(S)-Selectivity in Phenylacetyl Carbinol Synthesis Using the Wild-Type Enzyme Acetoin:Dichlorophenolindophenol Oxidoreductase from Bacillus licheniformis

Article abstract of DOI:10.1002/adsc.201600359

Thiamine diphosphate (ThDP)-dependent enzymes are well known biocatalysts for the asymmetric synthesis of α-hydroxy ketones with preferential (R)-selectivity. Pharmaceutically relevant phenylacetyl carbinol (PAC) has been prepared with absolute (S)-configuration only on a few occasions using enzyme variants suitably designed through rational site-directed mutagenesis approaches. Herein, we describe the synthesis of (S)-phenylacetyl carbinol products with extended reaction scope employing the readily available wild-type ThDP-dependent enzyme acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) from Bacillus licheniformis. On a semipreparative scale, cross-benzoin-like condensations of methylacetoin (donor) and differently substituted benzaldehydes proceed with almost complete chemoselectivity yielding the target (S)-1-hydroxy-1-phenylpropan-2-one derivatives with high conversion efficiencies (up to 95%) and good enantioselectivities (up to 99%). Ao:DCPIP OR accepts hydroxy- and nitrobenzaldehydes and also sterically demanding substrates such as 1-naphthaldehyde and 4-(tert-butyl)benzaldehyde, which are typically poor acceptors in enzymatic transformations. The explorative synthesis of (S)-phenylpropionyl carbinol mediated by Ao:DCPIP OR via carboligation of benzaldehyde with 3,4-hexanedione is also reported. (Figure presented.).

Full text of DOI:10.1002/adsc.201600359

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DOI: 10.1002/adsc.201600359
(S)-Selectivity in Phenylacetyl Carbinol Synthesis Using the Wild-
Type Enzyme Acetoin:Dichlorophenolindophenol Oxidoreductase
from Bacillus licheniformis
Pier Paolo Giovannini,a,* Lindomar Alberto Lerin,a Michael Mꢀller,b
Giovanni Bernacchia,c Morena De Bastiani,c Martina Catani,a
Graziano Di Carmine,a and Alessandro Massia,*
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Universitꢁ di Ferrara, Via Fossato di Mortara 17, I-44121 Ferrara,
Italy
Fax : (+39)-0532-240709; e-mail: gvnppl@unife.it or alessandro.massi@unife.it
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universitꢂt Freiburg, Albertstrasse 25, 79104 Freiburg, Germany
Dipartimento di Scienze della Vita e Biotecnologie, Universitꢁ di Ferrara, Via L. Borsari 46, I-44121 Ferrara, Italy
b
c
Received: April 8, 2016; Revised: June 14, 2016; Published online: && &&, 0000
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201600359.
Abstract: Thiamine diphosphate (ThDP)-dependent ently substituted benzaldehydes proceed with almost
enzymes are well known biocatalysts for the asym- complete chemoselectivity yielding the target (S)-1-
metric synthesis of a-hydroxy ketones with preferen- hydroxy-1-phenylpropan-2-one derivatives with high
tial (R)-selectivity. Pharmaceutically relevant phenyl- conversion efficiencies (up to 95%) and good enan-
acetyl carbinol (PAC) has been prepared with abso- tioselectivities (up to 99%). Ao:DCPIP OR accepts
lute (S)-configuration only on a few occasions using hydroxy- and nitrobenzaldehydes and also sterically
enzyme variants suitably designed through rational demanding substrates such as 1-naphthaldehyde and
site-directed mutagenesis approaches. Herein, we de- 4-(tert-butyl)benzaldehyde, which are typically poor
scribe the synthesis of (S)-phenylacetyl carbinol acceptors in enzymatic transformations. The explora-
products with extended reaction scope employing tive synthesis of (S)-phenylpropionyl carbinol medi-
the readily available wild-type ThDP-dependent ated by Ao:DCPIP OR via carboligation of benzal-
enzyme acetoin:dichlorophenolindophenol oxidore- dehyde with 3,4-hexanedione is also reported.
ductase (Ao:DCPIP OR) from Bacillus lichenifor-
À
mis. On a semipreparative scale, cross-benzoin-like Keywords: asymmetric synthesis; C C coupling;
condensations of methylacetoin (donor) and differ- enzyme catalysis; thiamine diphosphate; umpolung
Introduction
two aldehyde molecules by the umpolung (polarity re-
versal) strategy (benzoin condensation). Organocata-
Chiral a-hydroxy ketones are key synthons in prepa- lytic strategies for the cross-benzoin-like condensation
rative organic chemistry for the synthesis of important have been reported using azolium salt pre-catalysts
molecules such as chiral amino alcohols and diols,[1] with some level of success;[8] in general, however,
and are structural subunits of many natural products chemical approaches suffer from chemoselectivity
and pharmaceuticals including antidepressants, anti- issues and high enantioselectivities are typically
fungal agents, and antitumor antibiotics.[2] In virtue of rare.[9] Biocatalysis with thiamine diphosphate
their synthetic and biological relevance, several chem- (ThDP)-dependent enzymes constitutes a great op-
ical approaches have been reported for their synthe- portunity to overcome the above limitations allowing
sis; the most common strategies rely on the a-hydrox- the synthesis of chiral a-hydroxy ketones with high
ylation of ketones with chiral oxidants,[3] the ketohy- levels of chemo- and enantioselectivity under environ-
droxylation of olefins,[3,4] the asymmetric dihydroxyla- mentally benign conditions.[10] The production on an
tion of silyl enol ethers,[5] the mono-oxidation of 1,2- industrial scale of (R)-phenylacetyl carbinol [(R)-
diols,[6] the organocatalytic a-oxygenation of ke- PAC], the key precursor in the l-ephedrine synthesis,
tones,[7] and the direct asymmetric condensation of from pyruvic acid and benzaldehyde mediated by pyr-
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uvate decarboxylase (PDC) is an illustrative example
of the impact of ThDP-dependent enzymes on practi-
[11]
À
cal C C bond-forming reactions. A toolbox of dif-
ferent wild-type (wt) ThDP-dependent lyases, includ-
ing several PDCs, benzoylformate decarboxylase
(BFD), branched-chain keto acid decarboxylase
(KdcA) and benzaldehyde lyase (BAL), is nowadays
available for the chemoselective (cross-)benzoin con-
densation of various aliphatic and aromatic aldehydes
(or their equivalents) to afford a-hydroxy ketones
with high enantioselectivity and almost exclusive (R)-
configuration.[12] Indeed, access to the corresponding
(S)-isomers is almost precluded by using the above
wild-type enzymes of the decarboxylase-subfamily[13]
with two exceptions: the kinetic resolution of ben-
zoins by BAL from Pseudomonas fluorescens,[14] and
the formation of (S)-hydroxypropiophenone deriva-
tives [(S)-HPPs] through condensation of benzalde-
hydes (donors) and acetaldehyde (acceptor) promot-
ed by BFD from Pseudomonas putida.[15] Remarkably,
mutagenesis studies based on structural analysis of
the active-site architecture have permitted the design
of (S)-selective variants of ThDP-dependent enzymes,
thus paving the way for the formation of (S)-HPPs
with wider substrate tolerance,[12c,15c] for the synthesis
of (S)-5-hydroxy-4-oxo-5-phenylpentanoate deriva-
tives (from a-ketoglutarate donor and benzaldehydes
Figure 1. Enzymatic carboligations for the direct asymmetric
synthesis of (S)-phenylacetyl carbinols (PACs).
as acceptors),[16] and for the production of (S)-ben- Results and Discussion
zoins.[17] As far as the synthesis of pharmaceutically
relevant phenylacetyl carbinols (PACs) is concerned, Ao:DCPIP OR is the first enzyme of the bacterial
a variant of PDC from Acetobacter pasteurianus has acetoin dehydrogenase enzyme system (AoDH
been generated in a breakthrough study to produce ES);[22,23] its physiological role is the oxidative cleav-
PAC derivatives with (S)-selectivity for the first age of acetoin (1) with formation of acetaldehyde and
time.[18] While the mutant enzyme promoted the car- transfer of the (hydroxyethyl)thiamine diphosphate
boligation of benzaldehyde and acetaldehyde with intermediate 2 (acetyl anion equivalent) to the lipo-
modest efficiency,[12c,18a,19] (S)-PAC was suitably ob-
ACHTUNGTRENaNUNG mide cofactor of the second enzyme of the system
tained using the same enzyme and pyruvate as donor (Scheme 1, route a). We have recently demonstrated
(70% ee, 95% yield; Figure 1).[20] Very recently, a var- that Ao:DCPIP OR from Bacillus licheniformis,
iant of PDC from Zymomonas mobilis has also been cloned and overexpressed in E. coli, is also able to
introduced for the highly chemoselective formation of mediate the non-physiological 1,2-addition of methyl-
(S)-PAC (76% ee, 95% yield; Figure 1).[21]
acetoin (donor) to activated ketones (acceptors)
It can be seen from the above survey that expand- yielding chiral tertiary a-hydroxy ketones with high
ing the enzyme toolbox to efficiently access the valua- efficiency (Scheme 1, route b).[24]
ble (S)-PAC structural motif with extended reaction
Complete control of the chemoselectivity could be
scope and using readily available enzymes would be achieved in this transformation due to the hitherto
highly desirable. Herein, we describe the unprece- unreported use of methylacetoin, whose activation
dented (S)-selective synthesis of phenylacetyl carbi- occurs with elimination of unreactive acetone. In ad-
nols mediated by the wt ThDP-dependent enzyme dition, some of the reaction products were formed
acetoin:dichlorophenolindophenol
oxidoreductase with opposite stereochemistry compared to that ob-
(Ao:DCPIP OR; EC 2.3.1.190) via carboligation of tained using other ThDP-dependent enzymes.[24] As
methylacetoin (acetyl anion precursor) with a set of a logical extension of that study on the formal alde-
substituted benzaldehydes displaying different stereo- hyde-ketone cross-carboligation reaction, we planned
electronic properties. The results of this study clearly to examine the efficiency of the Ao:DCPIP OR-
indicate Ao:DCPIP OR as a suitable candidate to fill methylacetoin enzyme-substrate pair in the mixed
the gap in the stereocontrolled synthesis of (S)-PAC benzoin-like reaction with aromatic aldehydes to
derivatives by the catalysis of ThDP-dependent en- access the class of valuable phenylacetyl carbinols,
zymes.
eventually with unusual (S)-configuration. Gratifying-
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The (S)-configuration of 6 was further confirmed
by comparison with an authentic sample of (R)-PAC
prepared using the highly (R)-selective cyclohexane-
1,2-dione hydrolase (CDH).[25] Hence, the first impor-
tant result of this explorative study was the confirma-
tion of Ao:DCPIP OR peculiarity to promote, in
some occasions, carboligations with opposite stereo-
chemical outcome compared to other wt ThDP-de-
pendent enzymes. Moreover, the previously observed
high level of chemoselectivity induced by Ao:DCPIP
OR catalysis was established in the 3/5 coupling as
well; indeed, the methylacetoin homocoupling prod-
uct 7 was detected in very small amounts (2%) and in
a racemic form,[26] whereas benzoin and 2-HPP by-
products were not observed in the crude reaction mix-
ture (Scheme 2).
The effect of variation of the substrate molar ratio,
reaction time, and enzyme amount on the efficiency
of the model cross-benzoin-type condensation was
evaluated next. Using equimolar concentrations of 3
and 5, a decrease of benzaldehyde conversion was ob-
served for reaction times of approximately 8–10 h,
and this effect was more pronounced increasing the
enzyme concentration (from 0.5 to 4.0 mgmLÀ1,
Figure 2).
Intriguingly, the graph reporting the enantiomeric
excess values as a function of time at different
enzyme concentrations displayed a similar trend with
erosion of enantioselectivity at long reaction times
and high concentrations of enzyme (Figure 3).
Scheme 1. Physiological and non-physiological activities of
Ao:DCPIP OR. R=(4-amino-2-methylpyrimidin-5-yl)meth-
yl; R’=ethyl diphosphate. Cofactor=lipoamide covalently
bound to the second enzyme (E2) of the acetoin dehydro-
genase enzyme system (AoDH ES).
These results were rationalized assuming that
ly, condensation of methylacetoin (3) with benzalde- Ao:DCPIP OR could also catalyze the cleavage of
hyde (5) under similar conditions to those previously (S)-PAC (6) to yield benzaldehyde (5) and acetalde-
described for the synthesis of chiral tertiary alcohols hyde [Scheme 3, Eq. (a)]. This hypothesis was initially
1
4[24] [3 (20 mM), 5 (20 mM), phosphate buffer pH 6.5 confirmed by H NMR analysis of the crude reaction
(50 mM), DMSO (10% v/v), MgSO4 (0.9 mM), ThDP mixture (Ao:DCPIP OR: 4 mgmLÀ1; reaction time:
(0.4 mM), purified and lyophilized Ao:DCPIP OR 48 h) that showed, after extraction with CDCl3, the
0.5 mgmLÀ1, 308C, 12 h], afforded the enantioen-
riched (S)-1-hydroxy-1-phenylpropan-2-one [6, (S)-
PAC] with 81% conversion (73% isolated yield) and
88% enantiomeric excess (ee), as determined by
chiral-phase GC and optical rotation analyses
(Scheme 2).
Figure 2. Conversion of benzaldehyde (5) as a function of
À1
*
*
Scheme 2. Explorative study of the cross-benzoin-like reac-
tion of methylacetoin (3) and benzaldehyde (5) catalyzed by
Ao:DCPIP OR.
time at different enzyme cÀo1ncentrations ( : 0.5 mgmL
;
:
2.0 mgmLÀ1
triplicates.
;
: 4.0 mgmL ). Values are the mean ÆSD of
~
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servations and with the aim to limit the reverse activi-
ty of Ao:DCPIP OR, the model cross-benzoin-type
condensation was optimized by using a low amount of
enzyme (0.5 mgmLÀ1), an excess of methylacetoin (3,
2 equiv.), and by suitably controlling the reaction time
(24 h). Under these conditions, (S)-PAC (6) was ob-
tained in 84% yield (95% conversion) and 94% ee on
a semipreparative scale [0.5 mmol; Scheme 3, Eq.
(c)]. A brief solvent screen was also undertaken and
DMSO was found to be the best performing co-sol-
vent among those tested (THF, EtOH, methyl tert-
butyl ether), especially in terms of conversion effi-
ciency (Supporting Information, Figure S1).
The scope of the Ao:DCPIP OR-mediated cross-
benzoin reaction was further investigated by testing
the behavior of the ortho- (a), meta- (b), and para-
substituted (c) benzaldehydes 8a, b, c–13a, b,
c (Table 1). Preliminary experiments run on an analyt-
ical scale (enzyme concentration: 0.5 mgmLÀ1; equi-
molar donor/acceptor) showed no decrease of conver-
sion efficiency and enantioselectivity in the formation
of the corresponding PACs 14a, b, c–19a, b, c within
8–48 h (Supporting Information, Figure S2: methyl-
Figure 3. Enantiomeric excess of 6 as a function of time at
different enzyme conceÀn1trations ( : 0.5 mgmLÀ1
;
:
&
*
2.0 mgmLÀ1
triplicates.
;
: 4.0 mgmL ). Values are the mean ÆSD of
~
AHCTUNGTREGaNNNU cetoin/p-tolualdehyde condensation as representative
example). This result was in contrast with the trend
observed for 6, thus suggesting that the cleavage of
PAC derivatives with substituted aromatic portions is
a slow reaction. Therefore, the reaction time was set
at 48 h for the subsequent screening study on a semi-
preparative scale (0.5 mmol) using a slight excess
(1.3 equiv.) of methylacetoin (3). The absolute config-
uration of all the synthesized PAC products (Table 1)
was assigned to be (S) on the basis of circular dichro-
ism analysis (appearance of a positive band centered
at 270–290 nm)[25] and confirmed for products 14a–c,
15a–c, 17a, 17c and 26 by comparison of their optical
rotations with literature values (see the Experimental
Section).[27]
No racemization was observed for compounds 14–
19 under the reaction conditions; this was verified in
a control experiment with isolated 14c (92% ee),
which maintained its enantiomeric integrity over
a period of 48 h [14c (20 mM), phosphate buffer
pH 6.5 (50 mM), DMSO (10% v/v), MgSO4 (0.9 mM),
ThDP (0.4 mM), 308C]. Overall, the electronic prop-
erties and position of the substituent on the aromatic
ring were found to have little effect on the conversion
(52–99%) and enantioselectivity (68–99% ee) of the
Scheme 3. Cleavage of (S)- and (rac)-PAC [Eq. (a) and (b)]
and optimized conditions for the synthesis of (S)-PAC (6).
presence of trace amounts of acetaldehyde. To un- cross-benzoin processes, with the exceptions of o-
À
equivocally establish the postulated C C bond cleav- chloro- and p-bromobenzaldehydes 9a and 10c, which
age of 6, two control experiments were performed gave the corresponding PAC derivatives 15a and 16c
where racemic PAC was incubated for 48 h under with modest enantioselectivity (68% ee for both), and
standard conditions with or without Ao:DCPIP OR. p-nitrobenzaldehyde 12c. In this latter case, the full
Indeed, benzaldehyde was formed only in the pres- consumption of 3/12c substrates occurred with forma-
ence of the enzyme, which preferentially consumed tion of a quite complex reaction mixture not contain-
the (S)-enantiomer of PAC, as determined by chiral ing the expected PAC derivative 18c (1H NMR and
GC analysis [Scheme 3, Eq. (b)]. In light of these ob- MS analyses). It is worth emphasizing, however, that
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Table 1. Synthesis of PAC derivatives 14–19 catalyzed by Ao:DCPIP OR.[a]
[a]
Conditions: 3 (26 mM), aldehyde (20 mM), phosphate buffer pH 6.5 (50 mM), DMSO (10% v/v), MgSO4 (0.9 mM),
1
ThDP (0.4 mM), purified and lyophilized Ao:DCPIP OR (0.5 mgmLÀ1). Conversion determined by H NMR analysis;
enantiomeric excess determined by chiral-phase GC analysis. Yields refer to the isolated product after column chroma-
tography.
Ao:DCPIP OR accepted nitro- and hydroxybenzalde- benzaldehyde (5) with 3,4-hexanedione (30) for the
hydes 12 and 13, which are suitable substrates only challenging synthesis of 1-hydroxy-1-phenylbutan-2-
for a very limited number of ThDP-dependent en- one (phenylpropionyl carbinol, PPC) 31 with (S)-se-
zymes.[25,28]
lectivity (Scheme 4).[30] It is important to remember
Ao:DCPIP OR proved also to be an effective bio- that alkyl a-diketones are highly reactive donors in
catalyst in the condensation of methylacetoin (3) with Ao:DCPIP OR catalysis;[24,31] however, their utiliza-
the sterically demanding aromatic aldehydes 20–24
furnishing the PAC products 25–29 with good conver-
sions (67–97%) and enantioselectivities (86–99% ee;
Table 2).
The combinations of 3 with 1-naphthaldehyde (20)
and with 4-(tert-butyl)benzaldehyde (23) are of partic-
ular relevance as these acceptors are notoriously poor
substrates in enzymatic transformations for steric and
solubility reasons.[25,29]
The donor substrate range of the Ao:DCPIP OR-
catalyzed cross-benzoin reaction was briefly investi-
gated in an explorative study on the carboligation of binol derivatives (PPCs) by Ao:DCPIP OR catalysis.
Scheme 4. Approaching the class of (S)-phenylpropionyl car-
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Table 2. Synthesis of sterically demanding PAC derivatives
25–29 catalyzed by Ao:DCPIP OR.[a]
uable phenylacetyl carbinol products (PACs). Indeed,
the carboligation of methylacetoin (3) (acetyl anion
donor) with differently substituted benzaldehydes af-
forded the target a-hydroxy ketone derivatives with
good levels of conversion efficiency and enantioselec-
tivity and, significantly, with almost complete chemo-
selectivity. In anticipation of a future improvement
based on the use of the higher homolog of methylace-
toin, the propionyl anion transfer on benzaldehyde
was attempted in this study employing the a-diketone
3,4-hexanedione (30) as donor substrate; the (S)-se-
lectivity induced by Ao:DCPIP OR catalysis was es-
tablished in the formation of phenylpropionyl carbi-
nol (PPC) as well. Ao:DCPIP OR belongs to the
narrow group of ThDP-dependent enzymes able to
promote asymmetric aldehyde-ketone cross-couplings
yielding chiral tertiary alcohols;[24] this constitutes
a further evidence of the tremendous catalytic poten-
tial of Ao:DCPIP OR in asymmetric carboligation re-
actions. The straightforward production, robustness
and facile immobilization of Ao:DCPIP OR[31b] are
additional valuable features of this enzyme for the de-
velopment of novel synthetic applications and techno-
logical improvements. The evaluation of aliphatic al-
dehydes as acceptor substrates and the operation of
Ao:DCPIP OR-functionalized packed-bed reactors
for the continuous-flow cross-benzoin and aldehyde-
ketone cross-coupling reactions are currently under
investigation in our laboratories. Moreover, determi-
[a]
Conditions: 3 (26 mM), aldehyde (20 mM), phosphate
buffer pH 6.5 (50 mM), DMSO (10% v/v), MgSO4
(0.9 mM), ThDP (0.4 mM), purified and lyophilized
Ao:DCPIP OR (0.5 mgmLÀ1). Conversion determined nation of the three-dimensional structure of the
1
by H NMR analysis; enantiomeric excess determined by
chiral-phase GC analysis. Yields refer to the isolated
product after column chromatography.
enzyme will offer new perspectives on thiamine catal-
ysis in respect to mechanistic information and the
design of variants with novel catalytic activities.
tion in mixed condensations with carbonyl acceptors
is complicated by the occurrence of the a-diketone
homocoupling side-reaction, which reduces the che-
moselectivity of the coupling process. Nevertheless,
the cross-benzoin-type reaction of 30 and 5 was at-
tempted to gain information about the Ao:DCPIP
OR capability to promote the (S)-selective synthesis
of PPC derivatives. Satisfyingly, under standard condi-
tions [30 (40 mM), 5 (20 mM), phosphate buffer
pH 6.5 (50 mM), DMSO (10% v/v), MgSO4 (0.9 mM),
ThDP (0.4 mM), purified and lyophilized Ao:DCPIP
Experimental Section
General Methods
Reactions were monitored by TLC on silica gel 60 F254 with
detection by charring with phosphomolybdic acid. Flash
column chromatography was performed on silica gel 60
(230–400 mesh). Optical rotations were measured at 20Æ
28C in the stated solvent; [a]D values are given in 10À1 deg
cm2 gÀ1. 1H (300 MHz), 13C (75 MHz), and 19F (282 MHz)
NMR spectra were recorded in CDCl3 solutions at room
temperature. Peak assignments were aided by H-1H COSY
1
OR (0.5 mgmLÀ1); 308C, 24 h] the target (S)-1-hy- and gradient-HMQC experiments. For HR-MS measure-
ments, the compounds were analyzed in positive ion mode
by Agilent 6520 HPLC-Chip Q/TOF-MS (nanospray) using
a quadrupole, a hexapole, and a time-of-flight unit to pro-
duce spectra. The capillary source voltage was set at 1700 V;
the gas temperature and drying gas were kept at 3508C and
5 LminÀ1, respectively. The MS analyzer was externally cali-
brated with ESI-L low concentration tuning mix from m/z=
118 to 2700 to yield accuracy below 5 ppm. Accurate mass
data were collected by directly infusing samples in 40/60
H2O/ACN 0.1% TFA into the system at a flow rate of
droxy-1-phenylbutan-2-one (31) was prepared in 60%
isolated yield and 98% ee (Scheme 4).
Conclusions
In summary, we have demonstrated that the wild-type
ThDP-dependent acetoin:dichlorophenolindophenol
oxidoreductase (Ao:DCPIP OR) serves as an effi-
cient biocatalyst for the (S)-selective synthesis of val- 0.4 mLminÀ1. Chiral phase HPLC analyses were carried out
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on an HP 1100 chromatography system (Agilent) using
a Chiralpak ID column (250ꢄ4.6 mm, particle size: 5 mm)
for compounds 15a–c, 16a–c, 25, a Phenomenex Lux Cellu-
lose-1 column (250ꢄ4.6 mm, particle size: 5 mm) for com-
pounds 19a and 19b, and a Phenomenex Amylose-2 lux
(250ꢄ4.6 mm, particle size: 5 mm) for compounds 19c, 26
and 29. Analyses were performed using a detection wave-
length of 254 nm and hexane/2-propanol (90:10) as eluent
(flow rate: 1 mLminÀ1), apart from compound 19c (eluent:
hexane/2-propanol/acetic acid 185:14:1). GC analyses were
performed on a Carlo Erba 6000, equipped with an FID de-
tector and a Megadex 5 column (25 mꢄ0.25 mm) with the
temperature programs below specified. CD spectra of the
products dissolved in acetonitrile were recorded on a Jasco
J-810 spectrometer. Purified Ao:DCPIP OR was obtained
as described in ref.[24]
General procedure for the Synthesis of the PAC
Analogues 14a, b, c–19a, b, c on Analytical Scale
Lyophilized Ao:DCPIPOR (0.75 mg) was added to a solu-
tion of the substituted benzaldehyde 8a, b, c–13a, b,
c (30 mmol), methylacetoin (3) (3.2 mL, 30 mmol), ThDP
(0,4 mg, 0.6 mmol) and MgSO4 (0.16 mg, 1.3 mmol) in 50 mM
phosphate buffer at pH 6.5 (1.5 mL) containing DMSO
(10% v/v). After 2, 8, 24 and 48 h samples (0.5 mL) were
withdrawn and extracted with CDCl3 (1.0 mL). The organic
extracts were dried with anhydrous Na2SO4 and analyzed by
1H NMR and chiral-phase GC to determine conversion and
ee, respectively.
General Procedure for the Synthesis of the PAC
Analogues 14a, b, c–19a, b, c and 25–29 on
Semipreparative Scale
Effect of Enzyme Concentration and Reaction Time
on the Synthesis of (S)-1-Hydroxy-1-phenylpropane-
2-one (6) [(S)-PAC]
Lyophilized Ao:DCPIP OR (12 mg) was added to a solution
of the substituted benzaldehyde 7a, b, c–12a, b, c and 20–24
(0.50 mmol), methylacetoin (3) (63 mL, 0.60 mmol), ThDP
(4.5 mg, 10 mmol) and MgSO4 (2.7 mg, 20 mmol) in 50 mM
phosphate buffer at pH 6.5 (25 mL) containing DMSO
(10% v/v). The reaction mixture was gently shaken at 308C
for 48 h and then extracted with ethyl acetate (3ꢄ10 mL).
The combined extracts were dried with anhydrous Na2SO4
and evaporated under reduced pressure. The residue was
chromatographed on silica gel with the suitable elution
system.
Three reactions were performed by adding 0.75, 3 and 6 mg
of lyophilized Ao:DCPIPOR, respectively, to a solution of
benzaldehyde (5) (3.0 mL, 30 mmol), methylacetoin (3)
(3.2 mL, 30 mmol), ThDP (0,4 mg, 0.6 mmol) and MgSO4
(0.16 mg, 1.3 mmol) in 50 mM phosphate buffer at pH 6.5
(1.5 mL) containing DMSO (10% v/v). The reactions were
gently shaken at 308C and after 2, 8, 24 and 48 h samples
(0.5 mL) were withdrawn and extracted with CDCl3
(1.0 mL). The organic extracts were dried with anhydrous
1
(S)-1-(2-Fluorophenyl)-1-hydroxypropan-2-one (14a): Col-
orless oil; yield: 90%; [a]D: +176 (c 1.0, MeOH), lit. for
(R)-enantiomer:[32b] À186 (c 0.56, MeOH); GC (temperature
program from 100 to 2008C, rate 18C minÀ1): Rt 22.3 min
Na2SO4 and analyzed by H NMR and chiral-phase GC to
determine conversion and ee, respectively.
Optimized Synthesis of (S)-1-Hydroxy-1-phenyl-
propane-2-one (6) [(S)-PAC]
1
(R), 23.2 min (S); ee 83% (S); H NMR: d=7.34–7.25 (m,
2H, Ar), 7.19–7.07 (m, 2H, Ar), 5.41 (s, 1H, H-1), 4.26 (bs,
1H, OH), 2.13 (s, 3H, CH3); 13C NMR: d=206.2, 160.5 (d,
J=246 Hz), 130.5, 128.8, 125.1, 124.9, 116.0, 73.6, 25.0;
19F NMR: d=À118.3 (m); HR-MS (ESI/Q-TOF): m/z=
168.0531, calcd. for C9H9FO2 [M]+: 168.0587.
Lyophilized Ao:DCPIP OR (12 mg) was added to a solution
of benzaldehyde (5) (51 mL, 0.50 mmol), methylacetoin (3)
(105 mL, 1.00 mmol), ThDP (4.5 mg, 10 mmol) and MgSO4
(2.7 mg, 20 mmol) in 50 mM phosphate buffer at pH 6.5
(25 mL) containing DMSO (10% v/v). The reaction mixture
was gently shaken at 308C for 24 h and then extracted with
ethyl acetate (3ꢄ10 mL). The combined extracts were dried
with anhydrous Na2SO4 and evaporated under reduced pres-
sure. The residue was chromatographed on silica gel using
cyclohexane/ethyl acetate (4:1) as eluent to afford pure (S)-
6 (ee 94%) as a yellow pale oil; yield: 63 mg (84%); [a]D:
+313 (c 0.3, CHCl3), lit.:[32a] +413 (c 0.82, CHCl3); GC
(temperature program from 100 to 2008C rate 18C minÀ1):
Rt 28.6 min (R), 30.2 min (S); 1H NMR: d=7.46–7.28 (m,
5H, Ar), 5.09 (d, J=4.2 Hz, 1H, H-1), 4.30 (d, J=4.2 Hz,
1H, OH), 2.07 (s, 3H, CH3); 13C NMR: d=207.1, 137.9,
129.0 (2C), 128.7 (2C), 127.3, 80.1, 25.3; HR-MS (ESI/Q-
TOF): m/z=173.0605, calcd. for C9H10O2Na [M+Na]+:
173.0578.
(S)-1-(3-Fluorophenyl)-1-hydroxypropan-2-one
(14b):
Colorless oil; yields: 85%; [a]D: +242.2 (c 0.93, CHCl3), lit.
for (R)-enantiomer:[32b] À221 (c 0.14, MeOH); GC (temper-
ature program from 100 to 2008C, rate 58C minÀ1): Rt
1
13.1 min (R), 13.2 min (S); ee 80% (S); H NMR: d=7.40–
7.33 (m, 1H, Ar), 7.14 (dd, J=7.9, 0.8 Hz, 1H, Ar), 7.07–
7.01 (m, 2H), 5.09 (s, 1H, H-1), 4.33 (s, 1H, OH), 2.10 (s,
3H, CH3); 13C NMR: d=206.4, 163.2 (d, J=248 Hz), 140.4,
130.7, 123.1, 115.9, 114.3, 79.6, 25.3; 19F NMR: d=À111.83
(m); HR-MS (ESI/Q-TOF): m/z=168.0611, calcd. for
C9H9FO2 [M]+: 168.0587.
(S)-1-(4-Fluorophenyl)-1-hydroxypropan-2-one (14c): Col-
orless oil; yield: 88%; [a]D: +330.3 (c 0.35, CHCl3), lit. for
(R)-enantiomer:[32b] À197 (c 0.69, MeOH); GC (temperature
program from 100 to 2008C, rate 18C minÀ1): Rt 33.6 min
1
(R), 36.1 min (S); ee 92% (S); H NMR: d=7.38–7.18 (m,
3,4-Dihydroxy-3,4-dimethylpentan-2-one (7): Obtained as
described before from the reaction of equimolar amounts of
2H, Ar), 7.15–6.99 (m, 2H, Ar), 5.07 (s, 1H, H-1), 4.28 (bs,
1H, OH), 2.07 (s, 3H, CH3); 13C NMR: d=206.98, 163.0 (d,
J=247 Hz), 133.87, 129.2 (2C), 116.1 (2C), 79.5, 25.3;
19F NMR: d=À112.91 (m); HR-MS (ESI/Q-TOF): m/z=
168.0524, calcd. for C9H9FO2 [M]+: 168.0587.
1
3 and 5 (0.50 mmol). H NMR: d=2.24 [s, 3H, C(O)CH3],
1.38 [s, 3H, C(OH)CH3], 1.24 (s, 6H, 2CH3).
(S)-1-(2-Chlorophenyl)-1-hydroxypropan-2-one
(15a):
Colorless oil; yield: 81%; [a]D: +179.5 (c 0.22, MeOH), lit.
Adv. Synth. Catal. 0000, 000, 0 – 0
7
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for (R)-enantiomer:[32b] À207 (c 0.34, MeOH); HPLC: Rt
perature program from 100 to 2008C rate 18C minÀ1): Rt
34.3 min (R), 35.5 min (S); ee 91% (S); H NMR: d=7.33–
1
1
11.7 min (S), 12.7 min (R); ee 68% (S); H NMR: d=7.50–
7.35 (m, 1H, Ar), 7.35–7.18 (m, 3H, Ar), 5.58 (s, 1H, H-1),
4.35 (bs, 1H, OH), 2.13 (s, 3H, CH3); 13C NMR: d=206.3,
135.6, 133.4, 130.1, 129.9, 128.9, 127.5, 76.4, 25.3; HR-MS
(ESI/Q-TOF): m/z=207.0150, calcd. for C9H9ClO2Na [M+
Na]+: 207.0189.
7.20 (m, 1H, Ar), 7.20–7.03 (m, 3H, Ar), 5.05 (s, 1H, H-1),
4.26 (bs, 1H, OH), 2.35 (s, 3H, CH3), 2.08 (s, 3H, CH3);
13C NMR: d=207.3, 138.9, 137.9, 129.6, 128.9, 127.9, 124.7,
80.2, 25.4, 21.5; HR-MS (ESI/Q-TOF): m/z=164.0927 calcd.
for C10H12O2 [M]+: 164.0837.
(S)-1-(3-Chlorophenyl)-1-hydroxypropan-2-one
(15b):
(S)-1-Hydroxy-1-(p-tolyl)propan-2-one (17c): Yellow pale
oil; yield: 90%; [a]D: +394.0 (c 0.55, CHCl3), lit. for (R)-
enantiomer:[32d] À385 (c and solvent not reported); GC
(temperature program from 100 to 2008C rate 18C minÀ1):
Colorless oil; yield: 87%; [a]D: +218.8 (c 0.70, CHCl3), lit.
for (R)-enantiomer:[32c] À115 (c and solvent not reported);
1
HPLC: Rt 9.7 min (S), 12.3 min (R); ee 78% (S); H NMR:
1
d=7.34–7.31 (m, 3H, Ar), 7.25–7.19 (m, 1H, Ar), 5.06 (s,
1H, H-1), 4.31 (bs, 1H, OH), 2.11 (s, 3H, CH3); 13C NMR:
d=206.3, 139.9, 135.1, 130.4, 129.1, 127.6, 125.6, 79.6, 25.4;
Rt 37.5 min (R), 39.6 (S); ee 99% (S); H NMR: d=7.22–
7.17 (m, 4H, Ar), 5.06 (d, J=4.2 Hz, 1H, H-1), 4.25 (d, J=
4.2 Hz, 1H, OH), 2.35 (s, 3H, CH3), 2.07 (s, 3H, CH3);
13C NMR: d=207.4, 138.7, 135.1, 129.8 (2C), 127.4 (2C),
80.0, 25.4, 21.3. HR-MS (ESI/Q-TOF): m/z=164.0711,
calcd. for C10H12O2 [M]+: 164.0837.
HR-MS
C9H9ClO2Na [M+Na]+: 207.0189.
(S)-1-(4-Chlorophenyl)-1-hydroxypropan-2-one
(ESI/Q-TOF):
m/z=207.0212,
calcd.
for
(15c):
Colorless oil; yield: 55%; [a]D: +222.5 (c 0.16, CHCl3), lit.
(S)-1-Hydroxy-1-(2-nitrophenyl)propan-2-one
(18a):
for (R)-enantiomer:[32b] À158 (c 0.58, MeOH); HPLC: Rt
Yellow amorphous solid; yield: 79%; [a]D: +183.0 (c 1.40,
1
1
10.7 min (R), 11.6 min (S); ee 89% (S); H NMR: d=7.42–
CHCl3); H NMR: d=8.00 (d, J=8.1 Hz, 1H, Ar), 7.76–7.57
7.32 (m, 2H, Ar), 7.29–7.25 (m, 2H, Ar), 5.06 (s, 1H, H-1),
4.29 (s, 1H, OH), 2.08 (s, 3H, CH3); 13C NMR: d=206.6,
136.5, 134.8, 129.3 (2C), 128.7 (2C), 79.5, 25.3; HR-MS
(ESI/Q-TOF): m/z=207.0158, calcd. for C9H9ClO2Na [M+
Na]+: 207.0189.
(m, 1H, Ar), 7.59–7.39 (m, 2H, Ar), 5.61 (s, 1H, H-1), 4.38
(s, 1H, OH), 2.21 (s, 3H, CH3); 13C NMR: d=205.3, 133.7,
133.3, 130.6, 130.5, 129.6, 125.1, 76.0, 25.6; HR-MS (ESI/Q-
TOF): m/z=196.0697, calcd. for C9H10NO4 [M+H]+:
196.0610.
(S)-1-(2-Bromophenyl)-1-hydroxypropan-2-one
(16a):
(S)-1-Hydroxy-1-(3-nitrophenyl)propan-2-one
(18b):
Colorless oil; yield: 72%; [a]D: +157.8 (c 0.32, CHCl3);
HPLC: Rt 12.4 min (S), 13.3 min (R); ee 89% (S); H NMR:
Yellow amorphous solid; yield: 85%; [a]D: +59.0 (c 0.80,
CHCl3); H NMR: d=8.30–8.24 (m, 2H, Ar), 7.70–7.66 (m,
1
1
d=7.71–7.48 (m, 1H, Ar), 7.45–7.26 (m, 1H, Ar), 7.25–7.14
(m, 2H, Ar), 5.60 (d, J=3.5 Hz, 1H, H-1), 4.38 (d, J=
3.50 Hz, 1H, OH), 2.14 (s, 3H, CH3); 13C NMR: d=206.3,
137.3, 133.4, 130.2, 128.9, 128.2, 123.7, 78.6, 25.5; HR-MS
(ESI/Q-TOF): m/z=227.9762, calcd. for C9H9BrO2 [M]+:
227.9786.
1H, Ar), 7.60–7.55 (m, 1H, Ar), 5.21 (s, 1H, H-1), 4.39 (bs,
1H, OH), 2.14 (s, 3H, CH3); 13C NMR: d=205.5, 140.0,
133.1, 130.1, 125.0, 123.7, 122.3, 79.2, 77.3, 25.2; HR-MS
(ESI/Q-TOF): m/z=196.0671, calcd. for C9H10NO4 [M+
H]+: 196.0610.
(S)-1-Hydroxy-1-(2-hydroxyphenyl)propan-2-one
(19a):
(S)-1-(3-Bromophenyl)-1-hydroxypropan-2-one
Yellow pale oil; yield: 71%; [a]D: +223.5 (c 0.90, CHCl3);
HPLC: Rt 10.1 min (S), 12.3 min (R); ee 78% (S); H NMR:
d=7.58–7.36 (m, 2H, Ar), 7.34–7.10 (m, 2H, Ar), 5.05 (s,
1H, H-1), 4.31 (s, 1H, OH), 2.10 (s, 3H, CH3); 13C NMR:
d=206.2, 140.1, 131.9, 130.5, 130.3, 125.9, 123.1, 79.4, 25.3;
HR-MS (ESI/Q-TOF): m/z=227.9806, calcd. for C9H9BrO2
[M]+: 227.9786.
(16b):
Colorless oil; yield: 86%; [a]D: +275.6 (c 0.64, MeOH);
HPLC: Rt 20.0 min (R), 27.2 min (S); ee 99% (S); H NMR:
1
1
d=7.34–7.13 (m, 2H, Ar), 6.98–6.90 (m. 1H, Ar), 6.88–6.75
(m, 1H, Ar), 5.22 (s, 1H, H-1), 2.11 (s, 3H, CH3). 13C NMR:
d=207.4, 154.9, 130.3, 129.4, 122.1, 120.9, 117.2, 78.7, 25.1.
HR-MS (ESI/Q-TOF): m/z=189.0601, calcd. for C9H10O3Na
[M+Na]+: 189.0528.
(S)-1-Hydroxy-1-(3-hydroxyphenyl)propan-2-one
(19b):
(S)-1-(4-Bromophenyl)-1-hydroxypropan-2-one
(16c):
White amorphous solid; yield: 89%; [a]D: +101.2 (c 0.68,
MeOH); HPLC: Rt 42.7 min (S), 45.0 min (R); ee 85% (S);
1H NMR: d=7.28–7.06 (m, 1H, Ar), 6.92–6.79 (m, 2H, Ar),
6.74 (dd, J=8.1, 1.0 Hz, 1H, Ar), 5.07 (s, 1H, H-1), 2.04 (s,
3H, CH3); 13C NMR: d=209.4, 158.9, 141.4, 130.8, 119.3,
116.3, 114.8, 81.1, 25.3; HR-MS (ESI/Q-TOF): m/z=
189.0433, calcd. for C9H10O3Na [M+Na]+: 189.0528.
Colorless oil;[3b] yield: 88%; [a]D: +170.5 (c 1.19, CHCl3);
1
HPLC: Rt 11.1 min (R), 11.7 min (S); ee 68% (S); H NMR:
d=7.52 (d, J=8.4 Hz, 2H, Ar), 7.21 (d, J=8.4 Hz, 2H, Ar),
5.05 (s, 1H, H-1), 4.28 (bs, 1H, OH), 2.08 (s, 3H, CH3);
13C NMR: d=206.5, 137.0, 132.3 (2C), 129.1 (2C), 122.9,
79.6, 25.3; HR-MS (ESI/Q-TOF): m/z=227.9711, calcd. for
C9H9BrO2 [M]+: 227.9786.
(S)-1-Hydroxy-1-(4-hydroxyphenyl)propan-2-one
(19c):
(S)-1-Hydroxy-1-(o-tolyl)propan-2-one (17a): Colorless
oil; yield: 90% yield; [a]D: +173.0 (c 0.30, CHCl3), lit. for
(R)-enantiomer:[32d] À363 (c 0.25, CHCl3); GC (temperature
program from 100 to 2008C rate 58C minÀ1): Rt 14.1 min
White amorphous solid; yield: 44%; [a]D: +131.2 (c 0.42,
MeOH); HPLC: Rt 27.4 min (R), 31.9 min (S); ee 99% (S);
1H NMR (CD3OD): d=7.19 (d, J=8.6 Hz, 2H, Ar), 6.78 (d,
J=8.6 Hz, 2H, Ar), 5.07 (s, 1H, H.1), 3.31 (s, 1H, OH), 2.01
(s, 3H, CH3); 13C NMR (CD3OD): d=209.7, 158.9, 130.6,
129.6 (2C), 116.6 (2C), 80.7, 25.4; HR-MS (ESI/Q-TOF): m/
z=189.0645, calcd. for C9H10O3Na [M+Na]+: 189.0528.
1
(S), 14.2 min (R); ee 97% (S); H NMR: d=7.24–7.08 (m,
4H, Ar), 5.26 (d, J=3.8 Hz, 1H, H-1), 4.16 (d, J=3.8 Hz,
1H, OH), 2.40 (s, 3H, CH3), 2.05 (s, 3H, CH3); 13C NMR:
d=207.6, 136.4, 135.9, 131.3, 128.7, 128.3, 126.6, 78.1, 25.4,
19.3; HR-MS (ESI/Q-TOF): m/z=164.0912, calcd. for
C10H12O2 [M]+: 164.0837.
(S)-1-Hydroxy-1-(naphthalen-1-yl)propan-2-one
(25):
Yellow amorphous solid; yield: 68%; [a]D: +284.3 (c 1.30,
CHCl3); HPLC: Rt 10.1 min (S), 10.8 min (R); ee 99% (S);
1H NMR: d=8.10–7.95 (m, 1H, Ar), 7.90–7.80 (m, 2H, Ar),
7.60–7.40 (m, 4H, Ar), 5.61 (s, 1H, H-1), 2.05 (s, 3H, CH3);
(S)-1-Hydroxy-1-(m-tolyl)propan-2-one (17b): Yellow
pale oil; yield: 88%; [a]D: +351.1 (c 0.88, CHCl3); GC (tem-
Adv. Synth. Catal. 0000, 000, 0 – 0
8
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13C NMR: d=208.3, 134.3, 133.3, 131.1, 129.7, 129.0, 127.7,
126.9, 126.1, 125.4, 123.4, 79.3, 25.5; HR-MS (ESI/Q-TOF):
m/z=223.0829, calcd. for C13H12O2Na [M+Na]+: 223.0735.
(ESI/Q-TOF): m/z=164.0801, calcd. for C10H12O2 [M]+:
164.0837.
(S)-1-Hydroxy-1-(naphthalen-2-yl)propan-2-one
(26):
White solid; yield: 58%; [a]D: +308.5 (c 1.00, CHCl3), lit.
for (R)-enantiomer:[32d] À101 (c 0.08, CHCl3); HPLC: Rt
Acknowledgements
1
19.5 min (S), 22.7 min (R); ee 92% (S). H NMR: d=8.01–
7.72 (m, 4H, Ar), 7.61–7.43 (m, 2H, Ar), 7.37 (dd, J=
8.5 Hz, J=1.8, 1H, Ar), 5.26 (s, 1H, H-1), 4.42 (bs, 1H,
OH), 2.11 (s, 3H, CH3); 13C NMR: d=207.1, 135.2, 133.3,
133.2, 128.9, 127.9, 127.7, 127.10, 126.5, 126.4, 124.1, 80.2,
25.3; HR-MS (ESI/Q-TOF): m/z=223.0612, calcd. for
C13H12O2Na [M+Na]+: 223.0735.
We gratefully acknowledge the University of Ferrara (fondi
FAR) and the Deutsche Forschungsgemeinschaft (FOR1296)
for financial support. Thanks are also given to Mr Paolo For-
maglio for NMR experiments and to Mrs Tatiana Bernardi
for HR-MS analyses.
(S)-1-(4-Ethylphenyl)-1-hydroxypropan-2-one (27): Color-
less oil; yield: 80%; [a]D: +326.0 (c 1.00, CHCl3); GC (tem-
perature program from 100 to 2008C rate 18C minÀ1): Rt
1
References
45.4 min (R), 47.5 min (S); ee 87% (S); H NMR: d=7.26–
7.19 (m, 4H, Ar), 5.07 (s, 1H, H-1), 4.25 (bs, 1H, OH), 2.64
(q, J=7.6 Hz, 2H, CH2), 2.09 (s, 3H, CH3), 1.23 (t, J=
7.6 Hz, 3H, CH2CH3); 13C NMR: d=207.3, 144.9, 135.1,
128.5 (2C), 127.3 (2C), 79.9, 28.5, 25.3, 15.5; HR-MS (ESI/
Q-TOF): m/z=223.0628, calcd. for C11H14O2Na [M+Na]+:
223.0735.
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(S)-1-(4-(tert-Butyl)phenyl)-1-hydroxypropan-2-one (28):
Colorless oil; yield: 75%; [a]D: +195.8 (c 1.70, CHCl3); GC
(temperature program from 100 to 2008C, rate 18C minÀ1):
Rt 56.4 min (R), 57.9 min (S); ee 96% (S); 1H NMR d=
7.54–7.30 (m, 2H, Ar), 7.34–7.07 (m, 2H, Ar), 5.07 (s, 1H,
H-1), 4.23 (bs, 1H, OH), 2.09 (s, 3H, CH3), 1.31 (s, 9H,
C(CH3)3); 13C NMR: d=207.3, 151.8, 134.9, 127.0 (2C),
125.9 (2C), 79.8, 34.6, 31.3, 25.4; HR-MS (ESI/Q-TOF):
m/z=229.1098, calcd. for C13H18O2Na [M+Na]+: 229.1204.
(S)-1-([1,1’-Biphenyl]-4-yl)-1-hydroxypropan-2-one (29):
Yellow amorphous solid; yield: 82%; [a]D: +267.1 (c 2.10,
CHCl3); HPLC: Rt 29.7 min (S), 34.7 min (R); ee 91% (S);
1H NMR: d=7.71–7.50 (m, 4H, Ar), 7.54–7.28 (m, 5H, Ar),
5.15 (s, 1H, H-1), 4.32 (bs, 1H, OH), 2.14 (s, 3H, CH3);
13C NMR: d=207.0, 141.7, 140.4, 136.9, 128.8 (2C), 127.8
(2C), 127.7 (2C), 127.6, 127.1 (2C), 79.9, 25.4; HR-MS
(ESI/Q-TOF): m/z=226.0727, calcd. for C15H14O2 [M]+:
226.0994.
[6] O. Onomura, H. Arimoto, Y. Matsumura, Y. Demizu,
Tetrahedron Lett. 2007, 48, 8668–8672.
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Shoji, Angew. Chem. 2004, 116, 1132–1135; Angew.
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(S)-1-Hydroxy-1-phenylbutan-2-one (31)
Lyophilized Ao:DCPIP OR (12 mg) was added to a solution
of benzaldehyde 5 (0.50 mmol) and 3,4-hexandione (30)
(121 mL, 1.0 mmol), ThDP (4.5 mg, 10 mmol) and MgSO4
(2.7 mg, 20 mmol) in 50 mM phosphate buffer at pH 6.5
(25 mL) containing DMSO (10% v/v). The reaction mixture
was gently shaken at 308C for 48 h and then extracted with
ethyl acetate (3ꢄ10 mL). The combined extracts were dried
with anhydrous Na2SO4 and evaporated under reduced pres-
sure. After chromatography on silica gel with cyclohexane-
ethyl acetate (4:1) as eluent, the product 31 was obtained as
a colorless oil; yield: 60%; [a]D: +254 (c 0.3, CHCl3),
lit.:[32e] +389.8 (c 2.06, CHCl3); GC (temperature program
from 100 to 2008C rate 28C minÀ1): Rt 19.0 min (R),
1
19.1 min (S); ee 98% (S); H NMR: d=7.45–7.25 (m, 5H,
Ar), 5.10 (s, 1H, H-1), 4.35 (bs, 1H, OH), 2.40–2.15 (m, 2H,
CH2), 1.01 (t, 3H, J=7.0 Hz, CH3); 13C NMR: d=210.1,
138.2, 129.1 (2C), 128.7, 127.4 (2C), 79.5, 31.2, 7.7; HR-MS
Adv. Synth. Catal. 0000, 000, 0 – 0
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FULL PAPERS
asc.wiley-vch.de
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preparative reaction with the aim to fully characterize
this derivative and determine its GC behavior. The rac-
emic form of 7 was confirmed by chiral phase GC-MS
analysis that showed two peaks (retention times: 6.48
and 6.64 min) with the same area and MS (see the Sup-
porting Information).
[27] Taking advantage of the reverse activity of AO:DCPIP
OR [Scheme 3, Eq. (b)], the synthesis of 14c was at-
tempted using the (S)-PAC (6) as donor and 4-fluoro-
benzaldehyde (8c; 5 equiv.) as acceptor. The expected
PAC derivative 14c was obtained after 24 h in 63%
conversion and 90% ee (for the experimental details
see the Supporting Information and Figure S3). The
use of 6 as donor in cross-benzoin-type reactions is cur-
rently under investigation in our laboratories and it will
be the subject of a forthcoming publication.
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FULL PAPERS
(S)-Selectivity in Phenylacetyl Carbinol Synthesis Using the
Wild-Type Enzyme Acetoin:Dichlorophenolindophenol
Oxidoreductase from Bacillus licheniformis
11
Adv. Synth. Catal. 2016, 358, 1 – 11
Pier Paolo Giovannini,* Lindomar Alberto Lerin,
Michael Mꢀller, Giovanni Bernacchia, Morena De Bastiani,
Martina Catani, Graziano Di Carmine, Alessandro Massi*
Adv. Synth. Catal. 0000, 000, 0 – 0
11
ꢃ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÞÞ
These are not the final page numbers!