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Mechanistic insight into benzenethiol catalyzed amide bond formations from thioesters and primary amines† Nicolai Stuhr-Hansen,a,b Nicolai Bork*c,d and Kristian Strømgaarda The influence of arylthiols on cysteine-free ligation, i.e. the reaction between an alkyl thioester and a primary amine forming an amide bond, was studied in a polar aprotic solvent. We reacted the ethylthioester of hippuric acid with cyclohexylamine in the absence or presence of various quantities of thiophenol (PhSH) in a slurry of disodium hydrogen phosphate in dry DMF. Quantitative conversions into the resulting amide were observed within a few hours in the presence of equimolar amounts of thiophenol. Ab initio calculations showed that the reaction mechanism in DMF is similar to the well-known aqueous reaction mechanism. The energy barrier of the catalyzed amidation reaction is approximately 40 kJ mol−1 lower than the non-catalyzed amidation reaction. At least partially this can be explained by a hydrogen bond
Received 29th January 2014, Accepted 3rd June 2014
from the amine to the π-electrons of the thiophenol, stabilizing the transition state in the aromatic thioester amidation reaction. Under similar conditions, cysteine-free ligation was achieved by coupling a fully
DOI: 10.1039/c4ob00073k
side-chain protected 15 amino acid phosphopeptide thioester to the free N-terminal of a side-chain pro-
www.rsc.org/obc
tected 9 amino acid peptide producing the corresponding 24 amino acid phosphopeptide.
Introduction The concept of native chemical ligation (NCL) introduced by Kent and co-workers1 involves the reaction of a peptide C-terminal thioester with an N-terminal cysteine of another peptide. NCL has developed into a widely used method for coupling peptides and highly functionalized proteins such as synthetic erythropoietin (EPO).2,3 Overall, the reaction is an amide bond formation allowing for ligation of peptide fragments, with the primary restriction that a cysteine is present at the ligation site or in certain cases a few amino acids away.4 NCL based methods have almost exclusively been performed in aqueous media, but Dittmann and co-workers succeeded in performing NCL in DMF.5 Furthermore, Wong and co-workers6 reported cysteine-free ligation7 in a 1 : 4 mixture of N-methyl-2-pyrrolidone–water. The question arose whether the polar aprotic solvent facilitated the amide bond formation and
a Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark b Department of Chemistry, Faculty of Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark c Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark d Department of Physics, Division of Atmospheric Sciences, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ob00073k
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prompted studies of cysteine-free ligation in a polar aprotic solvent. In standard NCL protocols8 arylthiols must be present for catalyzing the amide bond formation when reacting a peptide alkyl thioester with a cysteine N-terminated peptide. This encouraged us to study amide formation in the absence and presence of arylthiols in different stoichiometric ratios to obtain mechanistic insight into cysteine-free ligation in organic solvents. Here, we report the kinetic studies of the reaction between a model peptide thioester and a primary amine in the presence of zero to two equivalents of thiophenol and Na2HPO4 in DMF. The mechanism was investigated using ab initio electronic structure calculations and a simple kinetic model. By selecting the most optimal cysteine-free ligation conditions, a fully protected phosphorylated peptide thioester was reacted with the free N-terminal of a side chain protected peptide, affording the coupled phosphorylated peptide after global deprotection.
Results and discussion Aiming at the best possible representation of the local structural environment in peptides at the ligation site, we chose two model compounds. As a representative of the peptide thioester, S-ethyl thiohippurate 1 was chosen as a glycine thioester mimicking a peptide-Gly-SEt. As a model for a free N-terminal of a peptide, cyclohexylamine 2 was selected as a slightly
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Scheme 1 Reaction of S-ethyl thiohippurate 1 with cyclohexylamine 2 performed in a slurry of Na2HPO4 in DMF forming N-cyclohexyl hippuramide 3 in the absence or presence of thiophenol.
Fig. 1 GC-MS of a representative quenched reaction between the thioester 1 and cyclohexylamine 2 in the presence of thiophenol forming N-cyclohexyl hippuramide 3.
hindered primary amine resembling an amino acid. Compounds 1 and 2 were reacted under formation of N-cyclohexyl hippuramide 3. Compounds 1, 2, and 3 are soluble in DMF and ethyl acetate, which facilitated analysis by GC-MS. The experiments were performed by reacting solutions of 0.05 M 1 and 0.06 M 2 in DMF forming 3 in the presence of PhSH in quantities ranging from zero to two equivalents relative to 1 (Scheme 1). As expected, no reaction was observed in the absence of an external base, nor did triethylamine or DIPEA have any noticeable effect on the reaction rate. Na2HPO4 is the most widely utilized base in aqueous ligations and although only minutely soluble in DMF, adding an excess of Na2HPO4 as a slurry was found to initiate the amide formation. Samples were quenched in water at different timepoints, extracted with ethyl acetate, and the extracts were analyzed by GC-MS (Fig. 1). Sufficiently good EI-signals for monitoring the progress of the reaction were hereby obtained. The thioester 1 and the amide 3 did not have the same ionization probabilities in the mass detector, which meant that the kinetic data based on relative ratios in chromatograms had to be correlated by a factor found by performing GC-MS experiments on samples containing known amounts of 1 and 3. In order to check whether the sum of 1 and 3 in the chromatograms consistently represented the entire amount of the hippuric moiety, internal calibrations with p-phenyl phenol were performed on selected quenched samples, since this inert compound was found to display an ionization probability factor in between 1 and 3. The amide 3 was isolated in 84% yield from reactions run in 1 M in DMF in the presence of two equivalents of thiophenol. From Fig. 2 it is clear that, as in aqueous media, PhSH has a dramatic effect on the reaction rate. In the absence of PhSH, practically no amide formation was observed. However, adding 0.5 equivalent of PhSH, t1/2 was reduced to ca. 70 minutes. Adding two equivalents of PhSH, t1/2 was
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Fig. 2 Plot of ln([1][2]−1) as a function of time (data tabulated in Table S1†). The linear dependence is in accordance with the catalyzed reaction mechanism with the amidation of the arylthioester as the rate limiting step. Within 90 minutes, the reaction with 2 eq. of PhSH has run to completion wherefore the linear dependence breaks down.
Scheme 2 Proposed mechanistic pathways. Uncatalyzed and thiophenol catalyzed amidation of S-ethyl thioacetate by methylamine.
below ten minutes and the reaction had run to completion within 90 minutes. Kinetics In aqueous media, both experimental and theoretical studies have confirmed that the catalytic mechanism proceeds via trans-thioesterification from an attack of arylthiolate, followed by amidation of the arylthioester9–12 (Scheme 2). We tested the mechanistic effect of DMF using ab initio calculations, which have successfully been employed in several related studies.2,12–14 We used the B3LYP15,16 density functional with the 6-31+G* basis set17 to optimize the reactants, products, and transition states. Solvent effects were accounted for through the polarized continuum model (PCM).18 Vibrational frequencies and thermal corrections to the electronic energy were calculated in the rigid rotor-harmonic oscillator approximation. In all cases, each reactant and product had only positive frequencies, while each transition state had exactly one imaginary frequency mode. Intrinsic reaction coordinate calculations confirmed that this mode corresponded to the reaction coordinate of interest. To improve the reliability of the calcu-
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lations, single point CCSD(T)/6-31+G* coupled cluster calculations were performed. The Gibbs free energy, ΔG, was then determined as
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ΔG ¼ ΔGB3LYP ΔEB3LYP ΔE*CCSDðTÞ
ð1Þ
where ΔE denotes the electronic energy and * indicates that the structure is not optimized at that level of theory. CCSD(T) is fundamentally more accurate than B3LYP, but due to computational expense, it imposes restrictions on the size of the system. The computational model system was thus restricted to S-ethyl thioacetate reacting with methylamine in the presence or absence of PhSH. Inevitably, the choice of a smaller model system imposes discrepancies, but these errors are more than compensated for by the improved ΔG from eqn (1). All calculations were performed using the Gaussian 09 software package.19 The calculated potential energy surfaces of the reactions RSEt þ CH3 NH2 ! RSNHCH3 þ EtSH
ðR1Þ
RSEt þ PhS ! RSPh þ EtS
ðR2Þ
RSPh þ CH3 NH2 ! RSNHCH3 þ PhSH
ðR3Þ
EtS þ PhSH ! EtSH þ PhS
ðR4Þ
where R = COCH3, and are shown in Fig. 3. Thermodynamic details are available as ESI.† The catalyzed reaction was confirmed to proceed via a two-step process, both steps having an activation energy of ca. 105 kJ mol−1, compared to ca. 140 kJ mol−1 from the non-catalyzed reaction. See also Scheme 2. The optimized structures of the transition states of reactions R1, R2 and R3 are shown in Fig. 4. Using the Atoms-In-Molecules (AIM) approach20 we performed a bond critical point analysis on the TS3 structure
Fig. 4 Optimized structures (B3LYP/6-31+G*) of the three transition states shown in Scheme 2 and Fig. 3. Some descriptive bond distances are given. The transition state of the catalyzed amidation reaction, TS3, is stabilized by hydrogen bonding (dashed line) from an amine proton to the π-electrons of the benzene ring. An AIM bond critical point analysis is shown in Fig. S1.†
(Fig. 4). We found a hydrogen to π-electron bond stabilizing the catalyzed transition state compared to the non-catalyzed transition state. At least partially, this may explain the catalytic effect of PhSH. The bond critical points are shown in Fig. S1.† Since the two barriers in the catalyzed reaction mechanism are of similar magnitude, we investigated which of the reactions was rate-limiting. The trans-thioesterification is a pseudo first order reaction due to the regeneration of the PhS− catalyst, but the kinetic data could not be fitted to a first order reaction rate expression. The amidation reaction, on the other hand, is overall second order and the kinetic data were readily fitted to the standard second order reaction rate expression lnð½1½21 Þ ¼ at þ logð½10 ½20 1 Þ where a is a parameter, t is the time and the subscript 0 denotes the concentration at t = 0. This confirms that the amidation is the rate-limiting step (Fig. 2). The catalytic effect was investigated using a standard kinetic model (see ESI†). We find that the catalytic effect can be expressed as r cat r nocat
Fig. 3 Potential energy surface of the amidation of 1’ by 2’ in the presence (red) and absence (green) of PhSH. See also Scheme 2. Energies are from B3LYP/6-31+G* with CCSD(T)/6-31+G* electronic energy corrections. Transition state structures are shown in Fig. 4. Reaction R4 is the proton transfer from PhSH to EtS−, regenerating the PhS− catalyst and preventing the back reaction over TS1 by removing EtS− from the mixture.
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¼
½PhSH0 ½RHNCH3 ΔG2 þ ΔG4 þ EA3 EA1 exp ½RNHCH3 kB T ð2Þ
where R denotes the –COCH3 group, kB the Boltzmann constant, and T the absolute temperature. EA1, ΔG2, EA3 and ΔG4 are Gibbs free reaction energies (ΔG) or Gibbs free energy barriers (EA) of reactions R1, R2, R3 and R4, respectively. Taking the experiment with 2 equivalents of PhSH as an example, the catalytic speed-up is then predicted to be at least 105.
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Scheme 3 Cysteine-free ligation between a side-chain protected peptide and a fully protected phosphorylated peptide thioester producing the corresponding phosphorylated 24-peptide. = Fmoc SPPS side-chain protection = 2-chlorotrityl resin HFIP = 1,1,1,3,3,3-Hexafluoroisopropanol TFA/TIPS = trifluoroacetic acid/triisopropylsilane COMU = (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate.
Synthesis of a phosphopeptide by cysteine-free ligation For broader application of the reaction conditions, these must be utilized for coupling of peptide fragments preferably containing post-translational modifications. The logical limitation to cysteine-free ligation is that free side-groups, especially lysines, also react with the thioester moiety and side-chain protection is therefore required. This was overcome using our approach21 for handling protected peptides in organic solvents, in which all amino acid side-chains remained protected with their respective protecting group from standard Fmoc-based SPPS (solid phase peptide synthesis). A 9 amino acid peptide FVTRQPNKV was prepared by standard Fmoc SPPS on 2-chlorotrityl resin. The N-terminal Fmocgroup was removed ( piperidine/DMF) followed by cleavage from the resin using 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) affording the N- and C-terminally non-protected fully sidechain protected nonapeptide 4. We recently reported the synthesis of a fully protected 15 amino acid phosphorylated peptide thioester 522 prepared by standard SPPS on 2-chlorotrityl resin of a phosphorylated tetradecapeptide FYWT(PO3H2)SRQPNKHEDV utilizing Fmoc-O-(benzylphospho)-L-threonine [Fmoc-Thr-(PO3BnH)-OH] as a means of introduction of phosphorylated threonine.23 Successive cleavage from the resin HFIP and (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate24 (COMU) coupling the free C-terminal with the amino thioester of alanine afforded the N-terminally Boc-protected fully sidechain protected peptide thioester 5 with negligible racemization at the C-terminal amino acids.25 Cysteine-free ligation of 4 and 5 was successfully performed under coupling conditions similar to the above procedure followed by global cleavage (TFA–TIPS–H2O) of protecting groups affording the corresponding free phosphorylated 24-peptide 6 after HPLC purification (Scheme 3, Fig. 5).
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Conclusion In conclusion, it was shown that thioesters could be coupled with primary amines in DMF in the presence of PhSH using Na2HPO4 as a base. The effect of PhSH concentration on the reaction rate was studied kinetically showing no ligation in the absence of PhSH and quantitative conversion within 90 minutes in the presence of two equivalents of PhSH. The reaction mechanism was studied using ab initio calculations. Similar to the aqueous mechanism, we found evidence of a two-step reaction mechanism where trans-thioesterification precedes amidation. Further, we found that the aromatic ring stabilizes the transition state through H–π bonding with an amine proton, at least partially explaining the catalytic effect of PhSH. Furthermore, it was possible to expand the use of the method to coupling of a protected peptide thioester containing a phosphoryl moiety with a free N-terminal of a side-chain protected peptide affording a coupled peptide with an intact phosphoryl group. The method is considered to be of broad scope, since it can be utilized for ligating cysteine-free post translationally modified peptides with side-chain protection preserved by cleaving peptides from 2-chlorotrityl resin using 1,1,1,3,3,3-hexafluoroisopropanol.
Experimental section Chemistry All reagents were obtained from commercial suppliers and used without further purification. Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance (300 MHz). TLC analysis was performed on silica gel F254 (Merck) and detection was carried out by examination under UV light and staining with potassium permanganate. Automated flash column chromatography was performed on a Tele-
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Fig. 5 HPLC chromatograms. (a) HPLC (LC-MS chromatogram) of the crude reaction mixture after reaction between N- and C-terminally non-protected fully side-chain protected Cys-terminated nonapeptide 4 and the N-terminally Boc-protected fully side-chain protected peptide thioester 5 (Scheme 3). (b) Analytical HPLC of the purified phosphorylated peptide 6.
dyne ISCO CombiFlash Rf using silica gel columns with solvent of HPLC grade. Elemental analyses were performed by Mr J. Theiner, Department of Physical Chemistry, University of Vienna, Austria. Preparative HPLC was performed on an Agilent 1100 system using a C18 reverse phase column (Zorbax 300 SB-C18, 21.2 × 250 mm) with a linear gradient of the binary solvent system of H2O–ACN–TFA (A: 95/5/0.1 and B: 5/95/0.1) with a flow rate of 20 mL min−1. Analytical HPLC was performed on an Agilent 1100 system with a C18 reverse phase column (Zorbax 300 SB-C18 column, 4.6 × 150 mm) with a flow rate of 1 mL min−1, and a linear gradient of the binary solvent system of H2O–ACN–TFA (A: 95/5/0.1 and B: 5/95/0.1). Mass spectra were obtained with an Agilent 6410 Triple Quadrupole Mass Spectrometer instrument using electron spray coupled to an Agilent 1200 HPLC system (ESI-LC/MS) with a C18 reverse phase column (Zorbax Eclipse XBD-C18, 4.6 × 50 mm), an autosampler and a diode-array detector using a linear gradient of the binary solvent system of H2O–ACN– formic acid (A: 95/5/0.1 and B: 5/95/0.086) with a flow rate of 1 mL min−1. During ESI-LC/MS analysis evaporative light scattering (ELS) traces were obtained with a Sedere Sedex 85 Light Scattering Detector. Compound identity of all tested compounds was confirmed by ESI-LC/MS, which also provided purity data (all >95%; UV and ELSD). High resolution mass spectra (HRMS) were obtained using a Micromass Q-Tof 2 instrument and were all within ±5 ppm of theoretical values.
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GC-MS was performed on an Agilent 6890 Series GC-MS instrument. Reaction between S-ethyl thiohippurate and cyclohexylamine in the presence or absence of thiophenol: kinetic studies. To a stirred solution of S-ethyl thiohippurate 1 (0.022 g, 0.1 mmol) and cyclohexylamine 2 (0.014 mL, 0.12 mmol) in DMF (2 mL) were added Na2HPO4 (0.13 g, 0.5 mmol) and an appropriate amount of thiophenol (from 0 mL to 0.019 mL, 0.2 mmol), and stirring was maintained at room temperature. At times the samples (0.1 mL) were quenched with water (2 mL) and extracted with ethyl acetate (1 mL) and the organic phase was analyzed by GC-MS. N-Cyclohexyl hippuramide 3 To a stirred solution of S-ethyl thiohippurate 1 (0.22 g, 1 mmol) and cyclohexylamine 2 (0.14 mL, 0.12 mmol) in DMF (2 mL) were added Na2HPO4 (1.3 g, 5 mmol) and benzenethiol (0.19 mL, 2 mmol). The reaction mixture was stirred at room temperature for 2 hours and poured into water (30 mL) and extracted with ethyl acetate (3 × 15 mL). The pooled organic phases were washed with water, dried over sodium sulfate, filtered and evaporated. Separation was performed on a silica column using automated flash chromatography with ethyl acetate, which gave the amide 3 (0.22 g, 84%) as a colorless crystalline material; mp 164–165 °C (lit.26 165 °C). Anal. Calcd for C15H20N2O2: C, 69.20; H, 7.74; N, 10.76. Found: C, 69.34;
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H, 7.96; N, 10.62. Purity >98% (GC-MS). Mass spectrum (EI; m/z, relative intensity): 260 (M+, 7), 179 (14), 162 (20), 134 (100), 105 (89). 1H NMR (300 MHz, CDCl3): δ 1.01–1.34 (s, 5H), 1.49–1.55 (m, 1H), 1.60–1.68 (m, 2H), 1.78–1.85 (m, 2H), 3.64–3.76 (m, 1H), 4.08 (d, J = 5.1 Hz, 2H), 6.83 (br-s, 1H), 7.32–7.46 (m, 3H) 7.58 (br-s, 1H), 7.77–7.81 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 24.74, 25.49, 33.06, 44.02, 48.50, 127.21, 128.54, 131.77, 133.61, 167.85, 168.12.
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H-Phe-Tyr-Trp-Thr(PO3H2)-Ser-Arg-Gln-Pro-Asn-Lys-His-GluAsp-Val-Ala-Phe-Val-Thr-Arg-Gln-Pro-Asn-Lys-Val-OH 6 The fully side chain protected peptide sequence FVTRQPNKV with free N- and C-terminals 4 was synthesized by Fmoc-based solid-phase peptide synthesis (SPPS) using a microwave peptide synthesizer: Liberty 1 (CEM) on 2-chlorotrityl chloride polystyrene resin preloaded with valine (loading 0.78 mmol g−1, 0.16 g, 0.125 mmol). Coupling of the consecutive amino acid was carried out with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and DIPEA (resin–amino acid–HBTU–DIPEA 1 : 4 : 4 : 4) in dry DMF (2 mL) for 30 min. After draining and flow wash with DMF, Fmoc deprotection was performed with 20% piperidine in DMF (1 × 5 and 1 × 15 min; 2 mL in each; DMF wash step in between). Fmoc-deprotection with piperidine was also carried out after attaching the last amino acid. The resin was treated with 1,1,1,3,3,3-hexafluoroisopropanol–dichloromethane (1 : 4, 2 mL) for 15 minutes, filtered, and evaporated. Co-evaporation twice with acetonitrile and drying in a vacuum oven (10 Pa, 35 °C, 12 hours) gave 4 as a fluffy white powder, which was added to a slurry of the fully protected 15 amino acid phosphorylated peptide thioester 5 (0.1 mmol), Na2HPO4 (0.13 g, 0.5 mmol) and thiophenol (0.019 mL, 0.2 mmol) in DMF (1 mL) and stirring was maintained at room temperature for 4 hours. After pouring into HCl (0.1 M, 10 mL, 1 mmol) a white precipitate is filtered off, washed thoroughly with water and dried in a vacuum oven (10 Pa, 25 °C, 12 hours). An inhomogeneous mixture of TFA–triisopropylsilane (TIPS)–H2O (18 : 1 : 1, 3 mL) was added and shaking was maintained for 1 h. After evaporation in a gentle stream of nitrogen the crude peptide was washed with cold ether (3 × 5 mL) to give a colorless waxy product. Separation by HPLC, evaporation and lyophilization gave the pure phosphorylated peptide 6 (0.18 g, 59%) as an off-white solid. Purity >97%, analytical HPLC. HRMS (ES+) calcd for C134H204N39O40P [M + 4H]4+, 757.6216; found, m/z 757.6211.
References 1 P. Dawson, T. Muir, I. Clark-Lewis and S. Kent, Synthesis of proteins by native chemical ligation, Science, 1994, 266, 776–779. 2 P. Wang, S. Dong, J. A. Brailsford, K. Iyer, S. D. Townsend, Q. Zhang, R. C. Hendrickson, J. Shieh, M. A. S. Moore and S. J. Danishefsky, At Last: Erythropoietin as a Single Glycoform, Angew. Chem., Int. Ed., 2012, 51, 11576–11584.
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Mechanistic insight into benzenethiol catalyzed amide bond formations from thioesters and primary amines† Nicolai Stuhr-Hansen,a,b Nicolai Bork*c,d and Kristian Strømgaarda The influence of arylthiols on cysteine-free ligation, i.e. the reaction between an alkyl thioester and a primary amine forming an amide bond, was studied in a polar aprotic solvent. We reacted the ethylthioester of hippuric acid with cyclohexylamine in the absence or presence of various quantities of thiophenol (PhSH) in a slurry of disodium hydrogen phosphate in dry DMF. Quantitative conversions into the resulting amide were observed within a few hours in the presence of equimolar amounts of thiophenol. Ab initio calculations showed that the reaction mechanism in DMF is similar to the well-known aqueous reaction mechanism. The energy barrier of the catalyzed amidation reaction is approximately 40 kJ mol−1 lower than the non-catalyzed amidation reaction. At least partially this can be explained by a hydrogen bond
Received 29th January 2014, Accepted 3rd June 2014
from the amine to the π-electrons of the thiophenol, stabilizing the transition state in the aromatic thioester amidation reaction. Under similar conditions, cysteine-free ligation was achieved by coupling a fully
DOI: 10.1039/c4ob00073k
side-chain protected 15 amino acid phosphopeptide thioester to the free N-terminal of a side-chain pro-
www.rsc.org/obc
tected 9 amino acid peptide producing the corresponding 24 amino acid phosphopeptide.
Introduction The concept of native chemical ligation (NCL) introduced by Kent and co-workers1 involves the reaction of a peptide C-terminal thioester with an N-terminal cysteine of another peptide. NCL has developed into a widely used method for coupling peptides and highly functionalized proteins such as synthetic erythropoietin (EPO).2,3 Overall, the reaction is an amide bond formation allowing for ligation of peptide fragments, with the primary restriction that a cysteine is present at the ligation site or in certain cases a few amino acids away.4 NCL based methods have almost exclusively been performed in aqueous media, but Dittmann and co-workers succeeded in performing NCL in DMF.5 Furthermore, Wong and co-workers6 reported cysteine-free ligation7 in a 1 : 4 mixture of N-methyl-2-pyrrolidone–water. The question arose whether the polar aprotic solvent facilitated the amide bond formation and
a Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark b Department of Chemistry, Faculty of Sciences, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark c Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen, Denmark d Department of Physics, Division of Atmospheric Sciences, University of Helsinki, P.O. Box 64, FI-00014 Helsinki, Finland. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ob00073k
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prompted studies of cysteine-free ligation in a polar aprotic solvent. In standard NCL protocols8 arylthiols must be present for catalyzing the amide bond formation when reacting a peptide alkyl thioester with a cysteine N-terminated peptide. This encouraged us to study amide formation in the absence and presence of arylthiols in different stoichiometric ratios to obtain mechanistic insight into cysteine-free ligation in organic solvents. Here, we report the kinetic studies of the reaction between a model peptide thioester and a primary amine in the presence of zero to two equivalents of thiophenol and Na2HPO4 in DMF. The mechanism was investigated using ab initio electronic structure calculations and a simple kinetic model. By selecting the most optimal cysteine-free ligation conditions, a fully protected phosphorylated peptide thioester was reacted with the free N-terminal of a side chain protected peptide, affording the coupled phosphorylated peptide after global deprotection.
Results and discussion Aiming at the best possible representation of the local structural environment in peptides at the ligation site, we chose two model compounds. As a representative of the peptide thioester, S-ethyl thiohippurate 1 was chosen as a glycine thioester mimicking a peptide-Gly-SEt. As a model for a free N-terminal of a peptide, cyclohexylamine 2 was selected as a slightly
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Scheme 1 Reaction of S-ethyl thiohippurate 1 with cyclohexylamine 2 performed in a slurry of Na2HPO4 in DMF forming N-cyclohexyl hippuramide 3 in the absence or presence of thiophenol.
Fig. 1 GC-MS of a representative quenched reaction between the thioester 1 and cyclohexylamine 2 in the presence of thiophenol forming N-cyclohexyl hippuramide 3.
hindered primary amine resembling an amino acid. Compounds 1 and 2 were reacted under formation of N-cyclohexyl hippuramide 3. Compounds 1, 2, and 3 are soluble in DMF and ethyl acetate, which facilitated analysis by GC-MS. The experiments were performed by reacting solutions of 0.05 M 1 and 0.06 M 2 in DMF forming 3 in the presence of PhSH in quantities ranging from zero to two equivalents relative to 1 (Scheme 1). As expected, no reaction was observed in the absence of an external base, nor did triethylamine or DIPEA have any noticeable effect on the reaction rate. Na2HPO4 is the most widely utilized base in aqueous ligations and although only minutely soluble in DMF, adding an excess of Na2HPO4 as a slurry was found to initiate the amide formation. Samples were quenched in water at different timepoints, extracted with ethyl acetate, and the extracts were analyzed by GC-MS (Fig. 1). Sufficiently good EI-signals for monitoring the progress of the reaction were hereby obtained. The thioester 1 and the amide 3 did not have the same ionization probabilities in the mass detector, which meant that the kinetic data based on relative ratios in chromatograms had to be correlated by a factor found by performing GC-MS experiments on samples containing known amounts of 1 and 3. In order to check whether the sum of 1 and 3 in the chromatograms consistently represented the entire amount of the hippuric moiety, internal calibrations with p-phenyl phenol were performed on selected quenched samples, since this inert compound was found to display an ionization probability factor in between 1 and 3. The amide 3 was isolated in 84% yield from reactions run in 1 M in DMF in the presence of two equivalents of thiophenol. From Fig. 2 it is clear that, as in aqueous media, PhSH has a dramatic effect on the reaction rate. In the absence of PhSH, practically no amide formation was observed. However, adding 0.5 equivalent of PhSH, t1/2 was reduced to ca. 70 minutes. Adding two equivalents of PhSH, t1/2 was
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Fig. 2 Plot of ln([1][2]−1) as a function of time (data tabulated in Table S1†). The linear dependence is in accordance with the catalyzed reaction mechanism with the amidation of the arylthioester as the rate limiting step. Within 90 minutes, the reaction with 2 eq. of PhSH has run to completion wherefore the linear dependence breaks down.
Scheme 2 Proposed mechanistic pathways. Uncatalyzed and thiophenol catalyzed amidation of S-ethyl thioacetate by methylamine.
below ten minutes and the reaction had run to completion within 90 minutes. Kinetics In aqueous media, both experimental and theoretical studies have confirmed that the catalytic mechanism proceeds via trans-thioesterification from an attack of arylthiolate, followed by amidation of the arylthioester9–12 (Scheme 2). We tested the mechanistic effect of DMF using ab initio calculations, which have successfully been employed in several related studies.2,12–14 We used the B3LYP15,16 density functional with the 6-31+G* basis set17 to optimize the reactants, products, and transition states. Solvent effects were accounted for through the polarized continuum model (PCM).18 Vibrational frequencies and thermal corrections to the electronic energy were calculated in the rigid rotor-harmonic oscillator approximation. In all cases, each reactant and product had only positive frequencies, while each transition state had exactly one imaginary frequency mode. Intrinsic reaction coordinate calculations confirmed that this mode corresponded to the reaction coordinate of interest. To improve the reliability of the calcu-
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lations, single point CCSD(T)/6-31+G* coupled cluster calculations were performed. The Gibbs free energy, ΔG, was then determined as
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ΔG ¼ ΔGB3LYP ΔEB3LYP ΔE*CCSDðTÞ
ð1Þ
where ΔE denotes the electronic energy and * indicates that the structure is not optimized at that level of theory. CCSD(T) is fundamentally more accurate than B3LYP, but due to computational expense, it imposes restrictions on the size of the system. The computational model system was thus restricted to S-ethyl thioacetate reacting with methylamine in the presence or absence of PhSH. Inevitably, the choice of a smaller model system imposes discrepancies, but these errors are more than compensated for by the improved ΔG from eqn (1). All calculations were performed using the Gaussian 09 software package.19 The calculated potential energy surfaces of the reactions RSEt þ CH3 NH2 ! RSNHCH3 þ EtSH
ðR1Þ
RSEt þ PhS ! RSPh þ EtS
ðR2Þ
RSPh þ CH3 NH2 ! RSNHCH3 þ PhSH
ðR3Þ
EtS þ PhSH ! EtSH þ PhS
ðR4Þ
where R = COCH3, and are shown in Fig. 3. Thermodynamic details are available as ESI.† The catalyzed reaction was confirmed to proceed via a two-step process, both steps having an activation energy of ca. 105 kJ mol−1, compared to ca. 140 kJ mol−1 from the non-catalyzed reaction. See also Scheme 2. The optimized structures of the transition states of reactions R1, R2 and R3 are shown in Fig. 4. Using the Atoms-In-Molecules (AIM) approach20 we performed a bond critical point analysis on the TS3 structure
Fig. 4 Optimized structures (B3LYP/6-31+G*) of the three transition states shown in Scheme 2 and Fig. 3. Some descriptive bond distances are given. The transition state of the catalyzed amidation reaction, TS3, is stabilized by hydrogen bonding (dashed line) from an amine proton to the π-electrons of the benzene ring. An AIM bond critical point analysis is shown in Fig. S1.†
(Fig. 4). We found a hydrogen to π-electron bond stabilizing the catalyzed transition state compared to the non-catalyzed transition state. At least partially, this may explain the catalytic effect of PhSH. The bond critical points are shown in Fig. S1.† Since the two barriers in the catalyzed reaction mechanism are of similar magnitude, we investigated which of the reactions was rate-limiting. The trans-thioesterification is a pseudo first order reaction due to the regeneration of the PhS− catalyst, but the kinetic data could not be fitted to a first order reaction rate expression. The amidation reaction, on the other hand, is overall second order and the kinetic data were readily fitted to the standard second order reaction rate expression lnð½1½21 Þ ¼ at þ logð½10 ½20 1 Þ where a is a parameter, t is the time and the subscript 0 denotes the concentration at t = 0. This confirms that the amidation is the rate-limiting step (Fig. 2). The catalytic effect was investigated using a standard kinetic model (see ESI†). We find that the catalytic effect can be expressed as r cat r nocat
Fig. 3 Potential energy surface of the amidation of 1’ by 2’ in the presence (red) and absence (green) of PhSH. See also Scheme 2. Energies are from B3LYP/6-31+G* with CCSD(T)/6-31+G* electronic energy corrections. Transition state structures are shown in Fig. 4. Reaction R4 is the proton transfer from PhSH to EtS−, regenerating the PhS− catalyst and preventing the back reaction over TS1 by removing EtS− from the mixture.
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¼
½PhSH0 ½RHNCH3 ΔG2 þ ΔG4 þ EA3 EA1 exp ½RNHCH3 kB T ð2Þ
where R denotes the –COCH3 group, kB the Boltzmann constant, and T the absolute temperature. EA1, ΔG2, EA3 and ΔG4 are Gibbs free reaction energies (ΔG) or Gibbs free energy barriers (EA) of reactions R1, R2, R3 and R4, respectively. Taking the experiment with 2 equivalents of PhSH as an example, the catalytic speed-up is then predicted to be at least 105.
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Scheme 3 Cysteine-free ligation between a side-chain protected peptide and a fully protected phosphorylated peptide thioester producing the corresponding phosphorylated 24-peptide. = Fmoc SPPS side-chain protection = 2-chlorotrityl resin HFIP = 1,1,1,3,3,3-Hexafluoroisopropanol TFA/TIPS = trifluoroacetic acid/triisopropylsilane COMU = (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate.
Synthesis of a phosphopeptide by cysteine-free ligation For broader application of the reaction conditions, these must be utilized for coupling of peptide fragments preferably containing post-translational modifications. The logical limitation to cysteine-free ligation is that free side-groups, especially lysines, also react with the thioester moiety and side-chain protection is therefore required. This was overcome using our approach21 for handling protected peptides in organic solvents, in which all amino acid side-chains remained protected with their respective protecting group from standard Fmoc-based SPPS (solid phase peptide synthesis). A 9 amino acid peptide FVTRQPNKV was prepared by standard Fmoc SPPS on 2-chlorotrityl resin. The N-terminal Fmocgroup was removed ( piperidine/DMF) followed by cleavage from the resin using 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) affording the N- and C-terminally non-protected fully sidechain protected nonapeptide 4. We recently reported the synthesis of a fully protected 15 amino acid phosphorylated peptide thioester 522 prepared by standard SPPS on 2-chlorotrityl resin of a phosphorylated tetradecapeptide FYWT(PO3H2)SRQPNKHEDV utilizing Fmoc-O-(benzylphospho)-L-threonine [Fmoc-Thr-(PO3BnH)-OH] as a means of introduction of phosphorylated threonine.23 Successive cleavage from the resin HFIP and (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate24 (COMU) coupling the free C-terminal with the amino thioester of alanine afforded the N-terminally Boc-protected fully sidechain protected peptide thioester 5 with negligible racemization at the C-terminal amino acids.25 Cysteine-free ligation of 4 and 5 was successfully performed under coupling conditions similar to the above procedure followed by global cleavage (TFA–TIPS–H2O) of protecting groups affording the corresponding free phosphorylated 24-peptide 6 after HPLC purification (Scheme 3, Fig. 5).
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Conclusion In conclusion, it was shown that thioesters could be coupled with primary amines in DMF in the presence of PhSH using Na2HPO4 as a base. The effect of PhSH concentration on the reaction rate was studied kinetically showing no ligation in the absence of PhSH and quantitative conversion within 90 minutes in the presence of two equivalents of PhSH. The reaction mechanism was studied using ab initio calculations. Similar to the aqueous mechanism, we found evidence of a two-step reaction mechanism where trans-thioesterification precedes amidation. Further, we found that the aromatic ring stabilizes the transition state through H–π bonding with an amine proton, at least partially explaining the catalytic effect of PhSH. Furthermore, it was possible to expand the use of the method to coupling of a protected peptide thioester containing a phosphoryl moiety with a free N-terminal of a side-chain protected peptide affording a coupled peptide with an intact phosphoryl group. The method is considered to be of broad scope, since it can be utilized for ligating cysteine-free post translationally modified peptides with side-chain protection preserved by cleaving peptides from 2-chlorotrityl resin using 1,1,1,3,3,3-hexafluoroisopropanol.
Experimental section Chemistry All reagents were obtained from commercial suppliers and used without further purification. Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance (300 MHz). TLC analysis was performed on silica gel F254 (Merck) and detection was carried out by examination under UV light and staining with potassium permanganate. Automated flash column chromatography was performed on a Tele-
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Fig. 5 HPLC chromatograms. (a) HPLC (LC-MS chromatogram) of the crude reaction mixture after reaction between N- and C-terminally non-protected fully side-chain protected Cys-terminated nonapeptide 4 and the N-terminally Boc-protected fully side-chain protected peptide thioester 5 (Scheme 3). (b) Analytical HPLC of the purified phosphorylated peptide 6.
dyne ISCO CombiFlash Rf using silica gel columns with solvent of HPLC grade. Elemental analyses were performed by Mr J. Theiner, Department of Physical Chemistry, University of Vienna, Austria. Preparative HPLC was performed on an Agilent 1100 system using a C18 reverse phase column (Zorbax 300 SB-C18, 21.2 × 250 mm) with a linear gradient of the binary solvent system of H2O–ACN–TFA (A: 95/5/0.1 and B: 5/95/0.1) with a flow rate of 20 mL min−1. Analytical HPLC was performed on an Agilent 1100 system with a C18 reverse phase column (Zorbax 300 SB-C18 column, 4.6 × 150 mm) with a flow rate of 1 mL min−1, and a linear gradient of the binary solvent system of H2O–ACN–TFA (A: 95/5/0.1 and B: 5/95/0.1). Mass spectra were obtained with an Agilent 6410 Triple Quadrupole Mass Spectrometer instrument using electron spray coupled to an Agilent 1200 HPLC system (ESI-LC/MS) with a C18 reverse phase column (Zorbax Eclipse XBD-C18, 4.6 × 50 mm), an autosampler and a diode-array detector using a linear gradient of the binary solvent system of H2O–ACN– formic acid (A: 95/5/0.1 and B: 5/95/0.086) with a flow rate of 1 mL min−1. During ESI-LC/MS analysis evaporative light scattering (ELS) traces were obtained with a Sedere Sedex 85 Light Scattering Detector. Compound identity of all tested compounds was confirmed by ESI-LC/MS, which also provided purity data (all >95%; UV and ELSD). High resolution mass spectra (HRMS) were obtained using a Micromass Q-Tof 2 instrument and were all within ±5 ppm of theoretical values.
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GC-MS was performed on an Agilent 6890 Series GC-MS instrument. Reaction between S-ethyl thiohippurate and cyclohexylamine in the presence or absence of thiophenol: kinetic studies. To a stirred solution of S-ethyl thiohippurate 1 (0.022 g, 0.1 mmol) and cyclohexylamine 2 (0.014 mL, 0.12 mmol) in DMF (2 mL) were added Na2HPO4 (0.13 g, 0.5 mmol) and an appropriate amount of thiophenol (from 0 mL to 0.019 mL, 0.2 mmol), and stirring was maintained at room temperature. At times the samples (0.1 mL) were quenched with water (2 mL) and extracted with ethyl acetate (1 mL) and the organic phase was analyzed by GC-MS. N-Cyclohexyl hippuramide 3 To a stirred solution of S-ethyl thiohippurate 1 (0.22 g, 1 mmol) and cyclohexylamine 2 (0.14 mL, 0.12 mmol) in DMF (2 mL) were added Na2HPO4 (1.3 g, 5 mmol) and benzenethiol (0.19 mL, 2 mmol). The reaction mixture was stirred at room temperature for 2 hours and poured into water (30 mL) and extracted with ethyl acetate (3 × 15 mL). The pooled organic phases were washed with water, dried over sodium sulfate, filtered and evaporated. Separation was performed on a silica column using automated flash chromatography with ethyl acetate, which gave the amide 3 (0.22 g, 84%) as a colorless crystalline material; mp 164–165 °C (lit.26 165 °C). Anal. Calcd for C15H20N2O2: C, 69.20; H, 7.74; N, 10.76. Found: C, 69.34;
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H, 7.96; N, 10.62. Purity >98% (GC-MS). Mass spectrum (EI; m/z, relative intensity): 260 (M+, 7), 179 (14), 162 (20), 134 (100), 105 (89). 1H NMR (300 MHz, CDCl3): δ 1.01–1.34 (s, 5H), 1.49–1.55 (m, 1H), 1.60–1.68 (m, 2H), 1.78–1.85 (m, 2H), 3.64–3.76 (m, 1H), 4.08 (d, J = 5.1 Hz, 2H), 6.83 (br-s, 1H), 7.32–7.46 (m, 3H) 7.58 (br-s, 1H), 7.77–7.81 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 24.74, 25.49, 33.06, 44.02, 48.50, 127.21, 128.54, 131.77, 133.61, 167.85, 168.12.
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H-Phe-Tyr-Trp-Thr(PO3H2)-Ser-Arg-Gln-Pro-Asn-Lys-His-GluAsp-Val-Ala-Phe-Val-Thr-Arg-Gln-Pro-Asn-Lys-Val-OH 6 The fully side chain protected peptide sequence FVTRQPNKV with free N- and C-terminals 4 was synthesized by Fmoc-based solid-phase peptide synthesis (SPPS) using a microwave peptide synthesizer: Liberty 1 (CEM) on 2-chlorotrityl chloride polystyrene resin preloaded with valine (loading 0.78 mmol g−1, 0.16 g, 0.125 mmol). Coupling of the consecutive amino acid was carried out with O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) and DIPEA (resin–amino acid–HBTU–DIPEA 1 : 4 : 4 : 4) in dry DMF (2 mL) for 30 min. After draining and flow wash with DMF, Fmoc deprotection was performed with 20% piperidine in DMF (1 × 5 and 1 × 15 min; 2 mL in each; DMF wash step in between). Fmoc-deprotection with piperidine was also carried out after attaching the last amino acid. The resin was treated with 1,1,1,3,3,3-hexafluoroisopropanol–dichloromethane (1 : 4, 2 mL) for 15 minutes, filtered, and evaporated. Co-evaporation twice with acetonitrile and drying in a vacuum oven (10 Pa, 35 °C, 12 hours) gave 4 as a fluffy white powder, which was added to a slurry of the fully protected 15 amino acid phosphorylated peptide thioester 5 (0.1 mmol), Na2HPO4 (0.13 g, 0.5 mmol) and thiophenol (0.019 mL, 0.2 mmol) in DMF (1 mL) and stirring was maintained at room temperature for 4 hours. After pouring into HCl (0.1 M, 10 mL, 1 mmol) a white precipitate is filtered off, washed thoroughly with water and dried in a vacuum oven (10 Pa, 25 °C, 12 hours). An inhomogeneous mixture of TFA–triisopropylsilane (TIPS)–H2O (18 : 1 : 1, 3 mL) was added and shaking was maintained for 1 h. After evaporation in a gentle stream of nitrogen the crude peptide was washed with cold ether (3 × 5 mL) to give a colorless waxy product. Separation by HPLC, evaporation and lyophilization gave the pure phosphorylated peptide 6 (0.18 g, 59%) as an off-white solid. Purity >97%, analytical HPLC. HRMS (ES+) calcd for C134H204N39O40P [M + 4H]4+, 757.6216; found, m/z 757.6211.
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