Enzymatic synthesis of d-sorbose and d-psicose with aldolase RhaD: Effect of acceptor configuration on enzyme stereoselectivity

Article abstract of DOI:10.1016/j.bmcl.2011.09.087

It was previously reported that DHAP-dependent aldolase RhaD selectively chooses l-glyceraldehyde from racemic glyceraldehyde to produce l-fructose exclusively. Contrastingly, we discovered that d-glyceraldehyde is also tolerated as an acceptor and the stereoselectivity of the enzyme is lost in the corresponding aldol addition. Furthermore, we applied this property to efficiently synthesize two rare sugars d-sorbose and d-psicose.

Full text of DOI:10.1016/j.bmcl.2011.09.087

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7081–7084
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Bioorganic & Medicinal Chemistry Letters
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Enzymatic synthesis of D-sorbose and D-psicose with aldolase RhaD: Effect
of acceptor configuration on enzyme stereoselectivity
Zijie Li a,c, Li Cai b, , Qingsheng Qi c, Peng George Wang a,c,
⇑
⇑
a Department of Chemistry, Georgia State University, Atlanta, GA 30302, USA
b University of South Carolina Salkehatchie, Walterboro, SC 29488, USA
c National Glycoengineering Research Center and The State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
It was previously reported that DHAP-dependent aldolase RhaD selectively chooses L-glyceraldehyde
Received 25 August 2011
Revised 19 September 2011
Accepted 21 September 2011
Available online 29 September 2011
from racemic glyceraldehyde to produce
-glyceraldehyde is also tolerated as an acceptor and the stereoselectivity of the enzyme is lost in the
corresponding aldol addition. Furthermore, we applied this property to efficiently synthesize two rare
sugars -sorbose and -psicose.
L-fructose exclusively. Contrastingly, we discovered that
D
D
D
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Aldolase
Rare sugar
Synthesis
RhaD
Stereochemically controlled carbon–carbon bond construction
by aldolases confers them tremendous utility as synthetic biocata-
lysts.1–4 Among the aldolase families, dihydroxyacetone phosphate
(DHAP)-dependent aldolases are remarkably useful and widely
investigated, as the two new stereocenters generated in the aldol
additions are predictable.5,6 Four reported DHAP-dependent aldol-
ases are stereocomplementary, so that a complete set of diastereo-
mers of vicinal diols can be achieved.5,7 Over the past two decades,
aldolases of this group have been applied successfully in producing
many rare monosaccharides and their derivatives.8–10 As a specific
example,
L
-rhamnulose-1-phosphate aldoalse (RhaD) was used to
couple DHAP with
produce
L-glyceraldehyde or racemic glyceraldehyde to
L
-fructose-1-phosphate. L-Fructose could then be obtained
by dephosphorylation with acid phosphatase (AP).9,11 (Scheme 1)
Furthermore, Wong and co-workers reported that RhaD preferen-
tially made
by-products. Moreover, they concluded that pure
was not necessary since -glyceraldehyde was not consumed as a
substrate by this enzyme. Contrastingly, here we report RhaD also
L-fructose and no diastereomers were found as
L
-glyceraldehyde
D
easily tolerates
D-glyceraldehyde to produce both
D-sorbose and
O
OH
OH
O
O
OH
O
OH
L
RhaD
AP
H2O3PO
OH
H2O3PO
or
HO
OH
OH
O
OH OH
DHAP
OH OH
L
-fructose-1-phosphate
L
-fructose
OH
OH
rac.
RhaD = L-rhamnulose-1-phosphate aldoalse; AP = acid phosphatase
Scheme 1. Enzymatic synthesis of L
-fructose with RhaD showing perfect stereoselectivity.11
⇑
Corresponding authors. Tel.: +1 843 549 6314x381 (L.C.), +1 404 413 3591 (P.G.W.).
E-mail addresses: CAILI@mailbox.sc.edu (L. Cai), pwang11@gsu.edu (P.G. Wang).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.087

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Z. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7081–7084
D-psicose, however, the stereoselectivity of this enzyme was totally
lost when D-glyceraldehyde was the acceptor.
D
-Sorbose and D-psicose are both rare sugars with many com-
mercial applications, especially in the food industry.12–14 They
are low-caloric sweeteners and functional food bulking agents.
Additionally, D-psicose has several potential medicinal properties,
such as inhibiting the elevation of the blood glucose level. Unfortu-
nately, both of them are quite expensive, and their synthetic routes
are limited and costly because they are usually carried out at the
expense of other valuable starting materials.14–16
Interestingly and contrasting to Wong’s results, in which
eraldehyde was separated as the remaining starting material, when
racemic glyceraldehyde was used, we discovered that - and
D-glyc-
D
L
-glyceraldehyde were both consumed in RhaD (from Escherichia
coli)17 catalyzed reactions to give a complex mixture of addition
products. Thus, we directly examined the RhaD reaction using pure
Figure 2. Separation profile of
D
-sorbose and
D-psicose with cation exchange resin
(Ca2+ form) eluted with ddH2O under 70 °C.
D-glyceraldehyde and found this substrate was mostly consumed
O
O
OH
O
OH
O
H2O3PO
HO
HO
H2O3PO
OH
OH
OH
DHAP
OH OH
OH OH
-sorbose
RhaD
rt. 22 h
pH = 7.0
AP
D
º
37 C, 22 h
O
pH ~ 5
OH
O
OH
Total yield
73 %
OH
H2O3PO
OH
OH
OH
-glyceraldehyde
D
OH OH
OH OH
D
-psicose
RhaD = L-rhamnulose-1-phosphate aldoalse; AP = acid phosphatase
Scheme 2. Enzymatic synthesis of D-sorbose and D-psicose with RhaD and AP.
Figure 1. HPLC (hydrogen form, sulfonated divinyl benzene–styrene copolymer support and eluted with 5 mM H2SO4, detected with refractive index (RI) detector) profile of
final reaction (after AP) mixture compared with authentic samples.

Z. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7081–7084
7083
O
2-
2-
OPO3
HO
OPO3
HO
OH
GPO
O
OH
-glycerol
3-phosphate
OH
-glyceraldehyde
L
H2O2
O2
DHAP
D
catalase
RhaD
rt. 22 h
pH = 7.0
O
OH
HO
HO
OH
OH OH
-sorbose
O
OH
D
AP
H2O3PO
OH
º
37 C,22 h
O
OH
pH = 4.7
OH OH
Total yield
48%
OH
OH OH
D
-psicose
RhaD = L-rhamnulose-1-phosphate aldoalse; AP = acid phosphatase;
GPO = glycerol phosphate oxidase
Scheme 3. One-pot four enzymes synthesis of D-sorbose and D-psicose.
within 16 h. Subsequently, after dephosphorylation with AP, exam-
ination by TLC revealed that the corresponding product spot had the
In summary, we report that
(RhaD) loses its stereoselectivity when accepting
for aldol addition. A possible explanation could be the different
enzyme concentrations used. While Fessner et al. previously
reported a diastereomeric ratio of 97:3 (syn:anti) of RhaD,23 our ob-
L
-rhamnulose-1-phosphate aldoalse
D
-glyceraldehyde
same Rf value as
D
-sorbose. A single peak (203.0526, [M+Na+]) on
high resolution mass spectrum was observed as well (Scheme 2).
However, cation exchange HPLC and NMR revealed that what
appeared to be one spot on TLC was actually two different products,
served results indicated that the anti product D-psicose may be
D
-sorbose and
exclusively produced
reaction), the production of both
ingly indicated aldolase RhaD had no stereo-preference when
-glyceraldehyde was the acceptor. Additionally, the stereoselectiv-
ity of this enzyme was affected by substrate configuration. The
product ratio ( -sorbose/
D
-psicose (Scheme 2, Fig. 1). While
-fructose (also proven in our one-pot
-sorbose and -psicose interest-
L
-glyceraldehyde
thermodynamically favored and is accumulated during reaction
time. Nonetheless, we utilized this property to synthesize two rare
L
D
D
sugars
that similar acceptor-controlled stereo preference was also
observed when another DHAP-dependent aldolase -fuculose-1-
phosphate aldolase from Thermus thermophilus HB8 (FucAT.HB8
was used: FucAT.HB8 seems to lose its stereoselectivity when
accepting
-glyceraldehyde.24 The enzymatically synthesized dia-
D-sorbose and D-psicose simultaneously. It is worth noting
D
L
)
D
D-psicose = ꢀ2/3) was determined by HPLC
after calibration with standard curves (see Supplementary data).
After silica gel chromatography and gel filtration separation, the
mixture containing only two rare sugars could be well isolated by
cation exchange resin (Ca2+ form) chromatography18,19 at 70 °C to
L
stereomers could then be easily isolated with cation exchange
resin (Ca2+ form) under elevated temperature. Lastly, with the dis-
covery of these features, the large scale production of
D-sorbose
provide pure
D-sorbose and
D-psicose. Figure 2 shows a typical
and -psicose using fermentation are currently underway.
D
separation profile of the products
D
-sorbose and -psicose with
D
the separation mainly resulting from different coordination affini-
ties of the monosaccharides to the Ca2+. Prior temperature studies
indicated that the best separation was achieved under 70 °C com-
pared with separation profiles under 50, 60, and 80 °C.20
Subsequently, to improve the practicality of the reaction by
avoiding the use of DHAP, a rather unstable and expensive sub-
strate,21 we optimized our experiment to provide DHAP in situ from
cheaper starting materials. A one-pot four enzyme system was used
Acknowledgments
P.G.W. acknowledges support from the NIH (R01 HD061935
and R01 GM085267) and financial support from Georgia State Uni-
versity. L.C. acknowledges support from University of South
Carolina Salkehatchie.
Supplementary data
in which DHAP was generated from oxidation of L-glycerol 3-phos-
phate by glycerol phosphate oxidase (GPO).22 The by-product of this
oxidation, H2O2, being harmful for GPO activity was thus selectively
degraded by adding catalase. The DHAP generated was coupled with
Supplementary data (experimental procedures and character-
ization of compounds) associated with this article can be found,
in the online version, at doi:10.1016/j.bmcl.2011.09.087.
D
-glyceraldehyde in situ by RhaD and following AP catalyzed
dephosphorylation furnished -sorbose and -psicose in moderate
yields in a one-pot fashion. (Scheme 3) The product ratio ( -sor-
bose/
-psicose = ꢀ1/1) was determined by HPLC. Under the same
conditions, when -glyceraldehyde was used instead of the -glycer-
aldehyde, -fructose was produced exclusively with 66% overall
yield (see Supplementary data).
D
D
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