Native Chemical Ligation at Serine Revisited

Letter Cite This: Org. Lett. 2018, 20, 7616−7619

pubs.acs.org/OrgLett

Native Chemical Ligation at Serine Revisited Benoît Snella, Vincent Diemer, Hervé Drobecq, Vangelis Agouridas,* and Oleg Melnyk* University of Lille, Pasteur Institute of Lille, UMR CNRS 8204, Centre d’Immunité et d’Infection de Lille, F-59000 Lille, France

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S Supporting Information *

ABSTRACT: Standard conditions for the formation of serylcysteinyl junctions by Native Chemical Ligation (NCL) can result in significant epimerization of the serine residue. Epimerization can be minimized to background level by adjusting peptide concentration and working at 4 °C.

N

ative Chemical Ligation (NCL1) is a chemoselective peptide bond forming reaction which enables the coupling of a peptide thioester 1 with a cysteinyl (Cys) peptide 2 in water at neutral pH (Scheme 1). By far, the most

the peptide thioester features a C-terminal Thr, Val, Ile, or Pro residue.5 On the other hand, byproduct formation is often observed during the assembly of junctions involving Asp, Glu, Asn, or Gln (isopeptides), Pro (two amino acids deletion), and sometimes Lys (lactam).6 In the case of Asn, Gln, and Glu, isopeptide byproduct formation could be significantly reduced by catalyzing the reaction with 4-mercaptophenylacetic acid (MPAA7), which is now the most popular catalyst of NCL.6e Beyond those reported side reactions, the NCL process is generally considered to be low epimerizing under classical conditions. During our investigations on the synthesis of Muc1 polypeptides, we however noticed that NCL at serine formed the expected ligation product 3a accompanied by significant amounts of epimerized product 4a, although canonical experimental conditions were applied for this reaction (Figure 1A). The ease of epimerization of peptidyl seryl thioesters or thioesters surrogates has been noticed by a few authors in the past, but was never reported as being a serious problem during NCL.8 Serine is one of the most frequent amino acids in proteins (7.66%) after Leu and Ala. Therefore, the search for epimerization-free conditions for the formation of Ser-Cys junctions by NCL (or Ser-Ala after desulfurization) is an important goal. Toward this end, we determined the role of the different reaction parameters on the extent of serine epimerization. This knowledge enables us to propose new conditions for NCL at serine that reduce this side reaction to background levels. The method was validated by the total synthesis of human defensin DEFB133. Since the HPLC peaks of Muc1 derived target peptide 3a and its epimerized byproduct 4a were resolved at baseline

Scheme 1. NCL Reaction Using MPAA as Catalyst

popular method used for protein chemical synthesis today, NCL has seen its scope considerably extended in recent years with the advent of derived methodologies based on new types of acyl donors2 or cysteine surrogates.3 With such a comprehensive toolbox at the disposal of the protein chemist, nearly all types of junctions have become accessible, as illustrated by the diversity and the complexity of the proteins produced by chemical synthesis currently.4 However, the possibility of choosing virtually any site of ligation requires, in some cases, an experimental design that is specifically adapted to the nature of the junction residues. Indeed, the performance and the impurity profile of the NCL reaction are closely tied to the nature of the C-terminal residue of the thioester component (residue X in Scheme 1). Several works have clarified the role of this terminal residue with regard to the kinetics of NCL and to the occurrence of side reactions. Typically, the rate of NCL is severely affected when © 2018 American Chemical Society

Received: October 20, 2018 Published: November 13, 2018 7616

DOI: 10.1021/acs.orglett.8b03355 Org. Lett. 2018, 20, 7616−7619

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catalyst. MPAA concentration had almost no influence on the extent of epimerization in the 50−200 mM range (Figure S11). Neither the concentration of TCEP (Figure S12) nor that of the phosphate buffer (Figure S13) had more influence on the extent of Ser epimerization. In the latter experiment, we however noticed that the yield of the D-epimer 4a was lower (∼6%) than expected from previous experiments (∼9−10%). We found that this was due to a change in the batch of Cys peptide 2 used in the reaction. Both batches were of high purity and could not be distinguished by LC-MS. Since the batch that served for the first series of experiments was not purified by HPLC (batch 1), we examined if thiol impurities such as ethanedithiol (EDT) coming from the peptide cleavage and deprotection cocktail might promote the epimerization of Ser during NCL. The addition of EDT (1 mM) to the ligation mixture indeed promoted the formation of epimerized byproduct 4a up to 16% (see Figure S14). Whether epimerized byproduct formation is caused by thiol impurities or other trace impurities, the peptide segments were systematically purified by HPLC in the rest of this study, even if this step was not required per se in light of their crude purity. By taking into account the D-Ser content in the starting peptide 1a, NCL at 1 mM peptide concentration is responsible for ∼4% of the epimerized Ser residue in the final product. Although epimerization is expected to be base-catalyzed whether it proceeds through enolization or 5(4H)-oxazolonemediated mechanisms, we found that the pH has no significant effect on the extent of Ser epimerization in the pH range 6.4− 8.0 (Figure 2, Figure S15). In contrast, the temperature has a major effect as shown in Figure 2 (see also Figure S16). An important observation is that working at 4 °C reduces byproduct 4a formation to background levels.

Figure 1. (A) Model ligation at serine and starting conditions used in this study. (B) Effect of peptide concentration on D-Ser content (UV 215 nm).

level, this model reaction was used for measuring the effect of all the reaction parameters on the extent of Ser epimerization. Control experiments with Muc 1 peptide thioester equipped with a C-terminal D -Ser residue (APDTRPAPGSTAPPAHGVTs-MPA, peptide thioester 1b) as well as the time course for all reactions can be found in the Supporting Information (SI). Figure 1B shows the HPLC yields for 3a/4a as a function of peptide thioester 1a concentration in the range 0.1−8 mM. The level of D-Ser in the starting peptide thioester 1a was ∼1.8% as determined by chiral GC-MS analysis after peptide hydrolysis9 so that NCL accounted for the epimerized byproduct 4a above this value. Figure 1B shows a marked increase of Ser epimerization by decreasing peptide concentration, with the yield of epimerized byproduct 4a approaching 18% at 0.1 mM (see also Figure S9). While the amount of byproduct 4a amounted to only 4% at 8 mM, such a high peptide concentration cannot be easily attained with large peptide segments. Therefore, peptide thioester 1a concentration was fixed at 1 mM for the remainder of this study which is more representative of that used for protein chemical synthesis. Regarding the nature of the catalyst, NCL catalyzed by MPAA proceeded significantly faster than with thiophenol10 (MPAA 200 mM t1/2 14 min; PhSH 200 mM t1/2 60 min) as previously reported,7 but the level of epimerized byproduct 4a was similar at the end (∼9%, Figure S10). Ligation catalyzed by methylthioglycolate (MTG11) proceeded nearly as fast as with MPAA (MTG 200 mM, t1/2 25 min), but the amount of epimerized byproduct 4a was significantly higher (14%). Therefore, the study was continued with MPAA as the

Figure 2. Effect of temperature on the extent of Ser epimerization during NCL. Peptide 1a 1 mM, peptide 2a (batch 2) 1.2 equiv, 6 M Gn·HCl, pH 7.2, MPAA 200 mM, TCEP 100 mM, under nitrogen. (A) Ligation product 3a. (B) Epimerized byproduct 4a. HPLC yield (UV 215 nm). 7617

DOI: 10.1021/acs.orglett.8b03355 Org. Lett. 2018, 20, 7616−7619

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be determined by chiral GC-MS after acid hydrolysis due to a lack of sensitivity. Therefore, the different DEFB133 polypeptides were alkylated with iodoacetamide, trypsinized, and analyzed by LC-MS. Luckily, the peptides DTYXC(CH2CONH2)FIMR (X = S or s) were well separated by LCMS from the other tryptic fragments and coeluted with synthetic references (Figure 3; see also SI). Therefore, the

We also checked the role of the denaturant on Ser epimerization at 4 °C (w/o 6 M Gn.HCl, 10 mM Noctylglucoside; Figure S17). Ser epimerization was slightly more pronounced in 6 M Gn·HCl (4a 3%) than in the absence of Gn·HCl (4a 2%), while N-octylglucoside had no effect (4a 2%). Given the capacity of 6 M Gn·HCl to solubilize a large diversity of peptide segments, such a small increase in byproduct formation can be tolerated. Last, but not least, we determined the extent of Ser epimerization in the absence of Cys peptide by dissolving the thioester 1a in phosphate buffer alone (Figure S18) or in the presence of MPAA (200 mM, Figure S19). In both cases, the peptide thioester epimerized but epimerization was dramatically promoted by MPAA.12 To resume at this point, the epimerization of Ser during NCL can be significantly reduced by using peptide concentrations above 1 mM and by working at 4 °C (MPAA 200 mM, 0.1 M phosphate pH 7.2, 6 M Gn·HCl). Presolubilization of the peptide thioester in phosphate buffer must be avoided, and presolubilization in the ligation buffer containing MPAA, even more. We recommend dissolving the peptide thioester directly in the mixture containing the Cys peptide and all additives at the appropriate pH. Using this procedure, peptide 3a was produced successfully on preparative scale (73% yield, D-Ser: 3.5% (by integration of UV peaks at 215 nm), 3.8% (by integration of ionic current for [M + H]3+, 2.1% based on the mass of isolated products). To illustrate the usefulness of the novel procedure with another example, we undertook the total synthesis of human defensin DEFB133, i.e. DEFB133a in Scheme 2. The defensin Scheme 2. Total Synthesis of Human Defensin DEFB133

Figure 3. LC-MS analysis of the trypsic digest of alkylated DEFB133 polypeptides. The D- or L-Ser content was determined by integrating the ionic current for m/z 597 (doubly charged species for peptides DTY(S/s)C*FIMR, where C* denotes the alkylated Cys residue).

was assembled in one pot by a sequential NCL/bis(2sulfanylethyl)amido (SEA)-mediated ligation sequence.13 Three different methods were used for the first ligation step depending on the temperature (37 °C for Methods A,B; 4 °C for Method C) and on the inclusion or not of a presolubilization step for the peptide thioester 1c (presolubilization for Method A, none for Methods B,C). As a control, the peptide thioester 1d featuring a C-terminal D-Ser residue was prepared and used for assembling the DEFB133b analog. Unfortunately, the D- or L-Ser content in DEFB133a could not

integration of the ionic current for doubly charged species (m/ z 597) furnished the extent of Ser epimerization in the final DEFB133 products (indicated in Scheme 2 and Figure 3). As expected, Ser epimerization was at its highest for the polypeptide DEFB133a produced according to Method A (7.3%) which involved an intentional presolubilization of the thioester segment in the ligation buffer and classical conditions for NCL (37 °C). The D-Ser content in DEFB133a produced according to Method B was 2.9% which must be compared to D-Ser content in the starting peptide thioester 1b, 0.37%. Thus, 7618

DOI: 10.1021/acs.orglett.8b03355 Org. Lett. 2018, 20, 7616−7619

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Organic Letters the NCL step accounts for ∼2.5% of the epimerized byproduct. Since the NCL was performed at 2.7 mM peptide concentration, the extent of epimerization is logically below those reported for peptide 1a used at 1 mM, i.e. 4%. The lowest epimerization level was obtained at 4 °C with Method C. Serine is one of the most prone to epimerization among all proteinogenic amino acids.14 The facile epimerization of serine derivatives has been the subject of extensive studies and has been ascribed to the electron-withdrawing power of the side chain hydroxyl group, which renders the α-proton more acidic. For example, activated esters of Z-Ser, where Z stands for the benzyloxycarbonyl group, epimerize 29 times faster than ZAla.15 The ease of epimerization of peptidyl seryl thioesters during NCL reported here is probably reminiscent of the propensity of Ser activated esters to epimerize faster than those derived from other amino acids. In conclusion, we report that the epimerization of peptidyl seryl thioesters can be significant during NCL under classical experimental conditions. The level of Ser epimerization can be reduced to background level by avoiding any presolubilization of the peptide thioester and by performing the ligation at 4 °C. Working at low temperature is not an issue in this case since Ser is a fast reacting amino acid. This work provides important information to the utilizers of NCL and a simple solution to minimize this side reaction.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b03355. Experimental procedures and characterization of all peptides (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Oleg Melnyk: 0000-0002-3863-5613 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank financial support from ANR (ANR-15-CE07-0020, CyProt). REFERENCES

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DOI: 10.1021/acs.orglett.8b03355 Org. Lett. 2018, 20, 7616−7619