On the kinetic resolution of sterically hindered myo-inositol derivatives in organic media by lipases

Article abstract of DOI:10.1016/j.tetasy.2012.01.005

Sterically hindered myo-inositol derivatives were assayed against different commercial lipases. It was found that dl-1,3,6-tri-O-benzyl-myo-inositol undergoes efficient kinetic resolutions mediated by Pseudomonas sp. lipases (PS-C, PS-IM) and CaLB (Novozy

Full text of DOI:10.1016/j.tetasy.2012.01.005

Tetrahedron: Asymmetry 23 (2012) 47–52
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
On the kinetic resolution of sterically hindered myo-inositol derivatives
in organic media by lipases
Evelin A. Manoel a, Karla C. Pais c, Aline G. Cunha b, Maria Alice Z. Coelho a, Denise M.G. Freire b,
Alessandro B.C. Simas c,
⇑
a Universidade Federal do Rio de Janeiro (UFRJ), Escola de Química, CT, Departamento de Engenharia Bioquímica, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
b UFRJ, Instituto de Química, CT, Programa de Pós-Graduação em Bioquímica, Departamento de Bioquímica, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
c UFRJ, Núcleo de Pesquisas de Produtos Naturais (NPPN), CCS, bloco H, Ilha do Fundão, Rio de Janeiro, RJ 21941-902, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Sterically hindered myo-inositol derivatives were assayed against different commercial lipases. It was
found that DL-1,3,6-tri-O-benzyl-myo-inositol undergoes efficient kinetic resolutions mediated by Pseudo-
monas sp. lipases (PS-C, PS-IM) and CaLB (Novozym 435). Under the best conditions, the O-acylated L-
enantiomorph was obtained in up to >99% ee with conversions of up to >49%. Differences in the immo-
bilization support of the Pseudomonas sp. lipases had a marked effect on their resolution performance.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 21 October 2011
Accepted 6 January 2012
Available online 11 February 2012
1. Introduction
pase A (Novozym 435) was very effective in the kinetic resolution
of DL-1,2-O-isopropylidene-3,6-di-O-benzyl-myo-inositol, a bulky
Lipases are now a part of synthetic chemists’ toolbox, in great
part due to the versatility that such enzymes show.1,2 In reactions
that they mediate, high chemo-, regio- and stereoselectivities are
attained under mild conditions and, notably, in organic solvents.3
In the present work, we discuss our results on the kinetic reso-
lution (KR) of selectively O-polyalkylated myo-inositols by lipases,
aiming at the efficient chemoenzymatic syntheses of precursors of
chiral myo-inositol derivatives. Knowledge on the roles of myo-ino-
sitol phosphates in intricate cellular signaling pathways continue
to evolve.4 Thus, novel derivatives of these compounds are needed
for bioassays, which may require new methodologies or improve-
ment of the existing routes.5,6 Moreover, more practical routes
for known inositol derivatives are welcome as they increase their
availability for biological studies. In this sense, the use of O-alkyl-
ated (i.e., O-benzylated) substrates combined with lipase-cata-
lyzed KRs offer alternative routes to these target molecules that
might prove rewarding. Benzyl groups (Bn = C6H5CH2) are particu-
larly important for the protection of hydroxyl groups owing to
their stability and mode of removal, allowing strategies based on
the so-called orthogonal lability.7
key precursor of different myo-inositol phosphates bearing two
benzyl groups.10
2. Results and discussion
2.1. Screening of lipases and substrates
We reinvestigated the resolution of tetraether DL-1 (Fig. 1). This
is an important precursor of naturally occurring inositol deriva-
tives.11 Ling and Ozaki9b reported that they did not succeed in
enzymatically acylating this compound or in hydrolyzing the cor-
responding acetate. However, they did not mention the enzymes
that were tried. Thus, we screened 13 commercial lipases against
DL-1 employing vinyl acetate as the acylating agent in solvent-free
media. In no case was conversion to an acetylated product ob-
served. Thus, we investigated the resolution of tetraallylated
myo-inositol DL-212 (Fig. 1) (allyl = CH2CHCH2) in the hope that a
significantly less sterically hindered substrate would yield to
The use of lipases in the syntheses of chiral inositols has been
limited, notwithstanding the clear advantages offered by such bio-
catalysts.8–10 Mostly, the known routes for such bioactive sub-
stances employ myo-inositol itself as a precursor and chiral
partially-protected derivatives thereof are subjected to resolutions
by derivatization. Recently, we showed that Candida antarctica li-
OH
BnO
OH
HO
OBn
OH
OR
OR
BnO
RO
OH
DL-3: R=Bn
OR
DL-1: R=Bn
DL-2: R=Allyl
⇑
Corresponding author.
E-mail address: abcsimas@nppn.ufrj.br (A.B.C. Simas).
Figure 1. myo-Inositol derivatives assayed in this study.
0957-4166/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2012.01.005

48
E. A. Manoel et al. / Tetrahedron: Asymmetry 23 (2012) 47–52
biocatalysis. This compound was assayed against the same set of
lipases. We did observe (TLC) what appeared to be monoacylated
product in the reactions with PS-IM, PS-C amano, Novozym 435,
PS amano, RM-IM, A amano 12, AK amano, 34P. However, as the
degree of conversion was very low, we did not pursue these reac-
tions further. This lack of reactivity may result from the combina-
tion of stereochemical (reaction on a cis 1,2-diol) and steric issues
(the additional hydroxyl groups are protected). In few reports in
the literature wherein lipase-catalyzed reaction occurs at C1-OH
in myo-inositol derivatives, the 1,2,3-triol moiety was free and
the remaining hydroxyl groups were only partially protected.8b,9c
Next, we proceeded to investigate the resolution of triether DL-3
(Fig. 1). The enzymatic resolution of tri-O-benzylated myo-inositol
derivatives or congeners had not been previously reported, to the
best of our knowledge. We wondered if such a bulky myo-inositol
i. Bu2SnO
OH
BnO
(1.05 mol. eq.),
MeOH, toluene,
100°C
OH
HO
1
OH
OH
OH
OH
BnO
HO
4
3
OBn
OH
ii. BnBr, toluene,
DIPEA, 130°C
DL-3
myo-inositol
Scheme 2. Alternative synthesis of DL-3 from myo-inositol.
Table 1
Screening of different enzymes in the enzymatic resolution of DL-3
b
c
Lípase
Time (h) Conversiona (%) ees
eep
E
CaLB
112
96
48
112
48
48
48
48
48
48
48
48
48
43.5
47.7
48.9
2.5
1.2
2.1
2.1
2.3
—
—
—
—
—
77
88
91
1
98
99
99
>200 (220)
>200 (645)
>200 (646)
would be resolved by lipases. The enantiomorphs D-(+)-3 and/or L-
Lipase PS-C Amano II
Lipase Amano PS-IM
Lipase Amano PS
Lipozyme RM IM
A ‘Amano’ 12
AK ‘amano’
Lipomod 34P
Lipozyme TL IM
F ‘Amano’ AP15
Lipase D Amano II
G ‘Amano’
(ꢀ)-3 had been employed in the synthesis of inositol phosphates
and had been prepared either by resolution via the formation of
diastereomeric mixtures13 or by lipase-catalyzed resolution of a
conduritol B derivative.14 We prepared DL-3 from diether DL-415
via a simplified procedure (Scheme 1).16 We had previously shown
that selective mono-O-benzylation of intermediate tetrol 5 at C-3
via stannylene acetal technology leads to DL-3.17 Thus, we antici-
pated the possibility of a one-pot preparation of DL-3 (from DL-4).
Hydrolysis of DL-4 in MeOH in the presence of a controlled amount
of H2O led to 5. Interestingly, in the absence of H2O, no reaction
took place when run at the same temperature. Such a simple
hydrolysis procedure allowed us to perform the following transfor-
n.d.
0
—
—
—
—
—
—
—
—
—
n.d. n.d.
n.d. n.d.
n.d. n.d.
n.d. n.d.
—
—
—
—
—
—
—
—
—
—
AY ‘Amano’ 30
n.d.: not determined.
a
Determined by HPLC.
b
Enantiomeric excess (ee) of unreacted substrate DL-3 determined by HPLC.
ee of monoacetate formed (after methanolysis) determined by HPLC.
mation in the same reaction vessel. Indeed, we found that crude DL
-
c
5 successfully undergoes selective O-monobenzylation to sub-
stance DL-3 via stannylene chemistry (67% for the two steps). The
presence of triethylammonium sulfonate in crude DL-5 did not
interfere with either stannylene acetal formation or its reaction
with BnBr. Recently, we reported on the development of the direct
controlled protection of multiple hydroxyl groups via iterative
regeneration of stannylene acetals (use of only 1.00–1.05 mol e-
quiv of Bu2SnO). By means of this methodology, triether DL-3 could
be obtained in reasonable yield from myo-inositol in a single step
(Scheme 2).15a As we had disclosed in that report, each individual
step in the dynamic iterative O-alkylations follows the regioselec-
tivity expected for the classical stannylene acetal protocol. Thus, in
this case, cis 1,2-diol moieties react first (selectively at C-1 and C-3
equatorial OH groups). Next, a third 1,2-diol moiety is mono-O-
alkylated, mostly at the OH group at C-4 (or C-6). This provided a
practical alternative access to substrate DL-3. The main advantage
of the first route is that it enables differentiation of the protecting
groups at C-1, C-4 from the one at C-3.
Different lipases were screened against DL-3 in reactions in vinyl
acetate (Table 1). This acylating reagent is known to lead to more
efficient lipase-catalyzed resolutions.10b,18 Lipases PS ‘Amano’
(lyophilized), Lipozyme RM-IM, A ‘Amano’ 12, AK ‘Amano’ e Lipo-
mod 34P were active but led to low conversions (ꢁ2.0%). Con-
versely, lipases CaLB (Novozym 435, 10 U/g), PS-C (836 U/g) e PS-
IM (17.763 U/g) mediated high conversions, with the reactions of
the latter two being interrupted earlier, as conversions approached
50%. Moreover, we observed a sharp decrease in rates as conver-
sions were close to 50%, what suggested high enantioselectivities.
In all cases, HPLC analysis showed the formation of a single regio-
isomeric acetate.
It is noteworthy that, while immobilized forms of the lipase
from Pseudomonas sp. (PS-C, PS-IM) performed very well, the
lyophilized form (Amano PS) displayed low activity. Such results
with a challenging and rather uncommon substrate DL-3 highlight
the importance of lipase immobilization. This may increase the
activity and stability of enzymes by a number of processes.19 For
instance, inclusion inside the support pores prevent the contact
of the enzyme with inhibitors, proteases from the lipase extract
and even air bubbles. Aggregation or simple inactivation is thus
avoided.
At this point, it is not clear whether DL-3 is reactive toward CaLB
due to diequatorial diol moiety, or to having one less benzyl group
or to both features. These are not shared by DL-1 and DL-2 (vide su-
pra). In a previous study,10 we showed that the same biocatalyst is
highly effective in resolving another challenging substrate, DL-1,2-
O-isopropylidene-3,6-di-O-benzyl-myo-inositol as well. Actually,
only CaLB was able to mediate this resolution. In that study, we
employed computational tools to rationalize the interactions in-
volved in this substrate with both CaLB and RM-IM lipase (RMl),
O
OH
BnO
i. H2O, p-TsOH
BnO
OH
OH
EtOH, 80°C
OH
OH
O
HO
ii. Et3N
iii. evaporation
OBn
OBn
5
4
not isolated
OH
BnO
i. Bu2SnO, MeOH, toluene,
120oC
OH
OH
BnO
ii. BnBr, toluene,
120oC
OBn
which was inactive. The L-enantiomorph of that myo-inositol deriv-
ative fitted well CaLB’s active site and formed a stable tetrahedral
intermediate. Conversely, Trp88 in RMl’s structure appears to hin-
DL-3
Scheme 1. One-pot synthesis of triether DL-3 from diether DL-4.

E. A. Manoel et al. / Tetrahedron: Asymmetry 23 (2012) 47–52
49
der the substrate binding to this enzyme. As DL-3 resembles this
inositol, the same interactions with both enzymes may account
for their performances in the reactions of DL-3.
latter solvent still affording high E-values. It is worth noting that
TBME made the conversions drop sharply. In fact, the use of co-sol-
vents did not improve the resolutions compared to the reaction
with vinyl acetate as solvent.
2.2. Product analysis
Such results further illustrate the impact of the mode of immo-
bilization on lipases reactivity. While PS-IM is immobilized on
diatomite, PS-C is on ceramic. Kanerva et al. reported that the
immobilization nature of two commercial PS lipases had a marked
effect on their performance in the kinetic resolution of 1,2,3,4-tet-
rahydroisoquinoline-1-acetic acid esters with water in DIPE.2m
After separation from the unreacted triol, the monoacylated
product was subjected to methanolysis (MeOH/K2CO3). The result-
ing triol (enantioenriched 3), as well as the unreacted substrate 3
were analyzed by HPLC for the determination of ee (Fig. 2). These
data showed that the three lipases mediated successful resolutions
(E >200 and high conversions), especially PS-IM (Table 1). A quan-
titative experiment of kinetic resolution of DL-3 with CaLB was car-
ried out. By comparing the specific rotation of unreacted 3 with the
data in the literature,13 this compound was determined to be the
2.3.2. Novozym 435
In the resolutions by CaLB with vinyl acetate, the use of hexane
and TBME improved conversions and selectivities, particularly ees.
(Table 4). Differently from the PS-IM assays, the reactions in EtOAc
enriched
D
-(+)-3 enantiomorph (Scheme 3), establishing the con-
(also employed as solvent and acylating agent) led to acetate
6 in good ee, albeit at low conversion. Even isopropenyl acetate
(solvent and acylation agent) afforded
-(ꢀ)-6 in high ee. In general,
CalB proved to be very consistent concerning the achieved ee of
acetate
L
-(ꢀ)-
figuration of the acylated product as L. NMR-analysis (Table 5 in
the Section 4) determined that the acylation of
at the C-5 hydroxyl group, leading to acetate
successful catalysts displayed the same enantiospecificity, favoring
the formation of
-(ꢀ)-6.
L
-(ꢀ)-3 took place
L
L-(ꢀ)-6. The three
L
-(ꢀ)-6. In general, less polar solvents led to better
L
performances.
Overall, our results show that hexane was more consistent
regarding conversions (Fig. 3). With the exception of the case of
PS-IM, TBME stood out as well as it provided both high conversions
and selectivities. Conversely, isopropenyl acetate proved not to be
suitable for these resolutions.
2.3. Effects of co-solvents and acylating agents
An investigation into the effect of solvents and acylating agent
on the performance of the resolution of DL-3 catalyzed by PS-C,
PS-IM, and CaLB (Novozym 435) was carried out. It is known that
the solvent affects both enzyme activity and selectivity in the com-
plex mechanism involving interactions among the enzyme, the
substrate and the reaction medium. Indeed, the literature has
shown the strong solvent influence on enantioselectivity.20 We
chose to investigate the effects of hexane, TBME (t-butylmethyl
ether), and EtOAc as solvents. Isopropenyl acetate and EtOAc as
acylating agents were assayed as well.
3. Conclusion
We have established that sterically hindered myo-inositol
derivative DL-3 could be resolved by lipases. This compound is a
known precursor of bioactive inositol phosphates (and a potential
one for other biphosphates and triphosphates). To the best of our
knowledge, tri-O-alkylated myo-inositols have not been reported
to successfully undergo such transformation.
As we noted, the non-reactivity of DL-1 and DL-2 may be due to
the additional alkyl group and/or the relative configuration of the
1,2-diol moiety (cis).
The differences in reactivity shown by PS-C, PS-IM, and their
lyophilized counterpart illustrate the importance of lipase immobi-
lization for their catalytic performance.
The efficient protocols for the resolution of DL-3 advanced in our
study make the synthesis of both enantiomorphs (and derivatives)
very practical. We believe that our study demonstrates that the
2.3.1. PS-C and PS-IM
In the case of reactions with PS-C (Table 2), compared to the
reaction in vinyl acetate, TBME and EtOAc led to better perfor-
mances (conversions, ee and E). Conversion was kept high with
hexane, but the enantioselectivities decreased, especially of the
acylated product (eep). The reaction in isopropenyl acetate did
not perform well.
As for the resolutions of DL-3 (Table 3) catalyzed by PS-IM, these
reactions remained rapid when run in hexane and EtOAc, with the
Figure 2. Determination of ee for resolution of DL-3. (a) Unreacted triol
D
-(+)-3. (b)
L
-(ꢀ)-3 obtained by methanolysis of acetate -(ꢀ)-6.
L

50
E. A. Manoel et al. / Tetrahedron: Asymmetry 23 (2012) 47–52
OH
BnO 1
OH
BnO 3
+
OH
BnO
6
OBn
OH
lipases
OAc
OH
OH
OH
BnO
OAc
BnO
BnO
OBn 5
1
3
OH
OBn
D-3
L-6
DL-3
Scheme 3. Kinetic resolution of DL-3.
Table 2
inositols. The use of selectively-acylated product,
larly attractive.
L-6, is particu-
Conversions, enantiomeric excess values and E-values obtained from kinetic resolu-
tion DL-3 catalyzed by PS-Ca
Acyl donor
Solvents
% CHPLC
ees
eep
E
4. Experimental
4.1. Materials
Vinyl acetate
Vinyl acetate
Hexane
TBME
Ethyl acetate
Isopropenyl acetate
47.7
47.9
49.9
46.0
23.0
88
89
97
97
20
>99
92
>99
>99
66
>200
71
>200
>200
Isopropenyl acetate
96 h-Reactions.
5.9
Enzymes: Lipozyme TL IM (Thermomyces lanuginosus), Lipozyme
RM IM (Rhizomucor miehei) e Novozym 435 (Candida antarctica B,
CaLB) were supplied by Novo Nordisk. The lipases PS-C (Pseudomo-
nas species), PS-IM (Pseudomonas species), PS amano (Burkholderia
cepacia) F-AP15 (Rhizopus javonicus), A-12 (Aspergillus niger), AK
a
Table 3
Conversions, enantiomeric excess values and E-values obtained from the kinetic
(Pseudomonas fluorescens), AY-30 (Candida rugosa), lipase
G
resolution of DL-3 catalyzed by PS-IMa
‘amano’ (Amano) and D (Rhizopus oryzae) were supplied by
Amano. Lipomod 34P was supplied by Biocatalysts. myo-Inositol
Acyl donor
Solvents
% CHPLC ees eep
E
derivatives DL-1,4,5,6-tetra-O-benzyl-myo-inositol DL-120 and DL
-
Vinyl acetate
Vinyl acetate
Hexane
TBME
Ethyl acetate
Ethyl acetate
48.9
48.0
34.0
45.0
2.3
91 99
88 92
49 95
80 98
>200 (646)
1,2-O-isopropylidene-3,6-di-O-benzyl-myo-inositol DL-415a were
prepared according to literature procedures. DL-1,4,5,6-Tetra-O-
allyl-myo-inositol (DL-2) was prepared by a procedure similar to
the one used in the preparation of DL-1. HPLC grade acetonitrile,
n-hexane and 2-propanol were purchased from Tedia. Vinyl
acetate was purchased from Fluka.
56
63
>200 (245)
2.7
Ethyl acetate
Isopropenyl acetate Isopropenyl acetate 11.6
1
45
28 70
6.2
a
48 h-Reactions.
4.2. Enzyme activity assay
Table 4
Conversions, enantiomeric excess values and E-values obtained from the kinetic
resolution DL-3 catalyzed by CaLBa
This assay was carried out by the absorbance increase (410 nm)
over time produced by p-nitrophenol release in the 0.25 mL of
2.5 mM pNPL (p-nitrophenol laurate) hydrolysis in 2.2 mL of
25 mM sodium phosphate buffer at pH 7.0 carried out at 30 °C.
The reaction was initialized by the addition of chosen amount of
the crude enzyme. One international unit (IU) of the lipase was de-
Acyl donor
Solvents
% CHPLC
ees
eep
E
Vinyl acetate
Vinyl acetate
Hexane
TBME
Ethyl acetate
Ethyl acetate
Isopropenyl acetate
43.5
48.3
46.7
29.4
12.2
38.9
77
88
99
43
26
61
98
97
96
93
92
90
>200
>200
>200
39
27
33
Ethyl acetate
Isopropenyl acetate
fined as the amount of enzyme necessary to hydrolyze 1 lmol of
pNPL per minute under assay conditions.21
a
112 h-Reactions.
4.3. Screening of substrates and lipases
The screening was performed in sealed reactors kept at a con-
stant, controlled temperature, and magnetically stirred. A mixture
of 5.0 mg of substrate, 50 mg of enzyme (immobilized or lyophi-
lized) and 2.5 mL vinyl acetate was kept at 30 °C under these con-
ditions. The reaction was monitored by TLC on Merck silica gel 60
F254 plates eluted with a 7:3 hexanes/ethyl acetate mixture.
4.3.1. Synthesis of DL-3 via acetonide DL-4
A mixture of DL-4 (0.4 g; 1.0 mmol), H2O (36 lL; 2.0 mmol) and
PTSA (8.6 mg; 0.05 mmol) in CH3OH (2.0 mL) was heated to 80 °C
for 1–1.5 h. The reaction mixture was then cooled to 40–50 °C
and Et3N (55 lL; 0.4 mmol) was added, after which the volatiles
were evaporated under vacuum. Additional CH3OH (2.0 mL) was
added and subsequently removed under vacuum to afford DL-5.
After that, a mixture of crude DL-5 and Bu2SnO (0.274 g, 1.1 mmol)
in CH3OH/toluene (1:1, 3 mL) was heated to 120 °C for 3 h. The sol-
vents were evaporated, dry toluene (4 mL) was added to the resi-
due and a second evaporation to dryness was effected, which is
completed under high vacuum. Then, a mixture of the crude stann-
ylene derivative, Bu4NBr (0.0645 g, 0.2 mmol) and BnBr (0.343 g,
Figure 3. Effect of the solvent on lipase-catalyzed kinetic resolutions of DL-3 after
48 h with vinyl acetate as acyl donor.
combination of selective multiple O-alkylations and lipase-cata-
lyzed resolutions could be considered in the synthesis of chiral

E. A. Manoel et al. / Tetrahedron: Asymmetry 23 (2012) 47–52
51
Table 5
2D NMR data of L-(ꢀ)-6
HSQC
HMBC
3JCH
COSY
13C
1H
2JCH
2JCH
CH-1
CH-2
CH-3
CH-4
CH-5
AcO-5
CH-6
CH2-7
C-8
79.3
66.9
79.8
78.8
74.5
170.8
70.5
3.27 (dd, J = 9.6, 2.6 Hz, 1H)
4.21 (t, J = 2.7 Hz, 1H)
3.45 (dd, J = 9.5, 2.7 Hz, 1H)
3.94 (t, J = 9.6 Hz, 1H)
4.96 (t, J = 9.8 Hz, 1H)
—
4.03 (t, J = 9.7 Hz, 1H)
4.87–4.61 (m)
—
H-2; H-6
H-1, H-3
H-2; H-4
H-3; H-5
H-4; H-6
Me/AcO-5
H-1; H-5
—
—
H-4
H-7
H-2
—
H-5
H-2
H-1, H-3 e H-4
H-2, H-6
H-1, H-3
H-2, H-4
H-3, H-5
H-4, H-6
—
H-1, H-5
-
—
75.5, 72.9, 72.4
138.5, 137.7, 137.4
128.6, 128.5, 128.3, 128.2, 127.99, 127.96, 127.91, 127.7, 127.6
21.0
H-7
H-8; H-7
—
—
—
—
CH-8
Me/AcO-5
7.28–7.41 (m)
1.99 (s)
—
—
2.0 mmol) in dry toluene (4 mL) under argon was heated to 120 °C
till the reaction went to completion (3 h). After solvent evapora-
tion, the residue was purified by flash chromatography (silica
gel) to yield triether DL-3 (0.302 g, 67%).
The enantiomeric ratio (E) was calculated by using the equation
of Chen et al.22
4.7. Quantitative resolution of DL-3: synthesis of
tri-O-benzyl-myo-inositol -1,3,6-tri-O-benzyl-myo-
-(ꢀ)-6 and
inositol -(+)-3
L-5-acetyl-1,3,6-
L
D
4.3.1.1. DL-1,3,6-tri-O-benzyl-myo-inositol, DL-315a
.
1H NMR
D
(300 MHz, CDCl3), d 2.47 (s, 1H), 2.61 (s, 2H), 3.23 (dd, 1H,
J = 2.67 9.52), 3.38–3.43 (m, 2H), 3.82 (t, 1H, J = 9.52), 3.96 (t, 1H,
J = 9.28), 4.24 (s, 1H), 4.63–4.98 (m, 6H), 7.25–7.35 (m, 15H); 13C
NMR (75.00 Hz, CDCl3), d 66.9, 71.8, 72.2, 72.4, 74.1, 75.5, 79.0,
79.8, 80.4, 127.8, 127.87, 127.9, 128.1, 128.5, 128.58, 137.6,
137.7, 138.6.
Following the protocol of Section 4.4, DL-3 (95 mg), CaLB
(750 mg), vinyl acetate (27 mL) and TBME (18 mL) were mixed at
30 °C for 120 h. The obtained residue was purified by flash chroma-
tography (column eluted with 5:95, 20:80, 30:70, 70:30, 80:20,
100:0 EtOAc/hexane mixtures) to give
-(+)-3 (45 mg, 43%):
L
-(ꢀ)-6 (37 mg, 39%) and
D
4.4. Resolution of DL-3
4.7.1. -5-O-Acetyl-1,3,6-tri-O-myo-inositol
L
-(ꢀ)-6
The enzymatic reactions were realized in closed thermostatized
flasks containing DL-3 (5 mg), acylating agent (1.0 mL) and lipase
(50 mg) in the solvent (1.5 mL) under stirring at 30 °C. Aliquots
were taken after 0.5, 6, 24, 48, 72, 96, and 112 h, dried in a Speed
Vac Concentrator (Savant SPD 1010-Thermo scientific) and re-sol-
ubilized in a 60:40 acetronitrile:H2O mixture (1 mL) and analyzed
by HPLC.
½
aꢃ2DL5 ¼ ꢀ24:2 (c 1.36, CHCl3); 1H NMR (400 Mhz, CDCl3), d 7.28–
7.41 (m, 15H), 4.96 (t, J = 9.8 Hz, 1H), 4.87–4.61 (m, 6H), 4.21 (t,
J = 2.7 Hz, 1H), 4.03 (t, J = 9.7 Hz, 1H), 3.94 (t, J = 9.6 Hz, 1H), 3.45
(dd, J = 9.5, 2.7 Hz, 1H); 3.27 (dd, J = 9.6, 2.6 Hz, 1H); 1.99 (s, 3H).
13C NMR (100 MHz, CDCl3), d 170.8, 138.5, 137.7, 137.4, 128.6,
128.5, 128.3, 128.2, 127.99, 127.96, 127.91, 127.7, 127.6, 79.8,
79.3, 78.8, 75.5, 74.5, 72.9, 72.4, 70.5, 66.9, 21.0. IR (film): 3417;
3062; 3030; 2922; 2872; 1732; 1494; 1454; 1359 cmꢀ1. EM-ESI:
m/z = 493.2220 [M+H]+; 515.2035 [M+Na]+.
4.5. HPLC analysis of conversion of DL-3 to
L-(ꢀ)-6
Conversion analyses were carried out via HPLC on a Kromasil
C18 column (40 °C in a CTO-20A oven), eluted with a acetoni-
trile–H2O (60:40) mixture (0.5 mL/min) by a Shimadzu LC-20AT
pump. A Shimadzu SPD-M20A variable-wavelength UV/vis detec-
tor was employed, with the detection set at 215 nm, and the Shi-
madzu LC solution software was used for chromatogram
integration. The samples to be analyzed were filtered through a
4.7.2. -1,3,6-Tri-O-benzyl-myo-inositol
D
-(+)-3
½
aꢃ2DD5 ¼ þ17:8 (c 0.45, CHCl3) Lit.13a
[a]D = +20 (c 1.00 CHCl3)
Lit.13b
[a]D = +16.2 (c 1.00 CHCl3).
Acknowledgments
FAPERJ, CNPq and CAPES for funding and/or fellowships; LA-
MAR/NPPN and Central Analítica/NPPN for analytical data.
0.45
and
lm PTFE filter. The retention times of the substrate D-(+)-3
L-(ꢀ)-6 were 8 and 13 min, respectively. The analysis of the
synthetic mixture (prepared by acylation of DL-3 with Ac2O) con-
taining DL-6 also showed peaks at 22 and 39 min, corresponding
to diacetylated DL-3 and triacetylated DL-3.
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