10.1002/anie.202003125
Angewandte Chemie International Edition
COMMUNICATION
i
was added 4.018 mL Tricine buffer (100 mM, pH 9,). PrOH was
Based on the proposed mechanism, we hypothesized that
alternative reduction mechanisms that proceed through identical
radical intermediates should provide products with the same
enantioselectivity as that of the alkene reduction. As a test for this
hypothesis, we explored the radical deacetoxylation of 7 to provide
4n. Mechanistically, this reaction should occur via reduction of the
pyridine to the radical anion, whereupon elimination of acetate leads
to the same benzylic radical formed in the reduction of 3n.
Stereodetermining quenching of the benzylic radical should proceed
via hydrogen atom transfer from flavin in both cases: therefore
enantioselectivity is expected to be unperturbed. Indeed, when
racemic mixture of 7 was subjected to otherwise identical reaction
conditions, the reduced product 4n was observed in 57% yield with
96:4 e.r. (Figure 3), identical to the selectivity observed via alkene
reduction.
added to substrate vial (such that concentration was 1 M). To each
reaction shell vial was added 450 µL of “master mix”, 30 µL of
substrate in iPrOH, and 3 aliquots of protein. A rubber septum was
affixed to the reaction vial, brought out of the Coy chamber and
irradiated with blue LED’s at 0 ºC for 48 hours.
Upon completion, the reaction vials were treated with MeCN (0.9
mL) and an internal standard (1,3,5-tribromobenzene, 100 µL of a
10 mg/mL solution in MeCN [1 mg total]). The resultant mixture was
centrifugated (10,000 xg, 5 min), and supernatant was partitioned
between H2O:DCM (3 mL : 3 mL). The aqueous layer was
separated and extracted with DCM. Combined organic layers were
dried over anhydrous Na2SO4, filtered and concentrated under
reduced pressure. The crude residue was dissolved in CDCl3 and
analyzed by 1H-NMR for yield calculation. The CDCl3 solution was
reconcentrated and dissolved in HPLC grade hexanes for HPLC
analysis.
NostocER (1.0 mol %)
Ru(bpy)3Cl2 (0.5 mol %)
H
OAc
Me
Me
N
NADP+ (1.0 mol %)
GDH-105, glucose (2 equiv)
Tricine (100 mM, pH = 9)
Blue LEDs
N
Ph
4n, 57%, 96:4 e.r.
Ph
Conflict of interest
(±)-7
4 °C, 48h
The authors declare no conflict of interest.
Single Electron
Transfer
Hydrogen Atom
Transfer
Me
Ph
Keywords: biocatalysis • catalysis • photoenzymatic • heteroaromatic
N
–
OAc
reduction • enantioselectivity • ‘ene’-reductase
Common intermediate in alkene reduction and deacetoxylation
Figure 3. Intercepting
deacetoxylation
a dynamically stable radical via radical
[1] a) E. Vitaku, D. T. Smith, J. T. Njardarson, J. Med. Chem. 2014, 57, 10257; b)
M. Gaba, C. Mohan, Med. Chem. Res. 2016, 25, 173; c) J. Akhtar, A. A. Khan,
Z. Ali, R. Haider, M. S. Yar, Eur. J. Med. Chem. 2017, 125, 143; d) Z.
Hosseinzadeh, A. Ramazani, N. Razzaghi-Asl, Org. Chem. 2018, 22, 2256.
[2] For common synthetic strategies, see: a) M. Baumann, I. R. Baxendale, S. V.
Ley, N. Nikbin, Beilstein J. Org. Chem. 2011, 7, 442; b) M. Baumann, I. R.
Baxendale, Beilstein J. Org. Chem. 2013, 9, 2265; c) A. P. Taylor, R. P.
Robinson, Y. M. Fobian, D. C. Blakemore, L. H. Jones, O. Fadeyi, Org.
Biomol. Chem. 2016, 14, 6599.
[3] a) B. Martin, S. L. Buchwald, Acc. Chem. Res. 2008, 41, 1461; b) P. Ruiz-
Castillo, S. L. Buchwald, Chem. Rev. 2016, 116, 12564.
[4] a) D. Best, H. W. Lam, J. Org. Chem. 2014, 79, 831; b) H. B. Hepburn, P.
Melchiorre, Chem. Commun. 2016, 52, 3520.
[5] a) H. Yu, H. Tang, P. Xu, Sci. Rep. 2014, 4, 5397; b) J. Zhou, G. Xu, R. Han,
J. Dong, W. Zhang, R. Zhang, Y. Ni, Catal. Sci. Technol. 2016, 6, 6320; c) J.
Xu, S. Zhou, Y. Zhao, J. Xia, X. Liu, J.-M. Xu, B. He, B. Wu, J. Zhang, Chem.
Eng. J. 2017, 316, 919; d) M. Hall, A. S. Bommarius, Chem. Rev. 2011, 111,
4088; e) A. Rajagopala, W. Kroutil, Materials Today, 2011, 14, 145; f) D. J.
Pollard, J. M. Woodley, TRENDS in Biotech. 2007, 25, 66.
In conclusion, we found that flavin-dependent ‘ene’-reductases
can reduce vinyl pyridines through the merger of enzymatic and
photoredox catalysis. Mechanistic studies suggest that radical
termination occurring via hydrogen atom transfer from flavin within
the enzyme active site. Preliminary investigations suggest the
transformation is general and tolerates several other heteroaromatic
motifs for photoenzymatic reduction. Unlocking unnatural enzyme
function through synergistic catalyst merger expands the breadth of
nature’s most powerful catalysts, thereby propelling biocatalysis from
specialization toward generalization.
[6] a) C. K. Winkler, G. Tasnádi, D. Clay, M. Hall, K. Faber, J. Biotechnol. 2012,
162, 381; b) C. K. Winkler, K. Faber, M. Hall, Curr. Opin. Chem. Biol. 2018,
43, 97; c) T. Knaus, H. S. Toogood, N. S. Scrutton, Green Biocatalysis, John
Wiley & Sons, Hoboken, NJ, 2016, p. 473. d) H. S. Toogood, N. S. Scrutton,
ACS Catal. 2018, 8, 3532.
[7] a) M. A. Emmanuel, N. R. Greenberg, D. G. Oblinsky, T. K. Hyster, Nature
2016, 540, 414; b) K. F. Biegasiewicz, S. J. Cooper, X. Gao, D. G. Oblinsky,
J. H. Kim, S. E. Garfinkle, L. A. Joyce, B. A. Sandoval, G. D. Scholes, T. K.
Hyster, Science 2019, 364, 1166; c) M. J. Black, K. F. Biegasiewicz, A. J.
Meichan, D. G. Oblinsky, B. Kudisch, G. D. Scholes, T. K. Hyster, Nat. Chem.
2020, 12, 71.
Acknowledgements
Financial support provided by NSF (1846861), Searle Scholar
Program, Sloan Scholar Program, and Princeton University. YN
thanks the Australian Government for an Endeavour Postdoctoral
Fellowship. We thank Phil Jeffrey for assistance with x-ray structure
determination and the NSLS-II AMX (17-ID-1) beam line staff for
assistance with X-ray data collection. This research used NSLS-II
AMX (17-ID-1), a U.S. Department of Energy (DOE) Office of
Science User Facility operated for the DOE Office of Science by
Brookhaven National Laboratory under contract No. DE-SC0012704.
[8] a) K. F. Biegasiewicz, S. J. Cooper, M. A. Emmanuel, D. C. Miller, T. K.
Hyster, Nature Chem. 2018, 10, 770; b) B. A. Sandoval, S. I. Kurtoic, M. M.
Chung, K. F. Biegasiewicz, T. K. Hyster, Angew. Chem. Int. Ed. 2019, 58,
8714.
[9] For examples involving synergistic biocatalytic/photoredox cascades, see: a)
S. H. Lee, D. S. Choi, M. Pesic, Y. W. Lee, C. E. Paul, F. Hollmann, C. B.
Park, Angew. Chem. Int. Ed. 2017, 129, 8807; b) Z. C. Litman, Y. Wang, H.
Zhao, J. F. Hartwig, Nature 2018, 560, 355; c) R. C. Betori, C. M. May, K. A.
Scheidt, Angew. Chem. Int. Ed. 2019, 58, 16490; d) X. Guo, Y. Okamoto, M.
R. Schreier, T. R. Ward, O. S. Wenger, Chem. Sci. 2018, 9, 5052l e) J. Xu, M.
Arkin, Y. Peng, W. Xu, H. Yu, X. Lin, Q. Wu, Green Chem. 2019, 21, 1907;
f) W. Zhang, E. Fernández-Fueyo, F. Hollman, L. L. Martin, M. Pesic, R.
Wardenga, M. Höhne, S. Schmidt, Eur. J. Org. Chem. 2019, 80; g) M. K.
Peers, H. S. Toogood, D. J. Heyes, D. Mansell, B. J. Coe, N. S. Scrutton, Catal.
Sci. Technol. 2016, 6, 169. For recent reviews on the topic, see: h) L.
Schmermund, V. Jurkaš, F. F. Özgen, G. D. Barone, H. C. Büchsenschütz, C.
K. Winkler, S. Schmidt, R. Kourist, W. Kroutil, ACS Catal. 2019, 9, 4115; i)
C. J. Seel, T. Gulder, ChemBioChem 2019, 20, 1871.
Experimental Procedure
General Reaction Procedure. In the Coy chamber was
introduced: protein aliquots (NostocER, in “OYE concentration
buffer” [20 mM KPi pH 7.4, 300 mM NaCl], generally 2~4 mM
concentration, 100 nmol total in each aliquot), shell vial with
magnetic cross stir-bar, substrate in a one-dram vial, and a “master
mix” vial containing 1.0 mg Ru(bpy)3Cl2•6H2O and 2.0 mg NADP+
and 10.7 mg GDH-105 and 96.0 mg D-glucose. To the “master mix”
4
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