Chinese Chemical Letters  2018, Vol. 29 Issue (7): 1139-1142   PDF    
Total synthesis of snake toxin α-bungarotoxin and its analogues by hydrazide-based native chemical ligation
Xiao-Qi Guoa,b, Jun Liangb, Ying Lic, Yong Zhangb, Dongliang Huanga, Changlin Tiana,b    
a High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei 230031, China;
b School of Life Science, University of Science and Technology of China, Hefei 230027, China;
c Department of Chemistry, University of Science and Technology of China, Hefei 230026, China
Abstract: Nicotinic acetylcholine receptors (nAChRs) play important roles in intercellular communications of nerve cells. α-Bungarotoxins (αBtx) is a moderator for the nAChRs. Chemical synthesis provides a promising way to access αBtx and their analogues. Here, we reported a new method for α-bungarotoxin by combining Fmoc-SPPS and peptide hydrazide based ligation strategy. The two-segment ligation method may enable efficient synthesis of αBtx analogues. These synthetic toxin peptides are useful tools for development of imaging or therapeutic reagents.
Key words: Chemical synthesis     Native chemical ligation     Peptide hydrazide     α-Bungarotoxin     Nicotinic acetylcholine receptors    

Nicotinic acetylcholine receptors (nAChRs) are one of pentameric ligand-gated ion channels, which can respond to the neurotransmitter acetylcholine. nAChRs are widely exist in the central and peripheral nervous system and essential to neurotransmission. They play crucial roles in intercellular communications in nervous system [1]. As one of the best-studies of the ionotropic receptors, many animal venom including α-bungarotoxins (αBtx) were found to regulate the receptors and have been applied for structure-function studies [2].

αBtx can strongly bind to nAChR and cause heart failure or neuro transduction disorder. αBtx contains 74 amino acid residues and 5 pairs of disulfide bonds to keep structural stability and potent bioactivity. The peptide is type Ⅱ α neurotoxin derived from snake venom. αBtx have been widely used on nAChR-related researches, such as analyzing the amount of muscle nAChRs in myasthenia gravis and detecting corresponding binding nicotinic subunits on Western blots [3]. Meanwhile, αBtx probes were used for monitoring receptor expression changes in neurodegenerative or psychiatric diseases [4]. In the process of studying the structural, functional mechanism or other related researches, adequate amount of homogeneous αBtx toxins or its analogues are urgently required.

In previous accessible methods, αBtx was usually obtained by natural extraction from snake poison gland. But the banded krait-Bungarus Multicinctus contains a number of toxins with polarity and structural similarity. Those impurities are difficult to separate from the final αBtx product even with the most advanced purification technology and then led to the impurity of αBtx [5]. Xu and co-workers reported a recombinant expression approach to produce αBtx with an additional methionine and a glycine at Nterminal [6]. But expression work needed delicate construction of expression vectors, tedious expression and purification steps. Besides, expression method is difficult to provide the αBtx sample with artificial modifications.

Herein, we reported a new total chemical synthesis method for the preparation of toxin peptide αBtx. Our new approach takes advantage of Fmoc solid-phase peptide synthesis (SPPS) for individual peptide segments and peptide-hydrazide-based native chemical ligation (NCL) for segment condensation [7]. Desired toxin peptides were successfully obtained after folding of linear peptides. With the newly developed method, we synthetized natural αBtx and its analogue αBtx(V31). This new method would provide a convenient strategy to produce biophysical probes (such as fluorescent or isotope labeling) for kinetic studies of toxin and related receptor binding process.

In the past decades, one of the most important breakthrough in peptide synthesis was SPPS technique, especially through the Fmoc-based SPPS method [8]. Fmoc-SPPS can introduce functional groups into the peptide at atom level precision, such as nonnatural amino acids or post-translational modifications [9]. A series of ligation approaches (Kent's native chemical ligation (NCL) [10], Liu's hydrazide-based NCL [7a, 11], Li's Ser/Thr ligation [12] and Bode's KAHA ligation [13], etc.) have been reported for the synthesis of larger proteins or polypeptides. Those advances have made great contributions to the chemical synthesis of proteins. Even the small-to-medium sized membrane protein could be chemically synthesized by the newly developed removable backbone modification (RBM) strategy [14]. Using chemical synthesis techniques, many toxins such as mambalgin [15], alpha scorpion toxin protein Ts3 [16] had been reported. Those progresses would greatly facilitate the chemical synthesis of αBtx.

The chemically synthetic route of αBtx is shown in Scheme 1. αBtx was divided into two segments, αBtx(Ile1-Ala31Phe32)- CONHNH2 (1) and αBtx(Cys33-Gly74)-COOH (2). The peptides 1 and 2 were first synthesized by Fmoc SPPS and then ligated by peptide-hydrazide-based NCL to afford the full-length sequence αBtx 3. Finally, αBtx 3 was folded to give the native αBtx 4.

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Scheme 1. Sequences, synthetic routes and pdb structure of αBtx toxins. A. Sequences and disulfide bonds of αBtx(A31) and αBtx(V31). Different residues are marked in red and ligation sites for NCL are marked in yellow. B. Synthesis route and conditions for αBtx.

The preparation of peptide hydrazide 1 began with the coupling of Phe32 to H2NHN-2-Trt-resin, which was prepared from 2-Cl-Trtresin by previous method [17]. The chain assembly of peptide 1 was performed by a peptide automatic synthesizer (CS-Bio CS136XT) under standard coupling (HCTU/DIEA) and Fmoc deprotection conditions (20% piperidine in DMF). After final cleavage from the resin under standard conditions, the crude peptide was analyzed by analytical high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry (MSI-MS). The HPLC result showed that peptide 1 was found as the major peak (Fig. S1 in Supporting information) which was determined by ESI-MS (calcd. 3532.1 Da vs. obs. 3530.0 Da). The crude peptide was purified by semi-prepared RP-HPLC and obtained 142 mg of purified 1 in a 20% isolated yield of product (Fig. 1A).

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Fig. 1. Analytical HPLC (λ = 214 nm) and ESI-MS data of (A) αBtx(Ile1-Ala31Phe32)- CONHNH2 (peptide 1) and (B) αBtx(Cys33-Gly74)-COOH (peptide 2). HPLC conditions: a linear gradient of 10%-69% acetonitrile (0.1% TFA) in water (0.1% TFA) over 30 min.

To prepare peptide 2, the coupling of Gly74 (HBTU/HOBt/DIEA with catalytic amount of DMAP) was firstly carried out on a HOWANG-resin. The following assembly of peptide 2 was performed by a similar procedure as peptide 1. However, the result showed that it was inefficient using the normal Fmoc-AA coupling strategy and no desired product was observed by analytical RP-HPLC (data not show). It was speculated that the embedding effect of peptide chain on the resin caused the low coupling efficiency. To address this problem, we use Fmoc-(Dmb)Gly-OH to replace Fmoc-Gly-OH at Gly43 during chain peptide assembly. The backbone modified Dmb group can perturb the secondary structure formation and promoting coupling efficiency [18]. The Dmb group can be completely cleaved under TFA cocktails cleavage conditions. For this time, αBtx(Cys33-Gly74)-COOH was synthesized in good efficiency. Analysis of the crude peptide by analytical RP-HPLC indicated that peptide 2 (Fig. S3 in Supporting information) to be the major peak as determined by ESI-MS (calcd. 4496.16 Da vs. obs. 4498.0 Da). The crude peptide was purified by semi-prepared RPHPLC and obtained 372 mg of purified 2 in a 21% yield of isolated product (Fig. 1B).

With the purified two peptide segments in hand, we carried out the ligation experiment. In general, peptide hydrazide 1 (1 equiv., 22.9 mg) was dissolved in 6 mol/L Gn·HCl buffer (2.5 mL, pH 3), after cooling to -15 ℃ by ice-salt bath, 10 equiv. NaNO2 was added and gently stirred for another 15–20 min to obtain peptide hydrazide. After that, 30 equiv. 4-carboxybenzenethio (MPAA) was added to transfer peptide hydrazide into the corresponding thioester 1' in vivo. Then, 0.8 equiv. (22.5 mg) of the peptide 2 with N-terminal Cys residue was added and the pH was adjusted to 6.7 for peptide ligation. The ligation reaction was stirred at room temperature and monitored by RP-HPLC. The reaction traces were shown in Fig. 2A. After 12 h, the reaction was completed and the production was purified by semi-prepared RP-HPLC. The ligation product peptide 3 (calcd. 7994.25 Da vs. obs. 7993.97 Da) was confirmed by mass spectrometry (Fig. 2B). The peptide 3 was thus obtained in an isolated yield of 57% (24.3 mg).

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Fig. 2. Chemical synthesis of αBtx. (A) The HPLC traces of the ligation between peptides 1 and 2; Analytical HPLC (λ = 214 nm) and ESI-MS data of purified linear αBtx(A31) peptide 3 (B) and purified natural αBtx (C); HPLC condition is as same as Fig. 1; (D) CD spectrum of natural αBtx.

After the lyophilization of the linear peptide 3, a folding step was conducted in aqueous buffer with 1 mmol/L GSH and 0.1 mmol/L GSSG, pH 8.0. The final concentration of linear peptide 3 was 7 μmol/L. After 3 days, the refolding was monitored by the HPLC traces. As shown in Fig. S4 in Supporting information, the folding process can be completed after two days. Folded product 4 with five pairs of disulfide bonds was verified by mass spectrometry (Fig. 2C, calcd. 7984.25 Da vs. obs. 7984.20 Da). And refolded αBtx was purified and lyophilized to obtain as white powder with 50%-60% yield.

The folded αBtx 4 was further characterized by circular dichroism (CD) spectrum. αBtx 4 was dissolved in ddH2O at 0.1 mg/mL. The CD spectrum showed a minimum at 213 nm and a maxmum at 197 nm, indicating the formation of a well-structured β-sheet. The results were coordinated with previous reports that the αBtx structure was mainly constituted by β-sheets [19]. It suggested the successful formation of correctly folded αBtx.

To further verify the validity of our newly established strategy, we synthesized αBtx(V31), an isoform of αBtx. Similarly, αBtx (V31) was divided into two segments αBtx(Ile1-Val31Phe32)- CONHNH2 (peptide 5) and peptide 2. The synthesis of peptide 5 was the same with peptide 1 except for coupling Fmoc-Val31-OH to replace Fmoc-Ala31-OH. Ligation conditions of peptides 5 and 2, and folding manipulations of αBtx(V31) are all the same as the natural αBtx 4. Analytical RP-HPLC and ESI-MS data of peptide 5, and the ligation product 6, and folded αBtx(V31) 7 are shown in Fig. 3. ESI-MS results indicated that we obtained the correct linear αBtx(V31) 6 and 5 pairs disulfide bonds were smoothly formed after folding procedure. Meanwhile, the CD spectrum of αBtx(V31) is similar to the natural αBtx 4. These results demonstrated that this new approach could efficiently prepare toxin derivatives.

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Fig. 3. Analytical HPLC (λ = 214 nm) and ESI-MS data related to αBtx(V31) synthesis. HPLC and MS data of αBtx(Ile1-Val31Phe32)-CONHNH2 (peptide 5) (A), linear αBtx(V31) (peptide 6) (B) and folded αBtx(V31) (C), respectively. (D) CD spectrum of αBtx(V31). HPLC conditions are as same as Fig. 1.

In conclusion, we have successfully synthesized two different isoforms of snake toxin αBtx on milligram scale by combining SPPS and peptide hydrazide based NCL. Considering that αBtx is a strong binder to nicotinic acetylcholine receptors, the new chemical synthetic method allows tailor-made αBtx peptides which will be of valuable biophysical, biochemical and pharmaceutical studies of αBtx and nAChRs.

Acknowledgments

We thank Prof. Ji-Shen Zheng for his suggestions for this work. This work was supported by the Science and Technological Fund of Anhui Province for Outstanding Youth (No. 1808085J04) and the Innovative Program Development Foundation Hefei Center Physical Science Technology (No. 2017FXCX002).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cclet.2018.05.005.

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