Clickable tryptophan modification for late-stage diversification of native peptides

Abstract

As the least abundant residue in proteins, tryptophan widely exists in peptide drugs and bioactive natural products and contributes to drug-target interactions in multiple ways. We report here a clickable tryptophan modification for late-stage diversification of native peptides, via catalyst-free C2-sulfenylation with 8-quinoline thiosulfonate reagents in trifluoroacetic acid (TFA). A wide range of groups including trifluoromethylthio (SCF₃), difluoromethylthio (SCF₂H), (ethoxycarbonyl)difluoromethylthio (SCF₂CO₂Et), alkylthio, and arylthio were readily incorporated. The rapid reaction kinetics of Trp modification and full tolerance with other 19 proteinogenic amino acids, as well as the super dissolving capability of TFA, render this method suitable for all kinds of Trp-containing peptides without limitations from sequences, hydrophobicity, and aggregation propensity. The late-stage modification of 15 therapeutic peptides (1.0 to 7.6 kilodaltons) and the improved bioactivity and serum stability of SCF₃- and SCF₂H-modified melittin analogs illustrated the effectiveness of this method and its potential in pharmacokinetic property improvement.

The clickable late-stage diversification of native peptides is achieved via catalyst-free C2-sulfenylation of tryptophan residues.

INTRODUCTION

Tryptophan (Trp) is a unique residue in peptides and proteins (1). Because the biosynthesis of Trp is the most energy consuming among the 20 canonical amino acids (2), it is not unexpected that Trp has the lowest abundancy in proteins (~1%). Trp is encoded by a single codon UGG without redundancy and always enriched in functional sites, which indicates that Trp is introduced only when absolutely needed (3). With the largest planar electron-rich π-system, Trp plays key roles in the bioactivities and biophysical properties of proteins via multiple interactions more than simple hydrophobic effect. It contributes to the stabilization of secondary structures and protein-protein interactions via intrachain and interchain cation-π interactions, respectively (4, 5), and stabilization of protein–small-molecule interactions via π-π stacking effect (Fig. 1A) (6). Out of the proteomes, Trp is also frequently involved in the bioactive peptides from different origins (Fig. 1A), including ribosomally synthesized and posttranslationally modified peptides (RiPPs, e.g., darobactin and tryptorubin A) (7–9) and peptide hormones (e.g., somatostatin and gonadotropin-releasing hormone) (10, 11). Just like in the case of proteins, the multiple contributions to ligand-receptor interactions render Trp indispensable in peptide drugs derived from natural products or native hormones (e.g., vapreotide and tirzepatide) (12, 13). Moreover, Trp is actively involved in the enzymatic degradation of peptide drugs by chymotrypsin (14). As the most rapid-growing category of new drugs, peptide drugs are currently emerging as middle-sized therapeutic agents in addressing unmet medical needs due to their intrinsic biochemical and physical characteristics as compared to small-molecule drugs and biologics (15–18). More than 100 peptide drugs that have been approved by the Food and Drug Administration (FDA) to reach the market since the insulin was developed in 1923, among which about 40 drugs for treating a wide range of diseases (e.g., cancer, cardiovascular, and metabolic diseases) contain at least one Trp residue (see the Supplementary Materials). Chemical modifications of Trp residues embedded in peptide drugs will not only regulate the drug-target interactions but also provide opportunities to improve drug stability, bioavailability, and pharmacokinetics. Rapid generation of Trp-modified drug analogs in a structural diversification manner, especially via site-selective late-stage modifications on full-length side-chain unprotected peptides (19, 20), is undoubtedly an appealing goal to pursue.

Fig. 1. Important roles of tryptophan in proteins and peptides and the development of late-stage tryptophan-selective peptide modification methods.

(A) Tryptophan residues in proteins, natural products, and peptide drugs. (B) Reported examples of late-stage Trp-selective peptide modifications. (C) Late-stage tryptophan-selective S-diversification of native peptides developed in this work.

Trp has unique chemical properties including low aromaticity and soft nucleophilicity of the indole ring, which set the basis for developing Trp-selective transformations. Though the selective modification of Trp by o-nitrophenylsulfenyl chloride can be dated back to 1960s (21), the last decade has witnessed the bursting development of Trp-selective modification reactions (22). By adopting the four criterions proposed by Zhang et al. (23) for late-stage aromatic C─H bond functionalization, here we defined the effective late-stage Trp modification as reactions fulfilling the following five rules: high reactivity that facilitates smooth conversion at 40°C or lower; high site selectivity to ensure that only Trp is modified without the assistance of auxiliary; high chemoselectivity to generate well defined structures without heterogeneity; wide substrate scope to allow for any unprotected peptide (linear and cyclic) regardless of sequences, side-chain functionalities, and physicochemical properties to react; and solvents to dissolve any unprotected peptide. In the case of peptide modification, the solvent issue is worth noting because peptide substrates are generally poorly soluble in many organic solvents (e.g., dichloromethane, dioxane, and xylene) favored by transition metal catalyzed functionalizations (24–27). A series of late-stage Trp-selective modifications have been found to fulfill some of these five rules (Fig. 1) (28–45). Most of the reactions generate C2-modified products via forming C─C (28–34), C─N (35–37), or C─S bonds (38, 39), while modifications via N-enamine formation (40), Cβ-H alkylation (41) and dearomatization (42–45) have also been reported. Mechanistically, radical reactions initiated via chemical/electrochemical/photochemical processes (28–37, 39, 41, 44) are dominant because of the compatibility with aqueous solvent systems and acidic protons in both side chains and backbones. Reactions based on polar processes where indole serves as soft nucleophiles are less developed (38) because the competition of other side-chain nucleophiles and solvents poses great challenges to the reaction design. Deliberate balance of reactivity and chemo/site selectivity by the reagent design with solvent as an additional tuning factor will be key to success.

Herein, we report the development of a clickable tryptophan modification for late-stage diversification of native peptides (including on-market peptide drugs) via catalyst-free electrophilic C2-sulfenylation, using S-substituted 8-quinoline thiosulfonates as the electrophilic sulfur donor under trifluoroacetic acid (TFA) conditions (Fig. 1C). In this strategy, TFA served as a universal solvent for both peptide dissolvation and reagent activation. With this strategy, a series of functional groups ranging from electron-withdrawing SCF₃/SCF₂H to electron-donating SAlkyl/SAryl were efficiently installed without any bias, which allowed us to modulate the bioactivities and pharmacokinetic properties of bioactive peptides.

RESULTS

Establishment of clickable tryptophan modification based on late-stage C2-sulfenylation

We started our adventure by developing late-stage Trp-selective C2-trifluoromethylsulfenylation and difluoromethylsulfenylation of peptides. The trifluoromethylthio group (−SCF₃) is considered to have large lipophilicity (π = 1.44) and strong electron-withdrawing ability (σ_m = 0.40 and σ_p = 0.50) which is promising to improve the metabolic stability and membrane permeability of pharmaceuticals (46–49). The difluoromethylthio group (−SCF₂H) which presents medium lipophilicity (π = 0.68) and is recognized as a highly lipophilic weak hydrogen bonding donor also provides possibility for drug structural optimization (50–53). Unlike perfluoroalkyaltion where the perfluoroalkyl radicals are generated as the reactive species, the electrophilic sulfur atom polarized by CF₃/CF₂H groups is expected to match the soft nucleophilicity of the indole ring of Trp and undergo electrophilic aromatic substitution. Though sulfenyl chloride compounds have been successfully used as an electrophilic sulfur donor in Trp modification (21), the ultrahigh reactivity of CF₃SCl makes it difficult to achieve selectivity for unprotected peptide modification, and the gaseous nature limits its application in small-scale transformations (54). The ArNHSCF₃ showed good reactivity in the trifluoromethylthiolation of electron-rich aromatic systems (55). However, in a recent report of peptide modification, both Trp and Tyr were modified with ArNHSCF₃ (56). Last, we turned to aryl thiosulfonate type reagent PhSO₂SCF₃ 1a, which is easily accessible and shelf stable. Reagent 1a has been widely used in the radical based or transition metal catalyzed reactions (57–59). Because two electron-withdrawing groups (ArSO₂ and CF₃) are linked to sulfur atom, the role of 1a in polar transformations is controversial. In early reports, 1a served as a sulfonylation reagent to give rise to sulfonamides and thiosulfonates accompanied by releasing CF₃SH when it reacted with amine and thiol, respectively (58, 59). However, in a recent report of ortho-trifluorosulfinylation of N-aryl hydroxylamines, the Ar-NH-O-SCF₃ species formed via O-sulfenylation of hydroxylamine was proposed as the key intermediate, which indicated the potential of 1a as electrophilic SCF₃ donor (60).

In our trials, when model peptide 2a was treated with reagent 1a (PhSO₂SCF₃), no Trp modification product 3a was observed under all the conditions. To further improve the electrophilicity of SCF₃, we tested reagent 4 (QSO₂SCF₃, Q = quinolinyl) containing a quinoline structure, which has been synthesized in the literature without any test of its reactivity (61). To our delight, through extensive condition screening, we observed rapid and clean conversion of peptide 2a (10 mM) to Trp-modified peptide 3a when it was treated with 4 (50 mM) in TFA within 1 hour (entry 1, Fig. 2A). The structure of 3a was confirmed by nuclear magnetic resonance (NMR) analysis of the purified compound, where the signal of the proton on C2 of indole ring disappeared (fig. S2). Using TFA [pK_a, 0.23 (where K_a is the acid dissociation constant) at 25°C in H₂O] as solvent was found to be critical for success, while changing solvent to less acidic HOAc (pK_a, 4.76) or strong hydrogen bond donors like 2,2,2-trifluoroethanol (TFE) and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) led to no conversion (entries 2 to 4). Adding water (5% v/v) to TFA was partially tolerated, but 25% v/v water led to a much inferior conversion (58%) as determined by ultra-performance liquid chromatography–mass spectrometry (UPLC-MS) analysis (entries 5 to 6). In cases of other TFA/organic solvent mixtures, varying TFA/TFE ratio from 9:1 to 3:1 and 1:1 v/v led to the decreasing conversions 74, 70, and 42%, respectively, while TFA/dimethyl sulfoxide (DMSO) and TFA/N,N′-dimethylformamide (DMF) mixtures (1:1, v/v) totally suppressed the reaction (entries 7 to 11). We also tested the reaction of peptide 2a with reagents 1b to 1d in TFA, and only 1d bearing electron-withdrawing 4-NO₂ substitution gave a moderate 29% conversion (entries 12 to 15). TFA is rarely used as a solvent in organic reactions except for acidic deprotection, but it is quite beneficial for unprotected peptide modifications because it has the strongest denature capability and can dissolve almost any peptide sequence even with high hydrophobicity and aggregation propensity. In Strömberg’s O/S-selective palmitoylation of pulmonary surfactant protein-C by palmitoyl chloride, TFA was used as the solvent that fully dissolved the hydrophobic protein bearing 2 palmitoyl groups and totally suppressed the undesired acylation on Lys and Arg side chains (62). In our reaction, using TFA allowed high peptide operational concentration (10 mM) that makes the late-stage modification easily scalable.

Fig. 2. Discovery and establishment of late-stage C2-trifluoromethylsulfenylation on tryptophan residues.

(A) Summary of condition optimization using peptide 2a and reagent 4. (B) Monitoring interaction between thiosulfonate reagent 4 and TFA by ¹H NMR. (C) Electron density (mapped with ESP) surface calculation was performed at the M06/6-31G(d) level of DFT theory using Gaussian 16. Red indicates region of negative charge and blue region of positive charge.

The outstanding performance of 4 in TFA was likely attributed to the H-bond interaction between TFA and the quinoline ring that enhanced the electrophilicity of SCF₃ group. In our NMR titration study, the down field shift of both ¹H and ¹⁹F signals was clearly observed by gradually increasing the TFA/4 molar ratio from 1.3:1 to 25:1, while no further shifting was observed at 50:1 ratio (Fig. 2B). On the basis of this result, as well as our failed attempts to obtain 4-TFA salt crystal for x-ray diffraction analysis, we proposed that the H-bond interaction here was highly dynamic and concentration dependent, which could also interpret why TFA must be used as solvent rather than additive without other H-bond acceptors (DMSO or DMF). To further confirm the important role of quinoline but not sulfonyl group in H-bond interaction, we did NMR titration of 1d and observed no signal shift in both ¹H and ¹⁹F NMR. To gain more insight on how the electrophilicity of sulfur atom was improved by the TFA-reagent interaction, we did DFT calculation of Mulliken charge distribution of reagent 4 and 4-TFA complex on M06/6-31G(d) theory level (63–64). As shown in Fig. 2C, the H-bond interaction substantially increased the length of S─S bond from 2.156 to 2.252 Å, which indicated the activation of the reagent. Moreover, the increased electropositivity of the sulfur atom was illustrated by the change of the Mulliken charge distribution from 0.052 to 0.068 (figs. S178 to S181).

Substrate scope of clickable tryptophan modification

With the optimized conditions in hand, we examined the scope and limitations of the Trp-selective C2-trifluoromethylthiolation by reagent 4 on a series of Trp-containing model peptides with random sequences or derived from proteins like erythropoietin, adiponectin, and interlukin-25 (Fig. 3). We were pleased to find that model peptides 2a to 2o containing unprotected all 20 amino acids were trifluoromethylsulfenylated selectively at Trp residues with satisfactory high-performance liquid chromatography (HPLC) isolation yields ranging from 43 to 83% (3a to 3o). Aromatic side-chain residues, including Tyr and Phe, were not affected during the reaction (3b, 3e and 3f, etc.). It is conceivable that the nucleophilicity of polar side chains of Ser, Thr, Lys, Arg, Asp, Glu, and His was inhibited in TFA and well tolerated under our conditions. In addition, sensitive amino acid Met (3d and 3j) and Cys (3k and 3l) were compatible with the reaction. No S-sulfenylation of Met or peptide cleavage was observed, while the thiosulfonate (S-SO₂Q) and disulfide (S-SCF₃) formed via the reaction of Cys side chain and reagent 4 under our conditions were readily reduced back to free Cys by treating with an excess amount of reductant [tris(2-carboxyethyl)phosphine (TCEP), 500 mM in aqueous] after Trp modification (figs. S65 to S68).

Fig. 3. Scope of random native peptide sequences coupled with diverse thiosulfonate reagents.

Reaction conditions: peptide 2 (10 mM), thiosulfonate reagent 4/5a and 5b/6a to 6g (50 mM), TFA as solvent at 30°C for 1 hour. ^aReagent 4 (12 mM). ^bTCEP (0.5 M, pH 7.0) incubation at room temperature for 30 min before preparative HPLC purification. ^cReaction time: 2 hours.

Fluorinated thiosulfonates (ArSO₂SR_F) with diverse SR_F structures [R_F = CF₃, CF₂H, CF₂SO₂R, CF₂PO(OEt)₂, etc.] were developed and widely applied for the direct introduction of fluorinated alkyl group onto small organic molecules in recent years (65, 66). We synthesized the S-difluoromethylated (SCF₂H) (67, 68) and S-(ethoxycarbonyl)difluoromethylated (SCF₂CO₂Et) (69) quinoline-containing thiosulfonates (5a and 5b) and tested in the late-stage modification of peptide 2a. Reagent 5a facilitated the C2-difluoromethylsulfenylation under standard conditions with similar efficiency to reagent 4 and gave rise to 3p in 76% yield, while 5b afforded 3q in a medium 48% yield where the installed ethyl ester was quantitatively hydrolyzed to carboxylic acid under mild acidic conditions. Encouraged by this, we further explored the S-alkylated quinoline-containing thiosulfonates reagents anchoring both simple methyl group (6a) and a plethora of functional groups including carboxylic acid (6b), alcohol (6c), azide (6d), and alkyne (6e) in our reaction. Under optimal conditions, medium to excellent isolated yields were obtained in all cases (3r to 3v). Our result was different from the reported case on simple indole molecules under Lewis acid catalysis where no C2-sufenyaltion took place on 3-methylindole substrate (70), which showcased the key activation effect of TFA solvent. For S-arylated reagents 6f and 6g, modified peptides 3w and 3x were isolated in 68 and 66% yields, respectively, which was comparable with the radical based photocatalytic Trp-selective C2-arylsulfenylation reported recently (39).

Late-stage diversification of peptide drugs

In model studies, our late-stage Trp-selective C2-sulfenylation showed broad substrate scope with full side-chain tolerability and S-substitution diversity. Both electron-withdrawing SCF₃/SCF₂H groups and electron-donating SAlkyl/SAryl could be installed without any bias. With confidence, we moved to the modification of on-market therapeutic peptides and biomolecules with more complicated structures. Thanks to the super dissolving capability of TFA, all the peptide substrates were modified at 10 mM concentration. As illustrated in Fig. 4, the late-stage modification of therapeutic cyclic peptides with a disulfide bond such as somatostatin 7 (1.4 kDa, 1 Trp), octreotide 8 (1.0 kDa, 1 Trp), lanreotide 9 (1.1 kDa, 1 Trp), and setmelanotide 10 (1.1 kDa, 1 Trp) under standard conditions provided C2-sulfenylated products bearing fluorinated (SCF₃ and SCF₂H) or nonfluorinated [SMe, S(CH₂)₅COOH, and S(4-Cl-Ph)] substitutions in good yields with disulfide linkage intact. In the case of cyclic lipodepsipeptide antibiotic daptomycin 11 (1.6 kDa, 1 Trp), the reaction with reagent 4 was conducted at 10-mg scale to afford 13.2 mg of trifluoromethylsulfenylated analog 11a in 68% yield. The nonproteinogenic amino acids kynurenine (Kyn) and ornithine (Orn) in daptomycin were fully compatible with the reaction. For long linear peptide drugs, we modified long-acting glucagon-like peptide-1 receptor agonist semaglutide 12 (4.1 kDa, 1 Trp) and glucose-dependent insulinotropic polypeptide analog tirzepatide 13 (4.8 kDa, 1 Trp) by reagents 4, 5a, and 6a. No erosion of the yields (49% to 93%) was caused by the long lipid modification on Lys. In addition, linear relin peptide family drugs like leuprorelin 14 (1 Trp), hisrtrelin 15 (1 Trp), and triptorelin 16 (2 Trp) were smoothly modified by reagents 4, 5a, and 6a with C2-sulfenylation on all Trp residues. As a special case, the tert-butyl group on D-Ser6 of goserelin 17 (1 Trp) was removed under TFA conditions and gave rise to modified products 17a and 17b. In the modification of imaging diagnostic agent precursors DOTA-GGNle-CycMSHhex 18 and DOTA-TATE 19, prolonged reaction time was needed to get high conversion.

Fig. 4. Late-stage Trp-selective C2-sulfenylation of therapeutic peptides.

Reaction conditions: peptide (10 mM), thiosulfonate reagent (50 mM), TFA as solvent at 30°C for 1 to 4 hours.

To further explore the boundary of this method, we explored the late-stage modification of bioactive glycopeptide hAdn-WM6877 20 derived from human adiponectin collagenous domain. Peptide 20 (7.4 kDa, 69 amino acids) containing two O-glycosylated hydroxylysine residues showed promising anticancer and metabolism-regulating activities in our recent study, where the glycosylation was found to be indispensable (71). Treating 20 with 1.2 equiv. of reagent 4 at 10 mM concentration in TFA at 30°C for 1 hour afforded product 21 with C2-SCF₃ modification on the N-terminal Trp residue in 53% isolated yield, which demonstrated the compatibility with O-glycosylations (Fig. 5). The fast reaction kinetics that allowed a full conversion to be achieved in 1 hour was beneficial in this case, where prolonged reaction time in TFA may lead to cleavage of labile glycosidic linkages.

Fig. 5. Late-stage Trp-selective C2-trifluoromethylsulfenylation of glycopeptide hAdn-WM6877.

Reaction conditions: hAdn-WM6877 20 (10 mM), 4 (12 mM), TFA as solvent at 30°C for 1 hour. *Side products from the reaction.

Improvement of the serum stability of melittin via late-stage Trp modification

Native bioactive peptides for drug development always encounter poor serum stability and poor membrane permeability problems during in vivo evaluations (15). Trifluoromethylthio (SCF₃) and difluoromethylthio (SCF₂H) groups that have been demonstrated as promising modifications for improving pharmacokinetic properties on bioactive small molecules (46, 47, 51) may also exert similar effects on peptide molecules. To investigate the effect of Trp-selective C2-SCF₃ and SCF₂H modifications on pharmacokinetic properties of bioactive peptides, we chose melittin 22 (26 amino acids, 1 Trp) as our target. Melittin is a short bioactive peptide containing 26 amino acids (sequence H-GIGAVLKVLTTGLPALISWIKRKRQQ-NH₂) found from the venom of honeybee, which accounts for 40 to 60% dry weight of the venom (72, 73). Apart from causing pain sensation, the bioactivity of melittin includes antibacterial, antiviral, antifungal, antiparasitic, and anticancer effects (74). The anticancer activities of melittin have been demonstrated in melanoma, non–small cell lung cancer, leukemia, ovarian cancer, cervical cancer, glioblastoma, and pancreatic cancer (75). Currently, melittin is undergoing many clinical trials of cancer treatment (76). However, the extremely short plasma half-life as well as the nonspecific toxicity (77) limit its therapeutic use.

The C2-trilfuormethylsulfenylated melittin-SCF₃ 23a and C2-difluoromethylsulfenylated melittin-SCF₂H 23b were obtained via our Trp-selective late-stage modification method in 69 and 58% yields, respectively (Fig. 6A). In cytotoxicity assay, analogs 23a and 23b showed comparable efficacy with native melittin 22 on both human epidermal growth factor receptor 2 (HER2)–enriched breast cancer cell line (SKBR3) and triple-negative breast cancer cell line (MDA-MB-231), which demonstrated the biological activity was unattenuated. In luminal breast cancer cell (MCF7) line, 23a showed higher activity than 22, which showcased the enhanced membrane-interfering effect caused by SCF₃ group (Fig. 6B). In the in vitro human serum degradation assay, to our surprise, the introduction of SCF₃ and SCF₂H groups to Trp19 substantially improved the serum stability and prolonged the half-life from 8 to >24 hours (Fig. 6C). Our analysis showed that the modification on Trp19 largely prevented the serum peptidase-catalyzed cleavage at the adjacent Arg24-Gln25 site (figs. S193 and S194).

Fig. 6. Late-stage Trp modification of melittin and biological evaluation.

(A) Late-stage C2-sulfenylation of Trp19 of melittin. (B) Cell-viability assay of breast cancer cell lines (MDA-MB-231, SKBR3, and MCF7) treated by melittin 22 and its analogs (23a and 23b) with the IC₅₀ values. Error bars represent ± SEM of three replicates. (C) In vitro degradation stability study of melittin 22 and its analogs (23a and 23b) in human serum. The data were analyzed by UPLC-MS, and error bars represent ± SEM of three replicates. P values were calculated by two-tailed Student’s t test. *P < 0.05 versus melittin 22 group; ns, not significant.

DISCUSSION

Peptides are gradually emerging as middle-sized therapeutic agents in addressing unmet medical needs because of their intrinsic biochemical characteristics as compared to small-molecule drugs and biologics. More than 100 peptide drugs have been approved by the FDA for a wide range of diseases including anticancer, cardiovascular, metabolic diseases since the insulin was developed in 1923. In this regard, late-stage structural modifications of native peptides and peptide drugs can readily increase the structural and functional diversities and can substantially enhance their therapeutic potential by improving their stability, bioavailability, pharmacokinetics, and pharmacodynamics, as well as enabling targeted delivery and reducing immunogenicity. Enabling transformations to edit functionally dense molecules require a high level of selectivity (i.e., chemoselectivity, regioselectivity, and stereoselectivity). Native peptides carry various nucleophilic functionalities (e.g., thiol, amines, carboxylic acid, etc.) and are sensitive to redox conditions, and the solvents to dissolve unprotected peptides are also limited. Thus development of site-specific late-stage peptide modifications is a dauting task (78). In this study, we developed a clickable Trp modification via late-stage catalyst-free C2-sulfenylation using S-modified quinoline-containing thiosulfonate reagents. Diverse functional groups including trifluoromethylthio (SCF₃), difluoromethylthio (SCF₂H), (ethoxycarbonyl)difluoromethylthio (SCF₂CO₂Et), alkylthio, and arylthio groups could be efficiently installed on Trp residues in native peptides. In this transformation, TFA was used as the optimal solvent and played an important role in the activation of the reagents via H-bond interaction. Moreover, the super dissolving capability of TFA for hydrophobic and aggregation-prone peptides ensures the applicability of this method to difficult molecules like lipopeptides and self-assembling peptides at relatively high concentration. This method was successfully applied to the late-stage modification of several on-market peptide drugs as well as the bioactive glycopeptide hAdn-WM6877, showcasing the applicability of this method on the diversity-oriented modification of peptide-based active pharmaceutical ingredients. The improved bioactivity and serum stability of modified melittin analogs demonstrated the great potential of this method in drug development. Because Trp widely exists in RiPP natural products like darobactin and chloropeptin I, and drug leads screened from phage display and mRNA display, this method will be also usable to the natural product late-stage diversification for making molecular libraries and functional probes. We believe this single-step clickable late-stage Trp modification method will provide a robust platform for generating structural analogs in cost-efficient manner to satisfy the demand for optimizing drug activities and pharmacokinetic properties and will become a precious tool for medicinal chemists, peptide chemists, and chemical biologists.

MATERIALS AND METHODS

General procedure of clickable tryptophan modification

Peptide (0.010 mmol, 1.0 equiv) and thiosulfonate reagent (0.050 mmol, 5.0 equiv) were dissolved in TFA (1.0 ml) with a final concentration of 10 and 50 mM, respectively. The reaction mixture was stirred at 30°C for 1 to 4 hours according to the UPLC-MS monitoring result. After complete conversion, the solvent was removed by a flow of compressed air, and the residue containing the crude Trp-modified peptide was precipitated by adding cold diethyl ether. After centrifugation, the residue was dissolved in 10% MeCN/H₂O (10 ml) and subjected to preparative reversed-phase HPLC purification. For peptide substrates containing Met residues, 1.2 equiv. of the thiosulfonate reagent was used to suppress side reaction. For peptide substrates containing Cys residues, after the Trp modification step, the crude product with Cys side chain modified were dissolved in 10% MeCN/H₂O (9 ml) followed by addition of TCEP (pH 7.0, 0.5 M, 1.0 ml, 50 equiv). The mixture was incubated at room temperature for 30 min to reduce the S─SO₂Q bonds and then subjected to the reversed-phase preparative HPLC purification.

Supplementary Materials

This PDF file includes:

Supplementary Text

Figs. S1 to S195

Tables S1 to S3

References