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

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Progress in Chemistry >

2023 , Vol. 35 >Issue 11: 1579 - 1594

DOI: https://doi.org/10.7536/PC230325

Review

Segment Solubilizing Strategy in Protein Chemical Synthesis

  • Deng Xiangyu 1, 2 ,
  • Zhang Baochang 1 ,
  • Qu Qian , 2, *
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  • 1 Tsinghua University Tsinghua-Peking Center for Life Sciences, Ministry of Education Key Laboratory of Bioorganic Phosphorus Chemistry and Chemical Biology, State Key Laboratory of Chemical Oncogenomics (Shenzhen), Department of Chemistry,Beijing 100084, China
  • 2 Shanghai Jiao Tong University Institute of Translational Medicine,Shanghai 200240, China
*Corresponding author e-mail:

Received date: 2023-03-27

  Revised date: 2023-07-10

  Online published: 2023-08-07

Supported by

National Natural Science Foundation of China(22277073)

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Abstract

Proteins play critical roles in various biological processes and biomedical researches. A significant task of such biochemical studies is to obtain protein samples of high homogeneity with respect to their atomic compositions. Chemical protein synthesis offers a much more robust and effective strategy over recombinant expression technology for accessing proteins that are precisely modified or even artificially designed. However, some important proteins that can be used as drug targets (such as human interleukin-2, K+ channel protein Kir5.1, etc.) suffer from limited solubility of peptide segments during the journey of protein synthesis. Such hydrophobic peptides pose difficulty for subsequent purification, characterization, chemical ligation and other operations. The main factors for these problems may be that the peptide segments are prone to self-assemble into secondary structures through hydrophobic interactions, hydrogen bond or other interaction modes, thus reducing the solubility. Addition of solubilizing tags is recognized as one of the effective methods to overcome such obstacles. In this review, strategies of attaching solubilizing tags to the main chain, side chain and backbone of peptides are introduced. Membrane protein FCER1G, co-chaperone protein GroES and other proteins are selected as examples to describe the applications of the solubilizing tags. Moreover, the future of solubilizing tags strategy is discussed and prospected.

Contents

1 Introduction

2 Main chain solubilizing tags

2.1 C-terminal solubilizing tags

2.2 N-terminal solubilizing tags

3 Side chain solubilizing tags

3.1 Cysteine(Cys)side chain solubilizing tags

3.2 Lysine(Lys)side chain solubilizing tags

3.3 Asparagine(Asn)/Glutamine(Gln)side chain solubilizing tags

4 Backbone modifications as solubilizing tags

4.1 Irreversible backbone modification

4.2 Removable backbone modification

5 Conclusion and outlook

Key words： chemical protein synthesis; peptide; native chemical ligation; segment solubilizing strategy; hydrophobic interactions

Cite this article

Deng Xiangyu , Zhang Baochang , Qu Qian . Segment Solubilizing Strategy in Protein Chemical Synthesis[J]. Progress in Chemistry, 2023 , 35(11) : 1579 -1594 . DOI: 10.7536/PC230325

1 Introduction

Peptides and proteins are the most ubiquitous biomolecules in living systems, which play a key role in a variety of biochemical processes in life, including metabolism, signal transduction, central dogma and so on[1][2]. Analysis of the molecular biological mechanism behind the function of protein machine is a hot issue in biomedical research, and the acquisition of functionally related protein samples is the key to such research, which lays the foundation for further research in the fields of protein biochemical mechanism analysis, polypeptide drugs, protein materials and so on[3⇓⇓⇓⇓~8][9⇓⇓⇓~13][14⇓⇓⇓~18]. recombinant expression technology (recombinant expression technology) has been widely used to obtain natural proteins, but it is still limited in obtaining site-specific post-translational modification proteins or non-natural proteins (such as anti-chiral proteins)[19][20⇓~22][23].
In order to solve these problems, protein chemical synthesis technology has made great progress in recent years. With solid phase peptide synthesis (SPPS), automated fast flow peptide synthesis (AFPS), a series of peptide fragment ligation reactions, such as native chemical ligation (NCL), Ser/Thr ligation, etc.STL), diselenide-selenoester ligation (DSL), Cys/Pen ligation (CPL), α-ketoacid-hydroxylamine ligation (KAHA) and various fragment splicing strategies have made it possible to synthesize proteins of more than 300 amino acids by chemical means, and many key protein samples have been obtained[24][25,26][27⇓⇓~30][31,32][33][34][35,36][37⇓~39][40⇓⇓~43].
In the process of protein chemical synthesis, the poor solubility of polypeptide fragments is a common problem[44⇓~46]. Taking the chemical synthesis of membrane proteins as an example, membrane proteins have physiological functions such as cell signal transduction and molecular transport, and are one of the important targets of modern drugs. However, the transmembrane segments of these proteins have poor solubility in almost any solvent.The main reason may be that these polypeptide fragments are easy to form secondary structures through hydrophobic interaction, ionic interaction, hydrogen bonding and other modes of action, which reduces the solubility of the fragments and makes the subsequent chromatographic purification, mass spectrometry characterization and ligation reaction difficult (Figure 1)[47⇓⇓⇓~51][52,53][54,55][56].
图1 多肽片段溶解度不佳的原因[56]

Fig.1 Reasons for poor solubility of peptide fragments[56]

In response to the challenge of poor solubility of polypeptide fragments, a series of strategies have been developed, including the use of organic cosolvents, O-acyl isopeptide bond migration strategies, and external solubilization tag strategies (Figure 2)[57⇓~59][60⇓~62][63].
图2 提升多肽溶解度的方法[57⇓⇓⇓⇓⇓~63]

Fig.2 Methods for improving the solubility of peptides[57⇓⇓⇓⇓⇓~63]

Among them, the solubilization tag strategy provides a series of means to modify the polypeptide structure with various installation sites, so that the hydrophobic fragment presents similar properties to the hydrophilic polypeptide, which is convenient for subsequent operation. This strategy has been applied to the synthesis of many difficult proteins (such as human interleukin-2, membrane protein FCER1G, K+ channel protein Kir5.1, etc.), which provides a paradigm for the synthesis of other similar targets and paves the way for further biochemical mechanism and biophysical research, but there is no systematic summary of the solubilization tag strategy.
In this paper, the strategy of adding solubilizing tags is reviewed. It has been reported that solubilizing tags can be permanently or temporarily attached to hydrophobic peptide fragments[64⇓~66]. In this paper, we mainly introduce the removable temporary solubilization tag, and summarize the methods of installing solubilization tag on the polypeptide backbone, side chain and skeleton.

2 Backbone solubilizing tag

2.1 C-terminal solubilization tag

Adding solubilization tags to the C-terminal of polypeptide fragments can effectively improve their solubility, and the typical strategy is the C-terminal thioester solubilization strategy developed by Kent et al[67]. Kent et al. Introduced oligoarginine into the thioester group by solid phase peptide synthesis, which significantly improved the synthetic purity and solubility of insoluble crude peptides. During the native chemical ligation reaction, the solubilizing tag is removed with an intermolecular thioester exchange reaction (Scheme 1). Kent et al. Used this strategy to synthesize the hydrophobic fragment of diacylglycerol kinase (DGK).
图式1 C端硫酯增溶标签[67]

Scheme 1 C terminal thioester solubilizing tag[67]

DGK is a 121-amino acid membrane protein from E. coli ()[68]. Kent et al. Found that the peptide fragments designed in the chemical synthesis of the protein had poor solubility, which made the subsequent purification and mass spectrometry identification difficult. To solve the synthesis problem caused by poor solubility, Kent et al. Divided DGK into six fragments (Fig. 3), and except for the last [Cys113-Gly121] fragment, solubilizing oligoarginine was added to the thioester group of the other five fragments, and the synthetic purity and solubility of the corresponding crude peptides were significantly improved. Subsequently, Kent et al. Used this strategy to achieve the chemical synthesis of human insulin-like growth factor 1 (IGF-1)[69].
图3 DGK亲水片段的划分[67]

Fig.3 Division of DGK hydrophilic peptides[67]

During the ligation reaction, the ligation product may aggregate or precipitate due to the removal of the thioester-solubilized tag. For example, when Kent et al. Synthesized HIV-1 protease (HIV-1 PR), they found that the ligation product of [Thz71-Gly94] fragment and [Cys95-Phe99] fragment containing the above thioester solubilization tag had poor solubility, which caused difficulties in the subsequent ligation reaction[70]. In order to meet such challenges and achieve multi-fragment assembly in the synthesis of larger proteins, a series of C-terminal solubilizing tags that remain stable during the ligation reaction and can be removed after the ligation is completed have been developed, including solubilizing tags that are sensitive to acids, bases, and enzymes (Fig. 4)[71][72][70].
图4 其他C端增溶标签[70⇓~72]

Fig.4 Other C-terminal solubilizing tags[70⇓~72]

2.2 N-terminal solubilization label

In addition, alkali- and enzyme-sensitive N-terminal solubilization strategies have been developed (Fig. 5)[73][74]. Enzyme-based N-terminal solubilization tagging strategy has the advantages of mild conditions and efficient reaction, but the recognition motif of protease has certain restrictions on the type of N-terminal amino acids. In contrast, the removal strategy under alkaline conditions does not have the problem of sequence dependence. In this section, the alkali-sensitive strategy developed by Englebretsen et al. Is selected for brief introduction.
图5 N端增溶标签[73,74]

Fig.5 N-terminal solubilizing tags[73,74]

In order to solve the problem of synthesis of insoluble transmembrane peptides, Englebretsen et al. Developed a C-terminal solubilization strategy based on the structure of [hydrophobic peptide-linker-solubilized peptide], but this strategy is not suitable for the acquisition of amide-terminal peptides[75⇓~77]. Therefore, Englebretsen et al. Designed a complementary N-terminal solubilization strategy based on the above model (Figure 2), using Boc solid-phase peptide synthesis to introduce alkali-sensitive linkers and solubilizing arginine fragments into the N-terminal of model peptides, and the solubility of peptides was significantly improved[73]. After separation and purification, the N-terminal solubilizing tag can be quickly removed by 0. 1 mol/L sodium hydroxide solution. However, under strong alkaline removal conditions, asparagine, glutamine, aspartic acid, glutamic acid and other residues may undergo deamidation or succinimidylation side reactions, which to some extent limits the application scenarios of this strategy.
图式2 碱敏感的N端增溶标签[73]

Scheme 2 Base-liable N-terminal solubilizing tags[73]

3 Side-chain-based solubilization tag

The above strategy of adding only a limited number of solubilizing groups at the end of polypeptide fragments may be limited in the application of multi-fragment splicing process for larger protein synthesis, and then the solubilizing tag strategy based on side chain has been developed, which provides us with more alternative modification sites (such as sulfhydryl, amino, etc.). This section is illustrated by the classification of the sites of solubilization tag installation (fig. 6).
图6 基于侧链的增溶标签

Fig.6 Side chain-based solubilizing tags

3.1 Cysteine (Cys) side chain solubilization tag

Cysteine is a kind of amino acid with high activity. For this amino acid residue, the strategies of using palladium chloride (PdCl2) to remove, acidic conditions to remove, and reducing conditions to remove the solubilizing tag have been developed.

3.1.1 Alloc-Phacm solubilization strategy for PdCl2 removal

Brik et al. Developed the Alloc-Phacm strategy (Scheme 3) based on the phenylacetylaminomethyl (Phacm) protecting group, which introduces a cysteine module preattached to the Alloc-Phacm group by Fmoc solid phase peptide synthesis, followed by the removal of the allyloxycarbonyl (Alloc) -linked oligoarginine[78]. The solubilization tag can significantly enhance the solubility of the polypeptide fragment, and can be efficiently removed by PdCl2 in 6 mol/L neutral guanidine hydrochloride solution. Brik et al. Used this strategy to achieve the chemical total synthesis of histone H4.
图式3 Brik等发展的Cys侧链增溶策略[78]

Scheme 3 Cys side chain solubilizing strategy developed by Brik et al[78]

Histone H4, together with H2A, H2B and H3, forms a two-copy core histone octamer, which winds around DNA to form nucleosomes as the basic unit of chromatin[79]. In order to realize the chemical synthesis of histone H4, the previous work adopted the three-fragment ligation strategy, and found that the middle fragment had the problem of poor solubility, and the synthesis yield was low (only 3. 0%)[80]. In order to increase the solubility of the fragment and improve the synthesis efficiency, Brik et al. Used the above fragment solubilization strategy to divide histone H4 into two fragments, and introduced a solubilization tag containing oligoarginine at the 69th cysteine of the C-terminal fragment to improve the solubility of the fragment, and obtained the C-terminal fragment with a yield of 20%. After fragment splicing, Brik et al. Obtained the target product histone H4 more efficiently through the removal of solubilizing tags and free radical desulfurization reaction (Figure 4).
图式4 组蛋白H4的化学全合成[78]

Scheme 4 Total chemical synthesis of histone H4[78]

3.1.2 Trityl solubilization strategy for acid removal

The Alloc-Phacm strategy can significantly improve the solubility of hydrophobic peptides, but this strategy has some shortcomings, such as the complexity of the amino acid module synthesis route, and some more convenient solubilization strategies still need to be developed.
Yoshiya et al. Designed a solubilization tag (Fig. 5) based on the structure of the trityl (Trt) protecting group, which can be prepared in large quantities by solid phase peptide synthesis and linked to cysteine residues of polypeptide fragments in 50% – 60% trifluoroacetic acid (TFA) solution or hexafluoroisopropanol (HFIP)[81]. The solubilization tag can significantly improve the solubility of polypeptide fragments, is compatible with natural chemical ligation reaction and high performance liquid chromatography purification conditions, and can be efficiently removed in trifluoroacetic acid solution containing triisopropylsilane (TIS). It is worth mentioning that this strategy is to achieve solubilization of hydrophobic polypeptide fragments after solid phase synthesis, which can greatly reduce the cost of re-synthesis of polypeptides. Yoshiya et al. Used this strategy to realize the chemical total synthesis of CP149, the assembly domain of hepatitis B virus core protein.
图式5 Yoshiya等发展的Cys侧链增溶策略[81]

Scheme 5 Cys side chain solubilizing strategy developed by Yoshiya et al[81]

The HBV core protein consists of a capsid-forming assembly domain, CP149, and a C-terminal domain that regulates viral replication[82]. In order to realize the chemical synthesis of CP149, Yoshiya et al divided it into three fragments of N/M/C, and found that the ligation product of M and C fragments (M + C) had poor solubility, which brought challenges to separation, purification and ligation reaction. In order to increase the solubility of the fragment and thus improve the synthesis efficiency, Yoshiya et al. Introduced the above solubilization tag containing oligolysine to the 107th cysteine, which significantly improved the solubility of the fragment. Yoshiya et al. Successfully obtained the target protein CP149 by ligation from the C terminus to the N terminus and removal of the solubilization tag (Figure 6).
图式6 CP149的化学全合成[81]

Scheme 6 Total chemical synthesis of CP149[81]ALC

Subsequently, Yoshiya et al extended the application site of this strategy to aspartic acid, asparagine, glutamic acid and glutamine residues, and realized the chemical total synthesis of amyloid β-protein (Aβ42)[83].

3.1.3 Solubilization strategy of reducing condition removal

In addition, a series of solubilized tags have been developed that are removed under reducing conditions through disulfide bond installation. Deber et al. Designed a disulfide reagent, PEG-A-Cys, to introduce a polyethylene glycol-containing solubilizing tag into a polypeptide through a disulfide exchange reaction in an aqueous phase (Scheme 7)[84]. This solubilizing tag enhances the solubility of the fragment, which is removed under reducing conditions with Tris (2-carbonylethyl) phosphonium hydrochloride (TCEP). Deber et al. Used this strategy to achieve solubilization of transmembrane protein model peptide ALC. However, Deber et al. Have not tested the stability of this strategy in various ligation reactions, and its application in protein synthesis needs to be further explored.
图式7 Deber等发展的Cys侧链增溶策略[84]

Scheme 7 Cys side chain solubilizing strategy developed by Deber et al[84]

Recently, based on a similar principle, Li et al. Developed a solubilization tag installed by a disulfide bond, which is compatible with serine/threonine ligation and cysteine/penicillamine ligation reaction conditions without reducing agents[85]. It is worth mentioning that this solubilization tag can not only be introduced into fragments by solid phase peptide synthesis (Scheme 8 (a)), but also be installed on proteins obtained by recombinant expression in liquid phase (Scheme 8 (B)). Li et al. Used this strategy to achieve the total synthesis of protein 2B4 cytoplasmic tail and human membrane protein FCER1G, and the semi-synthesis of human high mobility group protein B1 (HMGB1). Here, the membrane protein FCER1G is taken as an example to briefly introduce this strategy.
图式8 Li等发展的Cys侧链增溶策略[85]

Scheme 8 Cys side chain solubilizing strategy developed by Li et al[85]

The membrane protein FCER1G is an adaptor protein that signals allergic inflammation[86]. In order to realize the chemical synthesis of FCER1G, Li et al. Divided it into two fragments, and found that the [Leu19-Tyr43] fragment had the problem of poor solubility, which brought great difficulties to the separation and purification. Therefore, Li et al. Introduced the above solubilization tag containing oligolysine at the 25th cysteine of this fragment, the solubility of the fragment was significantly improved, and the [Leu19-Tyr43] fragment was isolated in 7.3% yield. After one-pot ligation of the two fragments and removal of the solubilization tag, Li et al. Successfully obtained the target protein FCER1G (Figure 9).
图式9 膜蛋白FCER1G的化学全合成[85]

Scheme 9 Total chemical synthesis of membrane protein FCER1G[85]

3.2 Lys side chain solubilization tag

Due to the low abundance of cysteine in proteins, the above strategy may need to undergo multi-step reactions such as site mutation, solubilization tag installation, free radical desulfurization and so on, which is cumbersome[87]. In contrast, lysine is more widely distributed in proteins, and a series of lysine-based solubilization strategies have been developed, which can be divided into two categories according to the installation method.One is the direct introduction of amino acid modules of preformed solubilization tags in solid phase peptide synthesis, and the other is the chemical modification of lysine residues in solid phase peptide synthesis.

3.2.1 Application of Preformed Solubilization Tag Amino Acid Module

In the process of chemical synthesis, the absence of natural conformation of polypeptide fragments may lead to aggregation and methionine oxidation, which brings challenges to synthesis[88]. In order to solve this problem, Danishefsky et al. Designed a class of lysine and glutamic acid modules with arginine linked to the side chain, and introduced the fragments by Fmoc solid phase peptide synthesis[89]. The guanidine group of the side chain of the amino acid module stabilizes the polypeptide fragment in solution by charge repulsion, preventing it from oligomerizing; This tag can be excised in situ by palladium reagent Pd(PPh3)4 (fig. 10). Danishefsky et al. Used this strategy to improve the physicochemical properties of homogeneously glycosylated human erythropoietin (hEPO) hydrophobic fragments.
图式10 Danishefsky等发展的Lys侧链增溶策略[89]

Scheme 10 Lys side chain solubilizing strategy developed by Danishefsky et al[89]

hEPO protein is composed of 165 amino acid residues and 4 glycoside side chains. Danishefsky et al. Encountered the problems of methionine residue oxidation and fragment aggregation in the process of synthesizing this protein fragment, which makes the synthesis of hEPO protein more challenging[90]. In order to reduce such side reactions and improve the synthesis efficiency, they first identified the peptide segment [Asp43-Gly77] that led to residue oxidation and aggregation, and then introduced the above lysine and glutamic acid modules into the fragment (Fig. 11). After a period of time, there was no aggregation and oxidation of the peptide chain, indicating that the physicochemical properties of the fragment in solution were significantly improved.
图式11 hEPO多肽片段物理化学性质的优化[89]

Scheme 11 Optimization of physical and chemical properties of hEPO peptide fragment[89]

Hojo et al. Also designed a class of side-chain picoline-esterified lysine and glutamic acid modules (Scheme 12), into which fragments were introduced by Fmoc solid-phase peptide synthesis[91]. The picolinate reverses the polarity of the glutamic acid side chain, thereby increasing the isoelectric point of the polypeptide fragment, which makes it easier to separate and purify under acidic conditions. After fragment splicing, this solubilizing tag can be removed under conditions of silver acetate and 50% acetic acid. Hojo et al. Used this strategy to realize the chemical total synthesis of human interleukin-2 (IL-2).
图式12 Hojo等发展的Lys侧链增溶策略[91]

Scheme 12 Lys side chain solubilizing strategy developed by Hojo et al[91]

IL-2 is a lymphokine produced by lectin- or antigen-activated T cells[92]. In the process of synthesizing IL-2, Hojo et al. Found that the C-terminal [Ser99-Thr133] fragment had poor solubility and was difficult to purify and connect. The introduction of isopeptide bonds at the [Ala112-Thr113] and [Ile122-Thr123] sites makes the fragments partially soluble in acetonitrile aqueous solution containing 6 mol/L guanidine hydrochloride, but difficult to separate from by-products. In order to increase the solubility of the fragment and improve the synthesis efficiency, Hojo et al. Introduced amino acid modules and isopeptide bonds into the fragment, which significantly improved its solubility, and isolated the C-terminal fragment in a yield of 3.4%. Subsequently, they introduced the amino acid module into other polypeptide fragments, and Hojo et al. Successfully achieved the total synthesis of the target protein IL-2 through convergent ligation and removal of picolinate (Figure 13).
图式13 IL-2的化学全合成[91]

Scheme 13 Total chemical synthesis of IL-2[91]

3.2.2 Modification of lysine residues in solid phase peptide synthesis

The amino acid module used in the above strategy needs to be prepared in advance, which limits the ability to adjust the structure of the solubilization tag according to the actual situation in application. In response to this challenge, Kay et al. Developed the Fmoc-Ddae-OH molecule based on the structure of the lysine dae protecting group, which was introduced into the polypeptide fragment by Fmoc solid-phase peptide synthesis, followed by the removal of the Fmoc protecting group to connect the oligolysine[93][94]. This solubilization tag is compatible with high performance liquid chromatography purification conditions and native chemical ligation conditions, and can be efficiently removed by 1 mol/L hydrazine solution under neutral denaturing conditions (Figure 14). Kay et al. Successfully used this strategy to achieve the total synthesis of ribosomal protein L31, native and mirror-image E. coli co-chaperone GroES, and the total synthesis of co-chaperone GroES is introduced as an example here.
图式14 Kay等发展的Lys侧链增溶策略[94]

Scheme 14 Lys side chain solubilizing strategy developed by Kay et al[94]

Molecular chaperones are a ubiquitous family of cellular proteins that prevent erroneous interactions between other molecular surfaces[95]. In order to realize the chemical synthesis of GroES, Kay et al. Adopted a two-fragment ligation strategy, and found that the C-terminal fragment had poor solubility and could not be separated and purified. Therefore, Kay et al. Introduced the above solubilization tag containing oligolysine at lysine 77 of the fragment, which significantly improved the solubility of the fragment and isolated the C-terminal fragment on a milligram scale. Subsequently, Kay et al. Successfully obtained native and mirror-image GroES proteins through native chemical ligation, radical desulfurization, and solubilization tag removal (Figure 15).
图式15 GroES的化学全合成[94]

Scheme 15 Total chemical synthesis of GroES[94]

Subsequently, Kay et al. Modified the structure of the Ddae molecule to change the PEG unit in the molecule into an all-carbon alkyl chain (Fig. 7) to obtain a solid Ddap (Ddae is a viscous liquid), which has better stability and operability in aqueous solution and can be quickly removed by hydroxylamine solution[96]. Kay et al. Realized the chemical total synthesis of Shiga toxin subunit B (StxB) by using Ddap molecule.
图7 Ddap的结构[96]

Fig.7 The structure of Ddap[96]

Yoshiya et al. Have developed the Trityl solubilization strategy, which has been successful on cysteine-containing proteins because of its ease of operation, while other alternative solubilization strategies remain to be developed due to the low natural abundance of cysteine. Therefore, Yoshiya et al. Designed a strategy to install a solubilization tag at the Lys site via the unnatural amino acid canaline[97]. This solubilization tag can be structurally extended by Fmoc solid-phase peptide synthesis, remains stable under high performance liquid chromatography purification conditions and native chemical ligation conditions, and can be removed by self-shearing reaction when the system is adjusted to weak acidic conditions (pH = 4-5) (Figure 16). However, this strategy is not compatible with the commonly used free radical desulfurization conditions, and will produce more side reactions when applied.
图式16 Yoshiya等发展的Lys侧链增溶策略[97]

Scheme 16 Lys side chain solubilizing strategy developed by Yoshiya et al[97]

3.3 Asn/Gln side chain solubilization tag

In addition, a series of strategies to install solubilizing tags at the amide end of the side chain have been developed. The self-assembly of peptides and proteins into fibers provides inspiration for the field of biomaterials, but such systems are often difficult to regulate, prepare and purify[98]. In order to achieve the regulation of self-assembling peptides, Imperiali et al. Developed a photosensitive tag strategy installed on the asparagine side chain (Scheme 17)[99]. This tag consists of photolabile 3-amino-3- (2-nitrophenyl) -propionic acid linked to a group containing the N, N-dimethylethylenediamine (DMDA) structure. The DMDA group can stabilize the amyloid polypeptide by charge repulsion in neutral solution; The label can be removed by 365 nm ultraviolet irradiation, and the self-assembly state of the system can be restored. Using this strategy, Imperiali et al. Regulated the fibrillation of amyloid peptide in human prion protein (PrP).
图式17 Imperiali等发展的策略[99]

Scheme 17 Strategy developed by Imperial et al[99]

Liu et al. Also designed a photosensitive solubilization tag installed on the glutamine side chain based on the structure of 3-amino-3- (2-nitrophenyl) -propionic acid (Figure 18), and realized the synthesis of autophagosomal marker protein LC3-II[100].
图式18 Liu等发展的Gln侧链增溶策略[100]

Scheme 18 Gln side chain solubilizing strategy developed by Liu et al[100]

Autophagosomal marker protein LC3-Ⅱ is a lipid-anchored protein formed by the C-terminal of cytosolic microtubule-associated protein 1A/1B light chain 3 (LC3-Ⅰ) linked to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), and its level may be related to autophagic activity[101]. In order to realize the chemical synthesis of LC3-Ⅱ protein, Liu et al. First obtained the N-terminal [Met1-Ala114]-MESNa thioester fragment by recombinant expression. When obtaining the [Cys115-Gly120]-PE fragment connecting the lipid chain, it was found that the fragment was almost insoluble in acetonitrile aqueous solution containing 0. 1% trifluoroacetic acid and the ligation solution of the expressed protein, which made it difficult to separate, purify and ligate. In order to increase the solubility of the fragment and thus improve the synthesis efficiency, Liu et al. Introduced the above solubilization tag containing oligoarginine into the side chain of glutamine at position 116, which significantly improved the solubility of the fragment and enabled the subsequent natural chemical ligation reaction in 6 mol/L guanidine hydrochloride solution without adding detergents. After fragment ligation, Liu et al. Successfully obtained the target protein LC3-II by removing the solubilization tag with 365 nm UV illumination (Figure 19).
图式19 LC3-Ⅱ的化学全合成[100]

Scheme 19 Total chemical synthesis of LC3-Ⅱ[100]

However, the above two light removal strategies still have some shortcomings in application. Ultraviolet irradiation can oxidize tryptophan, histidine, methionine, tyrosine and other amino acid residues, which limits the application scenarios of such strategies to a certain extent.

4 Skeleton-modified solubilization tag

Kent et al. Proposed an important hypothesis that the characteristics of transmembrane peptides that are difficult to manipulate are mainly due to their tendency to form secondary structures (such as α helices and β sheets) through the aggregation of hydrogen bond networks between amide bonds, which brings great difficulties to subsequent operations[102]. Modification of the amide bond N atom of the polypeptide backbone can destroy the hydrogen bond network between the chains, thereby blocking the formation of secondary structure and improving the solubility of the fragment. This section will briefly introduce the development of backbone modification strategies, focusing on the removable backbone modification (RBM) strategy.

4.1 Irreversible skeleton modification

Based on the above hypothesis, Kent et al. Proposed a strategy to mask the amide bond hydrogen atom by irreversible backbone modification. When synthesizing the fourth transmembrane domain of signal peptide peptidase (SPP), Kent et al. Found that a fragment containing 30 amino acids had poor solubility and was difficult to separate and purify[102]. In order to destroy the hydrophobic structure formed by polypeptide fragments, Kent et al. Designed three kinds of polypeptide fragments (Fig. 8), including N-methyl substitution of backbone amide bond, D-type amino acid mutation and proline mutation. Through chromatographic analysis, it was found that the solubility of transmembrane peptide was significantly improved, indicating that this kind of modification method can effectively break the secondary structure formed by polypeptide in aqueous solution. However, the effect of this strategy on the structure of the target protein is irreversible, and it may not be applicable to general protein synthesis.
图8 不可逆的骨架修饰[102]

Fig.8 Irreversible backbone modification[102]

4.2 Removable Skeleton Modification

Liu et al. Suggested that the properties of insoluble polypeptide fragments can be improved by using a removable backbone modification group, which should have the following characteristics[103]. First, this group can be introduced by Fmoc solid phase peptide synthesis and is compatible with conventional peptide fragment ligation and purification conditions; Secondly, this group can break the hydrogen bond network between peptide chains to inhibit the formation of secondary structure. Thirdly, this group should be able to easily introduce solubilizing groups such as oligoarginine and oligolysine. Finally, this group should be able to be quantitatively cleaved by reagents commonly used in protein synthesis, such as trifluoroacetic acid.
2-Hydroxy-4-methoxybenzyl (Hmb), 2,4-dimethoxybenzyl (Dmb) and their derivatives (Figure 9) developed by predecessors are a class of removable skeleton modification groups, which can effectively inhibit the formation of secondary structures[104][105]. Taking Hmb as an example, this group can be quantitatively cleaved under the condition of trifluoroacetic acid cleavage, while the protection of phenolic hydroxyl group (such as acetylation) can keep it stable under strong acidic conditions.
图9 Hmb及其衍生物[104,105]

Fig.9 Hmb and its derivatives[104,105]

Based on this characteristic of the Hmb group, Liu et al. Designed the first generation of RBM group, introduced the RBM-modified glycine into the polypeptide fragment by Fmoc solid-phase peptide synthesis, and removed the Alloc protecting group on the amino group to connect the oligoarginine, which significantly improved the solubility of the fragment[56]. This solubilization tag is compatible with conventional peptide synthesis and purification conditions, such as trifluoroacetic acid peptide cleavage conditions, high performance liquid chromatography purification, hydrazide method, etc., and can be quantitatively cleaved by trifluoroacetic acid after adjusting the pH of the solution to neutral (Figure 20 (a)). Using this strategy, Liu et al. Successfully synthesized the serine-64 phosphorylated M2 proton channel and the core transmembrane domain of the K+ channel protein Kir5.1.
图式20 RBM策略[56,106]

Scheme 20 The RBM strategy[56,106]

However, due to the low yield of the ligation reaction between the secondary amine of other Fmoc protected amino acids and RBM, this method is limited to glycine residues, and there are still difficulties in preparing insoluble peptides without glycine in the sequence. In order to solve this problem, Liu et al. Developed the second-generation RBM strategy (Figure 20 (B)), expanded the installation site of RBM group, and successfully realized the chemical total synthesis of hepatitis C virus (HCV) P7 ion channel and transmembrane protein EmrE by using this strategy. Here, the total synthesis of P7 ion channel protein is briefly introduced as an example[106].
The P7 ion channel is a 63-amino acid membrane protein whose homohexamer mediates intracellular conduction of the H+ and is critical for viral replication, assembly, and release[107]. In order to realize the chemical synthesis of P7 ion channel, the strategy of two-fragment ligation was adopted by predecessors, and it was found that the two fragments had the problem of poor solubility, which made it difficult to separate and purify, and then not enough products were obtained for subsequent studies[108]. In order to increase the solubility of the fragment and thus improve the synthesis efficiency, Liu et al. Introduced the above solubilizing tag containing oligoarginine near the N-terminal 17-histidine and C-terminal 53-leucine, which significantly improved the solubility of the fragment. Liu et al. Obtained the P7 ion channel protein in 26% yield by native chemical ligation of the two fragments and excision of the RBM group (Figure 21). The RBM strategy provides an effective means to obtain hydrophobic proteins. Liu et al. Recently described the technical route of membrane protein synthesis using RBM strategy in detail[109].
图式21 p7离子通道的化学全合成[106]

Scheme 21 Total chemical synthesis of p7 ion channel[106]

In addition, Liu et al. Also found that the RBM strategy could break the soluble colloidal particles formed under ligation conditions where the reaction sites between polypeptide fragments were masked, thus realizing the chemical synthesis of Haemophilus influenzae DNA ligase Hin-Lig[110].

5 Conclusion and prospect

Solubilization tag strategy provides an effective means of polypeptide structure modification, which can break the self-assembly structure formed by intramolecular or intermolecular hydrophobic interaction, ionic interaction, hydrogen bonding and other modes of action, and then improve the solubility of fragments, so that the subsequent purification, characterization and ligation reactions can be carried out smoothly. In recent years, a series of solubilization strategies have been developed, such as thioester solubilization strategy, Trityl solubilization strategy, Can solubiliza-tion strategy, RBM strategy and so on.Many proteins (such as histone H4, hepatitis B virus core protein assembly domain CP149, chaperone GroES, etc.) Can be successfully obtained, which lays the foundation for understanding the molecular biology mechanism behind the function of protein machine, and also provides help for drug design and protein material research and development.
The solubilization tag strategies developed at present still have their own application limitations, for example, the Alloc-Phacm strategy developed by Brik et al. Has the disadvantage of complex amino acid module synthesis route, the Can strategy developed by Yoshiya et al. Has the limitation of incompatible free radical desulfurization conditions, and the light-controlled removal strategy developed by Imperiali et al. May cause the oxidation of methionine and other residues. In addition, recent advances in flow synthesis technology have made small and medium-sized full-length proteins (about 200 amino acids) available at one time through flow synthesis, further enhancing the ability to obtain natural and artificially designed proteins by chemical synthesis[26]. In the process of obtaining larger proteins by chemical synthesis in the future, the poor solubility of larger polypeptide fragments is still a possible problem. Therefore, it is still worth exploring the development of new solubilizing tag strategies that are simple and direct, compatible with conventional peptide synthesis and purification conditions, and can be removed mildly and efficiently.
Enzymatic solubilization labeling has the outstanding advantages of aqueous phase operation, high reaction efficiency and good specificity, which make it expected to become a kind of efficient and mild solubilization labeling strategy.At present, solubilization tag strategies based on TEV enzyme and immobilized carboxypeptidase B (CPB) have been applied, while the application of enzymatic removal strategy in more difficult protein synthesis remains to be explored[74][111].
In the future, the development of solubilization tags and a series of efficient low-concentration ligation reactions will further enhance the ability of protein chemical synthesis and break through the existing synthetic limits[112⇓⇓~115].

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