Superfast desulfurization for protein chemical synthesis and modification

Abstract

•Add-and-done desulfurization by tetraethylborate under ambient conditions•Compatible with various peptides and proteins bearing multiple Cys residues•One-pot NCL-desulfurization facilitating chemical protein synthesis•Highly efficient radical generation from tetraethylborate oxidation The capability of chemically synthesizing and modifying biomacromolecules with homogeneity and structural precision opens avenues to discover new biology and generate novel therapeutic methods. However, the synthesis of peptides/proteins carrying diverse functionalities demands highly selective, effective, robust, and operationally simple chemical transformations. Herein, we report new reactivity of an old reagent, tetraethylborate (NaBEt4), which performs superfast and easy-to-use desulfurization for peptides/proteins without heating, irradiation, inert atmosphere, or odorous additives. This method can cleanly desulfurize various substrates and facilitate tandem applications with existing protein ligation technology. The new tetraethylborate chemistry provides a powerful and practical tool for generating tailor-made proteins with natural or unnatural modifications, which will be of great value for studying fundamental biological processes and developing novel therapeutics. Highly effective yet chemoselective chemical transformation strategies enable the facile access and precise modification of complicated biomacromolecules. In particular, the application of desulfurization chemistry expands the dimension of chemical protein synthesis with the cysteine-based peptide ligation. Considering the existing peptide desulfurization methods, a milder, faster, and easier strategy is still required for the increasing complexity of proteins by chemical synthesis. Herein, we report a superfast desulfurization strategy based on tetraethylborate for effectively and chemoselectively desulfurizing peptides/proteins containing cysteine or penicillamine in an add-and-done manner. This strategy can be simply applied under ambient conditions without requirement of inert atmosphere protection, irradiation, heating, or exogenous thiol additives. Such desulfurization can even overcome a certain amount of radical scavengers. Various peptide and protein substrates were examined, and a practical one-pot native chemical ligation (NCL)-desulfurization was developed for the synthesis of leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) cytoplasmic domain and semisynthesis of serotonylated histone H3 (H3Q5ser) protein. Highly effective yet chemoselective chemical transformation strategies enable the facile access and precise modification of complicated biomacromolecules. In particular, the application of desulfurization chemistry expands the dimension of chemical protein synthesis with the cysteine-based peptide ligation. Considering the existing peptide desulfurization methods, a milder, faster, and easier strategy is still required for the increasing complexity of proteins by chemical synthesis. Herein, we report a superfast desulfurization strategy based on tetraethylborate for effectively and chemoselectively desulfurizing peptides/proteins containing cysteine or penicillamine in an add-and-done manner. This strategy can be simply applied under ambient conditions without requirement of inert atmosphere protection, irradiation, heating, or exogenous thiol additives. Such desulfurization can even overcome a certain amount of radical scavengers. Various peptide and protein substrates were examined, and a practical one-pot native chemical ligation (NCL)-desulfurization was developed for the synthesis of leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) cytoplasmic domain and semisynthesis of serotonylated histone H3 (H3Q5ser) protein. IntroductionHighly effective organic transformations are of great value for accessing and modifying biomacromolecules. Such reactions require demanded chemoselectivity and reactivity, considering the complex functionalities present on biomacromolecules (e.g., proteins) and aqueous media used for the reaction. Native chemical ligation (NCL),1Dawson P.E. Muir T.W. Clark-Lewis I. Kent S.B. 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As the last step of protein synthesis after NCL, current desulfurization strategies normally require purification of ligated products to remove excess amount of the NCL catalyst 4-mercaptophenylacetic acid (MPAA), which would strongly inhibit the following desulfurization.11Moyal T. Hemantha H.P. Siman P. Refua M. Brik A. Highly efficient one-pot ligation and desulfurization.Chem. Sci. 2013; 4: 2496-2501https://doi.org/10.1039/c3sc50239bCrossref Scopus (69) Google Scholar To facilitate the synthesis of peptides/proteins bearing multiple posttranslational modifications (PTMs), the development of efficient desulfurization protocols featuring mild conditions and wide compatibility including MPAA is of continuing interest.Previously, our group reported that tris(2-carboxyethyl)phosphine (TCEP) premixed with superhydride (LiEt3BH) could mediate the aqueous chemoselective peptide desulfurization from hours to overnight.8Jin K. Li T. Chow H.Y. Liu H. Li X. P−B desulfurization: an enabling method for protein chemical synthesis and site-specific deuteration.Angew. Chem. Int. Ed. Engl. 2017; 56: 14607-14611https://doi.org/10.1002/anie.201709097Crossref PubMed Scopus (57) Google Scholar However, this method suffers from slow reaction kinetics and tricky procedure. We now discover the decomposition product of long-time stored superhydride, later identified as tetraethylborate (BEt4−), is a more promising reagent that realizes faster and cleaner peptide desulfurization in only 15–30 s. Although tetraethylborate was discovered and synthesized in the last century,12Honeycutt J.B. Riddle J.M. Preparation and reactions of sodium Tetraethylboron and related compounds 1.J. Am. Chem. Soc. 1961; 83: 369-373https://doi.org/10.1021/ja01463a027Crossref Scopus (48) Google Scholar,13Damico R. Preparation, characterization, and reactions of lithium and sodium Tetraalkylboron compounds.J. Org. Chem. 1964; 29: 1971-1976https://doi.org/10.1021/jo01030a077Crossref Scopus (97) Google Scholar its application on desulfurization chemistry has not been investigated to the best of our knowledge. Based on the commercially available sodium tetraethylborate NaBEt4, we introduced the “add-and-done desulfurization (ADD)” method, which robustly performs the superfast desulfurization of various peptide and protein substrates (Figure 1B). The ADD method shows remarkable compatibility with complicated proteins, orthogonal protecting groups, delicate PTMs, and even a certain amount of MPAA. A practical one-pot NCL-ADD strategy has been developed accordingly and successfully applied for two cases of protein synthesis where those substrates encountered problematic desulfurization with the classical VA-044 method,6Wan Q. Danishefsky S.J. Free-radical-based, specific desulfurization of cysteine: A powerful advance in the synthesis of polypeptides and glycopolypeptides.Angew. Chem. Int. Ed. Engl. 2007; 46: 9248-9252https://doi.org/10.1002/anie.200704195Crossref PubMed Scopus (559) Google Scholar namely the leukocyte-associated immunoglobulin-like receptor 1 (LAIR1) cytoplasmic domain14Chen L. Flies D.B. Molecular mechanisms of T cell co-stimulation and co-inhibition.Nat. Rev. Immunol. 2013; 13: 227-242https://doi.org/10.1038/nri3405Crossref PubMed Scopus (1743) Google Scholar, 15Müschen M. Autoimmunity checkpoints as therapeutic targets in B cell malignancies.Nat. Rev. Cancer. 2018; 18: 103-116https://doi.org/10.1038/nrc.2017.111Crossref PubMed Scopus (30) Google Scholar, 16Rumpret M. Drylewicz J. Ackermans L.J.E. Borghans J.A.M. Medzhitov R. Meyaard L. Functional categories of immune inhibitory receptors.Nat. Rev. Immunol. 2020; 20: 771-780https://doi.org/10.1038/s41577-020-0352-zCrossref PubMed Scopus (26) Google Scholar and the wild-type serotonylated histone H3 (H3Q5ser) protein.17Farrelly L.A. Thompson R.E. Zhao S. Lepack A.E. Lyu Y. Bhanu N.V. Zhang B. Loh Y.E. Ramakrishnan A. Vadodaria K.C. et al.Histone serotonylation is a permissive modification that enhances TFIID binding to H3K4me3.Nature. 2019; 567: 535-539https://doi.org/10.1038/s41586-019-1024-7Crossref PubMed Scopus (165) Google Scholar, 18Thompson R.E. Muir T.W. Chemoenzymatic semisynthesis of proteins.Chem. Rev. 2020; 120: 3051-3126https://doi.org/10.1021/acs.chemrev.9b00450Crossref PubMed Scopus (79) Google Scholar, 19Zhao S. Chuh K.N. Zhang B. Dul B.E. Thompson R.E. Farrelly L.A. Liu X. Xu N. Xue Y. Roeder R.G. et al.Histone H3Q5 serotonylation stabilizes H3K4 methylation and potentiates its readout.Proc. Natl. Acad. Sci. USA. 2021; 118: 1-7https://doi.org/10.1073/pnas.2016742118Crossref Scopus (9) Google Scholar Following mechanistic study unveiled the radical nature of this superfast desulfurization and its mechanism based on tetraethylborate oxidation is proposed.Results and discussionDiscovery and establishment of superfast desulfurizationTetraethylborate from decomposed superhydride could mediate efficient peptide desulfurizationWhen performing the TCEP/LiEt3BH desulfurization, we observed fluctuating performance between batches of superhydride. Interestingly, the old bottle of superhydride with precipitation inside always afforded faster and smoother desulfurization compared with the new one from the identical supplier (within 15 min versus overnight). This phenomenon was particularly significant in the case of peptide 1 with N-terminal Cys, where little desulfurized peptide 2 was observed together with many side products, probably due to the rapid formation of the stable borate complex with N-terminal Cys (Figures 2A, 2B , S1, and S2). On the sharp contrary, the stale superhydride resulted in complete and clean desulfurization in only several minutes with the same procedure. This dramatic difference indicated the presence of a more efficient desulfurization reagent than TCEP/LiEt3BH, and it prompted us to investigate the underlying reason.Figure 2Discovery and establishment of superfast desulfurizationShow full caption(A) Table of peptide 1 desulfurization by different batches of superhydride or NaBEt4.(B) Ultra performance liquid chromatography (UPLC) spectra before and after the desulfurization of 1 by new or old superhydride.(C) Table of glutathione 3 desulfurization as a model study.(D) Table of condition optimization using peptide 5.(E–G) Graph of desulfurization under (E) different concentrations of NaBEt4, (F) different concentrations of peptide 5, and (G) different temperatures. Conditions: 0.1 M TCEP, pH 4.5, quenched by 10 equiv of MPAA or 50 equiv of hydroquinone. VA-044 method: 100 μM or 1.0 mM 5, 40 mM VA-044, with or without 10 vol % tBuSH, pH 7.0, 37°C. [BEt3]: 0.1 M. Each time point represents an independent experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To identify the bona fide reactive species, 11B NMR spectra of both old and new superhydride samples were taken (Figure S3). To our surprise, although the spectrum of the new batch matched that of triethylborohydride [Et3BH]−,20Nöth H. Vahrenkamp H. NMR-spektroskopische Untersuchungen an Borverbindungen III. 1H-NMR-spektren von Methyl- und äthylboranen.J. Organomet. Chem. 1968; 12: 23-36https://doi.org/10.1016/S0022-328X(00)90894-5Crossref Scopus (67) Google Scholar the sharp singlet peak in the old superhydride (δ −16.3 ppm) indicated a highly symmetric boron species, which we suspected to be tetraethylborate [BEt4]− from disproportionation of [Et3BH]−.21Crestani M.G. Muñoz-Hernández M. Arévalo A. Acosta-Ramírez A. García J.J. σ-borane coordinated to nickel(0) and some related nickel(II) trihydride complexes.J. Am. Chem. Soc. 2005; 127: 18066-18073https://doi.org/10.1021/ja056000mCrossref PubMed Scopus (67) Google Scholar More importantly, the commercial NaBEt4 did achieve the same desulfurization result of peptide 1 compared with the old superhydride, confirming the mysterious boron species being tetraethylborate (Figure 2A, entries 2 and 3). Tetraethylborate showed remarkable stability in water compared with the Grignard reagent, and it was a superior ethylation reagent in aqueous phase for efficient derivatization of heavy metal cations.22Rapsomanikis S. Derivatization by ethylation with sodium tetraethylborate for the speciation of metals and organometallics in environmental samples.A review. Analyst. 1994; 119: 1429-1439https://doi.org/10.1007/s002160050072Crossref Scopus (43) Google Scholar,23Yu X. Pawliszyn J. Speciation of alkyllead and inorganic lead by derivatization with deuterium-labeled sodium tetraethylborate and SPME-GC/MS.Anal. Chem. 2000; 72: 1788-1792https://doi.org/10.1021/ac990699vCrossref PubMed Scopus (66) Google Scholar However, its application for desulfurization reactions remains unexplored. Based on the above results, we proposed that tetraethylborate could be repurposed as a novel reagent for rapid peptide/protein desulfurization, and the detailed mechanism requires further investigation.To better understand the tetraethylborate-mediated desulfurization, easily accessible glutathione (GSH) 3 was chosen as a model. The desulfurization under various conditions were performed in D2O to facilitate a direct NMR monitoring (Figures 2C and S4–S10; Table S1). In pH 6.0 D2O containing 2.0 equiv of TCEP, peptide 3 was desulfurized smoothly by 1.0 equiv of NaBEt4, generating the deuterated product 4 within 15 min (Figure 2C, entry 1). Interestingly, over 90% conversion was observed even when a substoichiometric 0.13 equiv of NaBEt4 was used, which indicated the involvement of chain reaction in the NaBEt4-desulfurization (entry 2). The combination of NaBEt4 and TCEP was found to be optimal already, whereas other boron reagents and phosphine compounds afforded much inferior results (entries 4–8). Although the reaction showed good tolerance to the addition of phenol (entry 9), adding stronger radical scavenger hydroquinone completely inhibited the reaction to only 3% conversion (entry 10). In addition, if the desulfurization was carried out under anaerobic conditions, only 21% of the desulfurized product could be obtained (entry 11), which demonstrated the critical role of O2 from the air. Moreover, the reaction can be adapted to a broad range of pH from 3.0 to 6.0 without decreasing conversion (entry 12). The above observations suggested that a rapid and efficient radical chain reaction may be involved in the tetraethylborate-desulfurization under aerobic conditions. It is worth noting that triethylborane BEt3 is known as an efficient free-radical initiator24Nozaki K. Oshima K. Utimoto K. Et3B induced radical addition of Ph3SnH to acetylenes and its application to cyclization reaction.Tetrahedron. 1989; 45: 923-933https://doi.org/10.1016/0040-4020(89)80004-3Crossref Scopus (90) Google Scholar and has been applied for aqueous radical cyclizations.25Yorimitsu H. Nakamura T. Shinokubo H. Oshima K. Omoto K. Fujimoto H. Powerful solvent effect of water in radical reaction: triethylborane-induced atom-transfer radical cyclization in water.J. Am. Chem. Soc. 2000; 122: 11041-11047https://doi.org/10.1021/ja0014281Crossref Scopus (185) Google Scholar However, the desulfurization of GSH 3 with BEt3 always showed incomplete conversion under different conditions, whereas a catalytic amount of NaBEt4 was enough to give a satisfactory desulfurization (entry 5 and Table S1).NaBEt4 desulfurization led to superfast desulfurization under various conditionsDue to the lower concentration of peptides or proteins (μM-mM), an excess amount of reagent is normally required for peptide/protein desulfurization. To tackle the problem, we performed peptide 5 desulfurization under various conditions for optimization (Figures 2D and S11–S17). Peptide 5 (1 mM) was treated with 2.5–100 mM NaBEt4 in pH 4.5 citrate buffer with 6 M guanidine and 0.1 M TCEP (Figure 2E). Impressively, peptide 5 was cleanly desulfurized to product 6 in only 15–30 s after the addition of over 50 mM NaBEt4, whereas the desulfurization with 25 mM NaBEt4 took slightly longer time (e.g., 60 s) to complete. When NaBEt4 lower than 10 mM was applied, incomplete conversion was observed ranging from 25% to 75%, probably due to the unproductive consumption of NaBEt4. It should be noted that in all cases the induction period was extremely short, and an exponential climbing stage was readily observed in the first few seconds. Even 2.5 mM NaBEt4 could lead to over 20% desulfurization in 10 s. Although similar kinetic behavior was observed as well when 100 mM BEt3 was used under the same conditions, the desulfurization was noticeably slower with around 30% conversion similarly with that by 2.5 mM NaBEt4. As for the control group where a typical VA-044 procedure was applied, the reaction was still in its induction period at the first 100 s, and only a trace amount of desulfurized product was detected (<1%). Next, the NaBEt4-desulfurization was examined with highly diluted peptide 5 (Figure 2F). To our surprise, this reaction did not compromise its ultrafast kinetics at all, resulting complete desulfurization in 15–30 s for 50–1,000 μM substrates. The same result was obtained even at 10 μM concentration of peptide 5 (Figure S14). Increasing temperature did not further improve the reaction; however, the reaction rate decayed earlier and caused incomplete desulfurization (e.g., 80%, Figure 2G). By contrast, slightly slow but complete desulfurization was achieved by NaBEt4 at lower temperature (0°C–4°C), which could be more beneficial in the treatment of delicate peptides or proteins. More importantly, the NaBEt4 could still mediate satisfactory desulfurization of peptide 5 in the presence of a small amount of MPAA (up to 2 mM, see Figure S17), which may be applied in the one-pot NCL-desulfurization strategy. Based on the remarkable desulfurization kinetics and simple experiment operations, we propose the concept of ADD, where the desulfurization can be completed in dozens of seconds after the addition of aqueous NaBEt4 to the TCEP-containing solution of peptide/protein substrate (Figure 1B).Substrate scope of ADDThe NaBEt4-mediated desulfurization of random peptides bearing Cys or PenTo examine the scope and limitations of the ADD by NaBEt4, we tested it on random peptides containing cysteine or penicillamine (Figures 3 and S18–S67). Under ambient air conditions, all examples (peptide 6, 9–32) were cleanly desulfurized in an add-and-done manner with satisfactory HPLC isolation yields (Figures 3A and 3B). Sensitive amino acid residues such as methionine and tryptophan were not affected at all during the reaction (peptide 10, 16, 17, etc.). In addition, the reaction rate was not affected by the number of cysteine residues, and up to five Cys residues were all be rapidly desulfurized after the addition of NaBEt4 reagent (peptide 10, 11, and 22). Furthermore, the penicillamine residue that was used in NCL26Haase C. Rohde H. Seitz O. Native chemical ligation at valine.Angew. Chem. Int. Ed. Engl. 2008; 47: 6807-6810https://doi.org/10.1002/anie.200801590Crossref PubMed Scopus (241) Google Scholar and in cysteine/penicillamine ligation (CPL)27Tan Y. Li J. Jin K. Liu J. Chen Z. Yang J. Li X. Cysteine/penicillamine ligation independent of terminal steric demands for chemical protein synthesis.Angew. Chem. Int. Ed. Engl. 2020; 59: 12741-12745https://doi.org/10.1002/anie.202003652Crossref PubMed Scopus (9) Google Scholar to achieve ligation at the Val site was smoothly desulfurized as well to the native valine residue by our ADD protocol (peptide 23–26). It is notable that in the reported desulfurization of peptides 24–26 using the VA-044 method, well-optimized conditions including excess amount of initiator (200 mM), higher substrates concentration (5 mM), elevated temperature (65°C), and more reactive thiol additive (t-BuSH to GSH) were required to suppress the side reactions from radical desulfurization (such as oxidation, fragmentation, etc.).26Haase C. Rohde H. Seitz O. Native chemical ligation at valine.Angew. Chem. Int. Ed. Engl. 2008; 47: 6807-6810https://doi.org/10.1002/anie.200801590Crossref PubMed Scopus (241) Google Scholar In contrast, our protocol did not need tedious optimization and directly afforded complete desulfurization in dozens of seconds without detectable side reactions (Figure 3C). For difficult Pen peptides, it was interesting that the omission of sacrificial thiols did not cause side reactions from the internal hydrogen abstraction, which would be further discussed in the following mechanistic study.Figure 3Add-and-done desulfurization (ADD) of random peptidesShow full caption(A) Desulfurization of random peptides with isolated yields by the ADD protocol. See Figures S18–S67 for more details.(B and C) UPLC spectra of selected examples before and after the addition of NaBEt4.(D) Comparison of VA-044 and ADD methods on the desulfurization of serotonylated 32.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Meanwhile, such ADD method also showed excellent compatibility to other functional groups (peptide 27–32). Only the free cysteine was desulfurized chemoselectively in the presence of other cysteine residues protected by Acm (27) or Thz (28). Recently, maleimide was reported as an alternative orthogonal Cys protecting group and was utilized for chemical/semisynthesis of proteins.28Vamisetti G.B. Satish G. Sulkshane P. Mann G. Glickman M.H. Brik A. On-demand detachment of succinimides on cysteine to facilitate (semi)synthesis of challenging proteins.J. Am. Chem. Soc. 2020; 142: 19558-19569https://doi.org/10.1021/jacs.0c07663Crossref PubMed Scopus (16) Google Scholar Expectedly, the ADD protocol also showed complete tolerance to such maleimide protection, which was demonstrated by the chemoselective desulfurization of N-methyl-maleimide protected peptide 30 in high isolated yield (77%). Furthermore, moieties like biotin and glycan were well tolerated (peptide 29 and 31). Notably, the serotonylation discovered recently as a novel PTM in histone H317 was found to disrupt the VA-044 desulfurization (peptide 32 and Figure 3D) probably because the 5-hydroxyindole moiety acted as a radical scavenger.29Huether G. Fettkötter I. Keilhoff G. Wolf G. Serotonin acts as a radical scavenger and is oxidized to a dimer during the respiratory burst of activated microglia.J. Neurochem. 1997; 69: 2096-2101https://doi.org/10.1046/j.1471-4159.1997.69052096.xCrossref PubMed Scopus (44) Google Scholar The VA-044-desulfurization of peptide 32 was sluggish, and no desired product was detected, accompanied by serious side products including radical addition to the serotonin and peptide degradation. Fortunately, the inhibition effect and other side reactions from serotonylation was overcome by the ADD. The product 31 was obtained instantly after the treatment of NaBEt4 with 70% HPLC isolated yield, providing an opportunity for the synthesis of native proteins with serotonylation. The above results suggest that our ADD protocol can be adapted to a broad substrate scope with excellent compatibility.ADD of proteins bearing multiple Cys residuesApart from random peptides, commercially available proteins with multiple cysteine residues were also included to explore the scope of the ADD (Figures 4 and S68–S80). After the disulfide-bond reduction of proteins in TCEP-containing denaturing buffer (0.2–0.5 M), all thiol groups of the protein substrates (1.0–10 mg/mL) were readily removed by addition of NaBEt4 aqueous solution. Cys-to-Ala mutants of proteins could be elegantly obtained in seconds to minutes, including aprotinin (34→35, 6 Cys), ribonuclease (36→37, 8 Cys), and lysozyme C (38→39, 8 Cys). In the case of lysozyme, two or more treatments of NaBEt4 were normally required for generating homogeneous desulfurized product, which was presumably caused by the low accessibility of deeply buried cysteine residues. Nevertheless, the reaction time for the NaBEt4 desulfurization was still incredibly short (≤5 min). Replacement of NaBEt4 with BEt3 or LiEt3BH resulted in insufficient desulfurization and heterogeneous mass spectra. Such simple method might offer another option to generate Cys-to-Ala protein mutants without the need for altering the expression plasmid. Furthermore, its application for chemical protein synthesis was examined. We first chose ubiquitin (1–76), which is a protein-size PTM (ubiquitination) regulating other proteins’ degradation, location, and activity and has been used as a model substrate in protein chemistry.8Jin K. Li T. Chow H.Y. Liu H. Li X. P−B desulfurization: an enabling method for protein chemical synthesis and site-specific deuteration.Angew. Chem. Int. Ed. Engl. 2017; 56: 14607-14611https://doi.org/10.1002/anie.201709097Crossref PubMed Scopus (57) Google Scholar,11Moyal T. Hemantha H.P. Siman P. Refua M. Brik A. Highly efficient one-pot ligation and desulfurization.Chem. Sci. 2013; 4: 2496-2501https://doi.org/10.1039/c3sc50239bCrossref Scopus (69) Google Scholar,28Vamisetti G.B. Satish G. Sulkshane P. Mann G. Glickman M.H. Brik A. On-demand detachment of succinimides on cysteine to facilitate (semi)synthesis of challenging proteins.J. Am. Chem. 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