Proteins and peptides are vital components of living organisms and play crucial roles in massive biological and physiological processes [1]. Solid phase peptide synthesis (SPPS) [2, 3] is widely applied in chemical synthesis of peptides and proteins after over 50-year development. For long peptides containing more than 50 amino acids in their sequences, native chemical ligation (NCL) [4-7] of two peptides was usually employed with C-terminal peptide thioesters and N-terminal cysteinyl peptides. Both Boc-protecting chemistry [8] and Fmoc-chemistry [9-14] could be used for preparation of the key intermediate peptide thioester in NCL. Liu group reported the peptide hydrazide ligation [15-18] using hydrazide as the stable precursor of thioester that facilitates its preparation via SPPS and successive ligation with N-terminal cysteinyl peptides.

On-resin synthesis of proteins is performing ligations on solid support that simplifies the purification procedure with solvent washing steps [19-23]. In these reported methods, the ligation usually was carried out from C- to N-terminal direction with a water compatible resin. In the case of peptide ligation from N- to Cterminal direction, purified unprotected N-terminal peptide carrying functional tags (such as His6 tag) was captured by reactive resins (like Nickel resin) then allowed its C-terminal thioester to ligate with another Cys-peptide [22]. The stability and side-reaction of thioester on resin are the major concerns for N- to C-terminal strategy.

Here, we report a new approach of on-resin peptide ligation using C-terminal benzyl ester as the stabilized precursor of thioester, which enables both N-terminal elongation and Cterminal peptide ligation on a Rink Amide resin. This method is compatible to both protected and unprotected peptide fragments and has potential application in poor water-soluble peptide ligation.

As shown in Scheme 1, we initiated the study by coupling of the Asn side-chain amide on a Rink Amide resin using Fmoc-AspOBn. In our previous work, we employed Asn side-chain benzyl ester [24] and hydrazide [25] as thioester precursors and synthesized a series of branched and cyclic peptides. Here, we chose C-terminal benzyl ester as a stabilized surrogate for onresin peptide ligation. It has been reported [15] that peptide hydrazide ligation method is highly compatible with 17 Cterminus amino acids except Asp, Asn, and Gln, because of the intramolecular cyclization during cleavage from Wang resin. Recently we solved this problem by replacing the Wang resin with the acid-sensitive CTC resin [26]. In the current work, we provided an alternative solution to C-terminal Asn hydrazide ligation by on-resin strategy.

After assembly of the first Asn side chain on resin, we elongated the N-terminus via the normal Fmoc-SPPS and synthesized model peptide 1. During this process, the benzyl ester indicated excellent stability and compatibility with SPPS procedures. Then, we started to investigate the on-resin peptide ligation by converting the C-terminal benzyl ester to thioester (Scheme 1). Peptide 1 was treated with 5% hydrazine monohydrate in DMF for 90 min at room temperature. After filtration and washing with DMF and DCM, the peptide hydrazide 2 on resin was obtained and small portion of 2 was cleaved and deprotected from resin with Cocktail R reagent (mixture of 90% TFA, 5% thioanisole, 3% EDT, 2% anisole) and analyzed by HPLC and mass spectrometry (MS). As shown in Fig. 1, it revealed that the "on-resin" strategy significantly reduced the cyclized side product and gave peptide hydrazide 2 in excellent yield. Thereafter, the peptide hydrazide 2 on resin was converted into carbonyl azide by treating with NaNO₂ and successive reaction with mercaptophenylacetic acid (MPAA) [15, 26] gave the peptide thioester 3 on resin in excellent yields (see Supporting information for monitoring of these procedures). In this approach, we employed the RINK amide resin which is not a water compatible resin, thus we performed the reaction in DMF solvent. The added aqueous NaNO₂ in the system contains limited amount of water and does not influence the thioester formation.

With the peptide thioester 3 in hands, we performed the onresin NCL with N-terminal cysteinyl peptides and the on-resin auxiliary-assisted peptide ligation [27] with auxiliary-linked amino acids (Aux-amino acids). The on-resin ligation products 4a-g were analyzed by HPLC and MS detection after cleavage. The typical HPLC profiles of crude product 4a, 4c (ligation with Auxamino acids) and 4g (ligation with a Cys-peptide) were indicated in Fig. 1. The on-resin NCL achieved almost 100% conversion rate as shown in the HPLC profiles (Fig. 1D and Fig. S5 in Supporting information) that implicated the success of the on-resin ligation strategy. Meanwhile, on-resin auxiliary-assisted ligation also showed good result with Aux-Gly (Fig. 1B) with >95% conversion rate. The ligation of thioester 3 with Aux-Asp indicated moderate reaction rate (Fig. 1C) that is a reasonable result since the auxiliaryassisted ligation was reported with low yields for hindered amino acids [27]. And, it is surprising that the unreacted thioester 3 on the resin are very stable after 12 h ligation that suggested the optimization of the ligation by extending reaction time is possible. These successful results of on-resin ligation using peptide benzyl ester validated our tentative idea and encouraged us to apply this approach to other hydrophobic peptides with poor water-solubility.

We synthesized two hydrophobic peptides NH₂-Ala-Val-ProIle-Asn(resin)-OBn 5a (containing the membrane penetrating peptide motif AVPI) and NH₂-Leu-Leu-Leu-Leu-Leu-Asn(resin)- OBn 5b (containing the leucine-rich moiety usually found in protein transmembrane domains) via Fmoc-SPPS. With the same on-resin strategy described above, the peptide benzyl esters 5a-b were firstly converted to peptide hydrazides 6a-b, then converted to peptide thioesters 7a-b, and ligated with Aux-Gly or Cyspeptides to give peptides 8a-h (Scheme 2). The HPLC chromatograms of crude peptides 8a-h were shown in Fig. 2. In all cases of on-resin ligation, thioesters 7a-b were almost completely consumed without side reactions of hydrolysis or intramolecular cyclization. The ligation products 8a-h were obtained in excellent yields (>90% based on their HPLC profiles). These results demonstrated that on-resin ligation is a useful approach to deal with the hydrophobic peptides with poor water-solubility.

The successful on-resin ligation using Aux-amino acids prompted us to carry out a comparison study of on-resin and in-solution auxiliary-assisted ligation. As shown in Scheme 3, we prepared onresin peptide benzyl ester 9 and converted it to hydrazide 10 and thioester 11 successively. The on-resin thioester 11 was ligated with Aux-amino acids as the on-resin reaction groups. For in-solution ligation groups, peptide hydrazide 12 was cleaved from on-resin peptide 10 then was converted to the thioester and reacted with Aux-amino acids. The comparison reactions were monitored by HPLC and MS detection. Fig. S15 (Supporting information) indicated the HPLC profiles of product peptides 13a-d in on-resin and insolution groups. It revealed that Aux-Gly and Aux-Ala both reacted well with peptide thioesters either on resin or in solution with over 90% conversion rates (Fig. S15 panel a vs. a', panel b vs. b'). From their HPLC profiles, it is obvious that the advantage of on-resin ligation lies in the simplified purification procedures since all reactants were easily removed by solvent wash. In case of Aux-Asp and Aux-Lys, the reactivity is poor as compared to Aux-Gly and Aux-Ala both on resin and in solution (Fig. S15 panel c vs. c', panel d vs. d'). It seems the onresin strategy did not show enhanced reactivity for hindered auxiliary-assisted ligations.

In conclusion, we have developed a new on-resin peptide ligation strategy using C-terminal benzyl ester as the stabilized precursor of peptide thioester. The benzyl ester is stable during normal SPPS procedures and could be efficiently converted to thioester for peptide ligation that provides flexibility in N-terminal elongation and C-terminal ligation via either native chemical ligation or auxiliary-assisted peptide ligation. The advantages of this on-resin ligation include high efficiency, simple purification procedure, compatibility with protected and unprotected peptides, and friendly conditions for peptides with poor water-solubility. In addition, this approach could be also useful for cyclic peptide preparation via intramolecular amidation between C-terminal and N-terminal on resin.

Acknowledgments

This work was supported by Hunan Provincial Innovation Foundation for Postgraduate (China) (No. CX2017B546) and the National Natural Science Foundation of China (No. 21572244). We thank Dr. Houchao Tao and Dr. Fei Zhao in iHuman Institute, ShanghaiTech University and Dr. Jingjing Shi in SIMM for their kind help in MS determination.

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.2018.03.021.