Efficient desymmetrization of 4,6-di-O-benzyl-myo-inositol by Lipozyme TL-IM

Article abstract of DOI:10.1016/j.carres.2013.11.005

The enantioselective enzymatic desymmetrization of 4,6-di-O-benzyl-myo- inositol, a myo-inositol derivative, was effectively catalyzed by Thermomyces lanuginosus lipase (TL-IM). The product 1D-1-O-acetyl-4,6-di-O-benzyl-myo- inositol, a useful precursor t

Full text of DOI:10.1016/j.carres.2013.11.005

Carbohydrate Research 386 (2014) 7–11
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Efficient desymmetrization of 4,6-di-O-benzyl-myo-inositol
by Lipozyme TL-IM
Marcela G. Vasconcelos a, Raissa H. C. Briggs a, Lucia C. S. Aguiar b, Denise M. G. Freire c,
Alessandro B. C. Simas a,
⇑
a Universidade Federal do Rio de Janeiro (UFRJ), Núcleo de Pesquisas de Produtos Naturais (NPPN), CCS, bloco H, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
b UFRJ, Instituto de Química, CT, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
c UFRJ, Instituto de Química, CT, Programa de Pós-Graduação em Bioquímica, Departamento de Bioquímica, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2013
Received in revised form 27 October 2013
Accepted 11 November 2013
Available online 23 November 2013
The enantioselective enzymatic desymmetrization of 4,6-di-O-benzyl-myo-inositol, a myo-inositol deriv-
ative, was effectively catalyzed by Thermomyces lanuginosus lipase (TL-IM). The product 1D-1-O-acetyl-
4,6-di-O-benzyl-myo-inositol, a useful precursor to inositol phosphates, was obtained in excellent yield
and enantiomeric excess. Through the investigation of the effects of solvent, biocatalyst load, and temper-
ature, a more economical procedure resulted. The feasibility of biocatalyst reuse was also shown.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Desymmetrization
TL-IM Lipase
Thermomyces lanuginosus
Inositol
Enantioselectivity
Chiral
1. Introduction
activity in organic solvents.8 By means of practical kinetic resolu-
tion of racemic substances or desymmetrization of prochiral com-
Chiral myo-inositol derivatives, such as phosphatidylinositols
and inositol phosphates (e.g., D-myo-inositol 1,4,5-trisphosphate)
are fundamental tools for the study on cellular signaling
processes.1
pounds, such biocatalysts allow the preparation of chiral synthetic
blocks in high ee.
Few reports have dealt with the application of lipases and other
hydrolases (EC 3.1.1.3) in the syntheses of inositols from myo-ino-
sitol itself via resolutions.9,10 Even fewer studies were devoted to
the desymmetrizations of myo-inositol derivatives via hydro-
lases.11,12 myo-Inositol derivatives are interesting subjects for the
study of desymmetrization protocols. Different protocols (based
upon catalytic reactions or the use of chiral auxiliaries)13,14 have
been devised with this intent. Enzyme-catalyzed desymmetriza-
tions are relevant means for the preparation of chiral substances
in high chemical yields and ee.15 Lipases and related biocatalysts
are particularly effective in carrying out such transformations. Nat-
urally, with the possibility of nearly complete conversions to the
desired products, the biocatalyzed desymmetrization of inositols
allows a better match between economy and practicality than
the corresponding kinetic resolutions. Laumen and Ghisalba devel-
oped an efficient procedure for the desymmetrization of 4,6-di-O-
benzyl-myo-inositol, 1 (Scheme 1).11 The authors reported that
Amano PS lipase (EC 3.1.1.3) (lyophilized) catalyzed this transfor-
mation in vinyl acetate, leading to the mono-O-acetylated
compound, 1D-1-O-acetyl-4,6-di-O-benzyl-myo-inositol, D-(ꢀ)-2,
in 89% (after recrystallization). The achieved enantioselectivity
Although much development has occurred in the chemistry of
inositols,2,3 most syntheses of these relevant substances still rely
on optical resolutions with chiral auxiliaries, with all disadvan-
tages that such procedures display. The use of myo-inositol itself
as precursor in the enantioselective syntheses of bioactive myo-
inositols derivatives is, in many cases, rewarding. All carbon atoms
in the carbocyclic backbone of that largely available, pro-chiral
starting material are stereoselectively hydroxylated, which may
be conducive to short synthetic routes. Nevertheless, this usually
implies the need of optical resolutions of racemic intermediates.
The use of enzymes as synthetic tools for the enantioselective
synthesis of natural products and other bioactive substances has
gained increased attention in recent years.4,5 In this context, li-
pases and other hydrolases, especially those from microbial
sources, have found numerous applications,6,7, due to high regio-,
chemo- and stereoselectivies, availability, practicality, and good
⇑
Corresponding author. Tel.: +55 21 2562 6792; fax: +55 21 2562 6512.
E-mail address: abcsimas@nppn.ufr.br (A.B.C. Simas).
0008-6215/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2013.11.005

8
M. G. Vasconcelos et al. / Carbohydrate Research 386 (2014) 7–11
OH
OH
HO
AcO
OBn
OH
OBn
HO
HO
OH
OBn
OBn
Scheme 1. Desymmetrisation of 4,6 -di-O-benzyl myo-inositol.
Table 1
(ee >99%) of desymmetrization itself was not determined, as they
chose to record it for the deacetylated acetal derived from D-2.
Moreover, the reaction time of such transformations was not dis-
closed. Compound 1 is a precursor of relevant bioactive myo-inosi-
tol derivatives, such as phosphoinositides.16
Herein, we report on an alternative, efficient, and fast desym-
metrization of inositol 1 by an immobilized, thermostable lipase.
A detailed investigation on the performance of the most active li-
pase under different conditions and on the reaction economy
was carried out. Moreover, the biocatalyst reuse was shown to be
feasible, upon selection of more compatible solvents. Such features
of synthesis of D-2 have not been addressed before. Furthermore,
our study established the enantioselectivity in this enzymatic
transformation for the first time.
Screening of lipases for the desymmetrization of 1 in vinyl acetate
Entry
Lipase
Conversion (%)a
1
2
3
TL-IM
RM-IM
Lipomod 34P
95
19
30
a
48h-reactions.
2.2. Determination of ee
The enantiomeric excesses were determined by chiral HPLC
analyses (Fig. 2). All three enzymes were highly enantioselective
(ee >99%; E >100). The racemic sample (DL-2) for these analyses
was obtained by controlled acylation of tetrol 1 with Ac2O (Et3N,
DMAP, CH2Cl2, ꢀ20 °C) which resulted in a mixture of mono- and
polyacetates. The fraction containing the monoacetates was fur-
ther resolved by HPLC, affording enough DL-2 to develop the ana-
lytical protocol for ee determinations (Fig. 2a). These analyses
showed that all lipases led to the same enantiomer, D-(ꢀ)-2. Due
to the high performance of Lipozyme TL-IM, we focused on this
lipase.
2. Results and discussion
2.1. Selection of lipases and initial analyses
Eight commercially available lipases (see experimental section)
were used as biocatalysts against myo-inositol derivative 1 in vinyl
acetate as solvent. Lipozyme TL-IM (Thermomyces lanuginosus) was
the only enzyme to afford nearly full conversion (Fig. 1) to the acyl-
ated product. RM-IM (Rhizomucor miehei) and Lipomod 34P (Can-
dida cylindracea) led to the monoacetylated product with low
conversions (Table 1). Essentially, a single regioisomer was pro-
duced with all active enzymes. Di- and poly-acylated products
were not formed.
A preparative reaction of acylation of substrate 1 under TL-IM
catalysis was undertaken. The isolated product was purified and
analyzed. Firstly, its 1H NMR spectrum showed that the acetylation
occurred at the hydroxyl group at C-1 (or C-3), thus yielding ace-
tate 2. Moreover, the spectrum of 2 was consistent with the data
in the literature for the same compound.11 The specific rotation
of 2 indicated that the D-enantiomer, 1D-1-O-acetyl-4,6-di-O-ben-
zyl-myo-inositol, (D-(ꢀ)-2) was produced and, by comparison to
the value recorded by Ghisalba and Laumen, it suggested that a
high enantioselectivity was achieved.
2.3. Solvent selection for the desymmetrization of myo-inositol
derivative 1 by Lipozyme TL-IM
Organic media have a strong influence on the enzymatic activity
and the enantioselectivity of lipases.17 Therefore, we assayed the
desymmetrization of 1 with vinyl acetate (1:1 ratio with solvent)
by Lipozyme TL-IM in three different solvents: TBME (tert-butyl
methyl ether), EtOAc, and hexanes (Table 2). The time course of
the acylation of 1 for each condition (besides the one with vinyl
acetate as solvent/acylating agent) was established. The experi-
ment employing vinyl acetate as solvent showed that under this
condition the desymmetrization of 1 was virtually complete after
10 h. High ee and E were achieved in all cases. The use of EtOAc
as solvent led to lower conversions in the first hours of the reac-
tion. However, with time, such conditions overcame the one with
Figure 1. HPLC analysis of conversion in the reaction of substrate 1 with vinyl acetate catalyzed by a lipase.

M. G. Vasconcelos et al. / Carbohydrate Research 386 (2014) 7–11
9
Figure 2. Chromatographic determination of ee for the desymmetrization of 1 in vinyl acetate by TL-IM: (a) DL-2, (b) D-(-)-2.
Table 2
Table 3
Time course of conversions in the resolution of 1 catalyzed by TL-IM at 30 °C with
vinyl acetate as acylating agent in different solvents
Effects of temperature and substrate/enzyme ratio on the desymmetrization of 1 by
TL-IM in hexane using vinyl acetatea
Entry
Solvents
Conversion (%)a
Stereoselectivityb
Entry
Time (h)
45 °Cb
60 °Cb
1 h
2 h
6 h
10 h
24 h
eep (%)
E
1:3c
1:5c
95.3
95.8
95.8
100.0
100.0
1:3c
1:5c
97.9
99.0
100.0
99.4
1
2
3
4
Vinyl acetate
Ethyl acetate
Hexane
55
44
52
86
77
70
88
97
95
96
97
97
99
100
100
98
95
96
96
96
>99
>99
>99
>99
>100
>100
>100
>100
1
2
3
4
5
1
2
6
12
24
55.0
88.9
95.8
99.2
95.5
83.0
94.8
99.4
98.9
98.3
TBME
98.5
a
Formation of D-2.
Stereoselectivity after 24 h.
b
a
b
c
eep >99% (E >100) in all cases (after 24 h).
% Conversions to D-2 at each temperature.
Inositol 1/TL-IM ratio.
vinyl acetate as solvent. Our data show that more hydrophobic sol-
vents (hexanes, TBME) improved the enzymatic reaction, due to
substantial accelerations.
The desymmetrization of tetrol 1 in hexanes surpassed the pro-
tocol with vinyl acetate as solvent in conversion (see Section 4.5)
after 6 h and was complete after 10 h. This effect was even more
intense with TBME as solvent, as very high conversion (higher than
that obtained with vinyl acetate as solvent after 10 h) was ob-
served after 2 h. In all conditions, conversion erosions (related to
reversal to 1, as no by-product was detected) were observed after
it reached a maximum.
The desymmetrization of 1 using ethyl acetate both as solvent
and acylating agent was also attempted but no conversion was ob-
served in 24 h. Therefore, taking into account that (compared to
the reaction in vinyl acetate) less acylating agent was available in
the reaction with EtOAc/vinyl acetate and that EtOAc was shown
not to be effective as acylating agent, it is clear that EtOAc as sol-
vent (in combination with vinyl acetate) had, in fact, a beneficial
effect.
Figure 3. Time course of conversion (to D-2) in the desymmetrization of 1 by TL-IM
at different temperatures and substrate/enzyme ratios.
the enzyme load (entries 3–5). At the same temperature, the reac-
tion was essentially complete after 12 h employing only the 1:3 the
ratio. On the other hand, at 60 °C, the acylation is virtually com-
plete after 6 h and 2 h when the substrate/enzyme ratio was 1:3
and 1:5, respectively (entries 2 and 3).
Thus, higher temperatures allow lower loads of biocatalyst in
the desymmetrization of inositol 1 and shorten such enantioselec-
tive transformation. These results also show the stability of TL-IM
lipase is maintained in this reaction.
2.4. Effects of temperature and enzyme ratio
Lipases from Thermomyces lanuginosus are known for their high
thermal stability.18 Such property encouraged us to investigate the
effect of temperature and enzyme concentration (substrate/en-
zyme ratio) on the enzymatic desymmetrization of 1 as a means
to minimize the enzyme load. We chose to use hexane as solvent
(instead of TBME) for economic and safety reasons. Thus, stirred
mixtures of myo-inositol 1 and lipase TL-IM (1:3 or 1:5 mass ra-
tios), vinyl acetate in hexane were heated to 45 °C or 60 °C and
the conversions were determined at different reaction times (Ta-
ble 3; Fig. 3). Increasing the temperature to 45 °C enabled a signif-
icant decrease of enzyme load. Actually, when the reaction was run
at 45 °C, virtually the same conversion was obtained with a 1:3 ra-
tio after 2 h (Table 3, entry 2) as was obtained with a 1:10 ratio in
the same time (see Table 2, entry 3). Interestingly, the assays at
45 °C showed that, after 6 h, the conversion does not depend on
2.5. Reusability of Lipozyme TL-IM
The reusability of immobilized lipase is important for industrial
applications since the cost of lipase can make the processes eco-
nomically unattractive. Aiming at a more economical protocol,
experiments were performed to examine the reusability and the
stability of Lipozyme TL-IM. After every 2 h-cycle of the acylation
reaction of inositol 1, the immobilized lipase was recovered by

10
M. G. Vasconcelos et al. / Carbohydrate Research 386 (2014) 7–11
filtration, washed (three times) with the vinyl acetate-solvent mix-
ture and subsequently reused. Three reuse cycles were carried out,
with the conversions determined before and after each one of them
(Table 4).
and/or an aqueous basic solution of KMnO4, and heat as developing
agents. Analyses by TLC were done after 12, 24, and 48 h of the
reaction.
As shown in Table 3, the TL-IM lipase can be reused at least
three times. As usual,19 there was a gradual decrease in conversion
under all conditions. Good activity was retained by TL-IM in TBME
and, especially, in hexane. Taking into account the results shown in
Table 3, higher conversions for the reuse experiments run in hex-
ane at longer reaction times are expected. In fact, TL-IM, even in
smaller loads and at higher temperatures, proved to be very stable
over time.
Since the more hydrophilic solvents lead to steeper declines of
activity, such losses may be caused by removal of water content
of the catalyst. It is believed that the essential water layer on en-
zyme bestows flexibility, and hence, higher activity.8 Loss of bio-
catalyst due to handling and mass transfer issues may also be
responsible for the activity decays.20
4.3. Enzyme activity assay
The Lipozyme TL-IM activity was 500.0 U/g. It was quantified by
UV spectrophotometry at 410 nm from the hydrolysis reaction of
p-nitrophenyl laurate (pNPL) by lipase-catalyzed product forma-
tion with the chromophore p-nitrophenol. A solution containing
2.5 mL of 2.5 mM p-nitrophenyl laurate in sodium phosphate buf-
fer (25 mM, pH 7.0) was maintained at 30 °C for a period of 10 min.
The reaction was initialized by the addition of 10 mg of the crude
immobilized enzyme. One international unit (U) of pNPL was de-
fined as the amount of enzyme necessary to hydrolyze 1 lmol of
pNPL per minute under assay conditions.
4.4. Preparation of racemic 1-O-acetyl-4,6-di-O-benzyl-myo-
inositol
3. Conclusion
A stirred solution of 4,6-di-O-benzyl-myo-inositol (1) (159 mg;
0.44 mmol) and DMAP (10.8 mg; 0.088 mmol) in CH2Cl2 (5.0 mL)
under N2 was cooled to ꢀ20 °C and treated with Ac2O (0.04 mL;
0.41 mmol). After 15 min, the reaction mixture was allowed to
warm to 0 °C, being kept at this temperature for 10 min. Then, a sat-
urated aqueous solution of NaHCO3 (10 mL) was added to the mix-
ture at this temperature. After 5 min, the resulting mixture was
allowed to warm to room temperature. The product was extracted
with AcOEt, the organic phase was dried over Na2SO4 and the vola-
tiles were evaporated. Finally, the obtained residue was purified by
flash chromatography (elution with ethyl acetate/hexane mixtures)
to yield a white solid (53 mg), containing the monoacetate DL-2.
We have disclosed a rapid and very efficient protocol for the
desymmetrisation of myo-inositol derivative 1 using Lipozyme
TL-IM. Thus, the desired product D-(ꢀ)-2 was formed in excellent
yield and enantiomeric excess (>99%) in a variety of solvents. The
developed protocol expedites the syntheses of relevant inositol
phosphates and other derivatives. It was demonstrated that the
use of solvent (in combination with vinyl acetate as acylating
agent) leads to a more efficient and economical transformation.
The feasibility of catalyst reuse and lower enzyme load was dem-
onstrated as well.
4. Experimental
4.1. General
4.5. Analytical conditions
All the analyses for the determination of reaction conversion
and enantiomeric excess were performed by HPLC (Pump: Shima-
dzu LC-20AT; Detector: Shimadzu SPD-M20A variable-wavelength
UV/vis, with the detection set at 215 nm). The Shimadzu LC solu-
tion software was used for chromatogram integration.
Enzymes: Lipozyme TL IM (Thermomyces lanuginosus), Lipo-
zyme RM IM (Rhizomucor miehei) and Novozyme 435 (Candida ant-
arctica B, CaLB) were supplied by Novo Nordisk. AY amano 30
(Candida rugosa), FAP 15 amano (Rhizopus javonicus), G amano
(Penicillium camembertis) and AK amano (Peseudomonas fluores-
cens) were supplied by Amano. Lipomod 34P (Candida cylindracea)
was supplied by Biocatalysts. The substrate 1 was prepared as re-
ported by Shashidhar´s group.21 HPLC grade acetonitrile, n-hexane
and 2-propanol were purchased from Tedia. Vinyl acetate was pur-
chased from Fluka.
The conversion determination (based upon the disappearance
of the starting material) was performed with a C18 column eluted
isocratically using acetonitrile: H2O (40:60) mixture (flow rate
0.5 mL/min). The retention times of substrate 1 and monoacetate
L-(ꢀ)-2 were 12 min and 19 min, respectively.
Chromatographic determinations of the enantiomeric excesses
was performed with
a
Chiralcel OD-H column (5 lm;
4.6 ꢁ 250 mm) eluted with 2-propanol:hexane (30:70) mixture
(flow rate 0.8 mL/min). The retention times of monoacetates D-
(+)-2 and L-(ꢀ)-2 were 43.6 min and 50.5 min, respectively.
4.2. Screening of lipases
The enzymatic reactions were realized at 30 °C in closed ther-
mostatized flasks containing the substrate 1 (5.0 mg; 0.014 mmol),
vinyl acetate (2.5 mL) as solvent and acylating agent and the li-
pases (50 mg). The screening reactions were monitored by thin-
layer chromatography (TLC), using UV light as visualizing agent,
4.6. Solvent selection for the desymmetrization of myo-inositol
derivative 1 by Lipozyme TL-IM
The assays were performed according to the same protocol of
Section 4.2 (screening of lipases), run at 30 °C, employing 1
(5.0 mg), TL-IM (50.0 mg), vinyl acetate (1.25 mL), and solvent
(1.25 mL) at 30 °C.
HPLC analyses of conversion were done at reactions times of 1,
2, 6, 10, and 24 h, by independent experiments. HPLC determina-
tions of enantiomeric excess were done after 24 h.
Table 4
Reusability of TL-IM in the desymmetrization of 1 after 2 h-cycles at 30 °C using vinyl
acetate and different solvents
Entry
Solvents /time (h)
2 hb
Reuse 1b
Reuse 2b
Reuse 3b
1
2
3
4
Vinyl acetate
Ethyl acetate
Hexane
79
74
89
95
68
62
82
89
52
53
74
74
43
46
69
65
4.7. Reusability of Lipozyme TL-IM
TBME
a
eep > 99% (E>100) in all cases (after three cycles).
Conversions to D-2 (%).
The reusability of Lipozyme TL-IM was studied under the same
conditions as described in Section 4.6 (solvent selection). After
b

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11
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(1.0 mL) solution to remove any remaining substrate and product
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The assays were performed according to the same protocol of
Section 4.2 (screening of lipases), run at 30 °C, employing 1
(5.0 mg), TL-IM (15 or 25 mg), vinyl acetate (1.25 mL), and hexane
(1.25 mL) at 45 or 60 °C.
HPLC analyses of conversion were done in 1, 2, 6, 10, and 24 h
and HPLC analyses of enantiomeric excess were done in 24 h.
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(7.7 mL: 7.7 mL). The suspension stirred at 45 °C for 12 h. The en-
zyme was filtered of through a silica plug and the filtrate volatiles
were evaporated to yield D-(ꢀ)-2 (33.9 mg; 99% yield). HPLC and
NMR analyses of the crude product showed no contamination by
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R. V.; Simas, A. B. C.; Freire, D. M. G.; Simas, A. B. C. J. Chem. Technol. Biotechnol.
2012, 88, 205–211; (f) Manoel, E. A.; Pais, K. C.; Flores, M. C.; Miranda, L. S. de
M.; Coelho, M. A. Z.; Simas, A. B. C.; Freire, D. M. G.; Souza, R. O. M. A. J. Mol.
Catal. B: Enzym. 2013, 87, 139–143.
regioisomers. D-(ꢀ)-2: mp: 96–97 °C; ½a D20
ꢀ39.3 (c 1.00, MeOH)
ꢂ
Lit.11
m
½
a 2D0
ꢂ
ꢀ39.1 (c 1.00, MeOH), Rf = 0.45 (CH2Cl2/MeOH = 9:1); IR
(KBr) cmꢀ1: 3460, 3100, 3060, 3030, 2919, 2881, 1740, 1490,
1450, 1360, 1249, 1238, 1130, 1111, 1090, 1070, 1050,900, 730,
700.; 1H NMR (400 MHz, CDCl3) d 7.38–7.26 (m, 10H); 4.92–4.83
(dd, 4H); 4.14(s, 1H), 3.69–3.65 (t, 2H); 3.57–3.53(m, 3H); 2.67
(s, 1H); 2.52–2.50 (t, 2H). 13C NMR (200 MHz, CDCl3) d 169.8;
138.9; 137.9; 137.7; 128.1; 127.7; 124.4; 127.2; 109.8; 80.8;
79.0; 78.4; 73.8; 73.5; 73.3; 72.9; 70.6; 27.8; 25.2; 20.7.
11. Laumen, K.; Ghisalba, O. Biosci., Biotechnol., Biochem. 1994, 58, 2046–2049.
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1988, 71, 1367–1378; (b) Andersch, P.; Schneider, M. P Tetrahedron: Asymmetry
1993, 4, 2135–2138.
Acknowledgments
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Chiles, T. C.; Roberts, M. F. J. Am. Chem. Soc. 2008, 130, 7746–7755; (b) Jordan, P.
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CAPES, FAPERJ and CNPq for funding and/or fellowships; LA-
MAR/NPPN and Central Analítica/NPPN for analytical data.
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