On-resin peptide ligation via C-terminus benzyl ester

Article abstract of DOI:10.1016/j.cclet.2018.03.021

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 C-terminal peptide ligation on a Rink Amide resin. On-resin native chemical ligation and auxiliary-assisted peptide ligation were successfully achieved. This method is compatible to both protected and unprotected peptide fragments and has potential application in poor water-soluble peptide ligation.

Full text of DOI:10.1016/j.cclet.2018.03.021

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Accepted Manuscript
Title: On-resin peptide ligation via C-terminus benzyl ester
Authors: Bin Zhou, Faridoon, Xiabo Tian, Jian Li, Donglian
Guan, Xing Zheng, Yu Guo, Wei Huang
PII:
DOI:
Reference:
S1001-8417(18)30122-0
https://doi.org/10.1016/j.cclet.2018.03.021
CCLET 4482
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Chinese Chemical Letters
Received date:
Revised date:
Accepted date:
8-2-2018
7-3-2018
16-3-2018
Please cite this article as: Bin Zhou, Faridoon, Xiabo Tian, Jian Li, Donglian Guan,
Xing Zheng, Yu Guo, Wei Huang, On-resin peptide ligation via C-terminus benzyl
ester, Chinese Chemical Letters https://doi.org/10.1016/j.cclet.2018.03.021
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Communication
On-resin peptide ligation via C-terminus benzyl ester
Bin Zhoua,b, Faridoonb,c,, Xiabo Tianb, Jian Lib,c, Donglian Guanb,c, Xing Zhenga, Yu Guoa,*, Wei Huangb,c,*
a Hunan Province Cooperative Innovation Center for Molecular Target New Drug Study, Institute of Pharmacy and Pharmacology, University of South China,
Hengyang 421001, China
b CAS Key Laboratory of Receptor Research, CAS Center for Excellence in Molecular Cell Science, Center for Biotherapeutics Discovery Research, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
c University of Chinese Academy of Sciences, Beijing 100049, China
ARTICLE INFO
Article history:
Received 8 February 2018
Received in revised form 7 March 2018
Accepted 15 March 2018
Available online

*Corresponding Authors.
E-mail addresses: huangwei@simm.ac.cn (W. Huang); guoyuhy@126.com (Y. Guo); faridoon619@yahoo.com (Faridoon)
Graphical Abstract
Here, we reported 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 C-terminal peptide ligation on a Rink Amide resin.
ABSTRACT
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 C-terminal peptide ligation on a Rink Amide resin. On-resin native chemical ligation and auxiliary-assisted peptide ligation
were successfully achieved. This method is compatible to both protected and unprotected peptide fragments and has potential application in poor
water-soluble peptide ligation.
Keywords:
On-resin peptide ligation
Peptide benzyl ester
Native chemical ligation
Auxiliary-assisted peptide ligation
Peptide elongation
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

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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 C-terminal 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 C-terminal 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-Asp-OBn.
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 on-resin peptide ligation. It has
been reported [15] that peptide hydrazide ligation method is highly compatible with 17 C-terminus 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 on-resin 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 Aux-amino 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 auxiliary-assisted 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-Pro-Ile-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 Cys-peptides 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 on-resin 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 in-solution groups. It revealed that Aux-Gly and Aux-Ala both reacted well

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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 on-resin 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 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.
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Figure captions
Scheme 1. Synthesis of peptides 4a-g via on-resin ligation. Reagents and conditions: i) Fmoc-SPPS, HATU, DIPEA, 20% piperidine; ii) 5% NH2NH2 in DMF,
r.t., 90 min; iii) NaNO2, pH 2, –10 °C, 15 min; iv) MPAA, pH 7, –10 °C, 15 min. v) Aux-amino acids or cysteinyl peptides, MPAA, pH 7, r.t., 12 h.