Rapid Enantioselective and Diastereoconvergent Hybrid Organic/Biocatalytic Entry into the Oseltamivir Core

Article abstract of DOI:10.1021/acs.joc.1c00326

A formal synthesis of the antiviral drug (-)-oseltamivir (Tamiflu) has been accomplished starting from m-anisic acid via a dissolving metal or electrochemical Birch reduction. The correct absolute stereochemistry is efficiently set through enzyme-catalyzed carbonyl reduction on the resultant racemic α,β-unsaturated ketone. A screen of a broad ketoreductase (KRED) library identified several that deliver the desired allylic alcohol with nearly perfect facial selectivity at the new center for each antipodal substrate, indicating that the enzyme also is able to completely override inherent diastereomeric bias in the substrate. Conversion is complete, with d-glucose serving as the terminal hydride donor (glucose dehydrogenase). For each resulting diastereomeric secondary alcohol, O/N-interconversion is then efficiently effected either by synfacial [3,3]-sigmatropic allylic imidate rearrangement or by direct, stereoinverting N-Mitsunobu chemistry. Both stereochemical outcomes have been confirmed crystallographically. The α,β-unsaturation is then introduced via an α-phenylselenylation/oxidation/pyrolysis sequence to yield the targeted (S)-N-acyl-protected 5-amino-1,3-cyclohexadiene carboxylates, key advanced intermediates for oseltamivir pioneered by Corey (N-Boc) and Trost (N-phthalamido), respectively.

Full text of DOI:10.1021/acs.joc.1c00326

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Article
Rapid Enantioselective and Diastereoconvergent Hybrid Organic/
Biocatalytic Entry into the Oseltamivir Core
Virendra K. Tiwari, Douglas R. Powell, Sylvain Broussy,* and David B. Berkowitz*
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ABSTRACT: A formal synthesis of the antiviral drug (−)-oseltamivir (Tamiflu) has been accomplished starting from m-anisic acid
via a dissolving metal or electrochemical Birch reduction. The correct absolute stereochemistry is efficiently set through enzyme-
catalyzed carbonyl reduction on the resultant racemic α,β-unsaturated ketone. A screen of a broad ketoreductase (KRED) library
identified several that deliver the desired allylic alcohol with nearly perfect facial selectivity at the new center for each antipodal
substrate, indicating that the enzyme also is able to completely override inherent diastereomeric bias in the substrate. Conversion is
complete, with D-glucose serving as the terminal hydride donor (glucose dehydrogenase). For each resulting diastereomeric
secondary alcohol, O/N-interconversion is then efficiently effected either by synfacial [3,3]-sigmatropic allylic imidate rearrangement
or by direct, stereoinverting N-Mitsunobu chemistry. Both stereochemical outcomes have been confirmed crystallographically. The
α,β-unsaturation is then introduced via an α-phenylselenylation/oxidation/pyrolysis sequence to yield the targeted (S)-N-acyl-
protected 5-amino-1,3-cyclohexadiene carboxylates, key advanced intermediates for oseltamivir pioneered by Corey (N-Boc) and
Trost (N-phthalamido), respectively.
enzyme, oseltamivir stabilizes the viral HA−host−receptor
interaction, thereby preventing virion release and slowing the
progression toward increased viral load. NA cleavage of the
terminal sialic acid linked to a galactose moiety through an α-
(2,3)- or an α-(2,6)-glycosidic linkage proceeds through a
putative oxocarbenium ion-like TS stabilized by interaction
with an enzymatic tyrosine residue (Scheme 1). The
INTRODUCTION
■
The current pandemic due to the SARS-CoV-2 virus has
highlighted the importance of developing both therapeutic
agents, such as remdesivir1 and dexamethasone,2 in this case
and prophylactic (e.g., mRNA-based vaccines) antiviral agents,
in parallel. In this communication, we describe an efficient new
route to the most established therapeutic agent for influenza
A−C, namely, oseltamivir (Tamiflu).3 To our knowledge, the
only previously reported biocatalytic entries into the
oseltamivir skeleton involve either lipase-/esterase-mediated
kinetic resolution4/desymmetrization5 or whole-cell-mediated
microbial arene dioxygenation.6 We report herein a hybrid
chemo/biocatalytic route into oseltamivir that involves the use
of an isolated enzyme to set the key stereocenter from a
prochiral center with nearly perfect stereoinduction and
complete conversion. This is also the first example of the use
of a dehydrogenase enzyme in oseltamivir synthesis, with D-
glucose serving as the stoichiometric reductant for the key
stereochemically defining step. This is of particular significance
given the current interest in biocatalytic routes in process
chemistry, in general, and toward antivirals, in particular.7
Viral neuraminidase (NA) cleaves a terminal sialic acid on
the host cell glycoprotein receptor to which the viral
hemagglutinin (HA) is bound, thereby releasing the budding
virion from the infected cell.8 By inhibiting the viral NA
4
oxocarbenium ion has a H5 half-chair geometry that maps
directly onto the geometry of oseltamivir, making this an ideal
TS analogue.9 Zanamivir (Relenza), another potent NA
inhibitor, displays an E5 geometry. Oseltamivir displays much
higher oral bioavailability and has been the prodrug of choice
for the treatment of influenza.10,11 Oseltamivir was developed
by Gilead and marketed by Roche as Tamiflu, and the first
generic version was approved by the FDA in 2016. The recent
discovery that amino-piperidine-based inhibitors of the
Received: February 9, 2021
Published: April 15, 2021
https://doi.org/10.1021/acs.joc.1c00326
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developed a chiral oxazaborolidinium-ion-catalyzed Diels−
Alder reaction-based synthesis. This route passes through a
chiral cyclohexenoid intermediate that then undergoes a key
iodo-lactamization reaction to give key chiral intermediate 2, a
major focus of this communication. Transformation of 2 into a
cyclic N-acyl aziridine, followed by ring opening, furnishes 1
(Scheme 2b).14 Okamura reported a convergent [4 + 2]-
cycloaddition-based route reaction for the synthesis of 2
(Scheme 2c).15 In the Trost synthesis of oseltamivir,
palladium-catalyzed asymmetric allylic alkylation and rho-
dium-catalyzed dienoate aziridination lead to intermediate 3
(Scheme 2d),16 another key target of the current study. Kann
(Scheme 2e) elegantly exploited a built-in Fe(CO)3-coordi-
nated moiety to at once effect diene activation and steer facial
selectivity.17
Scheme 1. Neuraminidase Reaction and Oseltamivir as the
TS Analogue: Putative Oxocarbenium Ion (4H5) and
Enzymatic Transition State (See 4ZJQ for the Resulting
Intermediate) for Zanamivir (E5; 1NNC) and Oseltamivir
(4H5; 3CL0)
RESULTS AND DISCUSSION
■
Consistent with our longstanding interest in the use of
enzymes as both stereoselective reporters18 and stereoselective
catalysts19 for advancing organic synthesis, we set out to
develop a new enzyme-assisted entry into the oseltamivir core.
While the Tamiflu scaffold continues to draw the close
attention of synthetic chemists,20 there have been few
approaches that exploit enzymatic chemistry. The Xu4 route
employs a diastereoselective Diels−Alder reaction and
diazidation, followed by classic lipase-catalyzed kinetic
resolution (Scheme 2f). Banwell6d,21 (Scheme 2g) and
Hudlicky6a,22 utilize microbial arene oxidation as a key step
to set the stereochemistry for their Tamiflu syntheses (Scheme
2g). To our knowledge, this report constitutes the first entry
into the oseltamivir framework that exploits dehydrogenase
chemistry and one that does so with essentially complete
stereocontrol.
influenza virus cell entry act synergistically with oseltamivir12
has greatly heightened interest in the drug today.
Notable synthetic entries into this scaffold are delineated in
Scheme 2, highlighting key transformations in each case. In the
original Roche process chemistry route, shikimic acid, a highly
functionalized, stereochemistry-rich chiron is converted to a
key homoallylic epoxide intermediate (Scheme 2a).13 Corey
Scheme 2. Stereocontrolled Routes to the Oseltamivir Core
The strategic approach taken here is presented in Scheme 3.
It was envisioned that one could rapidly arrive at a key cyclic
Scheme 3. New Hybrid Organic/Biocatalytic Approach into
the Corey and Trost Intermediates
α,β-unsaturated ketone via Birch reduction of m-anisic acid.
This would, in principle, allow for enzymatic installation of the
key stereocenter through facially selective carbonyl reduction,
despite the presence of a potentially confounding second
stereocenter, adjacent to the ester functionality. A major
challenge would therefore be to find not only a dehydrogenase
active site that would exhibit high facial selectivity for such a
substituted cyclohexenone system but also one that would be
highly promiscuous in tolerating functionality at C5 and be
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fully “stereotolerant” at that position. The next key step would
be to translate the enzymatically installed C−O absolute
stereochemistry into the requisite C−N stereochemistry for
Tamiflu. It was envisioned that this could be achieved either by
a direct stereoinvertive N-Mitsunobu transformation or via
O,N-allylic transposition through stereospecific synfacial [3,3]-
sigmatropic rearrangement (Scheme 3).
The initially required Birch reduction of m-anisic acid
proceeded smoothly to give compound 4 under traditional
dissolving metal conditions with Li metal serving as reductant
in liquid ammonia. Optimal yields were obtained using ice
(water added dropwise into liquid ammonia at −78 °C) as a
proton source as opposed to EtOH−H2O mixtures as
delineated in detail in the SI (see Table S1). Under these
conditions, one obtains high conversion to the desired 5-
carboxy-cyclohexenone, following acid-catalyzed enol ether
hydrolysis and conjugative alkene isomerization to 4 (Scheme
4A).23 Pleasingly, the recently described24 electrochemical
catalyzed facially selective cyclohexenone reduction as the key
step (Scheme 4 and Table 1; for details see Tables S2 and S3).
According to our synthetic design (Schemes 3 and 4), the
desired allylic alcohol 6 would be obtained by a regioselective
1,2-cyclohexenone reduction reaction and with R-stereo-
selectivity. We had hoped to find enzymes that would treat
the resident carboethoxy stereocenter at C5 in ( )-5 as a
“spectator” stereocenter and deliver a hydride to the correct
face of the ketone regardless of the configuration of that pre-
existing stereocenter. Toward this end, a set of 82 KRED
enzymes were screened under identical conditions: 0.025
mmol of ( )-5, 1 mg of enzyme, 24 h, 30 °C, in the presence
of glucose dehydrogenase (GDH) for NAD(P)H cofactor
regeneration from D-glucose (Table 1 and SI Tables S2 and
S3).
In the ideal case, a quantitative yield in combination with R-
stereoinduction would result in a 50:50 mixture of cis/trans
diastereomers, in a “diastereotolerant” or “catalyst-controlled”
reduction.26 During the screening, besides the remaining
1
Scheme 4. Efficient Birch Reduction/KRED-Mediated
starting ketone, three products were identified by H NMR:
Introduction of the Proper Absolute Stereochemistry
the desired allylic alcohol 6 in various cis/trans ratios, the
saturated alcohol 7 (resulting from 1,4- and subsequent 1,2-
reduction), and the saturated ketone 8 (1,4-reduction only).
Product distributions for all 82 enzymes were calculated based
on integration of characteristic signals on the 1H NMR spectra
(see Table 1 and the SI, particularly Tables S2 and S3). Among
the 61 NADPH-dependent KRED enzymes, only a few gave
acceptable yields of 6 together with a cis/trans ratio close to
50:50. For example, KRED-101 gave 69% yield of 6 with 48:52
cis/trans ratio, resulting in 78% and 86% ee for the cis and trans
diastereomers, respectively. The enantiomeric excess was
determined by chiral phase HPLC analysis on the p-
bromobenzoate derivatives of the enzymatic reduction
products vs racemic standards (see SI) obtained by Luche
reduction of ( )-5 (NaBH4/CeCl3; 9:1 cis/trans selectivity).
NADH-dependent KRED enzymes (Table 1) generally gave
better yields of 1,2-reduction and enantiomeric excess values
for the 6a and 6b products than NADPH-dependent ones
(Table 1 and see SI Table S2).
In particular, KRED-117, -123, and -126 gave complete
conversion, complete regioselectivity, and nearly perfect
diastereotolerance toward C5 and nearly perfect facial
selectivity at the carbonyl center (>99% ee for 6a and 6b)!
The R-absolute configuration was established by determination
of optical rotation values of 3 and 2 and comparison with
literature values (see the Experimental Section). The reaction
was also achieved on a gram scale with KRED-123, giving 98%
yield of combined cis/trans diastereomers 6 in 99% ee each.
Moreover, KRED-123 recycling has been shown to be possible
for at least three cycles, resulting in an estimated turnover
number of 20 000 cycles (see Experimental Section).
alternative to the Birch reduction also proved successful for
this transformation. Utilizing Mg as an anode and a galvanized
steel nail (Zn) as a cathode with dimethylurea as proton source
and tris(pyrrolidino)phosphoramide as an internal overcharge
protectant, one obtains the desired 5-carboxycyclohexenone
without the need for the acid-catalyzed isomerization step
(Scheme 4B)
The desired R-absolute configuration having been set, the
diastereomeric mixture of enantiopure alcohols 6 was
converted to building blocks 3 and 2. A nitrogen-Mitsunobu
reaction upon 6 with phthalimide and DIAD cleanly gave 9 as
a 1:1 mixture of diastereomers. The desired α,β-unsaturation
with respect to the carboethoxy group was then smoothly
installed by ester enolate formation, α-phenylselenation,
oxidation, and selenoxide elimination. Literature precedents
indicate that the Mitsunobu reaction on cyclic, allylic
secondary alcohols can proceed through either an SN2- or an
SN2′-mechanism, depending mostly on the substrate and to a
lesser extent on the nucleophile and the solvent. The related,
Next, we set out to explore a wide array of nicotinamide-
dependent dehydrogenases, for the sought-after facially
selective and diastereotolerant, substituted cyclohexenone
reduction. In this endeavor, we were particularly drawn to
the KRED (ketoreductase) family of enzymes that have proven
useful by both academic and industrial chemists.25 In our own
hands, KRED enzymes had already shown potential on both
sides of the house, as catalytic reporters for our in situ
enzymatic screening efforts18c and as chiral catalysts, in and of
themselves.19e In fact, we are delighted to report here
streamlined pathways to the key advanced intermediates for
olseltamivir, 2 (Corey) and 3 (Trost), that feature KRED-
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a
Table 1. Screening of KREDs
b
c
b
c
b
KRED enz. recovered SM of 5 (%) total (%) yield of 6 ratio of 6a/6b (cis/trans)
% ee of 6a/6b
% yield of 7 (cis/trans ratio) % yield of 8
NADPH-dependent KREDs
d
101
102
103
104
105
106
107
119
120
134
135
136
137
139
0
69
48:52
∼ 78 (R)/86 (R)
31 (71:28)
58 (88:12)
19
27 (81:19)
6
57 (86:14)
80 (34:66)
50 (53:47)
100 (50:50)
78 (11:89)
67 (12:88)
66 (62:38)
57 (34:66)
14
0
14
6
trace
0
60
0
0
trace
50
Trace
22
33
6
-
-
-
-
-
-
-
-
28
75
0
33
43
6
0
trace
0
13
61
trace
14
0
0
0
0
20:80
-
-
-
66:34
-
13:87
15:85
∼ 70 (R)/92 (R)
-
0
12
26
0
16
17
86
trace
0
-
-
-
-
NADH-dependent KREDs
101
104
105
107
117
118
119
122
123
125
126
129
3
89
53
65
8
100
48
56
10
100
3
46:54
80:20
80:20
15
50:50
48:52
>99:1
9
50:50
-
49:51
91:9
>99 (R)/>99 (R)
8
0
0
0
0
0
0
trace
0
0
34
25
77
0
43
36
81
0
13
10
15
0
9
8
9
0
14
trace
12
>99 (R)/>99 (R)
>99 (R)/>99 (R)
>99 (R)/>99 (R)
83
0
60
0
0
0
99
28
a
Reaction conditions: 0.025 mmol of ( )-5, 1 mg of enzyme, 24 h, 30 °C, in the presence of glucose dehydrogenase (GDH) for NAD(P)H
b
cofactor regeneration from D-glucose. Product distribution was established by integration of characteristic signals for each compound in the crude
NMR spectrum, followed by calculation of the percentage of each component. See the SI for details. The cis/trans ratio is given only when accurate
measurement was possible, typically for >20% product. 2 mg of enzyme were used.
c
d
but relatively sterically unencumbered, 2-cyclohexen-1-ol
system reacted mostly through an SN2 manifold with
carboxylic acid nucleophiles (8−28% of non-SN2 products
detected).27 More sterically hindered derivatives of conduritol
(cyclohex-5-ene-1,2,3,4-tetra-ols) react exclusively by an SN2
mechanism or with up to 73% SN2′-mechanism, depending on
the relative stereochemistry of the protected hydroxy groups.28
For the SN2′-mechanism, the participation of both oxygen
atoms of the carboxylate nucleophile was postulated. The
Mitsunobu reaction with nitrogen nucleophiles shows some
dependence on the steric demand of the putative SN2- vs SN2′-
transition states. The use of TsNHBoc as an N-Mitsunobu
nucleophile on 2-cyclohexen-1-ol induced a decrease in the
enantiomeric excess of the product, presumably through a
competing SN2′-mechanism.29 However, the use of
TsNHPMB on 4,5-bis((tert-butyldimethylsilyl)oxy)cyclohex-
2-en-1-ol gave exclusively the product resulting from an SN2-
mechanism.30
configuration, see below) and the very high enantiomeric
excess obtained for 3 suggest that the reaction likely proceeds
through an SN2 mechanism with complete inversion of
configuration. Some participation of an SN2′ pathway cannot
be excluded but would have had to proceed with complete
retention to give the S-stereoisomer (Scheme 5). The optical
rotation of 3 matched that reported previously by Trost for this
advanced intermediate, and the X-ray structure determination
for 9b (Scheme 5 and SI) verified the relative stereo-
chemistry.16b The phthalimide protecting group of 9 was
readily converted to a Boc group in two steps to give 12, and
α,β-unsaturation was introduced as above to give 2, the Corey
intermediate. Chiral HPLC analysis confirmed the preservation
of ee (98%) in the O,N-interconversion, and again the optical
rotation matched reported values, confirming the absolute
stereochemistry.14,20h,31
A complementary sigmatropic rearrangement route into the
Corey intermediate was pursued in parallel from enzymatic
products 6a/6b. This approach exploits the symmetry in this
system, whereby synfacial O,N-allylic transposition upon R-
configured alcohols 6a/6b provides positionally and stereo-
In our system, due to symmetry, SN2- and SN2′-mechanisms
would lead to the same positional isomer; only the stereo-
chemistry could differ. The absolute stereochemistry (S-
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(traditional or electrochemical) of a simple, inexpensive
aromatic precursor to enter the needed substituted cyclo-
hexenone scaffold, (b) use of enzymatic reduction to install
absolute stereochemistry in a nearly perfect process that
completely overrides inherent diastereofacial bias in the
antipodal substrates (98% yield, >99% ee), and (c)
complementary routes into the Corey intermediate through
N-Mitsunobu or allylic imidate sigmatropic rearrangement
manifolds. In particular, remarkable facial selectivity and
diastereotolerance of the KRED-mediated37 chemistry speaks
to the value of hybrid synthetic organic/biocatalytic routes in
support of both chemical biology and process chemis-
try.7a,25b,38
Scheme 5. Synthesis of Trost Intermediate 3 via Stereo-
Inverting N-Mitsunobu Transformation
EXPERIMENTAL SECTION
■
I. General Procedure. Unless otherwise noted, all reactions were
performed in oven-dried glassware. All air- or water-sensitive reactions
were conducted under an inert atmosphere (N2 or Ar) using oven-
dried glassware. THF was distilled from sodium benzophenone ketyl.
Methanol was distilled over magnesium and iodine. Dichloromethane
and diisopropylamine were distilled over CaH2. Other reagents were
obtained from commercial sources and used without further
purification. Reaction progress was monitored by TLC or GC-MS
(HP model 5890 GC with model 5972 MS). Flash chromatography
was performed using silica gel 60 (230−400 mesh). 1H NMR spectra
were recorded on Bruker-DRX-Avance-400 or 500 MHz instruments
with chemical shifts reported relative to residual CHCl3 (7.25 ppm).
Proton-decoupled 13C NMR spectra were acquired on Bruker-DRX-
Avance-400 or 500 MHz instruments with chemical shifts reported
relative to CDCl3 (77.0 ppm). High-resolution mass spectra were
acquired at the Nebraska Center for Mass Spectrometry (University
of Nebraska). Enzymes of the KRED (ketoreductase) family were
obtained from Codexis. Crystallography was performed by Douglas R.
Powell at the University of Oklahoma.
chemically equivalent S-amide products to those obtained
through the direct displacement stereoinverting the N-
Mitsunobu route (see Schemes 5 and 6). In the event, [3,3]-
Scheme 6. Access to the Corey Intermediate via Syn-Facial
[3,3]-Sigmatropic Rearrangement
II. Synthetic Procedures for the Synthesis of Ketone 5. 5-
Oxocyclohex-3-ene-1-carboxylic Acid (4). A. Birch Reduction of m-
Anisic Acid. A three-necked round-bottom flask, equipped with an
ammonia condenser, glass stopper, and septum, was charged with m-
anisic acid (10.0 g, 66 mmol) and H2O (10 mL). The condenser was
filled with dry ice and acetone, and ammonia was condensed to a total
volume of about 350 mL. The ammonia inlet was replaced with an Ar
inlet, and a slow stream of argon was introduced above the surface.
Pieces of Li metal (1.4 g, 200 mmol) were slowly added, upon stirring,
over a period of 30 min. After the blue color completely disappeared
(about 1 h), the condenser was removed, and ammonia was allowed
to evaporate under a stream of argon. The residue was acidified to pH
1 with 1 M HCl(aq) followed by 6 M HCl(aq) and heated to reflux
for 1 h using a silicon oil bath. After allowing the reaction flask to cool
to rt, the product was extracted with Et2O (5 times), and the
combined organic layers were dried over Na2SO4 and evaporated, to
sigmatropic allylic imidate rearrangement32 of trichloroaceti-
midate 10 proceeded efficiently, appropriately shifting the
chirality from the R-secondary alcohol to the key S-
trichloroacetamide 11 in 92% yield, with the absolute
stereochemistry being cemented by X-ray crystal structure
determination of 11b (Scheme 6 and SI). The thermal [3,3]-
sigmatropic rearrangement of allylic trichloroacetimidates33 is
known to proceed stereospecifically in a synfacial fashion on 6-
membered ring systems such as cyclohexenes30,34 and
dihydropyrans.35 DFT calculations suggest that such rearrange-
ments typically proceed asynchronously, via a pseudopericyclic
transition state (Scheme 6).36 The rearrangement product 11
could easily be converted to the Corey intermediate 12 by
amide cleavage (NaBH4 and EtOH) followed by Boc
protection (Boc2O).
1
give acid 4 (8.4 g, 91%) as a slightly yellow solid. H NMR and
13C{1H} NMR spectral data were consistent with those previously
reported.23 Both the use of water (ice) as a proton source and the
specific addition sequence proved important in the optimization of
this reaction (see SI, especially Table S1, for more details).
B. Electrochemical Reduction Reaction.24 A Mg ribbon anode and
galvanized-Zn cathode (commercial nail) were set up around a
number 24-sized septum (photographs for the experimental setup are
shown in SI). To an oven-dried 7-dram vial charged with a small
magnetic stir bar were added m-anisic acid (100 mg, 0.6 mmol), 1,3-
dimethylurea (DMU) (2.4 mmol), and tri(pyrrolidin-1-yl) phosphine
oxide (TPPA) (4.8 mmol). Then a solution of lithium bromide (2.4
mL; 2 M in THF) was added, and the resultant reaction mixture was
degassed for 5 min under Ar flow, followed by the addition of THF
(15 mL) under Ar. The reaction vial was placed at 0 °C, and
electrodes were connected via alligator clips to a BIO-RAD Model
3000 XI power supply. The reaction mixture was initially subjected to
10 mA constant current for 1.5 h, followed by a steady ramping up to
CONCLUSIONS
■
In summary, this new biocatalyst-assisted approach into the
therapeutically important oseltamivir core includes the
following distinguishing features: (a) use of Birch reduction
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20 mA for 2 h, 25 mA for 2 h, and finally 100 mA for the next 3 h.
The reaction was monitored by TLC and after 9 h; the power supply
was disconnected; and the reaction mixture was transferred into an
RB flask. The electrodes were washed with EtOAc, and the combined
organics were concentrated on a rotary evaporator. The residue was
taken up in saturated sodium potassium tartrate solution/EtOAc,
partitioned in a separatory funnel, and the organic layer was further
washed with brine. After drying over Na2SO4 and concentrating under
reduced pressure, the crude product was purified by SiO2 flash
column chromatography (99:1 EtOAc/CH3OH) to give 4 (30 mg,
71% brsm) and recovered m-anisic acid (50 mg) (for a view of the
experimental setup, see Figure S1 in the SI).
Ethyl 5-Oxocyclohex-3-ene-1-carboxylate (5). Esterification
Reaction. Thionyl chloride (SOCl2; 520 μL, 7.14 mmol) was
added dropwise to 30 mL of ethanol at 0 °C. After 10 min, acid 4 (1
g, 7.14 mmol) was added, as a solid, at 0 °C, and the reaction mixture
was allowed to warm to rt over 3 h. The reaction was quenched by
addition of NH4Cl (aq., sat’d), and the product was extracted with
Et2O. The organic layer was dried over Na2SO4 and evaporated, and
the residue was purified by flash column chromatography on silica gel
(20% EtOAc in hexanes) to give ester 5 as a yellow oil (980 mg,
82%). 1H NMR and 13C{1H} NMR spectral data matched with
reported data.39 HRMS (TOF MS ESI+) m/z: [M + Na]+ calcd for
C9H12NaO3 191.0678, found 191.0686.
( )-Ethyl 5-Hydroxycyclohex-3-enecarboxylate Diastereomers
(6a/6b). Luche Reduction. A solution of ketone 5 (680 mg, 4.05
mmol) and CeCl3−7H2O (ceric chloride−heptahydrate; 5.42 mmol,
2.02 g) in methanol (40 mL) was stirred at 0 °C for 15 min, followed
by the addition of NaBH4 (5.00 mmol, 189 mg). The resulting
mixture was stirred at 0 °C for 30 min and then quenched via
dropwise addition of NH4Cl (aq., saturated) solution. Following
extraction with CH2Cl2, the organic phase was dried (Na2SO4) and
evaporated. Flash column chromatography on silica gel (30% EtOAc
in hexanes) gave 6a/6b (570 mg, 86%) as a 9:1 mixture of racemic,
cis- and trans-diastereomers, respectively. See below for complete
spectral characterization of each diastereomer.
b. With KRED-117. Ketone 5 (1.00 g, 5.95 mmol, 100 mM), when
treated for 36 h with KRED-117 (2 mg), NAD+ (0.078 mmol, 1.3
mM), dithiothreitol (0.5 mM), magnesium sulfate (2 mM), glucose
dehydrogenase (600 U), and D-glucose (9.6 mmol, 160 mM) in
potassium phosphate buffer pH 7.0 (250 mM, 60 mL final volume)
with shaking at 30 °C for 36 h gave 6a/6b (678 mg, 88% yield brsm)
as a 1:1 mixture of cis/trans diastereomers after workup, with 240 mg
of ketone 5 being recovered. This product was used for the next step
without further purification.
Enzyme Recycling. A mixture of ketone 5 (33.6 mg, 0.2 mmol, 100
mM), KRED-123 (1 mg), dithiothreitol (0.5 mM), magnesium sulfate
(2 mM), NAD+ (1.3 mM), D-glucose (160 mM), and glucose
dehydrogenase (20 U) in 250 mM potassium phosphate buffer of pH
7.0 (2 mL final volume) was shaken at 30 °C for 12 h. The reaction
products were separated from the enzyme by filtration through a
semipermeable membrane using a Centricon cell (centrifugation at
8000 rpm, 10 kDa-MW-cutoff membrane, Millipore). Filtration to a
minimal volume was followed by the addition of water (500 μL) and a
second centrifugation cycle. The combined filtrates were extracted
with Et2O, and the organic phase was dried (Na2SO4) and evaporated
to give 31 mg (91%) of alcohols 6a/6b (1:1 mixture of
diastereomers). The solution of KRED-123 enzyme thereby recovered
was resubmitted twice to the same reaction and workup conditions to
give an additional 30 mg (88%, second run) and 32 mg (94%, third
run) of alcohols 6a/6b. The combined amount of isolated product
was 93 mg (550 μmol). The enzyme molecular weight was estimated
to be 36 kDa by SDS-PAGE (single band). This corresponds to ∼28
nmol of protein (1 mg) being employed here. This translates to a total
turnover of approximately 20,000 substrate equivalents per enzyme
active site over three cycles. Note: On a large scale (1 g) with a single
cycle, a 98% yield was obtained (vide supra).
IV. Derivatization of Allylic Alcohol Product for HPLC
Analysis (6aa and 6ba). To alcohol 6 (216 mg, 1.2 mmol)
dissolved in THF (0.2 M) under nitrogen flow and at 0 °C was added
NaH (52 mg, 2.16 mmol), followed by p-bromobenzoyl imidazole
(356 mg, 1.44 mmol). The resultant reaction mixture was allowed to
warm to rt and stirred for another 1 h. The reaction mixture was
diluted with EtOAc washed with brine, and the aq. layer was back-
extracted with EtOAc twice. The combined organic layers were dried
over anhydrous Na2SO4 and concentrated under the reduced
pressure, then purified by flash column chromatography using
EtOAc/hexane (1:9) as an eluent to give 6aa/6ba (333 mg, 89%).
Chiral HPLC conditions: column Chiralcel OD (250 mm length, 4.6
mm inner diameter, 5 μm, particle size), flow rate 0.8 mL/min, UV
detection at 254 nm, 0.8% isopropyl alcohol/99.2% hexanes. Both cis-
and trans-diastereomers were obtained with >99% ee: 15.06 min
(trans, 5R), 21.91 min (cis, 5R). It was found that the mixture of
diastereomers could also be separated by flash column chromatog-
raphy using toluene/CH2Cl2 (9:1) as eluent. (6aa) cis-diastereomer,
III. Ethyl 5-Hydroxycyclohex-3-enecarboxylate Diaster-
eomers (6a/6b). Reduction with a KRED Enzyme (Illustrated for
the NADH-Dependent KRED-123 Enzyme). A mixture of ketone 5
(16.8 mg, 0.1 mmol, 50 mM), KRED-123 (1 mg), dithiothreitol (0.5
mM), magnesium sulfate (2 mM), NAD+ (1.3 mM), D-glucose (80
mM), and glucose dehydrogenase in 250 mM potassium phosphate
buffer pH 7.0 (2 mL final volume) was shaken at 30 °C for 24 h. The
reaction mixture was extracted with Et2O, and the combined organics
were dried over Na2SO4 and were evaporated to give alcohol 6a/6b as
a colorless oil and 1:1 mixture of cis/trans diastereomers (16 mg,
94%). The alcohols can be separated by column chromatography on
1
silica gel (5 to 15% EtOAc in hexanes). 6a (cis-diastereomer). H
NMR (500 MHz, CDCl3): δ 5.76−5.71 (m, 2H), 4.27 (m, 1H), 4.13
(q, J = 7.0 Hz, 2H), 2.68 (ddt, J = 10.5, 7.0, 3.0, Hz, 1H), 2.27−2.21
(m, 4H), 1.71 (ddd, J = 13.0, 10.5, 8.0 Hz, 1H), 1.24 (t, J = 7.0 Hz,
3H). 13C {1H} NMR (100 MHz, CDCl3): δ 175.3; 130.8, 126.9, 66.0,
60.7, 37.9, 34.2, 27.4, 14.1; α]20D + 168.6° (c 0.2, CHCl3). 6b (trans-
1
colorless oil. H NMR (400 MHz, CDCl3): δ 7.91 (d, J = 8.6 Hz,
2H), 7.58 (d, J = 8.6 Hz, 2H), 6.08 (ddd, J = 9.8, 5.1, 2.2 Hz, 1H),
5.94 (ddd, J = 9.9, 3.2, 1.7 Hz, 1H), 5.54 (broad, 1H), 4.19 (q, J = 7.1
Hz, 2H), 2.92−2.80 (m, 1H), 2.48 (dt, J = 18.2, 5.1 Hz, 1H), 2.37−
2.18 (m, 2H), 1.97 (ddd, J = 14.3, 12.7, 4.3 Hz, 1H), 1.29 (t, J = 7.1
Hz, 3H). 13C {1H} NMR (100 MHz, CDCl3): δ 174.09, 165.42,
131.67, 131.17, 129.71, 129.25, 128.09, 126.46, 70.05, 60.68, 37.83,
30.53, 27.18, 14.15. HRMS (TOF MS ESI+) m/z: [M + Na]+ calcd
for C12H17O4NaBr 375.0208 (79Br), 377.0187 (81Br); found
375.0191(79Br), 377.0197(81Br). (6ba): 1H NMR (400 MHz,
CDCl3): trans-diastereomer, colorless oil δ 7.91 (d, J = 8.6 Hz,
2H), 7.59 (d, J = 8.6 Hz, 2H), 5.95 (ddd, J = 9.3, 5.0, 2.5 Hz, 1H),
5.83−5.70 (m, 1H), 5.68−5.59 (m, 1H), 4.21−4.04 (m, 2H), 2.85−
2.73 (m, 1H), 2.50 (ddd, J = 12.6, 5.8, 3.1 Hz, 1H), 2.42−2.30 (m,
2H), 1.94 (td, J = 12.2, 9.1 Hz, 1H), 1.24 (t, J = 7.1 Hz, 3H). 13C
{1H}NMR (100 MHz, CDCl3) cis-diastereomer: δ 174.86, 165.16,
131.98, 131.66, 131.18, 129.40, 128.02, 124.20, 66.84, 60.67, 35.29,
30.68, 27.72, 14.23. HRMS (TOF MS ESI+) m/z: [M + Na]+ calcd
for C12H17O4NaBr 375.0208 (79Br), 377.0187 (81Br); found 375.0195
(79Br), 377.0201 (81Br).
1
diastereomer). H NMR (500 MHz, CDCl3): δ 5.90−5.86 (m, 1H),
5.84−5.82 (m, 1H), 4.27 (m, 1H), 4.13 (q, J = 7.0 Hz, 2H), 2.76 (m,
1H), 2.34−2.18 (m, 3H), 2.09 (dt, J = 13.5, 3.0 Hz, 1H), 1.79 (ddd, J
= 14.0, 12.5, 4.5 Hz, 1H), 1.24 (t, J = 7.0 Hz, 3H). 13C {1H} NMR
(100 MHz, CDCl3): δ 175.5, 130.9, 127.9, 62.9, 60.3, 34.5, 33.6, 27.3,
14.0. [α]20D + 11.0° (c 0.6, CHCl3). HRMS (FAB, 3-NBA) m/z: [M
+ Li]+ calcd for C9H14O3Li 177.1103, found 175.1106.
Scale-Up of Enzymatic Transformation. a. With KRED-123.
Ketone 5 (1.00 g, 5.95 mmol, 100 mM) was treated for 24 h with
KRED-123 (20 mg), NAD+ (0.078 mmol, 1.3 mM), dithiothreitol
(0.5 mM), magnesium sulfate (2 mM), glucose dehydrogenase (600
U), and D-glucose (9.6 mmol, 160 mM) in potassium phosphate
buffer of pH 7.0 (250 mM, 60 mL final volume) with shaking at 30 °C
for 24 h to give products 6a/6b after workup as a 1:1 mixture of cis/
trans diastereomers (990 mg, 98% yield). This product was used for
the next step without further purification.
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V. Synthetic Procedure-Trost Intermediate via N-Mitsuno-
bu Rxn. (5S)-Ethyl 5-(1,3-dioxoisoindolin-2-yl)cyclohex-3-enecar-
boxylate Diastereomers (9). To a 1:1 mixture of alcohols 6a/6b (280
mg, 1.65 mmol), triphenylphosphine (694 mg, 2.65 mmol), and
phthalimide (390 mg, 2.65 mmol) in THF (20 mL) at 0 °C was
added, dropwise, DIAD (693 μL, 3.52 mmol). After 1 h at 0 °C and 1
h at rt, the solvent was evaporated, and the residue was purified by
column chromatography on silica gel (10% EtOAc in hexanes) to give
the title compound 9 (418 mg, 85%) as a white solid (1:1 mixture of
δ 8.27 (s, 1H), 6.05−6.10 (m, 1H), 6.0−5.95 (m, 1H), 5.43−5.40 (m,
1H), 4.14 (q, J = 6.8 Hz, 2H), 2.87−2.79 (m, 1H), 2.45 (dt, J = 18.4,
4.8 Hz, 1H), 2.35−2.30 (m, 1H), 2.25−2.16 (m, 1H), 1.91−1.84 (m,
1H), 1.25 (t, J = 7.2 Hz, 3H). 13C {1H}NMR (100 MHz, CDCl3) cis-
diastereomer: δ 174.0, 162.2, 129.6, 126.0, 91.6, 74.3, 60.7, 38.0, 29.8,
27.4, 14.2. trans-Diastereomer: δ 175.0, 161.8, 132.5, 123.4, 91.6,
70.7, 60.6, 35.1, 29.8, 27.8, 15.6. HRMS (FAB, 3-NBA) m/z: [M +
Na]+ calcd for C11H14NO3Cl3Na 335.9937, found 335.9931.
Ethyl (5S)-5-(2,2,2-Trichloroacetamido)cyclohex-3-ene-1-car-
boxylate (11). A solution of 10 (600 mg, 1.91 mmol) in 60 mL of
xylene was heated using a silicon oil bath at reflux for 1 day. The
solvent was evaporated, and the residue was purified by flash column
chromatography on silica gel (10 to 20% EtOAc in hexanes) to give
11 (549 mg, 92%) as a white solid and 1:1 mixture of cis/trans
diastereomers. The mixtures of diastereomers were separated by flash
1
cis/trans diastereomers). H and 13C{1H} NMR spectral data for the
cis-diastereomer matched reported data.16b 1H NMR (400 MHz,
CDCl3) cis-diastereomer: δ 7.80 (dd, J = 5.5, 3.0 Hz, 2H), 7.69 (dd, J
= 5.5, 3.0 Hz, 2H), 5.93−5.89 (m, 1H), 5.56 (dm, J = 10.2 Hz, 1H),
5.00−4.94 (m, 1H), 4.12 (q, J = 7.0 Hz, 2H), 2.80−2.73 (m, 1H),
2.41−2.37 (m, 2H), 2.27 (q, J = 12.4 Hz, 2H), 2.20 2−15 (m, 1H),
1.18 (t, J = 7.5 Hz, 3H). 13C {1H}NMR (100 MHz, CDCl3) cis-
diastereomer: δ 174.1, 167.9, 133.9, 131.9, 128.0, 126.6, 123.2, 60.6,
47.4, 39.5, 29.1, 27.1, 14.2. trans-Diastereomer: δ 7.84−7.79 (m, 2H),
7.68−7.24 (m, 2H), 6.04−5.99 (m, 1H), 5.59 (dm, J = 10.0 Hz, 1H),
5.00−4.96 (m, 1H), 4.23−4.1 (m, 2H), 3.15−3.08 (m, 1H), 2.54−
2.46 (m, 1H), 2.42−2.34 (m, 1H), 2.90 (ddd, J = 4.0, 6.0, 13.6 Hz,
1H), 2.17 (ddd, J = 13.6, 8.8, 6.4 Hz, 1H), 1.27 (t, J = 7.0 Hz, 3H).
13C {1H}NMR (100 MHz, CDCl3) trans-diastereomer: δ 174.8,
168.2, 133.9, 131.9, 129.2, 124.5, 123.1, 60.6, 44.3, 36.7, 29.7, 26.4,
14.2. HRMS: (TOF MS ESI+) m/z [M + Na]+ calcd for
C17H17NO4Na 322.1050; found 322.1042.
1
column chromatography using EtOAc:DCM (1:9) as an eluent. H
NMR (400 MHz, CDCl3) cis-diastereomer: δ 7.29 (br. d, J = 6.8 Hz,
1H), 5.91−5.86 (m, 1H), 5.62 (ddd, J = 10.0, 4.4, 2.4 Hz, 1H), 4.59−
4.52 (m, 1H), 4.14 (q, J = 7.2 Hz, 2H), 2.77 (dddd, J = 12.8, 10.0, 6.8,
3.6 Hz, 1H), 2.36−2.30 (m, 3H), 1.76 (ddd, J = 13.6, 9.2, 7.6 Hz,
1H), 1.24 (t, J = 7.2 Hz, 3H). trans-Diastereomer: δ 6.57 (br. d, J =
6.4 Hz, 1H), 6.03−5.99 (m, 1H), 5.73−5.70 (m, 1H), 4.51−4.45 (m,
1H), 4.14 (q, J = 7.2 Hz, 2H), 2.60−2.52 (m, 1H), 2.35−2.25 (m,
2H), 1.24 (t, J = 7.2 Hz, 3H), 2.13 (apt. dt, J = 3.2, 14.0 Hz, 1H), 1.94
(ddd, J = 13.6, 12.0, 4.8 Hz, 1H). 13C {1H} NMR (100 MHz, CDCl3)
cis-diastereomer: δ 175.1, 161.4, 129.0, 126.3, 92.7, 61.0, 46.4, 37.3,
30.3, 27.0, 14.2. trans-Diastereomer: δ 174.5, 161.0, 131.6, 124.4,
92.7, 60.8, 45.3, 35.5, 30.3, 27.3, 14.2. HRMS (TOF MS ESI+) m/z:
[M + Na]+ calcd for C11H14NO3Na 335.9937 (35Cl), 337.9925
(37Cl); found 335.9935, 337.9927.
A reaction using pure cis-alcohol 6a (purified by column
chromatography) gave pure trans-product 9b without epimerization:
[a]20 −218.4° (c = 0.35, CHCl3).
D
(S)-Ethyl 5-(1,3-Dioxoisoindolin-2-yl)cyclohexa-1,3-dienecarbox-
ylate (3). To a solution of ester 9 (200 mg, 0.67 mmol) in THF (6
mL) at −78 °C was added dropwise KHMDS (0.5 M in toluene, 2.0
mL, 1.0 mmol), and the mixture was stirred for 1 h. Solid diphenyl
diselenide (251 mg, 0.8 mmol) was added at −78 °C, and the
resulting solution was stirred for 3 h at the same temperature and then
quenched by addition of NH4Cl (aq, sat’d), followed by extraction
with Et2O. The organic phase was dried over Na2SO4 and evaporated,
and the residue was purified by column chromatography on silica gel
(15% EtOAc in hexanes) to give the phenyl selenide intermediate as a
nearly 2:1 mixture of diastereomers (236 mg, 78%). This mixture of
diastereomers (180 mg, 0.4 mmol) was then dissolved in THF (4
mL) at 0 °C, and H2O2 (95 μL of a 30% aqueous solution, 0.8 mmol)
and pyridine (65 μL, 0.8 mmol) were added. The mixture was allowed
to warm to rt over 3 h, and Et2O was added. The organic phase was
washed with CuSO4 (aq., sat’d) and brine, dried over Na2SO4, and
evaporated. The residue was purified by flash column chromatography
on silica gel (15% EtOAc in hexanes) to give 3 (114 mg, 96%) as a
white solid (10:1 mixture of regioisomers), and the overall yield of the
two-step reaction was 85%. HRMS (TOF MS ESI+) m/z: [M + Na]+
Ethyl (5S)-5-((tert-Butoxycarbonyl)amino)cyclohex-3-ene-1-car-
boxylate (12). A solution of 9 (380 mg, 1.27 mmol) and
methylhydrazine (335 μL, 6.35 mmol) in ethanol (25 mL) was
heated using a silicon oil bath to reflux overnight. The solvent was
then removed under vacuum, and the residue was dissolved in
dichloromethane (20 mL). Diisopropylethylamine (442 μL, 2.54
mmol) and Boc2O (416 mg, 1.91 mmol) were added, and the
resulting mixture was stirred 5 h at rt. The solvent was then
evaporated, and the crude product was purified by flash column
chromatography on silica gel (20% EtOAc in hexanes) to give the title
compound 12 (340 mg, 99%) as a 1:1 mixture of cis/trans
diastereomers. 1H NMR (400 MHz, CDCl3) cis-diastereomer: δ
5.77−5.72 (m, 1H), 5.54 (br. d, J = 10.0 Hz, 1H), 4.57 (br. d, J = 7.2
Hz, 1H), 4.24 (br. s, 1H), 4.09 (q, J = 7.2 Hz, 2H), 2.69−2.62 (m,
1H), 2.33−2.13 (m, 3H), 1.44 (m, 1H), 1.42 (s, 9H), 4.57 (br. d, J =
7.2 Hz, 1H), 1.23 (t, J = 7.2 Hz, 3H). trans-diastereomer: δ 5.86−5.82
(m, 1H), 5.66 (br. d, J = 8.8 Hz, 1H), 4.21 (br. s, 1H), 4.13 (q, J = 7.2
Hz, 2H), 2.51 (apt. t, J = 12.0 Hz, 1H), 2.30−2.11 (m, 2H), 2.06 (br.
d, J = 13.2 Hz, 1H), 1.79 (dt, J = 12.8, 4.8 Hz, 1H), 1.41 (s, 9H), 1.23
(t, J = 7.2 Hz, 3H). 13C {1H}NMR (100 MHz, CDCl3) cis- and trans-
diastereomers: δ 175.2, 174.8, 155.3, 154.8, 129.2, 128.9, 127.6, 126.3,
79.4, 79.3, 60.5, 46.8, 44.0, 38.5, 35.4, 32.4, 31.8, 28.4, 28.2, 27.3,
27.2, 14.2, 14.1. HRMS (FAB, 3-NBA) m/z: [M + H]+ calcd for
C14H24NO4 270.1705, found 270.1696.
To a solution of 11 (108 mg, 0.34 mmol) in ethanol at 0 °C was
added sodium borohydride (51 mg, 1.36 mmol), and the reaction
mixture was slowly allowed to warm to rt. Excess acetone was added,
and the solvents were evaporated. The residue was dissolved in 1 mL
of water and Boc2O (148 mg, 0.68 mmol) was added. After 1 h, the
product was extracted with EtOAc. The organic phase was dried over
Na2SO4 and evaporated, and the residue was purified by column
chromatography on silica gel (15% EtOAc in hexanes) to give 12 (61
mg, 67%).
Ethyl (S)-5-((tert-Butoxycarbonyl)amino)cyclohexa-1,3-diene-1-
carboxylate (2). To a solution of ester 12 (236 mg, 0.88 mmol) in
10 mL of THF at −78 °C was added, dropwise, KHMDS (0.5 M in
toluene, 4.5 mL, 2.25 mmol), and the mixture was stirred for 1 h.
Solid diphenyl diselenide (309 mg, 0.99 mmol) was added at −78 °C,
and the resulting solution was stirred for 3 h at the same temperature
and then quenched by addition of NH4Cl (aq., sat’d), followed by
1
calcd for C17H15NO4Na 320.0899; found 320.0899. H and 13C{1H}
NMR spectral data matched reported data,16b as well as the optical
rotation, within experimental uncertainty: [a]20 −167.5° (c = 2.0,
D
CHCl3), lit.: [a]23D −168.2° (c = 2.8, CHCl3). The correspondence of
our optical rotation readings with that reported for this compound
serves to establish the absolute stereochemistry of our compounds.
VI. Synthetic Procedure: Corey Intermediate via Imidate RR.
Ethyl (5R)-5-(2,2,2-Trichloro-1-iminoethoxy)cyclohex-3-ene-1-car-
boxylate (10). To a solution of alcohol 6a/6b (570 mg, 3.35
mmol) and DBU (601 μL, 4.02 mmol) in CH2Cl2 at −10 °C was
added Cl3CCN (438 μL, 4.36 mmol), and the mixture was allowed to
warm to rt over 2 h. Then NaHCO3 (aq., sat’d) solution was added;
the product was extracted with Et2O and CH2Cl2; and the organic
fractions were dried (Na2SO4) and evaporated. The crude product
was quickly purified by flash column chromatography on silica gel
(30% EtOAc-hexanes) to give 10 (1.02 g, 97%) as a 1:1 mixture of
cis/trans diastereomers. 1H NMR (400 MHz, CDCl3) cis-diaster-
eomer: δ 8.31 (s, 1H), 5.94−5.89 (m, 1H), 5.79 (dt, J = 10.4, 2.0 Hz,
1H), 5.58−5.52 (m, 1H), 4.14 (q, J = 6.8 Hz, 2H), 2.77−2.68 (m,
1H), 2.56 (ddd, J = 12.4, 5.6, 2.4 Hz, 1H), 2.35−2.30 (m, 2H), 1.84
(dt, J = 4.4, 7.6 Hz, 1H), 1.25 (t, J = 7.2 Hz, 3H). trans-Diastereomer:
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extraction with Et2O. The organic phase was dried over Na2SO4 and
evaporated, and the residue was purified by column chromatography
on silica gel (15% EtOAc in hexanes) to give the phenyl selenide
intermediate as a nearly 2:1 mixture of diastereomers (271 mg, 73%).
This mixture of diastereomers (80 mg, 0.19 mmol) was then dissolved
in THF (4 mL) at 0 °C, and H2O2 (46 μL of a 30% aqueous solution,
0.38 mmol) and pyridine (31 μL mg, 0.38 mmol) were added. The
mixture was allowed to warm to rt over 3 h, and Et2O was added. The
organic phase was washed with CuSO4 (aq., saturated) and brine,
dried over Na2SO4, and evaporated. The residue contained a 2:1
mixture of regioisomers which was easily purified by flash column
chromatography on silica gel (10% EtOAc in hexanes) to give the title
compound 2 (31 mg, 62%) as a white solid. Chiral HPLC conditions:
column Chiralcel OD (250 mm length, 4.6 mm inner diameter, 5 μm
particle size), flow rate 1 mL/min, UV detection at 254 nm, 2%
isopropyl alcohol/hexanes, 10.02 min (major, S), 11.43 (minor, R),
98% ee. Racemic 2 was obtained following the same procedure from
racemic 6 and used as a chiral HPLC standard (see below for the
HPLC chromatograms). HRMS (FAB, 3-NBA) m/z: [M + Li]+ calcd
for C14H21NO4Li 274.1631; found 274.1634. 1H and 13C {1H} NMR
spectral data matched reported data.14 The optical rotation matched
reported values: [α]20D −217.6° (c = 1.0, CHCl3), lit. [α]25D −141.2°
(c = 1.0, CHCl3),14 [α]20D −217° (c = 1.1, CHCl3),31 [α]20D −219.7°
(c = 0.2, CHCl3).20h
D program for DBB’s appointment at the NSF. The authors
thank the NIH (SIG-1-510-RR-06307) and the NSF (CHE-
0091975 and MRI-0079750) for NMR instrumentation and
for the X-ray diffractometer (CHE-1726630) and the NIH
(RR016544) for facilities.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.1c00326.
■
sı
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Procedural details, KRED screening, NMR spectra,
HPLC traces, and X-ray crystal structure details (PDF)
Accession Codes
CCDC 2038401−2038402 contain the supplementary crys-
tallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
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AUTHOR INFORMATION
Corresponding Authors
■
Sylvain Broussy − University of Paris, CiTCoM, 8038 CNRS,
U 1268 INSERM, F-75006 Paris, France; orcid.org/
0000-0003-3098-5317; Email: sylvain.broussy@u-paris.fr
David B. Berkowitz − Department of Chemistry, University of
Nebraska, Lincoln, Nebraska 68588, United States;
orcid.org/0000-0001-7550-0112; Email: dberkowitz1@
unl.edu
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Authors
Virendra K. Tiwari − Department of Chemistry, University of
Nebraska, Lincoln, Nebraska 68588, United States
Douglas R. Powell − Department of Chemistry and
Biochemistry, University of Oklahoma, Norman, Oklahoma
73019, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.1c00326
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by NSF grants CHE/CBET-1800076
and CBET-2023250. These studies were facilitated by the IR/
■
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Furlong, K.; Manicassamy, B.; Caffrey, M.; Thatcher, G. R. J.; Rong,
L. Optimization of 4-aminopiperidines as inhibitors of influenza a viral
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