Protocol Received: 2 October 2013
Accepted: 6 October 2013
Published online in Wiley Online Library: 19 November 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2580
Tidbits for the synthesis of bis(2-sulfanylethyl) amido (SEA) polystyrene resin, SEA peptides and peptide thioesters‡ Nathalie Ollivier, Laurent Raibaut, Annick Blanpain, Rémi Desmet, Julien Dheur, Reda Mhidia, Emmanuelle Boll, Hervé Drobecq, Silvain L. Pira and Oleg Melnyk* Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl) amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Keywords: bis(2-sulfanylethyl)amido (SEA); thioester; Fmoc-SPPS; thiol exchange
Scope and Comments
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Protein total chemical synthesis is an interesting complement and potential alternative to the use of living systems for producing small proteins and therefore is gaining increasing significance for studying protein function or for developing future protein therapeutics. One of the great advantages of chemical synthesis is the atom-by-atom control of the protein structure and the possibility to introduce a large diversity of
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* Correspondence to: Oleg Melnyk, UMR CNRS 8161, Pasteur Institute of Lille, Univ. Lille Nord de France, 1 rue du Pr Calmette, 59021 Lille, France. E-mail: [email protected] ‡
This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the Chemical Protein Synthesis Meeting, April 3-6, 2013, Vienna, edited by Christian Becker (University of Vienna, Austria). UMR CNRS 8161, Pasteur Institute of Lille, Univ. Lille Nord de France, 1 rue du Pr Calmette, 59021, Lille, France
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
SEA PS RESIN FOR PEPTIDE THIOESTER SYNTHESIS
Scheme 1. Native chemical ligation reaction of C-terminal 3mercaptopropionic acid thioesters 4 with N-terminal Cys peptides 5.
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Step 1. Preparation of SEA PS resin 1 The protocol allows the preparation of 4 mmol (28 g) of SEA PS resin 1 (200–400 mesh, 1% cross-linked with divinylbenzene) and was validated at a 500 g scale. It has been used by several people in the lab independently at the ~20 g scale with similar results and externalized successfully for large-scale synthesis. The grafting of the bis(2-sulfanylethyl)amine is almost quantitative, leading to a final loading of 0.14–0.16 mmol/g. The final loading should not exceed this value, because we have observed that above 0.16 mmol/g, the diffusion of the reagents within the beads can be altered, may be because of additional cross-linking of the PS resin by the bis(2-sulfanylethyl)amine. Some commercially available trityl chloride resins have a loading greater than 1.4 mmol/g. In this case, we recommend changing the stoichiometry of the grafting procedure to not exceed a final loading of 0.14–0.16 mmol/g. The swelling of SEA PS resin 1 is 4.5 ml/g in DMF, 5.3 ml/g in N-methylpyrrolidone and 5.4 ml/g in DCM. Step 2. Peptide elongation and cleavage Peptide elongation was performed using standard Fmoc/tertbutyl chemistry. The first amino acid was coupled manually using HATU/DIEA activation in DMF for 2 h. This allows to control the coupling efficiency of the amino acid to the secondary amine on the solid phase and to determine the loading of the solid support with the C-terminal amino acid of interest. Usually, a single coupling step is enough. The capping step with Ac2O/DIEA, which follows the coupling of the first residue, is mandatory to avoid the potential contamination of the target peptide by C-terminal deletion side-products that are difficult to separate by HPLC. The rest of the peptide elongation was performed using an automated batch peptide synthesizer and HBTU/DIEA activation (0.1–0.5 mmol scale). When present, cysteine was coupled using Fmoc-L-Cys(StBu)-OH, which is compatible with iodine oxidation used in step 3 to convert SEAon peptide 2 into SEAoff peptide 3. Fmoc-L-Cys(Trt)-OH can be used if the oxidation procedure used to convert SEAon group into SEAoff is skipped. Step 3. Oxidation and purification step, isolation of SEAoff peptide 3 Step 3 requires only a few minutes of preparation before the purification of the crude peptide by preparative HPLC. The oxidation of SEAon peptide 2 into SEAoff peptide 3 with iodine in aqueous acetic acid was performed successfully with a large diversity of peptide sequences. The preparation of an iodine
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native or non-native modifications for tailoring the physicochemical, biochemical or biological properties of the protein. In this context, efficient strategies for accessing to synthetic proteins are highly needed. Modern protein chemical synthesis usually involves the sequential ligation of unprotected peptide segments in aqueous solution [1]. The native chemical ligation (NCL) [2] reaction is a chemoselective peptide bond forming process that is central today to the field of protein total synthesis either in solution[3] or on the solid phase[4–7] (for a review, see Ref. [8]). NCL is based on the reaction of a C-terminal peptide thioester, usually an alkylthioester of type 4, with an N-terminal Cys peptide 5 in water at neutral pH (Scheme 1). The key role played by NCL in the field of protein total synthesis has stimulated the development of various methods giving access to C-terminal peptide thioesters of type 4 [9]. Historically, peptide alkylthioesters were first synthesized using Boc-SPPS methods and thioester linkers [10]. However, Fmoc-SPPS is by far the most popular technique for peptide synthesis today [11]. The use of Fmoc-SPPS and thioester linkers for peptide thioester synthesis is faced with the instability of the thioester group in the presence of the base used for Fmoc removal. One solution to this problem is to decrease the basicity of the mixture used for Fmoc removal with acidic additives such as HOBt [12,13]. Another approach is based on the stabilization of the thioester linker using steric effects [14]. However, the most frequently used strategy is to introduce the thioester functionality after the peptide elongation step to avoid the manipulation of thioester-linked peptidyl resins and their exposure to basic reagents. This can be carried out either on the solid phase before the cleavage and deprotection step [15–19] or during the cleavage from the solid support [20–28]. Another strategy that is more practical from the analytical point of view consists in the generation of the thioester functionality in solution starting from a stable, easy to produce peptide precursor. Among the various methods described, the use of C-terminal hydrazides [29] or N,S-acyl shift systems [25,30–36] has gained increasing importance in the field during the last few years. One of these N,S-acyl shift systems is the bis(2-sulfanylethyl) amido (SEA) group 2 or 3 shown in the General Scheme [34,35]. It enables various useful chemical transformations [37–40] among which the synthesis of peptide thioesters of type 4 by an exchange reaction of the bis(2-sulfanylethyl)amino moiety with an alkylthiol such as MPA (General Scheme) [38]. The SEA group differentiates from the other members of this family by its good reactivity at mildly acidic pH, its resistance to hydrolysis and its unique redox properties [34,37,41]. Indeed, the SEA peptide is deprotected and cleaved from the resin in the form of the reactive SEAon peptide 2. Dithiol 2 can be inactivated
by mild oxidation into SEAoff peptide 3, which is highly stable as expected for an N,N-dialkylamide. Conversion of SEAoff peptide 3 into peptide thioester 4 is carried out preferably at pH 4 in the presence of the reductant tris(2-carboxyethyl) phosphine [38]. The aim of this protocol is to provide the detailed experimental procedures for the synthesis and characterization of SEA polystyrene resin 1 and its use for the synthesis of peptide thioesters 4 through steps 1–4 (General Scheme). It has been used for the chemical synthesis of several functional proteins such as the Kringle 1 domain of hepatocyte growth factor (HGF) in its native[41] or biotinylated [42] form, the heparin binding N domain of HGF [43] and large polypeptides or SEA thioester surrogates on a water-compatible solid support [7].
OLLIVIER ET AL. solution in DMSO is very practical as it allows the addition of a precise amount of iodine (2 equiv only), even for small scales. Moreover, DMSO increases significantly the solubility of iodine in the aqueous reaction mixture. This allowed a decrease in the proportion of acetic acid from 80% (as it is often reported for iodine oxidations) to 20%, thereby simplifying the purification step. Note that the slight excess of iodine is decomposed in a few seconds by addition of dithiothreitol (DTT) prior to the injection on the HPLC system. Because of the acidity of the mixture, DTT is unable to reduce disulfide bonds. In particular, SEAoff group and Cys(StBu) residues are not affected during this step. Other oxidants can be used as well such as N,N,N′,N′tetramethyl-azodicarboxamide at neutral pH, which enabled the synthesis of a SEAoff peptide functionalized by an alkylazido group [7]. This method is compatible with the presence of Cys (StBu) residues too. Step 4. Synthesis of MPA thioester 4 The last step discussed here is the exchange reaction of the SEA group by MPA to yield a C-terminal peptide thioester. This reaction goes almost to completion at pH 4 within 24 h for most C-terminal amino acid residues (37 °C). The method is compatible with C-terminal Glu residue. Aspartic acid is a special case, and the procedure for the synthesis of peptidyl aspartyl thioesters will be reported later on [44]. Valine required longer reaction times (96 h) because of the steric bulk of this amino acid [38]. Importantly, we have shown recently that the procedure could be adapted to the synthesis of MPA peptidyl prolyl thioesters [45]. In this case, the exchange reaction was very slow at 37 °C but proceeded efficiently at 65 °C. As an example, peptides ILKEPVHXP-SCH2CH2CO2H (X = Ala, Gly, Ile) were isolated with a 41–53% yield after HPLC purification. The efficiency of the exchange reaction enabled the recent design of a solid-phase elongation cycle allowing the N-to-C sequential NCL of unprotected peptide segments. Three elongation cycles – each including a SEAoff-MPA thioester exchange reaction – permitted the solid-phase ligation of five peptide segments and the isolation of a 15 kDa polypeptide in high yield (6.5% overall, nine chemical steps, 74% yield per step) [7]. Limitations
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The protocol reported here for the synthesis of SEA PS resin 1 (200–400 mesh) is not appropriate for the preparation of 100–200 mesh resins for unknown reasons. SEA PS resin 1 is highly efficient for batch synthesis using DMF and standard Fmoc-SPPS and coupling times. It can be used also with column peptide synthesizers. However, in some configurations, we observed modest yields for the first couplings steps when using DMF as the solvent. Better coupling yields were obtained by using N-methylpyrrolidone instead of DMF, which allows a better swelling of the SEA PS resin 1. An alternative that was not tested would be to use mixtures of DMF and DCM. Oxidation of SEAon group into SEAoff is recommended but not mandatory. The oxidation step can be skipped if the HPLC purification step can be performed quickly. Indeed, in this case, the N,S-acyl shift of SEAon group can occur on the HPLC column in acidic eluents. The N,S-acyl shift of SEAon amide group –CON(CH2CH2SH)2 yields SEAon thioester group – COSCH2CH2NHCH2CH2SH featuring a secondary amino group. Usually, SEAon thioester group is significantly more hydrophilic
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than SEAon amide group, resulting in significant differences in retention times (sometimes a few minutes apart). The formation of the SEA thioester form is favored at the acidic pH used for the elution due to the protonation of the secondary amine. This equilibrium results in low purification yields, unless the HPLC purification is performed rapidly. Because the kinetic rate of the N,S-acyl shift depends on the nature of the C-terminal amino acid residue, the purification of a SEAon peptide must be optimized case by case. Finally, the exchange reaction with MPA is highly efficient at pH 4 and 37 °C, but heating up to 65 °C helps for difficult amino acids such as proline. Experimental Procedure Step 1. Preparation of SEA PS resin 1 (200–400 mesh) Deprotection of the bis(2-tritylsulfanylethyl)amine HN(CH2CH2STrt)2. The synthesis must be carried out in an efficient fume hood with the appropriate protection. The bis(2-tritylsulfanylethyl) amine was prepared as described previously by reacting bis (2-chloroethyl)amine hydrochloride with triphenylmethyl mercaptan in the presence of DBU [34,46]. Both bis(2chloroethyl)amine hydrochloride and triphenylmethyl mercaptan are commercially available. Once formed, the bis(2-sulfanylethyl) amine trifluoroacetate salt HN(CH2CH2SH)2. CF3CO2H must be used immediately. Step 1.1.1. Weigh the bis(2-tritylsulfanylethyl)amine (2.48 g, 4.00 mmol) in a 200 ml round bottom flask equipped with a magnetic bar. Step 1.1.2. Add a mixture of TFA/triisopropylsilane (TIS): 97.5/2.5 by volume (100 ml). Step 1.1.3. Stir the reaction mixture at room temperature for 30 min. Step 1.1.4. Then, evaporate the solvent using a rotary evaporator under reduced pressure to obtain a white solid. Step 1.1.5. Dissolve the white solid in cyclohexane (~50 ml) and evaporate the solvent again as previously mentioned and repeat this step twice. This step allows removal of the residual TFA. Step 1.1.6. Dry the bis(2-sulfanylethyl)amine trifluoroacetic acid salt HN(CH2CH2SH)2. CF3CO2H in vacuo for 20 min. Conditioning of the trityl chloride resin. Step 1.2.1. Weigh the trityl chloride resin (40 mmol, 28.57 g, 1.4 mmol/g from IRIS Biotech GmbH, 200–400 mesh, ref BR1145) in a glass reactor equipped with a sintered glass and a tap at the bottom and a skirted silicone stopper on top. Caution: Trityl chloride resin is sensitive to hydrolysis. A tenfold excess of trityl chloride functions relative to the bis(2sulfanylethyl)amine trifluoroacetate is used in this protocol. Step 1.2.2. Swell the resin in CH2Cl2 for 10 min. Step 1.2.3. Filter and flush immediately with argon from the bottom (the cap must be open during this operation). Stop the argon flush and close the tap and cap in this order. Step 1.2.3. Add 30 ml of DMF through the skirted silicone stopper with a syringe and shake for 5 min.
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
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SEA PS RESIN FOR PEPTIDE THIOESTER SYNTHESIS Step 1.2.4. Filter. Coupling of the bis(2-sulfanylethyl)amine trifluoroacetate salt to the resin. Step 1.3.1. Solubilize the bis(2-sulfanylethyl)amine trifluoroacetate salt in DMF (25 ml) and add this solution to the resin through the cap using a syringe. Wash the round bottom flask with DMF (5 ml) and add this solution to the resin as previously mentioned. Step 1.3.2. Shake overnight. Step 1.3.3. Mix MeOH (1.5 ml) and 2,5-lutidine (4.45 ml) and add the mixture to the aforementioned suspension. Working under argon atmosphere is no longer necessary. Step 1.3.4. Agitate for 30 min. Step 1.3.5. Wash the beads with DMF, MeOH, DMF, DIEA in DMF (5% by volume), DMF, CH2Cl2 and Et2O (2 × 2 min for each solvent). Step 1.3.6. Dry the beads under vacuum and store at 4 °C. The resin is very stable and can be stored for months at 4 °C. Characterization of SEA PS resin 1. Step 1.4.1. Perform an Ellman assay to detect the presence of free thiols. For this, prepare a solution of 5,5′-dithiobis(2nitrobenzoic acid) (DTNB) in DMF (2 mg/ml). Add one drop of the DTNB solution to an aliquot of the SEA PS resin 1 and then one drop of 5% DIEA in DMF. Agitate for 5 min. Use another resin for the negative control. A positive assay is characterized by the development of a yellow color in solution. This assay must be negative. Step 1.4.2. Perform a chloranil assay [47] to detect the presence of secondary amines. For this, prepare a chloranil solution in DMF (20 mg/ml) and an acetaldehyde solution in DMF (2% by volume). Add four drops of each solution to an aliquot of the SEA PS resin 1. Agitate for 5 min. This assay must be strongly positive, that is, the beads must be blue. Step 1.4.3. Determine the loading of the SEA PS resin 1. For this, couple Fmoc-Gly-OH (150 mg, 0.50 mmol) to the SEA resin (357 mg, expected 0.05 mmol) using standard HATU (190 mg, 0.49 mmol)/DIEA (260 μl, 1.5 mmol) activation in DMF (200 μl). Fmoc-Gly-OH was pre-activated 1 min before being added to the beads swelled in DMF. The beads were agitated for 1 h 30 min at room temperature and then washed with DMF (3 × 2 min). Wash the resin with CH2Cl2 (3 × 2 min) and Et2O (3 × 2 min) and dry in vacuo. Perform a chloranil assay, which must be negative. Weight three aliquots (m = 3–5 mg) of the resin and add a solution of piperidine in DMF (20% by volume, 1 ml). Transfer each solution to a 10 ml volumetric flask and dilute with DMF up to 10 ml. Measure the UV absorbance at 290 nm using the piperidine in DMF solution for the blank. Calculate the 290 loading using the equation: loading (mmol/g) = 2:02 Absm (where m is the mass of resin in milligrams). The loading should be 0.14–0.16 mmol/g.
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Step 2.2. Typical procedure for batchwise peptide elongation using Fmoc-SPPS. Peptide elongation was performed using standard Fmoc/tert-butyl SPPS protocols at room temperature on an automated batch peptide synthesizer (0.1–0.5 mmol scale). Couplings were performed using a fivefold molar excess of each Fmoc-L-amino acid, a 4.5-fold molar excess of HBTU and a tenfold molar excess of DIEA (30 min coupling time, double couplings). A capping step was performed after each coupling with a mixture of 10% Ac2O and 5% DIEA in DMF. At the end of the synthesis, the resin was washed with CH2Cl2 (2 × 2 min), diethylether (2 × 2 min) and dried in vacuo. Step 2.3. Typical procedure for the cleavage and deprotection step. Isolation of the crude SEAon peptide 2. Deprotection and cleavage were performed with TFA in the presence of the appropriate scavengers. TIS, DMS, thioanisole, anisole, thiophenol and water are compatible with the synthesis of SEA peptides. The mixture TFA/TIS/DMS/H2O/thioanisole: 90/2.5/2.5/ 2.5/2.5 by volume (40 ml for 0.5 mmol scale) is appropriate for a large diversity of peptide sequences and lengths. The beads were filtered, and the TFA solution was added dropwise to a cold mixture of Et2O/n-heptane: 1/1 by volume (500 ml). We recommend performing the cleavage/precipitation procedure at least twice. As an example, for the synthesis of peptide IRNC(StBu) IIGKGRSYKGT15V16SITKSGIK-SEAon, the peptidyl resin (0.5 mmol scale) was treated three times with the TFA mixture described earlier (3 h and 2 × 1 h) to give 797 (44.5%), 303 (17%) and 54 mg (3%) of the crude peptide (total crude yield 64.5%). In this case, the synthesis was complicated by a difficult coupling of threonine residue 15 on valine residue 16 (see later). Step 3. Typical procedure for the oxidation step and purification of SEAoff peptide 3 SEAon peptide 2 (25 μmol) was dissolved in AcOH/H2O: 1/4 by volume (final peptide concentration 0.5 mM). Iodine solution (200 mM in DMSO, 50 μmol, 250 μl) was added in one portion. After 30 s, DTT (65 mM in water, 50 μmol, 775 μl) was added to quench the excess of iodine. Once DTT was added, the mixture was immediately purified by RP HPLC using a linear water–acetonitrile gradient containing 0.05% of TFA to give SEAoff peptide 3. Using this procedure, we isolated 70 mg (4%) of the peptide IRNC(StBu)IIGKGRSYKGTVSITKSGIK-SEAoff (0.5 mmol scale). The modest yield is due to the presence, in significant amounts, of the acetylated peptide Ac-VSITKSGIK-SEAoff in the crude product. In a subsequent synthesis, we obtained much better yields for this SEAoff peptide by optimizing the peptide elongation step (27%, see Ref. [41]).
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Step 2. Fmoc-SPPS of the SEAon peptide 2
Step 2.1. Coupling of the first amino acid. This step was performed manually. SEA PS resin 1 (0.5 mmol, 0.16 mmol/g) was conditioned in CH2Cl2 (3 × 2 min) and DMF (3 × 2 min). Fmoc-Aa-OH (5.00 mmol, 2.43 g for Fmoc-L-Lys(Boc)-OH, see the example provided in §3) and HATU (4.90 mmol; 1.90 g) were dissolved in the minimal volume of DMF. DIEA (10 mmol, 1741 μl) were added to the aforementioned solution, which was agitated at room temperature (1 min) and then added to the beads. The resin was agitated during 2 h and washed with DMF (3 × 2 min). Control the coupling step using the chloranil assay and repeat the coupling step if necessary. The resin was then treated with Ac2O/DIEA/CH2Cl2: 10/5/85 by volume (10 ml, 2 × 10 min) to cap unreacted secondary amino groups. Finally, the resin was washed with DMF (3 × 2 min).
OLLIVIER ET AL. Step 4. Typical procedure for the synthesis of MPA thioester 4 By using the general optimized procedure, SEAoff peptide IRNC(StBu)IIGKGRSYKGTVSITKSGIK-SEAoff was converted into MPA thioester IRNCIIGKGRSYKGTVSITKSGIK-SCH2CH2CO2H with an isolated yield of 41% following HPLC purification (25 mg scale) [41]. In another experiment, we isolated the same MPA thioester in 66% yield by desalting the crude product on a C18 solid-phase extraction column. Most of the time, a high-resolution HPLC purification step is not required because (i) the starting SEAoff peptide 3 is purified in the previous step and (ii) the exchange reaction is very clean and proceeds in high yields. Note that Cys(StBu) residues are reduced into Cys during this step.
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References
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fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of sidechain and backbone anchoring strategies, and compatible protection for N-terminal cysteine. J. Pept. Res. 2005; 65: 395–410. Ingenito R, Bianchi E, Fattori D, Pessi A. Solid phase synthesis of peptide C-terminal thioesters by Fmoc/t-Bu chemistry. J. Am. Chem. Soc. 1999; 121: 11369–11374. Ohta Y, Itoh S, Shigenaga A, Shintaku S, Fujii N, Otaka A. Cysteine-derived S-protected oxazolidinones: potential chemical devices for the preparation of peptide thioesters. Org. Lett. 2006; 8: 467–470. Swinnen D, Hilvert D. Facile, Fmoc-compatible solid-phase synthesis of peptide C-terminal thioesters. Org. Lett. 2000; 2: 2439–2442. Quaderer R, Hilvert D. Improved synthesis of C-terminal peptide thioesters on “safety-catch” resins using LiBr/THF. Org. Lett. 2001; 3: 3181–3184. Sewing A, Hilvert D. Fmoc-compatible solid-phase peptide synthesis of long C-terminal peptide thioesters. Angew. Chem. Int. Ed. 2001; 40: 3395–3396. Nagaike F, Onuma Y, Kanazawa C, Hojo H, Ueki A, Nakahara Y, Nakahara Y. Efficient microwave-assisted tandem N- to S-acyl transfer and thioester exchange for the preparation of a glycosylated peptide thioester. Org. Lett. 2006; 8: 4465–4468. Blanco-Canosa JB, Dawson PE. An efficient Fmoc-SPPS approach for the generation of thioester peptide precursors for use in native chemical ligation. Angew. Chem. Int. Ed. 2008; 47: 6851–6855. Mende F, Beisswenger M, Seitz O. Automated Fmoc-based solidphase synthesis of peptide thioesters with self-purification effect and application in the construction of immobilized SH3 domains. J. Am. Chem. Soc. 2010; 132: 11110–11118. Tofteng AP, Sørensen KK, Conde-Frieboes KW, Hoeg-Jensen T, Jensen KJ. Fmoc solid-phase synthesis of C-terminal peptide thioesters by formation of a backbone pyroglutamyl imide moiety. Angew. Chem. Int. Ed. 2009; 48: 7411–7414. Fang G-M, Li Y-M, Shen F, Huang Y-C, Li J-B, Lin Y, Cui H-K, Liu L. Protein chemical synthesis by ligation of peptide hydrazides. Angew. Chem. Int. Ed. 2011; 50: 7645–7649. Hojo H, Onuma Y, Akimoto Y, Nakahara Y, Nakahara Y. N-Alkyl cysteine-assisted thioesterification of peptides. Tetrahedron Lett. 2007; 48: 25–28. Nakamura K, Mori H, Kawakami T, Hojo H, Nakahara Y, Aimoto S. Peptide thioester synthesis via an auxiliary-mediated N–S acyl shift reaction in solution. Int. J. Pep. Res. Ther. 2007; 13: 191–202. Nakamura K, Kanao T, Uesugi T, Hara T, Sato T, Kawakami T, Aimoto S. Synthesis of peptide thioesters via an N–S acyl shift reaction under mild acidic conditions on an N-4,5-dimethoxy-2-mercaptobenzyl auxiliary group. J. Pept. Sci. 2009; 15: 731–737. Tsuda S, Shigenaga A, Bando K, Otaka A. N,S Acyl-transfer-mediated synthesis of peptide thioesters using anilide derivatives. Org. Lett. 2009; 11: 823–826. Ollivier N, Dheur J, Mhidia R, Blanpain A, Melnyk O. Bis(2-sulfanylethyl) amino native peptide ligation. Org. Lett. 2010; 12: 5238–5241. Hou W, Zhang X, Li F, Liu CF. Peptidyl N,N-bis(2-mercaptoethyl)-amides as thioester precursors for native chemical ligation. Org. Lett. 2011; 13: 386–389. Taichi M, Hemu X, Qiu Y, Tam JP. A thioethylalkylamido (TEA) thioester surrogate in the synthesis of a cyclic peptide via a tandem acyl shift. Org. Lett. 2013; 15: 2620–2623. Boll E, Dheur J, Drobecq H, Melnyk O. Access to cyclic or branched peptides using bis(2-sulfanylethyl)amido side-chain derivatives of Asp and Glu. Org. Lett. 2012; 14: 2222–2225. Dheur J, Ollivier N, Vallin A, Melnyk O. Synthesis of peptide alkylthioesters using the intramolecular N,S-acyl shift properties of bis(2-sulfanylethyl)amido peptides. J. Org. Chem. 2011; 76: 3194–3202. Dheur J, Ollivier N, Melnyk O. Synthesis of thiazolidine thioester peptides and acceleration of native chemical ligation. Org. Lett. 2011; 13: 1560–1563. Pira SL, Boll E, Melnyk O. Synthesis of peptide thioacids at neutral pH using bis(2-sulfanylethyl)amido peptide precursors. Org. Lett. 2013; 15: 5346–5349. Ollivier N, Vicogne J, Vallin A, Drobecq H, Desmet R, El-Mahdi O, Leclercq B, Goormachtigh G, Fafeur V, Melnyk O. A one-pot threesegment ligation strategy for protein chemical synthesis. Angew. Chem. Int. Ed. 2012; 51: 209–213. Ancot F, Leroy C, Muharram G, Lefebvre J, Vicogne J, Lemiere A, Kherrouche Z, Foveau B, Pourtier A, Melnyk O, Giordano S, Chotteau-Lelievre A, Tulasne D. Shedding-generated Met receptor
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SEA PS RESIN FOR PEPTIDE THIOESTER SYNTHESIS fragments can be routed to either the proteasomal or the lysosomal degradation pathway. Traffic 2012; 13: 1261–1272. 43 Raibaut L, Vicogne J, Leclercq B, Drobecq H, Desmet R, Melnyk O. Total synthesis of biotinylated N domain of human hepatocyte growth factor. Bioorg. Med. Chem. 2013; 21: 3486–3494. 44 Dang B, Kubota T, Mandal K, Bezanilla F, Kent SB. Native chemical ligation at Asx-Cys, Glx-Cys: chemical synthesis and high-resolution X-ray structure of ShK toxin by racemic protein crystallography. J. Am. Chem. Soc. 2013; 135: 11911–11919.
45 Raibaut L, Seeberger P, Melnyk O. Bis(2-sulfanylethyl)amido peptides enable native chemical ligation at proline and minimize deletion side-product formation. Org. Lett. 2013; 15(21): 5516–5519. 46 Banerjee SR, Maresca KP, Stephenson KA, Valliant JF, Babich JW, Graham WA, Barzana M, Dong Q, Fischman AJ, Zubieta J. N,N-bis(2mercaptoethyl)methylamine a new coligand for Tc-99m labeling of hydrazinonicotinamide peptides. Bioconj. Chem. 2005; 16: 885–902. 47 Vojkovsky T. Detection of secondary amines on solid phase. Pept. Res. 1995; 8: 236–237.
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Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
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Accepted: 6 October 2013
Published online in Wiley Online Library: 19 November 2013
(wileyonlinelibrary.com) DOI 10.1002/psc.2580
Tidbits for the synthesis of bis(2-sulfanylethyl) amido (SEA) polystyrene resin, SEA peptides and peptide thioesters‡ Nathalie Ollivier, Laurent Raibaut, Annick Blanpain, Rémi Desmet, Julien Dheur, Reda Mhidia, Emmanuelle Boll, Hervé Drobecq, Silvain L. Pira and Oleg Melnyk* Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl) amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Keywords: bis(2-sulfanylethyl)amido (SEA); thioester; Fmoc-SPPS; thiol exchange
Scope and Comments
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Protein total chemical synthesis is an interesting complement and potential alternative to the use of living systems for producing small proteins and therefore is gaining increasing significance for studying protein function or for developing future protein therapeutics. One of the great advantages of chemical synthesis is the atom-by-atom control of the protein structure and the possibility to introduce a large diversity of
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* Correspondence to: Oleg Melnyk, UMR CNRS 8161, Pasteur Institute of Lille, Univ. Lille Nord de France, 1 rue du Pr Calmette, 59021 Lille, France. E-mail: [email protected] ‡
This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the Chemical Protein Synthesis Meeting, April 3-6, 2013, Vienna, edited by Christian Becker (University of Vienna, Austria). UMR CNRS 8161, Pasteur Institute of Lille, Univ. Lille Nord de France, 1 rue du Pr Calmette, 59021, Lille, France
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
SEA PS RESIN FOR PEPTIDE THIOESTER SYNTHESIS
Scheme 1. Native chemical ligation reaction of C-terminal 3mercaptopropionic acid thioesters 4 with N-terminal Cys peptides 5.
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Step 1. Preparation of SEA PS resin 1 The protocol allows the preparation of 4 mmol (28 g) of SEA PS resin 1 (200–400 mesh, 1% cross-linked with divinylbenzene) and was validated at a 500 g scale. It has been used by several people in the lab independently at the ~20 g scale with similar results and externalized successfully for large-scale synthesis. The grafting of the bis(2-sulfanylethyl)amine is almost quantitative, leading to a final loading of 0.14–0.16 mmol/g. The final loading should not exceed this value, because we have observed that above 0.16 mmol/g, the diffusion of the reagents within the beads can be altered, may be because of additional cross-linking of the PS resin by the bis(2-sulfanylethyl)amine. Some commercially available trityl chloride resins have a loading greater than 1.4 mmol/g. In this case, we recommend changing the stoichiometry of the grafting procedure to not exceed a final loading of 0.14–0.16 mmol/g. The swelling of SEA PS resin 1 is 4.5 ml/g in DMF, 5.3 ml/g in N-methylpyrrolidone and 5.4 ml/g in DCM. Step 2. Peptide elongation and cleavage Peptide elongation was performed using standard Fmoc/tertbutyl chemistry. The first amino acid was coupled manually using HATU/DIEA activation in DMF for 2 h. This allows to control the coupling efficiency of the amino acid to the secondary amine on the solid phase and to determine the loading of the solid support with the C-terminal amino acid of interest. Usually, a single coupling step is enough. The capping step with Ac2O/DIEA, which follows the coupling of the first residue, is mandatory to avoid the potential contamination of the target peptide by C-terminal deletion side-products that are difficult to separate by HPLC. The rest of the peptide elongation was performed using an automated batch peptide synthesizer and HBTU/DIEA activation (0.1–0.5 mmol scale). When present, cysteine was coupled using Fmoc-L-Cys(StBu)-OH, which is compatible with iodine oxidation used in step 3 to convert SEAon peptide 2 into SEAoff peptide 3. Fmoc-L-Cys(Trt)-OH can be used if the oxidation procedure used to convert SEAon group into SEAoff is skipped. Step 3. Oxidation and purification step, isolation of SEAoff peptide 3 Step 3 requires only a few minutes of preparation before the purification of the crude peptide by preparative HPLC. The oxidation of SEAon peptide 2 into SEAoff peptide 3 with iodine in aqueous acetic acid was performed successfully with a large diversity of peptide sequences. The preparation of an iodine
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native or non-native modifications for tailoring the physicochemical, biochemical or biological properties of the protein. In this context, efficient strategies for accessing to synthetic proteins are highly needed. Modern protein chemical synthesis usually involves the sequential ligation of unprotected peptide segments in aqueous solution [1]. The native chemical ligation (NCL) [2] reaction is a chemoselective peptide bond forming process that is central today to the field of protein total synthesis either in solution[3] or on the solid phase[4–7] (for a review, see Ref. [8]). NCL is based on the reaction of a C-terminal peptide thioester, usually an alkylthioester of type 4, with an N-terminal Cys peptide 5 in water at neutral pH (Scheme 1). The key role played by NCL in the field of protein total synthesis has stimulated the development of various methods giving access to C-terminal peptide thioesters of type 4 [9]. Historically, peptide alkylthioesters were first synthesized using Boc-SPPS methods and thioester linkers [10]. However, Fmoc-SPPS is by far the most popular technique for peptide synthesis today [11]. The use of Fmoc-SPPS and thioester linkers for peptide thioester synthesis is faced with the instability of the thioester group in the presence of the base used for Fmoc removal. One solution to this problem is to decrease the basicity of the mixture used for Fmoc removal with acidic additives such as HOBt [12,13]. Another approach is based on the stabilization of the thioester linker using steric effects [14]. However, the most frequently used strategy is to introduce the thioester functionality after the peptide elongation step to avoid the manipulation of thioester-linked peptidyl resins and their exposure to basic reagents. This can be carried out either on the solid phase before the cleavage and deprotection step [15–19] or during the cleavage from the solid support [20–28]. Another strategy that is more practical from the analytical point of view consists in the generation of the thioester functionality in solution starting from a stable, easy to produce peptide precursor. Among the various methods described, the use of C-terminal hydrazides [29] or N,S-acyl shift systems [25,30–36] has gained increasing importance in the field during the last few years. One of these N,S-acyl shift systems is the bis(2-sulfanylethyl) amido (SEA) group 2 or 3 shown in the General Scheme [34,35]. It enables various useful chemical transformations [37–40] among which the synthesis of peptide thioesters of type 4 by an exchange reaction of the bis(2-sulfanylethyl)amino moiety with an alkylthiol such as MPA (General Scheme) [38]. The SEA group differentiates from the other members of this family by its good reactivity at mildly acidic pH, its resistance to hydrolysis and its unique redox properties [34,37,41]. Indeed, the SEA peptide is deprotected and cleaved from the resin in the form of the reactive SEAon peptide 2. Dithiol 2 can be inactivated
by mild oxidation into SEAoff peptide 3, which is highly stable as expected for an N,N-dialkylamide. Conversion of SEAoff peptide 3 into peptide thioester 4 is carried out preferably at pH 4 in the presence of the reductant tris(2-carboxyethyl) phosphine [38]. The aim of this protocol is to provide the detailed experimental procedures for the synthesis and characterization of SEA polystyrene resin 1 and its use for the synthesis of peptide thioesters 4 through steps 1–4 (General Scheme). It has been used for the chemical synthesis of several functional proteins such as the Kringle 1 domain of hepatocyte growth factor (HGF) in its native[41] or biotinylated [42] form, the heparin binding N domain of HGF [43] and large polypeptides or SEA thioester surrogates on a water-compatible solid support [7].
OLLIVIER ET AL. solution in DMSO is very practical as it allows the addition of a precise amount of iodine (2 equiv only), even for small scales. Moreover, DMSO increases significantly the solubility of iodine in the aqueous reaction mixture. This allowed a decrease in the proportion of acetic acid from 80% (as it is often reported for iodine oxidations) to 20%, thereby simplifying the purification step. Note that the slight excess of iodine is decomposed in a few seconds by addition of dithiothreitol (DTT) prior to the injection on the HPLC system. Because of the acidity of the mixture, DTT is unable to reduce disulfide bonds. In particular, SEAoff group and Cys(StBu) residues are not affected during this step. Other oxidants can be used as well such as N,N,N′,N′tetramethyl-azodicarboxamide at neutral pH, which enabled the synthesis of a SEAoff peptide functionalized by an alkylazido group [7]. This method is compatible with the presence of Cys (StBu) residues too. Step 4. Synthesis of MPA thioester 4 The last step discussed here is the exchange reaction of the SEA group by MPA to yield a C-terminal peptide thioester. This reaction goes almost to completion at pH 4 within 24 h for most C-terminal amino acid residues (37 °C). The method is compatible with C-terminal Glu residue. Aspartic acid is a special case, and the procedure for the synthesis of peptidyl aspartyl thioesters will be reported later on [44]. Valine required longer reaction times (96 h) because of the steric bulk of this amino acid [38]. Importantly, we have shown recently that the procedure could be adapted to the synthesis of MPA peptidyl prolyl thioesters [45]. In this case, the exchange reaction was very slow at 37 °C but proceeded efficiently at 65 °C. As an example, peptides ILKEPVHXP-SCH2CH2CO2H (X = Ala, Gly, Ile) were isolated with a 41–53% yield after HPLC purification. The efficiency of the exchange reaction enabled the recent design of a solid-phase elongation cycle allowing the N-to-C sequential NCL of unprotected peptide segments. Three elongation cycles – each including a SEAoff-MPA thioester exchange reaction – permitted the solid-phase ligation of five peptide segments and the isolation of a 15 kDa polypeptide in high yield (6.5% overall, nine chemical steps, 74% yield per step) [7]. Limitations
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The protocol reported here for the synthesis of SEA PS resin 1 (200–400 mesh) is not appropriate for the preparation of 100–200 mesh resins for unknown reasons. SEA PS resin 1 is highly efficient for batch synthesis using DMF and standard Fmoc-SPPS and coupling times. It can be used also with column peptide synthesizers. However, in some configurations, we observed modest yields for the first couplings steps when using DMF as the solvent. Better coupling yields were obtained by using N-methylpyrrolidone instead of DMF, which allows a better swelling of the SEA PS resin 1. An alternative that was not tested would be to use mixtures of DMF and DCM. Oxidation of SEAon group into SEAoff is recommended but not mandatory. The oxidation step can be skipped if the HPLC purification step can be performed quickly. Indeed, in this case, the N,S-acyl shift of SEAon group can occur on the HPLC column in acidic eluents. The N,S-acyl shift of SEAon amide group –CON(CH2CH2SH)2 yields SEAon thioester group – COSCH2CH2NHCH2CH2SH featuring a secondary amino group. Usually, SEAon thioester group is significantly more hydrophilic
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than SEAon amide group, resulting in significant differences in retention times (sometimes a few minutes apart). The formation of the SEA thioester form is favored at the acidic pH used for the elution due to the protonation of the secondary amine. This equilibrium results in low purification yields, unless the HPLC purification is performed rapidly. Because the kinetic rate of the N,S-acyl shift depends on the nature of the C-terminal amino acid residue, the purification of a SEAon peptide must be optimized case by case. Finally, the exchange reaction with MPA is highly efficient at pH 4 and 37 °C, but heating up to 65 °C helps for difficult amino acids such as proline. Experimental Procedure Step 1. Preparation of SEA PS resin 1 (200–400 mesh) Deprotection of the bis(2-tritylsulfanylethyl)amine HN(CH2CH2STrt)2. The synthesis must be carried out in an efficient fume hood with the appropriate protection. The bis(2-tritylsulfanylethyl) amine was prepared as described previously by reacting bis (2-chloroethyl)amine hydrochloride with triphenylmethyl mercaptan in the presence of DBU [34,46]. Both bis(2chloroethyl)amine hydrochloride and triphenylmethyl mercaptan are commercially available. Once formed, the bis(2-sulfanylethyl) amine trifluoroacetate salt HN(CH2CH2SH)2. CF3CO2H must be used immediately. Step 1.1.1. Weigh the bis(2-tritylsulfanylethyl)amine (2.48 g, 4.00 mmol) in a 200 ml round bottom flask equipped with a magnetic bar. Step 1.1.2. Add a mixture of TFA/triisopropylsilane (TIS): 97.5/2.5 by volume (100 ml). Step 1.1.3. Stir the reaction mixture at room temperature for 30 min. Step 1.1.4. Then, evaporate the solvent using a rotary evaporator under reduced pressure to obtain a white solid. Step 1.1.5. Dissolve the white solid in cyclohexane (~50 ml) and evaporate the solvent again as previously mentioned and repeat this step twice. This step allows removal of the residual TFA. Step 1.1.6. Dry the bis(2-sulfanylethyl)amine trifluoroacetic acid salt HN(CH2CH2SH)2. CF3CO2H in vacuo for 20 min. Conditioning of the trityl chloride resin. Step 1.2.1. Weigh the trityl chloride resin (40 mmol, 28.57 g, 1.4 mmol/g from IRIS Biotech GmbH, 200–400 mesh, ref BR1145) in a glass reactor equipped with a sintered glass and a tap at the bottom and a skirted silicone stopper on top. Caution: Trityl chloride resin is sensitive to hydrolysis. A tenfold excess of trityl chloride functions relative to the bis(2sulfanylethyl)amine trifluoroacetate is used in this protocol. Step 1.2.2. Swell the resin in CH2Cl2 for 10 min. Step 1.2.3. Filter and flush immediately with argon from the bottom (the cap must be open during this operation). Stop the argon flush and close the tap and cap in this order. Step 1.2.3. Add 30 ml of DMF through the skirted silicone stopper with a syringe and shake for 5 min.
Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.
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SEA PS RESIN FOR PEPTIDE THIOESTER SYNTHESIS Step 1.2.4. Filter. Coupling of the bis(2-sulfanylethyl)amine trifluoroacetate salt to the resin. Step 1.3.1. Solubilize the bis(2-sulfanylethyl)amine trifluoroacetate salt in DMF (25 ml) and add this solution to the resin through the cap using a syringe. Wash the round bottom flask with DMF (5 ml) and add this solution to the resin as previously mentioned. Step 1.3.2. Shake overnight. Step 1.3.3. Mix MeOH (1.5 ml) and 2,5-lutidine (4.45 ml) and add the mixture to the aforementioned suspension. Working under argon atmosphere is no longer necessary. Step 1.3.4. Agitate for 30 min. Step 1.3.5. Wash the beads with DMF, MeOH, DMF, DIEA in DMF (5% by volume), DMF, CH2Cl2 and Et2O (2 × 2 min for each solvent). Step 1.3.6. Dry the beads under vacuum and store at 4 °C. The resin is very stable and can be stored for months at 4 °C. Characterization of SEA PS resin 1. Step 1.4.1. Perform an Ellman assay to detect the presence of free thiols. For this, prepare a solution of 5,5′-dithiobis(2nitrobenzoic acid) (DTNB) in DMF (2 mg/ml). Add one drop of the DTNB solution to an aliquot of the SEA PS resin 1 and then one drop of 5% DIEA in DMF. Agitate for 5 min. Use another resin for the negative control. A positive assay is characterized by the development of a yellow color in solution. This assay must be negative. Step 1.4.2. Perform a chloranil assay [47] to detect the presence of secondary amines. For this, prepare a chloranil solution in DMF (20 mg/ml) and an acetaldehyde solution in DMF (2% by volume). Add four drops of each solution to an aliquot of the SEA PS resin 1. Agitate for 5 min. This assay must be strongly positive, that is, the beads must be blue. Step 1.4.3. Determine the loading of the SEA PS resin 1. For this, couple Fmoc-Gly-OH (150 mg, 0.50 mmol) to the SEA resin (357 mg, expected 0.05 mmol) using standard HATU (190 mg, 0.49 mmol)/DIEA (260 μl, 1.5 mmol) activation in DMF (200 μl). Fmoc-Gly-OH was pre-activated 1 min before being added to the beads swelled in DMF. The beads were agitated for 1 h 30 min at room temperature and then washed with DMF (3 × 2 min). Wash the resin with CH2Cl2 (3 × 2 min) and Et2O (3 × 2 min) and dry in vacuo. Perform a chloranil assay, which must be negative. Weight three aliquots (m = 3–5 mg) of the resin and add a solution of piperidine in DMF (20% by volume, 1 ml). Transfer each solution to a 10 ml volumetric flask and dilute with DMF up to 10 ml. Measure the UV absorbance at 290 nm using the piperidine in DMF solution for the blank. Calculate the 290 loading using the equation: loading (mmol/g) = 2:02 Absm (where m is the mass of resin in milligrams). The loading should be 0.14–0.16 mmol/g.
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Step 2.2. Typical procedure for batchwise peptide elongation using Fmoc-SPPS. Peptide elongation was performed using standard Fmoc/tert-butyl SPPS protocols at room temperature on an automated batch peptide synthesizer (0.1–0.5 mmol scale). Couplings were performed using a fivefold molar excess of each Fmoc-L-amino acid, a 4.5-fold molar excess of HBTU and a tenfold molar excess of DIEA (30 min coupling time, double couplings). A capping step was performed after each coupling with a mixture of 10% Ac2O and 5% DIEA in DMF. At the end of the synthesis, the resin was washed with CH2Cl2 (2 × 2 min), diethylether (2 × 2 min) and dried in vacuo. Step 2.3. Typical procedure for the cleavage and deprotection step. Isolation of the crude SEAon peptide 2. Deprotection and cleavage were performed with TFA in the presence of the appropriate scavengers. TIS, DMS, thioanisole, anisole, thiophenol and water are compatible with the synthesis of SEA peptides. The mixture TFA/TIS/DMS/H2O/thioanisole: 90/2.5/2.5/ 2.5/2.5 by volume (40 ml for 0.5 mmol scale) is appropriate for a large diversity of peptide sequences and lengths. The beads were filtered, and the TFA solution was added dropwise to a cold mixture of Et2O/n-heptane: 1/1 by volume (500 ml). We recommend performing the cleavage/precipitation procedure at least twice. As an example, for the synthesis of peptide IRNC(StBu) IIGKGRSYKGT15V16SITKSGIK-SEAon, the peptidyl resin (0.5 mmol scale) was treated three times with the TFA mixture described earlier (3 h and 2 × 1 h) to give 797 (44.5%), 303 (17%) and 54 mg (3%) of the crude peptide (total crude yield 64.5%). In this case, the synthesis was complicated by a difficult coupling of threonine residue 15 on valine residue 16 (see later). Step 3. Typical procedure for the oxidation step and purification of SEAoff peptide 3 SEAon peptide 2 (25 μmol) was dissolved in AcOH/H2O: 1/4 by volume (final peptide concentration 0.5 mM). Iodine solution (200 mM in DMSO, 50 μmol, 250 μl) was added in one portion. After 30 s, DTT (65 mM in water, 50 μmol, 775 μl) was added to quench the excess of iodine. Once DTT was added, the mixture was immediately purified by RP HPLC using a linear water–acetonitrile gradient containing 0.05% of TFA to give SEAoff peptide 3. Using this procedure, we isolated 70 mg (4%) of the peptide IRNC(StBu)IIGKGRSYKGTVSITKSGIK-SEAoff (0.5 mmol scale). The modest yield is due to the presence, in significant amounts, of the acetylated peptide Ac-VSITKSGIK-SEAoff in the crude product. In a subsequent synthesis, we obtained much better yields for this SEAoff peptide by optimizing the peptide elongation step (27%, see Ref. [41]).
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Step 2. Fmoc-SPPS of the SEAon peptide 2
Step 2.1. Coupling of the first amino acid. This step was performed manually. SEA PS resin 1 (0.5 mmol, 0.16 mmol/g) was conditioned in CH2Cl2 (3 × 2 min) and DMF (3 × 2 min). Fmoc-Aa-OH (5.00 mmol, 2.43 g for Fmoc-L-Lys(Boc)-OH, see the example provided in §3) and HATU (4.90 mmol; 1.90 g) were dissolved in the minimal volume of DMF. DIEA (10 mmol, 1741 μl) were added to the aforementioned solution, which was agitated at room temperature (1 min) and then added to the beads. The resin was agitated during 2 h and washed with DMF (3 × 2 min). Control the coupling step using the chloranil assay and repeat the coupling step if necessary. The resin was then treated with Ac2O/DIEA/CH2Cl2: 10/5/85 by volume (10 ml, 2 × 10 min) to cap unreacted secondary amino groups. Finally, the resin was washed with DMF (3 × 2 min).
OLLIVIER ET AL. Step 4. Typical procedure for the synthesis of MPA thioester 4 By using the general optimized procedure, SEAoff peptide IRNC(StBu)IIGKGRSYKGTVSITKSGIK-SEAoff was converted into MPA thioester IRNCIIGKGRSYKGTVSITKSGIK-SCH2CH2CO2H with an isolated yield of 41% following HPLC purification (25 mg scale) [41]. In another experiment, we isolated the same MPA thioester in 66% yield by desalting the crude product on a C18 solid-phase extraction column. Most of the time, a high-resolution HPLC purification step is not required because (i) the starting SEAoff peptide 3 is purified in the previous step and (ii) the exchange reaction is very clean and proceeds in high yields. Note that Cys(StBu) residues are reduced into Cys during this step.
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22 23 24
25
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