Chemoselective Wittig and Michael Ligations of Unprotected Peptidyl

Letter pubs.acs.org/OrgLett

Chemoselective Wittig and Michael Ligations of Unprotected Peptidyl Phosphoranes in Water Furnish Potent Inhibitors of Caspase‑3 Kai Holland-Nell, María Isabel Fernández-Bachiller, Ahsanullah, and Jörg Rademann* Medicinal Chemistry, Free University Berlin, Königin-Luise-Strasse 2 + 4, 14195 Berlin, Germany Leibniz Institute for Molecular Pharmacology (FMP), Robert Rössle-Strasse 10, 13125 Berlin, Germany S Supporting Information *

ABSTRACT: Unprotected peptidyl phosphoranes 1 with sequence Ac-L-aspartyl-L-glutamyl-L-valinyl-L-aspartyl are released from polymer support and react with aliphatic and aromatic aldehydes in aqueous medium in a Wittig ligation. Obtained vinyl ketones 6−12 are potent inhibitors of caspase-3. Vinyl ketone 6, derived from formaldehyde, undergoes Michael ligations with thiol nucleophiles furnishing products 14−16, also in aqueous medium. The demonstrated ligation reactions enable the modification of complex functionalized peptides in water providing bioactive protein ligands without side-chain protection.

C

Starting from one known ligand of a protein, e.g., an electrophilic inhibitor fragment, DLS can be conducted on microtiter plates with chemical libraries of nucleophiles to extend the starting fragment iteratively to more potent fragment combination. We are highly interested in the discovery of novel chemoselective ligation reactions for application in biological assays as such reactions would be useful for the rapid structural variation and optimization of bioactive molecules. Recently, we have demonstrated that peptidyl phosphoranes can be synthesized on polymer support and cleaved off the polymer with aliphatic and aromatic aldehydes affording peptidyl vinyl ketones.9 In order to investigate the potential of this reaction as a “Wittig ligation” in aqueous medium, a protocol for the synthesis of soluble peptidyl phosphorane was developed.10 Here, application of soluble peptidyl phosphoranes in chemoselective ligation reactions in aqueous medium will be investigated and the obtained peptidyl vinyl ketones will be tested as inhibitors of the cysteine protease caspase-3. Caspase-3 was selected as a protein target due to its central role in the execution of programmed cell death, or apoptosis.11

hemical ligations have been defined as chemoselective reactions linking two building blocks in the presence of several other unprotected functionalities.1 In order to comply with this definition, chemical ligations require high-yielding reactions that tolerate the presence of water or other protic solvents and can be applied at room temperature or up to 37 °C. Until now, several chemical ligation reactions have been broadly employed in chemical biology and drug discovery including amide bond formations,2 for example, cysteinyl thioester ligations,3 Staudinger ligations,4 Cu-catalyzed alkyne−azide cycloadditions,5 cycloadditions with heteroaryls, or carbonyl condensations leading to hydrazones or oximes,6 respectively. The chemoselectivity of chemical ligation reactions has facilitated in situ screening, i.e., the chemical synthesis in microtiter plates with subsequent screening in bioassays.7 Moreover, ligation reactions have been conducted in even more complex biological experiments including cellular experiments and in vivo studies. Dynamic chemical ligations, i.e., reversible ligation reactions, have been the focus of research in recent years. As dynamic ligations proceed smoothly under the conditions of biochemical assays, the protein target can act as a molecular template shifting the equilibrium to the ligation reaction. The approach, denominated as dynamic ligation screening (DLS), enables the sensitized detection of cooperatively binding fragments.8 © 2014 American Chemical Society

Received: July 3, 2014 Published: August 20, 2014 4428

dx.doi.org/10.1021/ol501910x | Org. Lett. 2014, 16, 4428−4431

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with triphenylphosphine, or tris(4-methoxyphenyl)phosphine, respectively, furnishing the polymer-supported phosphonium salts 3a,b. These were deprotonated with triethylamine (Et3N) to the ylides, which were acylated with Fmoc-Asp-(O-t-Bu)OH employing fluoro-N,N,N′,N′-bis(tetramethylene)formamidinium hexafluorophosphate (BTFFH) as activating agent and DIPEA as a base, giving the polymer-supported 2acyl-2-phosphoranylidene esters 4a,b. Fmoc cleavage and peptide elongation were carried out with 20% piperidine/ DMF (v/v) and N,N-diisopropylcarbodiimide (DIC) with hydroxybenzotriazole (HOBt), respectively. The N-terminus of the obtained peptide was acetylated with acetic anhydride (Ac2O) to yield the N-acetyl protected resins 5a,b. Soluble tetrapeptidyl phosphonium salts 1a (81%) and 1b (46%) were obtained after cleavage, deprotection, and decarboxylation in a mixture of trifluoacetic acid/dichloromethane (TFA/CH2Cl2 (95:5)) in a single step. Both products, 1a and 1b, displayed only one set of well-resolved signals in the high-resolution 1H NMR spectrum, indicating formation of a single diastereomer as a product and the absence of epimerization (see the Supporting Information). Because of the higher reactivity of the tris(4-methoxy)phenylphosphonium salt 1b compared to 1a, 1b was selected for Wittig ligation reactions with various aliphatic and aromatic aldehydes in aqueous medium and at room temperature (22 °C) (Table 1).

Inhibition of caspase-3 is beneficial in several hypoxic situations leading to undesired cell death as encountered in heart attack,12 brain stroke, brain trauma, and inflammatory diseases13 and in reperfusion injury following organ transplantation. The tetrapeptide L-aspartyl-L-glutamyl-L-valinyl-L-aspartyl (DEVD) has been identified as a recognition sequence of the enzyme, and the peptide aldehyde Ac-DEVD-H is used as a high-affinity probe for the enzyme.14 Unfortunately, the C-terminal aldehyde functionality of this inhibitor lacks metabolic stability. Therefore, several other electrophilic warheads, including the more stable ketone moiety,8b,15 have been introduced into the sequence in order to optimize the biological effects of the probe. The ketone warhead, however, not only is advantageous due to increased stability but also enables elongation of inhibitors into the S1′-site of the protein, possibly enhancing the affinity of the molecule. We therefore designed the soluble, unprotected peptidyl phosphorane 1 (Scheme 1) containing the Ac-DEVD motif in order to investigate Wittig ligations and to demonstrate fragment variation in the S1′-pocket of potential caspase-3 inhibitors. For the synthesis, Wang resin 2 was acylated with bromoacetyl bromide in the presence of N,N-diisopropylethylamine (DIPEA). The obtained 2-bromoacetyl ester was reacted Scheme 1. Preparation of the Soluble Peptidyl Phosphoranes 1a,b Containing the Recognition Sequence of Caspase-3

Table 1. Wittig Ligation Reactions of Unprotected Peptidyl Phosphorane 1b

entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14

aldehyde R′ H

time (min) 90

methyl

120

ethyl

120

propyl

90

butyl

120

phenyl

7d

4-CH3−Ph

7d

product

yielda (%)

combined yield (%)

6a 6b 7a 7b 8a 8b 9a 9b 10a 10b 11a 11b 12a 12b

35 49 28 48 20 15 14 24 19 20 39 33 29 24

84 76 35 38 39 72 53

a

The yield for every isolated product was calculated after purification by HPLC. Conditions: semipreparative HPLC column (VP 250/10 Nucleodur 100-5 C18 ec Macherey-Nagel) employing a mixture of H2O (A, with 0.01% TFA) and CH3CN (B, with 0.01% TFA), gradient from 5% to −40% in 40 min.

Phosphonium salt 1b was readily soluble in a mixture of H2O and CH3CN (3:1). Addition of 4 equiv of Et3N or DIPEA as base was sufficient to deprotonate all three carboxylic acid residues and the phosphonium functionality to the ylide and induced the reactivity required for the ligation reaction with aldehydes. The rate of the ligation reaction depended on the 4429


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reactivity of the aldehyde used. While the reaction with formaldehyde was completed after 90 min furnishing peptidyl vinyl ketone 6, it took several days with the aromatic aldehydes, benzaldehyde, and 4-methylbenzaldehyde yielding products 11 and 12. All Wittig ligations furnished exclusively E-olefins 7−12 as indicated by the coupling constants of the olefinic protons around 16 Hz. Products were obtained in moderate to very good yields (35 to 84%) subsequent to isolation by RP-HPLC. Under the described reaction conditions, peptidyl vinyl ketones 6−12 were formed as equimolar mixtures of two diasteromers a,b. In all cases, both diastereomers could be cleanly separated by HPLC and fully assigned by NMR spectroscopy using a combination of mono- (1H and 13C) and two-dimensional (HMQC and HMBC) experiments. A comparison of the 1H NMR spectra of the diastereomers revealed the largest deviations between the signals of the Cterminal Asp and Val residues suggesting epimerization at the α-carbon of Asp-1 (Supporting Information, Figure S1). Redissolution of the enantiopure products in aqueous buffer at physiological pH of 7.4 led to the epimerization, indicating that the vinyl ketones are not configurationally stable under these experimental conditions. A different product was found for the ligation reaction between the phosphonium salt 1b and formaldehyde, if Et3N instead of DIPEA was used as a base. In this case, no formation of the vinyl ketones 6a,b was observed. Instead, under these experimental conditions, two molecules of formaldehyde were added to the phosphorane, yielding the Baylis−Hillman product 13 with 70% yield (Scheme 2).16 If, however, DIPEA was used as a base, vinyl ketones 6a and 6b were isolated in 84% yield.

Table 2. Michael Addition of Aliphatic and Aromatic Thiols into the Vinyl Ketone 6a

entry

conditions

compd

R

yieldd (%)

1 2 3 4 5

FeCl3 UVA none none none

14 14 14 15 16

Et Et Et Ph Bn

14b 26c 35 30 39

a

Reaction conditions: all of the reactions were performed with a mixture of diastereomers 6a,b (6.50 mg, 0.01 mmol, 1 equiv) and the corresponding thiol (0.50 mmol, 50 equiv) in a mixture of H2O:CH3CN (750 μL, 3:1 (v/v)). bIn the presence of FeCl3 (catalytic amount) for 20 h at room temperature. cUV. Irradiation at 365 nm for 2 h. dThe yield for every isolated product was calculated after purification by HPLC as a mixture of both diasteromers.

6a,b, yielding the products 15 and 16 in 30 and 39% yield, respectively. In all cases, the 1,4 Michael addition was confirmed by NMR spectroscopy. The inhibitory activity of the synthesized vinyl ketones 7−12 against recombinant caspase-3 was evaluated in a concentration-dependent manner using the substrate Ac-DEVD-AMC. This substrate provides the 7-amino-4-methylcoumarin (AMC) chromophore with a λemi at 450 nm after hydrolysis by caspase. The biochemical assay was validated by reproducing the KI value of the peptide aldehyde inhibitor Ac-DEVD-H for control.14,15b Vinyl ketones 7−12 were found to be potent reversible inhibitors of human recombinant caspase-3 with KI values in the low nanomolar range. Though ketones are less electrophilic and chemically more stable than the aldehyde inhibitor Ac-DEVD-H, the best vinyl ketone inhibitors were in the same range or even better than the control (Table 3). Vinyl ketones 9−12 with propyl, butyl, phenyl, or 4-methylphenyl substituents were most active with KI values between 3.5 and 1.7 nM, respectively, suggesting that these residues can occupy favorably the hydrophobic S1′-pocket of the enzyme. Vinyl ketones with small aliphatic substituents at the vinyl group were less potent than those with extended aliphatic or aromatic substituents. Isolated diastereomers of the same structure display only small differences in the biological activity, in agreement with an epimerization of the compounds under assay conditions. All vinyl ketones 7−12(a,b) show a reversible binding mode indicating that no irreversible addition of the active site cysteine to the double bonds occurs. In summary, we have demonstrated that soluble peptidyl phosphoranes containing unprotected amino acid side chains can ligate with aromatic and aliphatic aldehydes in aqueous medium affording peptidyl vinyl ketone 6−12 in good yields. If Et3N was used as a base, the reaction with formaldehyde furnished the Baylis−Hilman product 13. Peptidyl vinyl ketones containing the Ac-DEVD-motif are potent inhibitors of caspase-3; the 4-methylphenyl, phenyl, and butyl substituents provided the highest activity. The unsubstituted peptidyl vinyl ketone 6 was able to undergo a subsequent Michael ligation reaction yielding the thiol-addition products

Scheme 2. Formation of Baylis−Hillman Product 13 in the Presence of Et3N

Having the novel peptidyl vinyl ketones 6−13 in hand and considering the electron-deficient olefin, it was an obvious challenge to investigate the new compounds as Michael acceptors in a subsequent ligation reaction. For these Michael ligation reactions, the mixture of vinyl ketone 6a and 6b was selected as starting material. Taking into account that Michael addition reactions of thiols proceed readily under basic conditions, experimental protocols for the base-free addition of aliphatic and aromatic thiols in aqueous medium were studied (Table 2).17 The addition of ethanethiol to this α,β-unsaturated ketone with iron(III) chloride as catalyst afforded the desired compound 14 with low yield (14%, entry 1).17a When the reaction mixture was irradiated with an UVA lamp at 365 nm, the compound 14 was isolated with 26% yield (entry 2). The best results were obtained when the reaction was carried out in absence of Lewis acid and without irradiation (entry 3). Under these experimental conditions, the thio Michael product 14 was obtained with a yield of 35% after 20 h at room temperature. The last experimental conditions described above were used for the addition of thiophenol and benzylthiol to the vinyl ketone 4430


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Table 3. KI (nM) Determination of Peptidyl Vinyl Ketones 6−12 against Caspase-3a (See Scheme 2 for General Structure) entry

compd

1 2 3 4 5 6 7 8 9 10 11 12 13

7a 7b 8a 8b 9a 9b 10a 10b 11a 11b 12a 12b AcDEVD-H

R′ Me ethyl propyl butyl Ph 4-CH3Ph

(4) Schilling, C. I.; Jung, N.; Biskup, M.; Schepers, U.; Bräse, S. Chem. Soc. Rev. 2011, 40, 4840−4871. (5) Meldal, M.; Tornøe, C. W. Chem. Rev. 2008, 108, 2952−3015. (6) (a) Dirksen, A.; Dirksen, S.; Hackeng, T. M.; Dawson, P. E. J. Am. Chem. Soc. 2006, 128, 15602−15603. (b) Dirksen, A.; Hackeng, T. M.; Dawson, P. E. Angew. Chem., Int. Ed. 2006, 45, 7581−7584. (7) Brik, A.; Wu, C. Y.; Wong, C. H. Org. Biomol. Chem. 2006, 4, 1446−1457. (8) (a) Schmidt, M. F.; Isidro-Llobet, A.; Lisurek, M.; El-Dahshan, A.; Tan, J.; Hilgenfeld, R.; Rademann, J. Angew. Chem., Int. Ed. 2008, 47, 3275−3278. (b) Schmidt, M. F.; El-Dahsham, A.; Keller, S.; Rademann, J. Angew. Chem., Int. Ed. 2009, 48, 6346−6349. (c) Schmidt, M. F.; Groves, M. R.; Rademann, J. ChemBioChem 2011, 12, 2640−2646. (d) Fernández-Bachiller, M. I.; Horatscheck, A.; Lisurek, M.; Rademann, J. ChemMedChem 2013, 8, 1041−56. (9) El-Dahshan, A.; Nazir, S.; Ahsanullah; Ansari, F. L.; Rademann, J. Eur. J. Org. Chem. 2011, 4, 730−739. (10) Ahsanullah; Al-Gharabli, S. I.; Rademann, J. Org. Lett. 2012, 14, 14−17. (11) Elmore, S. Toxicol. Pathol. 2007, 35, 495−516. (12) Narula, J.; Panday, P.; Arbstini, E.; Haider, N.; Narula, N.; Kolodgie, F. D.; Dal Bello, B.; Semigran, M. J.; Bielsa-Masdeu, A.; Dec, G. W.; Israels, S.; Ballester, M.; Virmani, R.; Saxena, S.; Kjarbanda, S. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 8144−8149. (13) Cornelius, S.; Kersse, K.; Festjens, N.; Lamlanfi, M.; Vandenabeele, P. Curr. Pharm. Des. 2007, 13, 367−385. (14) Chéreau, D.; Kodandapani, L.; Tomaselli, K. J.; Spada, A. P.; Wu, J. C. Biochemistry 2003, 42, 4151−4160. (15) (a) Becker, J. W.; Rotonda, J.; Soisson, S. M.; Aspiotis, R.; Bayly, C.; Francoeur, S.; Gallant, M.; Garcia-Calvo, M.; Giroux, A.; Grimm, E.; Han, Y.; Mckay, D.; Nicholson, D. W.; Peterson, E.; Renaud, J.; Roy, S.; Thornberry, N.; Zamboni, R. J. Med. Chem. 2004, 47, 2466− 2474. (b) Goode, D. R.; Sharma, A. K.; Hergenrother, P. J. Org. Lett. 2005, 7, 3529−3532. (16) Basavaiah, D.; Veeraraghavaiah, G. Chem. Soc. Rev. 2012, 41, 68−78. (17) (a) Hui, X. P.; Yin, C.; Ma, J.; Xu, P. F. Synth. Commun. 2009, 39, 676−690. (b) Xu, L. W.; Yang, M. S.; Qiu, H. Y.; Lai, G. O.; Jiang, J. X. Synth. Commun. 2008, 7, 1011−1019. (18) Cheng, Y. C.; Prusoff, W. H. Biochem. Pharmacol. 1973, 22, 3099−3108.

KI (nM)b,c 24.7 24.9 14.9 5.5 3.3 3.5 3.5 3.1 2.7 2.8 1.9 1.7 4.1

± ± ± ± ± ± ± ± ± ± ± ± ±

1.5 1.3 1.7 2.2 0.5 0.6 0.8 0.6 0.2 0.6 0.1 0.3 1.8

a For KI determination, caspase-3 ([E] = 1U) and Ac-DEVD-AMC ([S] = 50 μM), and 50 mM HEPES (pH 7.4), containing 100 mM NaCl, 0.5% CHAPS, 10 mM DDT, 1 mM EDTA, and 10% glycerol, were used. bThe KI values are the mean (n = 3) ± SD; λabs = 350 nm, λemi = 450 nm. The assays were carried out with 5% DMSO (final concentration). cThe KI values for these inhibitors were calculated using the equation of Cheng and Prusoff (KI = IC50/(1 + [S]/KM)),18 taking into account the competitive binding mode. [S] = 50 μM and KM = 9.5 μM.

14−16. The reported chemoselective ligation reactions conducted with unprotected peptide reagents will enable the preparation of further peptidomimetics bearing potential as ligands for other protein targets and for application in ligation and in situ screening.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures, characterization data, and inhibition curves. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +4930 838459829. Tel: +4930 83853272. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Deutsche Forschungsgemeinschaft (DFG) (Grant Nos. Ra895/11, FOR 806 (Ra895/5), and SFB 765 (Ra895-6)) and by the EU project SILVER. We thank Dr. Jens Peter von Kries and Dr. André Horatscheck, Leibniz Institut für Molekulare Pharmakologie (FMP) Berlin, for the caspase-3 protein and for technical support.

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REFERENCES

(1) (a) Wagner, A.; King, M. Bioconjugate Chem. 2014, 25, 825−839. (b) Hackenberger, C. P. R.; Schwarzer, D. Angew. Chem., Int. Ed. 2008, 47, 10030−10074. (c) Ramil, C. P.; Lin, Q. Chem. Commun. 2013, 49, 11007−11022. (2) Bode, J. W. Curr. Opin. Drug Discovery Devel. 2006, 9, 765−775. (3) (a) Tam, J. P.; Yu, Q.; Yang, J. L. J. Am. Chem. Soc. 2001, 123, 2487−2494. (b) Simerska, P.; Toth, I. Molecules 2013, 18, 4373−4388. 4431


Chemoselective Wittig and Michael Ligations of Unprotected Peptidyl

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