2025 Volume 73 Issue 9 Pages 787-792
Conventional peptide synthesis involves multiple protection and deprotection steps, and typically relies on stoichiometric amounts of coupling reagents and additives. This makes the process cumbersome, and results in poor atom economy and hazardous waste generation. Therefore, direct peptide bond formation using unprotected amino acids is a promising alternative. However, this approach presents some challenges: 1) Solubility of unprotected amino acids in organic solvents; 2) Control of undesired side reactions; 3) Chemo-selective activation of the carboxylic acid group in the presence of an amine functionality; and 4) Epimerization. To address these challenges, we developed tris(2,2,2-trifluoroethoxy)silane [H-Si(OCH2CF3)3], a cost-effective and accessible coupling reagent. This single reagent efficiently synthesizes N-terminal free peptides from unprotected amino acids and amino acid tert-butyl esters, without the need for any additives. H-Si(OCH2CF3)3 enhances amino acid solubility through coordinating with both termini and plays a dual role, serving as a transient amine-protecting group and as a carboxylic acid activating or promoting reagent for peptide bond formation. This method is operationally simple and versatile, enabling the efficient synthesis of N-terminal free peptides from unprotected amino acids and amino acid tert-butyl esters, with good yields, high optical purity, and broad side-chain compatibility.
Peptides are low-molecular-weight compounds composed of fewer than 50 amino acids linked by peptide bonds, and they play a vital role in numerous physicochemical and biological processes.1–3) The chemical synthesis of peptides from amino acids dates back to the 19th century, and has continued to draw immense attention.4) Currently, synthetic peptides are integral to the treatment of cancer,5,6) diabetes,7) and cardiovascular diseases.8) Given their extensive therapeutic relevance, there is a growing demand for new, cost-effective laboratory methods capable of producing biologically active peptides in large quantities.
Conventionally, peptide synthesis was accomplished involving three steps: protection of the amino group, peptide bond formation using a stoichiometric amount of “coupling” reagents, and deprotection of the protecting group.9–11) In general, traditional peptide synthesis relies on protecting groups, such as 9-fluorenylmethyloxycarbonyl (Fmoc), tert-butoxycarbonyl (Boc), and benzyloxycarbonyl (Cbz), and often involves reagents with molecular weights exceeding those of most amino acids.12,13) This leads to poor atom economy and the generation of toxic waste and hazardous materials on a large scale. The process is time-consuming, requiring vast amounts of solvent for purification and separation12,13) (Chart 1a). Consequently, peptide synthesis using unprotected amino acids presents a superior alternative. This approach promises improved atom economy, fewer steps, reduced waste, and minimizes the production costs.14,15) This process is very rare and suffers from the following issues: 1) solubility of amino acids in organic solvents, 2) uncontrolled reactivity leading to dimerization or polymerization, 3) activation of acid functional group in the presence of amine functionality, and 4) epimerization or racemization12–15) (Chart 1).
The first N-terminal free peptide synthesis, with unprotected amino acids, was achieved by Leuchs using phosgene derivatives.16) Later, Burger and colleagues. employed hexafluroacetone,17) while Liskamp and colleagues. used dichlorosilane18) or boran trifluoride,19) (Chart 1b). More recently, Sheppard identified borate ester as an effective amidation reagent.20,21) In all these cases, the reaction proceeds via a cyclic intermediate16–21) (Chart 1b). In 2021, we developed an N-terminal peptide elongation method for unprotected amino acids, catalyzed by CsF and imidazole, and mediated by H-Si[OCH(CF3)3]3 and N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA)22) (Chart 1c). This method requires expensive two silicon reagents along with catalysts; these two silicon-based reagents serve distinct roles: H-Si[OCH(CF3)3]3 acts as a coupling reagent for peptide bond formation, whereas MTBSTFA serves as a transient protecting group for the amine group of unprotected amino acid. More recently, we established three additional approaches for peptide synthesis using unprotected amino acids. One approach employs AlMe323) another uses a SiMe2Im224) coupling reagent with Ta(OEt)5 as a catalyst (Chart 1c), and the third utilizes TMSIm and Ta(OEt)5 as catalysts.25)
In the previously reported methods, peptide synthesis was typically performed using two distinct and expensive silicon reagents, metal catalysts, or pyrophoric trimethylaluminum. As part of our ongoing efforts to develop novel methodologies for peptide synthesis employing unprotected amino acids, we aim to identify a single, cost-effective reagent capable of functioning as both a transient protecting group for the amine moiety and coupling agent. We propose that H-Si(OCH2CF3)3 is a promising candidate for N-terminal peptide elongation with unprotected amino acids, serving dual roles as a coupling reagent for peptide bond formation and as a transient amine-protecting group (Chart 1d). Significantly, H-Si(OCH2CF3)3 is readily prepared from inexpensive trichlorosilane and 2,2,2-trifluoroethanol, further enhancing its practical utility. This new methodology also greatly expands the strategies organic chemists can utilize for peptide bond formation from unprotected amino acids.
We performed a model reaction using l-phenylalanine 1a (1 equivalent (equiv.)) and l-alanine tert-butyl ester 2a (2 equiv.), and tris(2,2,2-trifluoroethoxy)silane [H-Si(OCH2CF3)3] (3 equiv.) in chloroform at 80°C. The reaction smoothly yielded the desired dipeptide, H-l-Phe-l-Ala-Ot-Bu (3a), in 84% yield with > 20:1 diastereoselectivity (Chart 2, entry 1). Inquisitive about this result, we planned to detect the plausible intermediates proposed in Chart 1d by treating l-phenylalanine with H-Si(OCH2-CF3)3 in chloroform; unfortunately, we were unable to find them either by 1H-NMR or mass analysis. This is possibly because the proposed intermediates might be sensitive to air, reactive, and easily decomposed. Conversely, using the previously developed tris((1,1,1,3,3,3-hexafluoropropan-2-yl)oxy)silane (H-Si[OCH(CF3)2]3) instead of H-Si(OCH2CF3)3 significantly decreased the desired product’s yield (entry 2). In this case, decrease in the yield of product was observed because the bulkier H-Si[OCH(CF3)3]3 may not be able to protect with amine of the unprotected amino acid as compared with the less bulkier H-Si(OCH2CF3)3. As a result, this possibly leads to polymerization by self-coupling of the amino acid under these reaction conditions. Changing the solvent to CPME or MeCN instead of chloroform was ineffective (entries 3 and 4). Altering the amount of H-Si(OCH2CF3)3 to either 2 or 4 equiv. (instead of 3 equiv.) negatively affected the reaction outcome (entries 5 and 6). Although the addition of Lewis acids such as B(C6F5)3, Ta(OEt)5, and Si(OMe)4 alongside H-Si(OCH2CF3)3 showed some activity, the yields obtained were lower than those under the standard conditions (entries 7–9). Additionally, lowering the reaction temperature or using microwave irradiation instead of conventional heating led to reduced efficiency (entries 9–12).
With optimized reaction conditions, we investigated the method’s scope and generality by reacting various unprotected amino acid electrophiles with l-alanine tert-butyl ester to synthesize N-unprotected dipeptides (Chart 3). Unprotected amino acids with alkyl side chains, such as H-l-Phe-OH, H-l-Ala-OH, and H-l-Leu-OH, provided the desired dipeptides in satisfactory yields and excellent diastereoselectivities (3a–3c). Importantly, the protocol proved compatible with functionally diverse amino acids, including those bearing thioether (H-l-Cys(Trt)-OH, H-l-Met-OH), ether (H-l-Ser(t-Bu)-OH, H-l-Thr(Bzl)-OH), heterocycle (H-l-Trp(Boc)-OH), ester (H-l-Glu(Ot-Bu)-OH), and amide (H-l-Asn(Trt)-OH) side chains. This yielded the corresponding dipeptides (3d–3j) in moderate to good yields while maintaining high optical purity. The reaction is sluggish with hydroxyl group unprotected H-l-Thr-OH and yields the desired product (3g′), in 24% yield. Hydroxyl unprotected H-l-Ser-OH completely failed to deliver the desired product. These results show that the amino acids with polar functionalities in their side chain need to be protected for the success of the reaction. Notably, unprotected amino acids with aromatic rings bearing electron-donating and electron-withdrawing groups (H-l-Tyr(t-Bu)-OH, H-l-Phe(4-F)-OH, H-l-Phe(4-CF3)-OH, H-l-Phe(4-Cl)-OH, and H-l-Phe(4-I)-OH) did not affect the reaction outcome. These reactions consistently yielded the desired products (3k–3o). Unnatural amino acids (H-l-Cha-OH, H-l-Ala(1-Nap)-OH, H-l-Ala(2-Nap)-OH) participated in the reaction, affording the corresponding products (3p–3r) in moderate yields with good optical purity. The optimized reaction conditions were also applied to the synthesis of dipeptides between amino acids with active functional groups (H-l-Asp(Ot-Bu)-OH) and an amino acid ester with a bulky functional group (H-l-Leu-Ot-Bu) in 91% yield and >20:1 dr.
We next explored the scope of amino acid tert-butyl esters as nucleophiles. A wide range of amino acid tert-butyl esters participated in the reacted with unprotected amino acid electrophiles to yield N-unprotected dipeptides (Chart 4). Esters with bulky alkyl and heterocyclic side chains gave satisfactory yields and high optical purities (3t–3w). Esters bearing alkyl, ether, thioether, ester, amine, or heterocyclic groups also reacted effectively with H-l-Cys(Bzl)–OH, affording dipeptides (3x–3ae) in good yield and optical purity. Interestingly, this method accommodated racemization-prone H-l-Phg-Ot-Bu and self-dimerization-prone H-l-Ala-Oi-Pr, affording the corresponding dipeptides (3af–3ag) in good yield with excellent optical purity. It is noteworthy to state that in both the scope and generality of electrophiles and nucleophiles, in the majority of cases, the dipeptides were isolated in more than 75% yields, although at a slight decrease of yields because of the lower solubility of unprotected amino acids in organic solvents, and partly self-condensation of amino acids.
We introduced a cost-effective, tris(2,2,2-trifluoroethoxy)silane as an efficient and single reagent for synthesizing N-terminal free peptides from unprotected amino acids and their tert-butyl esters and isopropyl esters. This method accommodates a broad substrate scope, by including both diverse side chains (alkyl, ether, thioether, ester, amine, amide, and heterocycles), yielding dipeptides in good yield and high selectivity. This single-reagent approach significantly simplifies peptide synthesis and expands strategies for direct dipeptide construction from unprotected amino acids.
Into a flame-dried 5 mL screw-cap vial, we charged unprotected amino acid 1 (0.5 mmol, 1 equiv.), dry chloroform (1.5 mL, 0.33 M), and tris(2,2,2-trifluoroethoxy)silane (0.490 g, 1.5 mmol, 3 equiv.) inside a glovebox. The sealed vial was stirred for 5 min. Subsequently, amino acid tert-butyl ester 2 (1.0 mmol, 2 equiv.) was added via syringe, maintaining the glovebox conditions. The vial was then sealed under argon, removed from the glovebox, and the mixture was stirred at 80°C in a preheated oil bath for 22 h. Upon completion, the reaction was diluted with chloroform (1.5 mL), and approximately 0.3 g of silica gel (SiO2) was added. The mixture was vigorously stirred at room temperature for 5 min, then filtered through a G4 sintered funnel with a celite pad. The celite pad was thoroughly washed with chloroform (150 mL). The volatiles were removed under reduced pressure, and the crude mixture was transferred onto a silica gel column using a pipette. Flash column chromatography with methanol in chloroform afforded the desired product 3.
This work was partially supported by Grant-in-Aid for Specially Promoted Research (23H05407) from the Japan Society for the Promotion of Science (JSPS). We would like to acknowledge Ms. Masae Kawase for assistance with the measurement of IR and optical rotation data of the compounds.
The authors declare no conflict of interest.
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