English

• Chemical protein synthesis enables producing proteins (e.g., post-translationally modified proteins, mirror-image proteins) that are difficult-to-obtain or even inaccessible by conventional recombinant means for basic biological, biomedical and synthetic biological studies [1-5]. In general, chemical synthesis of proteins relies on solid-phase peptide synthesis (SPPS) to prepare peptide segments and peptide ligation to join the peptides together [6-8]. However, chemical protein synthesis often suffers from unexpected challenges in the peptide synthesis and handling (e.g., purification, and ligation) due to the aggregation-prone and poorly soluble peptide segments of a protein [9].

  One solution to address these challenges is to introduce chemical modification groups onto peptides. For example, commercially available Dmb(Gly) and pseudoprolines derived from threonine/serine have been developed to facile the solid phase peptide synthesis due to their ability to disrupt secondary structure formation [10-12]. Meanwhile, semi-permanent solubilizing groups have also been installed into peptides to improve the handling properties (purification and ligation) of peptides [13-18]. Furthermore, another promising type of methods has been developed by installing the backbone semi-permanent groups to facilitate both peptide synthesis and ligation. For example, the depsipeptide strategy involves esterification at serine (Ser) or threonine (Thr) to form the corresponding depsipeptide which can disrupt aggregation during peptide synthesis, purification and ligation [19-22]. Meanwhile, the Hmb strategy incorporates the acid-labile 2-hydroxy-4-methoxybenzyl (Hmb) group into the backbone amide bond of peptide to beneficially disrupt peptide secondary structures, thereby minimizing peptide aggregation tendency and improving the coupling during solid phase peptide synthesis [23, 24]. Moreover, the Hmb strategy also takes an advantage in that the modified Hmb groups can be rendered temporarily acid-stable upon acylation, and the retained Hmb groups can increase peptide solubility and facilitate peptide purification and ligation during chemical protein synthesis. The Hmb strategy has been applied in the chemical synthesis of a variety of proteins such as interleukin-2, HMGA1a protein, oncogenic protein KRas and DNA ligase from Haemophilus influenzae [17, 25-28]. Nonetheless, the Hmb group is usually installed at Gly due to the poor coupling efficiency of the amino acid following the Hmb residue caused by an extra steric hindrance effect of the Hmb group at secondary amine.

  Recently, we and others developed a removable backbone modification strategy, where commercial 2-hydroxy-4-methoxy-5-nitrobenzaldehyde was used to install a backbone modified 2-hydroxy-4-methoxy-5-aminobenzyl (Hmab) group at any primary amino acid via an activated O-to-N acyl transfer [29-31]. However, the Hmab group and its acyl variants were more stable than Hmb to trifluoroacetic acid (TFA) acidolysis and the removal of Hmab requires the longer cleavage time or more acidic mixture which may cause peptide side products sometimes [31, 32]. In one of our model tests, it took 11 h to remove the Hmab group under cleavage conditions (TFA/H₂O/TIPS = 95/2.5/2.5, v/v/v), whereas the Hmb can be completely removed within 2 h (Figs. S1 and S2 in Supporting information). These issues motivated us to convert Hmab into Hmb to facilitate the final cleavage. Herein, we developed an improved installation of Hmb (iHmb) method which takes advantages of the flexible installation of Hmb at any primary amino acid and the convenient removal of Hmb (Scheme 1). The iHmb method facilitates the peptide synthesis and improves peptide purification and ligation.

  Scheme 1

  

  Scheme 1.  The improved installation of Hmb (iHmb) method.

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  Our study began with the synthesis of the resin-bound Hmab-modified model peptide 1 (GVKLL^Hmab-AcAQKFNG) by combining the 9-fluorenylmethyloxycarbonyl (Fmoc) based solid-phase peptide synthesis (SPPS) technique and our previously reported removable backbone modification strategy [33]. Analysis of a test cleavage by analytical reverse phase high performance liquid chromatography (RP-HPLC) and electrospray ionization mass spectrometry (ESI-MS) showed the target peptide 1 was obtained with high purity (Fig. S3 in Supporting information). Then, according to a previously reported work where the amino group in a substituted aniline can be replaced with hydrogen in a one-pot manner by using 5 equiv. sodium nitrite (NaNO₂) in a biphasic water/dichloromethane (DCM) solvent system (1:1) in the presence of 10% AcOH for 10 min at 4 ℃ [34], we tried the direct one-step on-resin deamination of Hmab to Hmb under the same conditions. The extension of the reaction was determined by RP-HPLC of a test cleavage and verified by ESI-MS. The result showed a vast majority of the diazonium intermediate (80%) with a small minority of the target Hmb-modified product 2 (GVKLL^Hmb-AcAQKFNG) (< 5%) (Table 1, entry 1). We reasoned that the electron-donating groups of Hmab can stabilize the diazotized intermediate to inhibit its decomposition.

  Table 1

  

  Table 1.  Oxidation of amino group of Hmab on peptide 1 into Hmdab.

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  We then tested the deamination of Hmab to Hmb by the sequential oxidative diazotization and reductive elimination. We tested other mild oxidants tert-butyl nitrite (t-BuNO₂) and isoamyl nitrite (IAN). The results were shown in Table 1 and Fig. S4 (Supporting information). The combination of tBuNO₂ (5 equiv.) with salicylic acid (1 equiv.) [35] gave less than half of diazotized products 3 (GVKLL^Hmdab-AcAQKFNG) (45%), with most of the unreacted reactants 1 (Table 1, entry 2). The increase of the amount of t-BuNO₂ and replacement of solvents can only increase the yield up to 75% (Table 1, entry 3). Fortunately, when IAN (10 equiv.) and HBF₄ (5 equiv.) [36] were used and the reaction performed in DMF at 37 ℃ for 15 min, the diazotized product 3 can be generated in 90% yield (Table 1, entry 4). Another optimized condition (5 equiv. IAN, 5 equiv. HBF₄, r.t., 15 min, DMF) can further increase the yield to 98% (Table 1, entry 5). The robustness of the optimized condition was further demonstrated by the quantitative conversion of a variety of Hmab-modified peptides into the corresponding Hmdab-modified peptides, including a diazotized peptide 4 (GVKDG^Hmdab-AcAQKFNG).

  Encouraged by the above results, we then tested reductive elimination of the Hmdab group on peptide 4. First, 10 mmol/L HCl and a reaction time of 30 min at 37 ℃ was used, but we found no target Hmb-Ac modified product 5 (GVKDG^Hmb-AcAQKFNG) (Table 2, entry 1). We tried HPO₂ and Cu₂O inorganic oxidants to convert Hmab- to Hmdab-modified peptides, but with low yields of 30%–40% (Table 2, entries 2 and 3, and Fig. S5 in Supporting information). When NaBH₄ (2–5 equiv.) was used as the source of hydrogen and the reaction performed at 37 ℃ for 15 min, the Hmb product 5 was only formed in a moderate yield (40%) due to the generation of byproducts with —NH₂, —NHNH₂, and —N═NH groups (Table 2, entry 4, and Figs. S6 and S7 in Supporting information). We then ran the reaction with a mild reducing agent NaBH₃CN, the yield increased to 60% (Table 2, entry 5). Increasing the reaction temperature from 37 ℃ to 60 ℃ significantly improved the yield (88%). It is noted that we observed the deprotection of acetyl groups of Hmb due to the alkalinity and reducibility of boron reagents. The released phenol group might stabilize the diazonium ion, making the decomposition of diazonium more difficult. Furthermore, other mild thiol reducing reagents such as ethane thiol (EtSH), 1,2-ethanedithiol (EDT), cysteine (Cys) and dithiothreitol (DTT) were employed instead to decompose the diazonium salts. The reaction with 50% EtSH in DMF at room temperature for 12 h can afford the target 5 in a 75% yield. An increase of the reaction temperature to 60 ℃ can give the target in an 85% yield within 1.5 h. The yield further increased to 92% when the oxidative diazotization with HBF₄ and reductive elimination with EtSH steps were repeated once (Table 2, entry 8). When EDT (30%, in DMF) was used as the hydrogen source (Table 2, entry 9), the Hmb-modified product 5 was generated in excellent yield (92%) at 70 ℃ for 1 h. Cys and DTT afforded the product 5 in low to moderate yields (Table 2, entries 10 and 11).

  Table 2

  

  Table 2.  On-resin decomposition of diazonium group.

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  We investigated the scope of the iHmb method for different amino acids using the optimized conditions (5 equiv. IAN, 5 equiv. HBF₄, r.t., 15 min, DMF) for oxidative diazotization and (30% EDT in DMF, 70 ℃, 1 h) for reductive elimination. We tested a series of peptides (6–16) where the Hmb was installed between various two amino acids (such as Asp-Gly, Asp-Glu, Asp-Asn, Leu-Leu, Val-Leu, Ile-Leu, Leu-Phe, Asp-Val, and Ala-Ile). As shown in Table 3, Fig. 1 and Figs. S8–S17 (Supporting information), most of the peptides can be readily prepared with good efficiency (purity of crude peptides = 83%–94%; isolated yields = 40%–55%). Even for highly sterically hindered sites such as Val-Leu and Ile-Leu, the Hmb group could be smoothly installed (Table 3, entries 6 and 7, crude peptide purities: 87% and 88%; isolated yields: 48% and 45%). Note that the aspartimide formation at Asp-Yaa motifs such as Asp-Gly was completely prevented by utilizing the iHmb method (Table 3, entries 1–4). Finally, we installed Hmb at the epimerization-prone Cys residue and prepared the GVKCLAQKFNG 15 and GVKcLAQKFNG 16 by the iHmb method. HPLC analysis of 16 showed that the epimerization was < 1% (Fig. S18 in Supporting information).

  Table 3

  

  Table 3.  Scope of the iHmb method using a model sequence H-Gly-Val-Lys-Xaa-Yaa^Hmb-Ala-Gln-Lys-Phe-Asn-Gly-NH₂.

  DownLoad: CSV

  Figure 1

  

  Figure 1.  The RP-HPLC analysis of crude peptides 6–14 (a–i), and 16–18 (j–l) synthesized by the iHmb method and their corresponding ESI-MS. ^a 17 was synthesized on Wang resin by the iHmb method; ^b 18 was synthesized on Trt hydrazine resin by the iHmb method.

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  In addition, we tested the compatibility of the iHmb method with other types of resin including Wang resin, Trt hydrazine resin and 2-Cl-Trt resin. The results showed that peptides 17 and 18 can be readily synthesized by the iHmb method on Wang resin and Trt hydrazine resin, respectively (Table 3, entries 12 and 13, Figs. S19 and S20 in Supporting information, crude peptide purities: 81% and 77%; isolated yields: 43% and 41%). However, the iHmb method was not compatible with the synthesis of C-terminal carboxyl peptides by using 2-Cl Trt resin due to the acidic lability of the resin towards the acidic conditions for the NO₂ reduction of RBM.

  We next tested the iHmb method for the synthesis of aspartimide-prone peptides which usually suffer from low yields and costly purification. For example, we attempted to synthesize the allyl-based cyclization method for lactam-bridged cyclic peptide 19 containing a macrolactam ring between the N-terminal Gly1 and Asp7 (Fig. 2a). The side-chain carboxyl group Allyl-protected Asp7 was used during microwave assisted Fmoc SPPS. However, the on-resin synthesis of lactam-bridged cyclic peptide 19 via the standard microwave assisted Fmoc SPPS gave substantial amounts (> 70%) of aspartamide formation owing to the aspartimide-prone Allyl-protected Asp-motif (Fig. S21 in Supporting information) [37]. Then, an Hmab group was installed at Val8. After finishing the peptide assembly by the standard Fmoc SPPS to give resin-bound peptide 19a, the Allyl group was removed by treatment with Pd(PPh₃)₄/PhSiH₃ to provide resin-bound peptide 19b. Intramolecular cyclization was then performed between the side-chain carboxyl group of Asp7 and the N-terminal amino group of the peptide chain by PyAOP/HOAt activation to afford peptide 19c. After that, the phenolic hydroxyl group was acetylated with acetic anhydride, followed by the treatment with 6 mol/L SnCl₂ to conversion the NO₂ group into an NH₂ group. Next, treatment with isoamyl nitrite (5 equiv.) and HBF₄ (5 equiv.) in DMF at room temperature for 15 min afforded expected diazonium salt 19d. Treating the diazonium salt with 30% EDT at 70 ℃ for 1 h gave a clearly deaminated Hmb-modified product 19e. Finally, the resin was treated with piperidine, and then TFA (TFA/H₂O/TIPS/EDT = 94/2.5/2.5/1) to afford the cyclized 19 in an isolated yield of 24%. The test cleavage of each step was analyzed by analytical RP-HPLC and verified by ESI-MS (Fig. 2b and Fig. S22 in Supporting information). It is noted that aspartimide formation was entirely avoided by using the iHmb method, resulting in a significant increase of synthetic yield. This iHmb method would also been used in the orthogonal solid-phase synthesis of N-glycopeptide to avoid aspartimide formation [38].

  Figure 2

  

  Figure 2.  Fmoc SPPS of cyclic peptide 19 by the iHmb method. (a) Synthetic route; (b) HPLC, ESI-MS and deconvoluted spectra of 19b, 19d, 19.

  DownLoad: Full-Size Img PowerPoint

  In addition, we challenged the iHmb method with the one-shot synthesis of the 76-residue ubiquitin (Ub) 20 (Fig. 3a). First, the full-length Ub with the Hmab(Ac) group at Glu34 was obtained by the microwave-assisted Fmoc SPPS. Then, the on-resin Ub-Hmab(Ac) peptide was successively treated with isoamyl nitrite/HBF₄ in DMF at room temperature for 15 min and 30% EDT at 70 ℃ for 1 h to convert the Hmab(Ac) group into the Hmb(Ac) group. After that, the deacetylation by piperidine and the cleavage by TFA cocktails were carried out to give the target Ub 20 in a 3% isolated yield which was purified by semi-preparation RP-HPLC and verified by ESI-MS (Fig. 3 and Fig. S23 in Supporting information).

  Figure 3

  

  Figure 3.  Fmoc SPPS of ubiquitin 20 by the iHmb method. (a) Synthetic route; (b) HPLC, ESI-MS and deconvoluted spectra of 20a, 20.

  DownLoad: Full-Size Img PowerPoint

  Next, we sought to demonstrate the potential of the iHmb method by synthesizing D-peptides composed of D-amino acids. D-Peptides have been considered attractive functional biomaterials and therapeutic agents due to their superior resistance to proteolysis over their natural L-peptide counterparts [1, 2]. We are interested in the mirror-image version of anchor peptides that bind to synthetic polymers such as polyethylene terephthalate (PET). Engineering the mirror-image version of anchor peptides with plastic degrading enzymes would enhance polymer absorption [39] and enzymatic degradation and therefore a robust synthesis of anchor D-peptides is of importance. However, our initial synthesis of anchor D-peptide 21 with conventional Fmoc SPPS gave no detectable product due to its hydrophobic nature. In addition, the popular pseudoproline dipeptides incorporation strategy for the synthesis of difficult L-peptides was largely limited because there have not yet been commercially available D-pseudoprolines. We then used the iHmb method to install the Hmab group at ^DAla20 (Fig. 4a). After that, the Hmab group was smoothly converted into the Hmb group to provide the desired product 21 as the major peak as shown in RP-HPLC of the crude peptide. The D-peptide was purified by HPLC with a 26% isolated yield, which was verified by ESI-MS (Fig. 4b and Fig. S24 in Supporting information). The above result showed that the iHmb method provides practical access to otherwise inaccessible mirror-image of difficult peptides.

  Figure 4

  

  Figure 4.  Chemical synthesis of D-peptide 21 by the iHmb method. (a) The synthetic route; (b) HPLC, ESI-MS and deconvoluted spectra of 21.

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  Finally, the iHmb method was applied to prepare a 31-residue single transmembrane protein rabbit sarcolipin (SLN). Our previous synthesis of S-palmitoylated SLN by the removable backbone modification method suffered from the incomplete removal of the backbone modified groups by TFA cocktails and a prolonged TFA treatment (5 h) caused substantial unwanted trifluoroacetyl peptides [40]. To improve the synthetic efficiency, we decided to use the iHmb method. SLN was divided into two segments: SLN(1–8)-MPAA (22), and SLN(9–31) (23) with an Hmb-modified group at SLN. Peptide 22 was readily prepared by Fmoc SPPS with a 30% isolated yield according to the previous protocol [41, 42]. The new iHmb method was used to synthesize peptide 23 (Fig. 5a). Thus, the resin-bounded Hmab(Ac)-modified peptide was treated with IAN/HBF₄ and EtSH conditions, respectively, to give the Hmb-modified peptide. After that, gaminobutyric acid (GABA) was used to temporarily protect the phenolic hydroxy group of Hmb, which can occur the autocyclization in neutral ligation conditions to active the Hmb group. Finally, a TFA cleavage step was performed to provide the crude peptide that was well soluble in CH₃CN aqueous. The RP-HPLC analysis of the crude Hmb-modified 23 showed main peak as the target. Peptide 23 was purified by semipreparative RP-HPLC using a water-acetonitrile mobile phase with a 21% isolated yield. Peptides 22 and 23 were determined by ESI-MS (Fig. 5b and Figs. S25 and S26 in Supporting information). Note that SLN(9–31) without Hmb modification could not be prepared owing to poor handling properties.

  Figure 5

  

  Figure 5.  Chemical synthesis of SLN 25 based on the iHmb method. (a) The synthetic route for the solid-phase peptide synthesis of 23 by the iHmb method; (b) HPLC and ESI-MS of 23; (c) Chemical synthesis for 25; (d) HPLC analysis of NCL; (e) ESI-MS and deconvoluted spectra of 24; (f) ESI-MS and deconvoluted spectra of 25.

  DownLoad: Full-Size Img PowerPoint

  Native chemical ligation of 22 (2 equiv.) and Hmb(GABA)-modified 23 (1.5 mmol/L, 1 equiv.) was carried out at 30 ℃ for 2 h. RP-HPLC analysis showed that the ligation proceeded smoothly accompanying with the autocyclization of GABA under the ligation conditions to afford the Hmb-modified ligation product 24 that was verified by ESI-MS (Figs. 5c–e). The ligation product was soluble and could be readily purified by RP-HPLC in 61% isolated yield. Finally, purified 24 was dissolved in TFA cocktails at room temperature for 0.5 h to remove Hmb group. Then, the TFA solution was concentrated by N₂ blowing and precipitated with chilled diethyl ether to give 25, which cannot be eluted by RP-HPLC (data not shown) but can be identified by ESI-MS (Fig. 5f). In comparison with our previous backbone modifications that removed slowly, Hmb was removed within 0.5 h at the same conditions and the undesired trifluoroacetylation side-reaction was largely avoided. Collectively, the results indicated that the iHmb method promotes the chemical synthesis of difficult proteins and difficult peptides.

  In summary, we developed a practical iHmb method to install the Hmb group into peptide at any primary amino acid via the sequential oxidative diazotization and reductive elimination on resin from the Hmab-containing peptide for the first time. In our study, the utility of commercially available 4-methoxy-5-nitrosalilaldehyde assisted peptide synthesis to allow the Hmab groups to be installed at any primary amino acids of peptides. More importantly, we found that the optimized conditions, that is IAN/HBF₄ for oxidation and EDT for reduction, can fully convert Hmab into Hmb which can be readily removed by standard TFA treatment to give the target peptides. Using the iHmb method, Hmb was installed into aspartimide-prone peptides to suppress aspartimide formation. Hmb was also used to prevent peptide aggregation and facilitate the one-shot synthesis of a long-chain peptide up to 76-residue and difficult peptides especially difficult D-peptides to avoid the use of commercially unavailable D-pseudoprolines. In addition, the use of Hmb for improving peptide purification and ligation was demonstrated with the synthesis of an insoluble membrane protein. In combination with the recent progress such as multi-segment condensation and glycosylation-assisted folding strategies for chemical protein synthesis, the iHmb method may find general applications for synthesis of more challenging peptides and proteins [43-48].

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  This work was supported by the National Key Research and Development Program of China (No. 2019YFA0706900), the National Natural Science Foundation of China (Nos. 22022703 and 22177108) and the Collaborative Innovation Program of Hefei Science Center, CAS (No. 2022HSC-CIP013).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.109033.

  
  
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  Scheme 1  The improved installation of Hmb (iHmb) method.

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  Figure 1  The RP-HPLC analysis of crude peptides 6–14 (a–i), and 16–18 (j–l) synthesized by the iHmb method and their corresponding ESI-MS. ^a 17 was synthesized on Wang resin by the iHmb method; ^b 18 was synthesized on Trt hydrazine resin by the iHmb method.

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  Figure 2  Fmoc SPPS of cyclic peptide 19 by the iHmb method. (a) Synthetic route; (b) HPLC, ESI-MS and deconvoluted spectra of 19b, 19d, 19.

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  Figure 3  Fmoc SPPS of ubiquitin 20 by the iHmb method. (a) Synthetic route; (b) HPLC, ESI-MS and deconvoluted spectra of 20a, 20.

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  Figure 4  Chemical synthesis of D-peptide 21 by the iHmb method. (a) The synthetic route; (b) HPLC, ESI-MS and deconvoluted spectra of 21.

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  Figure 5  Chemical synthesis of SLN 25 based on the iHmb method. (a) The synthetic route for the solid-phase peptide synthesis of 23 by the iHmb method; (b) HPLC and ESI-MS of 23; (c) Chemical synthesis for 25; (d) HPLC analysis of NCL; (e) ESI-MS and deconvoluted spectra of 24; (f) ESI-MS and deconvoluted spectra of 25.

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  Table 1.  Oxidation of amino group of Hmab on peptide 1 into Hmdab.

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  Table 2.  On-resin decomposition of diazonium group.

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  Table 3.  Scope of the iHmb method using a model sequence H-Gly-Val-Lys-Xaa-Yaa^Hmb-Ala-Gln-Lys-Phe-Asn-Gly-NH₂.

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