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Abbreviation (ISO4): Prog Chem      Editor in chief: Jincai ZHAO

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Progress in Chemistry >

2023 , Vol. 35 >Issue 4: 526 - 542

DOI: https://doi.org/10.7536/PC220930

Review

Chemical Synthesis of Peptides and Proteins

  • Xinyue Wang ,
  • Kang Jin , *
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  • Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmacy, Cheeloo College of Medicine, Shandong University,Jinan 250012, China
* Corresponding author e-mail:

Received date: 2022-09-26

  Revised date: 2022-11-07

  Online published: 2023-02-16

Supported by

Taishan Scholar Program in Shandong Province, the National Natural Science Foundation of China(22007059)

Shandong Provincial Key Research and Development Program (Major Technological Innovation Project)(2021CXGC010501)

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Abstract

As the material basis of active substances and life activities in living organisms, peptides and proteins play vital roles in basic physiological processes such as signal transmission, energy utilization, immune response, etc. And they are closely related to the occurrence of a variety of diseases. An important prerequisite for studying their structure and biological function and developing related drugs is to obtain a certain number of high pure peptides and proteins. The sources of natural peptides and proteins mainly include tissues and organs of animals and plants, secondary metabolites of microorganisms, etc. Natural extraction, recombinant technology, and chemical synthesis are the main methods to obtain peptides and proteins. Chemical synthesis can conveniently introduce unnatural amino acids or specific types of post-translational modification groups at any site of peptides and proteins compared with the former two, such as glycosylation, phosphorylation, fluorophores, and photorelinking reaction groups, which has greatly promoted the application and development of peptides and proteins in the field of medicine research. This review comprehensively introduces the various chemical synthesis strategies of peptides and proteins, along with the basic principles, advantages and disadvantages, and application values, aiming to provide a novel sight for synthesizing peptides and proteins.

Key words： peptide synthesis; difficult peptides; protein synthesis; chemical strategies

Cite this article

Xinyue Wang , Kang Jin . Chemical Synthesis of Peptides and Proteins[J]. Progress in Chemistry, 2023 , 35(4) : 526 -542 . DOI: 10.7536/PC220930

Contents

1 Introduction
2 Chemical synthesis of peptides
2.1 Solution-phase synthesis
2.2 Solid-phase synthesis
2.3 Chemical synthesis of difficult peptides
2.4 Other synthesis strategies
3 Chemical synthesis of proteins
3.1 Native chemical ligation
3.2 Diselenide-selenoester ligation
3.3 Ser/Thr ligation;Cys/Pen ligation
3.4α-Ketoacid hydroxylamine ligation
3.5 Chemical synthesis of difficult proteins
4 Conclusion and outlook

1 Introduction

The history of human peptide synthesis can be traced back to the beginning of the 20th century. In 1901, Fischer et al. Synthesized the first unprotected dipeptide structure (Gly-Gly), and proposed the technical term "peptide" the following year[1]. At first, there were two main methods for the synthesis of polypeptides: the acyl azide condensation reaction and the acyl chloride condensation reaction proposed by Curtius and Fischer, respectively[2][3]. Among them, the lack of removable temporary amino acid protecting groups is the main constraint for polypeptide synthesis. Therefore, a variety of protective groups have been invented and used, such as Cbz (benzyloxycarbonyl) protective group and Boc (tert-butoxycarbonyl) protective group, which have promoted the research process of polypeptide synthesis to varying degrees, and many famous polypeptides have been successfully synthesized with the help of these protective groups, such as octapeptide oxytocin and β-adrenocorticotropic hormone[4][5]. Nowadays, various amino acid protecting groups have been invented and used, which has become an important cornerstone of polypeptide synthesis research. Merrifield's Solid phase peptide synthesis (SPPS) method pushed peptide synthesis to a new height, and his outstanding contribution to the field also earned him the Nobel Prize in Chemistry in 1984[6,7]. Over the past decades, with the continuous development of resins and condensing agents, solid-phase peptide synthesis technology has become mature and has become the first choice for peptide synthesis. The rapid development of peptide synthesis has also promoted the study of protein synthesis. At present, the chemical synthesis of proteins is mainly divided into two parts: the synthesis of polypeptide fragments and the ligation of peptide fragments. Nowadays, various ligation methods have been developed rapidly, allowing unprotected peptide chains to polymerize under mild conditions, making the chemical synthesis of proteins more convenient and rapid.
Although there are many ways to obtain peptides and proteins: natural extraction, recombinant technology and chemical synthesis, only chemical synthesis allows the manipulation of peptides and proteins from the atomic level, thus achieving the introduction of unnatural amino acids or various complex post-translational modification processes. Chemical synthesis provides opportunities for unlimited variation in protein structure, which is extremely important for understanding the mechanism of protein structure and functional properties at the molecular level. This review is mainly carried out from two aspects. In the part of chemical synthesis of peptides, the synthesis strategies of peptides (especially difficult peptides) are introduced in detail by solid-phase synthesis. In the part of protein chemical synthesis, the current situation of modern protein chemical synthesis is introduced in detail, mainly by the solution phase ligation of various polypeptide fragments. This review not only introduces the principle, development and application steps of various methods, but also includes the synthesis methods of special peptides and difficult proteins, as well as the semi-synthesis methods of proteins.

2 Chemical synthesis of peptide

2.1 Liquid phase synthesis

Before the advent of solid phase synthesis, liquid phase synthesis was the only method for the chemical synthesis of polypeptides, and great achievements were made, such as the octapeptide oxytocin and the world's first synthetic crystalline bovine insulin[4][8]. Like most chemical reactions, it involves the condensation of amino acids in the liquid phase. Although solid-phase synthesis is the main method of peptide synthesis today, liquid-phase synthesis is still used because of its unique advantages, especially for some peptides with unconventional structures, which are often difficult to obtain by solid-phase synthesis. In 2016, Hattori et al. Successfully obtained Triostin A, a symmetrical bicyclic structure with disulfide bonds, which is also a special cyclic peptide containing two ester bonds[9]. In 2017, Zhang et al. Successfully synthesized acetyl hexapeptide-3 using the "3 + 2 + 1" liquid phase condensation strategy[10]. In 2014, Fuse et al. Achieved the total synthesis of mannamycin aglycone using a solution-phase method[11].
However, due to the time-consuming and laborious steps of repeated condensation, separation and purification, this liquid-phase reaction is not suitable for the acquisition of long-chain peptides or proteins, so solid-phase synthesis has become the mainstream method of peptide synthesis.

2.2 Solid phase synthesis

Since Merrifield reported the solid phase peptide synthesis in 1963, the research progress of peptide and protein chemical synthesis has been accelerated[6,7][12,13]. In the solid-phase synthesis process, the first amino acid at the end of the peptide chain is connected to an insoluble resin, and then the amino acids are connected in sequence through repeated coupling and deprotection steps, and the target molecule is cleaved from the resin after the peptide chain is extended. During the whole process, except for the peptide chain attached to the resin, other soluble impurities were removed by multiple washing and filtration. This repeated wash filtration replaces the purification step in the liquid phase reaction and greatly increases the rate of peptide synthesis. Moreover, the simple operation of impurity removal allows the addition of excessive reactants to promote the condensation process, which is also an important point that the solid phase reaction is superior to the liquid phase reaction.
Boc and Fmoc (9-fluorenylmethoxycarbonyl) are two protective groups suitable for α-NH2, which require two different deprotection conditions of strong acid and weak base, respectively[14]. Although many peptides have been obtained using Boc-SPPS technology, it requires the use of highly toxic hydrofluoric acid for cleavage, and many post-translational modifications are not compatible with hydrofluoric acid, which makes the milder Fmoc-SPPS dominate synthetic peptides[15,16][17]. At present, the operation steps of Fmoc-SPPS have been described in detail in many literatures. The basic process can be summarized as resin swelling, connecting the first amino acid, quenching the unreacted resin site with methanol, coupling the next amino acid, repeating the coupling washing until the chain elongation is completed, and treating the resin with lysate to obtain the polypeptide (Figure 1)[18,19]. Solid-phase peptide synthesis is different from ordinary liquid-phase reactions in that the progress of the reaction cannot be monitored by the convenient TLC (thin layer chromatography), but the coupling process can be monitored by the Kaiser test because free amino groups (except the secondary amine proline) that are not involved in coupling are stained (blue) with ninhydrin[20].
图1 固相多肽合成的基本过程

Fig.1 Basic procedures of Solid phase peptide synthesis

For decades, the core idea of solid-phase peptide synthesis technology (that is, its synthesis steps) has remained unchanged, but the resins and condensation reagents involved in the reaction have been continuously developed. The resin, as the source of the solid phase state, is composed of a solid support (composed of a cross-linked polymer) and a Linker (connecting the solid support and the target peptide chain). It must meet several requirements: chemical stability, insolubility, swellability, suitable loading equivalent. The solid phase support is important because the peptide synthesis reaction takes place within its swollen grid space, and this structure, which is insoluble in most solvents, can be well swollen in DCM (dichloromethane) and DMF (N, N-dimethylformamide), which is why DCM and DMF are used as solid phase reaction reagents. Since the synthetic direction of the peptide chain is mostly from the C terminus to the N terminus, most resins anchor the carboxyl group of the first amino acid at the C terminus of the peptide chain. Table 1 below lists the resins commonly used in the Fmoc-SPPS technique.
表1 Fmoc-SPPS过程中的常用树脂

Table 1 Common resins used in Fmoc-SPPS

Name Structure ref
Wang resin 21
2-Chlorotrityl
Chloride resin
(2-Cl-(Trt)-Cl resin)		 22
2-Cl-(Trt)-
NHNH2
Fmoc resin		 23
BAL resin 24
MeDbz-resin 25
Rink Amide
resin		 26
ChemMatrix
resin		 27
The peptide backbone is built up by the formation of amide bonds, which are not easy to form. Although the amino group of an amino acid is a good nucleophilic group at high pH, i.e., alkaline conditions, the hydroxyl group in the carboxyl group is not a good leaving group, so the carboxyl group must be activated (CO-X) to allow the acyl acceptor to accept the nucleophilic attack of the amino group, and this activating reagent is the condensation reagent. The following table lists some commonly used condensing agents (Table 2).
表2 常用的缩合试剂

Table 2 Common coupling reagents

Type Name Structure ref
carbodiimides DCC 28
DIC 29
EDC 30
Oxyma 31
phosphonium
salts		 PyBop 32
PyAop 33
PyOxim 34
Uronium/
aminium
salts		 HBTU 35
HATU 35
HAPy-U 36
COMU 37
Solid-phase peptide synthesis is now well developed and allows the successful synthesis of peptides containing up to 50 amino acids and some small proteins[38]. It has made great contributions to the acquisition of a large number of polypeptides and proteins. However, this method still has many shortcomings, such as the use of environmentally unfriendly solvents (DCM and DMF, etc.) And condensing agents in the condensation and washing process, which makes the cost too high and causes waste. Therefore, when large-scale short peptides are synthesized, the liquid phase synthesis method is relatively more suitable.

2.3 Synthetic Strategies for Difficult Peptides

At first, Difficult Peptides mainly refer to Peptides with aggregation tendency, which will affect the contact between the solution and the peptide chain on the resin during solid phase synthesis, resulting in incomplete or even retarded deprotection and condensation reactions[39,40]. Peptide chain folding (such as β sheet), which is caused by the formation of non-covalent hydrogen bonds between the amide hydrogen (— NH) and the carbonyl oxygen (— C = O), is the main cause of aggregation tendency (Fig. 2). Later, the scope of difficult polypeptides was extended to all polypeptides that are not easy to synthesize, such as poor solubility caused by hydrophobic amino acids, difficulty in condensation of an amino acid, and so on[41]. During Fmoc-SPPS, the aggregation of the peptide chain occludes the N terminus that should continue the reaction. However, in Boc-SPPS, TFA (trifluoroacetic acid) used for deprotection can effectively destroy the aggregation of peptide chains. Therefore, the synthetic strategy for difficult peptides in this section is mainly applied to Fmoc-SPPS. The applicability and characteristics of various synthetic strategies are summarized in Table 3.
图2 氢键的形成导致多肽聚集

Fig.2 Hydrogen bonds contribute to aggregates of peptide

表3 困难多肽合成策略的应用范围及其特点

Table 3 Application scopes and advantages of difficult peptides synthesis strategies

Synthesis
strategies		 Application scopes Advantages
Pseudo-Prolines Difficult peptides, cyclization of peptides Disrupting the formation of hydrogen bond; Inducing cis-amide conformation
ortho-Hydroxybenzyl structure Difficult peptides Disrupting the formation of hydrogen bond
O-acyl isopeptide Difficult peptides Preventing β-sheet interactions and subsequent aggregations;Stable in TFA condition
Pegylation Difficult peptides,non-
polar peptides		 Solubilization

2.3.1 Pseudoproline method

Pseudo-Prolines method was proposed by Mutter et al. In 1996[42]. Inspired by the structure of proline, they connected the α-NH2 of Thr (threonine), Ser (serine), Cys (cysteine) with the — OH (or — SH) of the side chain to form a five-membered ring. The pseudo-proline containing an oxazolidine or thiazolidine five-membered ring structure is used to replace a common amino acid to be connected to a peptide chain, so that the formation of a hydrogen bond can be prevented, the accumulation of the peptide chain is disturbed, and the purpose of solubilization is achieved. After peptide chain elongation is complete, the pseudo-proline structure can be restored to the native amino acid residue under deprotected TFA conditions (Figure 3A). Usually this pseudoproline will be attached to the resin peptide chain in the form of a more condensable dipeptide unit, such as AA-Ser(ψMe,Mepro)[43⇓~45]. The pseudo-proline method opens up new prospects for the synthesis of complex peptides and has been applied to the synthesis of many peptides and proteins. The successful synthesis of human islet amyloid polypeptide (hAmylin1-37) by Abedini et al. In 2005 is a strong proof of this method. The use of pseudo-proline dipeptide fragments not only improves the yield of solid-phase synthesis, but also facilitates the purification process, which simplifies the synthesis of this difficult polypeptide[45].
In addition to solubilization, there are many applications of this strategy. It was found that the change of cis-amide bond conformation by the pseudo-proline structure can help the cyclization of cyclic peptides (Fig. 3B)[46]. Taking advantage of this, Liu et al. Successfully synthesized cyclopentapeptides MZ602 and MZ568 using the method of pseudo-proline et al[47]. From another point of view, the pseudo-proline structure is a temporary protection for the hydroxyl group of the side chain. In 2012, when Wang et al. Synthesized glycopeptides, in order to prevent the formation of asparagine by-products, they used pseudo-proline dipeptide structure to synthesize peptide chains, and successfully synthesized two key glycopeptide fragments: EPO (79-124) and EPO (1-28)[48]. In 2020, Zeng et al. Also used the pseudo-proline dipeptide structure to inhibit the formation of asparagine by-products when synthesizing the peptide chain part of human E-cadherin N-glycopeptide[49]. In conclusion, this method is increasingly used in the synthesis of polypeptides as an effective solution.
图3 (a)伪脯氨酸结构在多肽全脱保护的酸性条件下即可方便除去;(b)正常的反式酰胺键和伪脯氨酸结构导致的顺势酰胺键,顺式构型有利于环肽的环化

Fig.3 (a)Pseudoproline removed conveniently during acidic global deprotection of peptides; (b)Normal trans-amide and cis-amide caused by pseudoproline, and the latter is preferred during cyclization of cyclic Peptides

2.3.2 O-hydroxybenzyl protection

Similar to the pseudo-proline method, o-hydroxybenzyl protection also perturbs hydrogen bond formation by protecting the backbone amide bond. The original o-hydroxybenzyl protecting group was N- (2-hydroxy-4-methoxybenzene) (Hmb) proposed by Johnson et al., which was also the first protection of backbone amide[50]. In general, secondary amines require more aggressive acylation conditions than primary amines, and are difficult to acylate completely even with repeated coupling and prolonged time. The o-hydroxyl group in the ingenious Hmb structure plays a role of co-acylation (non-steric hindrance) through O/N acyl transfer, which ensures the smooth coupling of the next amino acid. The deFmoc N-Hmb structure has two tautomers, A and B. Configuration B favors O-acylation, and once the ester bond is formed, O/N acyl transfer also occurs (Figure 4A). In 2016, Li et al. Successfully synthesized nucleoprotein HMGA1a using the STL (serine/threonine ligation) strategy with the help of the Hmb structure. The introduction of the Hmb structure improved the solubility of the protein, enabling the synthesis route to achieve an acceptable multi-milligram yield[51].
图4 (a)下一个氨基酸引入到末端N被Hmb保护的接树脂多肽链的机制;(b) Hmsb和Hmnb的结构

Fig.4 (a) Mechanism of next amino acid introduction into N(Hmb)-peptidyl-resin; (b) Hmsb and Hmnb

Although the Hmb protecting group ensures the completion of the acylation process, the speed is not satisfactory. Therefore, a series of modified protecting group structures based on Hmb were reported in order to accelerate the acylation process by introducing electron-withdrawing groups, such as Hmsb and Hmnb (Fig. 4B)[52][53]. At the same time, solvents with strong ability to swell the resin also accelerate acyl transfer, such as 1,4-dioxane[52].

2.3.3 O-acylated isopeptide method

The O-acyl isopeptide is different from the common polypeptide chain in that its backbone structure contains an ester bond rather than a single amide bond. This ester bond results from the condensation of the side chain OH of Thr or Ser, instead of α-NH2, with the COOH of the next amino acid. The presence of an ester bond disrupts the continuity of the amide bond in the backbone, thereby preventing aggregation by β-sheets and the formation of intra- or intermolecular hydrogen bonds. This method has two key steps, esterification and acyl O/N transfer (releasing the native amide bond)[54]. Considering that esterification condensation causes epimerization, the researchers condensed ester-linked dipeptide units (such as Boc-Ser (Fmoc-Val) -OH) onto the peptide chain rather than esterifying directly on the resin peptide chain (Figure 5A), and the commercially available O-isoacyl dipeptide units are now readily available[55].
图5 (a)使用二肽单元合成异肽;通过(b):pH和(c):光照射介导O/N酰基转移使异肽恢复为正常多肽的机制[57]

Fig.5 (a) Synthesis of isopeptide using dipeptide unit; Mechanism of isopeptide to peptide through O/N acyl shift mediated by (b): pH and (c): photoirradiation[57]

This O-acylation modification not only facilitates the solid phase synthesis of difficult peptides, but also facilitates the separation and purification of the liquid phase state after synthesis. Because the O-acylated isopeptide is stable under acidic conditions, it is not affected by the TFA lysate and is stable as a trifluoroacetate powder (fig. 5B). Therefore, the transfer of acyl O/N can be induced by adjusting pH[56]. In addition to pH, other methods can be used to induce acyl transfer, such as photoirradiation. The "light-triggered switch" method uses a special protecting group to protect the α-NH2 of Thr or Ser, which can be removed under light. In 2008, Taniguchi et al. Used this method to synthesize Aβ1-42, a peptide associated with Alzheimer's disease[57]. They used a coumarin derivative as an amino protecting group, which remained on the peptide chain after resin removal and participated in the liquid phase purification process, which was greatly improved due to the high water solubility of the protecting group. The purified O-acylated isopeptide was deprotected by coumarin derivatization under illumination, and the target peptide was obtained by acyl migration in neutral buffer (Figure 5C). In 2021, Hojo et al. Successfully synthesized Caveolin-1, a membrane protein composed of 177 amino acid residues, by using isopeptide structure to increase protein solubility[58].

2.3.4 PEGylation method

Polyethylene glycol (PEG) is a hydroxyl-terminated linear or branched polyether, so the high solubility brought by this structure can be used to modify biomolecules — Pegylation. Similarly, the introduction of polyethylene glycol structural units into the peptide chain will greatly increase the water solubility of the polypeptide chain, which is not only beneficial to the coupling extension of the peptide chain in the SPPS process, but also beneficial to the later liquid phase purification. Therefore, the successful insertion of polyethylene glycol into the polypeptide chain is the key. In 1994, Lu et al. Introduced the polyethylene glycol structure into the peptide sequence and used SPPS to synthesize PEGylated peptides[59]. They used different ligation methods to introduce polyethylene glycol into different sites :NH2 terminus, at the side chain (lysine/aspartic acid), COOH terminus. For example, for two methods of introduction to the side chain: coupling of a previously prepared PEG-linked amino acid (fig. 6) to the peptide chain; Or after the peptide chain is extended according to the standard procedure, PEG is added to the resin polypeptide. As an excellent solubilizer, PEG is an important member of the peptide optimization strategy. Its modification of natural peptides or proteins can effectively improve the structure and obtain better biological functions. For example, in 2021, Yang et al. Introduced a cysteine residue at position 24 of a certain glucagon to achieve PEGylation of the polypeptide[60]; In 2020, Wang et al. Attached PEG6 to the N-terminus of the target peptide with the help of glutamic acid to enhance peptide stability and targeting[61].
图6 连接PEG基团的氨基酸

Fig.6 Amino acids linked with PEG group

2.4 Other synthesis strategies

Because of their special structural and pharmacological characteristics, such as strong affinity and low toxicity, cyclic peptides provide a wider space for drug design and have become a hot research topic in recent years[62]. Linear peptides have been easily synthesized with the help of solid-phase synthesis technology, but the cyclization of linear peptides has brought many challenges to the acquisition of cyclic peptides. Head-to-tail condensation (or head and side chain, side chain and side chain, side chain and tail) is the most direct way of cyclization, but this direct condensation has unavoidable side reactions, such as intermolecular condensation, destruction of C-terminal chiral structure, etc[63]. The development of natural chemical ligation (NCL) provides a new method for the cyclization of cyclic peptides, which not only effectively reduces the epimerization of the C-terminus, but also allows the cyclization of linear peptides in the fully deprotected state. In addition to NCL, chemoselective methods such as STL and KAHA (α-keto acid/hydroxylamine) ligation can be applied to cyclic peptide synthesis, and these ligation techniques are described in detail below[64][65][66]. On-resin cyclization is also an important method for the synthesis of cyclic peptides, and its important advantage is that it can easily remove solvents and excess reagents. In 2018, Gless et al. Reported an efficient method for the synthesis of cyclic peptides by cyclization on resin using o-amino (methyl) -aniline (MeDbz) as a linker (Fig. 7)[67].
图7 使用MeDbz Linker进行接树脂的环化[67]

Fig.7 On-resin cyclization using the MeDbz Linker[67]

Glycosylation is a common and complex post-translational modification. There are two natural glycosylation processes: O-glycoside, in which the glycan is linked to the hydroxyl group of serine, threonine, or tyrosine to form an α or β linkage; N-glycoside, the glycan forms a β linkage with the side chain amide of aspartic acid. The complexity of this glycosylated structure brings many challenges to its synthesis, which is usually not available through biological expression, while chemical methods and chemoenzymatic intervention are effective for the synthesis of this particular polypeptide[68]. In 2022, Ma et al. Reported a solution-phase chemistry combined with chemoenzymatic synthesis of complex glycopeptides, and the facile synthesis of multiple glycopeptides demonstrated the broad applicability of this method[69]. In 2020, Zeng et al. Chemically synthesized human E-cadherin N-glycopeptide, in which the glycosidation process at the asparagine site was achieved by solid-phase synthesis and was chemoselective[49].

3 Chemical synthesis of protein

A variety of ingenious methods provide unlimited possibilities for the chemical synthesis of peptides. Liquid-phase and solid-phase synthesis are two ideas for the condensation and elongation of peptide chains. Various amide backbone protection strategies have effectively improved the problems of aggregation and insolubility in peptide chain synthesis, and these methods have also been widely used in the synthesis of special peptides such as cyclic peptides and glycopeptides. However, these common methods of polypeptide can only synthesize shorter peptide chains, and it is difficult to synthesize macromolecular proteins. Proteins not only have larger molecular weights (50 to 300 amino acid residues or more) than polypeptides, but also have more complex structures due to the further folding of linear polypeptide chains. Therefore, protein can not be simply understood as a larger polypeptide, and its chemical synthesis is more complex. The main idea is to use solid-phase synthesis to obtain several polypeptide fragments, and then connect these peptides in a liquid-phase reaction, so it is called combined solution/solid-phase synthesis. Solid-phase peptide synthesis has been described in detail above, and the main methods of peptide chemical ligation are summarized below. Table 4 summarizes the ligation methods of various peptides.
表4 多肽连接方法的应用范围及其特点

Table 4 Application scopes and advantages of peptide ligation methods

Peptide ligation methods Application scopes Advantages
NCL N-terminal residue is cysteine or thiol-derived amino acid Mild, aqueous conditions
Useing completely
unprotected peptide
fragments
DSL N-terminal residue is selenocystine diselenide Faster than NCL
STL N-terminal residue is
Ser or Thr		 Compatibility with most of the C-terminal residues except Asp, Glu, and Lys
Simple operation in pyridine/AcOH solution
CPL N-terminal residue is cysteine or Penicillamine Compatibility with sterically demanding amino acids at the C-terminus
KAHA ligation Ligation junctions such as Phe-Ala, Ala-Phe, Pro-Ala, and Ala-Ala
are viable		 Producing only
H2O and CO2 as
by-products
Without
additional reagents

3.1 NCL

The development of peptide ligation chemistry has been promoted by the proposal of Native chemical ligation (NCL), which allows the ligation of more soluble deprotected peptides under a common condition, and this convenient ligation has made a great contribution to protein synthesis[70]. The process of natural chemical ligation to form a natural amide bond is quite different from that in solid phase synthesis. It uses the side chain of the N-terminal Cys (soft base) to attack the acyl carbon of the C-terminal thioester (soft acid) to form a temporary acyl sulfide bond, and then forms a natural amide bond through intramolecular S/N acyl transfer (Fig. 8). The method involves two peptide fragments, a C-terminal thioester polypeptide fragment and a polypeptide fragment with a cysteine at the N-terminus. Aryl thioesters have been shown to be highly effective participants in natural chemical ligation, and many exogenous thioester activators have been developed, such as MPAA (4-mercaptophenylacetic acid)[71]. Therefore, many efficient methods are used to synthesize inert peptide precursors that can be converted to active aryl thioesters under NCL conditions to participate in ligation reactions.
图8 自然化学连接的机制

Fig.8 Mechanism of NCL

3.1.1 Polypeptide hydrazide structure

As an excellent precursor structure, polypeptide hydrazide can not only be easily generated under Fmoc-SPPS conditions, but also be easily activated into thioester structure to participate in NCL. In 2011, Fang et al. Reported this natural chemical ligation reaction with the hydrazide structure as the precursor (Figure 9 a)[72]. Peptide hydrazide was first synthesized by Fmoc-SPPS on the self-made hydrazide resin, and then oxidized to acyl azide in the buffer containing sodium nitrite. A thiol is then added to thiolate it to a thioester, so that the original hydrazide structure is activated and can be linked to the cysteine site of another fragment. The oxidation, thiolysis, and peptide ligation of the hydrazine structure in this method is a favorite "one-pot reaction.". Unfortunately, this method is not applicable when the C-terminal amino acids of polypeptide fragment 1 are Asp (aspartic acid)/Asn (asparagine) and Gln (glutamine), because the deprotected side chains of these three amino acids form a ring with the terminal hydrazine junction, preventing the formation of the C-terminal thioester.
图9 (a)自然化学连接中使用多肽酰肼作为硫酯供体[72];(b)使用2-Cl-(Trt)-NHNH2树脂合成多肽酰肼[73];(c)通过酰基吡唑结构产生多肽硫酯供体[72]

Fig.9 (a) Peptide hydrazide serves as thioester surrogate used in NCL[72]; (b) peptide hydrazide synthesis on 2-Cl-(Trt)-NHNH2 resin[73]; (c) generation of peptide thioester surrogate: acyl pyrazoles[72]

To overcome the problem of ring formation due to side reactions of Asp, Asn, and Gln, Tian et al. Used a new resin (2-Cl-(Trt)-NHNH2resin) to synthesize polypeptide hydrazide (Figure 9B)[73]. Different from the previous method, the resin can be cleaved under mild acidic conditions to obtain the side-chain protected polypeptide, and then deprotected, thus avoiding the side reaction of the first C-terminal amino acid side chain and the "hydrazine" structure. In order to prolong the storage life of the resin and reduce side reactions, the researchers introduced Fmoc protecting group to synthesize 2-Cl-(Trt)-NHNH2Fmoc resin[23].
The conversion of hydrazide to thioester is the key step in this process, which is achieved by the oxidation of NaNO2 as described above, however, some groups cannot adapt to the oxidation conditions containing NaNO2, such as N-terminal thiazole or other redox-sensitive groups, so more excellent and mild peptide hydrazide agonistic conditions need to be produced. In 2018, Flood et al. Reported a mild thioester synthesis condition for this limitation[74]. Acetylacetone is used to activate the peptide hydrazide to an acylpyrazole intermediate (a highly effective acylating agent), which is further thiolyzed to a polypeptide thioester to participate in the natural ligation reaction (Fig. 9c). Similarly, a series of ligation processes after the solid-phase synthesis are also carried out by a "one-pot reaction" without any intermediate purification and separation operations.
In addition to the use of SPPS to synthesize peptide hydrazide structures, expressed proteins and enzymatic catalysis can form such structures for application in NCL[23].

3.1.2 Cryptic peptide thioester

Terrier et al. Invented a conveniently synthesized stealthy thioester device N-Hnb-Cys (StBu), which can efficiently and rapidly obtain the peptide thioester fragments required by NCL under neutral pH conditions, and NCL is completely feasible in this method[75]. Compared with other methods, the method is simple and efficient, and the target polypeptide can be obtained by connecting the two polypeptide fragments through only one step of reaction after solid-phase synthesis. As shown in Figure 10, the protecting group StBu (tert-butyl sulfhydryl) of SH can be quickly removed under NCL conditions, and the next reaction will be triggered after the thiol is exposed; Intramolecular N/S acyl conversion is an efficient synthesis of peptide thioester structures with the help of Hnb protecting groups. This approach has already been successfully applied to the synthesis of two Cys-rich polypeptide sequences: MT7 and Cg-BigDef1.
图10 隐形肽硫酯N-Hnb-Cys(StBu)的合成及其在自然化学连接过程中转化为肽硫酯的机制[75]

Fig.10 Synthesis of peptide crypto-thioesters: N-Hnb-Cys(StBu) and its Mechanism for transformation to peptide thioester during NCL[75]

Stealth peptide thioester is an excellent thioester substitute, but when Ile (isoleucine), Val (valine) and Thr or Pro are used as the C-terminal amino acid of the stealth peptide thioester, the coupling yield of these special amino acids with the N-Hnb-Cys structure under Fmoc-SPPS conditions is extremely low. Lelievre et al. Showed that the slow O/N acyl transfer process was responsible for the difficulty in coupling these amino acids[76]. Therefore, they further optimized the coupling process, and when the first amino acid (such as Pro) was attached, the undesired esterification product was eliminated by the reaction of a mixed solution of hydroxylamine hydrochloride and imidazole, and the desired secondary amine acylation was increased. The NCL process was also optimized, the pH value was changed to 5.8, and the temperature was increased to 50 ℃.

3.1.3 Thiolated amino acid

The proposal of NCL is of great significance for protein synthesis, but this method can only be limited to peptides containing Cys residues, and the content of cysteine in natural proteins is very low (1.2%), which greatly limits its application. In order to expand the application range of NCL, various schemes have been further proposed. One of the most important solutions is to use Cys analogs for ligation, and then remove the sulfhydryl group by desulfurization to obtain the desired amino acid side chain. Desulfurization was initially carried out using Raney nickel or palladium/alumina (Pd/Al2O3), however, both of these conditions have some limitations[77]. Raney nickel causes epimerization of secondary alcohols and reduction of thiols, thioesters, and thioethers. The thiazolidine structure (Thz), which protects the N-terminal cysteine residue, is unstable under palladium/alumina conditions[78]. Therefore, mild desulfurization conditions need to be proposed urgently. In 2007, Danishefsky et al. Realized the desulfurization of cysteine to alanine by free radical-mediated reduction. This mild condition (TCEP, Tris (2-carboxyethyl) phosphine) is suitable for the synthesis of complex polypeptides and glycopeptides[78]. In 2016, Pasunooti et al. Used this desulfurization condition to convert thiolated Ile into natural amino acid residues (Fig. 11A), and similar desulfurization reactions are widely used in the field of protein synthesis[79][80,81].
图11 (a)使用硫醇化氨基酸进行自然化学连接并进一步脱硫[79];(b) γ-硫醇化的异亮氨酸、β-硫醇化的赖氨酸和γ-硫醇化的脯氨酸

Fig.11 (a) Using thiol-derived amino acids in NCL and further desulfurization[79]; (b) γ-thiol Ile, β-thiol Lys and γ-thiol Pro

An increasing number of thiolated amino acids have also been synthesized: γ-thiol Ile, β-thiol Lys, γ-thiol Pro (Fig. 11b), etc[79][82][83]. In 2020, Yin et al. Reported an efficient photocatalytic asymmetric reaction for the synthesis of β-thio (Cys analog)/seleno-amino acids, which achieved gram-scale preparation of β-thio/seleno-amino acids[84]. In conclusion, the efficient desulfurization reaction and the development of these thiolated amino acid tautomers have opened up new horizons for the application of NCL.

3.2 DSL

The inherent limitations of NCL have prompted the development of alternative chemoselective ligation techniques, such as DSL (Diselenide-selenoester ligation). DSL is very similar to NCL in that it is ligated between a C-terminal arylselenolipid peptide fragment and another N-terminal selenocysteine peptide fragment, followed by deselenization to obtain the target polypeptide (fig. 12). This method can be used for the rapid synthesis of proteins, including cases where other peptide ligation methods cannot be used or cannot be used efficiently. In 2019, Kulkarni et al. Used this method to successfully synthesize three polypeptide proteins: adiponectin (19-107), heme 1, and heme 2[18]. In 2022, Liczner et al. Reported an efficient method for the synthesis of peptide-oligonucleotide conjugates (modified oligonucleotides conjugated to peptides) using DSL and deselenylation strategies[85].
图12 DSL(Diselenide-selenoester ligation)的步骤[18]

Fig.12 Steps of DSL[18]

3.3 STL/CPL

STL (Ser/Thr ligation) is different from NCL in that salicylaldehyde ester is used as an acyl donor to ligate a polypeptide fragment with Ser or Thr at the N terminus[86]. First, the aldehyde group in the salicylaldehyde structure reacts with the hydroxyl and amino groups of Ser or Thr to form a five-membered thiazole ring, and the two peptide fragments are thus connected. Secondly, salicylaldehyde has an o-hydroxyl structure similar to Hub, forming a natural amide bond through O/N acyl transfer via a six-membered ring transition state (Fig. 13). What is more ingenious is that after the connection is completed, the target peptide chain can be obtained through appropriate acidification conditions, and salicylaldehyde is like a lock, which can be easily removed after the two peptide chains are connected. STL meets the chemoselectivity required for polypeptide ligation, because the process of imine formation between aldehyde group and amino group is reversible, and the amino site on the side chain hinders further reaction due to the lack of hydroxyl group after imine formation, thus achieving chemoselectivity. In terms of the scope of application, 20 natural amino acids, except Asp, Glu (glutamic acid) and Lys (lysine), can participate in the ligation reaction as the C-terminal[87]. In addition, the method can be connected in a mixture of pyridine and acetic acid with a suitable ratio without complex operation or equipment, so it is widely used in the synthesis of peptides and proteins[88,89].
图13 丝氨酸/苏氨酸连接的机制

Fig.13 Mechanism of Ser/Thr ligation

Similar to the peptide thioester in NCL, peptide salicylaldehyde ester (SAL ester) is also essential for STL, and there are many ways to prepare it. Direct coupling is the Direct condensation of a fully protected C-terminal carboxylic acid polypeptide with dimethoxy salicylaldehyde (the dimethoxy protecting group can restore the aldehyde group under acidic conditions) under the conditions of PyBop (benzotriazol-1-yl-oxy-tripyrrolidinophosphate hexafluorophosphate) and DIEA (N, N-diisopropylethylamine) to form an ester. However, this direct condensation can only be applied in the case of Gly and Pro at the C-terminus, because this condensation condition causes epimerization of the terminal amino acid[90]. The n + 1strategy successfully avoids the differentiation problem and allows the STL to connect from the N side to the C side (N to C ligation)[91]. As shown in Figure 14 a, n refers to the fully protected peptide synthesized by Fmoc-SPPS, 1 refers to the monoamino acid salicylaldehyde ester with the aldehyde group protected by semicarbazone, n and 1 are condensed and connected under appropriate conditions, and then the side chain protecting group and the aldehyde protecting group are removed by TFA and pyruvic acid (pyruvic acid), respectively.
图14 (a) 使用“n+1”策略合成肽水杨醛酯;(b) 末端氨基酸的高位阻使肽聚酯中的α-N羰基失活;(c) 半胱氨酸/青霉胺连接的步骤[93]

Fig.14 (a) Synthesis of SAL ester by “n+1” strategy; (b) High hindrance of terminal AA contributes to the deactivation roles of the α-N-carbonyl in a peptidyl prolylester; (c) steps of CPL[93]

Some sterically demanding amino acids, such as Pro, Val, Ile (isoleucine), and Thr, which account for up to 22% of proteins in nature, are often hindered when used as peptide terminal amino acids (Figure 14b), so better ligation schemes need to be proposed to solve these problems[92]. In 2020, Tan et al. Developed a chemostereoselective polypeptide fragment ligation method CPL (Cys/Pen ligation) based on STL (Fig. 14C)[93]. The overall ligation process does not require a thiol group as a nucleophilic group to attack the terminal acyl carbon and thus is not affected by the high steric hindrance of the C-terminal amino acid. In the report of Tan et al., the ligation rate of this method is as high as 95%, and there is no epimerization between the two amino acids at the ligation site during the ligation process. As a supplement to STL, CPL makes the application of this kind of connection method more extensive.

3.4 KAHA ligation

In 2006, Bode et al. invented and reported a new chemoselective ligation method, KAHA (α-Ketoacid hydroxylamine) ligation (Fig. 15)[94]. Different from other ligation methods, this method is based on the reaction of α-keto acid (KA) and hydroxylamine (HA) to form an amide bond. Studies have shown that the N-terminal peptide can have two variants: O-unsubstituted hydroxylamine and O-substituted hydroxylamine, of which five-membered cyclic hydroxylamine is the most commonly used[95][96]. In addition to the general advantages of good chemoselectivity, mild conditions and no catalyst, the most attractive advantage of KAHA connection is that the by-products are only carbon dioxide and water. The successful synthesis of a variety of proteins has demonstrated the utility of this method, and more details about the KAHA ligation method for protein synthesis are included in this review[97,98][99].
图15 KAHA连接发生于N末端多肽的α-酮酸与C末端多肽的羟胺之间

Fig.15 KAHA ligation: peptides ligate between an N-terminal peptide α-ketoacid and a C-terminal peptide hydroxylamine

3.5 Synthetic strategy of difficult protein

3.5.1 Solubilization label

membrane proteins, which account for about 20% to 30% of the proteins encoded by the human genome, are of great importance to biology, pharmacy and other fields. They are important mediators for cells to play their roles. Studies have shown that up to 50% of drug targets are related to membrane proteins[100][101]. Membrane protein is a kind of difficult protein, and its strong hydrophobicity has always been a problem in the process of chemical synthesis. Some additional amino acids with good solubility are introduced into the target peptide to achieve a fusion structure with desired properties (e.g., solubilization), and this additional group is called a "tag". This removable temporary tag is an important means to address the poor solubility of membrane proteins. When the solubilization tag is attached to the side chain, it can also play the role of backbone modification, so it can prevent the aggregation of branched peptides in the SPPS process while solubilizing. Common solubilization tags are Poly-arginine tag, Poly-lysine tag (fig. 16A), etc. This group composed of amino acids is easily attached to the target peptide, so it is widely used in the synthesis of membrane proteins[102].
图16 (a) 聚精氨酸和聚赖氨酸标签;(b)通过可移除的骨架修饰基团—RBM标签合成膜蛋白[103];(c)借助可被还原的增溶标签(RSTs)合成膜蛋白[90]

Fig.16 (a) Poly-arginine tag and Poly-lysine tag; (b) synthesis of membrane proteins through a removable backbone modification-RBM Tag[103]; (c) membrane protein synthesis via reducible solubilizing tags (RSTs)[90]

In 2016, Zheng et al. Introduced a general method for membrane protein synthesis using solubilizing tags[103]. They connected the polyarginine solubilization Tag to the polypeptide through the Hmnb structure to form a removable backbone modification group (RBM-Tag), which effectively solved the problems of protein aggregation and insolubility, and successfully synthesized the double/four-transmembrane proteins (ion channel protein HCV P7 and transporter EmrE) (Fig. 16b). Recently, Liu et al. Also reported a method to synthesize membrane proteins with the help of solubilizing tags, and proposed a reducible solubilizing tag (RST)[90]. RST is a convenient tool that is easy to install and remove. It is connected with Cys/Ala (alanine) side chain or salicylaldehyde ester through disulfide bond (thiolated Ala needs to be desulfurized again), and then removed by TCEP (Tris (2-chloroethyl) phosphate) or desulfurization environment (TCEP and tBuSH) (Fig. 16c). Finally, they used this strategy to successfully synthesize protein 2B4, a human protein encoded by the CD244 gene, and membrane protein FCER1G.

3.5.2 Semisynthesis of protein

Protein Semisynthesis is an efficient method between expressed Protein and chemical protein, in which at least one structural block is obtained by expression and the rest is synthesized chemically. Semisynthesis combines the simplicity and low cost of protein expression with the modifiability of chemical proteins to special sites, and can also avoid the problem that total synthesis can not fold proteins[104]. Semi-synthesis makes up for the deficiency of total chemical synthesis in obtaining large proteins. Statistics show that the average size of proteins obtained by total synthesis is about 90 amino acid residues, while semi-synthesis can obtain large proteins with more than 200 amino acid residues[105]. Although semisynthesis has many advantages for obtaining large proteins, there are still many shortcomings. For example, the lack of a suitable cysteine thiol protecting group prevents the desulfurization reaction from proceeding selectively in the presence of native cysteine residues, thereby hindering the ligation of expressed proteins and subsequent desulfurization reactions. In 2020, Wang et al. Reported a cysteine-thiol protection strategy with light-controlled release, which was successfully applied to the semi-synthesis of phosphorylated protein cystatin S (CST4)[80].
There are many strategies for Protein semisynthesis, including Expressed Protein Ligation (EPL), enzyme-catalyzed protein Ligation, enzyme-catalyzed EPL, etc[106,107][108][109]. With the help of various strategies, a variety of special proteins have been successfully synthesized, including various post-translationally modified proteins, cyclic peptides, histones, and so on[110][111][109]. Protein posttranslational modifications (PTMs) include glycosylation, Phosphorylation, Acetylation, etc. Chemical synthesis (including semi-synthesis and total synthesis) is an important tool to obtain these special proteins, and is helpful to study the mechanism of these PTMs[112,113]. When full synthesis cannot be used, semi-synthesis shows its unique advantages. In 2017, when Tan et al. Synthesized phosphorylated proteins, they found that a certain N-terminal peptide chain could not be obtained by solid-phase synthesis, and then transferred to semi-synthetic method (transpeptidase-catalyzed protein ligation)[23]. As shown in fig. 17, the N-terminal peptide chain is synthesized by biological expression, and then a hydrazide structure is formed under the catalysis of an enzyme, and then the hydrazide structure is connected with the peptide fragment synthesized by solid phase to obtain the target protein.
图17 酶催化介导的蛋白质半合成的步骤

Fig.17 Steps of Sortase-mediated protein Semisynthesis

4 Conclusion and prospect

The chemical synthesis of peptides and proteins is an important research focus in the field of contemporary synthetic chemistry and chemical biology, and has been highly concerned by academia and industry. In this review, the current chemical synthesis strategies and their applications are summarized and prospected from two aspects of peptide synthesis and protein synthesis. As an important means of peptide synthesis, SPPS has been widely used in related fields, and the development of new resins and condensing agents has made it more and more perfect. A variety of effective strategies have been proposed for the synthesis of complex peptides, such as the pseudo-proline method, the o-hydroxybenzyl protection method, the O-acylated isopeptide method, and the PEGylation method, which make the solid-phase synthesis of "difficult" peptides feasible. In addition, various effective synthetic methods for cyclic peptides and glycopeptides with special structures have been improved and innovated. For example, the peptide backbone amide bond protecting group pseudoproline can not only reduce the aggregation of polypeptide chains by destroying the formation of hydrogen bonds to achieve the purpose of solubilization, but also change the spatial structure of peptide chains, thus promoting the cyclization process of cyclic peptides. With the development of various efficient ligation technologies, such as NCL, DSL, STL, KAHA ligation, etc., most small and medium-sized protein synthesis can be successfully completed by the combination of solid phase and ligation methods, while some proteins with complex sequences or large scale need to adopt special strategies or switch to semi-synthetic methods.
The chemical synthesis of peptides and proteins not only has important theoretical significance, but also has a wide range of applications in clinical testing and drug development. The introduction of modification groups at specific sites by chemical synthesis can effectively improve the physical and chemical properties of polypeptide and protein molecules, improve biological activity and stability, and promote the preparation of drugs. Peptide synthesis technology combined with combinatorial chemistry can synthesize combinatorial peptide libraries containing a large number of compounds, which can be used to screen lead compounds with specific pharmacological activities. Although great progress has been made in the field of chemical synthesis of peptides and proteins, many inherent limitations still exist. For the synthesis of "difficult peptides", many strategies revolve around certain chemical modifications to achieve better solubility, thus facilitating various manipulations and purification processes. However, these strategies are narrow in scope and complex and time-consuming, so low-cost and general methods suitable for "difficult peptides" need to be further developed. It often takes several weeks to obtain a certain amount of high-purity common peptides containing less than 30 amino acid residues by solid-phase peptide synthesis, and it is difficult to reach the gram level. In recent years, the rapid development of flow chemistry has provided a new method for the chemical synthesis of peptides and proteins. This method of synthesis with the help of automated fast-flow instruments can allow the polypeptide chain to be extended in a few hours, greatly improving the efficiency of peptide and protein synthesis[114]. It is inevitable to use excessive condensing agents and environmentally unfriendly washing solvents (DCM, DMF) in the process of peptide synthesis, so the development of more efficient condensation methods and environmentally friendly solvents is a problem to be solved in the field of peptide and protein chemical synthesis. At present, various peptide ligation methods are limited to laboratory research, and most of them are not widely used in practical production, so more efficient chemical ligation methods are still in urgent need. In a word, the existing synthetic strategies can not meet the needs of people to explore peptides and proteins in depth, and more mature, universal and simple methods need to be further unlocked, so as to continue to promote the process of peptides and proteins in molecular biology and pharmacology.

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