An NAD(P)H-Dependent artificial transfer hydrogenase for multienzymatic cascades

Article abstract of DOI:10.1021/jacs.6b02470

Enzymes typically depend on either NAD(P)H or FADH₂ as hydride source for reduction purposes. In contrast, organometallic catalysts most often rely on isopropanol or formate to generate the reactive hydride moiety. Here we show that incorporati

Full text of DOI:10.1021/jacs.6b02470

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Communication
An NAD(P)H-dependent Artificial Transfer
Hydrogenase for Multi-enzymatic Cascades
Yasunori Okamoto, Valentin Köhler, and Thomas R. Ward
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b02470 • Publication Date (Web): 21 Apr 2016
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An NAD(P)H-dependent Artificial Transfer Hydrogenase for Multi-
enzymatic Cascades
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Yasunori Okamoto, Valentin Köhler and Thomas R. Ward*
Department of Chemistry, University of Basel, Spitalstrasse 51, CHꢀ4056 Basel Switzerland
Supporting Information Placeholder
enzymatic processes, where the hydride is utilized in a
productive fashion, have not been disclosed yet (Scheme
1a). We hypothesize that this may be traced back to the
mutual deactivation of both transition metal catalysts
and natural enzymes. To overcome this challenge, spaꢀ
tial separation of both catalytic partners has proven most
effective.13ꢀ15 In this context, artificial metalloenzymes
(AMEs) have received increasing attention as alternative
to both homogeneous catalysts and enzymes.16 To test
the versatility of AMEs for the implementation of enzyꢀ
matic cascades, we reported on the compartmentalizaꢀ
tion of a biotinylated Cp*Irꢀbased transfer hydrogenaꢀ
tion catalyst into streptavidin (Sav) variants (Scheme
1d).15 To drive the hydride transfer reactions at reasonaꢀ
ble rates however, concentrations of formate in the mo-
lar range were required.7,15,17 Such concentrations of
formate may lead to inactivation of natural enzymes and
are thus incompatible with in vivo applications. Here, we
show that the biological reducing agent NAD(P)H can
serve as an efficient hydride source for an Cp*Irꢀ
catalyzed hydride transfer in multienzymatic cascade
reactions at millimolar loadings and near perfect stoichiꢀ
ometric fidelity under concurrent enzymatic NAD(P)H
regeneration.
ABSTRACT: Enzymes typically depend on either
NAD(P)H or FADH2 as hydride source for reduction
purposes. In contrast, organometallic catalysts most ofꢀ
ten rely on isopropanol or formate to generate the reacꢀ
tive hydride moiety. Here we show that incorporation of
a Cp*Ir cofactor possessing a biotin moiety and 4,7ꢀ
dihydroxyꢀ1,10ꢀphenanthroline into streptavidin (Sav)
yields an NAD(P)Hꢀdependent artificial transfer hydroꢀ
genase (ATHase). This ATHase (0.1 mol%) catalyzes
imine reduction with 1 mM NADPH (2 mol%), which
can be concurrently regenerated by a glucose dehydroꢀ
genase (GDH) using only 1.2 equivalents glucose. A
four enzyme cascade consisting of the ATHase, the
GDH, a monoamine oxidase and a catalase leads to the
production of enantiopure amines.
The introduction of synthetic catalysts into a biologiꢀ
cal context is at the focus of current efforts in both synꢀ
theticꢀ and chemical biology.1,2 Goals include i) the supꢀ
plementation of existingꢀ or engineered metabolic pathꢀ
ways, ii) decaging inactive forms of enzymes to trigger
enzymatic cascades,2,3 iii) shifting the redox equilibrium
in cancer cells to induce apoptosis4 or iv) produce fuels
with the help of biological redox equivalents.3ꢀ5 Achievꢀ
ing high productivity of synthetic organometallic cataꢀ
lysts inside a living system remains challenging and
progress is likely to be incremental.2,6 In contrast, the
combination of isolated enzymes and transition metal
catalysts in carefully designed in vitro systems of modꢀ
est complexity experiences increasing success.7ꢀ9
Scheme 1. (a) NAD(P)H as a hydride source for imine
hydrogenation. Structure of (b) NAD+, (c) pianostool
complexes (L^L is a bidentate ligand) and (d) sche-
matic representation of a Sav-based ATHase.
Transition metalꢀmediated formal hydride transfer ocꢀ
cupies a prominent role in many of these initiatives.9
Remarkably, synthetic catalysts and enzymes have
gained common ground for the conversion of ketones to
alcohols, imines to amines (and vice versa), the reducꢀ
tion of activated double bonds and the racemisation of
secondary alcohols and amines.10 A few isolated studies
have shown that transition metal complexes can accept
NAD(P)H as a hydride source.5,11,12 To the best of our
knowledge however, their concurrent use coupled with
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In previous designs of artificial transfer hydrogenases shielding effect of the host protein (Scheme 3, entries 1
(ATHase) based on d6ꢀpianostool complexes, we relied
on aminosulfonamide or aminoamide ligands to catalyze
the transfer of hydride from formate to ketones, imines,
enones18 and Nꢀalkylated nicotinamides (Scheme
1c).15,19 Inspired by Sadler’s work on the NADH oxidaꢀ
tion catalysts,11 we screened a range of commercial biꢀ
dentate ligands (L^L 1ꢀ27) for their ability to act as ligꢀ
ands for Cp*Ir in the oxidation of dihydronicotinamides
(NADH) in the presence of an acceptor substrate
(Scheme 2a). The amount of consumed NADH was deꢀ
termined by absorbance spectroscopy in the presence of
1 eq. 1ꢀmethylꢀ3,4ꢀdihydroisoquinoline (MDQ A hereafꢀ
ter), [Cp*IrCl2]2 (12.5 mol%) and L^L (27.5 mol%) posꢀ
sessing various donor and acceptor properties (Scheme
2b and S1). Among the ligands displaying catalytic oxiꢀ
dation of NADH (> 25 % = 1 turnover), bisoxazoline 1
and phenanthroline ligands 2ꢀ5 gave the highest activiꢀ
ties (Scheme 2b and Scheme S1). To distinguish comꢀ
petitive hydrogen evolution from substrate reduction,
these ligands were employed in reactions with
[Cp*IrCl2]2 (0.2 mol%), ligand L^L (0.44 mol%) and
stoichiometric amount of NADH (25 mM). Formation of
1ꢀmethylꢀ1,2,3,4ꢀtetrahydroisoquinoline (MTQ B hereꢀ
after) was monitored by HPLC (Scheme 2c). The elecꢀ
tron rich 4,7ꢀdihydroxyꢀ1,10ꢀphenanthroline 5,20 and to
a lesser extent the bioxazoline 1, led to significantly imꢀ
proved rates with respect to the product formation.
and 2). Mutation at position S112 did not improve the
performance of the ATHase and no activity was obꢀ
served in the presence of coordinating aminoacids at
either S112 or K121 (Scheme 3, entries 3, 4 and Table
S7, entries 1ꢀ6). The conservative mutation K121R afꢀ
forded the most active ATHase [BiotꢀCp*Ir(L^L
1
2
3
4
5
6
7
8
5)Cl]⊂Sav K121R (Scheme 3, entry 5). Raising the
temperature from 25 to 37°C and replacing NAD+ by
NADP+ led to further improvements (Scheme 3, entries
5ꢀ7). Finally, extending the reaction time from 14 to 16
hours yielded full conversion, corresponding to TONIr =
1000 and to TONNADP+ = 50 (Scheme 3, entry 8). Under
these conditions, only 1.2 equivalents of glucose are
required to drive the imine reduction to completion.
Even under physiological NADP(H) concentrations (i.e.
120 ꢁM),24 a TONIr of 164 was obtained (Scheme 3, enꢀ
try 9). The previously reported HCOOHꢀdependent
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ATHase
[Cp*Ir(biotꢀpꢀL)Cl]⊂Sav
S112AꢀK121A
(Scheme 1c),25 afforded a modest TONIr of 69 (6.9 %
conversion) in the presence of 60 mM sodium formate
(i.e. 120 mol%) (Scheme 3, entry 10, compare Table
S7).
Scheme 3. Transfer-hydrogenation coupled with
NAD(P)H regeneration by a GDH and glucose (Glu).
Scheme 2. (a) NADH as hydride source for the reduc-
tion of MDQ A catalyzed by [Cp*Ir(L^L 1-27)Cl]. (b)
Selected ligands and corresponding conversion of
NADH into NAD+. (c) Time-course plot of MDQ A
hydrogenation by [Cp*IrCl2]2 with ligands 1 (blue), 2
(orange), 3 (green), 4 (red) and 5 (cyan).
Sav
isoform
Hydride
source
entry
TONIr
e.e. [%]
1
No Sav
WT
NAD+/Glu
NAD+/Glu
NAD+/Glu
NAD+/Glu
NAD+/Glu
NAD+/Glu
75
202
214
101
270
679
961
>999
164
1 (R)
17 (S)
8 (S)
2
3
S112A
K121A
K121R
K121R
K121R NADP+/Glu
K121R NADP+/Glu
K121R NADP+/Glue
4
6 (S)
5
13 (R)
13 (R)
13 (R)
14 (R)
13 (R)
6 a
7a
8a, b
9a ,c, d
Having identified the most promising bidentate ligand
L^L 5, the corresponding ATHase [BiotꢀCp*Ir(L^L
5)Cl]⊂
Sav was assembled (Scheme 1c, d).21ꢀ23 Next, we
S112Aꢀ
K121A
10a ,f
HCOONag
69
6 (R)
combined this ATHase with glucose dehydrogenase
(GDH) to regenerate the consumed NAD(P)H. This enaꢀ
bles the use of glucose as reductant, yielding NAD(P)H
and gluconoꢀδꢀlactone (Scheme 3). The performance of
the concurrent enzymatic cascade was optimized by
screening Sav variants focusing on closeꢀlying positions
S112 and K121.22 The reactions contained GDH (0.1
mg/ml), NAD+ (1 mM, 2 mol%), glucose (60 mM, 120
mol%), BiotꢀCp*Ir(L^L 5)Cl (50 µM, 0.1 mol%), Sav
free binding sites (100 µM) and MDQ A (50 mM).
Compared to [BiotꢀCp*Ir(L^L 5)Cl], the conversion by
a
b
Experiments were performed at 25 or 37 ºC for 14, 16
c
or 24 h; 50 ꢁM [BiotꢀCp*Ir(L^L 5)Cl]⊂Sav variant, 0.1
mg/ml GDH, 1 mM NAD(P)+, 50 mM MDQ A and 60 mM
glucose in 0.3 M MOPS (pH 7.9). d100 ꢁM [Biotꢀ
Cp*Ir(L^L 5)Cl]⊂
Sav. 120 ꢁM NADP+. [Cp*Ir(biotꢀpꢀ
e
f
L)Cl] was used instead of [BiotꢀCp*Ir(L^L 5)]Cl. g 60 mM
HCOONa. Average values for duplicate reactions are disꢀ
played; standard deviations were ≤ 4.0 %.
However, the enantiomeric excess obtained via the
above cascade was modest (Scheme 3 and Scheme 4,
[BiotꢀCp*Ir(L^L 5)Cl]⊂Sav doubled, highlighting the
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entry 4). To overcome this drawback, we integrated a
monoamine oxidase (MAO) and a catalase, into the
1
2
3
4
5
6
7
8
GDH/ATHase cascade for the production of MTQ B
(Scheme 4).15,26 Combining the highly enantioselective
MAOꢀcatalyzed oxidation of (S)ꢀMTQ B with simultaꢀ
neous ATHaseꢀdriven reduction resulted in perfect enanꢀ
tioselectivity to yield (R)ꢀMTQ B in full conversion with
only 2 equivalents of glucose required (Scheme 4, entry
3). In this multienzymatic cascade reaction, the benefit
of compartmentalizing the cofactor [BiotꢀCp*Ir(L^L
5)Cl] within Sav K121R is most prominent: the converꢀ
sion improved from a mere TONIr of at least 30 (6%
yield) for the free cofactor to a TONIr of greater than 445
(89% yield) with the ATHase (Scheme 4, entries 1, 2).
a)
Experiments were performed at 37 ºC; 100 ꢁM [Biotꢀ
9
Cp*Ir(L^L 5)Cl]⊂Sav(K121R), 0.25 mg/ml ADH, 1 mM
NADP+, 1.25 mM MgSO4, 10 mM substrate C in 0.3 M
MOPS (pH 7.9). Average values for duplicate reactions are
plotted; standard deviations were ≤ 0.23%.
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Finally, we investigated the transhydrogenase activity
of the ATHase [BiotꢀCp*Ir(L^L 5)Cl]⊂Sav K121R.
Scheme 4. Enzyme cascade for the deracemization of
cyclic amines.a)
Transhydrogenases rapidly equilibrate mixtures of
NAD(H) and NADP(H) in a situation where one of
NAD(H) and NADP(H) is more rapidly consumed than
the other.29 In the absence of ATHase, the redox equiliꢀ
bration between NADPH and NAD+ or NADH and
NADP+ was slow (Table 1, entries 2, 4). In contrast,
[BiotꢀCp*Ir(L^L 5)Cl]⊂Sav K121R significantly accelꢀ
erates the reaction to reach its equilibrium within 30 min
(Table 1, entries 1, 3 and Table S13). This transhydroꢀ
genase activity may find applications to equilibrate the
NAD(P)H levels in reaction cascades which combine
both NADHꢀ and NADPHꢀdependent enzymes. Examꢀ
ples include the biocatalytic synthesis of the potent drug
hydromorphone30 or oxidation reactions catalyzed by
P450 enzymes.31
Sav
MAO
time conv.
e.e.
[%]
entry
isoform [mg/ml]
[h]
12
12
24
24
[%]
1
2
3
4
No Sav
K121R
K121R
K121R
0.2
0.2
0.2
–
6
>99 (R)
75 (R)
89
>99
>99
>99 (R)
13 (R)
Table 1. Transhydrogenase activity of ATHase.a)
a)
reductant
/substrate
NADPH/NADH
(mM)
Experiments were performed under the following condiꢀ
entry
catalyst
tions; 100 ꢁM [BiotꢀCp*Ir(L^L 5)Cl], 0.1 mg/ml GDH, 0.2
mg/ml MAO, 500U catalase, 1 mM NADP+, 50 mM MDQ
A and 100 mM glucose in 0.3 M MOPS (pH 7.9) at 37 ºC.
Average values for duplicate reactions are displayed and
standard deviations were ≤ 1.0 %.
1
2
3
4
ATHase
NADPH/NAD+
NADPH/NAD+
NADH/NADP+
NADH/NADP+
1.9/2.4
4.6/0.1
1.9/2.4
0.1/4.7
–
ATHase
–
Instead of GDH, we also coupled an alcohol dehydroꢀ
genase (ADH), another representative NAD(P)H regenꢀ
eration system, with the ATHase [BiotꢀCp*Ir(L^L 5)Cl]
⊂Sav K121R. Relying on a hydrogenꢀborrowing strateꢀ
gy,27 we designed a cascade reaction to enable a two
step transformation of a linear aminoalcohol into a cyꢀ
clic amine via an imine. For this purpose, 4ꢀaminoꢀ1ꢀ
phenylꢀ1ꢀbutanol C was selected as a substrate (Scheme
5a). In view of the exquisite enantioselectivity of ADH,
one enantiomer of the aminoalcohol C is thought to be
transformed into the corresponding dihydropyrrole D
and pyrrolidine E in 12. 8% and 36.1 % yield respectiveꢀ
ly after 48 h (Scheme 5b and Table S11).28
a)
Experiments were performed at 37 ºC; 50 ꢁM [Biotꢀ
Cp*Ir(L^L 5)Cl]⊂Sav K121R, 5 mM NAD(P)H and 5 mM
NAD(P)+ in 0.3 M MOPS (pH 7.9) for 30 min. Average
values for duplicate reactions are displayed and standard
deviations were ≤ 1.0 %.
In summary, NAD(P)Hꢀdependent ATHases were sucꢀ
cessfully developed that allow the use of NAD(P)H as
hydride source for transferꢀhydrogenation under physioꢀ
logical conditions. Two representative enzymatic
NAD(P)H regeneration systems were shown to be comꢀ
patible with this ATHase. A chemogenetic optimization
strategy allowed to rapidly identify [BiotꢀCp*Ir(L^L
Scheme 5. (a) A hydrogen-borrowing cascade reac-
tion comprised of an ADH with the NAD(P)H-
dependent ATHase and (b) its reaction progress: of 2-
5)Cl]⊂Sav K121R as a versatile ATHase for the reducꢀ
tion of imines. Addition of a monoamine oxidase couꢀ
pled with a catalase allows to produce enantiopure
amines as a result of a four enzyme cascade. Further
researches to exploit this ATHase in vivo are in progress.
phenyl-3,4-dihydro-5H-pyrrole
D
(red),
2-
phenylpyrrolidine E (cyan), and their sum (black).a)
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(15) Köhler, V.; Wilson, Y. M.; Dürrenberger, M.; Ghislieri, D.;
Churakova, E.; Quinto, T.; Knörr, L.; Häussinger, D.; Hollmann, F.;
Turner, N. J.; Ward, T. R. Nat. Chem. 2013, 5, 93.
ASSOCIATED CONTENT
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The supporting information includes materials, instruments,
experimental procedures and additional data of Scheme 2ꢀ4
and Table 1. (PDF) This material is available free of charge
via the Internet at http://pubs.acs.org.
(16) (a) Wilson, M. E.; Whitesides, G. M. J. Am. Chem. Soc. 1978,
100, 306. (b) Yu, F.; Cangelosi, V. M.; Zastrow, M. L.; Tegoni, M.;
Plegaria, J. S.; Tebo, A. G.; Mocny, C. S.; Ruckthong, L.; Qayyum,
H.; Pecoraro, V. L. Chem. Rev. 2014, 114, 3495. (c) Ueno, T.; Abe,
S.; Yokoi, N.; Watanabe, Y. Coord. Chem. Rev. 2007, 251, 2717. (d)
Hayashi, T.; Sano, Y.; Onoda, A. Isr. J. Chem. 2014, 55, 76. (e)
Pàmies, O.; Diéguez, M.; Bäckvall, J. Adv. Synth. Catal. 2015, 357,
1567. (f) Bos, J.; Roelfes, G. Curr. Opin. Chem. Biol. 2014, 19, 135.
(17) (a) Ruppert, R.; Herrmann, S.; Steckhan, E. J. Chem. Soc.
Commun 1988, 1150. (b) Lo, H. C.; Buriez, O.; Kerr, J. B.; Fish, R.
H. Angew. Chem., Int. Ed. 1999, 38, 1429. (c) Lo, H. C.; Leiva, C.;
Buriez, O.; Kerr, J. B.; Olmstead, M. M.; Fish, R. H. Inorg. Chem.
2001, 40, 6705. (d) Ogo. S.; Abura, T.; Watanabe, Y. Organometal-
lics 2002, 21, 2964. (e) Hollmann, F.; Lin, P.ꢀC.; Witholt, B.; Schmid,
A. J. Am. Chem. Soc. 2003, 125, 8209. (f) Yan, Y. K.; Melchart, M.;
Habtemariam, A.; Peacock, A.; Sadler, P. J. J. Biol. Inorg. Chem.
2006, 11, 483. (g) Canivet, J.; SüssꢀFink, G.; Štěpnička, P. Eur. J.
Inorg. Chem. 2007, 4736.
(18) Heinisch, T.; Langowska, K.; Tanner, P.; Reymond, JꢀL.;
Meier, W.; Palivan, C.; Ward, T. R. ChemCatChem 2013, 5, 720.
(19) Dürrenberger, M.; Heinisch, T.; Wilson, Y. M.; Rossel, T.;
Nogueira, E.; Knörr, L.; Mutschler, A.; Kersten, K.; Zimbron, M. J.;
Pierron, J.; Schirmer, T.; Ward, T. R. Angew. Chem., Int. Ed. 2011,
50, 3026.
(20) (a) Himeda, Y.; OnozawaꢀKomatsuzaki, N.; Sugihara, H.;
Kasuga, K. Organometallics 2004, 23, 1480. (b) Himeda, Y.;
OnozawaꢀKomatsuzaki, N.; Sugihara, H.; Kasuga, K. J. Am. Chem.
Soc. 2005, 127, 13118.
AUTHOR INFORMATION
Corresponding Author
9
* Eꢀmail for T. R. W.; Thomas.ward@unibas.ch
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Funding Sources
Generous support from the Swiss National Science Foundaꢀ
tion (grant 200020_162348) and from the NCCR Molecular
Systems Engineering is gratefully acknowledged.
ACKNOWLEDGMENT
We thank Prof. Dr. N. J. Turner for providing the plasmid
coding MAO and Umicore for a loan of [Cp*IrCl2]2.
REFERENCES
(1) (a) Streu, C.; Meggers, E. Angew. Chem., Int. Ed. 2006, 45,
5645. (b) Li, N.; Lim, R. K. V.; Edwardraja, S.; Lin, Q. J. Am. Chem.
Soc. 2011, 133, 15316. (c) Yusop, R. M.; UncitiꢀBroceta, A.; Johansꢀ
son, E. M. V.; SánchezꢀMartín, R. M.; Bradley, M. Nat. Chem. 2011,
3, 239. (d) Spicer, C. D.; Triemer, T.; Davis, B. G. J. Am. Chem. Soc.
2012, 134, 800. (e) Völker, T.; Dempwolff, F.; Graumann, P. L.;
Meggers, E. Angew. Chem., Int. Ed. 2014, 53, 10536.
(2) Völker, T.; Meggers, E. Curr. Opin. Chem. Biol. 2015, 25, 48.
(3) Li, J.; Yu, J.; Zhao, J.; Wang, J.; Zheng, S.; Lin, S.; Chen, L.;
Yang, M.; Jia, S.; Zhang, X.; Chen, P. R. Nat. Chem. 2014, 6, 352.
(4) SoldevilaꢀBarreda, J. J.; RomeroꢀCanelón, I.; Habtemariam, A.;
Sadler, P. J. Nat. Commun. 2015, 6, 6582.
(5) (a) Maenaka, Y.; Suenobu, T.; Fukuzumi, S. J. Am. Chem. Soc.
2012, 134, 9417. (b) Maenaka, Y.; Suenobu, T.; Fukuzumi, S. J. Am.
Chem. Soc. 2012, 134, 367. (c) Anbarasan, P.; Baer, Z. C.; Sreekuꢀ
mar, S.; Gross, E.; Binder, J. B.; Blanch, H. W.; Clark, D. S.; Toste,
F. D. Nature 2012, 491, 235.
(6) (a) Spicer, C. D.; Davis, B. G. Nat Commun 2014, 5, 4740. (b)
Chankeshwara, S. V.; Indrigo, E.; Bradley, M. Curr. Opin. Chem.
Biol. 2014, 21, 128.
(21) Hyster, T. K.; Knörr, L.; Ward, T. R.; Rovis, T. Science 2012,
338, 500.
(22) Zimbron, J. M.; Heinisch, T.; Schmid, M.; Hamels, D.;
Nogueira, E. S.; Schirmer, T.; Ward, T. R. J. Am. Chem. Soc. 2013,
135, 5384.
(23) Quinto, T.; Schwizer, F.; Zimbron, J. M.; Morina, A.; Köhler,
V.; Ward, T. R. ChemCatChem 2014, 6, 1010.
(24) Bennett, B. D.; Kimball, E. H.; Gao, M.; Osterhout, R.; Van
Dien, S. J.; Rabinowitz, J. D. Nat. Chem. Biol. 2009, 5, 59.
(25) Schwizer, F.; Köhler, V.; Dürrenberger, M.; Knörr, L.; Ward,
T. R. ACS Catal. 2013, 3, 1752.
(26) (a) Turner, N. J. Nat. Chem. Biol. 2009, 5, 567. (b) Turner, N.
J. Chem. Rev. 2011, 111, 4073. (c) Ghislieri, D.; Green, A. P.; Ponꢀ
tini, M.; Willies, S. C.; Rowles, I.; Frank, A.; Grogan, G.; Turner, N.
J. J. Am. Chem. Soc. 2013, 135, 10863.
(27) (a) Hamid, M. H. S.; Slatford, P. A.; Williams, J. M. J. Adv.
Synth. Catal. 2007, 349, 1555. (b) Mutti, F. G.; Knaus, T.; Scrutton,
N. S.; Breuer, M.; Turner, N. J. Science 2015, 349, 1525. (c) Guillena,
G.; J Ramón, D.; Yus, M. Chem. Rev. 2010, 110, 1611. (d)
Dobereiner, G. E.; Crabtree, R. H. Chem. Rev. 2010, 110, 681. (e)
Sattler, J. H.; Fuchs, M.; Tauber, K.; Mutti, F. G.; Faber, K.; Pfeffer,
J.; Haas, T.; Kroutil, W. Angew. Chem., Int. Ed. 2012, 51, 9156. (f)
Zhang, Y.; Lim, C.ꢀS.; Sim, D. S. B.; Pan, H.ꢀJ.; Zhao, Y. Angew.
Chem., Int. Ed. 2014, 53, 1399. (g) Gunanathan, C.; Milstein, D. Sci-
ence 2013, 341, 1229712. (h) Oldenhuis, N. J.; Dong, V. M.; Guan, Z.
J. Am. Chem. Soc. 2014, 136, 12548. (i) Chen, F. F.; Liu, YꢀY.;
Zheng, GꢀW.; Xu, JꢀH.; ChemCatChem 2015, 7, 3838.
(28) The enantiopurity of the remaining 4ꢀaminoꢀ1ꢀphenylꢀ1ꢀ
butanol C could not be determined. According to the enzyme providꢀ
er Codexis however, the enantioselectivity of KREDꢀP1ꢀB12 toward
acetophenone reduction is 97% ee (R).
(29) (a) Hall, M.; Bommarius, A. S. Chem. Rev. 2011, 111, 4088.
(b) van der Donk, W. A.; Zhao, H. Curr. Opin. Biotechnol. 2003, 14,
421.
(7) (a) de Gonzalo, G.; Ottolina, G.; Carrea, G.; Fraaije, M. W.
Chem. Commun. 2005, 3724. (b) Hollmann, F.; Arends, I. W. C. E.;
Buehler, K. ChemCatChem 2010, 2, 762.
(8) Mifsud, M.; Gargiulo, S.; Iborra, S.; Arends, I. W. C. E.;
Hollmann, F.; Corma, A. Nat. Commun. 2014, 5, 3145.
(9) (a) Verho, O.; Bäckvall, J.ꢀE. J. Am. Chem. Soc. 2015, 137,
3996. (b) Köhler, V.; Turner, N. J. Chem. Commun. 2015, 51, 450. (c)
Denard, C. A.; Hartwig, J. F.; Zhao, H. ACS Catal. 2013, 3, 2856.
(10) (a) Wu, X.; Xiao, J. in Metal-Catalyzed Reactions in Water,
Wiley-VCH, 2013, pp. 173. (b) Schrittwieser, J. H.; Velikogne, S.;
Kroutil, W. Adv. Synth. Catal. 2015, 357, 1655. (c) Musa, M. M.;
Phillips, R. S.; Laivenieks, M.; Vieille, C.; Takahashi, M.; Hamdan,
S. M. Org. Biomol. Chem. 2013, 11, 2911.
(11) (a) BetanzosꢀLara, S.; Liu, Z.; Habtemariam, A.; Pizarro, A.
M.; Qamar, B.; Sadler, P. J. Angew. Chem., Int. Ed. 2012, 51, 3897.
(b) Liu, Z.; Deeth, R. J.; Butler, J. S.; Habtemariam, A.; Newton, M.
E.; Sadler, P. J. Angew. Chem., Int. Ed. 2013, 52, 4194.
(12) Tang, L.L; Gunderson, W. A.; Weitz, A. C.; Hendrich, M. P.;
Ryabov, A. D.; Collins, T. J. J. Am. Chem. Soc. 2015, 137, 9704.
(13) Poizat, M.; Arends, I.; Hollmann, F. J. Mol. Catal. B 2010, 63,
149.
(14) Wang, Z. J.; Clary, K. N.; Bergman, R. G.; Raymond, K. N.;
Toste, F. D. Nat. Chem. 2013, 5, 100.
(30) Boonstra, B.; Rathbone, D. A.; French, C. E.; Walker, E. H.;
Bruce, N. C. Appl. Environ. Microbiol. 2000, 66, 5161.
(31) Mouri, T.; Shimizu, T.; Kamiya, N.; Goto, M.; Ichinose, H.
Biotechnol. Prog. 2009, 25, 1372.
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