Kinetic resolution of a precursor for myo-inositol phosphates under continuous flow conditions

Article abstract of DOI:10.1016/j.molcatb.2012.11.006

In this work, we have investigated the biocatalytic continuous flow process with a packed-bed reactor for the kinetic resolution of (±)-1,3,6-tri-O- benzyl-myo-inositol ((±)-1) by alcoholysis using Novozym 435. Excellent conversions and stereselectivities were attained in short reaction time. We found that this enzymatic transformation under continuous flow in TBME with vinyl acetate (10:1 ratio with (±)-1) at 50°C, with a 3 min-residence time secured the best results (50% conversion and ee_p > 99%). The feasibility of a continuous operation of the Novozym 435-containing-packed-bed reactor over a longer period of time was demonstrated via a 9-cycle experiment wherein the lipase remained stable.

Full text of DOI:10.1016/j.molcatb.2012.11.006

Journal of Molecular Catalysis B: Enzymatic 87 (2013) 139–143
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Kinetic resolution of a precursor for myo-inositol phosphates under continuous
flow conditions
Evelin A. Manoela, Karla C. Paisb, Marcella C. Floresc, Leandro Soter de M. e Mirandac,
Maria Alice Zarur Coelhoa, Alessandro B.C. Simasb, Denise M.G. Freired,∗∗
,
Rodrigo Octavio M.A. de Souzac,∗
a EQ, Departamento de Engenharia Bioquímica, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
b Núcleo de Pesquisas de Produtos Naturais (NPPN), Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
c Instituto de Química, Departamento de Química Orgânica, Biocatalysis and Organic Synthesis Group, Universidade Federal do Rio de Janeiro, Ilha do Fundão, Rio de Janeiro, RJ, Brazil
d Programa de Pós-Graduac¸ ão em Bioquímica, Departamento de Bioquímica, Laboratório de Biotecnologia Microbiana, Universidade Federal do Rio de Janeiro, 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 9 July 2012
Received in revised form 26 October 2012
Accepted 7 November 2012
Available online 16 November 2012
In this work, we have investigated the biocatalytic continuous flow process with a packed-bed reactor
for the kinetic resolution of ( )-1,3,6-tri-O-benzyl-myo-inositol (( )-1) by alcoholysis using Novozym
435. Excellent conversions and stereselectivities were attained in short reaction time. We found that this
enzymatic transformation under continuous flow in TBME with vinyl acetate (10:1 ratio with ( )-1) at
50 ◦C, with a 3 min-residence time secured the best results (50% conversion and eep > 99%). The feasibility
of a continuous operation of the Novozym 435-containing-packed-bed reactor over a longer period of
time was demonstrated via a 9-cycle experiment wherein the lipase remained stable.
© 2012 Elsevier B.V. All rights reserved.
Keywords:
Flow chemistry
Kinetic resolution
Lipase
Myo-inositol
Packed bed reactor
1. Introduction
usually relies on the use of chiral auxiliaries yielding diastereoiso-
meric mixtures. In some cases, two molar equivalents of these
Myo-inositol is the scaffold of many important molecules with
fundamental roles in the physiology of living organisms. For exam-
ple, the mono- and polyphosphates of myo-inositol, including
inositol phospholipids, act as secondary messenger molecules,
involved in a number of biological processes, including insulin
signal transduction [1], intracellular calcium control [2], reduc-
ing blood cholesterol [3], among others. The understanding of the
biological roles of such myo-inositol derivatives in living orga-
nisms has greatly benefited from Chemical Synthesis which has
provided substantial amounts of these substances and analogues
as well. The continuing evolution in the molecular diversity of
inositols offers new possibilities for the development of synthetic
technology.
agents are incorporated. These processes are not catalytic and the
separation of the diastereoisomers may be, in some cases, a diffi-
cult process. In addition, the use of chiral auxiliaries imposes an
additional step to remove this chiral resolving agent, what makes
the process cost ineffective. On the other hand, enzyme-catalyzed
kinetic resolutions are well known for their practicality, high effi-
ciency and selectivity. Additionally, the separation of the formed
derivative from the unreacted substrate is more easily accom-
plished, naturally [4–7].
Among the many enzyme classes used as biocatalysts for syn-
thetic organic transformations, lipases (triacyl glycerol hydrolases
EC 3.1.1.3) stand out as they can be applied in both aqueous
and nonaqueous media, have wide substrate specificity, have
an excellent ability to recognize chirality, and do not require
labile cofactors [7]. Notwithstanding lipases efficiency and the
advantages that they offer, few reports in the literature deal
with their application to inositol syntheses. So far, the cases
of myo-inositol derivatives, with distinctive structural patterns
and including triether ( )-1 (Scheme 1), subjected to resolutions
by lipases amount to only six (five types if one only considers
reactions on the cyclic backbone) (vide infra) [8]. This is sur-
prising, in some sense, due to the many possibilities that the
Mostly, the syntheses of bioactive chiral myo-inositol deriva-
tives employ myo-inositol itself, due to its availability, what implies
the formation of racemic intermediates. The required resolution
∗
Corresponding author.
Corresponding author.
∗∗
E-mail addresses: freire@peq.coppe.ufrj.br (D.M.G. Freire),
souzarod21@gmail.com (R.O.M.A. de Souza).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.11.006

140
E.A. Manoel et al. / Journal of Molecular Catalysis B: Enzymatic 87 (2013) 139–143
Scheme 1. Kinetic resolution of ( )-1 by Novozym 435 (CaLB): (A) batch conditions and (B) continuous flow conditions.
myo-inositol structure offers, the importance of inositol chem-
istry and the broad commercial availability of lipases. A relevant
feature of the lipase-catalyzed resolutions of racemic inositols
is the high regioselectivities (especially in the case of acyla-
tions), which may foster synthetic flexibility and efficiency in the
following functional manipulations of the unprotected polyolic
moiety.
Despite the success of lipase-catalyzed reactions in batch reac-
tors, they may suffer from long reaction times and high catalyst
loadings. Such drawbacks are not found when these reactions are
performed in a continuous flow system. In fact, it is reported that
continuous flow systems improve the productivity of the method-
ology and reduce reaction times. Continuous flow systems are
preferred over batch reactors due to their greater process control,
high productivity and improvement of product quality/purity and
yield [9,10]. Among the different types of continuous-flow reac-
tors, packed-bed reactors (PBR) are the most popular due to their
high efficiency, low cost and ease of construction, operation and
maintenance [11–14].
Recently, we have developed kinetic resolutions of ( )-1,3,6-
tri-O-benzil-myo-inositol, ( )-1, and ( )-1,2-O-isopropylidene-3,
6-di-O-benzyl-myo-inositol using lipase B from Candida antarctica
(CaLB) with excellent yields and enantiomeric ratios [9,15]. Nev-
ertheless, these procedures suffer from long reaction times. Those
compounds are precursors of several important inositol phosphates
(e.g., 1,4,5-IP3) and analogues. The use of derivative ( )-1 is partic-
ularly interesting as it is available in only step from myo-inositol.
Due to the increasing demand for more economical synthetic pro-
cesses and the high productivity associated with continuous-flow
systems, we engaged in applying this technology [16] to the lipase-
mediated resolution of ( )-1,3,6-tri-O-benzyl-myo-inositol ( )-1.
The results of this study are reported herein. In the previous inves-
tigation on the kinetic resolutions this inositol [9], under catalysis
by CaLB in batch conditions, high selectivities were achieved when
vinyl acetate, hexane, TBME and, to a lesser extent, ethyl acetate
were used as solvents (vinyl acetate as acylating agent).
2. Results and discussion
Lipase-catalyzed kinetic resolutions in continuous-flow systems
have been addressed by different reports. However, the applica-
tion of this technology to the synthesis of inositols has not been
previously studied.
As a starting point, we pursued the kinetic resolution of ( )-1
CaLB employing ethyl acetate as solvent (vinyl acetate as acy-
lating agent) under continuous flow (Table 1). In batch reactors,
Table 1
Kinetic resolution of ( )-1 with vinyl acetate catalyzed by Novozym 435 in TBME at different temperatures and substrate-acyl donor ratios in the continuous flow system.
Entry
1
(
)-1/2a
Temp. (◦C)
30
Residence time (min)
Conv. 3 (%)a
eep (%)a
Ea
1:5
3
1.5
3
1.5
3
1.5
3
1.5
3
1.5
3
1.5
3
1.5
3
1.5
3
1.5
3
1.5
3
1.5
3
0.4
0.1
39
26
40
30
4
n.d.
n.d.
99
–
–
2
1:10
1:15
1:5
>200
68
>200
>200
24
96
3
>99
>99
92
n.d.
>99
97
>99
97
96
92
>99
99
>99
>99
95
4
40
50
60
2
–
5
1:10
1:15
1:5
41
30
47
33
8
>200
98
>200
105
53
6
7
4
24
8
1:10
1:15
1:5
50
41
50
40
12
5
50
44
50
42
>200
>200
>200
>200
25
9
10
11
12
92
25
1:10
1:15
>99
>99
>99
>99
>200
>200
>200
>200
1.5
a
Values based on HPLC analysis. See Section 4.

E.A. Manoel et al. / Journal of Molecular Catalysis B: Enzymatic 87 (2013) 139–143
141
Fig. 1. Integrity of the packed-bed reactor after nine 5 min-cycles on the kinetic resolution of ( )-1 using Novozym 435 as biocatalyst, in the best experimental conditions:
TBME as solvent, vinyl acetate as acyl donor (1:10 substrate/vinyl acetate ratio), at 50 ◦C. Residence time: 3 min.
Table 2
such condition led to a stereoselective reaction showing moderate
Kinetic resolution of ( )-1 with vinyl acetate in TBME catalyzed by Novozym
conversion (30%, E = 39 for the 112 h-reaction) [9]. We wondered
whether, under the new condition, both conversion and stereose-
435 at 50 ◦C under batch conditions. Reaction time: 24 h (productivity of
0.001 mgproduct/mgenzyme h in both cases).
lectivitywouldbe improved. Thus, asolutioncontainingthe inositol
Entry
(
)-1/2a
Conv. 3 (%)a
eep (%)a
Ea
(
)-1 and vinyl acetate in ethyl acetate was pumped through the
packed-bed reactor charged with CaLB (Novozym 435). Different
temperatures, residence times and proportions between ( )-1 and
vinyl acetate were tested. Unfortunately, as it had occurred in batch
experiments [9], ethyl acetate did not perform well as solvent in
the kinetic resolution of ( )-1 under flow conditions. Conversions
to acetate l-(−)-3 remained below 10% even in runs applying long
residence time (3 min) and large excess of vinyl acetate (1:15) at
60 ◦C.
We had previously shown that hexane or TBME as solvent sig-
nificantly improve this transformation [9]. Due to the very low
solubility of the substrate in hexane, we decided to run the contin-
uous flow reactions in TBME (Table 1). The batch condition (with
5 mg of the substrate ( )-1 and 0.5 U of the lipase) had led to a 47%
conversion (E > 200) after 112 h.
The use of TBME for the kinetic resolution of myo-inositol ( )-1
under continuous flow conditions had a marked effect on the effi-
ciency of this transformation. In general, proportions of 1:10 and
1:15 (substrate/vinyl acetate) gave the best conversions/selectivity
while the use of smaller excesses of vinyl acetate (1:5) led to poor
conversions to the desired product and lower enantiomeric ratios
(Entries 1, 4, 7 and 10, Table 1). Besides bringing about much higher
productivity (vide infra) the reactions in the continuous flow sys-
tem allowed the use of lower amounts of vinyl acetate compared
to the batch process [9] (1:300 substrate/vinyl acetate ratio), what
makes the process more economical, naturally [17].
1
2
1:10
1:15
49
49
>99
>99
>200
>200
a
Values based on HPLC analysis. See Section 4.
a residence time of 3 min gave good conversions to acetate l-(−)-3
and higher enantiomeric excess (eep) and enantiomeric ratios (E)
as long as a 1:10 ( )-1/vinyl acetate ratio, at least, was employed.
It noteworthy that, when 1:10 or 1:15 ( )-1/vinyl acetate ratios
at 50 ◦C under batch conditions were employed, the reaction took
24 h to achieve the same conversion and enantiomeric ratios as
those achieved under continuous flow conditions as shown in
Table 2.
The best condition obtained for the kinetic resolution of ( )-1
with vinyl acetate under continuous flow conditions (1:10 ratio,
50 ◦C and 3 min of residence time) was applied to resolution with
other acetylating agents in order to identify alternatives to vinyl
acetate. The results obtained are shown in Table 3.
The results obtained with the use of isopropenyl acetate and
acetic anhydride in TBME (Entries 1 and 2, Table 3) are good but,
Table 3
Kinetic resolution of ( )-1 with vinyl acetate in TBME catalyzed by Novozym 435 at
50 ◦C in the flow system (residence time of 3 min) using different acetylating agents.
The temperature also had an important effect on conversions,
with the best results occurring at 50 and 60 ◦C. On the other hand,
the resolutions run at 30 and 40 ◦C had the conversions improved
by the use of larger quantities of acetylating agent (Entries 3
and 6, Table 1) or by longer residence times, with a consequent
decrease on productivity. Irrespective of the chosen temperature,
Entry
Acylating agent
Conv. 3 (%)a
eep (%)a
Ea
1
2
3
Isopropenyl acetate (2b)
Acetic anhydride (2c)
Ethyl acetate (2d)
45
42
3
98
85
85
>200
23
12
a
Values based on HPLC analysis. See Section 4.

142
E.A. Manoel et al. / Journal of Molecular Catalysis B: Enzymatic 87 (2013) 139–143
when compared to our previous result using vinyl acetate (Entry
8, Table 1), show lower conversion and selectivity. In TBME, ethyl
acetate as acylating agent led to very low conversion and moderate
selectivity (Entry 3, Table 3).
The integrity of the biocatalyst over time was investigated by a
9-cycle assay with repeated determination of conversions (Fig. 1).
Satisfyingly, the reactor displayed high consistency.
detector was employed, with the detection set at 215 nm,
and the Shimadzu LC solution software was used for chro-
matogram integration. The samples to be analyzed were filtered
through a 0.22 ␮m PTFE filter. The retention times of the sub-
strate ( )-1 and the acetate l-(−)-3 were 8 min and 13 min,
respectively.
4.4. Determination of enantiomeric excesses (ee)
3. Conclusion
Unreacted substrate, d-(+)-1, and monoacetylated product, l-
(−)-3, were separated by HPLC. In the case of substance l-(−)-3, it
was subjected to methanolysis reaction (MeOH/K2CO3), to give triol
l-(−)-1, prior to the HPLC analyses [19]. Chromatographic deter-
minations of ee for l-(−)-3, eep (shown in the tables), (via l-(−)-1)
were carried out on the same equipment mentioned above carry-
ing a Chiralcel OD-H column (5 ␮m; 4.6 × 250 mm), eluted with a
7:3 hexane-2-propanol mixture (0.6 mL/min). The retention times
of d-(+)-1 and l-(−)-1 enantiomorphs were 24.5 min and 28.5 min,
respectively. The enantiomeric ratio (E) was calculated by using the
equation (with eep and conversion as inputs; see supplementary
material) of Chen et al. [20].
In conclusion, we have developed a very efficient continuous
flow approach for the kinetic resolution of ( )-1,3,6-tri-O-benzyl-
myo-inositol (( )-1) by which the reactions catalyzed by Novozym
435 using TBME as solvent and vinyl acetate as acetylating agent
could be performed in very short reaction times (3 min of resi-
dence time) with high conversions and enantiomeric ratios. The
operation stability of the biocatalyst was demonstrated via a 9-
cycle assay. The use of this protocol may lead to very practical
chemical syntheses of bioactive inositols. The best condition for
this flow process obtained by our screening allowed a productivity
531 times higher (3.188 mgproduct/mgenzyme h) than that of the opti-
mized condition for the corresponding batch process as we have
recently established (0.006 mgproduct/mgenzyme h) [21]. As a matter
of fact, the productivity issue in lipase/esterase-catalyzed stereos-
elective syntheses of myo-inositols has not been addressed in the
literature.
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2012.11.006.
Acknowledgments
4. Experimental
We thank CAPES (Coordenac¸ ão de Aperfeic¸ oamento de Pessoal
de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento
Científico e Tecnológico), FAPERJ (Fundac¸ ão Carlos Chagas Filho de
Amparo a Pesquisa do Estado do Rio de Janeiro) and FINEP (Agência
Financiadora de Estudos e Projetos) for financial support.
4.1. Enzyme
Novozym 435 (Lipase B of Candida antarctica immobilized
on a macroporous acrilic resin, 2720 U/g) was purchased from
Novozyme. The enzyme activity was determined as the initial rate
in esterification reactions between oleic acid and ethanol at a molar
ratio of 1:1, temperature of 40 ◦C and enzyme concentration of
5 wt% in relation to the substrates [18]. One lipase activity unit (U)
was defined as the amount of enzyme necessary to consume 1 ␮mol
of oleic acid per minute at the established experimental conditions
previously presented. All enzymatic activity determinations were
replicated at least three times.
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