Synlett 2020; 31(03): 237-247
DOI: 10.1055/s-0039-1691576
cluster
© Georg Thieme Verlag Stuttgart · New York

A Hammett Study of Clostridium acetobutylicum Alcohol Dehydrogenase (CaADH): An Enzyme with Remarkable Substrate Promiscuity and Utility for Organic Synthesis

Gaurav P. Kudalkar
,
Virendra K. Tiwari
,
Joshua D. Lee
,
David B. Berkowitz
Department of Chemistry, University of Nebraska, Lincoln, NE 68588, USA   Email: dberkowitz1@unl.edu
› Author Affiliations
This work was supported by the National Science Foundation (NSF), Division of Chemistry (Grant Nos. CHE-1500076 and CHE-1800574). These studies were facilitated by the independant research & development program for D.B.B.’s appointment at the NSF. The authors thank the National Institutes of Health (NIH) (Grant No. SIG-1-510-RR-06307) and the NSF (Grant Nos. CHE-0091975 and MRI-0079750) for NMR instrumentation and the NIH, National Center for Research Resources (Grant No. RR016544) for facilities.
Further Information

Publication History

Received: 11 December 2019

Accepted after revision: 08 January 2020

Publication Date:
16 January 2020 (online)


Published as part of the Cluster Biocatalysis

Abstract

Described is a physical organic study of the reduction of three sets of carbonyl compounds by the NADPH-dependent enzyme Clostridium acetobutylicum alcohol dehydrogenase (CaADH). Previous studies in our group have shown this enzyme to display broad substrate promiscuity, yet remarkable stereochemical fidelity, in the reduction of carbonyl compounds, including α-, β- and γ-keto esters (d-stereochemistry), as well as α,α-difluorinated-β-keto phosphonate esters (l-stereochemistry). To better mechanistically characterize this promising dehydrogenase enzyme, we report here the results of a Hammett linear free-energy relationship (LFER) study across three distinct classes of carbonyl substrates; namely aryl aldehydes, aryl β-keto esters and aryl trifluoromethyl ketones. Rates are measured by monitoring the decrease in NADPH fluorescence at 460 nm with time across a range of substrate concentrations for each member of each carbonyl compound class. The resulting v 0 versus [S] data are subjected to least-squares hyperbolic fitting to the Michaelis–Menton equation. Hammett plots of log(V max) versus σX yield the following Hammett parameters: (i) for p-substituted aldehydes, ρ = 0.99 ± 0.10, ρ = 0.40 ± 0.09; two domains observed, (ii) for p-substituted β-keto esters ρ = 1.02 ± 0.31, and (iii) for p-substituted aryl trifluoromethyl ketones ρ = –0.97 ± 0.12. The positive sign of ρ indicated for the first two compound classes suggests that the hydride transfer from the nicotinamide cofactor is at least partially rate-limiting, whereas the negative sign of ρ for the aryl trifluoromethyl ketone class suggests that dehydration of the ketone hydrate may be rate-limiting for this compound class. Consistent with this notion, examination of the 13C NMR spectra for the set of p-substituted aryl trifluo­romethyl ketones in 2% aqueous DMSO reveals significant formation of the hydrate (gem-diol) for this compound family, with compounds bearing the more electron-withdrawing groups showing greater degrees of hydration. This work also presents the first examples of the CaADH-mediated reduction of aryl trifluoromethyl ketones, and chiral HPLC analysis indicates that the parent compound α,α,α-trifluoroacetophenone is enzymatically reduced in 99% ee and 95% yield, providing the (S)-stereoisomer, suggesting yet another compound class for which this enzyme displays high enantioselectivity.

Supporting Information

 
  • References and Notes

    • 1a Sheldon RA, Woodley JM. Chem. Rev. 2018; 118: 801
    • 1b Hughes G, Lewis JC. Chem. Rev. 2018; 118: 1
    • 1c Clouthier CM, Pelletier JN. Chem. Soc. Rev. 2012; 41: 1585
    • 2a Zhang G, Quin MB, Schmidt-Dannert C. ACS Catal. 2018; 8: 5611
    • 2b Schrittwieser JH, Velikogne S, Hall M, Kroutil W. Chem. Rev. 2018; 118: 270
    • 2c Oeggl R, Massmann T, Jupke A, Rother D. ACS Sustainable Chem. Eng. 2018; 6: 11819
    • 2d Schmidt S, Scherkus C, Muschiol J, Menyes U, Winkler T, Hummel W, Groeger H, Liese A, Herz H.-G, Bornscheuer UT. Angew. Chem. Int. Ed. 2015; 54: 2784
    • 2e Mutti FG, Knaus T, Scrutton NS, Breuer M, Turner NJ. Science 2015; 349: 1525
    • 2f Heidlindemann M, Rulli G, Berkessel A, Hummel W, Groeger H. ACS Catal. 2014; 4: 1099
    • 2g Anderson M, Afewerki S, Berglund P, Cordova A. Adv. Synth. Catal. 2014; 356: 2113
  • 3 Turner NJ, O’Reilly E. Nat. Chem. Biol. 2013; 9: 285
  • 4 Bornscheuer U, Huisman G, Kazlauskas R, Lutz S, Moore J, Robins K. Nature 2012; 485: 185
    • 5a Wu S, Zhou Y, Gerngross D, Jeschek M, Ward TR. Nat. Commun. 2019; 10: 1
    • 5b Rudroff F. Curr. Opin. Chem. Biol. 2019; 49: 84
    • 5c Wu S, Li Z. ChemCatChem 2018; 10: 2164
    • 5d Both P, Busch H, Kelly PP, Mutti FG, Turner NJ, Flitsch SL. Angew. Chem. Int. Ed. 2016; 55: 1511
  • 6 Savile CK, Janey JM, Mundorff EC, Moore JC, Tam S, Jarvis WR, Colbeck JC, Krebber A, Fleitz FJ, Brands J, Devine PN, Huisman GW, Hughes GJ. Science 2010; 329: 305
  • 7 Huffman MA, Fryszkowska A, Alvizo O, Borra-Garske M, Campos KR, Canada KA, Devine PN, Duan D, Forstater JH, Grosser ST, Halsey HM, Hughes GJ, Jo J, Joyce LA, Kolev JN, Liang J, Maloney KM, Mann BF, Marshall NM, McLaughlin M, Moore JC, Murphy GS, Nawrat CC, Nazor J, Novick S, Patel NR, Rodriguez-Granillo A, Robaire SA, Sherer EC, Truppo MD, Whittaker AM, Verma D, Xiao L, Xu Y, Yang H. Science 2019; 366: 1255
  • 8 Broussy S, Cheloha RW, Berkowitz DB. Org. Lett. 2009; 11: 305
  • 9 Tassano E, Faber K, Hall M. Adv. Synth. Catal. 2018; 360: 2742
  • 10 Tassano E, Hall M. Chem. Soc. Rev. 2019; 48: 5596
    • 11a Xu G.-C, Shang Y.-P, Yu H.-L, Xu J.-H. Chem. Commun. 2015; 51: 15728
    • 11b Nealon CM, Musa MM, Patel JM, Phillips RS. ACS Catal. 2015; 5: 2100
    • 11c Wang S, Nie Y, Xu Y, Zhang R, Ko T.-P, Huang C.-H, Chan H.-C, Guo R.-T, Xiao R. Chem. Commun. 2014; 50: 7770
    • 11d Borzecka W, Lavandera I, Gotor V. J. Org. Chem. 2013; 78: 7312
    • 11e Truppo MD, Escalettes F, Turner NJ. Angew. Chem. Int. Ed. 2008; 47: 2639
  • 12 Moore JC, Pollard DJ, Kosjek B, Devine PN. Acc. Chem. Res. 2007; 40: 1412
  • 13 Friest JA, Maezato Y, Broussy S, Blum P, Berkowitz DB. J. Am. Chem. Soc. 2010; 132: 5930
  • 14 Applegate GA, Berkowitz DB. Adv. Synth. Catal. 2015; 357: 1619
  • 15 Brenna E, Crotti M, Gatti FG, Monti D, Parmeggiani F, Pugliese A. Molecules 2017; 22: 1591
    • 16a Valencia LE, Zhang Z, Cepeda AJ, Keatinge-Clay AT. Org. Biomol. Chem. 2019; 17: 1375
    • 16b Ren Y, Hu L, Ramstroem O. Mol. Catal. 2019; 468: 52
    • 16c Klaus T, Seifert A, Häbe T, Nestl BM, Hauer B. Catalysts 2019; 9: 252
    • 16d van Rantwijk F, Stolz A. J. Mol. Catal. B: Enzym. 2015; 114: 25
    • 16e Brenna E, Crotti M, Gatti FG, Monti D, Parmeggiani F, Pugliese A, Santangelo S. J. Mol. Catal. B: Enzym. 2015; 114: 37
    • 16f Babich L, van Hemert LJ. C, Bury A, Hartog AF, Falcicchio P, van der Oost J, van Herk T, Wever R, Rutjes FP. J. T. Green Chem. 2011; 13: 2895
  • 17 Berkowitz DB, Pumphrey JA, Shen Q. Tetrahedron Lett. 1994; 35: 8743
    • 18a Berkowitz DB, Choi S, Maeng J.-H. J. Org. Chem. 2000; 65: 847
    • 18b Berkowitz DB, Maeng J.-H, Dantzig AH, Shepard RL, Norman BH. J. Am. Chem. Soc. 1996; 118: 9426
    • 18c Berkowitz DB, Maeng J.-H. Tetrahedron: Asymmetry 1996; 7: 1577
    • 19a Malik G, Swyka RA, Tiwari VK, Fei X, Applegate GA, Berkowitz DB. Chem. Sci. 2017; 8: 8050
    • 19b Ginotra SK, Friest JA, Berkowitz DB. Org. Lett. 2012; 14: 968
    • 19c Friest JA, Broussy S, Chung WJ, Berkowitz DB. Angew. Chem. Int. Ed. 2011; 50: 8895
    • 20a Berkowitz DB, Shen W, Maiti G. Tetrahedron: Asymmetry 2004; 15: 2845
    • 20b Berkowitz DB, Maiti G. Org. Lett. 2004; 6: 2661
    • 20c Berkowitz DB, Bose M, Choi S. Angew. Chem. Int. Ed. 2002; 41: 1603
    • 21a Karukurichi KR, Fei X, Swyka RA, Broussy S, Shen W, Dey S, Roy SK, Berkowitz DB. Sci. Adv. 2015; 1: e1500066/1
    • 21b Dey S, Powell DR, Hu C, Berkowitz DB. Angew. Chem. Int. Ed. 2007; 46: 7010
    • 21c Dey S, Karukurichi KR, Shen W, Berkowitz DB. J. Am. Chem. Soc. 2005; 127: 8610
  • 22 Swyka RA, Berkowitz DB. Curr. Prot. Chem. Biol. 2017; 9: 285
  • 23 Nolling J, Breton G, Omelchenko MV, Makarova KS, Zeng Q, Gibson R, Lee HM, Dubois J, Qiu D, Hitti J, Wolf YI, Tatusov RL, Sabathe F, Doucette-Stamm L, Soucaille P, Daly MJ, Bennett GN, Koonin EV, Smith DR, Aldredge T, Ayers M, Bashirzadeh R, Bochner H, Boivin M, Bross S, Bush D, Butler C, Caron A, Caruso A, Cook R, Daggett P, Deloughery C, Egan J, Ellston D, Engelstein M, Ezedi J, Gilbert K, Goyal A, Guerin J, Ho T, Holtham K, Joseph P, Keagle P, Kozlovsky J, LaPlante M, LeBlanc G, Lumm W, Majeski A, McDougall S, Mank P, Mao J.-I, Nocco D, Patwell D, Phillips J, Pothier B, Prabhakar S, Richterich P, Rice P, Rosetti D, Rossetti M, Rubenfield M, Sachdeva M, Snell P, Spadafora R, Spitzer L, Shimer G, Thomann H.-U, Vicaire R, Wall K, Wang Y, Weinstock K, Wong LP, Wonsey A, Xu Q, Zhang L. J. Bacteriol. 2001; 183: 4823
    • 24a Nimbalkar PR, Khedkar MA, Chavan PV, Bankar SB. ACS Omega 2019; 4: 12978
    • 24b Lim J, Byun H.-E, Kim B, Lee JH. Ind. Eng. Chem. Res. 2019; in press; DOI: DOI: 10.1021/acs.iecr.9b03016.
    • 24c Steen EJ, Chan R, Prasad N, Myers S, Petzold CJ, Redding A, Ouellet M, Keasling JD. Microb. Cell Fact. 2008; 7: 36
  • 25 Applegate GA, Cheloha RW, Nelson DL, Berkowitz DB. Chem. Commun. 2011; 47: 2420
    • 26a Panigrahi K, Fei X, Kitamura M, Berkowitz DB. Org. Lett. 2019; 21: 9846
    • 26b Loranger MW, Forget SM, McCormick NE, Syvitski RT, Jakeman DL. J. Org. Chem. 2013; 78: 9822
    • 26c Diab SA, De Schutter C, Muzard M, Plantier-Royon R, Pfund E, Lequeux T. J. Med. Chem. 2012; 55: 2758
    • 26d Panigrahi K, Eggen M, Maeng J.-H, Shen Q, Berkowitz DB. Chem. Biol. 2009; 16: 928
    • 26e Romanenko VD, Kukhar VP. Chem. Rev. 2006; 106: 3868
    • 26f Lopin C, Gautier A, Gouhier G, Piettre SR. J. Am. Chem. Soc. 2002; 124: 14668
    • 26g Berkowitz DB, Bose M, Asher NG. Org. Lett. 2001; 3: 2009
    • 26h Berkowitz DB, Bose M, Pfannenstiel TJ, Doukov T. J. Org. Chem. 2000; 65: 4498
    • 26i Berkowitz DB, Eggen M, Shen Q, Shoemaker RK. J. Org. Chem. 1996; 61: 4666
    • 26j Shen QR, Sloss DG, Berkowitz DB. Synth. Commun. 1994; 24: 1519
  • 27 Panigrahi K, Applegate GA, Malik G, Berkowitz DB. J. Am. Chem. Soc. 2015; 137: 3600
  • 28 Hammett LP. Chem. Rev. 1935; 17: 125
  • 29 Hansch C, Leo A, Taft RW. Chem. Rev. 1991; 91: 165
  • 30 Santiago CB, Guo J.-Y, Sigman MS. Chem. Sci. 2018; 9: 2398
    • 31a Hansch C, Deutsch EW, Smith RN. J. Am. Chem. Soc. 1965; 87: 2738
    • 31b Kirsch JF. Linear Free Energy Relationships in Enzymology. In Advances in Linear Free Energy Relationships. Chapman NB. Shorter J. Plenum Press; London: 1972. Chap. 10, 369
    • 31c Fife TH, Rikihisa T, Benjamin BM. Biochemistry 1971; 10: 3875
    • 31d Zeller EA, Palmberg PF, Babu BH. Biochem. J. 1967; 105: 41P
    • 33a Shalan H, Colbert A, Nguyen TT, Kato M, Cheruzel L. Inorg. Chem. 2017; 56: 6558
    • 33b Yu X, Perez B, Zhang Z, Gao R, Guo Z. Green Chem. 2016; 18: 2753
    • 33c Banerjee S, Goyal S, Mazumdar S. Bioorg. Chem. 2015; 62: 94
    • 33d Garcia Linares G, Arroyo Manez P, Baldessari A. Eur. J. Org. Chem. 2014; 6439
    • 33e Zhiryakova D, Ivanov I, Ilieva S, Guncheva M, Galunsky B, Stambolieva N. FEBS J. 2009; 276: 2589
    • 33f Piens K, Stahlberg J, Nerinckx W, Teeri TT, Claeyssens M. ACS Symp. Ser. 2004; 889: 207
    • 33g Lin G. J. Chin. Chem. Soc. 2004; 51: 423
    • 33h Speare DM, Olf P, Bugg TD. H. Chem. Commun. 2002; 2304
    • 33i Lin G. J. Phys. Org. Chem. 2000; 13: 313
    • 33j Capeillere-Blandin C, Martin C, Gaggero N, Pasta P, Carrea G, Colonna S. Biochem. J. 1998; 335: 27
    • 33k Shan S.-o, Herschlag D. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 14474
    • 33l Golly I, Hlavica P. Arch. Biochem. Biophys. 1992; 292: 287
    • 33m Davidson VL, Jones LH, Graichen ME. Biochemistry 1992; 31: 3385
    • 33n Kanerva LT, Klibanov AM. J. Am. Chem. Soc. 1989; 111: 6864
    • 34a Schmitke JL, Stern LJ, Klibanov AM. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 12918
    • 34b Fitzpatrick PA, Steinmetz AC. U, Ringe D, Klibanov AM. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 8653
  • 35 Davies DD, Ugochukwu EN, Patil KD, Towers GH. N. Phytochemistry 1973; 12: 531
  • 36 Mayr P, Nidetzky B. Biochem. J. 2002; 366: 889
  • 37 Klinman JP. J. Biol. Chem. 1972; 247: 7977
  • 38 Tsai CS, Tang JY, Subbarao SC. Biochem. J. 1969; 114: 529
  • 40 Nie J, Guo H.-C, Cahard D, Ma J.-A. Chem. Rev. 2011; 111: 455
    • 41a Guanti G, Banfi L, Guaragna A, Narisano E. J. Chem. Soc., Chem. Commun. 1986; 138
    • 41b Bucciarelli M, Forni A, Moretti I, Torre G. Synthesis 1983; 897
    • 41c Bucciarelli M, Forni A, Moretti I, Torre G. J. Chem. Soc., Chem. Commun. 1978; 456
    • 42a Choudhury S, Baeg J.-O, Park N.-J, Yadav RK. Green Chem. 2014; 16: 4389
    • 42b Hibino A, Ohtake H. Process Biochem. 2013; 48: 838
    • 42c Itoh K.-i, Nakamura K, Aoyama T, Matsuba R, Kakimoto T, Murakami M, Yamanaka R, Muranaka T, Sakamaki H, Takido T. Biotechnol. Lett. 2012; 34: 2083
    • 42d Nakamura K, Matsuda T, Itoh T, Ohno A. Tetrahedron Lett. 1996; 37: 5727
    • 42e Bradshaw CW, Hummel W, Wong CH. J. Org. Chem. 1992; 57: 1532
    • 42f Qin F, Qin B, Zhang W, Liu Y, Su X, Zhu T, Ouyang J, Guo J, Li Y, Zhang F, Tang J, Jia X, You S. ACS Catal. 2018; 8: 6012
    • 42g Qin F, Qin B, Mori T, Wang Y, Meng L, Zhang X, Jia X, Abe I, You S. ACS Catal. 2016; 6: 6135
    • 42h Li A, Ye L, Yang X, Yang C, Gu J, Yu H. Chem. Commun. 2016; 52: 6284
    • 42i Li A, Ye L, Wu H, Yang X, Yu H. J. Mol. Catal. B: Enzym. 2015; 122: 179
    • 42j Liu Y, Tang T.-X, Pei X.-Q, Zhang C, Wu Z.-L. J. Mol. Catal. B: Enzym. 2014; 102: 1
    • 42k Nie Y, Xiao R, Xu Y, Montelione GT. Org. Biomol. Chem. 2011; 9: 4070
    • 42l Musa MM, Lott N, Laivenieks M, Watanabe L, Vieille C, Phillips RS. ChemCatChem 2009; 1: 89
    • 42m De Wildeman SM. A, Sonke T, Schoemaker HE, May O. Acc. Chem. Res. 2007; 40: 1260
    • 43a Stewart R, Teo KC. Can. J. Chem. 1980; 58: 2491
    • 43b Yang JS, Liu KT, Su YO. J. Phys. Org. Chem. 1990; 3: 723
    • 44a Ohno A, Kobayashi H, Oka S. Tetrahedron Lett. 1983; 24: 5123
    • 44b Ohno A, Yamamoto H, Oka S. J. Am. Chem. Soc. 1981; 103: 2041
  • 45 Stewart R, Van Dyke JD. Can. J. Chem. 1970; 48: 3961
  • 46 Thompson JD, Higgins DG, Gibson TJ. Nucleic Acids Res. 1994; 22: 4673
  • 47 Hess B, Kutzner C, Van Der Spoel D, Lindahl E. J. Chem. Theory Comput. 2008; 4: 435
  • 48 Trott O, Olson AJ. J. Comput. Chem. 2010; 31: 455
  • 49 Krieger E, Koraimann G, Vriend G. Proteins 2002; 47: 393
  • 50 Kavanagh KL, Joernvall H, Persson B, Oppermann U. Cell. Mol. Life Sci. 2008; 65: 3895
    • 51a Hyster TK. Synlett 2019; in press; DOI: DOI: 10.1055/s-0037-1611818.
    • 51b Biegasiewicz KF, Cooper SJ, Emmanuel MA, Miller DC, Hyster TK. Nat. Chem. 2018; 10: 770
    • 51c Emmanuel MA, Greenberg NR, Oblinsky DG, Hyster TK. Nature 2016; 540: 414
  • 52 Enzymatic Reduction/Preparation of (S)-2,2,2-Trifluoro-1-phenylethanol; Typical ProcedureTo a solution of NADPH (21 mg, 0.0287 mmol, 0.01 equiv), d-glucose (3.09 g, 17.2 mmol, 6 equiv), CaADH (50 units) and NADP+-dependent glucose dehydrogenase (2% w/w) in KPO4 buffer (100 mM, pH 8.0) was added trifluoromethylphenyl ketone (500 mg, 2.87 mmol) in DMSO so that the final DMSO concentration was 10% (v/v). The reaction mixture was allowed to stir at rt for 8–10 h and the reaction progress was monitored by TLC. The product was extracted with EtOAc and dried over Na2SO4. Following vacuum filtration and concentration, the crude residue was purified by flash column chromatography on silica gel (EtOAc/hexane, 1:4) to give the title alcohol as a colorless oil (484 mg, 96%) in homogeneous form. The ee was determined to be 99.6% (S) by HPLC with a chiral stationary phase (see the Supporting Information).1H NMR (400 MHz, CDCl3): δ = 7.55–7.47 (m, 2 H), 7.47–7.41 (m, 3 H), 5.11–4.97 (m, 1 H), 2.66 (d, J = 4.5 Hz, 1 H). 13C NMR (100 MHz, CDCl3): δ = 180.88 (q, J = 35 Hz), 135.85, 130.47 (q, J = 2.8 Hz), 128.91, 107.01 (q, J = 294 Hz). 19F NMR (376 MHz, CDCl3): δ = –78.36 (d, J = 6.7 Hz).