Paper
RSC Advances
Generalitat de Catalunya (2009 SGR 00281) and ERA-IB
MICINN. PIM2010EEI-00607. Authors would also like to
acknowledge the support from COST action: CM0701 and the
new COST action CM1303 Systems Biocatalysis, the Croatian
Ministry of Science Education and Sports for PhD scholarship of
Martina Sudar and Susana Amezqueta from Bioglane S.L.N.E.
for the assessment of purity of the Bioglane S.L.N.E. materials
used in this work. The help of Apl. Prof. Dr Martina Pohl in
Conclusion
In this work, a novel cascade reaction consisting of three
enzymes operating simultaneously in one-pot was successfully
developed to produce the aldol adduct (3S,4R)-6-[(benzyloxy-
carbonyl)amino]-5,6-dideoxyhex-2-ulose (3), precursor of the
iminocyclitol D-fagomine in good yields (79%). ADH from horse
liver was used for the oxidation of 1 to produce 2 which is
concomitantly used, i.e. in the same pot, as a substrate in the
aldol addition catalyzed by FSA A129S to produce 3. In this
reaction, NOX from L. lactis was successfully applied as a
regenerating enzyme for the in situ regeneration of coenzyme
NAD+. The use of a regeneration system is essential to make a
process economically feasible. This cascade presents an inter-
esting way to conduct aldol addition reactions using stable and
readily available starting materials such as amino alcohols.
Because of high instability of 2, as well as 1, used as the main
reactant in this cascade, it would not be possible to carry out
successfully this cascade reaction without the protecting group.
Optimization of initial conditions of cascade reaction
showed that higher concentrations of coenzyme and HLADH
favor both the formation of 3 and 4. Maximal yield of 3 achieved
in this cascade reaction was 79%. At the same time yield of 4
was 10%. The system might be further improved by nding the
appropriate ADH which would not catalyze the over-oxidation of
amino aldehyde to amino acid.
¨
providing HLADH puried in the lab of Research Centre Julich
is greatly acknowledged.
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c
kd
Molar concentration, mM
Operational stability decay rate constant, hꢂ1
Specic activity, U mgꢂ1
Reaction time, min
S.A.
T
t1/2
V.A.
Venz
Vr
Half-life time, h
Volume activity, U mLꢂ1
Enzyme volume, mL
Reactor volume, mL
Reaction yield, %
Y
g
Mass concentration, mg mLꢂ1
Horse liver alcohol dehydrogenase
Dihydroxyacetone
HLADH
DHA
FSA
HCl
NOX
TEA
TFA
D-Fructose-6-phosphate aldolase
Hydrochloric acid
NADH oxidase
Triethanolamine
Triuoroacetic acid
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This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 69819–69828 | 69827