Protection-Free Site-Directed Peptide or Protein <i>S</i> -Glycosylation and Its Application in the Glycosylation of Glucagon-like Peptide 1
Gefei Li, Yuankun Dao, Juan Mo, Suwei Dong, Shin‐ichiro Shoda, Xin‐Shan Ye
Abstract
Open AccessCCS ChemistryCOMMUNICATION6 Jun 2022Protection-Free Site-Directed Peptide or Protein S-Glycosylation and Its Application in the Glycosylation of Glucagon-like Peptide 1 Gefei Li, Yuankun Dao, Juan Mo, Suwei Dong, Shin-ichiro Shoda and Xin-Shan Ye Gefei Li State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191 , Yuankun Dao State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191 , Juan Mo State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191 , Suwei Dong State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191 , Shin-ichiro Shoda Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-8579 and Xin-Shan Ye *Corresponding author: E-mail Address: [email protected] State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191 https://doi.org/10.31635/ccschem.021.202101115 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The development of a facile cysteine-directed S-glycosylation strategy would facilitate the intensive investigation of the effect of glycosylation on protein translational modification. Herein, we introduce glycosyl Bunte salt as an efficient glycosyl donor for site-selective peptide/protein modification. The coupling reaction with cysteine thiols under alkaline buffer conditions proceeded chemoselectively, delivering homogeneous glycoconjugates and inorganic salt as the only byproduct. A series of sugar moieties, including monosaccharides and oligosaccharides, were successfully conjugated to the peptides and protein via the disulfide bond. Furthermore, this protocol was applied to the glycosylation of the glucagon-like peptide 1 (GLP-1) variant, and its glycosylated effect on blood glucose control was also studied. Download figure Download PowerPoint Introduction Site-directed protein glycosylation commonly referred to as one of the post-translational modification (PTM) routes, is often responsible for protein chemodiversity, physicochemical properties, and metabolic fate in vivo.1 Additionally, glycosylation has proven to be an effective approach to overcome the intrinsic obstacles of natural peptide drugs such as inadequate absorption and rapid degradation by proteolytic enzymes.2 Consequently, various efficient and controlled PTM strategies have been developed for studying the precise functionality of glycopeptides and glycoproteins.3,4 Of them, conjugation of carbohydrate moieties to the cysteine thiol of protein with the unusual S-glycosidic bond brings many advantages such as high chemo- and regioselectivity due to the unique nucleophilicity and low natural abundance of cysteine in a protein sequence.5,6 Furthermore, for those proteins that have no natural cysteine in their sequence, a cysteine can be inserted at a predetermined position via site-directed mutagenesis, which can be subsequently modified by S-glycosylation.7 Apart from that, thioglycosides are hydrolase-resistant mimics of O-linked glycosides that can offer improved antimicrobial activity and stronger immune response.8–10 For example, Donk and co-workers11,12 studied the effect of S-glycosylation on the antimicrobial activity of sublancin, indicating that these natural S-linked glycopeptide analogues carrying different saccharides can retain their bioactivity at diverse minimum inhibitory concentrations (MICs). In another case, Corzana and co-workers9 found that the antitumor immune response of the tumor-associated glycopeptide antigen could be improved by O → S replacement at the glycosidic linkage. Recently, glycomimetics with a disulfide bond as useful tools in the study of glycobiology have also attracted attention.13 The increased distance between the carbohydrate and aglycone adds more flexibility to the molecules with respect to the corresponding natural glycosides. As a result, glycosyl disulfides can potentially better adjust to an active conformation and consequently and positively influence biological activity. The growing interest in peptide glycosylation with a newly formed disulfide bond has triggered the development of advanced methods for their preparation. The most common way to access the disulfide linkage between the peptide and sugar moiety relies on an SN2 process by the use of a glycosulfenyl reagent with a good leaving group. This strategy has the advantage of installing the aglycone without changing the anomeric configuration. Despite successful development of useful glycosyl donors by Davis and co-workers, including glycomethane/phenyl-thiosulfonates and glycosyl selenenylsulfide (Glyco-SSePh) for cysteine-directed modification (Scheme 1a),14–18 ample room for future improvements such as processing chemistry and green chemistry still exist.19 For example, during the preparation of glycosyl donors, the synthetic efficiency in the displacement reaction of protected glycosyl halides with sodium alkylthiosulfonate is low. Therefore, there remains an urgent need for developing mild and simplified peptide or protein glycosylation protocols. Scheme 1 | (a and b) Strategies for the cysteine-selective peptide glycosylation. Download figure Download PowerPoint Herein, we report an improved approach for mild and cysteine-selective S-glycosylation employing glycosyl Bunte salts. As an alternative donor to the previous work, glycosyl Bunte salts20 synthesized directly from native sugars in water can be used for site-selective peptide S-glycosylations featuring many attractive advantages (Scheme 1b). Especially, this protocol can be applied to the chemical modification of glucagon-like peptide 1 (GLP-1), which enables us to study the effect of glycosylation on its activity and stability. Furthermore, this method can be extended to the site-selective protein S-glycosylation. These synthetic homogeneous glycoforms are important for the study of fundamental biological and therapeutic strategies of peptides/proteins. Experimental Methods To a solution of native sugars and Et3N in H2O/CH3CN at 0 °C, 2-chloro-1,3-dimethylimidazolinium chloride (DMC) and sodium thiosulfate pentahydrate were added. The resulting mixture was stirred at the same temperature for 90 min and purified by ion-exchange chromatography and activated carbon column, giving rise to the isolated unprotected glycosyl Bunte salt. The peptide was dissolved in 1.0 mL of degassed buffer solution (Tris–HCl, pH 8.9 or 70 mM 2-(cyclohexylamino)ethanesulfonic acid (CHES), 5 mM 4-morpholineethanesulfonic acid (MES), 2 mM CaCl2, pH 9.5) and stirred at 30 °C for 10 min under Ar atmosphere. Thereafter, a solution of glycosyl Bunte salt (5.0 equiv) in degassed water (50 μL) was added to the peptide solution. The resulting mixture was stirred at 30 °C for another 6 h under Ar atmosphere. Purified S-glycosylated peptide was obtained by high-performance liquid chromatography (HPLC). Animal raising and experimental protocols were approved by the Institutional Animal Care and Use Committee at Peking University Health Science Center and complied with Experimental Animal Regulations. Results and Discussion In our previous research, the glucosyl Bunte salt was found to be reactive with excess thiophenol in the presence of triethylamine, affording the corresponding glucosyl disulfide in good yield.17 This result showed that glycosyl Bunte salts could behave as potential glycosyl donors for thiol-directed modification. To check the possibility of glycosyl Bunte salts on protein glycosylation, we initially screened the S-coupling process using N-acetyl-l-cysteine methyl ester in the preliminary model reaction (Table 1). In a typical procedure, the methanol solution of N-acetyl-l-cysteine methyl ester was added dropwise into a buffer solution of the unprotected glycosyl Bunte salt, and the resulting mixture was stirred at 30 °C for several hours under an argon atmosphere. When the cysteine derivative was treated with equivalent β-glucosyl Bunte salt under a pH 7.7 condition, the β-S-glucosylated product was obtained in 50% yield (entry 1), accompanied by the dimerized byproduct (Cys-S)2 in 40% yield. Encouraged by this result, we further optimized the reaction conditions. As expected, with the increased amount of glucosyl Bunte salt and higher pH values, generation of the dimerized byproduct could be considerably inhibited, which led to higher conversion of the target product (entries 2 and 3). Next, the attractive one-pot reaction was also examined using the unpurified glucosyl Bunte salt directly (entry 4). However, the coupling yield was decreased in this complicated system. A large excess of sodium thiosulfate in the reaction mixture seems to be troublesome. Thus, purified glycosyl Bunte salts should be used for better conversion. Thereafter, other representative monosaccharides were screened in this model reaction. The β-galactosylated Cys derivative was isolated smoothly in a satisfactory yield at pH 8.0 (entry 5). A higher pH value (pH 8.9) was required for the efficient coupling reaction when aminosugar-type Bunte salts were employed (entries 6–8). Nevertheless, the use of α-mannosyl Bunte salt led to the S-glycosylation product in low yields even under pH 9.5 conditions (entries 9–11). This method appeared to work well with most carbohydrate types although mannose conversion remained low. Table 1 | S-Glycosylation of l-Cysteine Derivative Using Different Glycosyl Bunte Salts Entry Donor Conditiona Yield (%)b 1 β-Glc-SSO3− (1.0 equiv) pH 7.7, 5 h 50 2 β-Glc-SSO3− (2.0 equiv) pH 7.7, 5 h 70 3 β-Glc-SSO3− (2.0 equiv) pH 8.0, 5 h 75 4c β-Glc-SSO3− (3.0 equiv) pH 8.0, 5 h 60 5 β-Glc-SSO3− (3.0 equiv) pH 8.0, 2 h 75 6 β-GlcNAc-SSO3− (3.0 equiv) pH 8.0, 6 h 40 7 β-GlcNAc-SSO3− (3.0 equiv) pH 8.9, 6 h 75 8 β-GalNAc-SSO3− (3.0 equiv) pH 8.9, 6 h 80 9 α-Man-SSO3− (3.0 equiv) pH 8.0, 6 h — 10 α-Man-SSO3− (3.0 equiv) pH 8.9, 7 h 10 11 α-Man-SSO3− (3.0 equiv) pH 9.5, 6 h 37 aThe reaction was carried out in Tris–HCl buffer; N-acetyl-l-cysteine methyl ester was predissolved in methanol and added dropwise. bIsolated yield. cOne-pot reaction: 2-chloro-1,3-dimethylimidazolinium chloride (DMC, 203 mg, dissolved in 0.4 mL H2O) was added to a solution of glucose (0.3 mmol), sodium thiosulfate pentahydrate (225 mg), and Et3N (504 μL) in H2O/MeCN (1.6 mL/2.0 mL) at 0 °C. After the reaction was stirred for 90 min at 0 °C, the reaction mixture was concentrated and washed with CH2Cl2. The resulting mixture was then employed for the subsequent S-glycosylation directly. To ensure cysteine thiol selectivity, we then focused our attention on applying the glycosyl Bunte salts to the reaction of a cysteine-containing peptide. In a general procedure, peptide 1 (Ala-Gly-Cys-Ser-Ala [AGCSA]) was dissolved in degassed buffer, which was followed by incubation with various glycosyl Bunte salts for 6 h at 30 °C. The reaction was monitored by HPLC and mass spectrographic (MS) analysis, in which we found that glucose (Glc), galactose, and melibiose (Meli) units could be coupled to the peptide cysteine through nucleophilic substitution at a pH of 8.9, providing the corresponding glycopeptides 2a, 2b, and 2e in good yields (95%). Consistent with the above cysteine coupling, the efficient glycosylation with N-acetylglucosamine (GlcNAc) and GalNAc-type Bunte salts should be carried out at higher pH value (pH 9.5) to obtain glycosylated products 2c and 2d, probably due to the reduced reactivity of these aminosugar donors. However, a sluggish reaction (the formation of 2f) was observed when using 6′-sialyllactosyl (SiaLac) Bunte salt as the donor. HPLC-MS analysis revealed the quantitative appearance of the peptide-SSO3− intermediate after adding SiaLac-SSO3− (5.0 equiv) to the peptide solution, so it seems that the peptide intermediate is formed quickly through a sulfonate metathesis process between the sugar-SSO3− and peptide cysteine thiol in an alkaline medium. Subsequently, the peptide intermediate, which is more susceptible to nucleophilic attack, further reacted with the formed glycosyl thiolate (sugar-S−) to produce the corresponding sialyllactosyl peptide 2f (Figure 1, route A). Since only one equivalent of glycosyl thiolate was formed during the sulfonate metathesis, the following transformation to glycopeptide became a rate-limiting step. A similar experimental result was also reported by Davis and co-workers17 in the protein glycosylation reaction using Glyco-SSePh as the donor. In this case, the protein-SSePh intermediate was produced via treatment of the protein with a large excess of Glyco-SSePh at pH 8.5. Also, efficient glycosylation was only observed at pH 9.5 for the further transformation, which was defined as a pH-dependent manner. Nevertheless, in our case, when the weakly reactive donor SiaLac Bunte salt was used, the yield of peptide glycosylation was poor at pH 9.5 (Table 2, 2f). Figure 1 | Optimization of peptide glycosylation using glycosyl Bunte salts. Download figure Download PowerPoint Table 2 | Cysteine-Selective Peptide Glycosylation Using Different Glycosyl Bunte Salts Reaction details: see the Supporting Information. Yield was determined by HPLC. To improve reaction efficiency, we presumed that the amount of glycosyl thiolate was one of the key factors to accelerating further conversion. Due to the limited formation of glycosyl thiolate during the sulfonate exchange process, it is likely that the external addition of glycosyl thiolate is required. Recently, we reported a simple protocol for the generation of glycosyl thiolates from glycosyl Bunte salts in the presence of sodium sulfide (Na2S).21 Hence, in the following experiment, some SiaLac Bunte salts were premixed with an equivalent of Na2S to form the corresponding sialyllactosyl thiolate, and it was then added to the peptide glycosylation solution (Figure 1, route B). We detected a 95% yield of the sialyllactosyl peptide 2f in a shorter time at a milder pH value (pH 8.5), thereby offering an improved two-step one-pot protocol for the peptide glycosylation. Taken together, these results indicated that the current reaction is site-selective, compatible with a variety of sugar moieties, and proceeds with retention of the anomeric configuration. To demonstrate the applicability of this methodology toward medicinal peptides, sublancin ( 3), a natural S-linked antimicrobial glycopeptide containing two disulfide bonds and one free cysteine at position 22, was chosen as the substrate.11 Originally, the direct conjugation of sublancin with glucosyl Bunte salt at pH 9.5 gave only the sublancin-Bunte salt 4a as a stable intermediate (Figure 2, route A). Hence, the improved procedure as described in route B in Figure 1 was carried out to afford the desired S-glucosylated sublancin 4b in 90% yield (Figure 2, route B) without cleaving the native disulfide bonds on the peptide structure, displaying another advantage of this mild ligation strategy over other reported methods.22 However, in the case of using SiaLac Bunte salt, the efficiency of the sulfonate exchange process was low, which could be attributed to the low reactivity of the sialyl-type donor. Therefore, we attempted to first treat the sublancin with the reactive glucosyl Bunte salt to facilitate the generation of sublancin-SSO3− intermediate, followed by the addition of the preincubated SiaLac thiolate reagent. Based on this optimized manipulation, 6′-sialyllactose (SiaLac)-modified sublancin 4c was successfully prepared in 85% yield at pH 8.5 (Figure 2, route C). It should be noted here that both the disulfide-linked product (peptide-SS-sugar, 55%) and thioether-linked byproduct (peptide-S-sugar, 35%) would be observed if a higher pH value (pH 9.5) was used ( Supporting Information, page S22). This interesting phenomenon could be explained by the base-mediated cysteine elimination, resulting in the generation of the dehydroalanine (Dha) structure on the peptide.23 Subsequently, the thioether-linked product was formed via Michael addition of the glycosyl thiol nucleophile. However, the unexpected pH-dependent Michael addition was only observed in the case of using sublancin as the substrate with long reaction time (>6 h). Overall, based on the reactivities of peptide substrates and donors, different routes (A, B, or C) were adopted in this glycosylation reaction. Figure 2 | The comparison of sublancin glycosylation using glycosyl Bunte salts in different ways. Download figure Download PowerPoint With the improved cysteine-selective glycosylation protocol in hand, we next wanted to apply this method to study the glycosylated effect of medically valuable peptides. GLP-1 is considered to be the most potent insulinotropic hormone and a therapeutic candidate for Type II diabetes.24 However, the clinical use of native GLP-1 is limited by its short half-life toward dipeptidyl peptidase-4 (DPP-IV) enzyme and low activity in vivo.25 Glycoengineering strategy has been demonstrated as a useful approach to improve its proteolytic stability and bioactivity.26 Recently, glycosylation at residue 18 was proven to be effective for better Glc clearance and serum stability.27 To meet the requirements of the current method, the cysteine residue was introduced to replace the serine residue at position 18 on GLP-1 through chemical synthesis, providing the mutated GLP-SH. To obtain insights into the glycosylated effect on GLP-1, several saccharides, including monosaccharides and oligosaccharides, were conjugated to the GLP variant through a two-step protocol (route C). In a general process, the sulfonated intermediate GLP-SSO3− was initially prepared by treating the GLP-SH with GlcNAc-SSO3− at pH 9.0, followed by the addition of various glycosyl thiolates to produce the corresponding glycopeptides. The in vivo bioactivities of these synthetic glycopeptides were evaluated by detecting the blood Glc levels in mice on time. It has been shown that the GlcNAc-attached analogue, via the newly formed disulfide linkage, exhibited better blood Glc-lowering activity and higher enzymatic stability in vivo compared with the native GLP-1. This finding is consistent with the reported result,27 suggesting the effect of S-glycosylation modification with disulfide linkage is similar to the natural O-glycosylation to increase the serum stability of peptides. In comparison, the conjugation of other sugars has not always been accompanied by the enhanced pharmacological activity of these glycosylated peptides (Figure 3). In fact, the disulfide bond, as a reversible linker, can be cleaved via an enzymatic reduction both in vivo and in vitro.28 To avoid this undesired cleavage, Davis and co-workers14 have developed a desulfurization reaction that enables the conversion of readily synthesized disulfide-linked glycopeptides into the corresponding thioether-linked glycoconjugates by via treatment with organophosphorus agent. However, in our experiments, the stability of the disulfide-linked GLP-GlcNAc in vivo was indeed improved compared with the reduced GLP-SH, indicating that the disulfide modification is stable enough under physiological conditions. This information should be useful for the future design of glycosylated GLP-1 analogues. Moreover, the chemically attached GlcNAc residue would be useful for the further enzymatic ligation of eukaryotic N-glycans.29,30 The circular dichroism (CD) spectra of the modified and native GLP-1 peptides were also examined, and it was found that there are no dramatic differences ( Supporting Information Figure S8.2), indicating that the S-glycosylated modification does not affect its α-helix secondary structure of GLP-1.31 Figure 3 | Comparable S-glycosylation stabilizes GLP-1 from proteolysis and improves Glc clearance in vivo. Sugar type: Glc, GlcNAc, Meli, SiaLac; Blood Glc tolerance test: X axis indicates time (min), Y axis indicates blood Glc value (mmol/L). Mice were injected intraperitoneally (i.p.) with various GLP peptides (100 μg/kg, n = 5) 20 min prior to i.p. injection of Glc at 1g/kg body weight. Glc was i.p. injected to mice at 0, 105, and 180 min, respectively. Blood Glc levels were then measured after the indicated lengths of time. Download figure Download PowerPoint Finally, the optimized strategy was applied in native protein modification using bovine serum albumin (BSA; 66.5 kDa) featuring a single reduced cysteine thiol as the model substrate,32 as BSA could be used as a protein carrier and the glycosylated BSA is able to find applications in carbohydrate-based vaccine development. The reaction was carried out in Tris–HCl buffer at 30 °C under argon atmosphere. The preincubated melibiosyl Bunte salt and Na2S were added portionwise to afford the efficient glycosylation (route B). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was employed to confirm the protein modification, indicating 95% labeling of BSA (Figure 4), thus implying the potential of this strategy used in the protein PTM. Figure 4 | BSA glycosylation using melibiosyl Bunte salt. Download figure Download PowerPoint Conclusions We have described the unprotected glycosyl Bunte salts as attractive donors for the cysteine-selective peptide/protein glycosylation under mild aqueous conditions. Notably, this protocol allows an improved two-step reaction (route B or C) for the efficient conjugation between low-reactive donors and peptides/proteins with the generation of nontoxic inorganic salt as the only byproduct. In this process, the easily available monosaccharide-derived Bunte salt appears to be an efficient sulfonating reagent to activate the peptide cysteine. Furthermore, the optimized strategy can be applied to study the glycosylated effect of medically useful peptides, and the in vivo blood Glc-lowering activity and metabolic stability of GLP-1 can be improved by glycosyl conjugation. Our results suggested a similar glycosylated effect with disulfide linkage compared with the natural O-linkage. Based on this facile method, other PTMs are now under investigation. Supporting Information Supporting Information is available and includes parts of the HPLC/MS studies, detailed experimental procedures, characterization data, and copies of 1H and 13C NMR spectra for new compounds and additional Figures S5.4–S8.2. Conflict of Interest There is no conflict of interest to report. Funding Information This work was financially supported by grants from the National Natural Science Foundation of China (nos. 21907004 and 81821004), the National Key R&D Program of China (no. 2018YFA0507602), and the Beijing Outstanding Young Scientist Program (no. BJJWZYJH01201910001001). Acknowledgments The authors are grateful to Dr. Yiqun Geng from the Institute of Materia Medica at the Chinese Academy of Medical Sciences & Peking Union Medical College for her insightful discussion and support. References 1. Boutureira O.; in Protein an to the of Davis of in Protein Davis A for Protein Protein through Davis Protein Davis of at in and of and S-Glycosylation Boutureira O.; Corzana of of Peptide by or at the of to and Using a Donk a an Donk into the of of and Application to and Davis to Davis Glycosylation of by a Site-Directed and Davis for Protein Davis Protein in Davis for 1, Li Shoda Bunte A of for Sugar Li Shoda of from Glycosyl Bunte Salts and Its Application to for Peptide and Protein Boutureira O.; Davis for to on and 2, of a of Glucagon-like Glucagon-like Peptide and for in of Glucagon-like Peptide of Glycosylation on and in Blood of and in of in of a Li of and Li Li Li Glycosylation of with of Glucagon-like in with the of the Glucagon-like of in and with Information & Chinese Bunte authors are grateful to Dr. Yiqun Geng from the Institute of Materia Medica at the Chinese Academy of Medical Sciences & Peking Union Medical College for her insightful discussion and support.