Enzymatic Cross-Benzoin-Type Condensation of Aliphatic Aldehydes: Enantioselective Synthesis of 1-Alkyl-1-hydroxypropan-2-ones and 1-Alkyl-1-hydroxybutan-2-ones

Article abstract of DOI:10.1002/adsc.201800357

Benzoin-type reactions have been intensively exploited as a synthetic strategy for the preparation of α-hydroxy ketones. Thiamine diphosphate (ThDP) dependent enzymes are excellent catalysts for asymmetric versions of such reaction types. In particular, in cross-benzoin condensations of aromatic reactants and mixed aromatic/aliphatic reactions, use of these enzymes has resulted in high levels of chemo-, regio- and stereoselectivity. The present work, which confirms this trend for aliphatic reactants, outlines results obtained in the formal cross-benzoin-type condensation of the ‘umpoled’ acetaldehyde and propionaldehyde with various aliphatic aldehydes catalyzed by the ThDP-dependent enzyme acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR). In these reactions, 3-hydroxy-3-methylbutan-2-one (methylacetoin) was used as the activated acetaldehyde donor, while 4-hydroxy-4-methylhexan-3-one was employed for the first time as the precursor of activated propionaldehyde. With the exception of 3-hydroxypentan-2-one and 3-hydroxyhexan-2-one, which were obtained in almost racemic form by the condensation of methylacetoin with propanal and butanal, respectively, all other products achieved from reactions performed using the same donor with more hindered aldehyde acceptors were obtained with high conversions (89–99%) and in good to high enantiomeric excess (72–99% ee). In a similar way, high conversions (75–99%) and good ee (76–99%) were observed in reactions performed with the same set of aldehyde acceptors, but using 4-hydroxy-4-methylhexan-3-one as propionyl anion donor. This is the first time that most of the products described herein have been prepared via benzoin-type condensation. (Figure presented.).

Full text of DOI:10.1002/adsc.201800357

Title: Enzymatic Cross-Benzoin-Type Condensation of Aliphatic
Aldehydes: Enantioselective Synthesis of 1-Alkyl-1-
hydroxypropan-2-ones and 1-Alkyl-1-hydroxybutan-2-ones
Authors: Graziano Di Carmine, Olga Bortolini, Alessandro Massi,
Michael Müller, Giovanni Bernacchia, Giancarlo Fantin,
Daniele Ragno, and Pier Paolo Giovannini
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201800357
Link to VoR: http://dx.doi.org/10.1002/adsc.201800357

10.1002/adsc.201800357
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Enzymatic Cross-Benzoin-Type Condensation of Aliphatic
Aldehydes: Enantioselective Synthesis of 1-Alkyl-1-
hydroxypropan-2-ones and 1-Alkyl-1-hydroxybutan-2-ones
Graziano Di Carmine,a Olga Bortolini,a Alessandro Massi,a Michael Müller,b Giovanni
Bernacchia,c Giancarlo Fantin,a Daniele Ragno,a and Pier Paolo Giovanninia,*
a
Dipartimento di Scienze Chimiche e Farmaceutiche, Università di Ferrara, Via Fossato di Mortara 17, 44121 Ferrara,
Italy
Fax: (+39) 0532 240709; e-mail: pierpaolo.giovannini@unife.it
Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104 Freiburg, Germany
Dipartimento di Scienze della Vita e Biotecnologie, Università di Ferrara, Via L. Borsari 46, 44121 Ferrara, Italy
b
c
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.((Please
delete if not appropriate))
Abstract. Benzoin-type reactions have been intensively With the exception of 3-hydroxypentan-2-one and 3-
exploited as a synthetic strategy for the preparation of - hydroxyhexan-2-one, which were obtained in almost
hydroxy ketones. Thiamine diphosphate (ThDP) dependent racemic form by the condensation of methylacetoin with
enzymes are excellent catalysts for asymmetric versions of propanal and butanal, respectively, all other products
such reaction types. In particular, in cross-benzoin achieved from reactions performed using the same donor
condensations of aromatic reactants and mixed with more hindered aldehyde acceptors were obtained with
aromatic/aliphatic reactions, use of these enzymes has high conversions (89–99%) and in good to high
resulted in high levels of chemo-, regio- and enantiomeric excess (72–99% ee). In a similar way, high
stereoselectivity. The present work, which confirms this conversions (75–99%) and good ee (76–99%) were
trend for aliphatic reactants, outlines results obtained in the observed in reactions performed with the same set of
formal cross-benzoin-type condensation of the ‘umpoled’ aldehyde acceptors, but using 4-hydroxy-4-methylhexan-3-
acetaldehyde and propionaldehyde with various aliphatic one as propionyl anion donor. This is the first time that
aldehydes catalyzed by the ThDP-dependent enzyme most of the products described herein have been prepared
acetoin:dichlorophenolindophenol
oxidoreductase via benzoin-type condensation.
(Ao:DCPIP OR). In these reactions, 3-hydroxy-3-
methylbutan-2-one (methylacetoin) was used as the activated
acetaldehyde donor, while 4-hydroxy-4-methylhexan-3-one
was employed for the first time as the precursor of activated
propionaldehyde.
Keywords: asymmetric synthesis; C–C coupling; enzyme
catalysis; thiamine diphosphate; umpolung
umpolung coupling of aldehydes (benzoin-type
condensation) can be added to this list of
methodologies.
Introduction
The development of efficient asymmetric synthetic
methodologies is of fundamental importance for the
production of chiral bioactive compounds.[1] Huge
efforts in this area are devoted to the production of
enantiopure building blocks, the availability of which
allows for the directed control of the stereochemical
course of complex synthetic pathways.[2] Many
bioactive molecules contain an -hydroxy ketone
motif, in which the configuration of the carbinolic
center is often critical for the biological activity.[3]
Thus, the development of new catalytic strategies for
the asymmetric synthesis of -hydroxy ketones is of
high interest.[4] Significant results in this area have
been achieved through the -oxidation of ketones[5]
or of their enolate and enol ether derivatives.[6]
Alternative approaches are the ketohydroxylation of
olefins,[7] and the monooxidation of diols.[8]
Furthermore, thanks to the recent development of
chiral N-heterocyclic carbene (NHC) catalysts,[9] the
Concurrently with the introduction of these
chemocatalytic strategies, several biocatalytic
methodologies
have
been
developed.[3a]
Enantioenriched -hydroxy ketones have been
obtained via the kinetic resolution of their racemates
by enantioselective acylation catalyzed by lipases.[10]
Alternative enzymatic
monoreduction of
approaches
-diketones
are
and
the
the
monooxidation of vicinal diols, both catalyzed in a
stereoselective manner by NAD(P)-dependent
dehydrogenases.[11] Also worthy of mention is the
unique example of the enantioselective -oxidation
of ketones promoted by a cytochrome P450
enzyme.[12]
An alternative, straightforward biocatalytic
approach is offered by catalysis using thiamine
diphosphate (ThDP) dependent lyases. Thanks to
their tightly bound cofactor ThDP, the enzymes of
this family catalyze benzoin-type condensations with
1
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10.1002/adsc.201800357
Advanced Synthesis & Catalysis
O
the same umpolung mechanism as the above-noted
NHC catalysts, often with a high level of enantio-
selectivity.[13] Furthermore, while the NHC catalysts
need at least one aromatic aldehyde in order to direct
the chemo- and regioselectivity of the cross-benzoin
condensation,[9] various ThDP-dependent lyases are
known to efficiently catalyze the chemo- and
stereoselective formation of fully aliphatic
acyloins.[14] Scheme 1 shows generic structures of the
more significant products achieved via these
enzymatic approaches. Although the efficiency of
acetaldehyde and propionaldehyde as both donor and
acceptor has been demonstrated in ThDP-dependent
enzyme-catalyzed homo-benzoin-type reactions,[15]
there are only rare examples of cross-benzoin-type
reactions involving these aldehydes or their -keto
acids precursors (pyruvate and 2-oxobutyrate,
respectively) as donors with aliphatic acceptors and
few of the resulting products have been
characterized.[16]
HO
R
O
R
OH
OH
O
O
HO
-
CO2
(Ref. 14a-c)
(Ref. 14d)
O
-
O
HO2C
CO2
O
R
(Ref. 14e,f)
R = alkyl group
O
HO2C
R
OH
O
R1
Ph
-
R1
CO2
(Ref. 14g)
OH
R1 = Me or Et
(this work)
O
Ph
R
OH
O
R
R1
OH
We herein report the results obtained in benzoin-
type condensations of 3-hydroxy-3-methylbutan-2-
one (Scheme 1, R1 = Me) and 4-hydroxy-4-
methylhexan-3-one (Scheme 1, R1 = Et), which act as
Scheme 1. Generic structures of the main aliphatic
acyloins achievable via ThDP-dependent enzyme catalysis.
precursors
of
activated
acetaldehyde
and
The activity of the Ao:DCPIP OR–methylacetoin
enzyme–substrate system was tested on analytical
scale reactions (1.5 mL reaction volume), using the
linear aliphatic aldehydes propanal and butanal (7a
and 7b, Table 1) as acceptors. By employing reaction
conditions similar to those adopted for the synthesis
of the PAC analogues 6[18] [methylacetoin (1; 30
mM), 7a or 7b (20 mM), phosphate buffer pH 6.5 (50
mM), MgSO4 (0.9 mM), ThDP (0.4 mM), purified
and lyophilized Ao:DCPIP OR (0.5 mg mL–1)], after
48 hours at 30 °C we observed the formation of the
expected products 8a and 8b with complete and 57%
conversion, respectively (Table 1, entries 1 and 2). 1H
NMR analyses of the reaction mixtures did not reveal
the presence of products arising from the homo-
coupling of aldehydes 7a or 7b. The non-occurrence
of such a reaction was further confirmed by
experiments performed without donor 1. These
results agree with those reported in previous synthetic
applications of Ao:DCPIP OR,[18] namely that
although aldehydes are good acceptors, they cannot
act as acyl-anion donors since this enzyme needs -
hydroxy ketones or -diketones for this role. Chiral-
phase GC analyses of crude 8a and 8b indicated low
ee for both products (43% and 29%, respectively).
Previous studies on the synthesis of the same
products, conducted using PDC as catalyst and
pyruvate as donor, reported low conversions for
8a[16b] and 8b[16a] formation (41% and 2%,
respectively) and did not provide information about
the optical purity of the products. The results
achieved with the short-chain linear aldehydes 7a and
7b were encouraging concerning the chemo- and
regioselectivity of the enzymatic approach. Hence,
we investigated the activity of Ao:DCPIP OR in the
condensation of methylacetoin (1) with more
hindered acceptors, such as the -branched aliphatic
and alicyclic aldehydes 7c–g (Table 2). Gratifyingly,
propionaldehyde, respectively, with various aliphatic
aldehydes as acceptors catalyzed by the ThDP-
dependent enzyme acetoin:dichlorophenolindophenol
oxidoreductase (Ao:DCPIP OR).
Results and Discussion
We recently highlighted the synthetic value of the
substrate–enzyme pair constituted by methylacetoin
(1, Scheme 2) and Ao:DCPIP OR from Bacillus
licheniformis by demonstrating its efficiency in the
enantioselective synthesis of tertiary acyloins 4 and
phenylacetylcarbinol (PAC) analogues 6 through the
addition of the activated acetaldehyde 2, derived from
1, to various activated methyl ketones 3 (Scheme 2,
route a)[17] and aromatic aldehydes 5 (Scheme 2, route
b),[18] respectively. Most of the resulting products
were highly enantioenriched. Noteworthy, reactions
with the aromatic aldehydes afforded the (S)-
enantiomers of the PAC analogues, a behavior not
observed with other wild-type ThDP-dependent
enzymes.[19]
These encouraging results prompted us to
investigate the activity of Ao:DCPIP OR in benzoin-
type reactions between donor 1 and various aliphatic
aldehydes 7 as acceptors (Scheme 2, route c). In fact,
apart from acetoin, which represents a case
study,[13a,b] few other -hydroxy ketones of type 8
(Scheme 2, route c) have been produced exploiting
the ThDP-dependent enzyme pyruvate decarboxylase
(PDC). Furthermore, the conversion levels were low
and, for most products, the enantiomeric excess (ee)
was not determined.[16]
2
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10.1002/adsc.201800357
Advanced Synthesis & Catalysis
in a preliminary study conducted on an analytical
scale (1.5 mL reaction volume) with different donor
to acceptor molar ratios,
substrates. By virtue of their protected alcoholic and
aldehydic functionalities, the expected products 8h
and 8i (Table
all the aldehydes 7c–g were accepted as substrates
by Ao:DCPIP OR.
Table 1. Ao:DCPIP OR catalyzed synthesis of 1-alkyl-1-
hydroxypropan-2-ones 8a–j using methylacetoin (1) as
donor.a)
O
Conversion
[%]b)
(yield [%])c)
Product
Acceptor
ee
[%]d)
Entry
EWG
route a)
OH
Ref 17
11 examples
4
O
EWG
OH
O
1e)
99
43
29
99
3
route b)
O
(n.d.)f)
O
7a
8a
Ao:DCPIP OR,
RII
O
O
R
5
ThDP
R
OH
O
2e)
57
(n.d.)f)
N
S
RI
OH
OH
O
O
7b
HO
6
Ref 18
24 examples
1
8b
2
OH
O
99
(49)
O
3
4
O
O
Alkyl
Alkyl
7c
8c
7
OH
OH
O
route c)
99(S)
99
(42)
this work
8
O
8d
Scheme 2. Ao:DCPIP OR catalyzed benzoin-type
condensation of methylacetoin (1) with activated ketones
(route a), aromatic aldehydes (route b) and aliphatic
aldehydes (route c). RI = (4-amino-2-methylpyrimidin-5-
yl)methyl, RII = 2-diphosphate ethyl.
7d
OH
O
72
86
99
(81)
5
6
O
8e
7e
OH
O
89
(82)
A slight excess of the donor (1.5 equivalents) was
sufficient to obtain almost complete conversion of the
acceptors (see Supporting Information). Products 8c–
g have not been obtained previously via ThDP-
dependent enzyme-catalyzed benzoin-type reactions
(aldehydes 7e–g have been used as acceptors in
condensations with hydroxypyruvate catalyzed by
engineered transketolases).[14b] Reactions repeated on
a preparative scale (Table 1, entries 3–7) confirmed
the high conversion levels (89–99%) observed on the
smaller scale. After purification by column
chromatography, products 8e–g were obtained in high
yields (81–89%), while part of products 8c and 8d
was lost during the purification procedure, probably
due to their high volatility (49% and 42% isolated
yield for 8c and 8d, respectively). Attempts to use
volatile solvents like pentane, diethyl ether,
chloroform or dichloromethane did not afford
appreciable results in terms of separation and
recovery of the products as well. Unlike the short-
chain linear products 8a and 8b, acyloins 8c–g were
highly enantioenriched, with ee values ranging from
72% to 99%.
O
7f
8f
OH
O
99
(89)
92(S)
7
8
O
8g
7g
72(S)g)
OH
O
99
(92)
BnO
MeO
BnO
O
7h
8h
OH
O
n.d.f)
99
(61)
MeO
9
MeO
O
MeO
7i
8i
O
O
HO
99
(93)
84(S)g)
10
N
Boc
N
Boc
8j
7j
a)
Conditions: reaction volume 25 mL, 1 (30 mM),
aldehyde 7 (20 mM), phosphate buffer pH 6.5 (50 mM),
These results indicate that the -branched
structure of the substrates does not limit their access
to the enzyme active site, but orientates them within
this site, thus ensuring highly stereoselective attack of
the activated acetaldehyde 2. To assess the robustness
of the enzymatic procedure, the -oxygenated
aldehydes 7h and 7i were employed as acceptor
MgSO4 (0.9 mM), ThDP (0.4 mM), purified and
lyophilized Ao:DCPIP OR (0.5 mg mL–1). Determined
b)
by 1H NMR analysis. c) Refers to the isolated product after
d)
column chromatography.
GC or HPLC analysis.
Determined by chiral-phase
Reaction performed on an
e)
3
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10.1002/adsc.201800357
Advanced Synthesis & Catalysis
f)
g)
analytical scale only.
Not determined.
Absolute
The successful results thus far were thanks to the
employment of the non-natural donor 1 whose higher
potential in carboligation reactions, with respect to
the natural substrate acetoin, is due to the release of
unreactive acetone during formation of the activated
acetaldehyde 2 (Scheme 2).[17] On the other hand, as
we have demonstrated previously, Ao:DCPIP OR is
also able to catalyze the cleavage of hexane-3,4-dione
(9) and addition of the resulting activated
propionaldehyde 10 to various activated ketones[17]
and benzaldehydes[18] (Scheme 3, route a). The
synthetic efficiency of 9 as a donor, however, is
negatively affected by the unavoidable formation of
byproduct 11 (homo-coupling of 9), which
necessitates the use of an excess of the donor with the
configuration determined by 1H-NMR analysis of the
Mosher’s esters derivatives (see Supporting Information).
1, entries 8 and 9) are interesting building blocks for
asymmetric synthesis.
The two aldehydes 7h and 7i have been used as
donor and acceptor, respectively, in a cross-benzoin-
type reaction catalyzed by benzaldehyde lyase;[20]
however, neither has been used as an acceptor of the
activated acetaldehyde 2.
Gratifyingly, the reactions conducted with donor
1 and Ao:DCPIP OR as catalyst afforded the
corresponding products 8h and 8i with almost
complete conversion and a satisfactory 72% ee for 8h. consequent complication of product purification.
Likewise what described in previous work[14d] for
Table 2. Ao:DCPIP OR catalyzed synthesis of 1-alkyl-1-
hydroxybutan-2-ones 13c–j using 12 as donor.a)
analogous products, also for 8i a low isolated yield
(34%) was achieved after chromatography on
conventional silica gel. This drawback, probably due
to the low stability of the acetal group on the acidic
stationary phase, has been overcome by performing
the chromatographic purification on neutral silica gel.
Under this conditions a considerably higher yield of
61% was obtained (Table 1, entry 9). Finally, the
tolerance of the procedure for bulky substrates was
confirmed by the use of 1-Boc-substituted piperidine-
4-carboxaldehyde 7j as acceptor. In this case, the
enzymatic reaction provided the expected product 8j
in high isolated yield (93%) and good ee (84%).
Conversion
[%]b)
(yield [%])c)
Product
ee
[%]d)
Acceptor
Entry
OH
O
O
1
90
99
(52)
7c
13c
OH
O
99(S)
n.d.e)
95
(45)
2
3
7d
O
13d
13e
13f
OH
O
83
(70)
O
O
O
X
OH
O
7e
Y
+
O
OH
O
OH
O
4' (X = Me, Y = EWG)17
6' (X = H, Y = Ph)18
9
78
90
(82)
4
5
6
11
O
7f
OH
OH
O
O
Y
O
85
(78)
59(S)
O
X
13g
13h
13i
7g
route a)
RII
OH
O
75
(68)
92(S)f)
BnO
MeO
BnO
N
S
RI
MeMgBr
THF
7h
O
HO
route b)
OH
O
n.d.e)
10
99
(62)
O
MeO
MeO
7
8
MeO
O
6
7i
O
O
HO
99
(91)
76
O
O
O
this work
N
Boc
N
OH
13j
7j
OH
13f
Boc
a)
12
Conditions: reaction volume 25 mL, 12 (30 mM),
aldehyde 7 (20 mM), phosphate buffer pH 6.5 (50 mM),
MgSO4 (0.9 mM), ThDP (0.4 mM), purified and
Scheme 3. Synthesis and exploitation of 4-hydroxy-4-
methylhexan-3-one (12) as a propionyl anion donor
alternative to hexane-3,4-dione (9). RI = (4-amino-2-
methylpyrimidin-5-yl)methyl, RII = 2-diphosphate ethyl.
b)
lyophilized Ao:DCPIP OR (0.5 mg mL–1). Determined
by 1H NMR analysis. c) Refers to the isolated product after
d)
column chromatography.
Determined by chiral-phase
GC or HPLC analysis, apart from 13g.[21] e) Not determined.
Absolute configuration determined by H-NMR analysis
f)
1
of the Mosher’s esters derivatives (see Supporting
Information).
4
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10.1002/adsc.201800357
Advanced Synthesis & Catalysis
R
Searching for a more efficient propionyl anion donor
reminiscent of the structure of 1, we aimed to convert
diketone 9 into 4-hydroxy-4-methylhexan-3-one (12)
through reaction with a methyl Grignard reagent. In
the event, the addition of methylmagnesium bromide
(1.2 equivalent) to 9, followed by refluxing of the
resulting mixture (2 h) and aqueous workup, afforded
the expected racemic product 12. Under these
conditions we did not observe the formation of the
diol and the product 12 was purified by distillation
(48% yield). The so-obtained acyloin 12 was tested as
donor (1.5 equivalents) in the model reaction with
acceptor 7f (Scheme 3, route b). Gratifyingly, this
afforded a 90% conversion and the expected product
13f was enantioenriched (78% ee) (Table 2, entry 4).
Noteworthy, in forming the activated aldehyde 10,
Ao:DCPIP OR catalyzes the cleavage of 12 with
comparable rates for both enantiomers, as
demonstrated by chiral-phase GC analysis of the
residual donor in the reaction mixture (see Supporting
OH
O
S
S
S
S
R
OH
8
14
1) n-BuLi; THF
2) aldehydes 7
16
HgO/HBF4
EtOH
R
OH
S
S
O
S
S
R
OH
17
13
15
Scheme 4. Synthesis of racemic samples of products
8c,d,g,h,j and 13c,d,f,h,j.
Finally, preparation of racemic samples to assess
the optical purity of the enzymatic products by chiral-
phase chromatographic analysis was required. Initial
attempts to racemize the optically active products by
acidic or basic treatment afforded the expected
samples only for products 8e and 8f, albeit with very
low conversions.[27] Therefore, most of the acyloins
were synthesized in their racemic form by addition of
the carbanions generated from 2-methyl-1,3-
benzodithiole (14) and 2-ethyl-1,3-benzodithiole
(15)[28] to the aldehyde acceptors 7, followed by
hydrolysis of the resulting adducts[29] 16 and 17, as
depicted in Scheme 4 (see Supporting Information).
1
Information). Additionally, H NMR analysis of the
same mixture excluded the presence of byproducts
derived from donor homo-coupling. These features,
added to the complete non-reactivity of the methyl
ethyl ketone released upon donor cleavage,[22]
contribute to increasing the synthetic value of 12 as
propionyl anion donor and encouraged us to extend
the methodology to the complete set of acceptors 7c–
j successfully used with donor 1. As observed for the
reactions with methylacetoin (1), a study conducted
with different donor 12 to acceptor 7f molar ratios
(see Supporting Information) indicated that the best
conversion and optical purity of product 13f were
achieved using 1.5 equivalents of donor. Hence, these
conditions were adopted in all reactions performed on
a preparative scale (25 mL) with donor 12 and
acceptor substrates 7c–j, the results of which are
summarized in Table 2. The conversions and yield
and ee values of products 13c–j are comparable with
the results obtained for the lower homologues 8c–j;
therefore, 12 is a suitable propionyl anion donor in
reactions catalyzed by Ao:DCPIP OR. The absolute
configuration of products 8d,[23] 8g,[24] 8h, 13d[25]
13g[21] and 13h show that, with acceptors 7d, 7g or
7h, the enzyme is (S)-stereoselective independently
from the use of 1 or 12 as donor.[26] These results are
congruent with those we previously reported using
aromatic aldehydes as acceptors.[18]
Conclusion
We have demonstrated that the benzoin-type coupling
of methylacetoin (1) with -substituted aliphatic
aldehydes catalyzed by the ThDP-dependent enzyme
Ao:DCPIP OR is a valuable synthetic strategy to
produce a broad range of enantioenriched 1-alkyl-1-
hydroxypropan-2-ones 8c–j. Most of these products
have not been synthesized previously via enzymatic
or
organocatalytic
benzoin-type
reactions.
Additionally, we have disclosed the unprecedented
use of 4-hydroxy-4-methylhexan-3-one (12) as
propionyl anion donor that has allowed us to
significantly extend the range of optically active
aliphatic acyloins obtainable through Ao:DCPIP OR
catalysis. Surprisingly, this enzyme is sterically
unselective in the cleavage of the racemic compound
12, while being highly stereoselective in the addition
of the resulting activated propionaldehyde 10 to the
-substituted aliphatic aldehydes. The results
obtained with both donors 1 and 12 seem to confirm
the (S)-enantioselectivity observed in previous
biocatalytic exploitations of Ao:DCPIP OR.[17,18]
Additionally, since neither 1 and 12 nor the ketones
released from their cleavage (acetone and methyl
ethyl ketone, respectively) act as acceptors,
methylacetoin (1) and 4-hydroxy-4-methylhexan-3-
one (12) become valuable alternatives to pyruvate
and 2-oxobutyrate, respectively, in ThDP catalysis.
Our results provide further evidence for the general
5
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10.1002/adsc.201800357
Advanced Synthesis & Catalysis
1
min–1): tR (min) = 23.8 (minor), 25.1 (major); 99% ee; H
NMR (300 MHz):  = 4.24 (s, 1H, H-3), 3.38 (bs, 1H, OH),
2.19 (s, 3H, CH3), 1.69–1.36 (m, 3H), 1.30–1.06 (m, 2H),
applicability of what can be defined as the
‘methylacetoin approach’ which can be successfully
employed to catalyze the asymmetric addition of
activated acetaldehyde and propionaldehyde to the
carbonyl group of various kinds of acceptors, namely
1.01 (t, J = 7.2 Hz, 3H, CH3), 0.89–0.79 (m, 3H, CH3); 13
NMR (101 MHz):  = 210.9, 78.3, 44.7, 25.4, 23.3, 21.4,
12.2, 12.0; HRMS (ESI/Q-TOF): m/z = 167.1048, calcd for
C8H16NaO2 [M+Na]+: 167.1060.
C
activated
ketones,
variously
substituted
benzaldehydes and, as here demonstrated, also -
substituted aliphatic aldehydes.
(S)-3-Hydroxy-4,4-dimethylpentan-2-one [(S)-8d]
Column chromatography with cyclohexane/EtOAc 7:3
afforded 8d as a colorless oil, 42% yield. []D +93 (c 1.0,
CHCl3), lit. for (S)-enantiomer +133.2 (c 1.0, CHCl3);[23]
GC (temperature program: 80 to 200 °C, rate 2 °C min–1):
tR (min) = 16.7 (R), 17.8 (S); 99% ee; 1H NMR (300 MHz):
 = 3.88 (d, J = 6.3 Hz, 1H, H-3), 3.25 (d, J = 6.3 Hz, 1H,
OH), 2.24 (s, 3H, CH3), 0.99 (s, 9H); 13C NMR (101
MHz):  = 211.0, 84.5, 35.5, 29.4, 26.3; HRMS (ESI/Q-
TOF): m/z = 153.0891, calcd for C7H14NaO2 [M+Na]+:
153.0879.
Experimental Section
General Methods
All commercially available reagents were used as received
without further purification, unless otherwise stated.
Liquid aldehydes were freshly distilled before use.
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
1-Cyclopropyl-1-hydroxypropan-2-one (8e)
1
60 (230–400 mesh) or on Florisil (60–100 mesh). H and
Column chromatography with cyclohexane/EtOAc 8:2
afforded 8e as a colorless oil, 81% yield. []D +112 (c 1.0,
CHCl3); GC (temperature program: 80 to 200 °C, rate 2 °C
13C NMR spectra were recorded on 300 and 400 MHz
spectrometers at room temperature using CDCl3 as solvent.
Chemical shifts () are reported in ppm relative to residual
solvent signals. High-resolution mass spectra (HRMS)
were recorded in positive ion mode with an Agilent 6520
HPLC-Chip Q/TOF-MS nanospray system using a time-of-
flight, quadrupole or hexapole unit to produce spectra.
Optical rotations were measured at 20 ± 2 °C in the stated
solvent; []D values are given in 10–1 deg cm2 g–1. The
enantiomeric excess (ee) of products was determined by
chiral-phase HPLC or GC analysis. For the HPLC analyses,
a Phenomenex Amylose-2 Lux (250 × 4.6 mm, 5 m
particle size) or a Phenomenex Lux Cellulose-1 (250 × 4.6
mm, 5 m particle size) column was used, together with a
UV detector operating at 254 nm. GC analyses were
performed using a flame ionization detector and a
Megadex 5 column (25 m × 0.25 mm), with the
temperature programs as specified. Purified Ao:DCPIP OR
was obtained as previously described.[17]
1
min–1): tR (min) = 6.3 (minor), 6.7 (major); 72% ee; H
NMR (300 MHz):  = 3.64 (dd, J = 7.7, 4.3 Hz, 1H, H-1),
3.50 (d, J = 4.3 Hz, 1H, OH), 2.31 (s, 3H, CH3), 1.05–0.88
(m, 1H, CHcprop), 0.73–0.35 (m, 4H); 13C NMR (101
MHz):  = 209.1, 79.1, 25.6, 14.2, 2.9, 2.0; HRMS (ESI/Q-
TOF): m/z = 137.0578, calcd for C6H10NaO2 [M+Na]+:
137.0590.
1-Cyclopentyl-1-hydroxypropan-2-one (8f)
Column chromatography with cyclohexane/EtOAc 7:3
afforded 8f as a colorless oil, 82% yield. []D +47 (c 1.0,
CHCl3); GC (temperature program: 80 to 200 °C, rate 2 °C
1
min–1): tR (min) = 17.7 (minor), 19.0 (major); 86% ee; H
NMR (300 MHz):  = 4.23 (dd, J = 4.8, 3.4 Hz, 1H, H-1),
3.42 (d, J = 4.8 Hz, 1H, OH), 2.38–2.23 (m, 1H, CHCpent),
2.20 (s, 3H, CH3), 1.72–1.49 (m, 5H), 1.40–1.16 (m, 3H);
13C NMR (101 MHz):  = 209.8, 78.5, 42.0, 29.5, 27.0,
26.0, 25.5, 24.7; HRMS (ESI/Q-TOF): m/z = 165.0891,
calcd for C8H14NaO2 [M+Na]+: 165.0878.
General Procedure for the Synthesis of Products 8a–j
on an Analytical Scale
Lyophilized Ao:DCPIP OR (0.75 mg) was added to a
solution of aldehyde 7a–j (30 mol), methylacetoin (1; 4.7
L, 45 mol), ThDP (0.4 mg, 0.9 mol) and MgSO4 (0.16
mg, 1.3 mol) in 50 mM phosphate buffer at pH 6.5 (1.5
mL). The reaction mixture was gently shaken at 30 °C and,
after 8, 24 and 48 h, samples (0.5 mL) were withdrawn and
extracted with CDCl3 (1.0 mL). The organic extracts were
(S)-1-Cyclohexyl-1-hydroxypropan-2-one [(S)-8g]
Column chromatography with cyclohexane/EtOAc 7:3
afforded 8g as a colorless oil, 89% yield. []D +146 (c 1.0,
CHCl3), lit. for (S)-enantiomer +54.9 (c 1.0, CHCl3);[24]
92% ee (determined by chiral-phase HPLC analysis after
conversion into 16g, as described in the Supporting
Information); 1H NMR (300 MHz):  = 4.04 (d, J = 2.4 Hz,
1H, H-1), 3.36 (bs, 1H, OH), 2.19 (s, 3H, CH3), 1.88–1.58
(m, 5H), 1.55–1.39 (m, 1H), 1.37–1.09 (m, 5H); 13C NMR
(101 MHz):  = 209.9, 81.1, 41.1, 30.1, 26.5, 26.0, 25.8,
25.5, 25.0; HRMS (ESI/Q-TOF): m/z = 179.1048, calcd for
C9H16NaO2 [M+Na]+: 179.1058.
1
dried with anhydrous Na2SO4 and analyzed by H NMR
spectroscopy and chiral-phase GC to determine the
conversion and the ee, respectively.
General Procedure for the Synthesis of Products 8c–j
on a Semipreparative Scale
Lyophilized Ao:DCPIP OR (12 mg) was added to a
solution of aldehyde 7c–j (0.50 mmol), methylacetoin (1;
79 L, 0.75 mmol), ThDP (4.5 mg, 10 mol) and MgSO4
(2.7 mg, 20 mol) in 50 mM phosphate buffer at pH 6.5
(25 mL). The reaction mixture was gently shaken at 30 °C
for 48 h and then extracted with Et2O (3 × 5 mL). The
combined extracts were dried with anhydrous Na2SO4 and
concentrated under reduced pressure. The residue was
chromatographed on silica gel with the noted elution
system.
4-(Benzyloxy)-3-hydroxybutan-2-one (8h)
Column chromatography with cyclohexane/EtOAc 8:2
afforded 8h as a colorless oil, 92% yield. []D +17 (c 1.0,
CHCl3); GC (temperature program: 120 to 210 °C, rate
2 °C min–1): tR (min) = 33.0 (minor), 34.0 (major); 72% ee;
1H NMR (300 MHz):  = 7.43–7.27 (m, 5H), 4.61 (d, J =
12.0 Hz, 1H, HaBn), 4.49 (d, J = 12.0 Hz, 1H, HbBn), 4.26
(m, 1H, H-3), 3.83 (dd, J = 10.4, 3.7 Hz, 1H, OCHa), 3.72
(dd, J = 10.4, 3.7 Hz, 1H, OCHb), 3.63 (d, J = 5.1 Hz, 1H,
OH), 2.22 (s, 3H, CH3); 13C NMR (101 MHz):  = 207.8,
137.4, 128.5, 127.9, 127.8, 77.2, 76.9, 73.7, 70.8, 25.7;
4-Ethyl-3-hydroxyhexan-2-one (8c)
HRMS (ESI/Q-TOF): m/z
=
217.0841, calcd for
Column chromatography with cyclohexane/EtOAc 7:3
afforded 8c as a colorless oil, 49% yield. []D +117 (c 1.0,
CHCl3); GC (temperature program: 80 to 200 °C, rate 2 °C
C11H14NaO3 [M+Na]+: 217.0856.
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.201800357
Advanced Synthesis & Catalysis
3-Hydroxy-4,4-dimethoxybutan-2-one (8i)
(ESI/Q-TOF): m/z = 181.1204, calcd for C9H18NaO2
[M+Na]+: 181.1217.
Column chromatography
on florisil gel with
cyclohexane/EtOAc 10:3 afforded 8i as a colorless oil,
(S)-4-Hydroxy-5,5-dimethylhexan-3-one [(S)-13d]
1
61% yield. []D +76 (c 1.0, CHCl3); H NMR (300 MHz):
 = 4.40 (d, J = 3.2 Hz, 1H, H-4), 4.21 (m, 1H, H-3), 3.64
(bs, 1H, OH), 3.49 (s, 3H, CH3O), 3.46 (s, 3H, CH3O),
2.29 (s, 3H, CH3); 13C NMR (101 MHz):  = 207.2, 106.2,
77.7, 57.3, 55.8, 27.3; HRMS (ESI/Q-TOF): m/z =
171.0633, calcd for C6H12NaO4 [M+Na]+: 171.0647.
Column chromatography with cyclohexane/EtOAc 7:3
afforded 13d as a colorless oil, 45% yield. []D +135 (c 0.6,
CHCl3), lit. for (S)-enantiomer +94.5 (c 2.2, CHCl3);[25]
GC (temperature program: 80 to 200 °C, rate 2 °C min–1):
tR (min) = 25.0 (R), 26.0 (S); 99% ee; 1H NMR (300 MHz):
 = 3.87 (s, 1H, H-4), 2.66–2.37 (m, 2H, CH2), 1.10 (t, J =
7.2 Hz, 3H, CH3), 0.98 (s, 9H, 3 × CH3); 13C NMR (76
MHz):  = 214.2, 84.0, 35.6, 27.1, 26.5, 8.0; HRMS
(ESI/Q-TOF): m/z = 167.1048, calcd for C8H16NaO2
[M+Na]+: 167.1062.
tert-Butyl 4-(1-Hydroxy-2-oxopropyl)piperidine-1-
carboxylate (8j)
Column chromatography with cyclohexane/EtOAc 6:4
afforded 8j as a white waxy solid, 93% yield. []D +91 (c
1.0, CHCl3); 84% ee (determined by chiral-phase HPLC
analysis after conversion into 16j, as described in the
1-Cyclopropyl-1-hydroxybutan-2-one (13e)
1
Supporting Information); H NMR (300 MHz):  = 4.25–
Column chromatography with cyclohexane/EtOAc 8:2
afforded 13e as a colorless oil, slightly contaminated with
unknown compounds, 70% yield. 1H NMR (300 MHz):  =
3.64 (d, J = 7.7 Hz, 1H), 3.55 (bs, 1H, OH), 2.89–2.70 (m,
1H, HaCH2), 2.60–2.40 (m, 1H, HbCH2), 2.06–1.89 (m, 1H,
CHcprop), 1.16 (t, J = 7.1 Hz, 3H, CH3); 13C NMR (76
MHz):  = 201.8, 78.8, 37.1; HRMS (ESI/Q-TOF): m/z =
151.0735, calcd for C7H12NaO2 [M+Na]+: 151.0723.
4.10 (m, 2H, CH2), 4.10–4.06 (m, 1H, H-1), 3.39 (bs, 1H,
OH), 2.79–2.50 (m, 2H, CH2), 2.21 (s, 3H, CH3), 2.01–
1.81 (m, 1H, H-4), 1.65 (m, 4H, 2 × CH2), 1.43 (s, 9H,
3 × CH3); 13C NMR (101 MHz):  = 209.1, 154.6, 80.0,
79.5, 43.7, 39.4, 28.9, 28.4, 25.7, 24.4; HRMS (ESI/Q-
TOF): m/z = 258.1705, calcd for C13H24NO4 [M+H]+:
258.1721.
Synthesis of 4-Hydroxy-4-methylhexan-3-one (12)
1-Cyclopentyl-1-hydroxybutan-2-one (13f)
To a stirred solution of hexane-3,4-dione (9; 9.4 g, 82.4
mmol) in anhydrous THF (30 mL), a 3.0 M MeMgBr
solution in Et2O (33 mL, 99 mmol) was added dropwise, at
room temperature. The resulting mixture was refluxed for
2 h and then a saturated aqueous solution of NH4Cl (50
mL) was slowly added. The organic layer was separated
and the aqueous phase was extracted with Et2O (2 × 20
mL). The combined organic layers were washed with brine
and dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure and the residue (6.3 g, 6.6
mL) was purified by vacuum distillation (70 °C/10 mmHg)
to obtain the expected product 12 as a yellow oil, 5.2 g (40
Column chromatography with cyclohexane/EtOAc 7:3
afforded 13f as a colorless oil, 82% yield. []D +60.0 (c 0.5,
CHCl3); 78% ee (determined by chiral-phase HPLC
analysis after conversion into 17f, as described in the
Supporting Information); 1H NMR (300 MHz):  = 4.24 (d,
J = 3.2 Hz, 1H, H-1), 3.46 (bs, 1H, OH), 2.63–2.38 (m, 2H,
CH2), 2.37–2.21 (m, 1H, CH), 1.85–1.42 (m, 6H), 1.39–
1.19 (m, 2H), 1.15–1.09 (m, 3H, CH3); 13C NMR (76
MHz):  = 212.8, 78.0, 42.4, 31.6, 29.7, 26.2, 26.1, 25.0,
7.9; HRMS (ESI/Q-TOF): m/z = 179.1048, calcd for
C9H16NaO2 [M+Na]+: 179.1039.
1
mmol, 48% yield). H NMR (400 MHz):  = 3.89 (bs, 1H,
(S)-1-Cyclohexyl-1-hydroxybutan-2-one [(S)-13g]
OH), 2.60–2.41 (m, 2H, CH2), 1.79–1.66 (m, 2H, CH2),
1.34 (s, 3H, CH3), 1.10 (t, J = 7.3 Hz, 3H, CH3), 0.78 (t, J
= 7.4 Hz, 3H, CH3); 13C NMR (101 MHz):  = 215.1, 78.9,
32.5, 28.9, 25.3, 7.8, 7.7; HRMS (ESI/Q-TOF): m/z =
153.0891, calcd for C7H14NaO2 [M+Na]+: 153.0905.
Column chromatography with cyclohexane/EtOAc 7:3
afforded 13g as a colorless oil, 78% yield. []D +73.6 (c
1.0, CHCl3), lit. for (R)-enantiomer –112.5 (c 1.0,
CHCl3);[21] 59% ee;[21] 1H NMR (300 MHz):  = 4.24 (d, J
= 1.7 Hz, 1H, H-1), 2.56–2.39 (m, 2H, CH2), 1.89–1.56 (m,
10H), 1.55–1.41 (m, 1H), 1.11 (t, J = 7.2 Hz, 3H, CH3);
13C NMR (101 MHz):  = 212.8, 80.5, 41.4, 31.4, 30.1,
29.7, 26.6, 26.0, 25.8, 25.1, 7.6; HRMS (ESI/Q-TOF): m/z
= 193.1204, calcd for C10H18NaO2 [M+Na]+: 193.1219.
General Procedure for the Synthesis of Products 13c–j
on an Analytical Scale
The reactions were performed and analyzed as described
for the synthesis of products 8a–j on an analytical scale,
using 4-hydroxy-4-methylhexan-3-one (12; 6.2 L, 45
mol) instead of methylacetoin.
1-(Benzyloxy)-2-hydroxypentan-3-one (13h)
Column chromatography with cyclohexane/EtOAc 8:2
afforded 13h as a colorless oil, 68% yield. []D +48.1 (c
1.0, CHCl3); chiral-phase HPLC (Phenomenex Amylose-2
Lux column, n-hexane/propan-2-ol 9:1, flow 1.0 mL min–
1): tR (min) = 14.9 (major), 34.0 (minor); 92% ee; 1H NMR
(300 MHz):  = 7.38–7.23 (m, 5H), 4.60 (d, J = 12.1 Hz,
1H, HaBn), 4.49 (d, J = 12.1 Hz, 1H, HbBn), 4.30–4.22 (m,
1H, H-2), 3.81 (dd, J = 10.4, 3.7 Hz, 1H, OCHa), 3.71 (dd,
J = 10.4, 3.7 Hz, 1H, OCHb), 2.69–2.34 (m, 2H, CH2),
1.10 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (76 MHz):  =
210.8, 137.7, 128.7, 128.1, 128.0, 76.5, 73.9, 71.3, 71.2,
31.8, 7.6; HRMS (ESI/Q-TOF): m/z = 231.0997, calcd for
C12H16NaO3 [M+Na]+: 231.0985.
General Procedure for the Synthesis of Products 13c–j
on a Semipreparative Scale
The reactions were performed and worked up as described
for the synthesis products 8c–j on a semipreparative scale,
using 4-hydroxy-4-methylhexan-3-one (12; 103 L, 0.75
mmol) instead of methylacetoin.
5-Ethyl-4-hydroxyheptan-3-one (13c)
Column chromatography with cyclohexane/EtOAc 7:3
afforded 13c as a colorless oil, 52% yield. []D +60.8 (c
0.5, CHCl3); 90% ee (determined by chiral-phase HPLC
analysis after conversion into 17c, as described in the
2-Hydroxy-1,1-dimethoxypentan-3-one (13i)
1
Supporting Information); H NMR (300 MHz):  = 4.28–
4.22 (m, 1H, H-4), 3.41 (d, J = 4.5 Hz, 1H, OH), 2.60–2.33
(m, 2H, CH2), 1.67–1.37 (m, 4H), 1.29–1.15 (m, 1H), 1.12
(t, J = 7.3 Hz, 3H, CH3), 1.01 (t, J = 7.2 Hz, 3H, CH3),
0.83 (t, J = 7.2 Hz, 3H, CH3); 13C NMR (76 MHz):  =
213.9, 77.8, 45.3, 31.4, 23.6, 21.6, 12.4, 12.3, 8.0; HRMS
Column chromatography
on florisil gel with
cyclohexane/EtOAc 12:3 afforded 13i as a colorless oil,
62% yield. []D +72.7 (c 0.8, CHCl3); 1H NMR (300
MHz):  = 4.37 (d, J = 3.2 Hz, 1H, H-1), 4.19 (dd, J = 5.3,
3.2 Hz, 1H, H-2), 3.62 (d, J = 5.3 Hz, 1H, OH), 3.45 (s, 3H,
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.201800357
Advanced Synthesis & Catalysis
CH3O), 3.42 (s, 3H, CH3O), 2.82–2.65 (m, 1H, HaCH2),
2.55–2.37 (m, 1H, HbCH2), 1.05 (t, J = 7.2 Hz, 3H, CH3);
13C NMR (76 MHz, CDCl3):  = 210.2, 106.4, 76.8, 57.4,
55.9, 33.4, 7.5; HRMS (ESI/Q-TOF): m/z = 185.0790,
calcd for C7H14NaO4 [M+Na]+: 185.0803.
F. Franz, S. J. Connon, K. Zeitler, Chem. Sci. 2012, 3,
735–740.
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Möller, Tetrahedron: Asymmetry 1996, 7, 2207–2210;
b) P. Hoyos, M. Fernandez, J. V. Sinisterra, A. R.
Alcántara, J. Org. Chem. 2006, 71, 7632–7637; c) P.
Hoyos, P. Domínguez de María, J. V. Sinisterra, A. R.
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Asymmetry 2006, 17, 1125–1128; e) G. Scheid, E.
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tert-Butyl
carboxylate (13j)
4-(1-Hydroxy-2-oxobutyl)piperidine-1-
Column chromatography with cyclohexane/EtOAc 6:4
afforded 13j as a white waxy solid, 91% yield. []D +34.6
(c 1.0, CHCl3); 76% ee (determined by chiral-phase HPLC
analysis after conversion into 17j, as described in the
1
Supporting Information); H NMR (300 MHz):  = 4.16
(bs, 2H), 4.09 (bs, 1H, H-1), 3.40 (bs, 1H, OH), 2.82–2.38
(m, 5H), 1.97–1.80 (m, 1H), 1.71–1.57 (m, 3H), 1.43 (s,
9H, 3 × CH3), 1.12 (t, J = 7.3 Hz, 3H, CH3); 13C NMR
(101 MHz):  = 212.0, 154.6, 79.5, 79.4, 43.6, 39.7, 31.6,
29.7, 28.9, 28.4, 24.4, 7.6; HRMS (ESI/Q-TOF): m/z =
272.1862, calcd for C14H26NO4 [M+H]+: 272.1879.
Acknowledgements
[11] a) M. Kurina-Sanz, F. R. Bisogno, I. Lavandera, A. A.
Orden, V. Gotor, Adv. Synth. Catal. 2009, 351, 1842–
1848; b) K. Edegger, W. Stampfer, B. Seisser, K. Faber,
S. F. Mayer, R. Oehrlein, A. Hafner, W. Kroutil, Eur. J.
Org. Chem. 2006, 1904–1909; c) R. Médici, H.
Stammes, S. Kwakernaak, L. G. Otten, U. Hanefeld,
Catal. Sci. Technol. 2017, 7, 1831–1837; d) J. Zhang, T.
Xu, Z. Li, Adv. Synth. Catal. 2013, 355, 3147–3153; e)
P. P. Giovannini, G. Fantin, A. Massi, V. Venturi, P.
Pedrini, Org. Biomol. Chem. 2011, 9, 8038–8045.
We gratefully acknowledge the University of Ferrara (fondi FAR)
for financial support. Thanks are also given to Mr Paolo
Formaglio for NMR experiments and to Mrs Morena De Bastiani
for the production of the recombinant enzyme.
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10.1002/adsc.201800357
Advanced Synthesis & Catalysis
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9
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10.1002/adsc.201800357
Advanced Synthesis & Catalysis
FULL PAPER
Enzymatic Cross-Benzoin-Type Condensation of
Aliphatic Aldehydes: Enantioselective Synthesis of
1-Alkyl-1-hydroxypropan-2-ones and 1-Alkyl-1-
hydroxybutan-2-ones
Adv. Synth. Catal. Year, Volume, Page – Page
O
R1
AO:DCPIP OR
(ThDP-dependent lyase)
O
O
R2
OH
+
R2
OH
aq. buffer pH 6.5
48 h, 30 °C
TOC Graphic
R1
R1
eight -hindered
aliphatic aldehydes
two acyl anion donors
R1 = H or Me)
16 examples
3493% yield
7299% ee
Et
OMe
R2
=
;
;
;
;
;
;
NBoc
;
OBn
Et
OMe
ThDP = thiamine diphosphate
Graziano Di Carmine, Olga Bortolini, Alessandro
Massi, Michael Müller, Giovanni Bernacchia,
Giancarlo Fantin, Daniele Ragno, and Pier Paolo
Giovannini*
10
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