High-Throughput Preparation of Optically Active Cyanohydrins Mediated by Lipases

Article abstract of DOI:10.1002/ejoc.201601028

Cyanohydrins are versatile compounds with high applicability in organic synthesis; they are used as starting materials for the synthesis of other chemical targets with high industrial added value. Lipase-mediated kinetic resolution reactions are a promising route for the synthesis of optically active cyanohydrins. These reactions can be carried out through the acylation of cyanohydrins or the deacylation of cyanohydrin esters, with different biocatalysts and under different reaction conditions. Unfortunately, depending on the substrate structure, long reaction times can be required to achieve suitable enantiomeric excesses. In this context, we present a high-throughput protocol for the production of optically active cyanohydrins in continuous-flow mode. The products were obtained with moderate to good enantioselectivity (E values from 8 up to >200) and with productivity values from 2.4 to 8.7 times higher in continuous-flow mode than in batch mode. Moreover, the reaction times were reduced from hours in batch mode to minutes in continuous-flow mode.

Full text of DOI:10.1002/ejoc.201601028

A Journal of
Accepted Article
Title: High-Throughput Preparation of Optically Active Cyanohydrins Mediated by
Lipases
Authors: Leandro Piovan; Juliana Christina Thomas; Bruno Bernardi Aggio; Alfredo
Ricardo Marques De Oliveira
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of the final Version of Record (VoR). This work is currently citable by using the Digital
Object Identifier (DOI) given below. The VoR will be published online in Early View as
soon as possible and may be different to this Accepted Article as a result of editing.
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601028
Link to VoR: http://dx.doi.org/10.1002/ejoc.201601028
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High-throughput preparation of optically active cyanohydrins
mediated by lipases
Juliana Christina Thomas,[a] Bruno Bernardi Aggio,[a] Alfredo Ricardo Marques de Oliveira,[a] Leandro
Piovan*[a]
Abstract: Cyanohydrins are versatile compounds with high
applicability in organic synthesis, being employed as starting
materials for other chemical targets with high industrial added value.
Lipase-mediated kinetic resolution reactions are a promising route
for the synthesis of optically active cyanohydrins. These reactions
can be performed via cyanohydrin acylation or deacylation of
cyanohydrin esters, with different biocatalysts and reaction
conditions. Unfortunately, depending on substrate structure, high
reaction times are required to achieve suitable enantiomeric
excesses. In this context, we present a high throughput protocol to
produce optically active cyanohydrins in continuous-flow mode.
Compounds were obtained with moderate to good enantioselectivity
(E values from 8 up to >200) and with productivity values 2.4 to 8.7-
fold higher in continuous-flow than in batch mode. Moreover,
reaction time was reduced from hours, in batch mode, to minutes in
continuous-flow mode.
Introduction
Scheme 1. Cyanohydrins EKR via acylation (A), deacylation (B)
as chiral building blocks;[1] in this context, they can be employed
and deacylation in continuous-flow mode (C).
Cyanohydrins are versatile compounds with several applications
as precursors of β-aminoalcohols,[2] α-hydroxyacids,[3] α-
hydroxyketones[4] and other compounds with high added value
Several examples of EKR reactions involving cyanohydrins have
in the pharmaceutical industry.[5] Cyanohydrins have already
already been described; their involve acylation by Burkholderia
cepacia[13] and Candida antarctica lipase B;[14] hydrolysis by
been employed as precursors of different drugs, such as
Bacillus coagulans,[15] Bacillus licheniformis,[16] Candida
rugosa,[17] Pseudomonas aeruginosa,[18] metagenome-derived
esterases[19] and several other proteases;[20] alcoholysis by
Candida antarctica lipase A,[21] Candida antarctica lipase B,[22]
Candida rugosa[23] and Pseudomonas fluorescens;[24] and
aminolysis by Candida antarctica lipase A.[25] Results indicate
that, depending on substrate structure, despite of good
enantioselectivity, long reaction times are required to achieve
high enantiomeric excesses.
fluoxetine, duloxetine and derivatives,[6] capuramycin,[7]
bufuralol,[8] denopamine and salbutamol.[9]
Optically active cyanohydrins can be prepared either via
asymmetric catalysis  mediated by metal-organic frameworks[8]
and metal complexes[10]  or biocatalytic methods based on the
use of oxynitrilases[11] and peptide based catalysis.[12] Another
relevant way to achieve optically active cyanohydrins employs
lipase-mediated kinetic resolution (EKR) reactions, which can be
performed via acylation of cyanohydrins (Scheme 1 – A) or by
hydrolysis/alcoholysis of cyanohydrin esters (Scheme1 - B), in
both cases with classical[13-25] or dynamic resolution.[26]
Promising dynamic kinetic resolution (DKR) reactions of
cyanohydrins have also been reported. They involve
a
racemization agent and have the theoretical advantage of total
conversion. For them, different racemization agents and
biocatalysts have been described in the literature.[26] In this case
reaction times are even larger than those observed for EKR
reactions and high enantiomeric excess are strongly dependent
of racemization agent and substrate structure.
Currently,
a trend in biocatalytic transformations is their
[a]
J. C.Thomas, B. B. Aggio, A. R. M. de Oliveira, L. Piovan
Department of Chemistry
Universidade Federal do Paraná
Paraná, Brazil
performance in continuous-flow mode and several enzymes
have already been employed as biocatalysts in this mode, such
as aldolases,[27] amidases,[28] transaminases,[29] oxidases,[30]
peroxidases[31] and especially lipases, in esterification,[32]
interesterification,[33] transesterification[34] and EKR reactions [35]
Although resolution reactions have been increasingly carried out
in continuous-flow (CF) mode,[36] cyanohydrins have so far been
E-mail: lpiovan@quimica.ufpr.br
Supporting information for this article is given via a link at the end of
the document.
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European Journal of Organic Chemistry
10.1002/ejoc.201601028
FULL PAPER
underexplored as CF substrates.[37] Considering that continuous-
flow can offer several advantages over batchwise reactions,
including reproducibility, efficient control of reaction parameters,
fast/homogeneous heating, lower costs in reaction optimization
and the observation of green chemistry principles,[38] the
development of fast CF protocols for cyanohydrin resolution is
highly desirable.
corresponding aldehydes and then submitted to acylation with
acetic
anhydride
as
acyl
donor
and
4-(N,N-
dimethylamino)pyridine (DMAP) as catalyst (see experimental
section and supporting information for details).
Enzymatic Kinetic Resolution (EKR) Reactions
This work investigates the performance of the two modes in the
biocatalytic resolution of benzylic, aliphatic and heterocyclic
cyanohydrins; for this, we employed deacylation reactions with
Candida antarctica lipase B (Scheme 1 – A and B). Calculated
enantioselectivity and productivity parameters revealed that the
continuous-flow mode provides higher productivity despite some
loss of enantioselectivity. All reactions carried out in CF
presented lower reactions time than batch mode. Particularly
good results were obtained with benzylic cyanohydrins
employing both batch and CF modes, with a surprising increase
on enantioselectivity in CF mode for halogenated compounds.
Cyano(phenyl)methyl acetate (1) was chosen as a model
compound for preliminary assays. It was employed as acyl
donor and n-butanol as nucleophile in the acylation reaction
mediated by Candida antarctica lipase B. Cyanohydrin product
was derivatized to its corresponding propionate in order to
determine enantioselectivity and conversion parameters.
After optimization of reaction conditions, EKR of 1 reached 50%
of conversion with high enantioselectivity (E > 200) in 8 h (Table
1 – entry 1). When the same reaction was performed in
continuous-flow, results were even better, since the productivity
parameter was higher than in batch mode (12.5 in continuous-
flow and 5.2 in batch; Table 1 – entry 1) and reaction time was
48-fold lower in continuous-flow than in batch mode.
Based on the high enantioselectivy and productivity, as well as
the short reaction time observed in EKR reactions employing
cyano(phenyl)methyl acetate (1) as acyl donor, the strategy was
expanded to include the cyanohydrin esters 2-9 (Table 1).
Results and Discussion
Selection of Substrates
A series of compounds was synthesized in order to investigate
the effect of aromatic (1-5), aliphatic (6 and 7) and heterocyclic
(8) substituents attached to stereogenic center (Figure 1) on the
lipase-mediated EKR. For aromatic compounds, the effect of
electron donor and electron withdrawing groups was also
investigated.
Figure 1. Cyanohydrin esters (1-8) employed as substrates and
their cyanohydrin parent compounds (1a-8a).
Chemical Synthesis
Cyanohydrin esters (1-8) were synthetized in two consecutive
reactions. Firstly, cyanohydrins were prepared from their
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European Journal of Organic Chemistry
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Table 1. EKR reactions with cyanohydrin esters (1-9) in batch and continuous-flow modes
Batch
Flow
r [e]
µmol
min-1
g-1
/
r [g]
/
µmol
min-1
g-1
e.e. [b] / %
e.e. [b] / %
#
R
t /
h
c [a]
/ %
f / mL
C
Y
c [a]
/ %
E [d]
t [f] / h
E [d]
min-1
e.e.p
[c]
e.e.s
e.e.p
e.e.s
[c]
1
2
3
4
8
50
50
50
62
>99
98
98
>200
>200
>200
9
5.2
3.5
3.5
1.8
0.1
0.1
0.1
0.1
2
2
2
2
0.14
0.14
0.14
0.14
50
50
50
52
97
97
96
90
98
>200
116
127
31
12.5
12.8
12.5
13.0
12
12
24
97
97
53
93
94
82
98
86
5
24
53
97
96
55
1.5
0.1
2
0.14
52
96
90
90
74
13.0
0.1
(0.1)
1
(2)
0.07
(0.13)
66
(51)
47
(83)
8
(31)
33.0
(25.5)
6
7
4
54
54
99
98
84
88
60
71
11.3
1.9
(88)
0.1
(0.1)
2
(2)
0.14
(0.13)
54
(28)
92
(37)
78
(96)
26
(70)
13.5
(14.0)
24
8
12
60
90
61
12
4.2
0.1
1
0.07
53
88
78
23
26.5
Reaction conditions: batch mode: cyanohydrin ester (0.1 mmol), n-butanol (0.4 mmol), toluene (2 mL) and Novozym 435® (20 mg). Flow mode: cyanohydrin
ester (0.1 mol L-1), n-butanol (0.4 equivalents), toluene (5 mL) and Novozym 435® (200 mg). Temperature for both modes: 50 oC. [a] Conversion: ees / (ees +
eep). [b] Enantiomeric excess: (R – S) / (R + S) x 100 (determined by chiral GC analysis). [c] Determined by derivatization to corresponding propionate. [d]
Enantiomeric ratio: E = ln {[eep (1 - ees)] / (eep + ees)} / ln {[eep (1 + ees)] / (eep + ees)}. [e] Productivity (batch): nP / t me. [f] Residence time: reactor volume /
flow rate. [g] Productivity (flow): [P] f / me. CY: cycles of elution
5). This fact could be justified by a possible lipase inhibition by
EKR of cyano(phenyl)methyl acetate 1 (Table 1 – entry 1), as
already described, presented high E values in both modes and
productivity higher in continuous-flow than in batch. For cyano(4-
methoxyphenyl)methyl acetate (2) and cyano(p-tolyl)methyl
acetate (3), the presence of the methoxy or methyl groups led to
higher reaction times (Table 1 – entries 2 and 3). This was
already expected, because electron donor substituents such as
–OMe and -Me, usually slow down this type of reaction. There
are also steric effects when compounds 2 and 3 are compared
to model compound 1, which can also justify the lower reaction
rate for p-substituted compounds. Selectivity in continuous-flow
and batch mode was comparable for 2 and 3, with higher
productivity observed in continuous-flow. Halogenated
compounds (4-chlorophenyl)(cyano)methyl acetate (4) and
cyano(4-fluorophenyl)methyl acetate (5), in turn, presented the
most significant differences between their corresponding results
with higher selectivity in continuous-flow (Table 1 – entries 4 and
reaction products (chloro- and fluoro-cyanohydrin and aldehyde)
as already reported for other lipases[39] and other classes of
enzymes.[40] Although this effect is strongly felt in batch mode, it
can be minimized in continuous-flow, since the products are
quickly removed from the contact with the biocatalyst. It is
important to highlight that compounds 4 and 5 presented the
longest reaction times in batch mode, corroborating the
hypothesis of enzyme inhibition.
For aliphatic compounds 1-cyanobutyl acetate (6) and 1-
cyanoheptyl acetate (7), continuous-flow EKR presented less
selectivity than batch reactions (Table 1 – entries 6 and 7).
Considering that the cyanohydrin EKR of aliphatic compounds is
usually more challenging than EKR of benzylic compounds, E
values are, usually, lower for these compounds,[41] and that the
continuous-flow approach employs a larger amount of enzyme
than batch mode, is possible that both enantiomers to be
transformed. Accordingly, when compounds 6 and 7 were
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European Journal of Organic Chemistry
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submitted to EKR using a smaller amount (100 mg) of
biocatalyst, E values were higher than those obtained with 200
mg (Table 1 – entries 6 and 7, values in parenthesis), although
they were still lower than those obtained in batch mode. On the
other hand, despite this lower enantioselectivity in continuous-
flow, high productivity values were observed for both
compounds 6 and 7. Results gathered for the heterocyclic
cyanohydrin ester 8, in turn, were all very similar to those given
by the chloro-substituted substrate 4, also suggesting some
degree of impairing interaction between the biocatalyst and the
substrate (Table 1 – entry 8).
and decrease for 6 and 7 - depending on steric and electronic
demands of the substituent groups attached to the chiral center.
Conclusions
A successful protocol to achieve optically active cyanohydrins in
continuous-flow mode was developed. Reaction times
considerably decreased compared to batch mode and even with
values found in literature. With this novel protocol, different
optically active cyanohydrin esters can be achieved in few
minutes of reaction. For compounds that may present inhibitory
activity, the use of continuous-flow reactions is even more
interesting, since compounds are quickly removed from contact
with biocatalyst, as observed for halogenated cyanohydrin
esters.
In general, the greatest difference between EKR in batch and
CF modes was evidenced by the productivity parameter (r),
which considers the quantity of biocatalyst and measure how
much product can be obtained in 1 min using 1 g of enzyme.[42]
For cyanohydrin esters 1-8, productivity values were always
higher in continuous-flow than in the batch mode (Figure 2). The
most significant difference between productivities in batch and
continuous-flow was observed for halogenated cyanohydrins 4
and 5, which presented productivities 7.2 and 8.7-fold higher in
continuous-flow than in batch, respectively.
Experimental Section
General Procedure for the Syntheses of Racemic Cyanohydrin Esters 1-
5 and 8
A solution of Na2S2O5 (6 mmol, 1.141 g) in 5 mL of water was kept in an
ice bath and received the dropwise addition of the suitable aldehyde (10
mmol). After magnetic stirring for 10 min, a solution of KCN (10 mmol,
0.652 g) in cold water (5 mL) was added dropwise, the ice bath was
removed and the mixture was stirred at room temperature for 24 h. It was
then extracted with dicloromethane (3 x 10 mL), dried over MgSO4,
filtered and evaporated under reduced pressure.[43] The resulting
cyanohydrin was immediately submitted to a sequent acylation reaction,
in which it was solubilized in dichloromethane (10 mL) and received the
addition of both acetic anhydride (15 mmol, 1.42 mL) and DMAP (one
crystal). This reaction medium was left stirring overnight at room
temperature, after which it was washed with a solution of NaHCO3 up to
pH 8. The organic phase was dried over MgSO4, filtered and evaporated
under reduced pressure. Residual aldehyde was removed by
crystallization as its bisulphite salt.
Figure 2. Comparative productivity values for EKR of
cyanohydrin esters 1-8 in batch and continuous-flow modes.
Finally, a scaled-up reaction was performed in order to evaluate
the reproducibility of results obtained in analytical scale and to
demonstrate the robustness of our homemade continuous-flow
system. For this purpose 1.051 g of cyanohydrin ester 1, was
resolved employing same reaction conditions described in Table
1 (entry 1). After the resolution reaction and compounds
chromatographic separation, (R)-1 and (S)-1a were obtained in
34% and 37% isolated yield, respectively. It is important to
highlight that no significant changes in estereoselectivity
parameters were observed since (R)-1 and (S)-1a were isolated
with 97% and 95% enantiomeric excess, respectively, in
accordance with analytical scale.
In summary, a high-throughput protocol to achieve optically
active cyanohydrins, together with a comparison between EKR
reactions in continuous-flow and batch modes, have been
presented in this work. Although EKR of all substrates presented
here have already been described in literature, it is the first time
they were performed in continuous-flow mode.
Cyano(phenyl)methyl acetate (1). Yield: 62%. GC-MS (70 eV), m/z
(relative intensity): 175 (M•+, 16%), 133 (76%), 115 (74%), 105 (35%), 89
(30%), 77 (21%), 63 (16%), 51 (19%), 43 (100%). 1H NMR (200 MHz,
CDCl3, TMS), δ (ppm): 2.17 (s, 3H); 6.41 (s, 1H); 7.42-7.54 (m, 5H). 13C
NMR (50 MHz, CDCl3), δ (ppm): 20.4; 62.9; 116.1; 127.9; 129.2; 130.4;
131.8; 168.9. IR (cm-1): 3067, 3037, 2944, 1754, 1496, 1458, 1369, 1216,
1024, 756, 697.
Cyano(4-methoxyphenyl)methyl acetate (2). Yield: 35%. GC-MS (70 eV),
m/z (relative intensity): 205 (M•+, 30%), 163 (25%), 146 (100%), 135
(20%), 116 (23%), 103 (17%), 91 (23%), 76 (21%), 63 (9%), 50 (10%),
1
43 (32%). H NMR (200 MHz, CDCl3, TMS), δ (ppm): 2.15 (s, 3H); 3.83
(s, 3H); 6.36 (s, 1H); 6.95 (d, J = 8.7 Hz, 2H); 7.45 (d, J = 8.7 Hz, 2H).
13C NMR (50 MHz, CDCl3), δ (ppm): 20.5; 55.4; 62.6; 114.6; 116.3;
123.9; 129.6; 161.1; 169.0. IR (cm-1): 3006, 2960, 2938, 2840, 1752,
1611, 1514, 1467, 1371, 1258, 1211, 1175, 1026, 960, 829.
Cyano(p-tolyl)methyl acetate (3). Yield: 63%. GC-MS (70 eV), m/z
For all substrates employed, continuous-flow reactions
presented higher productivities than their batch counterparts.
Changes in enantioselectivity on going from the batch to the CF
approach were observed - increase for compounds 4, 5 and 8
(relative intensity): 189 (M•+, 37%); 147 (85%); 129 (100%); 119 (36%);
1
103 (44%); 91 (27%); 77 (24%); 65 (17%); 51 (11%); 43 (48%). H NMR
(200 MHz, CDCl3, TMS), δ (ppm): 2.16 (s, 3H); 2.39 (s, 3H), 6.37 (s, 1H)
7.25 (d, J = 8.2, 2H); 7.41 (d, J = 8.2 Hz, 2H). 13C NMR (50 MHz, CDCl3),
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European Journal of Organic Chemistry
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δ (ppm): 20.5; 21.3; 62.7; 116.2; 127.9; 128.9; 129.9; 140.7; 169.0. IR
Acylations with propionic anhydride (5 µL) and DMAP (one crystal) were
carried out directly in aliquots taken from reaction mixture and followed a
procedure similar to those described in the previous sections for
reactions with acetic anhydride. After acylation, the reaction mixtures
were stirred at room temperature for 5 min and neutralized with aqueous
NaHCO3. The organic layers were then dried over anhydrous MgSO4
before analysis.
(cm-1): 3032, 2926, 2862, 1754, 1613, 1515, 1372, 1214, 1020, 963, 812..
(4-chlorophenyl)(cyano)methyl acetate (4). Yield: 30%. GC-MS (70 eV),
m/z (relative intensity): 209 (M•+, 17%); 167 (50%); 149 (63%); 139
(24%); 132 (10%); 123 (17%); 114 (45%); 88 (9%); 75 (16%); 63 (8%);
50 (8%); 43 (100%). 1H NMR (200 MHz, CDCl3, TMS), δ (ppm): 13C NMR
(50 MHz, CDCl3), δ (ppm): 2.18 (s, 3H); 6.39 (s, 1H); 7.40-7.54 (m, 4H)
IR (cm-1): 3092, 3069, 2942, 1754, 1596, 1492, 1416, 1370, 1212, 1090,
1014, 964, 819.
General Procedure for EKR Reactions of Cyanohydrin Esters in Batch
Mode
Cyano(4-fluorophenyl)methyl acetate (5). Yield: 30%. GC-MS (70 eV),
m/z (relative intensity): 193 (M•+, 12%); 151 (54%); 133 (100%); 123
(30%); 107 (35%); 95 (15%); 75 (12%); 57 (12%); 43 (94%). 1H NMR
(200 MHz, CDCl3, TMS), δ (ppm): 2.17 (s, 3H); 6.39 (s, 1H); 7.09-7.20 (m,
2H); 7.48-7.57 (m, 2H). 13C NMR (50 MHz, CDCl3), δ (ppm): 20.4; 62.2;
116.0; 116.4 (d, J = 22.3 Hz); 127.8 (d, J = 3.0 Hz); 130.1 (d, J = 8.8 Hz);
163.8 (d, J = 250.1 Hz); 168.8. IR (cm-1): 3079, 2947, 1755, 1606, 1511,
1425, 1373, 1218, 1161, 1023, 963, 832.
In a 4 mL sealed vial, cyanohydrin esters 1-8 (0.1 mmol) were solubilized
in toluene (2 mL). To this solution, n-butanol (0.4 mmol, 37 µL) and the
supported enzyme CAL-B (Novozym 435®, 20 mg) were added. From the
reaction mixture kept stirring at constant temperature (50 ºC), aliquots
were periodically taken for analysis by chiral GC.
Continuous-Flow (CF) System
Our continuous-flow system consisted of a syringe pump connected to
the reactor through a Teflon cannula. The reactor itself was an empty
HPLC stainless steel column (74.0 x 4.6 mm) that was previously
washed to remove the stationary phase. It was then filled with the
biocatalyst, the supported lipase Novozym 435® (200 mg or 100 mg,
internal volume 0.43 mL and 0.40 mL, respectively), and deactivated
glass wool in both ends, in order to prevent enzyme agglomeration on
the top of reactor. A homemade heating block (8.5 x 5.0 x 2.0 cm)
controlled by a commercial thermostat was used to set the reaction
temperatures.
Cyano(furan-2-yl)methyl acetate (8). Yield: 39%. GC-MS (70 eV), m/z
(relative intensity): 166 (8%); 139 (28%); 124 (4%); 108 (4%); 97 (100%);
81 (10%); 69 (15%); 52 (18%); 43 (69%). 1H NMR (200 MHz, CDCl3,
TMS), δ (ppm): 2.18 (s, 3H); 6.45 (dd, J = 3.4; 1.9 Hz, 1H); 6.48 (s, 1H);
6.69 (dm, J = 3.4 Hz, 1H); 7.52 (dd, J = 1.9; 0.8 Hz, 1H). 13C NMR (50
MHz, CDCl3), δ (ppm): 20.3; 55.7; 111.1; 112.5; 114.1; 145.5; 168.7. IR
(cm-1): 3154, 3129, 2955, 2922, 2851, 1749, 1496, 1368, 1209, 1016,
753.
General Procedure for the Syntheses of Racemic Cyanohydrin Esters 6
and 7
General Procedure for EKR Reactions in Continuous-Flow Mode
A solution of KCN (15 mmol, 0.977 g) in 20 mL of methanol was cooled
down to 0 oC and received the dropwise addition of the corresponding
aldehyde (10 mmol) in methanol (2 mL). After stirring for 15 min, glacial
acetic acid (20 mmol, 1.15 mL) was added dropwise, the ice bath was
removed and the mixture was warmed up to room temperature while
stirring for 45 min. Dicloromethane (10 mL) was then added and the
reaction medium was washed with a solution of NaHCO3 up to pH 8. The
solvent was dried over MgSO4, filtered and evaporated under reduced
pressure.[44] This reaction product was submitted to a subsequent
acylation reaction, according to the previous experimental procedure.
The cyanohydrins obtained from these syntheses were employed in EKR
reactions without further purification.
The cyanohydrin esters 1-8 (0.5 mmol) and n-butanol (2 mmol, 0.18 mL)
were dissolved in toluene (5 mL) and then eluted through a packed-bed
column with the biocatalyst (200 mg) in a flow rate ranging from 0.1 to 1
mL min-1. Aliquots (0.5 mL) collected for each flow rate were analysed by
chiral GC. The reactor internal volume was equal to 0.43 mL.
General Procedure for Scaled-up EKR of Cyanohydrin Ester 1
Cyanohydrin ester 1 (6 mmol, 1.051 g) and n-butanol (24 mmol, 2.20 mL)
were dissolved in toluene (60 mL) and then eluted through the reactor in
a 0.1 mL min-1 flow rate. After 2 cycles, toluene was removed under
reduced pressure and the crude material was chromatographed (10:1
hexanes/ethyl acetate). After solvent removing, (R)-1 and (S)-1a were
recovered in 34% and 37% isolated yield, respectively.
1-cyanobutyl acetate (6). Yield: 49%. GC-MS (70 eV), m/z (relative
intensity): 112 (1%); 99 (9%); 87 (2%); 81 (6%); 71 (4%); 61 (4%); 57
(11%); 43 (100%). 1H NMR (200 MHz, CDCl3, TMS), δ (ppm): 1.00 (t, J =
7.4 Hz, 3H); 1.45-1.63 (m, 2H); 1.84-1.95 (m, 2H); 2.14 (s, 3H); 5.33 (t, J
= 6.7 Hz, 1H). 13C NMR (50 MHz, CDCl3), δ (ppm): 13.3; 17.9; 20.3; 34.2;
60.9; 116.9; 169.2. IR (cm-1): 2965, 2878, 1757, 1467, 1372, 1220, 1111,
1035.
Absolute configuration assignment
Absolute configurations of compounds were attributed via optical rotation
and comparison with the literature data. For this purpose, compounds 1-8
were submitted to EKR in preparative scale, employing continuous-flow
mode, and compounds were separated via flash column (10:1
hexanes/ethyl acetate). Enantiomeric excesses were determined via
chiral GC analyses.
1-cyanoheptyl acetate (7). Yield: 85%. GC-MS (70 eV), m/z (relative
intensity): 184 (M•+, 1%), 154 (1%), 140 (2%), 122 (2%), 112 (7%), 95
(14%), 81 (23%), 70 (8%), 55 (27%), 43 (100%). 1H NMR (200 MHz,
CDCl3, TMS), δ (ppm): 0.89 (t, J = 6.6 Hz, 3H); 1.26-1.53 (m, 8H); 1.85-
1.95 (m, 2H); 2.14 (s, 1H); 5.31 (t, J = 6.8 Hz, 1H). 13C NMR (50 MHz,
CDCl3), δ (ppm): 13.9; 20.4; 22.4; 24.4; 28.4; 31.4; 32.2; 61.1; 116.9;
169.2. IR (cm-1): 2956, 2932, 2861, 1755, 1464, 1373, 1222, 1037.
(R)-cyano(phenyl)methyl acetate [(R)-1]. 3.71 (c = 0.5, CHCl3); e.e.
45
93%. Lit.:
4.1 (c = 1.16, CHCl3); e.e. 85%.
(S)-2-hydroxy-2-phenylacetonitrile [(S)-1a]. -27.29 (c = 0.5, CHCl3);
46
e.e. 96%. Lit.: -8.6 (c = 0.50, CHCl3); e.e. 81%.
General Procedure for the Syntheses of Propionates
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European Journal of Organic Chemistry
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(R)-cyano(4-methoxyphenyl)methyl acetate [(R)-2]. -15.11 (c = 0.5,
[4]
Krepski, L. R.; Jensen, K. M.; Heilmann, S. M.; Rasmussen, J. K.
Synthesis 1986, 1986, 301-303.
45
CHCl3); e.e. 97%. Lit.:
-4.7 (c = 1.14, CHCl3); e.e. 27%.
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(S)-2-hydroxy-2-(4-methoxyphenyl)acetonitrile [(S)-2a]. -31.07 (c =
47
0.5, CHCl3); e.e. 99%. Lit.: -43.6 (c = 1.25, CHCl3); e.e. 93%.
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(R)-cyano(p-tolyl)methyl acetate [(R)-3]. -6.09 (c = 0.5, CHCl3); e.e.
45
97%. Lit.:
-2.5 (c = 1.27, CHCl3); e.e. 30%.
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(S)-2-hydroxy-2-(p-tolyl)acetonitrile [(S)-3a]. -40.17 (c = 0.5, CHCl3);
48
e.e. 96%. Lit.: -31.5 (c = 0.51, CHCl3); e.e. 68%.
(R)-(4-chlorophenyl)(cyano)methyl acetate [(R)-4]. -8.04 (c = 0.5,
45
CHCl3); e.e. 95%. Lit.:
-2.5 (c = 1.11, CHCl3); e.e. 21%.
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(S)-2-(4-chlorophenyl)-2-hydroxyacetonitrile [(S)-4a]. -33.10 (c = 0.5,
49
CHCl3); e.e. 96%. Lit.: -28.5 (c = 1.10, CHCl3); e.e. 70%.
(R)-cyano(4-fluorophenyl)methyl acetate [(R)-5]. 2.49 (c = 0.5,
CHCl3); e.e. 73%. Lit.: for (S) enantiomer -6.7 (c = 0.012, CHCl3);
e.e. 92%.14
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(S)-2-(4-fluorophenyl)-2-hydroxyacetonitrile [(S)-5a]. -11.65 (c = 0.5,
46
CHCl3); e.e. 97%. Lit.: -16.2 (c = 0.79, CHCl3); e.e. 74%.
(R)-1-cyanobutyl acetate [(R)-6]. 81.48 (c = 0.5, CHCl3); e.e. 85%.
Lit.: for (S) enantiomer -56.8 (c = 0.08, CHCl3); e.e. 87%.
50
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(R)-1-cyanoheptyl acetate [(R)-7]. 40.94 (c = 0.5, CHCl3); e.e. 65%.
(S)-2-hydroxyoctanenitrile [(S)-7a]. -10.27(c = 0.5, CHCl3); e.e. 90%.
51
Lit.: -13.3 (c = 1.00, CHCl3); e.e. 98%.
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Keywords: biocatalysis, continuous-flow, cyanohydrins, kinetic
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FULL PAPER
Cyanohydrins continuous-flow
resolution*
Juliana Christina Thomas, Bruno
Bernardi Aggio, Alfredo Ricardo
Marques de Oliveira, Leandro Piovan*
Page No. – Page No.
A novel protocol to achieve optically active cyanohydrins in continuous-flow mode
was developed. Cyanohydrin esters were employed as substrates in lipase-
mediated enzymatic kinetic resolution (EKR) reactions. EKRs presented lower
reaction times in continuous-flow than batch mode, as well as higher productivity in
continuous-flow.
High-throughput preparation of
optically active cyanohydrins
mediated by lipases
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