Polypeptide Preparation by β-Lactone-Mediated Chemical Ligation

Native chemical ligation (NCL) represents a cornerstone strategy in accessing synthetic peptides and proteins, remaining one of the most efficacious methodologies in this domain. The fundamental requisites for achieving a proficient NCL reaction involve chemoselective coupling between a C-terminal thioester peptide and a thiol-bearing N-terminal peptide. However, achieving coupling at sterically congested residues remains challenging. In addition, while most NCLs proceed without epimerization, β-branched (e.g., Ile, Thr, Val) and Pro-derived C-terminal thioesters react slowly and can be susceptible to significant epimerization and hydrolysis. Herein, we report an epimerization-free NCL reaction via β-lactone-mediated native chemical ligation which constructs sterically congested Thr residues. The constrained ring from the β-lactone allows rapid peptide ligation without detectable epimerization. The method has a broad side-chain tolerance and was applied to the preparation of cyclic peptides and polypeptidyl thioester, which could be difficult to obtained otherwise.


■ INTRODUCTION
−7 NCL not only aids in native protein assembly but also efficiently enables the creation of peptide and protein analogues containing noncanonical residues. 8The cornerstone of successful NCL lies in the incorporation of a C-terminal thioester as an acyl donor, which enables chemoselective transthioesterification between two peptidyl fragments.One significant challenge encountered during peptide transthioesterification is the linking of amino acid residues with steric hindrance.Amino acid residues featuring bulky side chains at the reacting C-terminal thioester demand extended reaction periods, often accompanied by low yields and epimerization, thereby constraining the efficiency and outcomes of the ligations.The thioesters derived from Pro, Val, Ile, Leu, and Thr are particularly difficult to ligate. 9−11 For example, it was reported that the ligation between a Thr thioester and Cys residue requires more than 48 h to complete 12 (Figure 1a).In addition, Ser residues have been reported to incur substantial epimerization. 13In search of more capable peptidyl C-terminal preactivation, more effective thioesters to assist NCL have been extensively investigated. 9,14,15Among all proteinogenic amino acid residues, the occurrences of Thr-Cys and Thr-Ala connections in protein peptide bonds are 5.57% and 6.02%, respectively. 16erefore, developing a new methodology to enable Thr-Xaa junctions is crucial.We present a distinct C-terminal activator derived from Thr residues: a strained β-lactone moiety.The efficiency and practicality of β-lactone-mediated ligations were demonstrated as a one-pot ligation method at the Thr site and significantly accelerated the NCL reaction time without epimerization (Figure 1b).

■ RESULTS AND DISCUSSION
In a previous study, we illustrated the employment of constrained moieties in the form of β-thiolactones as thioester surrogates.This approach effectively addresses the ligation hurdle encountered at the C-terminal valine residue, facilitating the swift connection of two peptidyl fragments through onepot ligation and subsequent desulfurization (Val-Xxx).Releasing ring constraints not only markedly accelerates ligation rates but also permits the linking of sterically hindered amino acid residues at ligation sites.However, converting residues such as Thr to β-thiolactone scaffolds is not viable without eroding the Thr side chain stereochemistry.−19 Investigation began with the synthesis of polypeptides bearing a Thr-derived β-lactone from commercially available amino acid (Scheme 1). 20β-Lactone TFA salt 2 could be produced after two steps in high yield (67%) from Boc-L-threonine 1. Installation of C-terminal activated polypeptide 3 was accomplished by coupling 2 with Boc-Pro-Ala-Val-OH under EDC/HOOBt conditions, 21 then subsequent global deprotection followed by HPLC purification furnished peptide 3 in decent yield over two steps (39%).
The initial assessment of chemical ligation between peptide 3 and cysteine-bearing peptide 4 was conducted in PBS buffer (Table 1).At pH = 7.8, only hydrolysis of β-lactone can be found (6).We suspected that reducing the solution pH might render ligated product formation.Unfortunately, decreasing the pH to 6.8 furnished hydrolysis product 6 exclusively.The attempt to use a water-soluble organic base (Et 3 N, pH 8, entry 3) as the reaction medium failed to generate any product.It is evident that the aqueous solution outcompetes the desired thiolysis, rapidly opening the β-lactone and yielding Thr at the C-terminus.Based on literature, cysteine thiolate is 10,000 times more nucleophilic than other amino acid residues in basic buffer. 22,23We speculate that at elevated pH levels, thiolate may rival solvolysis.Subsequently, increase reaction pH to 10 (Et 3 N, entry 4) furnished the desired ligation, and product 5 was generated in 82% yield, whereas byproduct 6 was observed in less than 5%.Although Et 3 N is commonly regarded as non-nucleophilic, we conducted experiments to eliminate the possibility of β-lactone ring opening by Et 3 N before transthioesterification. Ligations conducted under basic conditions using NaOH yielded similar results (entry 5), while attempts with DABCO (entry 6) and imidazole (entry 7) 24 did not yield the desired product at the same pH.We suspected that the (albeit marginal) increased nucleophilicity of DABCO and imidazole might impede transthioesterification through nucleophilic ring opening of the Thr β-lactone.However, no ring-opening products derived from DABCO or imidazole were detected during the reaction.Ultimately, we observed that increasing the temperature did not enhance ligation yield (entry 8) and reducing the pH to 8 in the presence of Et 3 N failed to yield the desired product (entry 3).Based on the observed data, at a higher pH level (∼10), the formation of cysteine thiolate (SH → S − ) facilitates the opening of the βlactone ring, thus promoting ligation.However, the success of this ligation is not solely dependent on the reaction pH.The presence of nucleophilic additives in solution can disrupt the desired ligation process and result in hydrolysis.
With optimized reaction conditions in hand (Et 3 N, pH = 8 in water), the reaction scope was explored (Table 2).The βlactone-incorporated peptides were synthesized according to the method outlined in Scheme 1, and C-terminal epimerization was not detected during the preparation of polypeptide 7. The coupling of peptide 3 with cysteine-bearing peptides 9 and 11 resulted in the production of polypeptides 10 and 12 in excellent yields (89% and 93%, respectively).Subsequently, our methods facilitated the successful coupling of more intricate polypeptide sequences, resulting in ligated products with consistently high yields (entries 3−5).Furthermore, we observed that the 4-mercapto-threonine-bearing peptide 20 successfully enabled ligation at Thr-Thr when reacted with βlactone peptide 3, followed by metal-free desulfurization, yielding a good overall yield of 58% after two steps (entry 6).An identical protocol was employed for the one-pot synthesis  Reaction conditions: 0.1 eq of 5 mixed with 1 eq of 6 in different solutions to make the concentration 10 mM at 25 °C in 1 h.b Same ligation parameters at 35 °C in 0.5 h. of the Thr-Leu linkage between octapeptides 22 and 24, resulting in a yield of 38% over two steps.The fair reaction yield was due to the low-efficiency desulfurization step, which hampered the overall outcome.In general, reactive residues, such as Lys, Asp, Thr, His, Arg, Tyr, Met, and Ser, do not interfere with the ligation process.Beyond conventional NCL, β-lactone-mediated ligation offers a method for connecting noncanonical peptides.For instance, the ligation between a βlactone-bearing peptide 26 and a non-natural peptide 27 (with 2-mercaptoacetic acid at the N-terminus) successfully produced thioester 29-mer 28 in good yield (entry 9).These findings highlight the potential utility of the C-terminal βlactone in peptide chemical synthesis.Although serine-derived β-lactone was initially considered as a candidate for C-terminal activation, it proved to be too labile for ligation.The compound decomposed during the EDC coupling step and could not be used in polypeptide synthesis.Thiolysis exclusively took place between the carbonyl of β-lactone (C1) and the cysteine thiol group.No ring opening at C3 was observed, 25 likely due to the steric hindrance of the methyl group.
The activation of the C-terminus may induce epimerization at the α-center.We conducted an evaluation of potential epimerization at the Thr ligation site, considering the constraints and reactivity associated with epimerization occurred during the β-lactone-mediated ligation.This may be attributable to the near-flat geometry of the βlactone moiety, which is unlikely to permit enolization and subsequent stereocenter erosion. 26o confirm the chemoselectivity of the C-terminal β-lactone, a competition experiment was devised.Alongside the desired intermolecular thiolysis, competing hydrolysis 15 and aminolysis 27 processes could potentially take place.Tetrapeptides Ala-Val-Ala-Pro 31 and Ser-Val-Ala-Pro 33 (analogue of 4) were obtained.The experiment was carried out by mixing β-lactone peptide 3 with three different scaffolds bearing N-terminal Ala (31), Ser (33), and Cys (4) under standard conditions, which may generate three distinct ligated peptides 32, 34, and 5 (Scheme 2).After 10 min in the aqueous solution (Et 3 N, pH = 10), the LCMS trace of the crude reaction suggested the formation of exclusive Cys ligation product 5 at 12.5 min, which suggested the chemoselectivity between Thr β-lactone and Cys residues.No intermolecular hydrolysis or aminolysis products were detected, and the only additional substrate observed in LC was a small fraction of the β-lactone hydrolysis product 6.
Utilizing the developed methodology, we showcased the applicability of β-lactone-mediated peptide ligation by preparing cyclic peptides (Scheme 3).Linear peptides 35 and 37 were obtained, and intramolecular ring closure of 34 and 36 was achieved under optimized conditions.Within 2 h, the corresponding cyclic peptides 36 and 38 were obtained in good yields (72% and 63% respectively).

■ CONCLUSION
In summary, we introduced a novel method for C-terminal activation in peptide ligation.The β-lactone ring strain release overcomes residue steric hurdles and enables shorter ligation times (30 min to 6 h).The thiolysis of β-lactone is completely chemoselective to generate peptidyl bonds.This protocol allows Thr-Xaa ligation sites after one-pot ligation and desulfurization with broad residue tolerance.β-Lactonemediated ligation can be utilized for both intermolecular and intramolecular peptide ligations, allowing for the rapid synthesis of elongated polypeptides and cyclic peptides.Lastly, β-lactone chemistry offers a solution for coupling at sterically congested Thr residues without epimerization and facilitates the efficient introduction of noncanonical residues into the peptidyl backbone, such as midsequence peptide thioesters.

■ ASSOCIATED CONTENT Data Availability Statement
The data underlying this study are available in the published article and its Supporting Information.
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Figure 2 . 1 H
Figure 2. 1 H NMR spectra of ligation product L-and D-isomers.