Straightforward enzymatic process based on HNL CLEA-catalysis towards cyanohydrin derivatives

Article abstract of DOI:10.1021/op900207n

An efficient enzymatic process based on HNL-CLEA catalysis in buffer-saturated organic media was developed to limit the amount of overall waste generated and the number of safety issues incurred by handling HCN-containing waste. Starting from benzaldehyde as a model substrate, (R)- and (S)-mandelonitrile can be obtained under these reaction conditions without an extraction step being required. The crude cyanohydrin intermediate was sufficiently pure to be used as starting material in a second step towards a range of derivatives.

Full text of DOI:10.1021/op900207n

Organic Process Research & Development 2010, 14, 114–118
Straightforward Enzymatic Process Based on HNL CLEA-Catalysis towards
Cyanohydrin Derivatives
Fabien L. Cabirol,†,‡,§ Angela E. C. Lim,‡ Ulf Hanefeld,† and Roger A. Sheldon*,†
Department of Biotechnology, Laboratory of Biocatalysis and Organic Chemistry, Delft UniVersity of Technology, Julianalaan
136, 2628 BL Delft, The Netherlands, and Biocatalysis Laboratory, Institute of Chemical and Engineering Sciences, 1 Pesek
Road, Jurong Island S(627833), Singapore
Abstract:
acetates, followed by the corresponding downstream chemistry
of the free cyanohydrin, has been reported.10 However, in such
kinetic resolutions yields are limited to 50%. Earlier attempts
to combine a HNL with a lipase in one pot failed due to the
hydrolysis of the acylating reagent. The acetic acid released
then denatured the HNL.11 The direct enantioselective synthesis
of cyanohydrins from inexpensive HCN and readily available
prochiral carbonyl compounds is nonetheless remarkably at-
tractive in terms of atom economy. An integrated multistep
synthetic strategy towards cyanohydrin derivatives would
therefore answer a need for step economy12,13 in order to
“efficaciously deliver a meaningful supply of target” (a cyano-
hydrin derivative) as Wender described.14
An efficient enzymatic process based on HNL-CLEA catalysis in
buffer-saturated organic media was developed to limit the amount
of overall waste generated and the number of safety issues incurred
by handling HCN-containing waste. Starting from benzaldehyde
as a model substrate, (R)- and (S)-mandelonitrile can be obtained
under these reaction conditions without an extraction step being
required. The crude cyanohydrin intermediate was sufficiently
pure to be used as starting material in a second step towards a
range of derivatives.
Introduction
A century ago, in 1908, Rosenthaler reported the preparation
of (R)-mandelonitrile from benzaldehyde and hydrogen cyanide
using emulsin as catalyst.1 This publication arguably marks the
birth of asymmetric biocatalysis. Hydroxynitrile lyases (HNLs)
and the preparation of enantioenriched cyanohydrins have been
one of the key topics in this field of research ever since.2-7
Chiral cyanohydrins are mostly used as intermediates in
synthetic chemistry.2-4,8 One of the main drawbacks of cyano-
hydrins as commercial products is the possible release of HCN
upon decomposition.9 This potential source of hazard incurs
additional costs in handling, shipping, and storage of bulk
quantities. As a result, cyanohydrins are generally seen as in-
house intermediates towards more stable and marketable chiral
products such as R-hydroxy-amines, R-hydroxy-acids, and
R-hydroxy-esters. O-Protected cyanohydrins are also of par-
ticular interest when further derivatization involves a basic
medium that would otherwise lead to the decomposition of
unprotected cyanohydrins.
Both (R)- and (S)-HNLs are naturally occurring, stable, and
relatively inexpensive enzymes with a wide substrate range. The
HNL-catalysed preparation of cyanohydrins is typically carried
out in aqueous buffer or in a biphasic (buffer:organic) type
medium. This type of medium is not suitable for the develop-
ment of cost-effective multistep syntheses since the reagents
required to derivatize the cyanohydrin would decompose in
water. Purification of the cyanohydrin is therefore required and
is usually carried out by extraction in organic media followed
by distillation of the organic layer (Figure 1).
The extraction step was identified as a significant drawback
in terms of waste generation since the aqueous layer needs to
be disposed of and large amounts of organic solvent are usually
required. Furthermore, each layer from the extraction step can
potentially contain residual HCN from the reaction mixture and
therefore requires appropriate handling and waste disposal
procedures. In order to efficiently implement a multistep strategy
towards cyanohydrin derivatives, the HNL-catalysed formation
of cyanohydrins should be carried out in an organic medium
(Figure 1).
The preparation of cyanohydrin esters, THP-protected, and
trialkylsilyl derivatives by kinetic resolution of cyanohydrin
Engineering the reaction medium for the HNL-catalysed
synthesis of cyanohydrins typically aims at limiting the con-
tribution of the noncatalysed addition of HCN to the substrate
which decreases the practical enantiopurity of the product.15-21
To this end, the development of immobilized HNLs as cross-
* To whom correspondence should be addressed. Fax: (+31)-15-278-1415.
E-mail: r.a.sheldon@tudelft.nl.
† Delft University of Technology, The Netherlands.
‡ Institute of Chemical and Engineering Sciences, Singapore.
§ Current Address: Codexis Laboratories Singapore, 61 Science Park Road,
#03-15/24, The Galen, Singapore Science Park II S(117525), Singapore.
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(2) Gregory, R. J. H. Chem. ReV. 1999, 99, 3649.
(3) Effenberger, F.; Fo¨rster, S.; Wajant, H. Curr. Opin. Biotechnol. 2000,
11, 532.
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Eur. J. Org. Chem. 2002, 1516.
(11) Hanefeld, U.; Straathof, A. J. J.; Heijnen, J. J. J. Mol. Cat. B: Enzym.
2001, 11, 213.
(4) Holt, J.; Hanefeld, U. Curr. Org. Synth. 2009, 6, 15.
(5) Griengl, H.; Schwab, H.; Fechter, M. Trends Biotechnol. 2000, 18,
252.
(12) Wender, P. A.; Handy, S. C.; Wright, D. L. Chem. Ind. 1997, (6
October), 765.
(6) North, M. Tetrahedron: Asymmetry 2003, 14, 147.
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10.1021/op900207n  2010 American Chemical Society
Published on Web 11/25/2009

Figure 1. Downstream processing of the reaction mixture depending on the choice of reaction media.
linked-enzyme-aggregates(CLEA)wasparticularlysuccessful.21-24
CLEAs of the HNLs from Linum usitatissimum (LuHNL),25
Manihot esculenta (MeHNL),21,26 and Prunus amygdalus
(PaHNL)21,27,28 showed high volumetric activity and improved
stability in organic media. Similar to earlier observations for
PaHNL29 and other HNLs immobilized on different carriers17,20,30
the HNL CLEAs were catalytically active in buffer-saturated
organic solvents.
we selected benzaldehyde as model substrate and four subse-
quent mandelonitrile derivatization methods to be discussed
herein.
Results and Discussion
We first established reaction conditions under which the
enzymatic step reached more than 95% conversion with at least
97% ee in buffer-saturated diisopropyl ether (DIPE) within 4 h
at room temperature. This could be achieved using 4 g/L CLEA
at 52 g/L substrate loading (0.49 M) and 4 equiv HCN for both
MeCLEA and PaCLEA with (S)-mandelonitrile and (R)-
mandelonitrile, respectively. Saturation of the organic solvent
with aqueous buffer (50 mM citrate/phosphate pH ) 5.5) was
absolutely necessary for optimum catalytic performance since
deactivation of the biocatalyst occurs under conditions where
the medium can still remove water from the enzyme. Water
plays an important part in the structure of HNLs and is present
even in the active site.31 The ability of the enzyme to retain
structural water molecules therefore dictates the biocatalyst
stability in the medium.23
Downstream processing of the cyanohydrin was then con-
ducted as described in Figure 1. The immobilized biocatalyst
could be filtered easily through cotton wool and recycled directly
for the next batch of cyanohydrin. We typically reused the
catalyst three times without noticeable loss of activity. Distil-
lation was avoided since cyanohydrins decompose upon heating
and a distillation step would require additives such as citric acid
to stabilize the product.7 The volatiles were therefore removed
under reduced pressure and recondensed carefully since the
reaction mixture still contains excess HCN. This overall process
fulfilled the target of limiting the amount of waste generated
since no extraction step was required. Extraction would indeed
In order to illustrate the key advantages of a HNL CLEA-
catalysed cyanohydrin synthesis in organic solvent as a part of
multistep synthesis strategy towards cyanohydrin derivatives
(16) Bauer, M.; Griengl, H.; Steiner, W. Enzyme Microb. Technol. 1999,
24, 514.
(17) Costes, D.; Wehtje, E.; Adlercreutz, P. Enzyme Microb. Technol. 1999,
25, 384.
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Microb. Technol. 2000, 27, 423.
(19) Gerrits, P. J.; Marcus, J.; Birikaki, L.; van der Gen, A. Tetrahedron:
Asymmetry 2001, 12, 971.
(20) Persson, M.; Costes, D.; Wehtje, E.; Adlercreutz, P. Enzyme Microb.
Technol. 2002, 30, 916.
(21) Cabirol, F. L.; Hanefeld, U.; Sheldon, R. A. AdV. Synth. Catal. 2006,
348, 1645.
(22) Sheldon, R. A. AdV. Synth. Catal. 2007, 349, 1289.
(23) Avi, M.; Griengl, H. Organic Synthesis with Enzymes in Non-Aqueous
Media; Carrea, G., Riva, S., Eds.; Wiley-VCH: Weinheim, 2008, p
211.
(24) Hanefeld, U.; Gardossi, L.; Magner, E. Chem. Soc. ReV. 2009, 38,
453.
(25) Cabirol, F. L.; Tan, P. L.; Tay, B.; Cheng, S.; Hanefeld, U.; Sheldon,
R. A. AdV. Synth. Catal. 2008, 350, 2329.
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van Rantwijk, F.; Sheldon, R. A. AdV. Synth. Catal. 2006, 348, 1655.
(27) van Langen, L. M.; Selassa, R. P.; van Rantwijk, F.; Sheldon, R. A.
Org. Lett. 2005, 7, 327.
(28) Roberge, C.; Fleitz, F.; Pollard, D.; Devine, P. Tetrahedron Lett. 2007,
48, 1473.
(29) Effenberger, F.; Ziegler, T.; Fo¨rster, S. Angew. Chem., Int. Ed. Engl.
1987, 26, 458.
(30) Costes, D.; Rotcenkovs, G.; Wehtje, E.; Adlercreutz, P. Biocatal.
Biotransform. 2001, 19, 119.
(31) Gruber, K.; Kratky, C. J. Polym. Sci., Part A: Polym. Chem. 2004,
42, 479.
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Figure 2. Multistep syntheses investigated.
require ∼5 volumes of organic solvent with respect to the
amount of water used as reaction media. This solvent is later
removed during the distillation step (Figure 1), and its handling
and possible recycling are difficult due to residual HCN. In the
process described here only one volume of organic solvent is
used (the reaction media). Moreover, we minimized potential
hazard due to residual HCN by limited handling with a very
straightforward downstream processing procedure.
slight excess of isocyanate (1.5 equiv) and CuBr ·Me2S (1.5
equiv) was necessary under these conditions. After purification
by crystallisation good yields (82%) and excellent enantiopurity
(99.5%) were obtained.
Derivatisation of the nitrile functionality in cyanohydrin
chemistry leads to a range of very useful intermediates in
organic synthesis such as R-hydroxy-acids, R-hydroxy-esters,
and R-hydroxy-amides.2-6
Water from the media was the main impurity in the crude
cyanohydrin obtained. This residual water can have a significant
impact on the second step. We therefore investigated four
subsequent derivatization methods to determine if the crude
cyanohydrin obtained after this downstream process could be
used without further purification (Figure 2).
The formation of R-hydroxy-N-tert-butyl-amide from cy-
anohydrins Via the Ritter reaction has been reported earlier.34
The reaction is performed in a mixture of 60% H2SO4 and tert-
butanol as reagents/solvent. Starting from crude (R)-mande-
lonitrile prepared by our method, the reaction proceeded
smoothly to afford the corresponding R-hydroxy-amide (R)-5
in good yield (70%) and very good ee (96%).
As a first example of follow-up chemistry, we investigated
the derivatization of the crude cyanohydrin obtained upon the
catalysis of either PaCLEA or MeCLEA into the TMS-
derivatives (R)-3 and (S)-3, respectively. Only a moderate excess
(2.5 equiv) of chlorotrimethylsilane (TMSCl) and base (2 equiv)
were needed to achieve good yields (>80%) and maintain the
excellent enantiopurity (>99%) obtained in the enzymatic step.
The deleterious effect of residual water was therefore not so
significant as to preclude further reactions with water-sensitive
reagents such as TMSCl. We also noticed that, when TMSCN
was used as the sole protecting reagent, a slight drop in the
enantiopurity of the protected cyanohydrin was observed. This
effect was attributed to the racemic (noncatalysed) addition of
TMSCN to residual benzaldehyde and could be suppressed
completely when TMSCl was used as the protecting reagent.
The carbamoylation of chiral alcohols using isocyanates and
copper bromide has been reported earlier for the determination
of enantiopurity by NMR.32 This derivatization method employs
a different type of reagent to the protecting-group chemistry
(typically base- or acid-catalysed) discussed earlier and was
therefore suitable to further probe the robustness of our multistep
approach toward cyanohydrin derivatives. Coupling of freshly
prepared isocyanate33 with (R)-mandelonitrile in THF proceeded
smoothly toward the corresponding carbamate (R)-4. Only a
As a final example of successful application of our HNL-
CLEA-based multistep strategy toward cyanohydrin derivatives
we selected the synthesis of (R)-mandelic acid from benzalde-
hyde on a multigram scale. R-Hydroxy-acids are indeed
prepared industrially on a multiton scale from the corresponding
aldehyde using PaHNL as catalyst in a biphasic medium
followed by acid hydrolysis.7 Moreover, we recently reported
the highly efficient synthesis of (R)-2-hydroxy-2-methyl-butyric
acid from 2-butanone using a similar strategy based on CLEAs
of the HNL from Linum usitatissimum (LuHNL) as biocatalyst.25
Using the PaCLEA-catalysed approach developed here, recrys-
tallized (R)-mandelic acid (R)-6 was obtained in very good
yields (80%) and excellent ee (99%).
Conclusions
The combination of immobilized biocatalysts and organic
solvent as reaction media allowed the development of a
straightforward overall process towards derivatives of either (R)-
or (S)-mandelonitrile from benzaldehyde. In particular, down-
stream processing of the cyanohydrin intermediate did not
require an extraction step, thereby reducing the amount of waste
when compared to reactions in aqueous or biphasic media.
Impurities in the crude cyanohydrin obtained, especially water
and residual benzaldehyde, were minimal and did not limit a
range of derivatization to be conducted successfully. These
(32) See for instance: Blanco, M. J.; Sardina, F. J. J. Org. Chem. 1996,
61, 4748.
(33) Caira, M. R.; Hunter, R.; Nassimbeni, L. R.; Stevens, A. T.
Tetrahedron: Asymmetry 1999, 11, 2175.
(34) Effenberger, F.; Ja¨ger, J. J. Org. Chem. 1997, 62, 3867.
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expedient procedures toward enantiopure cyanohydrin deriva-
tives also facilitated the recycling of the catalyst and enabled
high volumetric yields, which are all essential to the successful
application in the laboratory and the implementation of cost-
efficient industrial processes.
filtered through silica using hexane as eluent. The solvent was
removed under reduced pressure to give (R)-TMS-mandeloni-
trile (R)-3 (339.3 mg, 1.65 mmol) in 85% yield (from benzal-
dehyde) and 99% ee and (S)-TMS-mandelonitrile (S)-3 (475.3
mg, 2.31 mmol) in 83% yield (from benzaldehyde) and 99%
ee respectively, as clear liquids. The ee of TMS-mandelonitrile
was determined by HPLC (Chiralpak AD; Hex:iPA (99.8:0.2);
1.00 mL/min; Rt(R) ) 10.27 min, Rt(S) ) 12.17 min). 1H and
13C NMR spectra of the products were consistent with literature
data: 1H NMR36 (CDCl3, 400 MHz): δ ) 0.24 (s, 9H, TMS),
5.50 (s, 1H, CH), 7.37-7.48 (m, 5H, Ph). 13C NMR36 (CDCl3,
100.65 MHz): δ ) -0.3, 64.0, 119.4, 126.8, 129.7, 129.8,
135.9.
(1-(S)-Phenyl-ethyl) Carbamic Acid (R)-Cyano-phenyl-
methyl Ester. (R)-Mandelonitrile was prepared from benzal-
dehyde (255.7 mg, 2.41 mmol) using PaCLEA (14.9 mg) as
described in the general procedure. The crude cyanohydrin was
cooled to 0 °C and THF (5 mL) was added. A solution of (S)-
1-phenylethyl isocyanate33 (534.2 mg, 1.5 equiv) in THF (5
mL) was added at this temperature followed by CuBr ·Me2S
(736.2 mg, 1.5 equiv), and the mixture was allowed to warm
up slowly to r.t.. After overnight stirring, the solids were filtered
off and rinsed with diethyl ether (20 mL). The green solution
was then concentrated in Vacuo, and the residue was stirred
vigorously in diethyl ether (20 mL). The insoluble materials
were filtered off and rinsed with diethyl ether (20 mL); the
volatiles were removed under reduced pressure to give the crude
carbamate (722.4 mg). The pure carbamate derivative (555.5
mg, 1.98 mmol) was obtained after recrystallisation from
pentane/iPA as a white solid in 82% yield (from benzaldehyde)
and 99.5% ee. The ee of the carbamate was determined by
HPLC (Chiralpak AD; Hex/iPA (70:30); 1.50 mL/min; Rt(S)
) 3.95 min, Rt(R) ) 5.13 min). 1H NMR37 (CDCl3, 400 MHz):
δ ) 1.48, (d, 3H), 4.84 (mc, 1H), 5.18 (bs, 1H), 6.41 (s, 1H),
7.29-7.53 (m, 10H). 13C NMR37 (CDCl3, 100.65 MHz): δ )
22.1, 51.3, 63.6, 118.9, 125.6, 125.9, 127.7, 128.8, 129.1, 130.2,
132.1, 132.9, 154.4.
(R)-N-tert-Butyl-2-hydroxy-2-phenylacetamide. (R)-Man-
delonitrile was prepared from benzaldehyde (319.9 mg, 3.01
mmol) using PaCLEA (15.4 mg) as described in the general
procedure. The crude cyanohydrin was dissolved in tert-butanol
(2 mL), then aqueous 60% H2SO4 (2 mL) was added slowly to
the mixture at r.t., and stirring was continued for 24 h. The
solution was diluted with 10 mL of water and extracted with 3
× 50 mL of methylene chloride. The combined organic layers
were dried over MgSO4 and evaporated under reduced pressure
to give the crude product. Traces of tert-butanol were removed
by stirring the crude product under high vacuum, and (R)-N-
tert-butyl-2-hydroxy-2-phenylacetamide (436.1 mg, 2.10 mmol)
was obtained as a white solid in 70% yield (from benzaldehyde)
and 96% ee. Enantiopure product (ee > 99%) could be obtained
by recrystallisation from a minimum amount of hexane. The
ee of the product was determined by HPLC (Chiralpak AD;
Hex/iPA (90:10); 1.00 mL/min; Rt(R) ) 6.35 min, Rt(S) ) 7.06
min). 1H and 13C NMR spectra of the products were consistent
Experimental Section
CAUTION. All procedures involving hydrogen cyanide
were performed in a well-ventilated fume hood equipped with
a HCN detector. HCN-containing wastes were neutralized using
commercial bleach and stored independently over a large excess
of bleach for disposal.
General Methods. Chemicals and reagents were com-
mercially available and used without further purification unless
indicated otherwise. Benzaldehyde was distilled before use to
remove traces of benzoic acid. Solutions of the hydroxynitrile
lyases from Prunus amygdalus (PaHnL, 690 U/mL) and
Manihot esculenta (MeHnL, 2.23 kU/mL) were purchased from
Codexis. The corresponding CLEAs were prepared from these
solutions as previously reported.21,26,27 1H and 13C NMR spectra
were recorded on a Bruker 400 Avance Ultrashield spectrom-
eter. Gas chromatography (GC) was performed using an Agilent
Technologies 6890N chromatograph equipped with a HP-5
column or a ꢀ-Dex 225 chiral column and a FID detector. High
performance liquid chromatography (HPLC) was performed on
an Agilent Technologies 1100 series chromatograph equipped
with a diode array detector. Optical rotations were measured at
589 nm in a 10 cm sample tube. A 2 M HCN stock solution in
buffer-saturated DIPE was prepared as reported previously21
and used without further purification for reaction.
CLEA-Catalysed Formation of (R)- and (S)-Mandeloni-
trile: General Method. The corresponding CLEA (∼15 mg)
was suspended in a 2 M stock solution of HCN in DIPE (4
mL, 8 mmol), and benzaldehyde (200 µL, 1.96 mmol) was
added. After stirring gently (150 rpm) for 4 h at r.t. the CLEA
was filtered off, and the volatiles were removed under reduced
pressure to yield the crude cyanohydrin. Conversion of ben-
zaldehyde into mandelonitrile was greater than 95%, and the
ee of the corresponding cyanohydrin was greater than 97%.
Conversion and ee were determined by chiral HPLC (Chiralcel
OJ; Hex/iPA (90:10); 1.00 mL/min; Rt(benzaldehyde) ) 3.76
min, Rt(R) ) 12.18 min, Rt(S) ) 15.62 min). 1H and 13C NMR
spectra of the products were consistent with literature data: 1H
NMR35 (CDCl3, 400 MHz): δ ) 3.46 (bs, 1H, OH), 5.54 (s,
1H, CH), 7.41-7.56 (m, 5H, Ph).13C NMR35 (CDCl3, 100.65
MHz): δ ) 63.3, 119.0, 126.8, 129.1, 129.8, 135.1.
TMS-Protected-(R)- and -(S)-Mandelonitrile. (R)-Man-
delonitrile was prepared from benzaldehyde (206.2 mg, 1.94
mmol) using PaCLEA (15.3 mg) and (S)-mandelonitrile from
benzaldehyde (293.8 mg, 2.79 mmol) using MeCLEA (17.8
mg) as described in the general procedure. The crude cyano-
hydrin was cooled to 0 °C and TMSCl (635 µL, 2.5 equiv)
was added to the residue at 0 °C under argon followed by a
solution of pyridine (322 µL, 2 equiv) in methylene chloride
(2 mL) dropwise. The mixture was stirred for 5 min at 0 °C
and the ice water bath was removed. After 3 h at r.t., the
volatiles were evaporated carefully and the crude product was
(36) Denmark, S. E.; Chung, W. J. J. Org. Chem. 2006, 71, 4002.
(37) Although this compound has not been reported previously, the 154.4
ppm peak in 13C NMR was consistent with that of a carbamate carbon
as described in J. Am. Chem. Soc. 2003, 125, 7307.
(35) Shen, Z. L.; Ji, S. J.; Loh, T. P. Tetrahedron Lett. 2005, 46, 3137.
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with literature data: 1H NMR38 (CDCl3, 400 MHz): δ ) 1.32
(s, 9H), 2.50 (bs, 1H), 4.90 (s, 1H), 5.83 (bs, 1H), 7.31-7.40
toluene (150 mL) to give pure (R)-mandelic acid (10.34 g, 68
mmol) in 80% yield (from benzaldehyde) and 99% ee. The ee
of the product was determined by comparison of the sample
13
(m, 5H). C NMR38 (CDCl3, 100.65 MHz): δ ) 28.6, 51.9,
73.8, 127.0, 128.6, 129.0, 139.1, 170.4.
specific optical rotatory power to the literature data39 [R]22
D
(R)-Mandelic Acid: Gram-Scale Preparation. (R)-Man-
delonitrile was prepared from benzaldehyde (8.65 mL, 85
mmol) using PaCLEA (677.8 mg) as described in the general
procedure. A mixture (1:1) of conc. HCl/water (30 mL) was
added to the crude cyanohydrin, the solution was heated to
65-70 °C, and the reaction was allowed to proceed upon
vigorous stirring at this temperature for 40 h. The mixture was
allowed to cool to r.t.; diethyl ether (200 mL) was added, and
vigorous stirring was continued for 30 min. The insoluble
materials were filtered off, and the phases were separated. The
aqueous fraction was extracted with diethyl ether (2 × 200 mL),
and the combined ethereal layers were evaporated under reduced
pressure. The crude product (12.40 g) was recrystallised from
-151.9° (c 2.52, MeOH), (lit. [R]24 D -152° (c 2.52, MeOH)).
1H NMR spectrum of the product was consistent with literature
1
data: H NMR40 (CDCl3, 400 MHz): δ ) 5.12 (s, 1H),
7.27-7.36 (m, 3H), 7.46-7.49 (m, 2H).
Acknowledgment
We thank ICES Ltd, Singapore, for financial support.
Received for review August 5, 2009.
OP900207N
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