Benzaldehyde lyase catalyzed enantioselective self and cross condensation reactions of acetaldehyde derivatives

Article abstract of DOI:10.1039/c0ob01121e

Flexible protected 1,3,4-trihydroxy-2-butanone is synthesized in high enantiomeric excesses by using asymmetric homo- and cross- acyloin coupling of aliphatic aldehydes catalyzed by benzaldehyde lyase.

Full text of DOI:10.1039/c0ob01121e

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Benzaldehyde lyase catalyzed enantioselective self and cross condensation
reactions of acetaldehyde derivatives†
˙
Peruze Ayhan, Ilke S¸ims¸ek, Burc¸e C¸ ifc¸i and Ayhan S. Demir*
Received 6th December 2010, Accepted 21st February 2011
DOI: 10.1039/c0ob01121e
Flexible protected 1,3,4-trihydroxy-2-butanone is synthesized
in high enantiomeric excesses by using asymmetric homo- and
cross- acyloin coupling of aliphatic aldehydes catalyzed by
benzaldehyde lyase.
Synthetically useful asymmetric cross acyloin reactions with
functionalized aliphatic aldehydes, such as 1, have a broad range
of application, for which no general and efficient enantioselective
system is currently available. The a,a¢-dihydroxy ketone subunits,
as shown in 2, can be found in many biologically important
compounds and are used for the synthesis of a wide range of com-
pounds, such as ketosugars and aminodiols.1 1,3,4-Trihydroxy-
2-butanone (erythrulose) (2) (Scheme 1) is one of the trioses
and tetroses that are widely used as a self-tanning substance in
cosmetic and dermatological formulations.2 Erythritols (3) are
used as sweeteners, and its mannosyl derivatives form a promising
class of biosurfactants.3
Scheme 2 BAL catalyzed cross coupling reactions of aromatic aldehydes
with mono- & dimethoxy acetaldehydes.
in the asymmetric condensation reactions of aliphatic aldehydes in
low enantioselectivities. However, the products that were formed
lacked functionality, as the groups on the molecule cannot be
easily converted to other functionalities, and aliphatic aldehydes
were only used in self condensation reactions.10
Up to now, only phosphate buffer has been used for BAL
mediated reactions, and the improvement of the enantioselectivity
of the targeted reaction has been attempted by using increased
amounts of enzyme. However, the screening of reaction parameters
such as the solvent and buffer for the improvement of the desired
activity is an important strategy.
By searching for the ability of BAL for the cross aliphatic
acyloin condensation study, BAL was used for the self and
cross condensation reactions of functionalized acetaldehyde where
aliphatic aldehydes were used as donors. A new two phase system
was developed for efficient aliphatic acyloin reactions.
Scheme 1 Polyhydroxy compounds.
The self-condensation of benzyloxyacetaldehyde was firstly
achieved with the classical methodology where the substrate
was dissolved in potassium phosphate buffer (pH 7, containing
MgSO4 and ThDP) containing 20% DMSO. By the addition
of BAL (50 U), the reaction was started at 37 ◦C. The same
amount of enzyme was added on a daily basis. After 4 days,
the reaction was terminated. The ee of the self-condensation
product of benzyloxyacetaldehyde was 50%. Various solvents,
such as dimethyl formamide (DMF), toluene, ether, benzene,
acetonitrile, and tetrahydrofuran (THF) were tested to increase the
selectivity. With acetonitrile and THF, product formation could
not be achieved. It was found that when toluene was used with
phosphate buffer (two-phase reaction), the condensation product
was obtained with 61% ee and 54% yield. A further improvement
was achieved with employing diisopropyl ether where a 75% ee
and 71% yield were attained.
Lyases are an important class of enzymes that achieve several
important reactions, including C–C bond formations.4 The thi-
amine diphosphate dependent enzyme, benzaldehyde lyase (BAL,
EC 4.1.2.38),5 has been shown to catalyze a broad range of C–
C bond formation reactions.6 BAL has been used extensively
to achieve aromatic-aromatic,7 aromatic-aliphatic,7–9 and non-
functionalized aliphatic-aliphatic acyloin derivatives.10 Recently,
BAL has been used in asymmetric carboligation reactions using
a,b-unsaturated aldehydes.11 BAL is believed to need aromatic
aldehydes as donors while acceptors can be aromatic and aliphatic
aldehydes.12 For the first time, we published the aromatic-aliphatic
cross coupling reaction with functionalized acetaldehyde deriva-
tives as shown in Scheme 2.8 Recently, this enzyme has been used
Department of Chemistry, Middle East Technical University, 06531, Ankara,
Turkey. E-mail: asdemir@metu.edu.tr; Fax: +90 312 2103200; Tel: +90 312
2103242
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c0ob01121e
The reaction was finally optimized with the selection of the
proper buffer. The buffer type has also been shown to be effective
on both enantioselectivity and the yield of the reaction, as seen
in Table 1. Several buffers (Good buffers12 such as MES, MOPS,
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Table 1 Effect of the buffer type on enantiomeric excess and conversion
Scheme 4 BAL mediated cross condensation of 4 with 10.
Buffera
% Conversion
% eeb
the previous cross condensation reaction, only one cross conden-
sation product [1-(benzyloxy)-3-hydroxy-4,4-dimethoxybutan-2-
one, 13] was observed in the reaction medium out of four
theoretical products in 93% ee (Scheme 3). The self-condensation
products (5 & 7) were again only detectable when one of the
substrates was used in excess amounts. In all cases, (R)-configured
products are obtained.
Phosphatea
MESb
71
88
91
85
73
75
93
95
87
91
MOPSa
HEPESc
Trisa
a Self condensation reactions were performed at specific pH values accord-
ing to the buffer type; (a) pH 7, (b) pH 6.5, (c) pH 8 and diisopropyl ether
was used as cosolvent. b The ee value was determined with a Chiralpakꢀ
R
OA column, 80 : 20/hexane : isopropanol, 0.8 mL min-1, 220 nm, Rt: 12.036
min for (S), 13.934 min for (R).
HEPES, and other previously used buffers such as phosphate and
tris) were screened for increasing the enantioselectivity and yield.
Good buffers were employed at varying pH values to observe the
effect of pH together with the buffer type. However, the pH was
found to have a negligible effect on the enantioselectivity and yield.
Although MES buffer was previously shown to be inappropriate
for BAL13 with the findings of lower residual activity, and via
regular enzyme addition, the lower residual activity limitation
could be overcome.
After the optimization of the reaction conditions, cross con-
densation reactions of benzyloxyacetaldehyde with 2-furan-2-
carbaldehyde and dimethoxyacetaldehyde were performed. When
two different aldehydes are reacted under the catalysis of BAL
in a cross condensation manner, four products can be expected
(Scheme 3); two self-condensation (5 & 7) and two cross conden-
sation products (8 & 9).
Scheme 5 BAL mediated cross condensation of 4 with 12.
The changing of the reaction conditions and co-solvents change
the substrate spectrum and selectivity. It seems that the aromatic
aldehydes are the preferred substrate as donor aldehyde when
they are present. It has been shown that aliphatic aldehydes are
both suitable donors and acceptors. In this case, the active side
differs regarding the aliphatic aldehydes whether to be a donor
or acceptor. This interaction could be the summary of the steric
and electronic interaction. The orientation of the carbonyl moiety
should be the same due to the results of the absolute configuration
of the product. These results also show that the substrate spectra
of these C–C-ligating enzymes is broader than described in the
literature so far, so that further interesting substrates could be
expected to be applied in this attractive carboligating reaction.
More studies are essential for the explanation of the mechanism.
With this novel system, it is possible to synthesize 1 with
different protecting groups, which also makes it possible to have
both enantiomers of the products in one reaction after further
modifications.
Experimental
Scheme 3 Carboligation of benzyloxyacetaldehyde, 4 with different
aldehydes.
General
When benzyloxyacetaldehyde is reacted with furan-2-
carbaldehyde under BAL catalysis at a 1 : 1 ratio in DIPE (diiso-
propyl ether)-MOPS in a two phase system, only one cross conden-
sation product [3-(benzyloxy)-1-(furan-2-yl)-2-hydroxypropan-1-
one, 11] was obtained with 90% ee, Scheme 4. Furan-2-
carbaldehyde acted as donor while benzyoxyacetaldehyde was
used by the enzyme as an acceptor. Self-condensation products
(5 & 7) were only detectable when one of the substrates was used
in excess amounts.
The preference for acceptor and donor changed when ben-
zyloxyacetaldehyde was reacted with dimethoxyacetaldehyde,
Scheme 5. This substrate acted as a donor in the reaction with
dimethoxyacetaldehyde while it was not accepted as a donor when
furan-2-carbaldehyde was employed as a cosubstrate. Similar to
NMR spectra were recorded with Brucker-Spectrospin DPX-400,
Ultra Shield, High Performance Digital FT-NMR spectrometer
using tetramethylsilane (TMS) as an internal standard and deuter-
ated chloroform (CDCl3) as a solvent. Mass spectra were recorded
with Thermo Quest GC-MS equipped with a Phenomenex Zebron
capillary GC column (60 m length, 0.25 mm ID, 0.25 mm film
thickness). Flash column chromatography was performed by
using Merck Silica Gel 60 (particle size: 40–63 mm, 230–400
mesh ASTM). Enantiomeric excess values were determined by an
Agilent 1100 series HPLC device using a Chiralpak OA column,
Chiralpak OD column and Chiralpak OJ column.
The E.coli BL21(DE3)pLysS strain purchased from
R
Invitrogenꢀ was used as a host to produce the recombinant
enzyme (BALHIS). Enzyme production was performed in the
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New Brunswick BioFlo110 Fermentor, equipped with pH and
temperature probes as well stirring rate controls. The purification
1H), 3.8 (dd, J = 3.7, 10.1 Hz, 1H), 4.21 (d, J = 17.2 Hz, 1H),
4.28 (d, J = 17.2 Hz, 1H), 4.58–4.30 (m, 5H), 7.37–7.09 (m,
10H). 13C-NMR (100 MHz, 2.5/1: CDCl3/CCl4): d ppm 70.86,
73.21, 73.53, 73.63, 75.07, 127.80, 127.93, 128.12, 128.45, 128.54,
132.75, 134.12, 204.90. HRMS for C18H20NaO4 (M + Na+): calcd.
323.1252, found: 323.1263.
of the hexa-histidine tagged enzyme was performed with an
2+
R
Ni -NTA affinity column (Invitrogenꢀ).
Preparation of benzaldehyde lyase
E.coli BL21(DE3)pLysS carrying pUC19-BALHIS construct was
used for BAL (EC. 4.1.2.38) production. The cultures were
maintained on LB agar slants. The cells from the newly prepared
slants were inoculated into the preculture Luria broth (LB)
where it was grown for 8 h at 37 ◦C, and then transferred
to the production (LB) medium with an inoculation ratio of
1/10 (1.65 L in a 2 L fermentor). 6 h after the induction with
isopropyl-b-D-thiogalactopyranoside (IPTG), cells were harvested
by centrifugation. The enzyme was either used as a crude form
without purification (the pelleted cells were transferred to a Petri
dish and lyophilized for 36 h) or as a purified enzyme.
One unit (U) of BAL activity is defined as the amount of
enzyme that catalyzes the cleavage of 1 mmol benzoin in potassium
phosphate buffer (50 mM, pH 7) containing MgSO4 (2.5 mmol
L-1), ThDP (0.15 mmol L-1), and DMSO (20 vol-%) at 30 ◦C per
minute at 30 ◦C.
(R)-3-(Benzyloxy)-1-(furan-2-yl)-2-hydroxypropan-1-one (11)
25
R
Yellow oil, [a]D : +70.3 (c 0.4, CHCl3)90% ee, Chiralpakꢀ OD
column, 98 : 2/hexane: isopropanol, 1 mL min-1, 254 nm, Rt:
68.688 min for (S), 75.499 min (R). H NMR (400 MHz, 2.5/1:
1
CDCl3/CCl4): 3.72 (dd, J = 3.26, 10.21 Hz, 1H), 3.81 (dd, J = 3.26,
10.21 Hz, 1H), 4.38 (d, J = 12.3 Hz, 1H), 4.48 (d, J = 12.3 Hz, 1H),
4.82 (t, J = 3.57 Hz, 1H), 6.47 (m, 1H), 7.06–7.09 (m, 2H), 7.11–
7.19 (m, 3H), 7.24 (d, 1H), 7.47 (m, 1H). 13C NMR (100 MHz,
2.5/1: CDCl3/CCl4): 72.26, 75.92, 95.07, 111.36, 117.70, 126, 127,
128, 137, 145.52, 145.59, 149.59, 186.32. HRMS for C14H14NaO4
(M + Na+): calcd. 269.0790, found: 269.0784.
(R)-1-(Benzyloxy)-3-hydroxy-4,4-dimethoxybutan-2-one (13)
25
R
Yellow oil, [a]D : -14.5 (c 0.4, CHCl3). 93% ee, Chiralpakꢀ OJ
column, 90 : 10/hexane: isopropanol, 0,8 mL min-1, 220 nm, Rt:
18.833 min for (S), 65.387 min (R). H-NMR (400 MHz, 2.5/1:
1
General procedure for the BAL catalyzed self condensation of
benzyloxyacetaldehyde
CDCl3/CCl4): d = 3.37 (s, 6H), 4.18 (d, J = 18.2 Hz, 2H), 4.35
(m, 2H), 4.49 (dd, J = 11.8 Hz, 1H), 4.49 (dd, J = 11.8 Hz, 1H),
7.22–7.26 (m, 5H). 13C-NMR (100 MHz, 2.5/1: CDCl3/CCl4):
d ppm 54.69, 55.971, 72.482, 72.561, 104.397, 127.009, 127.281,
127.465, 136.201, 205.274. HRMS for C13H18NaO5 (M + Na+):
calcd. 277.1052, found: 277.1045.
Benzyloxyacetaldehyde 4 (150 mg, 1 mmol) was dissolved in 10 mL
diisopropylether (25 vol-%), and then 30 mL (75 vol-%) MOPS
buffer (50mM, pH 7) containing 0.15 mM THDP and 2.5 mM
MgSO4 was added to this solution. The reaction was started with
the addition of BAL (50 U) at 30 ◦C (120 rpm). BAL was added
(50 U) on a daily basis. The reaction was monitored with TLC
and GC-MS and concluded after 96 h. The reaction mixture was
extracted with chloroform (3 ¥ 50 mL) and the combined organic
layers were dried over MgSO4, and the solvent was removed under
reduced pressure. The product was purified with flash column
chromatography.
Conclusions
BAL catalyzed the cross acyloin reactions with functionalized
acetaldehydes with the same and different protecting groups
that were achieved in high yields and enantioselectivities. All of
the products obtained in this study can be employed as chiral
synthons, as they are amenable to further modifications.
General procedure for BAL catalyzed cross condensation reactions
of benzyloxyacetaldehyde
Acknowledgements
The financial support from the Middle East Technical University,
the Scientific and Technological Research Council of Turkey
(TUBITAK), the Turkish Academy of Sciences, and the COST
CM0701 is gratefully acknowledged.
Benzyloxyacetaldehyde 4 (150 mg, 1 mmol) and the corresponding
aldehyde (1 mmol) were dissolved in 10 mL diisopropylether
(25 vol-%) then 30 mL (75 vol-%) MOPS buffer (50mM, pH 7)
containing 0.15 mM THDP and 2.5 mM MgSO4 was added to
this solution. The reaction was started with the addition of BAL
(50 U) at 30 ◦C (120 rpm). BAL was added (50 U) on a daily basis.
The reaction was monitored with TLC and GC-MS and concluded
after 96 h. The reaction mixture was extracted with chloroform (3 ¥
50 mL) and the combined organic layers were dried over MgSO4,
and the solvent was removed under reduced pressure. The product
was purified with flash column chromatography.
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(R)-1,4-Bis(benzyloxy)-3-hydroxybutan-2-one (5)
25
R
Yellowish oil, [a]D : -1.3 (c 0.9, CH2Cl2) 95% ee, Chiralpakꢀ OA
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