Enzymatic diastereo- and enantioselective synthesis of α-alkyl- α,β-dihydroxyketones

Article abstract of DOI:10.1039/c1ob05928a

An enzymatic strategy for the preparation of optically pure α-alkyl-α,β-dihydroxyketones is reported. Homo- and cross-coupling reactions of α-diketones catalyzed by acetylacetoin synthase (AAS) produce a set of α-alkyl-α-hydroxy-β-diketones (30-60%, ee 67

Full text of DOI:10.1039/c1ob05928a

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PAPER
Enzymatic diastereo- and enantioselective synthesis of
a-alkyl-a,b-dihydroxyketones†
Pier Paolo Giovannini,*a Giancarlo Fantin,a Alessandro Massi,a Valentina Venturib and Paola Pedrinib
Received 9th June 2011, Accepted 22nd August 2011
DOI: 10.1039/c1ob05928a
An enzymatic strategy for the preparation of optically pure a-alkyl-a,b-dihydroxyketones is reported.
Homo- and cross-coupling reactions of a-diketones catalyzed by acetylacetoin synthase (AAS) produce
a set of a-alkyl-a-hydroxy-b-diketones (30–60%, ee 67–90%), which in turn are reduced regio-,
diastereo-, and enantioselectively to the corresponding chiral a-alkyl-a,b-dihydroxyketones (60–70%,
ee >95%) using acetylacetoin reductase (AAR) as catalyst. Both enzymes are obtained from Bacillus
licheniformis and used in a crude form. The relative syn stereochemistry of the enantiopure
a,b-dihydroxy products is assigned by NOE experiments, whereas their absolute configuration is
determined by conversion of the selected 3,4-dihydroxy-3-methyl-pentan-2-one to the natural product
(+)-citreodiol.
Introduction
Enantiopure tertiary alcohols are very valuable building blocks
for the synthesis of many different natural products and pharma-
ceuticals. As a consequence, many efforts have been devoted to
the development of chemical1 and enzymatic2 strategies towards
these valuable structures. Various biocatalysts, such as epoxide
hydrolases,3 halohydrin dehalogenases,4 hydroxynitrile lyases,5
lipases, and esterases6 have been widely used to this purpose.
Surprisingly, the use of thiamine diphosphate (ThDP)-dependent
enzymes in this field appears to be scanty. In 2010 Mu¨ller and co-
workers described the utilization of YerE, a recombinant ThDP-
dependent flavoenzyme, for umpolung couplings of pyruvate
with different ketone acceptors to give enantioenriched tertiary
alcohols.7 In the same year, we reported a parallel study in
which we demonstrated that a-diketones may serve as acyl anion
equivalents when ThDP-dependent acetylacetoin synthase (AAS)
from Bacillus licheniformis8 is used as catalyst in the homo- and
cross-couplings of a-diketones 1 (Scheme 1).
By the disclosed carboligation reaction, which is virtually an in-
termolecular aldehyde–ketone coupling, we were able to prepare a
set of chiral a-alkyl-a-hydroxy-b-diketones 2 (30–45%) with good
enantioselectivity (ee 67–91%) together with the regioisomeric
prochiral derivatives 39 (Scheme 1). Tertiary alcohols 2 and 3 are
promising building blocks for asymmetric synthesis because they
display a structure with two additional carbonyl groups which
Scheme 1 Synthesis of the syn-a-alkyl-a,b-dihydroxy-ketones 4a–e,g.
can be selectively elaborated. In this regard, the enantio- and
stereoselective reduction of a-hydroxy-b-diketones 2 towards a,b-
dihydroxy-ketones 4 is an opportunity (Scheme 1).
On the other hand, enantiopure diols of type 4 have already
proven to be valuable precursors for the synthesis of chiral
polysubstituted tetrahydrofurans10 and natural products.11 Indeed,
various dehydrogenases have been successfully employed for the
asymmetric reduction of b-diketones,12 but only few of them
showed activity toward a-disubstituted substrates.13
Herein we report the highly regio-, diastereo-, and enan-
tioselective reduction of a-hydroxy-b-diketones 2, obtained by
the previously described AAS-based strategy, to syn-a-alkyl-a,b-
dihydroxy-ketones 4 using B. licheniformis acetylacetoin reductase
(AAR) as catalyst.14 A crucial issue, in the present work, is the
stereochemical assignment of the resulting a,b-dihydroxyketones
4 that we addressed by means of NOE experiments and by
aDipartimento di Chimica, Universita` di Ferrara, Via L. Borsari 46, I-44121
Ferrara, Italy. E-mail: gvnppl@unife.it; Fax: +39 0532 240709; Tel: +39
0532 455173
bDipartimento di Biologia ed Evoluzione, Universita` di Ferrara, C.so Ercole
I d’Este 32, I-44121 Ferrara, Italy
† Electronic supplementary information (ESI) available: copies of 1H and
13C NMR spectra. See DOI: 10.1039/c1ob05928a
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Table 1 AAR-mediated reduction of compounds 2a–g
Entry
1
a-Hydroxy-b-diketone (ee%)a
Time (h)
3
a,b-Dihydroxyketone yield%b (ee%)a
2
3
3
4
4
4
5
5
6
7
12
12
—
a Determined by chiral GC-analysis (see experimental section). b Isolated yield. c Isolated yield based on a-hydroxy-b-diketone 2.
the stereoconservative conversion of a selected product into the
natural compound (+)-citreodiol.
study showed the capability of bacterial AAR to promote the
selective reduction of acetylacetoin 2a to acetylbutanediol 4a on
an analytical scale.16 This evidence prompted us to optimize a
preparative procedure for the same transformation and investigate
AAR activity on all diketones 2 and 3. Accordingly, B. licheni-
formis was grown on high sucrose concentration broth to induce
a high expression level of AAR,17 thus allowing the use of the
cell free extract in its crude form. The enzymatic reduction of 2a
was carried out in phosphate buffer at pH 6.5 in the presence of
sodium formate (5 equiv.) and formate dehydrogenase (FDH) for
NADH recycling (Scheme 2).
Results and discussion
We recently synthesised a number of a-alkyl-a-hydroxy-b-
diketones 2 and 3 by coupling reactions of a-diketones 1 using
acetylacetoin synthase from Bacillus licheniformis (Scheme 1 and
Table 1 for complete structures).9 More precisely, compounds 2a–f
were obtained by homo-couplings of the corresponding diketones
1a–f, while 2g was prepared by cross-coupling of 1a as donor and
diketone 1f as acceptor. In the case of unsymmetrically substituted
diketones 1b–d, the formation of chiral b-diketones 2b–d was
accompanied by the generation of variable amounts (24–45%) of
the regioisomeric prochiral derivatives 3b–d.15
After
3
h
shaking, 3-methyl-3,4-dihydroxy-2-pentanone
(acetylbutanediol) 4a was recovered in 60% yield as a single
diastereoisomer and high enantiomeric purity (ee >95%), as
detected by 1H NMR and chiral GC analyses. It is worth
noting that the use of excess sodium formate for reaction rate
acceleration was possible due to the complete inactivity of AAR
on 4a. Enzyme selectivity was further confirmed by extending
With diketones 2 and 3 in hand, we could begin our investigation
on their asymmetric reduction to acyl diols through selective re-
duction of one carbonyl group. A previous explorative mechanistic
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Scheme 2 Acetylacetoin reductase (AAR)-based reduction of acetylace-
toin 2a.
the reaction time and/or adding excess of enzyme. The relative
stereochemistry of compound 4a was next investigated by NOE
experiments performed on the isopropylidene derivative 5a, which
was obtained by treating 4a with dimethoxypropane (DMP) under
acidic conditions (Scheme 3).
Scheme 4 Synthesis of (+)-citreodiol 8. Reagents and conditions: (i)
NaBH4, Et2O, MeOH; (ii) Tf2O, pyridine, CH2Cl2; (iii) DBU, CH2Cl2; (iv)
p-TsOH, CH2Cl2, MeOH; (v) Hoveyda-Grubbs 2nd gen. catal., acrolein,
CH2Cl2, THF, reflux; (vi) H3C(P(C6H5)3)CCOOCH3, benzene, reflux.
The synthesis began with the reduction of syn-5a with sodium
borohydride to afford the corresponding alcohol 6 (85% yield).
This compound was next converted to the allyl diol 7 in 40%
yield via a three-step one-pot procedure. This involved the initial
activation of 6 with triflic anhydride (Tf2O), followed by elimi-
nation to the corresponding terminal alkene and isopropylidene
removal promoted by 1,8-diazabicyclo[5.4.0] undec-7-ene (DBU)
and p-toluenesulfonic acid (p-TsOH), respectively. The next two
steps were run in a single flask as well. Hence, cross-metathesis
of pure 7 with acrolein promoted by Hoveyda-Grubbs 2nd
generation catalyst was followed by Wittig olefination with the 2-
(triphenylphosphoranylidene)-2-propanoic acid methyl ester18 to
give (+)-citreodiol 8 in 67% yield (two steps). The spectral data,
and optical rotation of 8 ([a]D = +6.8, c 0.7; lit.19 [a]D = +4.3, c
1.0) were consistent with those reported for (+)-citreodiol, thus
allowing us to confirm the syn stereochemistry of 4a and establish
the 3(R) and 4(S) absolute configurations of its chiral centers.
Importantly, this stereochemical assignment is in agreement with
the (S)-stereospecificity previously observed for AAR-catalyzed
reductions.14
Encouraged by the excellent stereochemical outcome detected
in the reduction of 2a, we next examined the substrate scope
of the proposed enzymatic reduction by applying the optimized
procedure to the previously synthesized a-hydroxy-b-diketones 2
and 3. The results of this investigation are summarized in Table 1.
Due to their difficult purification, chiral diketones 2b–d were
subjected to the AAR-catalyzed reduction together with the
corresponding regioisomers 3b–d (entries 2–4). In spite of this, a-
methyl-a,b-dihydroxy-ketones 4b–d were obtained in satisfactory
yields (61–70%) and excellent diastereo- and enantiomeric excesses
(de >95%; ee >95%). It is worth noting that prochiral diketones
3b–d were unreactive under these conditions. However, this out-
come can be exploited as a method for the kinetic resolution of 2/3
regioisomeric mixtures, though the recovery by chromatography
of symmetric diketones 3b–d was complicated by their intrinsic
instability on silica.20 The enzymatic reduction of pure diketone
2e proceeded smoothly in 5 h furnishing the diol 4e in a 63% yield
with high stereoselectivity (entry 5). By contrast, no reduction
product was obtained with symmetric diketone 2f (entry 6). The
optically pure a,b-dihydroxy-ketone 4g was finally obtained in
Scheme 3 Synthesis of syn- and anti-5a and NOE signals.
These measurements showed the presence of a signal between
the C4- and C5-methyl groups whereas no NOE was observed
between the C5-hydrogen and C4-methyl group directly bound to
the dioxolane ring. These results were in accordance with a syn
stereochemistry that was unequivocally confirmed by performing
the same NOE measurements on the diastereomeric anti-5a. The
mixture of syn- and anti-5a was readily prepared by reduction of
2a with NaBH4 and treatment of the resulting diols with DMP
(Scheme 3).
As expected, opposite results were detected in the NOE
measurements of anti-5a, namely the presence of a signal between
C5-hydrogen and C4-methyl group and lack of signal between
C4- and C5-methyl groups. On the other hand, an optimized
chiral GC analysis of the syn/anti-5a mixture displays all the
four stereoisomers, one of which corresponds in retention time
to the one obtained from the enzymatic reaction. Overall, these
results confirmed the essentially complete regio-, diastereo-, and
enantioselectivity of the AAR-catalyzed reduction of 2a (de >95%,
ee >95%).
In order to assign the absolute configuration of the two chiral
centres of 4a, the isopropylidene derivative syn-5a was transformed
to (+)-citreodiol 8, the antipode of a metabolite of Penicillium
citreoviride B (IFO 6200), which is related to the inhibitor of ATP-
synthesis and ATP-hydrolysis citreoviridine11 (Scheme 4).
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Table 2 Absolute configuration and optical rotation of compounds 4, 5 and 2
Compound
ee (%)
[a]D
4a
5a
4b
5b
4c
5c
4d
5d
4e
5e
4g
5g
2b
2c
2d
2e
2g
3R,4S-dihydroxy-3-methyl-2-pentanone
4R,5S-1-(2,2,4,5-tetramethyl-1,3-dioxolan-4-yl)-ethanone
4R,5S-dihydroxy-3-methyl-2-hexanone
4R,5S-1-(2,2,4,5-tetramethyl-1,3-dioxolan-4-yl)-propanone
2S,3R-dihydroxy-3-methyl-4-heptanone
4R,5S-1-(2,2,4,5-tetramethyl-1,3-dioxolan-4-yl)-butanone
2S,3R-dihydroxy-3-methyl-4-octanone
4R,5S-1-(2,2,4,5-tetramethyl-1,3-dioxolan-4-yl)-pentanone
2R,3S-dihydroxy-2-methyl-1-phenyl-1-butanone
4R,5S-1-(2,2,4,5-tetramethyl-1,3-dioxolan-4-yl)-benzophenone
4R,5S-dihydroxy-4-ethyl-3-hexanone
4R,5S-1-(4-ethyl-2,2,5-trimethyl-1,3-dioxolan-4-yl)-propanone
3R-hydroxy-3-methyl-2,4-hexandione
>95
>95
>95
>95
>95
>95
>95
>95
>95
>95
>95
>95
70
-10.7
+46.9
-8.6
+5.2
-10.0
+46.5
-8.2
+46.4
+8
+8.1
+6.6
+51.9
+15a
+3a
+7.3
+13a
+49.8a
3R-hydroxy-3-methyl-2,4-heptanedione
3R-hydroxy-3-methyl-2,4-octandione
2R-hydroxy-2-methyl-1-phenyl-1,3-butandione
3R-hydroxy-3-ethyl-1,3-hexandione
67
72
76
91
a The optical rotations are reported in ref. 15
good yield (72%) by the optimized procedure although with a
longer reaction time (12 h; entry 7).
conversion of acetyl groups to (S)-methyl carbinols or with respect
to the (R)-configuration of the a-quaternary carbon. This last
feature is particularly interesting as it is known that dehydro-
genases’ stereoselectivity for the a-stereocenter is often modest.
Most of the enantiomerically pure ketodiols herein synthesized
are new compounds and their complete characterization including
their stereochemical assignment is duly reported. Finally, the
present investigation has also allowed us to determine the absolute
configuration of the chiral diketone precursor.
A common feature of the AAR-catalyzed reduction of chiral
diketones 2b–e and 2g was the complete selectivity of the enzyme
toward the corresponding major (R)-enantiomers. The minor (S)-
enantiomers of 2b–e and 2g were, in fact, recovered unaltered in
the crude mixtures after reduction as determined by chiral GC
analysis. Moreover, on the basis of the above substrate study it
can be pointed out that: (i) AAR exhibits complete specificity for
the reduction of acetyl group in a-hydroxy-a-methyl-b-diketones
2; (ii) the presence of substituents bulkier than a methyl group on
the quaternary carbon of 2 inhibits the catalysis, as demonstrated
by the unreactivity of 2f and the lower reaction rate observed for
2g reduction; (iii) as already mentioned, the enzyme exhibits a high
selectivity with respect to the configuration of the a-quaternary
carbon.
As previously described for 4a, the relative syn stereochemistry
of the reduction products 4b–e and 4g was confirmed by their
conversion to the corresponding dioxolanes 5b–e and 5g. In all
1,3-dioxolanes, in fact, a NOE was observed between C5-methyl
group and R1-hydrogens, while no NOE interaction was detected
between C5- and R1-hydrogens. The absolute configuration of 4b–e
and 4g was finally assigned by assuming that the AAR-mediated
hydride attack on the acetyl group of 2b–e and 2g occurs at the Re-
face furnishing a methylcarbinol moiety with (S)-configuration.
As a consequence it is possible to deduce the (R) configuration of
the a-quaternary chiral centre. The absolute configurations and
the optical rotation of the newly synthesized chiral products are
summarized in Table 2.
Experimental
General information
Diketones 1a–f, formate dehydrogenase (FDH) from Candida
boidinii, nicotinamide adenine dinucleotide (NADH), thiamine
diphosphate (ThDP) and sodium formate were purchased from
Sigma-Aldrich. Products 2a, 2e, 2f, 2g and the mixtures of
regioisomers 2b–3b (ratio 1.2 : 1) and 2c–3c (ratio 2.2 : 1) were
obtained as described.15 TLC were run on precoated silica pates
(thickness 0.25 mm, Merck), and silica gel (Fluka, Kieselgel 60,
70–230 mesh) was used for column chromatography. ESI mass
spectra were obtained using a LCQ Duo (ThermoQuest, San Jose,
CA, USA), in positive-ion mode by introducing the sample as
10-6 M solution in methanol. Instrumental parameter: capillary
voltage, spray voltage 4.6 kV, capillary temperature 200 ◦C, mass
scan range was from m/z 100 to 1000 amu for 30000 ms scan time;
N2 was used as sheath gas. NMR spectra were recorded on a Varian
Gemini 300 spectrometer. Chemical shifts are given in parts per
million from Me4Si as internal standard. Optical rotations were
measured on a Perkin–Elmer Model 241 polarimeter.
Conclusions
GC analyses
In summary, we have shown that the combined use of the two en-
zymes AAS and AAR, readily available from the same bacterium
B. licheniformis, allows the preparation of enantiopure a-alkyl-
a,b-dihydroxyketones starting from inexpensive and commercially
available a-diketones. The high enantiopurity of the products is
mainly due to the high stereospecificity of AAR either for the
Gas chromatographic analyses were performed on a Carlo Erba
6000, equipped with a FID detector and a fused capillary column
Megadex 5 (25 m X 0.25 mm) containing dimethyl-n-pentyl-b-
cyclodextrin on OV 1701 (from Mega snc), helium as carrier gas
(80 kPa).
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◦
For AAR reduction of 2a: 90–200 ◦C (2 C min-1), retention
3-Hydroxy-3-methyl-2,4-octanedione (2d)
time (min): 2a, 7.1; 4a, 15.5.
Colourless oil (48%, ee 72%). [a]2D0 +7.3 (c 0.2, CHCl3). 1H NMR
(300 MHz, CDCl3) dH: 0.93 (t, 3H, J = 7.5 Hz, CH3) 1.22–1.36 (m,
2H, CH2) 1.48–1.62 (m, 2H, CH2) 1.56 (s, 3H, CH3) 2.25 (s, 3H,
CH3CO) 2.51 (dt, 1H, J = 17.5 and 7.5 Hz, CH2CO) 2.70 (dt, 1H,
J = 17.5 and 7.5 Hz CH2CO) 4.75 (br s, 1H, OH). 13C NMR (300
MHz, CDCl3) dC: 13.8, 22.5, 22,7, 24.6, 25.5, 36.5, 87.6, 207.4,
209.6; ESI [MNa]+ m/z 195.1. Anal calcd for C9H16O3: C, 62.77%;
H, 9.36%. Found: C, 62.69%; H, 9.29%.
For NaBH4 reduction of 2a: 90–200 ◦C (2 ◦C min-1), retention
time (min): 2a, 7.1; 4a, 15.5 and 15.9.
For AAR reduction of 2b: temp 80–100 ◦C (1.5 ◦C min-1), 100–
200 ◦C (10 ◦C min-1) retention time (min): 3b, 10.7; 2b, 12.5 and
12.8; 4b, 19.8.
For AAR reduction of 2c: temp 80–100 ◦C (1.5 ◦C min-1), 100–
◦
200 C (10 ◦C min-1) retention time (min): 3c, 14.6; 2c, 15.9 and
16.0; 4c, 21.0.
For AAR reduction of 2d: temp 80–100 ◦C (1.5 ◦C min-1), 100–
200 ◦C (10 ◦C min-1) retention time (min): 3d, 17.5; 2d, 18.3 and
18.4; 4d, 22.3.
3-Hydroxy-3-butyl-2,4-pentanedione (3d)
For AAR reduction of 2e: temp 100–200 ◦C (5 C min-1),
◦
Colourless oil (15%). 1H NMR (300 MHz, CDCl3) dH: 0.91 (t, 3H,
J = 7.5 Hz, CH3) 1.10–1.40 (m, 4H, 2CH2) 1.95–2.05 (m, 2H, CH2)
2.25 (s, 6H, 2CH3CO) 4.65 (br s, 1H, OH). 13C NMR (300 MHz,
CDCl3) dC: 13.8; 22.7, 25.2, 25.3, 29.4, 29.7, 36.1, 91.0, 207; ESI
[MNa]+ m/z 195.1. Anal calcd for C9H16O3: C, 62.77%; H, 9.36%.
Found: C, 62.71%; H, 9.32%.
retention time (min): 2e, 17.1 and 17.2; 4e, 16.5.
For AAR reduction of 2g: temp 80–200 ◦C (1.5 C min-1),
◦
retention time (min): 2g, 16.0 and 16.3; 4g, 33.6.
For the synthesis of dioxolane 5a–e and 5g: temp 60–200 ◦C
◦
(2 C min-1) retention time (min): 5a from enzymatic reduction,
16.1; syn-5a from NaBH4 reduction, 15.5 and 16.1; anti-5a from
NaBH4 reduction, 17.1 and 17.4; 5b, 21.1; 5c, 26.3; 5d, 30.9; 5e,
52.8; 5g, 29.6.
General procedure for the enzymatic reduction of
a-alkyl-a-hydroxy-b-diketones 2a–g
To a solution of NAD+ (10 mg, 15 mmol), sodium formate (335
mg, 5 mmol), formate dehydrogenase (1.0 mg, 10 U) and AAR cell
free extract (15 mL) in phosphate buffer 50 mM at pH 6.5 (35 mL)
containing EDTA (0.1 mM) and 1 mM 2-mercapto-ethanol, the
selected diketones 2 (1.0 mmol) were added. For compounds 2b–
d, the mixtures 2b/3b, 2c/3c and 2d/3d were used in the proper
amount to have 1.0 mmol of the diketone 2. The mixture was
gently shaken overnight at 30 ◦C and than warmed up at 80 ◦C for
20 min. The proteins were removed by centrifugation (9000 rpm,
20 min) and the supernatant was extracted with ethyl acetate (3
¥ 30 mL). The organic layer was dried over anhydrous Na2SO4
and concentrated to afford a pale yellow oil that was purified
by column chromatography on silica gel using cyclohexane–ethyl
acetate 2 : 1 as eluent.
Enzymes preparation
B. licheniformis was cultured in two different media in order to
obtain crude AAS or AAR respectively. For the preparation of
AAS, the medium was composed of meat extract (10 g L-1),
polypeptone (10 g L-1), NaCl (5 g L-1) and 3-hydroxy-2-butanone
(5 g L-1), while in order to obtain BSDR sucrose (40 g L-1),
peptone (20 g L-1), yeast extract (10 g L-1) Na2HPO4·6H2O (6.8 g
L-1), K2SO4 (2.6 g L-1) were present in the medium. The culture
conditions and the cells’ treatment were the same for both the
enzymes: the bacteria was grown for 48 h at 38–40 ◦C and 110 rpm,
then the cells were harvested by centrifugation (6000 rpm, 20 min)
and washed with 150 mM NaCl solution (50 mL). Wet cells
obtained from the two different cultures (2.0 g) were suspended
in 50 mM phosphate buffer at pH 6.5 containing EDTA (0.1
mM) and b-mercaptoethanol (1 mM) (20 mL), the suspensions
were treated at high pressure (1280 bar) with a French press and
then centrifuged (9000 rpm, 20 min, 10 ◦C). The cell free extracts
(16 mL) were used without further purification.
3R,4S-Dihydroxy-3-methyl-2-pentanone (4a)
Colourless oil (79 mg, 60%, ee >95%). [a]2D0 -10.7 (c 1.4, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 1.25 (s, 3H, CH3) 1.27 (d, 3H, J =
7.5 Hz, CH3) 1.98 (d, 1H, J = 9.0 Hz, OH) 2.30 (s, 3H, CH3CO)
4.02 (br s, 1H, OH) 4.05 (m, 1H, CHOH). 13C NMR (300 MHz,
CDCl3) dC: 13.8, 21.8, 24.0, 71.3; 81.6, 211.7, ESI [MNa]+ m/z
155.2. Anal calcd for C6H12O3: C, 54.53%; H, 9.15%. Found: C,
54.38%; H, 9.21%.
3-Hydroxy-3-methyl-2,4-heptanedione (2d) and
3-butyl-3-hydroxy-2,4-pentanedione (3d)
The AAS cell free extract (16 mL) was added to a solution of
diketone 1d (384 mg, 3 mmol), ThDP (17 mg, 39 mmol) and
magnesium sulfate (15 mg, 125 mmol) in 50 mM phosphate buffer
at pH 6.5 (50 mL). The reaction was gently shaken at 30 ◦C for 14
h and then heated (80 ◦C, 20 min). After removing the precipitate
by centrifugation (9000 rpm, 20 min) the solution was extracted
with ethyl acetate (3 ¥ 30 mL). The combined organic layers were
washed with saturated NaHCO3 (40 mL) and dried over anhydrous
Na2SO4. The solvent was evaporated to afford 168 mg (0.98 mmol,
65%) of a mixture of compounds 2d and 3d in a 3 : 1 ratio. Pure
samples of 2d and 3d were obtained by flash chromatography on
silica gel (n-hexane–AcOEt 15 : 1 as eluent).
4R,5S-Dihydroxy-4-methyl-3-hexanone (4b)
Colourless oil (89 mg, 61%, ee >95%). [a]2D0 -8.6 (c 1.5, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 1.13 (t, 3H, J = 7.0 Hz, CH3)
1.25 (s, 3H, CH3) 1.27 (d, 3H, J = 7.0 Hz, CH3) 2.05 (br s, 1H,
OH) 2.60 (dq, 1H, J = 7.0 and 18.0 Hz, CH2CO) 2.68 (dq, 1H, J =
7.0 and 18.0 Hz, CH2CO) 4.05 (m, 1H, OH) 4.10 (br s, 1H, OH).
13C NMR (300 MHz, CDCl3) dC: 7.6, 16.9, 21,7, 29.2, 71.1, 81.0,
214.3; ESI [MNa]+ m/z 169.2. Anal calcd for C7H14O3: C, 57.51%;
H, 9.65%. Found: C, 57.45%; H, 9.51%.
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2S,3R-Dihydroxy-3-methyl-4-heptanone (4c)
NMR (300 MHz, CDCl3) dH: 1.08 (d, 3H, J = 7.5 Hz, CH3),1.45
(s, 3H, CH3), 1.85 (d, 1H, J = 14 Hz, OH), 2.25 (s, 3H, CH3CO),
3.80 (br s, 1H, OH), 3.90 (dq, 1H, J = 7.0 and 10 Hz, CHOH).
The diastereomeric mixture of syn and anti-4a was dissolved in
dichloromethane (5 mL) and treated successively as described
in the general procedure for the synthesis of the corresponding
dioxolane. Column chromatography (silica, cyclohexane–diethyl
ether 10 : 1 as eluent) afforded the mixture of syn and anti-5a (130
mg, 92%). The signals of 1-(2,2,4,5-tetramethyl-1,3-dioxolan-4-
yl)-ethanone (anti-5a) were identified from 1H NMR spectrum of
the mixture: 1H NMR (300 MHz, CDCl3) dH: 1.16 (d, 3H, J = 7.0
Hz, CH3), 1.38 (s, 3H, CH3), 1.44 (s, 3H, CH3), 1.58 (s, 3H, CH3),
2.24 (s, 3H, CH3CO), 4.14 (q, 1H, J = 7.0 Hz, CH-OR).
Colourless oil (104 mg, 65%, ee >95%). [a]2D0 -10.0 (c 0.8, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 0.94 (t, 3H, J = 7.0 Hz, CH3)
1.23 (s, 3H, CH3) 1.27 (d, 3H, J = 7.0 Hz, CH3) 1.68 (sex, 2H, J =
7.0 Hz, CH2) 2.10 (br s, 1H, OH) 2.53 (dt, 1H, J = 7.0 and 18.0
Hz, CH2CO) 2.60 (dt, 1H, J = 7.0 and 18.0 Hz, CH2CO) 4.05 (m,
1H, CHOH) 4.10 (br s, 1H, OH); 13C NMR (300 MHz, CDCl3)
dC: 13.6, 16.8, 16.9, 21.6, 37.8, 71.0, 81.0, 213.6; ESI [MNa]+ m/z
183.0. Anal calcd for C8H16O3: C, 59.97%; H, 10.07%. Found: C,
60.01%; H, 10.11%.
2S,3R-Dihydroxy-3-methyl-4-octanone (4d)
Colourless oil (122 mg, 70%, ee >95%). [a]2D0 -8.2 (c 1.0, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 0.94 (t, 3H, J = 7.0 Hz, CH3)
1.25 (s, 3H, CH3) 1.28 (d, 3H, J = 7.0 Hz, CH3) 1.28–1.42 (m, 2H,
CH2) 1.58–1.70 (m, 2H, CH2) 1.88 (br s, 1H, OH) 2.58 (dt, 1H,
J = 7.0 and 18.0 Hz, CH2CO) 2.65 (dt, 1H, J = 7.0 and 18.0 Hz,
CH2CO) 4.05 (m, 1H, CHOH), 4.08 (br s, 1H, OH). 13C NMR
(300 MHz, CDCl3) dC: 13.8, 17.0, 21.7, 22.3, 25.5, 35.6, 71.0, 81.0,
213,6; ESI [MNa]+ m/z 198.1. Anal calcd for C9H18O3: C, 62.04%;
H, 10.41%. Found: C, 61.99%; H, 10.13%
General procedure for the synthesis of 1,3-dioxolane derivatives 5
To a solution of the selected a-alkyl-a,b-dihydroxy-ketones 4
(0.5 mmol) in CH2Cl2 (2 mL), 2,2-dimethoxypropane (312 mg,
0.37 mL, 3 mmol) and p-toluenesulfonic acid (5 mg, 0.03 mmol)
were added. After 2 h at room temperature the mixture was
diluted with a saturated solution of NaHCO3 (10 mL) and
extracted with dichloromethane (3 ¥ 10 mL). The organic layer
was dried over anhydrous Na2SO4 and evaporated. The residue
was chromatographed (silica gel, cyclohexane–diethyl ether 10 : 1
as eluent).
2R,3S-Dihydroxy-2-methyl-1-phenyl-1-butanone (4e)
Yellow pale oil (122 mg, 63%, ee >95%). [a]2D0 +8.0 (c 0.4, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 1.30 (d, 1H, J = 7.0 Hz, CH3)
1.54 (s, 1H, CH3) 2.10 (br s, 1H, OH) 4.22 (br s, 1H, OH) 4.38 (m,
1H, CHOH) 7.43–8.05 (m, 5H, Ph). 13C NMR (300 MHz, CDCl3)
dC: 16.8, 23.1, 71.4, 81.4, 128.5, 129.3, 132.8, 204.8; ESI [MNa]+
m/z 217.1. Anal calcd for C11H14O3: C, 68.02%; H, 7.27%. Found:
C, 68.15%; H, 7.23%.
4R,5S-1-(2,2,4,5-Tetramethyl-1,3-dioxolan-4-yl)-ethanone
(syn-5a)
Colourless oil (69 mg, 80%, ee >95%). [a]2D0 +46.9 (c 1.3, CHCl3);
1H NMR (300 MHz, CDCl3) dH: 1.20 (s, 3H, CH3) 1.28 (d, 3H,
J = 7.0 Hz, CH3) 1.40 (s, 3H, CH3) 1.50 (s, 3H, CH3) 2.30 (s, 3H,
CH3CO) 4.14 (q, 1H, CHOC). 13C NMR (300 MHz, CDCl3) dC:
14.5, 18.7, 25.5, 25.9, 28.4, 74.4, 87.5, 108.3, 211.7; ESI [MNa]+
m/z 195.1. Anal calcd for C9H16O3: C, 62.77%; H, 9,36%. Found:
C, 62.41%; H, 9.53%.
4R,5S-Dihydroxy-4-ethyl-3-hexanone (4g)
Colourless oil (115 mg, 72%, ee >95%). [a]2D0 +6.6 (c 0.5, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 0.75 (t, 3H, J = 7.0 Hz, CH3) 1.15
(t, 3H, J = 7.0 Hz, CH3) 1.27 (d, 3H, J = 7.0 Hz, CH3) 1.65–1.75
(m, 2H, CH2) 2.54 (dq, 1H, J = 7.0 and 18.0 Hz, CH2CO) 2.68
(dq, 1H, J = 7.0 and 18.0 Hz, CH2CO) 4.05 (m, 1H, CHOH) 4.13
(br s, 1H, OH). 13C NMR (300 MHz, CDCl3) dC: 7.4, 7.5, 17.5,
27.8, 29.5, 71,2, 84.5, 213.8; ESI [MNa]+ m/z 183.1 Anal calcd for
C8H16O3: C, 59.97%; H, 10.07%. Found: C, 60.11%; H, 10.15%.
1-(2,2,4,5-Tetramethyl-1,3-dioxolan-4-yl)-propanone (5b)
Colourless oil (79 mg, 85%, ee >95%). [a]2D0 +5.2 (c 1.0, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 1.03 (t, 3H, J = 7.0 Hz, CH3)
1.2 (s, 3H, CH3) 1.28 (d, 3H, J = 7.0 Hz, CH3) 1.39 (s, 3H, CH3)
1.48 (s, 3H, CH3) 2.65–2.80 (m, 2H, CH2CO), 4.08 (q, J = 7.0 Hz,
CHOR). 13C NMR (300 MHz, CDCl3) dC: 7.2, 14.5, 19.1, 26.0,
28.4, 30.6, 74.6, 87.6, 108.2, 214.0; ESI [MNa]+ m/z 209.3 Anal
calcd for C10H18O3: C, 64,49%; H, 9,74%. Found: C, 64.14%; H,
9.66%.
Reduction of acetylacetoin (2a) with NaBH4
Acetylacetoin 2a (130 mg, 1.0 mmol) was dissolved in diethyl
ether–methanol 5 : 1 (8 mL), the solution was cooled (ice bath)
and NaBH4 (38 mg, 1.0 mmol) was added. The ice bath was
removed and the mixture was maintained at r. t. The reaction was
monitored by TLC (cyclohexane–ethyl acetate 1 : 1) and when the
conversion was complete 1 M HCl (5 mL) was added. The organic
layer was separated and the aqueous solution was extracted with
diethyl ether (2 ¥ 10 mL). The combined organic phases were
dried (Na2SO4) and the solvent was removed under vacuum. The
products were purified by short column chromatography (silica gel,
cyclohexane–ethyl acetate 2 : 1 as eluent) to obtain the mixture of
syn- and anti-4a (110 mg, 83%).
1-(2,2,4,5-Tetramethyl-1,3-dioxolan-4-yl)-buranone (5c)
Colourless oil (87 mg, 87%, ee >95%). [a]2D0 +46.5 (c 1.5, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 0.93 (t, 3H, J = 7.0 Hz, CH3) 1.21
(s, 3H, CH3) 1.28 (d, 3H, J = 7.0 Hz, CH3) 1.40 (s, 3H, CH3) 1.50
(s. 3H, CH3) 1.60 (sex, 2H, J = 7.0 Hz, CH2) 2.68 (t, 2H, CH2CO)
4.10 (q, 1H, J = 7.0 Hz, CHOR). 13C NMR (300 MHz, CDCl3)
dC: 13.7, 14.5, 16.4, 19.0, 25.9, 28.4, 39.2, 74.5, 87.5, 108.1, 213.4;
ESI [MNa]+ m/z 223.0. Anal calcd for C11H20O3: C, 65,97%; H,
10,07%. Found: C, 65.62%; H, 9.96%.
1
The H NMR spectrum of the mixture allows us to identify
1
the signals of anti-2,3-dihydroxy-3-methyl-pentan-2-one (4a): H
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1-(2,2,4,5-Tetramethyl-1,3-dioxolan-4-yl)-pentanone (5d)
dried (anhydrous Na2SO4) and evaporated under nitrogen. The
crude 2,2,4,5-tetramethyl-4-vinyl-1,3-dioxolane [selected data 1H-
NMR (CDCl3) dH: 3.96 (dd, 1H, J = 6.5 Hz, CHOR), 5.15 (dd,
1H, J = 1.5 and 11.0 Hz, CH2), 5.33 (dd, 1H, J = 1.5 and 17.5 Hz,
CH2), 5.88 (dd, 1H, J = 11.0 and 17.5 Hz, CH)] was dissolved in
anhydrous methanol (7 mL) and acidified with p-toluenesulfonic
acid. The reaction was stirred at room temperature and checked
by TLC (eluent: cyclohexane–ethyl acetate 1.5 : 1). When the
conversion was complete silica gel was added to the mixture in
order to absorb the product and the solvent was removed under
reduced pressure. Short column chromatography of the residue
using cyclohexane–ethyl acetate 2 : 1 as eluent afforded product 7
slightly contaminated (45 mg, 45%): 1H NMR (300 MHz, CDCl3):
d 1.18 (d, 3H, J = 6.5 Hz, CH3), 1.26 (s, 3H, CH3), 3,68 (q, 1H,
J = 6.5 Hz, CHOH), 5.22 (dd, 1H, J = 1.3 and 11.0 Hz, CH2), 5.38
(dd, 1H, J = 1.3 and 17.0 Hz, CH2), 5.92 (dd, 1H, J = 11.0 and 17.0
Hz, CH), 13C NMR (300 MHz, CDCl3): d 16.5, 21.5, 72.8, 75.6,
114.3, 142.73; ESI [MNa]+ m/z 139.2.
Colourless oil (92 mg, 86%, ee >95%). [a]2D0 +46.4 (c 0.8, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 0.92 (t, 3H, J = 7.0 Hz, CH3)
1.20 (s, 3H, CH3) 1.29 (d, 3H, J = 7.0 Hz, CH3), 1.30–1.36 (m, 2H,
CH2), 1.38 (s, 3H, CH3) 1.47 (s, 3H, CH3) 1.60–1.40 (m, 2H, CH2)
2.70 (t, 2H, J = 7.0 Hz, CH2CO) 4.10 (q, 1H, J = 7.0 Hz, CHOR).
13C NMR (300 MHz, CDCl3) dC: 13.9, 14.5, 19.1, 22.3, 25.1, 25.9,
28.4, 37.0, 74.5, 87.5, 108.1, 213.5; ESI [MNa]+ m/z 237.0. Anal
calcd for C12H22O3: C, 67,26%; H, 10,35%. Found: C, 67.17%; H,
10.51%.
1-(2,2,4,5-Tetramethyl-1,3-dioxolan-4-yl)-benzophenone (5e)
Colourless oil (106 mg, 91%, ee >95%). [a]2D0 +8.1 (c 0.8, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 1.20 (s, 3H, CH3) 1.43 (d, 3H,
J = 7.0 Hz, CH3) 1.48 (s, 3H, CH3) 1.55 (s, 3H, CH3) 4.56 (q,
1H, J = 7.0 Hz, CHOR) 7.40–8.25 (m, 5H, Ph). 13C NMR (300
MHz, CDCl3) dC: 15.5, 21.7, 25.8, 28.5, 75.7, 87.5, 108.0, 128.1,
130.4, 132.9, 134.7, 202.5; ESI [MNa]+ m/z 257.0. Anal calcd for
C14H18O3: C, 71,77%; H, 7,74%. Found: C, 71.32%; H, 7.81%.
Synthesis of (+)-citreodiol (8)
The diol 7 (45 mg, 0.39 mmol) was dissolved in anhydrous
dichloromethane–tetrahydrofuran 4 : 1 (10 mL) with acrolein (0.22
mL, 3.1 mmol) and Hoveyda-Grubbs 2nd generation catalyst
(23 mg, 7 mol%). The solution was heated to reflux for 6 h
and then the solvent was removed under reduced pressure. The
residue was dissolved in benzene (8 mL) together with methyl-2-
(triphenylphosphoranylidene)-propanoate (0.3 g, 0.86 mmol) and
the mixture was refluxed for 6 h. After evaporation, the residue was
purified by column chromatography (silica gel, cyclohexane–ethyl
acetate 2 : 1 as eluent) affording citreodiol 8 as a colourless oil:
[a]D20 +6.8 (c 0.7, CHCl3), lit19 +4.3 (c 1.0; CHCl3); 1H NMR (300
MHz, CDCl3): d 1.20 (d, 3H, J = 6.5 Hz, CH3), 1.30 (s, 3H, CH3),
2.0 (s, 1H, CH3), 3.75 (m, 1H, CHOH), 3.77 (s, 3H, CH3), 6.12
(d, 1H, J = 15 Hz, CH), 6.70 (dd, 1H, J = 11.0 and 15.0 Hz, CH),
7.20 (1H, J = 11.0 Hz, CH), 13C NMR (300 MHz, CDCl3): d 12.9,
17.0, 22.0, 52.0, 73.1, 75.7, 76.6, 124.8, 127.6, 137.5, 145.2, 168.9.
13.8, 17.0, 21.7, 22.3, 25.5, 35.6, 71.0, 81.0, 213.6. ESI [MNa]+
m/z 237.3.
1-(4-Ethyl-2,2,5-trimethyl-1,3-dioxolan-4-yl)-propanone (5g)
Colourless oil (85 mg, 85%, ee >95%). [a]2D0 +51.9 (c 0.5, CHCl3).
1H NMR (300 MHz, CDCl3) dH: 0.81 (t, 3H, J = 7.5 Hz, CH3)
1.02 (t, 3H, J = 7.0 Hz, CH3) 1.30 (d, 3H, J = 7.0 Hz, CH3) 1.42 (s,
3H, CH3), 1.40–1.55 (m, 1H, CH2), 1.49 (s, 3H, CH3), 1.82–1.95
(m, 1H, CH2), 2.27 (dq, J = 7.0 and 19.0 Hz, CH2CO) 2.64 (dq,
J = 7.0 and 19.0 Hz, CH2CO) 3.97 (q, 1H, J = 7.0 Hz, CHOC). 13
C
NMR (300 MHz, CDCl3) dC: 7.0, 7.7, 13.8, 25.0, 26.2, 28.4, 32.4,
75.0, 90.9, 108.4, 214.4; ESI [MNa]+ m/z 223.1, Anal calcd for
C11H20O3: C, 65,97%; H, 10,07%. Found: C, 70.12%; H, 10.13%.
Synthesis of 1-(2,2,4,5-tetramethyl-1,3-dioxolan-4-yl)-ethanol (6)
Compound 5a (150 mg, 0.87 mmol) was dissolved in diethyl
ether–methanol 8 : 1 (5 mL), the solution was cooled (ice bath)
and NaBH4 (10 mg, 0.26 mmol) was added. After the addition
the mixture was stirred at room temperature for 1 h and then
diluted with 1 M HCl (5 mL). The organic phase was separated
and the aqueous layer was extracted with diethyl ether (2 ¥
10 mL). The combined organic phases were dried (anhydrous
Na2SO4), evaporated and the residue was purified by column
chromatography (silica gel, cyclohexane–ethyl acetate 2 : 1 as
eluent) affording an equimolecular mixture of (4R,5S,6S)- and
(4R,5S,6R)-6 diastereomers (136 mg, 90%): 1H NMR (300 MHz,
CDCl3) selected data: d 1.04, 1.08, 1.35, 1.36, 1.43 and 1.44 (6 s, 6
¥ 3H, 6CH3), 3.58 and 3.75 (2q, 2 ¥ 1H, J = 6.5 Hz, CHOH), 4.14
and 4.24 (2q, 2 ¥ 1H, J = 6.5 Hz, CHOR). ESI [MNa]+ m/z 197.1.
Acknowledgements
We gratefully acknowledge Cambrex-IEP GmbH Wiesbaden
Germany and ICE srl Reggio Emilia Italy for the financial support.
Thanks are due to Dr Tatiana Bernardi and Mr. Paolo Formaglio
for technical assistance in ESI-MS and NMR analysis.
Notes and references
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Org. Biomol. Chem., 2011, 9, 8038–8045 | 8045
©