Pyridoxal-Catalyzed Racemization of α-Aminoketones Enables the Stereodivergent Synthesis of 1,2-Amino Alcohols Using Ketoreductases

Article abstract of DOI:10.1021/acscatal.0c01502

Differentially substituted 1,2-amino alcohols are a prevalent motif in a variety of pharmaceutical and agrochemical molecules. Dynamic kinetic resolutions (DKRs) that involve the asymmetric reduction of α-amino ketones are attractive for preparing this motif; however, methods for racemizing the stereogenic α-carbon under mild conditions are underdeveloped. Here we report a chemoenzymatic DKR involving ketoreductases (KREDs), in which pyridoxal-5-phosphate (PLP) is used to catalyze racemization of the starting racemic α-aminoketone. This strategy enables access to a variety of 1,2-amino alcohols with high levels of diastereo- and enantioselectivity. Using commercially available KREDs, all four possible stereoisomers can be accessed, highlighting a benefit to this approach.

Full text of DOI:10.1021/acscatal.0c01502

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Letter
Pyridoxal-Catalyzed Racemization of #-Aminoketones Enables the
Stereodivergent Synthesis of 1,2-Amino Alcohols Using Ketoreductases
Jingzhe (Bill) Cao, and Todd K. Hyster
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.0c01502 • Publication Date (Web): 11 May 2020
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ACS Catalysis
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Pyridoxal-Catalyzed Racemization of α-Aminoketones Enables
the Stereodivergent Synthesis of 1,2-Amino Alcohols Using
Ketoreductases
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Jingzhe (Bill) Cao, and Todd K. Hyster*
Department of Chemistry, Princeton University, Frick Chemical Laboratory, Princeton, NJ 08544
variety of synthetic scenarios,11 including in dynamic
ABSTRACT: Differentially substituted 1,2-amino alcohols are a
kinetic resolutions. KREDs generally operate in a relatively
narrow pH window (pH 6-8), consequently substrates for
DKRs require acidic α-protons site with pKa’s between 7-12
(Figure 1a).12 As most ꢀ-aminoketones have less acidic α-
protons, only substrates containing addition electron
withdrawing groups can be racemized under these
conditions.13
prevalent motif in a variety of pharmaceutical and agrochemical
molecules. Dynamic kinetic resolutions (DKRs) that involve the
asymmetric reduction of -amino ketones are an attractive strategy
for preparing this motif, however, methods for racemizing the
stereogenic α-carbon under mild conditions are underdeveloped.
Here we report a chemoenzymatic DKR involving ketoreductases
(KREDs), where pyridoxal-5-phosphate (PLP) is used to catalyze
racemization of the starting racemic -aminoketone. This strategy
enables access to a variety of 1,2-amino alcohols with high levels
of diastereo- and enantioselectivity. Using commercial available
KREDs, all four possible stereoisomers can be accessed,
highlighting a benefit to this approach.
A. Substrates for Biocatalyt ic Dynamic Kinetic Resolutions
Challenging to
Readily Epimerized Under KRED Compatible Conditions
Epimerize
O
O
O
O
O
O
CN
H
R
Me
Me
Me
OEt
Me
R
H
R H
H2N
H
Keywords: biocatalysis, dynamic kinetic resolution,
racemization, pyridoxal, ketoreductase
pKa = 7.8 (H2O)
pKa = 9 (H2O)
pKa = 11 (H2O)
pKa > 20 (H2O)
B. Existing Strategy - Engineer for Enhanced Act ivity
Vicinal amino alcohols are ubiquitous in natural products
and pharmaceutically important molecules.1 The
prevalence of this motif has spurred the development of
catalytic methods for preparing 1,2-amino alcohols with
high levels of stereoselectivity. Among these methods, the
aminohydroxylation of alkenes and nucleophilic opening of
epoxides and aziridines catalyzed by enzymes and small
molecule catalysts are the most well developed. 2,3,4 While
these methods are highly effective, they involve
stereospecific reaction mechanisms, limiting their use to
the synthesis of specific stereoisomers. Alternatively,
asymmetric reductive amination. The asymmetric
reduction of 훼-aminoketones is an attractive alternative to
these approaches because it is not mechanistically
restricted to providing certain stereochemical outcomes.5
When coupled to a mechanism for 훼-aminoketone
racemization,6 stereoselective reduction catalysts can, in
theory, be employed in to access all possible stereochemical
outcomes from racemic starting materials.7,8 To achieve this
goal, however, catalysts scaffolds need to be identified that
are capable of furnishing all possible stereoisomers while
also tolerating under the basic conditions required for
racemization.
Protein
Engineering
Starting Ketoreductase
Evolved Ketoreductase
✓
Enantioselective
✓
Enantioselective
Tolerant to Basic Conditions
✓
Tolerant to Basic Conditions
C. This Work - Pyridoxal-Catalyzed Racemizat ion
O
O
O
Ph
Me
2-O3PO
Ph
Me
Ph
Me
N
H
H2N
H
H2N
H
(S)
OH
(R)
enhanced
acidity
N
Static α
Stereocenter
Static α
Stereocenter
Me
Dynamic Stereocenter
Can PLP catalyze the epimerization of �-aminoketones at neutral reaction conditions?
Figure 1. Biocatalytic Dynamic Kinetic Resolutions and
Pyridoxal-5-Phosphate Catalysis (as a strategy for labializing
the 훼-amino proton). A. Substrates for Biocatalytic Dynamic
Kinetic Resolutions. B. Existing Protein Engineering Strategy. C.
This Work using Pyridoxal-Catalyzed Racemization.
Efforts to overcome the pKa limitation have primarily
focused on engineering KREDs to function under more basic
conditions (Figure 1b). In their synthesis of Vibegron,
Merck & Co. partnered with Codexis to demonstrate that a
KRED could be evolved to operate at pH =10.0, enabling
racemization of Boc-protected 훼-aminoarylketones.14 We
imagined that the need for protein engineering could be
overcome by developing a strategy for racemizing 훼-
Ketoreductases (KREDs) are ideal catalysts for
stereoselective carbonyl reductions because of their
substrate promiscuity and ability to be tuned, using
directed evolution, to provide desired stereochemical
outcomes.9 Thanks to these features, collections of
structurally diverse KREDs have been compiled for use in
chemical synthesis.10 These panels have been applied to a
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aminoketones at neutral pH. This would enable
commercially available kits of structurally diverse KREDs to
be rapidly deployed in DKRs to prepare 1,2-amino alcohols,
thereby eliminating the need to engineer a base-tolerant
KREDs.15
Page 2 of 6
pyrazine side product.22 To test this hypothesis, we
explored the reduction of phenylalanine-derived methyl
ketone 1 with a panel of KREDs purchased from Codexis®
under aerobic conditions and observed pyrazine 3 as the
major product, with < 5% formation of the desired 1,2-
amino alcohol 2 (Figure 2). When the same reaction was
tested under anaerobic conditions, KRED P2-D11 provided
the desired 1,2-amino alcohol 2 in 32% yield with 6:1 dr and
>99:1 er, with the remaining mass balance being unreacted
starting material (Figure 2).
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Amine activation via aldehyde catalysis is widely used
in nature to facilitate amine metabolism. Nature’s aldehyde
of choice is pyridoxal-5-phosphate (PLP), a cofactor found
in a host of enzyme classes,16 including transaminase,
tryptophan synthase, threonine aldolase, tyrosine
decarboxylase, and alanine racemase.17 In these enzymes,
PLP forms a Schiff base with the amine enabling
stabilization of negative charge generated at the α-position
of the amine (Figure 1c).18 It is calculated that PLP can
increase the acidity of the α-position of glycine by 18 orders
of magnitude.19 Inspired by this profound effect, we
questioned whether PLP could catalyze the racemization of
-aminoketones in the absence of protein at neutral pH.
Previous reports demonstrate that PLP can mediate the
racemization of -aminoesters in 95% organic solvent.20 In
the presence of solvent stable hydrolases, a dynamic kinetic
resolution is achieved to prepare enantioenriched amino
acids which precipitated from solution to prevent further
racemization.21We hypothesized that PLP should also be
able catalyze the racemization of α-aminoketones under
aqueous conditions. When run in the presence of KREDs, an
enantio- and diastereoselective synthesis of 1,2-amino
alcohols would be achieved, with the decreased acidity of
the α-amino proton in the product precluding product
epimerization.
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Figure 3. PLP-Catalyzed Substrate Racemization
With a promising enzyme in hand, we shifted our
attention to developing conditions to racemize the -
aminoketone 1. In preliminary experiments, we found that
the starting material does not racemize in buffer at pH’s
ranging from 6.5-9.5 (Figure 3). Upon addition of PLP (10
mol %) to 2-(N-morpholino)ethanesulfonic acid buffer
(MES) at pH 6.5, racemization of the starting material from
96% ee to 20% ee occurred within two hours (Figure 3). We
recognized that enhanced rates of racemization could be
necessary for more kinetically active KREDs. Snell and
coworkers previously demonstrated that metal salts can
enhance the rate of pyridoxal catalyzed -aminoester
racemization by forming stable metal chelates.23 After
testing a small collection of metal salt additives, we found
that addition of catalytic NiSO4 (15 mol %) enabled
complete racemization within 90 minutes. With ideal
racemization conditions in hand, we explored the impact
these conditions have on the stereochemical integrity of
amino alcohol 2. We found that the product did not
decompose or epimerize under the reaction conditions,
presumably due to the insufficient acidity of the α-amino
proton (Scheme S1). Having identified optimal conditions
for substrate racemization, we tested the racemization
conditions in the presence of ketoreductase P2-D11 and
Figure 2. Pyrazine Formation and Dynamic Kinetic Resolution
Development
At the outset, we recognized that a challenge inherent
to the proposed reactivity was dimerization of the α-
aminoketone to afford a dihydropyrazine, which upon
oxidation would provide the pyrazine (Figure 2). We
hypothesized that under aerobic reaction conditions,
formation of the dihydropyrazine is reversible, while the
oxidation to form the pyrazine is irreversible. Based on this
understanding, we reasoned that anaerobic reaction
conditions should diminish formation of the undesired
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achieved an effective dynamic kinetic resolution of 1 to
afford 1,2-aminoalcohol 2 in 85% yield as an 11:1 dr and
99:1 er (Figure 3).
Next, we tested a series of substrates lacking aromatic
substituents. While a different KRED was required for high
levels of selectivity, n-hexyl glycine and derived c-
hexylalanine derived -aminoketone afforded product in
high yields, diastereoselectivities and enantioselectivities.
(17, 19). Leucine-derived -aminoketone is also an
effective substrate for this reaction, although product is
formed with slightly diminished diastereoselectivity 18
(90% yield, 8:1 dr, >99:1 er).
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Table 1. Substrate Scope
An attractive feature of this approach is the possibility
to access all four possible stereoisomeric products simply
by exchanging the KRED involved in the kinetic resolution.
We found that by screening the collection of the 24
ketoreductases in the Codexis® kit, enzymes can be
identified that provide all possible stereoisomers in high
yield and enantioselectivity and with good to excellent
levels of diastereoselectivity (Figure 4). The general
selectivity trends with these enzymes are observed with
structurally related substrates (Figure S1). These examples
demonstrate the possibility of novel racemization
mechanisms to be paired with curated collections of
enzymes to provide rapid access to the desired 1,2-amino
alcohol stereoisomer.
As a demonstration of the versatility of this method,
we sought to utilize this reaction to prepare amino alcohols
found in medicinally important molecules. We recognized
that amino alcohol motif found in HIV antiviral drugs
Darunavir24 and Atazanavir25 could be accessed using this
approach via conversion of a chloroaminoketone into an
amino alcohol. After screening the commercial collection of
KREDs, we found that KRED P1-B10 provided product in
excellent yield, enantioselectivity and diastereoselectivity.
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▪ ✓Stereodivergent Synthesis - Accessing All Possible Stereoisomers
KRED P1 B10
95 % yield
12:1 dr
KRED P1 B05
82 % yield
6:1 dr
OH
Et
OH
Et
O
Ph
Ph
Ph
Et
NH2
NH2
>99:1 er
99:1 er
NH3
20’
Cl
The scope and limitations of the transformation were
tested on a variety of substrates (Table 1). Codexis® KRED
P1-B05 proved most effective for phenylalanine-derived
methyl ketones, with various functional groups appended
to the aromatic ring being well-tolerated in this reaction.
Electron donating groups provided slightly higher levels of
diastereoselectivity by comparison to electron withdrawing
(2S, 3R)-20
(2S, 3S)-20
Racemic
KRED P2 C02
95 % yield
>20:1 dr
KRED 101
93 % yield
7:1 dr
OH
OH
PLP
Et
Et
Ph
Ph
NH2
NH2
>99:1 er
99:1 er
(2R, 3S)-20
(2R, 3R)-20
▪ ✓Toward the Synthesis of Darunavir
O
H
O
O
H
O
OH
substituents (Table 1, 4-10).
Differences in
Bn
Cl
Bn
Cl
HN
O
PLP
SO2C6H4NH2
NH3
NH2
21
diastereoselectivity are due to either a change in the rate of
racemization or variations in the rate and selectivity of
ketone reduction. Substrates with ortho- substituents are
also compatible with the reaction conditions. Beyond
simple aromatics, heteroaromatics, such as pyridine and
thiophene, are well-tolerated, affording product with good
yields and selectivities. More sterically demanding naphthyl
substituted ketones are also effective substrates for this
reaction.
Bn
N
Cl
OH
i-Pr
21’
95 % yield
>20:1 dr
>99:1 er
Darunavir
Figure 4. Stereodivergent Enzymes and Synthetic Applications
In conclusion, by merging nonenzymatic PLP
racemization and diastereoselective reduction using
ketoreductases, we developed a dynamic kinetic resolution
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process for the preparation of chiral aminoalcohols from -
Page 4 of 6
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aminoketone starting materials. Utilizing commercially
available KREDs, all four possible stereoisomers can be
accessed using the same protocol, highlight the advantage
of this approach by comparison to existing amino alcohols
syntheses. As this racemization strategy operates at neutral
conditions, it should be compatible with many evolved and
naturally occurring KREDs, enabling it to be applied to the
2
(a) Heravi, M. M.; Lashaki, T. B.; Fattahi, B.; Zadsirjan, V. Application
of Asymmetric Sharpless Aminohydroxylation in Total Synthesis of
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Sharpless Asymmetric Aminohydroxylation. J. Chem. Soc., Perkin Trans.
1 2002, 2733-2746.
3
synthesis of
a
range of structurally diverse -
(a) Jacobsen, E. N. Asymmetric Catalysis of Epoxide Ring-Opening
9
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G. The Chemistry of Transition Metals with Three-Membered Ring
Heterocycles. Chem. Rev. 2014, 114, 8153-8198.
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Enantioselective Aminohydroxylation of Styrenyl Olefins Catalyzed by
ASSOCIATED CONTENT
an Engineered Hemoprotein. Cho, I.; Prier. C. K.; Jia, Z. J.; Zhang, R. K.;
Görbe, T.; Arnold, F. H. Angew. Chem. Int. Ed. 2019, 58, 3138-3142.
For a complementary biocatalytic approach to prepare vicinal amino
Supporting Information
5
alcohols form 훼-hydroxyketones; see: (a) Enantioselective Synthesis of
Chiral Vicinal Amino Alcohols Using Amine Dehydrogenases. Chen, F. F.;
Cosgrove, S. C.; Birmingham, W. R.; Mangas-Sanchez, J.; Citoler, J.;
Thompson, M. P.; Zheng, G. W.; Xu, J. H.; Turner, N. J. ACS Catal. 2019,
9, 11813-11818. (b) Enantioselective Synthesis of Enantiopure β-
This material is available free of charge via the Internet at
http://pubs.acs.org
General experimental information, experimental procedure
with chemical structures and characterization data,
experiments, experimental procedure for the synthetic
applications with chemical structures and characterization
data, and NMR spectra (PDF)
Aminoalcohols
via
Kinetic
Resolution
and
Asymmetric
Reductiveamination by a Robust Transaminase From Mycobacterium
Vanbaalenii. Zhang, J. D.; Zhao, J. W.; Gao, L. L; Chang, H. H; Wei, W.
L.; Xu, J. H. J. Biotechnol. 2019, 290, 24–32.
6
(a) Hibino, T.; Makino, K.; Sugiyama, T.; Hamada, Y. Homogenous
AUTHOR INFORMATION
Corresponding Author
Chiral Nickel-Catalyzed Asymmetric Hydrogenation of Substituted
Aromatic 훼-Aminoketone Hydrochlorides through Dynamic Kinetic
Resolution. ChemCatChem 2009, 1, 237-240. (b) Ohkuma, T.; Ishii, D.;
Takeno, H.; Noyori, R. Asymmetric Hydrogenation of Amino Ketones
Using Chiral RuCl2(diphosphine)(1,2-diamine) Complexes. J. Am. Chem.
Soc. 2000, 122, 6510-6511. (c) Wu, W.; You, C.; Yin, C.; Liu, Y.; Dong, X.-
Q., Zhang, X. Enantioselective and Diastereoselective Construction of
Chiral Amino Alcohols by Iridium–f-Amphox-Catalyzed Asymmetric
Hydrogenation via Dynamic Kinetic Resolution. Org. Lett. 2017, 19,
2548-2551. (d) Liu, S.; Xie, J.-H.; Wang, L.-X.; Zhou, Q.-L. Dynamic
Kinetic Resolution Allows a Highly Enantioselective Synthesis of cis-α-
Todd K. Hyster - Department of Chemistry, Princeton University,
Frick Chemical Laboratory, Princeton, NJ 08544
*E-mail: thyster@princeton.edu. Phone: 612-716-2115
Present Addresses
Department of Chemistry, Princeton University, Frick Chemical
Laboratory, Princeton, NJ 08544
Aminocycloalkanols
by
Ruthenium-Catalyzed
Asymmetric
Hydrogenation. Angew. Chem. Int. Ed. 2007, 46, 7506-7508.
7
(a) Seashore-Ludlow, B.; Sait-Dizier, F.; Somfai, P. Asymmetric
Transfer Hydrogenation Coupled with Dynamic Kinetic Resolution in
Water: Synthesis of anti-β-Hydroxy-α-amino Acid Derivatives. Org. Lett.
2012, 14, 6334-6337. (b) Ohkima, T.; Ishii, D.; Takeno, H.; Noyori, R.
Asymmetric Hydrogenation of Amino Ketones Using Chiral
RuCl2(diphophine)(1,2-diamine) Complexes. J. Am. Chem. Soc. 2000,
122, 6510-6511. (c) Schwarz, J. L.; Kleinmans, R.; Paulisch, T.O.; Glorius,
F. 1,2-Amino Alcohols via Cr/Photoredox Dual-Catalyzed Addition of α-
Amino Carbanion Equivalents to Carbonyls. J. Am. Chem. Soc. 2020, 142,
2168−2174. (d) Ye, C.-X.; Yohannes, Y.; Li, M.; Cheng, J.-T.; Zhang, T.-T.;
Ruan, Y.-P.; Zheng, X.; Lu, X.; Huang, P.-Q. Dual Catalysis for
Enantioselective Convergent Synthesis of Enantiopure Vicinal Amino
Alcohols. Nat Commun, 2018, 9, 410.
Notes
The authors declare no competing financial interests.
Funding Sources
Searle Scholars Award (SSP-2017-1741), Sloan Research
Fellowship, ACS-PRF, and Princeton University for support
ACKNOWLEDGMENT
T.K.H thanks the Searle Scholars Award (SSP-2017-1741),
Sloan Research Fellowship, ACS-PRF, and Princeton University
for support.
8
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Enzymatic Reduction of Ketones. Acc. Chem. Res. 2007, 40, 1412-1419.
(b) Huisman, G. W.; Liang, J.; Krebber, A. Practical Chiral Alcohol
Manufacture Using Ketoreductases. Curr. Opin. Chem. Biol. 2010, 14,
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ACS Paragon Plus Environment

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Access to All Possible Stereoisomers
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