First stereoselective acylation of a primary diol possessing a prochiral quaternary center mediated by lipase TL from Pseudomonas stutzeri

Article abstract of DOI:10.1016/j.tet.2015.09.056

The first described acylation of a primary diol possessing a prochiral quaternary center catalyzed by lipase TL from Pseudomonas stutzeri is described. Optimized conditions were designed by testing different experimental conditions on model substrates (cy

Full text of DOI:10.1016/j.tet.2015.09.056

Tetrahedron 71 (2015) 9172e9176
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First stereoselective acylation of a primary diol possessing
a prochiral quaternary center mediated by lipase TL from
Pseudomonas stutzeri
,
,
y
b
b
b
b,
*
A. Pizzilli a y, R. Zoppi b , P. Hoyos , S. Gomez , F.G. Gatti , M.J. Hernaiz
,
ꢀ
ꢀ
b,
*
ꢀ
A.R. Alcantara
a Giulio Natta Politecnico di Milano, Department of Chemistry, Materials & Chemical Engineering, Piazza Leonardo Da Vinci 32, I-20133, Milan, Italy
b
ꢀ
Biotransformations Group, Department of Organic and Pharmaceutical Chemistry, Pharmacy Faculty, Complutense University of Madrid, Plaza Ramon
y Cajal s/n, Madrid, E-28040, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2015
Received in revised form 21 September 2015
Accepted 22 September 2015
Available online 9 October 2015
The first described acylation of a primary diol possessing a prochiral quaternary center catalyzed by li-
pase TL from Pseudomonas stutzeri is described. Optimized conditions were designed by testing different
experimental conditions on model substrates (cyclopentylmethanol, cyclohexylmethanol, cyclopentane-
1,1-diyldimethanol or cyclohexane-1,1-diyldimethanol) to find best organic solvent, optimal acyl donor
and temperature, as well as the optimal substrates/enzyme ratio. Lipase TL resulted the best biocatalyst,
while vinyl butyrate as acylating agent and a mixture of isooctane/THF 8/2, (v/v) resulted the best ex-
perimental conditions. Under these conditions, reaction were monitorized by chiral HPLC (diffraction
index detector). The enantiomeric excess in the acylation of target substrate, (tetrahydro-2H-pyran-2,2-
diyl)dimethanol, was measured by derivatization of monoesters with Mosher’s R-MTPA-Cl, which also
was useful to determine the S absolute configuration of the major reaction product of the lipase-
catalyzed monoacylation.
Keywords:
Acylation
Biocatalysis
Chirality
Lipase
Configuration determination
NMR spectroscopy
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
1, 1) is becoming a fashionable trend in biocatalysis,17e20 using
mainly hydrolases or lyases, the most common strategy consists in
The stereoselective synthesis of molecules bearing a quaternary
stereocenter is one of the most challenging tasks1e6 of organic
chemistry, being this situation particularly problematic for acyclic
compounds, because of the high number of degrees of freedom
associated to these structures;4 thus, the enantioselective desym-
metrization of either prochiral precursors or meso compounds is
generally the most effective methodology.
using a spacer moiety for separating the bulky quaternary stereo-
center from the point in which the enzyme is acting.
Although the exquisite stereodiscrimination ability of enzymes
is well known7e11 and it has been recurrently reported,12,13 espe-
cially in desymmetrization processes involving either meso or
prochiral structures,14,15 in most cases it has been applied to com-
pounds possessing tertiary carbon chiral or prochiral centers, be-
cause most of the typical hydrolytic enzymes are unable to accept
sterically hindered substrates bearing quaternary carbon atoms.16
However, since the kinetic resolution of tertiary alcohols (Scheme
Scheme 1. Different compounds possessing quaternary centers recognized by
enzymes.
Accordingly, some biocatalyzed resolutions of primary alcohols
(Scheme 1, 2) with remote quaternary stereocenters, either through
enzyme-catalyzed
acylation
or
hydrolysis,
have
been
described.21e33 In the same way, sterically crowded diesters
(Scheme 1, 4) have been enzymatically resolved as well.34e37
Perhaps one of the most attractive research area is the resolution
of diols possessing quaternary carbon atoms (Scheme 1, 3). For this
type of compounds, two different cases must be considered,
* Corresponding authors. Tel.: þ34 91 394 1820 (A.R.A.), þ34 91 394 1821
ꢀ
(M.J.H.); fax: þ34 91 394 1822; e-mail addresses: mjhernai@ucm.es (M.J. Hernaiz),
ꢀ
andalcan@ucm.es (A.R. Alcantara).
y
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.tet.2015.09.056
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

A. Pizzilli et al. / Tetrahedron 71 (2015) 9172e9176
9173
depending on the symmetry: i) 1,1-disubstituted 1,2-diols, molecules
possessing a primary and a secondary hydroxyl group (n¼0, ms0)
and ii) molecules having two primary hydroxyl groups (ns0, ms0).
However, for the second ones (Scheme 2, 5), due to the fact that
they do not possess any stereogenic center, conversion is not lim-
ited up to theoretical 50%, so that the enantiomeric composition of
the product (R or S-6) is independent on the conversion, and thus
the reaction may be run to completion. In that sense, different
examples can be found in literature of desymmetrization of pro-
chiral 2,2-disubstitued-1,3-diols generally through stereoselective
mono-acylation of 535,38e44 or less frequently by mono-hydrolysis
of diesters 7.45,46 In all cases, conversion and enantiomeric excess
values reported varied noticeably, as well as the absolute configu-
ration of the monoester 6.
Scheme 4. Desymmetrization of (tetrahydro-2H-pyran-2,2-diyl)dimethanol 15.
2. Results and discussion
Thus, as the first step in order to select the best conditions, the
acylation of primary cycloalkyl methanols 19 and 20 with different
vinyl esters 16 (Scheme 5) was carried out as a qualitative test to
establish the best lipase (See Supplementary data).
Scheme 2. Desymmetrization of prochiral 2,2-disubstitued 1,3-diols.
In this sense, Fukuyama and co-workers41 reported the total
synthesis of Leustroducsin, a colony stimulating factor inducer
isolated from of Streptomyces platensis SANK 60191. In their strat-
egy, the desymmetrization of the glycerol derivative 8 (Scheme 3, a)
was catalyzed by lipase AK-mediated acylation. An ulterior pro-
tection of the monoacetate 9 with tert-butylchlorodimethylsilane
yielded the O-protected monoalcohol R-10 in good ee and high
Scheme 5. Transesterification of primary alcohols 19 and 21.
Biocatalysts tested in this test reactions were lipases from
Pseudomonas stutzeri (lipase TL) Alcaligenes sp. (lipase QLC),
Candida rugosa, Pig Pancreatic lipase and Amano lipase PS (see
Supplementary data). Lipase TL and lipase QLC were apparently
the best catalysts, while vinyl butyrate 16a seemed to lead to
better overall conversion after 24 h. These qualitative results
were confirmed once the reaction was repeated after a shorter
reaction time (200 min) for lipase TL, lipase QLC and C. rugosa
lipase, also confirming the best performance of 16a as acyl donor.
Solvent tested were isooctane, THF and 2-MeTHF, being the first
one the best.
yield. Similarly, S-a-tocotrienol 14, a very interesting molecule with
many therapeutical effects,47 has been prepared in 19% overall yield
through a multistep synthesis44,48 that involves the CALB-catalyzed
acetylation of the bicyclic triol 11 (Scheme 3, b). The monoester S-
12 was obtained in 98% ee and 60% yield, which in principle could
be increased, since the diester 13 (27%) can be easily recycled by
a simple chemical hydrolysis. The assignation of the absolute con-
figuration of S-12 was also done through derivatization with
Mosher’s esters.48
In order to quantify the activity of lipases, acylation of cyclo-
pentylmethanol 19 was used as test for determining the acyl-
transfer activity of Lipase TL, using isooctane as solvent. As a re-
sult of calculation, activity resulted in a value of 8.15ꢀ10ꢁ4 units
mgꢁ1, defining one unit as the amount of enzyme necessary to
synthesize 1 mmol of 20b per minute under the described reaction
conditions (see Supplementary data). This enzyme has been fre-
quently used in our group, showing excellent results in many
biotransformations.51,52
In a second step, all the information gathered up to this point
about the catalytic performance of Lipase TL was applied to the
acylation of a primary symmetrical diol such as the cyclopentane-
1,1-diyldimethanol 23 (Scheme 6), for order to determining the
best conditions for attempting the stereoselective monoacylation of
our target molecule 15. Nevertheless, a previous solubility problem
had to be taken into account provided that 23 turned out to be
Scheme 3. Some acylation of prochiral quaternary diols described in literature.
In this paper we report the first results obtained in the
desymmetrization of a very simple prochiral 2,2-disubstitued 1,3-
diol such as (tetrahydro-2H-pyran-2,2-diyl)dimethanol 15
(Scheme 4) through lipase-catalyzed acylation. This diol has been
used as starting material in the preparation of different compounds
for treating hair loss from the human scalp,49 as well as for the
synthesis of compounds useful as TNF-alpha inhibitors.50 Thus,
different parameters were studied in order to optimize the re-
action, such as lipase nature, type of organic solvent, type of acyl
donor, etc.
Scheme 6. Acylation of cyclopentane-1,1-diyldimethanol 23.

9174
A. Pizzilli et al. / Tetrahedron 71 (2015) 9172e9176
insoluble in isooctane, the best organic solvent thus far, while THF
proved to be the best option to solubilize 23.
First reactions were then carried out using a low concentration
of acyl donor in order to limit monoacylation of 23 (0.5/1, M/M) in
different reaction media, ranging from 100% THF up to a binary
mixture 80/20 isoctane/THF (v/v), the higher possible percentage of
isooctane to ensure solubility of 23. Nevertheless, very low con-
versions were obtained (not higher than 10% in the best case) as
well as very low reaction rates, when using standard amount of
lipase TL (10 mg). Thus, acyl donor ratio was increased from 0.5/1
up to 1.5/1, showing a clear improvement of reaction rate and
conversion by increasing the amount of acyl donor. Finally, we
doubled up the amount of lipase for accelerating reaction rate, and
monitored the reaction upon a longer time (24 h) to control the
relative amount of diacylation. As it can be seen in Fig. 1, the re-
action rate is increased, although diacylated compound 25 becomes
considerably noticeable at relatively short reaction times, avoiding
the accumulation of 24 by a second entry of 24 at the lipase active
site leading to diacylation, as described in our group in the acyla-
tion of prochiral 2-phenyl-1,3-propanediol.53,54
Scheme 7. Derivatization of monester 17 by reaction with R-MTPA-Cl R-26.
Reaction yields were very similar (54% yield of 17, at room
temperature, and 42% yield at 37 ꢂC), and the derivatization was
done as described in Experimental Section. To measure the enan-
tiomeric excess, the relative proportion of the signals between 4.3
and 4.0 ppm, corresponding to the methylene groups near the
oxygen atom of the esters was analyzed. According to literature,55
the preferred conformation of a S-Mosher’s ester for secondary
alcohols, keeping H, carbonyl and CF3 group in a coplanar ar-
rangement, is the one shown in Scheme 8, a).
Scheme 8. a) Preferred conformation for the S-Mosher’s ester. b) Assumed minimum
energy conformation of S,S-27.
In fact, if we extrapolate that model to S,S-diastereomer,
(Scheme 8, b), the shielding effect of Ph group would affect more
specifically those diasterotopic methylene hydrogens in red. In
Fig. 2 we show an ampliation of the NMR spectra of Mosher’s esters
S,S-27 and S,R-27 obtained in the scaled reaction, as well as the
chromatograms obtained in each case.
Fig. 1. Acylation of 23. Experimental conditions as described in Experimental Section.
Finally, the acylation of the target prochiral substrate (tetrahy-
dro-2H-pyran-2,2-diyl)dimethanol 15 was undertaken, as depicted
in Scheme 4, considering all the previous results. Nevertheless, the
chiral column used so far (Chiralcel OD-RH) could not properly
separate R-17 and S-17, so that HPLC analysis were only useful for
determining overall conversion. Following the results obtained in
the acylation of model substrate 23, solvent used was a mixture of
isooctane and THF (80/20 v/v), 16a was added in a 1.5 M excess, and
20 mgs of lipase was used. Under these conditions, after 180 min no
traces of 15 were detected, and 54% of conversion of monoester 17
and 46% of diester 18 were present at that mentioned reaction time
at room temperature.
Fig. 2. NMR spectra of reaction obtained after derivatization and HPLC peak corre-
sponding to reactions with chemically synthesized rac-17 (a) and enzymatic reactions
at room temperature (b) and 37 ꢂC (c).
Raising up temperature to 37 ꢂC (optimal for acylation of 18)
resulted in a decrease in the formation of monoester 17 (37%) and
a concomitant increase in diester production (63%).
In order to determine the enantiopurity of monoester 17, the
same reactions carried out for analytical purposes were scaled-up
five times and R-17 and S-17 were converted to the correspond-
ing Mosher’s esters S,S-27 and R,S-27, as shown in Scheme 7.
Considering integration for determining enantiomeric excess
values, based on the signals at
d¼4.01e4.04 and
d¼3.96e3.94
(assigned to diastereotopic hydrogens at methylene closer to the O-
MTPA moiety), it can be deduced that for acylation of 15 at room

A. Pizzilli et al. / Tetrahedron 71 (2015) 9172e9176
9175
temperature (Fig. 2, a), the ee value of monoester 17 is 53%, while
3.2. Acylation procedures for diols
3.2.1. Chemical acylation of cyclopentane-1,1-diyldimethanol 23. To
increasing reaction temperature up to 37 ꢂC (Fig. 2, b) not only has
a deleterious effect on the yield, as commented previously, but also
on the ee value, which is reduced to 44%.
a stirred solution of triethylamine (1.5 mmol,156
m
L, d¼0.727 g/mL)
On the other hand, for assigning the absolute configuration of
in dichloromethane (15 mL) at ꢁ78 ꢂC, cyclopentane-1,1-
the products, the higher intensity of signals at
4.30e4.28 (diasteromer R,S-27) compared to those other ones at
d
¼3.96e3.94 and
diyldimethanol 23 (200 mg, 1.5 mmol) and butyryl chloride
(2.25 mmol, 233.6
mL, d¼1.026 g/mL) were added. The reaction was
d
¼4.01e4.04 and 4.22e4.20 (S,S-27) indicates that R,S-27 is the
quenched after 3 h by addition of a phosphate buffer (pH 7.0). The
organic materials were extracted with ether and combined extracts
were dried over sodium sulfate and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(AcOEt/hexane, 1/6, v/v) to yield 159 mg of (1-(hydroxymethyl)
cyclopentyl)methyl butyrate 24 (49.0%) and 19.5 mg of cyclo-
pentane-1,1-diylbis(methylene) dibutyrate 25 (4.8%).
major component, so that the enzyme is primarily generating S-17
with moderate ee, as schematized in Scheme 9.
3.2.1.1. (1-(hydroxymethyl)cyclopentyl)methyl butyrate 24. 1H
NMR (250 MHz, CDCl3): 4.05 (s, 2H), 3.35 (s, 2H), 2.85 (s, 1H), 2.36
(t, J¼7.3 Hz, 2H), 1.75e1.58 (m, 6H), 1.49e1.37 (m, 4H), 0.90 (t,
J¼7.3 Hz, 3H); 13C NMR (63 MHz, CDCl3): 175.0, 68.2, 66.8, 48.3,
36.6, 32.3, 25.6, 18.9, 14.0. Anal. Calcd for C11H20O3: C, 66.13%; H,
10.07%; found: C, 66.25%; H, 10.05%.
3.2.1.2. Cyclopentane-1,1-diylbis(methylene) dibutyrate 25. 1H
NMR (250 MHz, CDCl3): 4.0 (s, 4H), 2.4 (t, J¼7.4 Hz, 4H), 1.82e1.63
(m, 8H), 1.61e1.48 (m, 4H), 1.02 (t, J¼7.4 Hz, 6H); 13C NMR (63 MHz,
CDCl3): 174.2, 67.7, 46.0, 36.6, 32.8, 25.6, 18.9, 14.1. Anal. Calcd for
Scheme 9. Stereochemical course of acylation of 15.
C15H26O4: C, 66.64%; H, 9.69%; found: C, 66.83%; H, 9.66%.
This slightly higher acylation in the pro-S hydroxymethyl group
of 15 is consistent with the reported pro-S preference of other li-
pases, as shown in Scheme 3. The higher enantiomeric excess de-
scribed for those reported cases should be attributed to the higher
rigidity of 8 and 11 compared to 15, so different experiments are
being conducted to prove the efficiency of lipase TL upon less
flexible substrates.
To conclude, this is the first reported example of the use of lipase
TL from P. stutzeri in the stereoselective acylation of prochiral pri-
mary diol possessing a quaternary stereo center. The results shown
are opening the door for further application of this very interesting
catalyst in the preparation of optically enriched building blocks.
3.2.2. Enzymatic acylation of cyclopentane-1,1-diyldimethanol
23. Different amounts of vinyl butyrate 16a and 0.1 mmol of
cyclopentane-1,1-diyldimethanol 23 were dissolved in 1 mL of
several organic solvents, and reactions were started at 37 ꢂC by
adding a fixed amount of lipase. The reactions were qualitatively
followed by TLC (mobile phase n-hexane/ethyl acetate, 5/1, v/v). To
quantify reaction conversion, aliquots of 20 mL were taken at dif-
ferent reaction times, microfiltered (0.2 nm), re-dissolved in 1 mL
n-hexane, evaporated by speed-back and then dissolved again in
a 500 mL mixture CH3OH/CH3CN (50/50, v/v), to be analyzed by
HPLC (Chiralcel-OD-RH column, RI detector), using a mobile phase
H2O/CH3CN (60/40, v/v) and toluene as internal standard (calibra-
tion curves for qualitative determination of 24 and 25 in
Supplementary data); flow rate: 0.6 mL/min (tr: 6.7 min).
3. Experimental section
3.1. General
3.3. Chemical synthesis of (2-(hydroxymethyl)tetrahydro-2H-
pyran-2-yl)methyl butyrate 17 and (tetrahydro-2H-pyran-2,2-
diyl)bis(methylene) dibutyrate 18
Lipases from P. stutzeri (Lipase TLÒ) and immobilized lipase from
Alcaligenes sp (Lipase QLC) were kindly donated by Meito & Sangyo
Ltd, Japan. Lipases from C. rugosa, Amano Lipase PS and porcine
pancreas were purchased from SigmaeAldrich. All other reagents
and solvents were obtained from SigmaeAldrich and used as
received.
110 mg (0.75 mmol) of (tetrahydro-2H-pyran-2,2-diyl)dime-
thanol 15 and butyryl chloride (117
were added to a stirred solution of triethylamine (0.75 mmol,
m
L, 13 mmol, d¼1.026 g/mL)
NMR spectra were recorded on a Bruker AC-250 or Bruker AV
104
m
L, d¼0.727 g/mL) in dichloromethane (10 mL) at ꢁ78 ꢂC. The
500 MHz. Chemical shifts (
(ppm) relative to CHCl3 (1H:
77.0 ppm).
TLC was carried out on aluminium sheets precoated with silica
gel; the spots were visualized under UV light (
¼254 nm). Column
chromatography purifications were conducted on silica gel 60
(40e63 m). All enzymatic reactions were monitored by HPLC. For
d
) are reported in parts per million
reaction was quenched after 3 h at ꢁ78 ꢂC by addition of a phos-
phate buffer (pH 7). The organic materials were extracted with
ether and combined extracts were dried over sodium sulfate and
concentrated under vacuum. The residue was purified by column
chromatography on silica gel (AcOEt/hexane, 1/6, v/v) to yield
71.6 mg of (tetrahydro-2H-pyran-2,2-diyl)bis(methylene) dibuty-
rate 18 (33.4% yield).
d
7.27 ppm) and CDCl3
(
13C:
d
l
m
reagents containing chromophoric groups, chiral HPLC analysis
were performed on a chromatograph equipped with a Chiralcel-OD
column and Diode Array detector; mobile phase: n-hexane/i-PrOH
(98/2, v/v); flow rate: 0.6 mL/min. For the rest of compounds, HPLC
analysis were performed on a chromatograph equipped with
a Chiralcel-OD-RH column and a Refractive Index (RI) detector;
mobile phase: H2O/CH3CN (60/40, v/v); flow rate: 0.6 mL/min.
Same reaction was carried out at 1 h with a 1/1 M ratio of butyril
chloride and 15 to isolate racemic monoester 17 (46 mg, 48.1%).
3.3.1. (2-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)methyl butyrate
17. 1H NMR (250 MHz, CDCl3): 4.19 (s, 2H), 3.65 (t, J¼5.3 Hz, 2H),
3.53 (s, 2H), 2.28 (t, J¼7.5 Hz, 2H), 1.67e1.38 (m, 8H), 0.90 (t,
J¼7.4 Hz, 3H); 13C NMR (63 MHz, CDCl3): 174.3, 74.3, 64.6, 63.6, 62.7,

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A. Pizzilli et al. / Tetrahedron 71 (2015) 9172e9176
36.5, 27.3, 25.7, 19.0, 18.9, 14.0. Anal. Calcd for C11H20O4: C, 61.09%;
H, 9.32%; found: C, 61.19%; H, 9.35%.
References and notes
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3.3.2. (Tetrahydro-2H-pyran-2,2-diyl)bis(methylene) dibutyrate 18. 1H
NMR (250 MHz, CDCl3): 4.10 (dd, J¼10.1 Hz, J¼31.5 Hz, 4H), 3.65
(t, J¼5.8 Hz, 2H), 2.25 (q, J¼7.3 Hz, 4H), 1.68e1.41 (m, 10H), 0.87
(t, J¼7.3 Hz, 6H); 13C NMR (63 MHz, CDCl3): 173.8, 73.2, 64.2, 62.7,
36.5, 32.8, 28.0, 25.6, 18.8, 14.0. Anal. Calcd for C15H26O5: C, 62.91%;
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62.7, 55.8, 36.4, 28.0, 25.5, 19.0, 18.8, 18.8, 14.0.
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Acknowledgements
This work was supported by a research project of the Spanish
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MINECO (Ministerio de Ciencia e Innovacion de Espana) CTQ2012-
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2015.09.056.
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