridate Reagents for Chemoselective Histidine Bioconjugation. J. Am. Chem. Soc. 141, 7294-7301 (2019).

[9] a) Lin, S. et al. Redox-based reagents for chemoselective methionine bioconjugation. Science 355, 597-602, (2017); b) Taylor, M. T., Nelson, J. E., Suero, M. G. & Gaunt, M. J. A protein functionalization platform based on selective reactions at methionine residues. Nature 562, 563-568 (2018); c) Kim, J. et al. Site-Selective Functionalization of Methionine Residues via Photoredox Catalysis. J. Am. Chem. Soc. 142, 21260-21266 (2020); d) Zang, J., Chen, Y., Zhu, W. & Lin, S. Chemoselective Methionine Bioconjugation on a Polypeptide, Protein, and Proteome. Biochemistry (Mosc.) 59, 132-138 (2020).

[10] Flood, D. T. et al. Selenomethionine as an expressible handle for bioconjugations. Proc. Natl. Acad. Sci. U. S. A. 118, e2005164118 (2021).

[11] a) Alvarez, D., Deniaud, D., Mevel, M. & Gouin, S. G. Tyrosine Conjugation Methods for Protein Labelling. Chem. Eur. J. 26, 14257-14269 (2020); b) Sato, S. et al. Site-Selective Protein Chemical Modification of Exposed Tyrosine Residues Using Tyrosine Click Reaction. Bioconjugate Chem. 31, 1417-1424 (2020); c) Sato, S. et al. Site-Selective Protein Chemical Modification of Exposed Tyrosine Residues Using Tyrosine Click Reaction. Bioconjugate Chem. 31, 1417-1424 (2020); d) Li, B. X. et al. Site-selective tyrosine bioconjugation via photoredox catalysis for native-to-bioorthogonal protein transformation. Nat. Chem. 13, 902-908 (2021); e) Choi, E. J., Jung, D., Kim, J. S., Lee, Y. & Kim, B. M. Chemoselective Tyrosine Bioconjugation through Sulfate Click Reaction. Chem. Eur. J. 24, 10948-10952 (2018).

[12] a) Tower, S. J., Hetcher, W. J., Myers, T. E., Kuehl, N. J. & Taylor, M. T. Selective Modification of Tryptophan Residues in Peptides and Proteins Using a Biomimetic Electron Transfer Process. J. Am. Chem. Soc. 142, 9112-9118 (2020); b) Hansen, M. B., Hubalek, F., Skrydstrup, T. & Hoeg-Jensen, T. Chemo- and Regioselective Ethynylation of Tryptophan-Containing Peptides and Proteins. Chem. Eur. J. 22, 1572-1576 (2016).

27 Seki, Y. et al. Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins. J. Am. Chem. Soc. 138, 10798-10801 (2016); c) Seki, Y. et al. Transition Metal-Free Tryptophan-Selective Bioconjugation of Proteins. J. Am. Chem. Soc. 138, 10798-10801 (2016).

[13] a) Dempsey, D. R., Jiang, H., Kalin, J. H., Chen, Z. & Cole, P. A. Site-Specific Protein Labeling with N-Hydroxysuccinimide-Esters and the Analysis of Ubiquitin Ligase Mechanisms. J. Am. Chem. Soc. 140, 9374-9378 (2018).; b) Matos, M. J. et al. Chemo- and Regioselective Lysine Modification on Native Proteins. J. Am. Chem. Soc. 140, 4004-4017 (2018); c) Apel, C., Kasper, M. A., Stieger, C. E., Hackenberger, C. P. R. & Christmann, M. Protein Modification of Lysine with 2-(2-Styrylcyclopropyl)ethanal. Org. Lett. 21, 10043-10047 (2019); d) Adusumalli, S. R. et al. Chemoselective and Site-Selective Lysine-Directed Lysine Modification Enables Single-Site Labeling of Native Proteins. Angew. Chem. Int. Ed. 59, 10332-10336 (2020); e) Hymel, D. & Liu, F. Selective Lysine Modification Enabled by Intramolecular Acyl Transfer. Org. Lett. 22, 3067-3071 (2020); f) Liu, J. et al. A Genetically Encoded Fluorosulfonyloxybenzoyl-l-lysine for Expansive Covalent Bonding of Proteins via SuFEx Chemistry. J. Am. Chem. Soc. 143, 10341-10351 (2021).

[14] a) Ochtrop, P. & Hackenberger, C. P. R. Recent advances of thiol-selective bioconjugation reactions. Curr. Opin. Chem. Biol. 58, 28-36 (2020); b) Abbas, A., Xing, B. & Loh, T.-P. Allenamides as Orthogonal Handles for Selective Modification of Cysteine in Peptides and Proteins. Angew. Chem. Int. Ed. 53, 7491-7494 (2014); c) Kang, M. S., Kong, T. W. S., Khoo, J. Y. X. & Loh, T.-P. Recent developments in chemical conjugation strategies targeting native amino acids in proteins and their applications in antibody–drug conjugates. Chem. Sci. 12, 13613-13647 (2021).

[15] a) Deng, J. R. et al. Chemoselective and photocleavable cysteine modification of peptides and proteins using isoxazoliniums. Commun. Chem. 2, 93 (2019); b) Reddy, N. C., Kumar, M., Molla, R. & Rai, V. Chemical methods for modification of proteins. Org. Biomol. Chem. 18, 4669-4691 (2020).

[16] Hacker, S. M. et al. Global profiling of lysine reactivity and ligandability in the human proteome. Nat. Chem. 9, 1181-1190 (2017).

[17] Marino, S. M. & Gladyshev, V. N. Cysteine function governs its conservation and degeneration and restricts its utilization on protein surfaces. J. Mol. Biol. 404, 902-916 (2010).

[18] a) Bernardim, B. et al. Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents. Nat. Commun. 7, 13128 (2016); b) Seki, H. et al. Rapid and robust cysteine bioconjugation with vinylheteroarenes. Chem. Sci. 12, 9060-9068 (2021); c) Zhang, Y. et al. Thiol Specific and Tracelessly Removable Bioconjugation via Michael Addition to 5-Methylene Pyrrolones. J. Am. Chem. Soc. 139, 6146-6151 (2017); d) Shi, M. et al. A Protein-Binding Molecular Photothermal Agent for Tumor Ablation. Angewandte Chemie International Edition 60, 13564-13568 (2021); e) S. Ariyasu, S., Hayashi, H., Xing, B. & Chiba, S. Site-Specific Dual Functionalization of Cysteine Residue in Peptides and Proteins with 2-Azidoacrylates. Bioconjugate Chem. 28, 897-902 (2017); f) Seki, H. Walsh, S. J. Bargh, J. D. Parker, J. S. Carroll, J. & Spring, D. R. Rapid and robust cysteine bioconjugation with vinylheteroarenes. Chem. Sci. 12, 9060-9068 (2021)..

[19] a) Allouche, E. M. D., Grinhagena, E. & Waser, J. Hypervalent Iodine-Mediated Late-Stage Peptide and Protein Functionalization. Angew. Chem. Int. Ed. 61, e202112287 (2022); b) Byrne, S. A. et al. Late-stage modification of peptides and proteins at cysteine with diaryliodonium salts. Chem. Sci. 12, 14159-14166 (2021).

[20] a) Messina, M. S. et al. Organometallic Gold(III) Reagents for Cysteine Arylation. J. Am. Chem. Soc. 140, 7065-7069 (2018); b) Vinogradova, E. V., Zhang, C., Spokoyny, A. M., Pentelute, B. L. & Buchwald, S. L. Organometallic palladium reagents for cysteine bioconjugation. Nature 526, 687-691 (2015); c) Rodriguez, J. & Martinez-Calvo, M. Transition-Metal-Mediated Modification of Biomolecules. Chem. Eur. J. 26, 9792-9813 (2020); d) Schlatzer, T. et al. Labeling and Natural Post-Translational Modification of Peptides and Proteins via Chemoselective Pd-Catalyzed Prenylation of Cysteine. J. Am. Chem. Soc. 141, 14931-14937 (2019).

[21] a) Embaby, A. M., Schoffelen, S., Kofoed, C., Meldal, M. & Diness, F. Rational Tuning of Fluorobenzene Probes for Cysteine-Selective Protein Modification. Angew. Chem. Int. Ed. 57, 8022-8026 (2018); b) Zhang, C., Vinogradova, E. V., Spokoyny, A. M., Buchwald, S. L. & Pentelute, B. L. Arylation Chemistry for Bioconjugation. Angew. Chem. Int. Ed. 58, 4810-4839 (2019); c) Xu, L., Silva, M., Gois, P. M. P., Kuan, S. L. & Weil, T. Chemoselective cysteine or disulfide modification via single atom substitution in chloromethyl acryl reagents. Chem Sci 12, 13321-13330 (2021); d) Zhang, Y. et al. Cysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylene pyrrolones. Nat. Commun. 11, 1015 (2020); e) Wan, L. Q. et al. Nonenzymatic Stereoselective S-Glycosylation of Polypeptides and Proteins. J. Am. Chem. Soc. 143, 11919-11926 (2021); f) Chu, G.-C. et al. Cysteine-Aminoethylation-Assisted Chemical Ubiquitination of Recombinant Histones. J. Am. Chem. Soc. 141, 3654-3663 (2019).

[22] a) Choi, H., Kim, M., Jang, J. & Hong, S. Visible-Light-Induced Cysteine-Specific Bioconjugation: Biocompatible Thiol-Ene Click Chemistry. Angew. Chem. Int. Ed. 59, 22514-22522 (2020); b) Sato, S. & Nakamura, H. Protein Chemical Labeling Using Biomimetic Radical Chemistry. Molecules 24, 3980 (2019); c) Hu, K. et al. An In-tether Chiral Center Modulates the Helicity, Cell Permeability, and Target Binding Affinity of a Peptide. Angew Chem Int Ed 55, 8013-8017 (2016); d) McLean, J. T., Benny, A., Nolan, M. D., Swinand, G. & Scanlan, E. M. Cysteinyl radicals in chemical synthesis and in nature. Chem. Soc. Rev. 50, 10857-10894 (2021); e) Arumugam, S. Guo, J. Mbua, N. E. Friscourt, F. Lin, N. Nekongo, E. Boons, G.-J. & Popik, V. V. Selective and reversible photochemical derivatization of cysteine residues in peptides and proteins. Chem. Sci. 5, 1591-1598 (2014).

[23] a) Moore, J. E. & Ward, W. H. Cross-linking of Bovine Plasma Albumin and Wool Keratin. J. Am. Chem. Soc. 78, 2414-2418, (2002); b) Kalia, D., Malekar, P. V. & Parthasarathy, M. Exocyclic Olefinic Maleimides: Synthesis and Application for Stable and Thiol-Selective Bioconjugation. Angew. Chem. Int. Ed. 55, 1432-1435 (2016).

[24] a) Chalker, J. M., Bernardes, G. J., Lin, Y. A. & Davis, B. G. Chemical modification of proteins at cysteine: opportunities in chemistry and biology. Chem. Asian J. 4, 630-640 (2009); b) Gunnoo, S. B. & Madder, A. Chemical Protein Modification through Cysteine. Chembiochem 17, 529-553 (2016); c) Xu, L., Kuan, S. L. & Weil, T. Contemporary Approaches for Site-Selective Dual Functionalization of Proteins. Angew. Chem. Int. Ed. 60, 13757-13777 (2021); d) Baumann, A. L. et al. Chemically Induced Vinylphosphonothiolate Electrophiles for Thiol-Thiol Bioconjugations. J. Am. Chem. Soc. 142, 9544-9552 (2020).

[25] Sauerland, M. et al. Kinetic assessment of Michael addition reactions of alpha, beta-unsaturated carbonyl compounds to amino acid and protein thiols. Free Radic. Biol. Med. 169, 1-11 (2021).

[26] a) Shiu, H. Y. et al. Electron-deficient alkynes as cleavable reagents for the modification of cysteine-containing peptides in aqueous medium. Chem. Eur. J. 15, 3839-3850 (2009); b) Yu, J., Yang, X., Sun, Y. & Yin, Z. Highly Reactive and Tracelessly Cleavable Cysteine-Specific Modification of Proteins via 4-Substituted Cyclopentenone. Angew. Chem. Int. Ed. 57, 11598-11602 (2018); c) Shen, B. Q. et al. Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates. Nat. Biotechnol. 30, 184-189 (2012).

[27] Cameron, A. J., Harris, P. W. R. & Brimble, M. A. On-Resin Preparation of Allenamidyl Peptides: A Versatile Chemoselective Conjugation and Intramolecular Cyclisation Tool. Angew Chem Int Ed 59, 18054-18061 (2020).

[28] a) Kasper, M. A. et al. Cysteine-Selective Phosphonamidate Electrophiles for Modular Protein Bioconjugations. Angew. Chem. Int. Ed. 58, 11625-11630 (2019); b) Stieger, C. E., Franz, L., Korlin, F. & Hackenberger, C. P. R. Diethynyl Phosphinates for Cysteine-Selective Protein Labeling and Disulfide Rebridging. Angew. Chem. Int. Ed. 60, 15359-15364 (2021).

[29] DeKorver, K. A. et al. Ynamides: a modern functional group for the new millennium. Chem. Rev. 110, 5064-5106 (2010).

[30] Evano, G., Coste, A. & Jouvin, K. Ynamides: versatile tools in organic synthesis. Angew. Chem. Int. Ed. 49, 2840-2859 (2010).

[31] Koniev, O. & Wagner, A. Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chem. Soc. Rev. 44, 5495-5551 (2015).

[32] a) Yang, J., Wang, C., Xu, S. & Zhao, J. Ynamide-Mediated Thiopeptide Synthesis. Angew. Chem. Int. Ed. 58, 1382-1386 (2019); b) Hu, L. et al. Ynamides as Racemization-Free Coupling Reagents for Amide and Peptide Synthesis. J. Am. Chem. Soc. 138, 13135-13138 (2016).

[33] Yang, M., Wang, X. W. & Zhao, J. F. Ynamide-Mediated Macrolactonization. ACS Catal. 10, 5230-5235 (2020).

[34] a) Peng, Z., Zhang, Z., Tu, Y., Zeng, X. & Zhao, J. Regio- and Stereo-Selective Intermolecular Hydroamidation of Ynamides: An Approach to (Z)-Ethene-1,2-Diamides. Org. Lett. 20, 5688-5691 (2018); b) Peng, Z., Zhang, Z., Zeng, X., Tu, Y. & Zhao, J. Regio‐ and Stereoselective Hydrophosphorylation of Ynamides: A Facile Approach to (Z)‐β‐Phosphor‐Enamides. Adv. Synth. Catal. 361, 4489-4494 (2019); c) Hentz, A., Retailleau, P., Gandon, V., Cariou, K. & Dodd, R. H. Transition-Metal-Free Tunable Chemoselective N Functionalization of Indoles with Ynamides. Angew. Chem. Int. Ed. 53, 8333-8337 (2014).

[35] Grant-Mackie, E. S., Williams, E. T., Harris, P. W. R. & Brimble, M. A. Aminovinyl Cysteine Containing Peptides: A Unique Motif That Imparts Key Biological Activity. JACS Au 1, 1527-1540 (2021).

[36] Evano, G., Michelet, B. & Zhang, C. The anionic chemistry of ynamides: A review. Comptes Rendus Chimie 20, 648-664 (2017).

[37] a) Sato, A., Yorimitsu, H. & Oshima, K. Thieme Chemistry Journal Awardees - Where are They Now? Regio- and Stereoselective Radical Additions of Thiols to Ynamides. Synlett 2009, 28-31 (2009); b) Sato, A., Yorimitsu, H. & Oshima, K. Radical Additions of Arenethiols to Ynamides for the Selective Synthesis of N-[(Z)-2-(Arylsulfanyl)-1-alkenyl]amides. Bull. Korean Chem. Soc. 31, 570-576 (2010).

[38] Banerjee, B., Litvinov, D. N., Kang, J., Bettale, J. D. & Castle, S. L. Stereoselective additions of thiyl radicals to terminal ynamides. Org. Lett. 12, 2650-2652 (2010).

[39] Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click Chemistry: Diverse Chemical Function from a Few Good Reactions. Angew Chem Int Ed 40, 2004-2021 (2001).

[40] a) Merrifield, R. B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 85, 2149-2154 (1963); b) Carpino, L. A. & Han, G. Y. 9-Fluorenylmethoxycarbonyl function, a new base-sensitive amino-protecting group. J. Am. Chem. Soc. 92, 5748-5749 (1970); c) Dawson, P. E., Muir, T. W., Clarklewis, I. & Kent, S. B. H. Synthesis of Proteins by Native Chemical Ligation. Science 266, 776-779 (1994); d) Mousa, R., Lansky, S., Shoham, G. & Metanis, N. BPTI folding revisited: switching a disulfide into methylene thioacetal reveals a previously hidden path. Chem. Sci. 9, 4814-4820 (2018).

[41] Ascenzi, P. et al. The bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor): a milestone protein. Curr. Protein. Pept. Sci. 4, 231-251 (2003).

[42] Hudis, C. A. Trastuzumab--mechanism of action and use in clinical practice. N. Engl. J. Med. 357, 39-51 (2007).

Tables 1-6 are available in the supplementary files section.

There is NO Competing Interest.

Download PDF

Version 1

posted

You are reading this latest preprint version

Peptide and Protein Cysteine Modification by Virtue of Highly Chemo-, Regio- and Stereoselective Hydrosulfuration of Ynamide

Status:

Version 1

Abstract

Figures

Introduction

Results And Discussion

Conclusion

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1