De Novo Design To Synthesize Lanthipeptides Involving Cascade

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A de novo Design to Synthesize Lanthipeptides Involving Cascade Cysteine Reactions: SapB Synthesis as an Example Huai Chen, Yuan Zhang, Qian-Qian Li, Yu-Fen Zhao, Yong-Xiang Chen, and Yan-Mei Li J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b00259 • Publication Date (Web): 12 Jun 2018 Downloaded from http://pubs.acs.org on June 12, 2018

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The Journal of Organic Chemistry

A de novo Design to Synthesize Lanthipeptides Involving Cascade Cysteine Reactions: SapB Synthesis as an Example Huai Chen†, Yuan Zhang†, Qian-Qian Li†, Yu-Fen Zhao†, Yong-Xiang Chen†, Yan-Mei Li*, †, § † Key Lab of Bioorganic Phosphorus Chemistry & Chemical Biology, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China § Beijing Institute for Brain Disorders, Beijing 100069, P. R. China * Corresponding Author: E-mail: [email protected]

ABSTRACT: Lanthipeptides are a family of ribosomally synthesized peptides, which have crucial biological functions. However, due to their complicated structures, it’s challenging to total synthesize lanthipeptides. Here, a novel strategy to construct lanthipeptides is described, which involved cascade reactions of cysteine, including Cys disalkylation elimination, Michael reaction and native chemical ligation. We utilized this strategy to synthesize a lanthipeptide SapB as an example. This methodology is potential to obtain lanthipeptides and their analogues for biological research and drug discovery. Ribosomally synthesized and post-translationally modified peptides (RiPPs), which have a variety of structures and functions, have attracted increasing attention for biological research and drug discovery in recent years1. Lanthipeptides, which are widely produced by various bacteria, are one of the largest and best-studied families of RiPPs2. Lanthionine (Lan) and/or 3methyl-lanthionine (MeLan) motifs are the characteristic structural features in lanthipeptides (Figure 1a)3. These thioether-containing cross-links result in the complicated polycyclic topologies of lanthipeptides. These thioether-bridges are formed by the Michael-type addition reaction between cysteine (Cys) and dehydroalanine (Dha) or dehydrobutyrine (Dhb), which is usually generated from dehydration of serine (Ser) or threonine (Thr) with the assist of enzymes in the producing organisms (Figure 1a)2b,4. Many lanthipeptides have antimicrobial ability against various gram-positive bacteria, even some antibiotic-resistant bacteria2b,5. Besides, some lanthipeptides have some other biofunctions2b,6. For example, duramycin and cinnamycin can inhibit phospholipase A2 in eukaryotic organisms to maintain normal lipid metabolism7; labyrinthopeptin A1 manifests favorable activities against HIV and HSV8; SapB and SapT may function as biosurfactants to promote the growth of aerial hyphae9. Therefore, it’s a promising field on obtaining lanthipeptides for biological and medicinal research.

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Figure 1: (a) Structures of Dha, Dhb, Lan, and MeLan. Construction of Lan/MeLan residues in biological systems. (b) Structure of SapB (1). The green residues are Dha, and the red residues are Lan. The stereochemistry of the sites with a blue star hasn’t been determined. Scheme 1: Strategies for Total Synthesis of Lanthipeptides

Nevertheless, it is difficult to isolate lanthipeptides from natural sources for in-depth study and therapeutic use due to the small amount of production10. Therefore, varieties of methods have been developed, both biologically and chemically, to produce lanthipeptides and their analogues3,11. Among these methods, total chemical synthesis provides a powerful approach to bypass the biosynthetic system, expanding a more extensive chemical space for biological research and drug development. However, because of the complicated structures, total synthesis of lanthipeptides remains a formidable challenge. To tackle the challenge, several advances have been reported. Shiba and coworkers reported a pioneer work to total synthesize a natural lanthipeptide nisin A in solution-phase in 1988 (Scheme 1a)12. However, the approach was impeded for further development due to its tedious procedure and low yield. Chemical synthesis of lanthipeptides on solid resins has also been explored. Tabor and coworkers reported several approaches to achieve orthogonally protected Lan/MeLan derivatives, which could be used to


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synthesize lanthipeptides fragments via 9-Fluorenylmethoxycarbonyl solid-phase peptide synthesis (Fmoc-SPPS)13. Vederas’ group and van der Donk’s group also utilized the orthogonally protected Lan/MeLan to construct several full-length lanthipeptides, such as lacticin S, lacticin 481, epilancin 15X and their analogues (Scheme 1b)10,14. However, the overall yields were not high enough and the synthesis of orthogonally protected building blocks was still a challenging work. The biomimetic method, which forms the Lan/MeLan by intermolecular Michael addition reaction, has also been approached both in solid-phase and in solution15. Whereas, when there are multiple Cys and Dha/Dhb in the peptide sequence, it’s difficult to construct the correct polycyclic structure of the target lanthipeptide by this method15c. To overcome these obstacles, it is necessary to develop a facile and effective method to achieve the synthesis. We found that the generation of Dha16 and Lan11a could be both formed chemically in peptides through cysteine-based reactions. Thus, we de novo designed a novel strategy to synthesize full-length lanthipeptides utilizing several commercially available orthogonal protected Cys building blocks. Through cascade cysteine-based reactions, including Cys elimination to Dha in situ16, biomimetic Michael addition reaction15,17 and native chemical ligation (NCL)18, multiple Lan-bridge cycles and Dha residues in the peptides can be achieved (Scheme 1c). With the ability of orthogonal deprotections, we can accurately control the reaction sites of Cys and Dha to form the desired structures. Besides, all the amino acid building blocks are commercially available. Therefore, this strategy has the potential to construct complicated polycyclic topologies of lanthipeptides and their analogues through de novo design. The model lanthipeptide we chose was SapB, which was discovered from Streptomyces coelicolor in 199119. Although the exact structure of SapB hasn’t been reported4d, this 21 amino acid peptide was determined to have two Lan-bridge rings and two Dha residues in its molecule (Figure 1b)9a. The relatively simple topology and post-translational modifications of SapB make it a good model to verify the viability of our strategy. Based on our design, SapB was divided into two segments: SapB(1-9) and SapB(10-21). During Fmoc-SPPS of the two segments, we used four kinds of Cys building blocks with orthogonal protecting groups (PGs). The other amino acid building blocks were all commonly used in Fmoc-SPPS. The special Dha residues were formed from Cys in situ through the elimination reaction under bisalkylating conditions either on the solid support or in solution-phase20. The synthesis of SapB(10-21) began with the loading of Fmoc-Asn(Trt)-OH onto 2Chlorotrityl resin, followed by standard coupling of the next eleven residues sequentially, with a Fmoc-Cys(Trt)-OH at site 20, a Fmoc-Cys(Acm)-OH at site 16, a Fmoc-Cys(StBu)-OH at site 13, and a Boc-Thz-OH at site 10 (Figure 2a). These cysteine residues could be deprotected orthogonally when we need to achieve different purposes. With the full sequence assembled on the solid resin, we firstly chose the residue Cys13 with a StBu group as the precursor of Dha, as the StBu could be easily removed by reducing reagent on resin. 2 was treated with Dithiothreitol (DTT) and N, N-diisopropylethylamine (DIPEA) in DMF to afford the free thiol. Afterwards, 2,5-dibromoadipamide (DBAA) was used to convert the free thiol into the desired Dha16. Followed by the standard global deprotection and resin cleavage using TFA cocktail, the Dha13 containing peptide 3 was obtained. The crude peptide was purified with semipreparative RP-HPLC to give the purified 3. Since the Trt group was removed by the TFA



cocktail, the second free Cys20 was released. Thus, there was a Cys and a Dha existing simultaneously in 3, and Michael addition reaction could be induced. We dissolved 3 in an alkaline aqueous buffer A (50 mM Na2HPO4, pH 8.5) at room temperature and shook it for 1.5 h to induce the Michael reaction to form the first Lan-bridge ring. Since the molecular weight of 3 and 4 was the same, these two molecules could not be identified by MS. We monitored the reaction with analytical RP-HPLC and observed that the retention time had a small shift detected with the absorption at 215 nm, which suggested a new compound was produced. Besides, when detected at 254 nm, the absorption peak almost disappeared compared to 3, which might be due to the consumption of Dha (Figure 2c). We also affirmed that there was no free thiol in the new compound by testing it with Ellman reagent (Figure S7). The circular dichroism (CD) spectra showed that the new compound had a more helical structure than peptide 3 (Figure 2d). All together, these results indicated that 3 was fully converted undergoing a Michael reaction between Cys20 and Dha13 to form a cyclic peptide 4. We further analyzed 4 with a C18 analytical column and observed two diastereoisomers at a ratio about 6:1 (Figure S8). The stereoselectivity might due to the generation of an endocyclic enolate with an adjacent chiral center4c,21. We treated the major product with MeONH2·HCl and Tris(2carboxyethyl)phosphine Hydrochloride (TCEP·HCl) at pH 4.0, and the Thz in 4 was deprotected to release the third Cys10 (Figure 2e). This newly generated N-terminal free Cys could be used for the following NCL reaction. Separately, SapB(1-9) peptide hydrazide22 was prepared through Fmoc-SPPS with a Fmoc-Cys(StBu)-OH at site 3 and a Fmoc-Cys(Acm)-OH at site 6. The StBu group on 6 was removed and then converted to Dha3 by the above method, followed by treatment with TFA cocktail and purification with RP-HPLC to give purified 7(Figure 2b and f).


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Figure 2: (a) Synthesis of SapB(10-21). (b) Synthesis of SapB(1-9) peptide hydrazide. (c) HPLC traces of 3 and 4 at 215 nm and 254 nm. (d) CD spectra of 3 and 4. (e) HPLC trace (215 nm) and ESI-MS data of 5. (f) HPLC trace (215 nm) and ESI-MS data of 7.



Figure 3: (a) NCL of peptide 5 and 7, and following reactions. (b) HPLC trace(215 nm) and ESI-MS data of 1 (SapB). With the two requisite peptide segments in hand, we adopted the method reported by Liu23 to approach the NCL. However, we found several by-products appeared under the conditions. By identifying them with ESI-MS, we considered that these by-products were derived from the Michael addition of 4-Mercaptophenylacetic acid (MPAA) with 8 and N-terminal peptide MPAA thioester (Figure S9). To reduce the side reactions, we adjusted the pH of the reaction system from 6.5 to 5.5. Favorably, with the conversion maintained, the side reactions were significantly inhibited. After NCL, the second Lan-bridge (Dha3 and Cys10) was formed by dissolving 8 in the alkaline aqueous buffer A and shaking it for 2 h. We observed two equal peaks in the HPLC trace, which indicated that the newly generated chiral center was racemized (Figure S10). The difference between two Michael addition steps might due to the different chemical environment4c,15d,21. The former diastereoisomer in HPLC was selected as 9 for the following reactions. The last two Cys residues (Cys6 and Cys16) with Acm groups were deprotected with CH3COOAg in a solution of H2O/CH3COOH (v/v = 1:1) at 37℃ for 6 h followed by adding DTT24. The obtained peptide 10 was treated with DBAA to convert the two free Cys to Dha (Figure 3a). The reaction was monitored with analytical HPLC and ESI-MS (Figure 3b). After reaction and purification, the full-length lanthipeptide 1 (SapB) was obtained, with a yield of 9.8% based on peptide 3 over 6 steps.


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In summary, we de novo designed a strategy based on cascade reactions of Cys residues to synthesize full length multi-cyclic lanthipeptides. The multiple Cys residues could be deprotected orthogonally when we need to achieve desired reactions. We have demonstrated a facile synthesis of SapB by this strategy. To the best of our knowledge, this is the first example to synthesize SapB. This strategy has several advantages: 1) all the amino acid building blocks needed to synthesize lanthipeptides are commercially available; 2) with the controllable orthogonal deprotections, it has the potential to construct complicated polycyclic topologies that extensively exist in lanthipeptides by de novo design; 3) given the reasonable yield of each step, it can be used to obtain milligram scale of lanthipeptides. However, the major flaw of this strategy is the stereochemistry of the Michael addition. It’s known that addition reactions to Dha in peptides and proteins will lead to epimeric mixtures16,20a,25. The diastereomeric ratio may be affected by local chemical environment, but how to control the stereoselectivity needs to be further explored. In spite of this, the strategy provides a potential and promising methodology to obtain lanthipeptides and their analogues for biological research and drug development.

EXPERIMENTAL SECTION General. All the chemical reagents used here except DBAA were purchased from commercial resources and used without further purification. Amino acids mentioned below were used for peptide synthesis: Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-Cys(Acm)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(StBu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Boc-Thz-OH, Fmoc-Ala-OH, Fmoc-Arg(Pdf)-OH. HPLC grade CH3CN, CH3OH, TFA were purchased from commercial supplies and used for peptide purification and analysis. Peptide synthesis was completed manually using a peptide synthesis bubbler vessel. Peptides were purified using semi-preparative reverse-phase high performance liquid chromatography (RP-HPLC) at a flow rate of 10 mL/min after cleaved from resins. The semi-preparative column was a reversed-phase C18 column (YMC, 5 µm, 250 mm × 20 mm). All the peptides were analyzed using analytical HPLC with a reversed-phase C8 or C18 column (YMC, 5 µm, 150 mm × 4.6 mm) at a flow rate of 0.8 mL/min. The two solutions for HPLC are (A) water with 0.06% TFA and (B) CH3CN/H2O = 4:1 with 0.06% TFA. Peptides were identified by ESI-MS using a Thermo MSQ Plus single quadrupole electrospray ionization mass spectrometer. CH3CN/H2O = 1:1 with 0.06% formic acid was utilized as the solution at a flow rate of 0.4 mL/min. Reaction buffers used in the procedure: buffer A: 50 mM Na2HPO4, pH 8.5; buffer B: 6 M Gn·HCl, 200 mM Na2HPO4, pH 3.0; buffer C: 6 M Gn·HCl, 200 mM Na2HPO4, pH 7.4. Synthesis of peptide 5: 1.01 g 2-Chlorotrityl resin (0.911 mmol/g) was swelled in 6 mL DCM for 0.5 h. 0.25 mmol Fmoc-Asn(Trt)-OH was dissolved in 5 mL DCM with 3 drops of DMF and 220 µL DIPEA, and this solution was incubated with the resin in peptide synthesis bubbler vessel for 2 h. After washed with DCM for 3 times, a solution of DCM/CH3OH/DIPEA = 17:2:1 was used to cap the unreacted sites for 5 min ×3 times. Fmoc was removed using 20% piperidine solution in DMF. The following amino acids (4 equiv) were coupled with HATU/HOAt/DIPEA (4 equiv/4 equiv/ 8 equiv) activation, and monitored by Kaiser test reagent. The coupling reaction was carried out for 45 min, and the Fmoc-deprotection was for 5 min + 10 min. When finishing the last Boc-Thz-OH coupling, the resin was incubated with a solution of DTT (5 equiv) and DIPEA (10 equiv) in 5 mL DMF at 37℃ for 6 h to remove the



StBu group. After washed with DCM × 3 and DMF × 3, 4 mL DMF, K2CO3 (5 equiv) and DBAA (4 equiv) were added to the resin, and incubated at 37℃ overnight. 15 mL TFA cocktail including TFA/TIPS/H2O = 95:2.5:2.5 was used to deprotect and cleave the peptide from the resin. After precipitated from cold ether, the precipitate was dissolved in HFIP at a concentration of 10 mg/mL, purified with semi-preparative RP-HPLC at a gradient of 15-50% B over 30 min, and then lyophilized. 12.02 mg 3 was obtained with a yield of 3.8% based on resin loading. 2.00 mg peptide 3 was dissolved in 1 mL buffer A, and then divided equally in two 2 mL Eppendorf tubes. The tubes were placed on at 25℃ thermostatic metal bath and shaken at 300 rpm for 1.5 h. The reaction solution was monitored with analytical C8 column RP-HPLC at a gradient of 10-50% B over 30 min, and full conversion of 3 was observed. The major diastereoisomer was separated with a C18 analytical column at a gradient of 15-45% B over 30 min. And then it was treated with MeONH2·HCl (150 equiv) and TCEP·HCl (40 equiv), followed by adjusting pH to 4.0 , and then the resulting solution was shaken at 37℃ for another 24 h. After purification with analytical RP-HPLC at a gradient of 10-50% B over 30 min, 1.51 mg pure peptide 5 was obtained with the isolated yield 75.7%. 3, 4, 5 was identified with RPHPLC and ESI-MS. MS (ESI+) calcd for 3: C49H80N14O20S3 [M + H]+ 1282.4, found: 1281.7; [M + 2H]2+ 641.7, found: 641.3. MS (ESI+) calcd for 4: C49H80N14O20S3 [M + H]+ 1282.4, found: 1282.8; [M + 2H]2+ 641.7, found: 641.4. MS (ESI+) calcd for 5: C48H80N14O20S3 [M + H]+ 1270.4, found: 1269.8; [M + 2H]2+ 635.7, found: 635.3. Synthesis of peptide 7: 0.80 g 2-Chlorotrityl resin (0.911 mmol/g) was washed with DCM × 3 and DMF × 3, and then incubated with 5% N2H4·H2O/DMF (v/v) for 30 min twice. After washing, 5% CH3OH/DMF (v/v) was used to cap the unreacted sites on the resin for 10 min. After wash with DCM ×3 and DMF ×3, 0.2 mmol Fmoc-Leu-OH was used to couple the resin with 0.2 mmol HATU, 0.2 mmol HOAt and 0.4 mmol DIPEA for 60 min. A solution of DMF/Ac2O/pyridine = 3:2:1 was used to cap the unreacted sites for 30 min after coupling. Fmoc was removed using 20% piperidine solution in DMF. The following amino acids (4 equiv) were coupled with HATU/HOAt/DIPEA (4 equiv/4 equiv/8 equiv) activation, and monitored by Kaiser test reagent. The coupling reaction was carried out for 45 min, and the Fmoc-deprotection was for 5 min + 10 min. When finishing the last Fmoc-Thr(tBu)-OH coupling and Fmoc-deprotection, the resin was incubated with a solution of DTT (5 equiv) and DIPEA (10 equiv) in 5 mL DMF in 37℃ for 6 h to remove the StBu group. After washing with DCM × 3 and DMF × 3, 4 mL DMF, K2CO3 (5 equiv) and DBAA (4 equiv) were added to the resin, and incubated at 37℃ overnight. 15 mL TFA cocktail including TFA/TIPS/H2O = 95:2.5:2.5 was used to cleave the peptide from the resin. After precipitated from cold ether, the precipitate was dissolved in CH3CN/H2O = 1:1 at a concentration of 5 mg/mL, purified with semi-preparative RP-HPLC at a gradient of 15-50% B over 30 min, and then lyophilized. 15.25 mg 7 was obtained with a yield of 7.6% based on resin loading. 7 was identified with analytical RP-HPLC at a gradient of 10-50% B over 30 min and ESI-MS. MS (ESI+) calcd for 7: C42H77N15O11S [M + H]+ 1001.2, found: 1000.7; [M + 2H]2+ 501.1, found: 500.9. Synthesis of SapB (1): 0.85 mg peptide 7 (0.85 µmol, 1.55 equiv) was dissolved in 200 µL buffer B, and then placed in -20℃ ice-salt bath. 10 µL NaNO2 aqueous solution (0.85 M, 15.5 equiv) was added into the peptide solution and reacted at -20℃ for 20 min. 2.52 mg MPAA (14.8 µmol, 27 equiv) was dissolved in 200 µL buffer C, and then added to the peptide solution. After 10 s vortex, the pH was adjusted to 5.5, and then 0.70 mg peptide 5 (0.55 µmol, 1 equiv)


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was added. The reaction was carried out on 37℃ thermostatic metal bath with 300 rpm for 4 h. 100 µL TCEP·HCl aqueous solution (0.45 M, 50 equiv) was added to the reaction solution and incubated for 5 min. The reaction solution was monitored using analytical RP-HPLC at a gradient of 15-50% B over 30 min. With purification using analytical RP-HPLC at a gradient of 15-50% B over 30 min, 0.51 mg peptide 8 was obtained after lyophilized. 8 was dissolved and cyclized in 300 µL buffer A in a 2 mL Eppendorf tube at 25℃ for 2 h. With purification using analytical RP-HPLC at a gradient of 15-50% B over 30 min, the former diasetereoisomer in HPLC was selected as 9 and lyophilized. 9 was dissolved in 300 µL H2O/CH3COOH = 1:1 solution and 50 equiv CH3COOAg were added to the solution and incubated at 37℃ for 6 h. 150 equiv DTT was used to quench the reaction by incubating with the reaction solution for 5 min and then centrifuged 5 min at 6000 rpm. The supernatant was purified using analytical RPHPLC at a gradient of 15-50% B over 30 min. The obtained 10 was dissolved in 200 µL buffer A, and 20 µL DBAA (0.5 M in DMF) was added to the peptide solution. The resulting solution reacted at 25℃ for 1 h, and then 37℃ for 3 h. With purification using analytical RP-HPLC at a gradient of 15-50% B over 30 min, 0.17 mg purified SapB (peptide 1) was obtained after lyophilized. The yield was about 9.8% based on the amount of 3. 8, 9, 10 and 1 was identified with RP-HPLC and ESI-MS. MS (ESI+) calcd for 8: C90H153N27O31S4 [M + 2H]2+ 1119.8, found: 1119.6. MS (ESI+) calcd for 9: C90H153N27O31S4 [M + 2H]2+ 1119.8, found: 1119.8. MS (ESI+) calcd for 10: C84H143N25O29S4 [M + 2H]2+ 1048.7, found: 1048.6. MS (ESI+) calcd for 1: C84H139N25O29S2 [M + 2H]2+ 1014.6, found: 1014.6; [M + 3H]3+ 676.7, found: 676.7. Synthesis of 2,5-dibromoadipamide (DBAA): DBAA was synthesized using the reported method16 with a yield of 43.2%. 1H NMR (d6-DMSO, 400MHz): δ = 1.81-2.11 (4H, m, CH2CH2), 4.37 (2H, m, 2 × CHBr), 7.35 (2H, s), 7.73 (2H, s) (2 × NH2). 13C NMR (d6DMSO, 400MHz): δ = 32.47,32.58 (2 × CH2), 48.23,48.51 (2 × CHBr), 169.79,169.85 (2 × C=O). MS (ESI+) calcd for DBAA: C6H10Br2N2O2 [M + H]+ 302.9, found: 302.9. CD analysis of 3 and 4: The peptide solutions were prepared at a concentration of 0.5 mg/mL in buffer (10 mM NaH2PO4/Na2HPO4, pH 5.0). The CD spectra were measured in the standard procedure with a Chirascan Plus CD. Ellman reagent test of 3 and 4: 0.10 mg peptide (3 or 4) was dissolved in 200 μL buffer (50 mM NaH2PO4/Na2HPO4, pH 5.0), respectively. 10 equiv 5,5’-Dithiobis(2-nitrobenzoic acid) (DTNB, Ellman reagent) was added to each peptide solution respectively, and then shaken at 25℃ for 30 min. The reactions were monitored using analytical RP-HPLC at a gradient of 10-50% B over 30 min and ESI-MS.

ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (21332006, 21672126, 81661148047).

Supporting Information Copies of the 1H NMR and 13C NMR spectra of DBAA. Analytical HPLC and ESI-MS data of peptide 3, 4, 8, 9, 10. Analytical HPLC and ESI-MS data of Ellman reagent test. Analytical HPLC and ESI-MS data of the NCL reaction between 5 and 7. Analytical HPLC trace for the Michael addition reaction of 3 and 8.



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De Novo Design To Synthesize Lanthipeptides Involving Cascade

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