Development of protein chemical synthesis using peptide thioester synthetic blocks

Abstract

Emil Fischer was a pioneer in peptide chemistry, striving to elucidate the chemical nature of peptides and proteins. In 1901, he and his colleague published the first paper on the subject. Since then, peptide chemistry has advanced steadily to the point that it is now possible to synthesize polypeptides, including enzymes. In addition, chemical synthesis is flexible and not constrained by the limitations of protein biosynthetic systems. Thus, proteins with various modifications, including post-translational modifications, can be synthesized. Current protein synthesis uses peptide thioesters synthesized by solid-phase methods as building blocks. This review will explain why peptide thioesters are utilized as building blocks for polypeptide synthesis and discuss the evolution of thioester preparation methods, as well as their applications in protein synthesis.

Peptide thioesters as building blocks for chemical protein synthesis

1. Introduction

1.1. The beginning of peptide synthetic chemistry.

Fischer entered the field of protein chemistry in 1899 and began research into elucidating the chemical structure of proteins from a synthetic chemistry perspective.¹⁾ His research was based on the assumption that proteins are composed of amino acids linked via amide bonds (peptide bonds). First, he partially hydrolyzed the diketopiperazine (DKP) of glycine to obtain glycylglycine, prepared its derivatives and clarified the properties of these substances in detail. Together with his collaborator, he published these findings in 1901.²⁾ That paper is regarded as the paper that marked the beginning of peptide synthetic chemistry. At that time, there was still no exact knowledge of the molecular weight of proteins, and it was speculated that if approximately 20 to 30 amino acids were joined together, they would reach the realm of compounds with protein-like properties.

In 1932, Fischer’s disciples, Bergmann and Zervas, introduced the benzyloxycarbonyl (Z) group as a protective group for amino groups.³⁾ This group could be introduced or removed under mild conditions. This research laid the groundwork for modern peptide synthesis, dramatically advancing the field. After World War II, polypeptides such as oxytocin, a 9-residue peptide amide, were synthesized in 1953,⁴⁾ and β-corticotropin (adrenocorticotropic hormone), a 39-residue peptide, in 1963.⁵⁾ The total synthesis and crystallization of bovine insulin were also achieved in 1965.⁶⁾

While a wide variety of peptides could be synthesized chemically, as Fischer predicted, synthesizing high-molecular-weight peptides by the solution-phase method require a high degree of skill and experience.

1.2. Development of the solid-phase method.

Since the invention of the amino acid sequence analysis method for peptides by Edman and Sanger, the amino acid sequences of isolated bioactive peptides have been elucidated one after another. These peptides can only be extracted in small quantities from living organisms. The demand for chemically synthesized peptides has rapidly increased to promote life science research.

In 1963, Merrifield developed the solid-phase peptide synthesis (SPPS) method, making it possible to synthesize peptides quickly and efficiently.⁷⁾ First, N-protected amino acids corresponding to the C-terminus of the desired peptide are attached to a bead of cross-linked polystyrene resin. Then, the amino-protecting group is removed, and an excess of N-protected amino acids and condensing agent is added to form peptide bonds. The excess reagent is removed by washing the resin. Next, the reagent that removes the amino-protecting group is added again, followed by the addition of a solution of amino-protected amino acid and a coupling reagent to form peptide bonds. This process is repeated to elongate the peptide chain from the C-terminus to the N-terminus. Once elongation is complete, an acid reagent is added to release the peptide from the resin. Finally, he successfully obtained a model tetrapeptide after ion-exchange column chromatography.

This epoch-making method enabled the rapid synthesis of target peptides by repeating a simple operation. This SPPS method can also be applied to DNA synthesis. In 1984, Merrifield was awarded the Nobel Prize in Chemistry for his contribution to the development of a methodology for chemical synthesis on solid matrices.

In 1971, Gutte and Merrifield attempted to synthesize bovine RNase A, consisting of 124 amino acids, using an SPPS method.⁸⁾ They obtained a preparation with 78% of the activity of pure natural RNase A. However, the analytical techniques available at the time did not allow for a detailed molecular-level analysis of the sample.

In 1988, Schneider and Kent successfully synthesized HIV-1 protease consisting of 99 amino acids using a state-of-the-art SPPS method.⁹⁾ The synthesized protein could specifically cleave the gag precursor fragment of HIV-1, and pepstatin A, a specific aspartate protease inhibitor, inhibited its activity. Kent and his colleagues also successfully crystallized the chemically synthesized HIV-1 protease and determined its three-dimensional structure using X-ray diffraction.¹⁰⁾ They also crystallized synthetic HIV-1 protease complexes with an inhibitor, revealing detailed atomic-level interactions between the inhibitor and the enzyme.¹¹⁾ These findings provided important insights for designing HIV-1 protease inhibitors.

In this review, we present our work on the development of a method for protein chemical synthesis using peptide thioesters as synthetic building blocks and its application to various proteins.

2. Integration of solution- and solid-phase methods in protein synthesis

In the 1970s, genetic engineering technology was developed, enabling the cloning of genes that encode proteins in living organisms. This technology makes it possible to produce proteins using living organisms’ protein synthesis systems. In the 1980s, protein engineering emerged as a new technology that enabled the genetic modification of any gene using chemically synthesized DNA. It has evolved into a technology for producing proteins with novel structures and functions that do not exist in nature. Protein engineering has revolutionized protein preparation methods. Unlike biological protein synthesis systems, chemical protein synthesis methods are not subject to intrinsic limitations. This allows for the synthesis of a wide variety of proteins, including those with post-translational modifications (PTMs) and those labeled with stable isotopes in a site-specific manner. Realizing such protein synthesis is expected to advance the life sciences significantly. In response to this expectation, new chemical protein synthesis methods have been developed since the 1980s.^12)–16)

2.1. Synthesis of BPTI by the minimum-protection strategy.

Our first step was to verify the effectiveness of this strategy through the synthesis of bovine pancreatic trypsin inhibitor (BPTI), a small protein consisting of 58 amino acids.¹⁷⁾ We prepared partially protected synthetic blocks using a solid-phase method and sequentially condensed them to obtain the target product.

As shown in Fig. 1(a), BPTI was divided into three synthetic blocks, BPTI(1–13), BPTI(14–36), and BPTI(37–58). To avoid racemization during segment condensation, the carboxyl-terminal amino acid residues of BPTI(1–13) and BPTI(14–36) were split at glycine and proline residues, respectively. Because BPTI(1–13) contains Asp and Glu residues, the C-terminal carboxyl group of this peptide segment was prepared as the free form, keeping the side-chain carboxyl groups protected. To achieve this, we used (3-nitro-2-pyridyl)sulfenyl (Npys)-amino acids¹⁸⁾ and 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids¹⁹⁾ in the SPPS method. After completion of the peptide chain elongation, the peptide resin was treated with trifluoroacetic acid (TFA) to obtain peptide [Arg(Tos)¹, Asp(OcHex)³, Cys(Acm)⁵, Glu(OcHex)⁷]-BPTI(1–13) (1) (Acm: acetamidomethyl; cHex: cyclohexyl; Tos: tosyl). Troc-[Cys(Acm)^14,30]-BPTI(14–36) (2) and Troc-[Cys(Acm)^38,51,55]-BPTI(37–58) (3) (Troc: 2,2,2-trichloroethoxycarbonyl) were prepared by the standard tert-butoxycarbonyl (Boc) SPPS method.⁷⁾ The peptide resins were treated with anhydrous hydrogen fluoride (HF)²⁰⁾ to obtain peptides 2 and 3. The purified products 1, 2, and 3 were subjected to the introduction of necessary protecting groups and removal of unnecessary protecting groups to obtain synthetic building blocks 4, 5, and 6 required for segment condensation. The peptides prepared by solid-phase methods and partially protected peptides for synthetic building blocks are summarized in Table 1. (See the Supplementary Materials, Figs. S1, S2, and S3 in Section 1 for the synthesis of compounds 1 to 6, and Table S1 in Section 4 for the chemical structures of protecting groups.)

Fig. 1.

Synthesis of BPTI by the minimum protection strategy. (a) The primary sequence of BPTI. Arrows indicate the sites where segment couplings were carried out. The position of disulfide bridges is between Cys5–Cys55, Cys14–Cys38, and Cys30–Cys51. (b) The synthesis of BPTI(1–58) via the segment coupling method and the induction of the natural form of BPTI through folding. (c) Ion-exchange chromatogram of synthetic BPTI on TSK gel CM-5PW. (Upper panel) The elution profile of crude synthetic BPTI obtained by oxidation with the oxidized form of DTT. (Lower panel) The elution profile of native BPTI. Broken line indicates the concentration of AcONH₄ in the buffer. The elution profile in (c) was taken and revised from Ref. 17) (Fig. 7) with permission from the publisher. 1 M = 1 mol dm⁻³.

Table 1. Peptide segments 1, 2, and 3 prepared by the SPPS method, and derived building blocks 4, 5, and 6 for BPTI synthesis

Compound	Peptide structure
1	H-Arg(Tos)-Pro-Asp(OcHex)-Phe-Cys(Acm)-Leu-Glu(OcHex)-Pro-Pro-Tyr-Thr-Gly-Pro-OH
2	Troc-Cys(Acm)-Lys-Ala-Arg-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys-Ala-Gly-Leu-Cys(Acm)-Gln-Thr-Phe-Val-Tyr-Gly-OH
3	Troc-Gly-Cys(Acm)-Arg-Ala-Lys-Arg-Asn-Asn-Phe-Lys-Ser-Ala-Glu-Asp-Cys(Acm)-Met-Arg-Thr-Cys(Acm)-Gly-Gly-Ala-OH
4	Z-Arg(Tos)-Pro-Asp(OcHex)-Phe-Cys(Acm)-Leu-Glu(OcHex)-Pro-Pro-Tyr-Thr-Gly-Pro-OH
5	Boc-Cys(Acm)-Lys(Z)-Ala-Arg-Ile-Ile-Arg-Tyr-Phe-Tyr-Asn-Ala-Lys(Z)-Ala-Gly-Leu-Cys(Acm)-Gln-Thr-Phe-Val-Tyr-Gly-OH
6	H-Gly-Cys(Acm)-Arg-Ala-Lys(Z)-Arg-Asn-Asn-Phe-Lys(Z)-Ser-Ala-Glu-Asp-Cys(Acm)-Met-Arg-Thr-Cys(Acm)-Gly-Gly-Ala-OH

BPTI(1–58) was prepared according to the scheme shown in Fig. 1(b). Peptide 5 was converted to the corresponding active ester in the presence of N-hydroxysuccinimide (HOSu) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (WSCI·HCl). The active ester, peptide 6, and 4-methylmorpholine (NMM) were mixed in dimethyl sulfoxide (DMSO), yielding product 7 with an approximate yield of 50%. (7: Amino acid analysis: Asp_3.92Thr_1.61Ser_0.92Glu_1.98Gly_4.99Ala_6.001/2Cys_0.57Val_0.83Met_0.83Ile_0.73Leu_0.98Tyr_2.42Phe_2.69Lys_3.70Arg_4.53) The active ester of peptide 4, prepared as described above, was condensed with the amino component of the peptide, obtained by the TFA treatment of peptide 7, in DMSO. The amino component reacted with the active ester to produce protected BPTI(1–58) (8) in a yield of 30% based on compound 7. (8: Amino acid analysis: Asp_5.56Thr_3.35Ser_0.90Glu_3.67Pro_4.40Gly_6.51Ala_6.00l/2Cys_0.00Val_0.83Met_0.88Ile_0.77Leu_2.69Tyr_4.28Phe_4.50Lys_3.63Arg_5.36)

Protected BPTI (1–58) (8) was treated successively with HF, Hg(AcO)₂, and dithiothreitol (DTT). The reduced form of synthetic BPTI (9) was oxidized in the presence of 1,2-dithiane-4,5-diol (the oxidized form of DTT), as described by Creighton.²¹⁾ The native form of synthetic BPTI was generated, adsorbed on TSK gel CM-5PW, and eluted with a AcONH₄ (pH 8.6) linear gradient (0.01–1 M AcONH₄), as shown in Fig. 1(c). The main product was eluted at the same position as that of native BPTI. The amino acid analysis data of the product 10 agreed well with that of native BPTI. (10: Amino acid analysis: Asp_5.33Thr_2.98Ser_1.19Glu_3.29Pro_4.02Gly_6.16Ala₆l/2Cys_5.11Val_1.10Met_0.94Ile_1.24Leu_2.07Tyr_3.96Phe_4.00Lys_3.82Arg_5.84) The inhibitory activity of the product was measured using the method described by Kassell.²²⁾ The results showed that the product had practically the same activity (98 ± 4%) against trypsin as native BPTI. The data conclusively demonstrate the successful synthesis of highly pure BPTI. The synthetic BPTI (10) yielded 13% by amino acid analysis. As a control experiment, natural BPTI was reduced and oxidized under identical conditions, and the BPTI recovery rate after purification using the same procedure was 14%.

The BPTI synthesis was completed in two months. Therefore, the examined method enabled rapid polypeptide synthesis, an important practical advantage. The minimum-protection strategy effectively enabled segment coupling without undesirable side reactions. The results indicated that this method is effective for preparing polypeptides of this size. However, the recovery yield of peptides after reversed-phase high-performance liquid chromatography (RP-HPLC) purification decreased with an increase in the number of hydrophobic protecting groups, likely due to reduced solubility of the peptide segment in eluent solvents. Consequently, protein synthesis based on partially protected peptide segments is expected to be challenging for high-molecular-weight proteins.

2.2. The thiocarboxyl segment condensation strategy.

Of the methods that utilize peptide segments prepared by the SPPS method, the thiocarboxyl segment condensation strategy developed by Blake et al. is appealing, as shown in Fig. 2.¹²⁾

Fig. 2.

Peptide bond formation by the thiocarboxyl segment-coupling method.

In this method, the side-chain carboxyl groups in peptides 11 and 12 do not require protecting groups because the thiocarboxyl group at the C-terminus is selectively activated by silver ions to yield peptide 13.

In addition, this method has the following strengths:

• -    Elimination of side-chain protection: The peptide’s side-chain carboxyl group does not require protection, simplifying the synthesis of building blocks.
• -    Improved solubility in RP-HPLC: Fewer protecting groups enhance solubility in the solvent, facilitating purification.
• -    Proven applicability: Successful synthesis of polypeptides such as [Gly17]-β-endorphin (1–31)¹³⁾ and α-inhibin-92¹⁴⁾ demonstrates the method’s reliability.

However, there are some limitations to using the thiocarboxyl group:

• -    Chemical instability: Thiocarboxylic acid can decompose upon exposure to oxygen or water, requiring careful handling.
• -    Risk of side reactions: The high nucleophilicity of the thiol group leads to undesired reactions with common amino-protecting agents like Z-OSu.
• -    Limited protective group options: Only citraconic anhydride is usable, but it yields two isomers, complicating RP-HPLC purification.
• -    Poor solubility enhancement: The citraconyl group does not improve solubility in polar organic solvents such as DMSO, potentially reducing condensation efficiency.

2.3. Design of a partially protected peptide thioester as a building block.

As a result of comparing the two strategies above, we concluded that creating synthetic building blocks with a thioester group instead of a thiocarboxyl group at the C-terminus of the peptide would solve all the problems associated with the two strategies. Furthermore, if peptides with a thioester group can be synthesized, it will be possible to use peptides with unprotected functional groups on side chains other than cysteine residues as raw materials for synthetic building blocks. These peptides are purified by RP-HPLC, and then the side-chain amino groups are protected with a Boc group to finalize the synthetic building blocks. These peptide chains can easily be synthesized using the Boc SPPS method. Furthermore, if the thioester remains stable during the purification process and can be converted into an active ester with silver ions, we could propose a promising method for protein synthesis.

2.4. Use of partially protected peptide thioesters as synthetic building blocks.

To examine the efficiency of this strategy, we attempted to synthesize the 52-residue DNA-binding domain of the c-Myb protein²³⁾ (Fig. 3(a)) according to the scheme shown in Fig. 3(b).²⁴⁾

Fig. 3.

The synthesis of c-Myb protein(142–193). (a) Primary sequence of c-Myb protein(142–193) deduced from the cDNA clone of murine c-myb mRNA.²²⁾ The arrow indicates the site of segment coupling. (b) The synthesis of c-Myb protein(142–193)-NH₂ by the peptide thioester coupling method. (c) The RP-HPLC elution profile of the reaction mixture of peptide amide 19 after a 3-day reaction. Chromatography was carried out on Cosmosil 5C18 at a flow rate of 1 mL/min at 40 °C. The broken line indicates the acetonitrile concentration in a 0.1% aq. TFA solution. Arrows a, b, and c indicate the elution positions of peptide amide 17, the active ester of peptide 18, and the hydrolysate of active ester 18, respectively. (d) The ion-exchange chromatogram of RP-HPLC-purified 20 on TSK gel CM-5PW. The broken line indicates the concentration of NaCl in the buffer. The elution profiles in (c) and (d) were taken and revised from Ref. 24) (Figs. 5 and 7) with permission from the publisher.

Peptide segments 14 and 15 were prepared using the Boc SPPS method. The amino groups in peptide 14 were protected with Boc groups to produce building block 16. (See the Supplementary Materials, Fig. S4, in Section 2 for the synthesis of compound 16.) Boc groups were also introduced to peptide 15. Then, the Troc group was removed to produce building block 17. (See the Supplementary Materials, Fig. S5, in Section 2 for the synthesis of compound 17.) Next, we performed peptide segment coupling by converting thioester 16 to p-nitrophenyl ester 18 in the presence of p-nitrophenol (HONp) and AgNO₃. Following a three-day mixing process with building block 17, the condensation reaction was almost complete, forming peptide 19, as shown in Fig. 3(c). The final compound 20 was isolated by RP-HPLC after treating compound 19 with TFA containing 1,4-butanedithiol. (20: Amino acid analysis: Asp_6.08Thr_3.93Ser_1.78Glu_5.13Pro_0.93Gly_2.15Ala₄Val_2.05Met_1.09Ile_3.19Leu_3.03Tyr_1.01His_2.16Lys_6.11Trp_2.34Arg_6.07) The purity of the isolated 20 was confirmed once more by TSK-gel CM-5PW ion-exchange chromatography. As demonstrated in Fig. 3(d), a single symmetrical peak was observed. Using compound 20, the solution structure of the DNA-binding unit of the c-Myb protein (142–193) has been elucidated by NMR spectroscopy, performed in a potassium phosphate buffer at a neutral pH.²⁵⁾ The thioester in the peptide remained stable during purification by RP-HPLC and prolonged storage at 4 °C. Peptides 14 and 15 have no or one protecting group when purified using RP-HPLC. Consequently, purification was much easier compared with BPTI synthesis.¹⁷⁾ Consequently, the preparation of long peptide segments was straightforward. Introducing Boc groups to the peptide proceeded almost quantitatively without side reactions when using Boc-OSu as the protective reagent. Protecting groups such as the Boc group have enhanced the solubility of a partially protected peptide segment in DMSO or N,N-dimethylformamide (DMF) on segment condensation. No serious side reactions occurred during segment coupling under the minimum protection strategy. The yield of segment coupling was also satisfactory.

In conclusion, the partially protected peptide thioester is a promising building block for polypeptide synthesis. However, the yield of peptide H-c-Myb protein(142–163)-SCH₂CH₂CONH₂ was approximately half that of Troc-c-Myb protein(164–193)-NH₂, which was synthesized on 4-methylbenzhydrylamine (MBHA) resin. This must be improved.

2.5. Development of a thioester linker with enhanced stability.

One disadvantage of using peptide thioesters as building blocks is that they have a lower yield than peptide acids or amides. Next, we investigated the mechanism that reduces the stability of the resin during peptide thioester synthesis.²⁶⁾ To analyze the factors responsible for the low yield of peptide thioesters, we treated the Boc-Gly-SC(CH₃)₂CH₂CONH-MBHA resin under the conditions used for peptide chain elongation cycles. We then analyzed the compound liberated from the resin using an amino acid analyzer and mass spectrometry. When the resin was treated with 50% TFA in dichloromethane (DCM) (v/v), H-Gly-SC(CH₃)₂CH₂CONH₂ was liberated. The decomposition rate was more than 20 times higher than that of peptide amide synthesis on the MBHA resin. However, the peptide thioester resin remained stable in the presence of 5% N,N-diisopropylethylamine (DIEA) in N,N-dimethylformamide (DMF). These results suggest that the sulfur atom in the thioester accelerates the cleavage of the N–C bond in the MBHA moiety via neighboring effects on the carbon atom at the cleavage site during TFA treatment.

To test this hypothesis, an amino acid such as norleucine (Nle) or β-Ala was inserted as a spacer between the thioester moiety and the MBHA resin to keep the sulfur atom in the thioester moiety away from the carbon atom in the N–C of the MBHA moiety. (See the Supplementary Materials, Fig. S6, in Section 3.)

Starting from the four different thioester MBHA resins as shown in Table 2, HU-type DNA-binding protein (HBs(16–39)) was constructed using ABI 430A Peptide Synthesizer system software version 1.40 HOBt/NMP t-Boc. The yields of Troc-HBs(16–39)-SR became nearly double by the insertion of one amino acid residue, such as Nle or β-Ala, between the thioester moiety and the MBHA resin. The yield of the peptide thioesters was nearly the same level as that of peptide amide synthesis. However, no significant difference in yield was observed among primary, secondary, and tertiary alkyl thioesters with respect to the chemical structure of the thioester linkers.

Table 2. A comparison of the yields for Troc-HBs (16–39)-SR synthesized using resins with four types of thioester linkers

Troc-Leu-Ser-Lys-Lys-Asp-Ala-Thr-Lys-Ala-Val-Asp-Ala-Val- Phe-Asp-Ser-Ile-Thr-Glu-Ala-Leu-Arg-Lys-Gly-SR
-SR	Yield/%
-SCH2CH2CONH2	15
-SC(CH3)2CH2CONH2	15
-SC(CH3)2CH2CO-β-Ala-NH2	26
-SC(CH3)2CH2CO-Nle-NH2	28

Based on these data, four peptide building blocks that cover the whole sequence of a stable isotope-labelled HBs were synthesized as shown in Table 3.

Table 3. The yield of the building blocks prepared for the synthesis of a stable isotope-labeled HU-type DNA-binding protein using a resin with a Nle-inserted thioester linker

Peptide segments	Yield/%
Boc-[Lys(Boc)3]-HBs(1–15)-SC(CH3)2CH2CO-Nle-NH2    Boc-Met-Asn-Lys(Boc)-Thr-Glu-Leu-Ile-Asn-Ala-Val-Ala-Glu-Thr-Ser-Gly-SC(CH3)2CH2CO-Nle-NH2	22 (14)
Troc-[Lys(Boc)18,19,23,38]-HBs(16–39)-SC(CH3)2CH2CO-Nle-NH2    Troc-Leu-Ser-Lys(Boc)-Lys(Boc)-Asp-Ala-Thr-Lys(Boc)-Ala-Val-Asp-Ala-Val-Phe-Asp-Ser-Ile-Thr-Glu-Ala-Leu-Arg-Lys(Boc)-Gly-SC(CH3)2CH2CO-Nle-NH2	22 (10)
Troc-[Lys(Boc)41,59,(2-13C)Phe47,(1-13C)Ala56,(2-13C)Gly60]-HBs(40–60)-SC(CH3)2CH2CO-Nle-NH2    Troc-Asp-Lys(Boc)-Val-Gln-Leu-Ile-Gly-Phe*-Gly-Asn-Phe-Glu-Val-Arg-Glu-Arg-Ala*-Ala-Arg-Lys(Boc)-Gly*-SC(CH3)2CH2CO-Nle-NH2	41 (17)
[Lys(Boc)75,80,83,86,90,(guanidino-N2,3-15N2)Arg61,(methyl-2H3)Met69,(ε-15N)Lys80]-HBs(61–90)    Arg*-Asn-Pro-Gln-Thr-Gly-Glu-Glu-Met*-Glu-Ile-Pro-Ala-Ser-Lys(Boc)-Val-Pro-Ala-Phe-Lys*(Boc)-Pro-Gly-Lys(Boc)-Ala-Leu-Lys(Boc)-Asp-Ala-Val-Lys(Boc)	20 (19)

The yields in parentheses refer to the synthesis using an -SCH₂CH₂CONH₂ group as the thioester linker and an iNoc group for the terminal amino protection. The asterisk marks indicate the stable isotope-labeled amino acids.

The yield of peptide thioester building blocks increased by twofold compared with the yields of building blocks, in which Boc-Gly-SCH₂CH₂CO-MBHA-resin was used as the starting resin. The yields of peptide amide were consistent between the two syntheses. This result strongly suggests that the neighboring effect of the sulfur atom in the thioester is the probable cause of the peptide loss at the TFA treatment step during the peptide chain elongation cycle. Please refer to the Supplementary Materials, Fig. S6, in Section 3 for our working hypothesis.

2.6. Synthesis of a cysteine-containing polypeptide by the thioester method.

The Acm group on the cysteine residue remains stable under HF treatment conditions. Therefore, S-Acm cysteine-containing peptide thioesters can easily be synthesized using the Boc SPPS method. However, the S-Acm group is known to be partially removed under a wide range of conditions in the presence of silver ions. The thioester method uses silver compounds to activate the thioester moiety in segment coupling. Thus, we investigated the conditions under which segment coupling proceeds while retaining the Acm groups on the cysteine residues in the presence of silver compounds.

The thioester group converts to the corresponding active ester in the presence of silver ions and an active ester component. To evaluate the effect of the active ester components on Acm group stability, we examined four different components (HONp, HOSu, 1-hydroxybenzotriazole (HOBt), and 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOObt)) and their influence on the Acm group in the presence of AgNO₃ and DIEA. Both HOBt and HOObt enabled efficient segment condensation without decomposing Acm. When mixed, they immediately formed precipitates with AgNO₃. This is believed to be due to the formation of a complex between silver ions and HOBt and HOObt. The silver ions in the complex allow them to coordinate with sulfur atoms in the thioester but not strongly with the sulfur atoms in the thioether in the S-Acm. This prevents the removal of the Acm group. Under these conditions, we successfully synthesized adrenomedullin with two cysteine residues.²⁷⁾ Thus, we developed a thioester method to condense Cys(Acm)-containing peptides. It was later discovered that the combination of HOObt and AgCl enables safer condensation of Cys(Acm)-containing peptides. In contrast, the combination of AgNO₃ and HONp or HOSu decomposed the Acm groups.

2.7. The advent of the native chemical ligation method.

In 1953, Wieland et al. reported that valine thioester and cysteine condense via an intermolecular thiol–thioester exchange reaction, followed by an S–N acyl shift reaction to form Val-Cys in a neutral aqueous solution, as shown in Fig. 4(a).²⁸⁾

Fig. 4.

Chemo-selective peptide bond formation through an intermolecular thiol–thioester exchange reaction, followed by an S–N acyl shift reaction. (a) The ligation between a phenyl thioester and a cysteine. (b) Ligation between a peptide thioester and a cysteinyl peptide.

Building on this phenomenon, Kent et al. examined the reaction between peptide thioesters and cysteinyl peptides, which also formed amide bonds chemoselectively, as shown in Fig. 4(b).²⁹⁾ They named this condensation reaction “native chemical ligation” (NCL) and published their findings in 1994. This greatly impacted methods of chemical protein synthesis. Because the reaction proceeded chemoselectively between the thioester moiety and the cysteinyl residue at the N-terminus, the synthetic block required no protecting groups. Consequently, protein synthesis shifted rapidly from the SPPS method of sequential amino acid condensation to the use of peptide thioesters, which are synthetic building blocks prepared by the SPPS method. However, with this method, if the cysteine residue is not in the appropriate position, the amino acid residue must be modified at the ligation site to enable the NCL reaction. Peptide thioesters have been widely used to synthesize polypeptides and proteins since the development of the NCL method.

The other ligation methods such as Ser/Thr ligation³⁰⁾ and KAHA ligation,³¹⁾ in which peptide thioesters are not used, and enzyme-mediated ligation (e.g. sortase,³²⁾ butelase,³³⁾ OaAEP1³⁴⁾) were reported. Although those methods are important in chemical protein synthesis, the details are not shown here.

3. Investigation for the preparation of peptide thioesters by the Fmoc SPPS method

Peptide thioesters have become widely used as synthetic building blocks in the ligation method. The Boc SPPS method has been used to easily synthesize unmodified peptide thioesters. However, synthesizing glycosylated³⁵⁾ or phosphorylated³⁶⁾ peptides using the Boc SPPS method remains challenging. Conversely, the Fmoc SPPS method is suitable for synthesizing peptides with PTMs. Nevertheless, synthesizing peptide thioesters with the Fmoc SPPS method requires ingenuity. This is because thioesters decompose in the presence of piperidine, a compound commonly used to remove Fmoc groups.

To establish chemical synthesis methods for proteins as complementary techniques to protein engineering methods, it is essential to develop methods for synthesizing modified proteins. To achieve this goal, we worked on developing a peptide thioester synthesis method using the Fmoc SPPS method.

The authors discovered that using a weak nucleophilic base to remove the Fmoc group prevents the thioester group from decomposing.³⁷⁾ This enables the direct synthesis of peptide thioesters using the Fmoc method. However, when the amino acid bonded to the thioester is chiral, partial racemization occurs.³⁸⁾

Indirect methods using linkers, such as sulfonamides,³⁹⁾ N-acylureas,⁴⁰⁾ and hydrazides,⁴¹⁾ have been reported. Each method may overcome some of the difficulties associated with peptide thioester synthesis by the Fmoc SPPS procedure. However, we decided to investigate a process in which the peptide chain extension reaction is completed first, followed by converting the amide bond into a thioester via an N–S acyl shift reaction.⁴²⁾

3.1. The intramolecular N–S acyl shift reaction of peptides containing a thiol group.

3.1.1. The N–S acyl shift reaction at the thiol auxiliary Dmmb.

Originally developed as a thiol-mediated ligation auxiliary, the 4,5-dimethoxy-2-mercaptobenzyl (Dmmb) group can be removed by treating it with 1 M trifluoromethanesulfonic acid (TFMSA) in TFA after ligation is complete (Fig. 5).⁴³⁾ However, when peptide 24 containing the Dmmb group was treated with TFA alone, a new compound, X, was produced. Compound X eluted earlier than peptide 24 during RP-HPLC and had an identical mass to peptide 24.⁴⁴⁾ Compound X disappeared when the pH of the solution increased to 8. This suggests that compound X corresponds to the thioester intermediate 23, which is formed by reversing the ligation reaction, thus undergoing an intramolecular N–S acyl shift.

Fig. 5.

Thiol auxiliary Dmmb-mediated ligation.

Use of the Dmmb-containing peptide confirmed thioester formation. The ¹³C-labeled peptide, Fmoc-Gly(1-¹³C)-D,L-(Dmmb)Ala-OCH₃ (26) (Fig. 6), was treated with TFA and analyzed by ¹³C nuclear magnetic resonance spectroscopy (NMR) and RP-HPLC (Fig. 6).⁴⁵⁾ The ¹³C NMR signal at 172.8 ppm, which corresponds to amide 26, shifted to signals at 204.6 and 205.3 ppm, which correspond to thioester 27. The presence of two signals for the thioester indicates the existence of distinct conformers. Under the conditions used, a small amount of peptide 28 without the Dmmb group was observed.

Fig. 6.

Reaction of Dmmb-containing ¹³C-labeled peptide 26 in a TFA solution (14% CDCl₃, 0.5% tris(2-carboxyethyl)phosphine (TCEP)•HCl (v/v/w)) (a). (b–d): ¹³C NMR spectra after the indicated reaction time. (e–g): RP-HPLC elution profile. Column: YMC-Pack ProC18 (4:6 × 150 mm); eluent: 0.1% TFA in aq. CH₃CN, 1.0 mL/min. NMR spectra (b–d) and elution profiles (e–g) were taken and revised from Ref. 45) (Fig. 6) with permission.

3.1.2. N–S acyl shift reaction at the cysteine residue.

A cysteine residue can also play a role in forming a thioester. It has been suggested that thioesters form during the final deprotection process of cysteine-containing peptides using HF.⁴⁶⁾ In fact, the thioester signal was observed during the acid treatment of a cysteine-containing peptide.⁴⁵⁾ The ¹³C-labeled peptide Fmoc-Ile-Ala-Gly(1-¹³C)-Cys-Arg-NH₂ (29) was treated with TFA and analyzed by ¹³C NMR and RP-HPLC (Fig. 7). The signals at 172.5 and 172.7 ppm, which corresponded to amide 29, shifted to 201.8 ppm, which corresponded to thioester 30 (Fig. 7). The amide and thioester mixture reached equilibrium (1:4) after nearly one month.

Fig. 7.

Reaction of cysteine-containing ¹³C-labeled peptide 29 in a TFA solution (29% CDCl₃, 0.5% TCEP (v/v/w)) (a). (b–d): ¹³C NMR spectra after the indicated reaction time. (e–g): RP-HPLC elution profile. Column: YMC-Pack ProC18 (4:6 × 150 mm); eluent: aq. acetonitrile containing 0.1% TFA, flow rate: 1.0 mL/min. NMR spectra (b–d) and elution profiles (e–g) were taken and revised from Ref. 45) (Fig. 2) with permission.

Using RP-HPLC, we observed a peak corresponding to thioester 30 eluting earlier than amide 29 (Fig. 7). However, the ratio of amide to thioester (4:1) differed greatly from the NMR results. These results suggest that the cysteine-containing peptide exists in equilibrium between the amide and thioester due to an intramolecular acyl shift. The thioester predominates in concentrated TFA solutions but transforms into an amide under weakly acidic conditions, such as 0.1% TFA in aqueous acetonitrile, which was used in the RP-HPLC analysis.

3.2. Peptide thioester synthesis via an intramolecular N–S acyl shift.

3.2.1. Thiol auxiliary, Dmmb-assisted peptide thioester preparation.

The Dmmb group can mediate thioester formation via an intramolecular N–S acyl shift reaction, as described above. This thioester is relatively stable under RP-HPLC conditions using aqueous 0.1% TFA as the eluent. However, under neutral conditions, it easily transforms into an amide. Moreover, upon treatment with an excess amount of thiol under neutral conditions, this thioester intermediate can be transformed into a “stable thioester” that contains no amino group in the vicinity by intermolecular transthioesterification before the intramolecular S–N acyl shift. This corresponds to the reverse scheme for ligation (Fig. 5). This process was applied to peptide thioester synthesis via Fmoc SPPS (Fig. 8).^45),47),48) A Dmmb-containing peptide does not contain a thioester bond, and the peptide chain elongates on the Dmmb-containing resin 31 with the TFA-stable linker using a standard Fmoc SPPS methodology. After the assembly of the peptide chain, resin 32 is treated with an acid, such as TFA or hydrochloric acid, to remove protecting groups and promote an intramolecular N–S acyl shift. This results in the formation of the thioester intermediate 33 on the resin. This intermediate can be transformed into peptide thioester 21 by treating it with an excess amount of a thiol such as sodium 2-mercaptoethanesulfonate (MESNa) under neutral conditions. Under optimized conditions, a peptide thioester containing 29 amino acids (BPTI(1–29)-SCH₂CH₂SO₃H (35)) was obtained with a yield of 15% (Fig. 8(b)).⁴⁸⁾

Fig. 8.

Dmmb-mediated peptide thioester formation via Fmoc SPPS on-resin N–S acyl shift reaction. (a) Synthetic scheme. (b) The RP-HPLC elution profile of the reaction mixture of BPTI(1–29)-SCH₂CH₂SO₃H (35). RP-HPLC conditions: column: YMC-Pack ProC18 RS (4.6 × 150 mm); eluent: aq. acetonitrile containing 0.1% TFA, flow rate: 1.0 mL/min. Elution profile (b) was taken and revised from Ref. 48) (Fig. 3) with permission.

3.2.2. NAC-assisted peptide thioester preparation.

In the reaction with the Dmmb group, an N–S acyl shift occurs at the tertiary amide, resulting in the formation of a secondary amine. Secondary amines are less reactive than primary amines to an S–N acyl shift. tert-Amide structures can be simplified by using N-alkyl cysteine (NAC) residues, such as N-ethyl cysteine (Fig. 9).⁴⁹⁾ N-Ethyl cysteine can be easily prepared by the reductive amination of acetaldehyde with S-trityl (Trt) cysteine. Introducing an amino acid residue to an N-ethyl cysteine residue on a solid support results in a low yield; therefore, dipeptides (Fmoc-Xaa-(Et)Cys(Trt)) are first prepared and then introduced to the solid support.⁵⁰⁾ The thioester intermediate 37 is formed under acidic conditions from peptide 36 containing the NAC moiety. The thioester 37 is in equilibrium with the starting amide 36. The intermediate 37 is then converted into the “stable thioester” 21, which lacks a β-amino group. This conversion occurs in the presence of an excess amount of an “external thiol”, such as 3-mercaptopropionic acid (MPA) and MESNa.

Fig. 9.

Peptide thioester formation at the NAC.

The other structures such as N,N-bis(2-sulfanylethyl)amide (SEA) were also reported for taking an N–S acyl shift at the tertiary amide.⁵¹⁾

3.2.3. Preparation of peptide thioesters by introducing a CPE to the C-terminus.

In the equilibrium, the amide form is more stable than the thioester at Cys positions. We designed a structure to stabilize the thioester after the N–S acyl shift reaction based on DKP formation at the Cys positions. In 1985, Zanotti et al. reported DKP formation in which acyl cysteinylproline active ester 38 transformed into DKP thioester 39 via an intramolecular reaction (Fig. 10).⁵²⁾

Fig. 10.

Formation of DKP thioester.

Based on this structure, we designed a cysteinylproline ester (CPE) for thioester formation (Fig. 11).^53),54) Peptide 40, which contains a CPE sequence at the C-terminus, spontaneously transforms into DKP thioester 42 via an intramolecular N–S acyl shift followed by DKP formation under neutral aqueous conditions. The DKP thioester can transform into the corresponding peptide thioester 43 in the presence of an additional thiol, such as MESNa. Furthermore, the ligation product 45 is obtained in a one-pot reaction in the presence of the cysteinyl peptide 44.

Fig. 11.

DKP thioester formation and ligation of CPE peptide.

A CPE peptide can be prepared using the Fmoc SPPS procedure. In this synthesis, a dipeptide unit, Fmoc-Xaa-Cys(Trt) (47), must be prepared beforehand and introduced after the glycolic acid and proline residues are attached to the resin because the N-terminal Cys(Trt)-Pro ester sequence can form the DKP structure. Then, the peptide chain is elongated by the standard Fmoc SPPS procedure.

The rate of peptide thioester formation is enhanced when bulky amino acid residues, such as tert-leucine (Tle) and Val, are added after the ester moiety of the CPE group (e.g., peptide-Cys-Pro-OCH₂CO-Tle-NH₂).⁵⁵⁾ The proline residue can also be replaced with an N-substituted glycine residue, such as N-methylglycine.⁵⁵⁾

It was later shown that peptides containing special sequences such as Gly–Cys, His–Cys, and Cys–Cys can be transformed into the corresponding glycine, histidine, or cysteine thioester in the presence of thiols under acidic conditions.^56),57) Ligation via an N–S acyl shift at the α-methylcysteine site has also been reported.⁵⁸⁾ Other methods were also proposed to prepare the thioester using the N–S acyl shift reaction, and some are shown in another review.⁴²⁾

4. Chemical synthesis of proteins with various modifications

PTMs play a crucial role in regulating many biological processes. Fmoc SPPS is an effective method for synthesizing modified peptides, such as phosphorylated or glycosylated peptides. In addition, an intramolecular N–S acyl shift reaction has made it possible to synthesize peptide thioesters via Fmoc SPPS. Building on these achievements, we have established synthetic routes for various complex proteins, including post-translationally modified proteins that are difficult to prepare using methods involving living cells. We have synthesized a series of modified proteins to demonstrate the complementarity of chemical synthesis and protein engineering techniques.

4.1. Synthesis of modified histone proteins.

Histones are components of nucleosomes. An octamer containing two copies each of the four different histone proteins (H2A, H2B, H3, and H4) is wrapped by a DNA molecule to form the basic unit of chromatin, called a nucleosome. The PTM of histones, such as acetylation, methylation, and phosphorylation, play an important role in gene regulation.⁵⁹⁾ The chemical synthesis of histones with PTMs is important for studying their function and structure.

4.1.1. Synthesis of trimethyllysine-containing histone H3.

We applied our strategy for preparing peptide thioesters using the N–S acyl shift reaction to the synthesis of modified histones. First, H3 (55), which contains 135 amino acids including an N^ε-trimethyl Lys⁹ residue, was synthesized (Fig. 12).⁶⁰⁾ H3 was divided into three segments for synthetic purposes: [Lys(Me₃)⁹]H3(1–43)-Cys-Pro-OCH₂CONH₂ (48), Fmoc-H3(44–95)-Cys-Pro-OCH₂CONH₂ (50), and H3(96–135) (51). These segments were sequentially ligated by the NCL in the presence of 4-mercaptophenylacetic acid (MPAA), then the thioester method. H3 55 (MS (MALDI): m/z 15320.5, calcd for M⁺ 15315.9) was obtained after RP-HPLC purification in a yield of 6.3% based on the segment 50.

Fig. 12.

Synthesis of trimethyllysine-containing histone H3 by sequential ligation using CPE peptides. (a) Amino acid sequence of histone H3. (b) Synthetic scheme. (c) RP-HPLC of the reaction mixture after final removal of the protecting groups. Column: YMCPack C8 (4.6 × 150 mm), eluent: aq. acetonitrile containing 0.1% TFA, flow rate: 1.0 mL/min. Elution profile (c) was taken and revised from Ref. 60) (Fig. 8) with permission.

4.1.2. Synthesis of trimethyllysine-containing histone H3 using recombinant peptides.

Recombinant peptides can be used as building blocks for ligation. Histones H3 and H4 were synthesized using recombinant peptides as C-terminal building blocks.⁶¹⁾ A synthetic scheme for histone H3 based on NCL is shown in Fig. 13. In this synthesis, H3 55 (MS (MALDI): m/z 15313.6, calcd for M⁺ 15315.9) was obtained after RP-HPLC purification in a yield of 18% based on the recombinant peptide 57. Combining NCL and desulfurization with recombinant peptides as C-terminal building blocks is a useful and convenient strategy that can be applied to synthesize all core histones (H2A, H2B, H3, and H4) with various modifications. Therefore, it has the potential to prepare libraries of histones with PTMs.

Fig. 13.

Synthesis of trimethyllysine-containing histone H3 by using a recombinant peptide. (a) Synthetic scheme. (b) RP-HPLC of the reaction mixture after final removal of the protecting groups. Column: YMCPack C8 (4.6 × 150 mm), eluent: aq. acetonitrile containing 0.1% TFA, flow rate: 1.0 mL/min. Elution profile (b) was taken and revised from Ref. 61) (Fig. 5) with permission.

4.1.3. Synthesis of ubiquitinated histone H3.

Ubiquitination is another important PTM. Ubiquitin (Ub), a 76-amino acid peptide, forms an isopeptide bond with a lysine side-chain amino group at its C-terminus. Histones have also been reported to be ubiquitinated.^62),63) The ubiquitinated form of histone H3 (68) has been synthesized, as shown in Fig. 14,⁶⁴⁾ in which the δ-mercaptolysine derivative⁶⁵⁾ was used to introduce the native isopeptide structure. In this synthesis, ubiquitinated H3 68 (MS (MALDI): m/z 23917.0, calcd for [M + H]⁺ 23962.7) was obtained after RP-HPLC purification in a yield of 5.3% based on the segment 63.

Fig. 14.

Synthesis of ubiquitinated histone H3. (a) Synthetic scheme. (b) RP-HPLC of reaction mixture after final removal of the protecting groups. Column: YMCPack C8 (4.6 × 150 mm), eluent: aq. acetonitrile containing 0.1% TFA, flow rate: 1.0 mL/min. Elution profile (b) was taken and revised from Ref. 64) (Fig. 5) with permission.

The folding of Ub was confirmed by the CD measurement of the ubiquitinated H3 peptide (1–35) linked with the thiirane linker.⁶⁶⁾ Using these model peptides, it was shown that the ubiquitination of H3 stimulates DNA methyltransferase activity.^63),67) Furthermore, the nucleosome was prepared with recombinant histones H2A, H2B, and H4 from the ubiquitinated H3 linked with the thiirane linker.⁶⁷⁾

We also synthesized other histones, such as H2A with glycosylation, by combining the thioester-forming devices NAC and CPE, which are described in other sections. By combining these strategies, we can synthesize many kinds of histones with PTMs, which facilitates progress in their functional and structural studies.

4.2. Glycoprotein synthesis.

It is estimated that approximately half of natural proteins retain glycan chains at serine, threonine, and/or asparagine residues, which are referred to as O-linked and N-linked glycan, respectively. These glycan chains are inherently highly heterogeneous, which has posed challenges for their structural and functional analyses. To overcome this problem, we applied our method for the synthesis of homogeneous glycoproteins.

4.2.1. Chemoenzymatic synthesis of saposin C with N-glycan.

Chemoenzymatic synthesis can be used for N-glycopolypeptides.⁶⁸⁾ First, a polypeptide bearing the reducing end N-acetylglucosamine (GlcNAc) residue at the glycosylation site is synthesized using SPPS. If necessary for larger proteins, segment ligation follows. Next, the resulting GlcNAc-containing polypeptide undergoes glycan chain transfer using a glycosynthase. This chemoenzymatic strategy was successfully used to synthesize the extracellular domain of emmprin.⁶⁹⁾ However, in the case of saposin C, a lipid binding protein, a serious solubility problem arose.

The sequence of saposin C was divided at Ala³⁴–Cys³⁵, and NCL was attempted to couple the two segments (Fig. 15). However, the preliminary synthesis revealed that the N-terminal segment was highly hydrophobic and poorly soluble in aqueous acetonitrile and other organic solvents. To improve solubility, we introduced an O-acyl isopeptide structure developed by Kiso et al.⁷⁰⁾ at Ala²²–Thr²³ to enhance peptide solubility. The NAC method⁴⁹⁾ was employed to generate a C-terminal thioester. At the glycosylation site (Asn²¹), Fmoc-Asn bearing a GlcNAc unit was incorporated. The re-synthesized N-terminal segment 69 exhibited significantly improved solubility, and the NCL reaction with the C-terminal segment 70 proceeded efficiently, yielding saposin C with a GlcNAc unit. After the disulfide bonds were formed by DMSO oxidation, the octasaccharide oxazoline 75 was introduced to the GlcNAc residue using glycosynthase, yielding saposin C with a nonasaccharide structure (76), which was confirmed by MALDI-TOF MS analysis (m/z 10156.0, calcd for [M + H]⁺ 10154.5).⁷¹⁾ The O-acyl isopeptide strategy was also effective for synthesizing the extracellular domain of TIM-3 by the thioester method.⁷²⁾

Fig. 15.

Chemoenzymatic synthesis of glycosylated saposin C. (a) Structure of saposin C, (b) Synthetic procedure, (c) RP-HPLC profile of purified 76. The arrow and $\fbox{N}$ in the sequence indicate the ligation and the glycosylation sites, respectively. AT is the site where the isopeptide structure is formed. RP-HPLC condition: column YMC-Pack Protein RP (4.6 × 150 mm) at the flow rate of 1 mL/min and 50 °C; eluent, A, 0.1% aqueous TFA, B, 0.1% TFA in acetonitrile.

4.2.2. Chemical synthesis of O-glycosylated IL-2.

No efficient chemoenzymatic synthetic method has been established for O-glycoproteins. In the case of O-glycosylated interleukin-2 (IL-2), we attempted the total chemical synthesis by the thioester method.⁷³⁾ The full sequence was divided into four segments (Fig. 16). However, in this case also, a serious solubility problem occurred: the C-terminal segment (99–133) exhibited extremely poor solubility in common (aq.) organic solvents. Only TFA retained sufficient solubilizing ability. An initial attempt to improve solubility by introducing O-acyl isopeptide structures following saposin C synthesis was unsuccessful. Subsequently, we developed a strategy aimed at altering the isoelectric point of the segment: we protected the carboxy groups of Glu residues with basic picolyl groups, which effectively shifted the isoelectric point. This modification successfully overcame the solubility issue to obtain the C-terminal segment 80.

Fig. 16.

Synthesis of human IL-2 carrying core 1 sugar. (a) Structure of IL-2, (b) Synthetic procedure, (c) RP-HPLC profile of 84. The arrows and $\fbox{T}$ in the sequence indicate the ligation and the glycosylation sites, respectively. in the sequence shows Glu having picolyl ester. AT and IT are the sites where the isopeptide structures are formed. RP-HPLC condition: column YMC-Pack Protein RP (4.6 × 150 mm) at the flow rate of 1 mL/min; eluent, A, 0.1% aqueous TFA, B, 0.1% TFA in acetonitrile.

Glycopeptide thioester segment 77 was synthesized by incorporating Fmoc-Thr bearing a substituted benzyl-protected core 1 sugar⁷⁴⁾ during chain elongation. Once all segments were prepared, the ligation reaction was initiated using the thioester method with segments 79 and 80. Due to the improved solubility of the C-terminal segment 80, the reaction proceeded efficiently, yielding peptide 82. The entire IL-2 sequence was assembled in a one-pot process using the reactivity differences of peptide aryl and alkyl thioesters. After deprotection of all protecting groups, including picolyl ester, and oxidative folding, fully active IL-2 with a core 1 sugar 84, having the desired mass number (ESI mass, 1973.5, 1754.4, 1579.2, 1435.8, 1315.9, calcd for [M + 8H]⁸⁺: 1973.7, [M + 9H]⁹⁺: 1754.5, [M + 10H]¹⁰⁺: 1579.1, [M + 11H]¹¹⁺: 1435.7, [M + 12H]¹²⁺: 1316.1), was obtained (Fig. 16).

5. One-pot segment condensation strategy for protein synthesis

When synthesizing longer polypeptides or proteins, multiple ligation reactions are necessary. In the conventional sequential ligation approach, which starts at the C-terminus and ends at the N-terminus, the N-terminal protecting group must be removed after each ligation to allow for the next coupling step. This deprotection step requires isolating the ligated product with a free N-terminus, which is usually achieved by RP-HPLC, to remove the deprotection reagents. However, HPLC purification often results in low recovery yields due to the non-specific adsorption of polypeptides onto the column. Therefore, one-pot ligation procedures, in which peptide segments are sequentially ligated in the same reaction vessel without the isolation of the intermediates, are highly valuable.

5.1. Synthesis of human SOD by the one-pot thioester method.

For the synthesis of superoxide dismutase (SOD), we designed a one-pot method by relying on the reactivity difference of peptidyl esters from the highest selenoester, middle aryl thioester, to the lowest alkyl thioester. Three peptide esters were prepared using the NAC method.⁴⁹⁾ The ligation was initiated between the selenoester of the N-terminal peptide 85 and the next segment 86 with the aryl thioester. The selenoester selectively reacted, producing the desired intermediate with aryl thioester 87. The peptide alkyl thioester 88 was then added to the reaction mixture to perform the second ligation. Finally, the C-terminal segment 90 was added to perform the third ligation. After completing the ligation and deprotection, zinc and copper ions were coordinated, yielding fully active SOD 93 having the desired mass number (ESI mass found: m/z 3195.3, 2904.8, 2662.9, calcd for [M + 2Cu + 2Zn + 2H]¹⁰⁺: 3194.9, [M + 2Cu + 2Zn + 3H]¹¹⁺: 2904.6, [M + 2Cu + 2Zn + 4H]¹²⁺: 2662.6) (Fig. 17).⁷⁵⁾

Fig. 17.

A scheme for the synthesis of SOD by a one-pot four-segment coupling method based on the thioester method. (a) Structure of apo-SOD. (b) Synthetic procedure. (c) Size-exclusion chromatography of 93. Peptide 90 was synthesized by the condensation of two segments, (73–108) and (109–153). The arrows indicate the ligation sites. Elution conditions: column, Bio SEC-3 (3 µm, 100 Å, 2.1 × 250 mm, Agilent) at the flow rate of 80 µL/min; eluent, 100 mM ammonium acetate containing 5% CH₃CN (pH 7.0). The elution profile in (c) was taken and revised from Ref. 75) supporting information Fig. S10 with permission.

5.2. Synthesis of histone H2A with O-GlcNAc using a one-pot NCL strategy.

We also developed one-pot four-segment ligation by the NCL method based on the high reactivity of peptide aryl thioesters and two orthogonally activatable thioester precursors: peptidyl CPE and peptidyl NAC. First, the peptide aryl thioester 94 and the peptidyl CPE 95 underwent ligation (Fig. 18). Under these conditions, the aryl thioester reacted selectively with the cysteinyl peptidyl CPE to give intermediate 96. Then, cysteinyl peptidyl NAC 97 was added to the reaction mixture, and the pH was adjusted to 7.8. At this pH, the CPE converted to the thioester and underwent the ligation reaction while keeping the NAC moiety intact. Lastly, the C-terminal cysteinyl peptide 99 was added, and the pH was adjusted to 5.2. The NAC moiety was activated, and the ligation with the C-terminal peptide proceeded to obtain histone H2A with O-GlcNAc, having the desired mass number (ESI mass found: m/z 835.9, 789.4, 748.1, 710.7, 676.9, 646.2, 618.2, 592.5, calcd. for [M + 17H]¹⁷⁺: 835.9, [M + 18H]¹⁸⁺: 789.5, [M + 19H]¹⁹⁺: 748.0, [M + 20H]²⁰⁺: 710.7, [M + 21H]²¹⁺: 676.9, [M + 22H]²²⁺: 646.2, [M + 23H]²³⁺: 618.1, [M + 24H]²⁴⁺: 592.4) (101).⁷⁶⁾

Fig. 18.

Synthesis of histone H2A using a new one-pot ligation method based on the NCL method. (a) Structure of histone H2A. (b) Synthetic procedure. (c) RP-HPLC profile of crude mixture of the final desulfurization reaction. $\fbox{S}$ and the arrows indicate the O-glycosylation and ligation sites, respectively. RP-HPLC condition: column YMC-Protein RP (4.6 × 150 mm) at the flow rate of 1 mL/min; eluent, A, 0.1% aqueous TFA, B, 0.1% TFA in acetonitrile.

This one-pot procedure eliminates the time-consuming and low-recovery purification process, facilitating protein synthesis. It also allows for the efficient functional elucidation of proteins and their PTMs.

6. Conclusions

Over the past 40 years, methods for chemically synthesizing proteins, as well as the importance of the synthesis, have undergone significant changes. The authors have continued their research aimed at developing synthesis methods that leverage the advantages of chemical synthesis to produce various modified proteins, including those with PTMs. Peptide thioesters, designed as building blocks for protein synthesis, have proven to be excellent synthetic intermediates and are widely used in the synthesis of various polypeptides. Furthermore, we have successfully developed a method to synthesize peptide thioesters or their precursors with modified groups using the Fmoc SPPS method. As a result, it has become possible to synthesize a variety of modified proteins. In addition, we have developed a method to synthesize target proteins by sequentially adding synthesis blocks to the reaction solution for condensation. This method does not require the separation of condensation reaction products. Instead, it employs a set of peptide thioesters or their precursors with different condensation reaction conditions. Methods of chemical protein synthesis continue to evolve. We strongly anticipate that these proteins will contribute to advancements in the life sciences.

Conflict of interest

The authors declare no conflicts of interest.

Acknowledgements

The authors would like to express their sincere gratitude to Professor Keisuke Suzuki, Professor Emeritus at the Tokyo Institute of Technology and Member of the Japan Academy, for the opportunity to write this review. They would also like to thank Dr. Shunpei Sakakibara for his invaluable guidance in their peptide chemistry research. Finally, they would like to thank Professor Yoshiaki Nakahara of Tokai University, Professor Kenichi Akaji, president of Kyoto Pharmaceutical University, and all current and former collaborators and students for their support and contributions to the study of protein synthesis chemistry. This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from MEXT (Ministry of Education, Sports, Science, and Technology of Japan) and JSPS (Japan Society for the Promotion of Science) [Grant Nos. JP62540407, JP63540425, JP01740322, JP02263101, JP04254101, JP03259103, JP06276102 [to S.A.], JP14380287, JP15083204, JP18310145 [to S.A. and T.K.], JP15750143, JP17750158, JP21550155, JP24550187, JP15K05565, JP18K05316 [to T.K.], JP20380069, JP15K21124, JP16H04180, JP23380065 [to H.H.]], research grants from Hoansha Foundation [to T.K.] and the Naito Foundation [to T.K.].

Supplementary materials

Supplementary materials are available at https://doi.org/10.2183/pjab.101.034.

Notes

Edited by Shigekazu NAGATA, M.J.A.

Correspondence should be addressed to: S. Aimoto, Institute for Protein Research, The University of Osaka, Yamadaoka 3-2, Suita, Osaka 565-0871, Japan (e-mail: [e-mail]).

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Non-standard abbreviation list

Acm

acetamidomethyl

Boc

tert-butoxycarbonyl

BPTI

bovine pancreatic trypsin inhibitor

cHex

cyclohexyl

CPE

cysteinylproline ester

DCM

dichloromethane

DIEA

N,N-diisopropylethylamine

DKP

diketopiperazine

DMF

N,N-dimethylformamide

Dmmb

4,5-dimethoxy-2-mercaptobenzyl

DMSO

dimethyl sulfoxide

DTT

dithiothreitol

Fmoc

9-fluorenylmethoxycarbonyl

GlcNAc

N-acetylglucosamine

HF

anhydrous hydrogen fluoride

HOBt

1-hydroxybenzotriazole

HONp

p-nitrophenol

HOObt

3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

HOSu

N-hydroxysuccinimide

iNoc

4-pyridylmethoxycarbonyl

MBHA

4-methylbenzhydrylamine

MESNa

sodium 2-mercaptoethanesulfonate

MPA

3-mercaptopropionic acid

MPAA

4-mercaptophenylacetic acid

NAC

N-alkyl cysteine

NCL

native chemical ligation

Nle

norleucine

NMM

4-methylmorpholine

NMR

nuclear magnetic resonance spectroscopy

Npys

(3-nitro-2-pyridyl)sulfenyl

p-HTP

p-hydroxythiophenol

PTM

post-translational modification

RP-HPLC

reversed-phase high-performance liquid chromatography

SOD

superoxide dismutase

SPPS

solid-phase peptide synthesis

TCEP

tris(2-carboxyethyl)phosphine

TFA

trifluoroacetic acid

TFMSA

trifluoromethanesulfonic acid

Tle

tert-leucine

Tris

(hydroxymethyl)aminomethane

Troc

2,2,2-trichloroethoxycarbonyl

Tos

tosyl

Trt

trityl

Ub

ubiquitin

VA-044

2,2′-azobis[2-(2-imidazoline-2-yl)propane]·dihydrochloride

WSCI·HCl

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

Z

benzyloxycarbonyl

Profile

Saburo Aimoto was born in Yamaguchi Prefecture, Japan, in 1947, and graduated from the Faculty of Science, Osaka University (now The University of Osaka) in 1970. He received his Doctor of Science in 1977 from Osaka University under the supervision of Professor Yoshiharu Izumi. He joined Associate Professor Yasutsugu Shimonishi’s lab at the Institute for Protein Research, Osaka University, as an assistant professor (1972–1987). During this time, he worked as a postdoctoral fellow in the laboratories of Professor Gopinath Kartha at the Roswell Park Memorial Institute (1978–1979) and Professor Frederic M. Richards at the Department of Molecular Biophysics and Biochemistry, Yale University (1979–1980). Returning to the Institute for Protein Research, he was appointed as an associate professor (1987–1993) and then professor (1994–2011). He majored in organic chemistry, with a particular focus on synthetic protein chemistry. He has made significant contributions to initiating current innovations in protein synthesis chemistry. He was appointed Director of the Institute for Protein Research from 2008 to 2010 and served as an Executive Vice President of Osaka University from 2011 to 2015. He moved to the Protein Research Foundation in 2015 and became President from 2016 to 2021. He received the Japanese Peptide Society Award in 2007, the Xiaoyu Hu Memorial Award in 2010 from the Chinese International Peptide Symposium, and the Chemical Society of Japan Award 2010.

Toru Kawakami was born in Okayama Prefecture, Japan, in 1969. He graduated from the Faculty of Engineering Science at Osaka University (now The University of Osaka) in 1991, going on to obtain his Ph.D. in Chemistry from the university’s Graduate School of Engineering Science in 1995 under the supervision of Professor Shun-Ichi Murahashi. The title of his doctoral thesis was “Studies on the asymmetric synthesis of β-amino acids and related compounds”. From 1995 to 2003, he worked as an assistant professor in Professor Aimoto’s group at the Institute for Protein Research at Osaka University. He then worked as an associate professor until 2022. During this period, he spent a year (2001–2002) as a research associate at the University of California, San Francisco, collaborating with Professors James A. Wells and Robert M. Stroud on the project “Stabilization of proteins by covalently tethering small molecules”. In 2005, he received the Young Investigator Award from the Japanese Peptide Society for his work titled “Polypeptide synthesis based on ligation chemistry: Development of auxiliaries for peptide ligation”. He left the Institute for Protein Research at Osaka University in 2022. His research focuses on the chemical synthesis of proteins, particularly the development of ligation auxiliaries and peptide thioester synthesis based on the N–S acyl shift reaction. He also studied the chemical synthesis of histones with post-translational modifications. He currently works at Ooda Kasei Co., Ltd., and serves as a visiting associate professor at the Institute for Protein Research.

Hironobu Hojo was born in Nagano Prefecture, Japan, in 1963. He graduated from the Faculty of Science, Osaka University (now The University of Osaka), in 1985. He obtained his Ph.D. in Chemistry from Osaka University in 1994 under the guidance of Professor Saburo Aimoto. In 1994, he moved to Osaka City University as a lecturer and engaged in the development of efficient biomaterials with Professor Masayoshi Kinoshita and Dr. Kiyoshi Yamauchi. In 1998, he moved to Tokai University and started to develop a facile method for glycoprotein synthesis in collaboration with Professor Yoshiaki Nakahara. He moved to his present position, Professor of the Institute for Protein Research, Osaka University, in 2013 and is developing a chemical approach toward the understanding of the function of post-translationally modified proteins. He received the Young Investigator Award from the Japanese Peptide Society in 2000 for his work titled “Development of a method for polypeptide synthesis using peptide thioesters”.