Synthesis of rare sugars with l-fuculose-1-phosphate aldolase (FucA) from Thermus thermophilus HB8

Article abstract of DOI:10.1016/j.bmcl.2011.03.072

We report herein a one-pot four-enzyme approach for the synthesis of the rare sugars d-psicose, d-sorbose, l-tagatose, and l-fructose with aldolase FucA from a thermophilic source (Thermus thermophilus HB8). Importantly, the cheap starting material DL-GP (DL-glycerol 3-phosphate), was used to significantly reduce the synthetic cost.

Full text of DOI:10.1016/j.bmcl.2011.03.072

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5084–5087
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of rare sugars with L-fuculose-1-phosphate aldolase (FucA)
from Thermus thermophilus HB8
Zijie Li a,b, , Li Cai b, , Qingsheng Qi a, Thomas J. Styslinger b, Guohui Zhao b, Peng George Wang b,
⇑
a National Glycoengineering Research Center and the State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong 250100, China
b Department of Chemistry and Biochemistry, The Ohio State University, 484 West 12th Avenue, Columbus, OH 43210, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We report herein a one-pot four-enzyme approach for the synthesis of the rare sugars
D
-psicose, D-sor-
Received 24 February 2011
Revised 15 March 2011
Accepted 17 March 2011
Available online 23 March 2011
bose, L-tagatose, and L-fructose with aldolase FucA from a thermophilic source (Thermus thermophilus
HB8). Importantly, the cheap starting material DL-GP (DL-glycerol 3-phosphate), was used to signifi-
cantly reduce the synthetic cost.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Aldolase
Rare sugar
Synthesis
FucA
The aldol reaction has long been recognized as one of the most
powerful methods for new carbon–carbon bond formation.1,2 The
high stereoselectivity of aldolases in C–C bond construction confers
upon them tremendous applications as synthetic biocatalysts.3–5
Among the aldolases, dihydroxyacetone phosphate (DHAP)-depen-
dent aldolases are particularly attractive as a set of four possible
diastereomers of vicinal diols can be synthesized conferring upon
them the potential to be used in the synthesis of rare sugars and
other hydroxylated natural products.1,6,7 Unfortunately, the strict
requirement for the donor substrate DHAP, a rather expensive
and unstable compound, limits aldolase use in large-scale prepara-
tion.6,8,9 Therefore, the capability to generate DHAP from inexpen-
sive sources could ultimately broaden the scope of aldolase
reactions making it an attractive challenge.10,11
Rare sugars are monosaccharides, and their derivatives, that are
particularly uncommon in nature.12 Importantly, rare sugars pos-
sess many potential applications in the food, pharmaceutical and
nutrition industries.13 In addition, rare sugars can be used as start-
ing materials for the synthesis of intriguing natural products with
important biological activities.13,14 Unfortunately, most rare sugars
are quite expensive, and their synthetic routes are both limited and
costly due to the expense of costly starting materials. As a specific
with sucrose, it has 70% the sweetness but provides no energy due
to its suppressive effect toward hepatic lipogenic enzymes.15,16
Furthermore, it has been observed that foods supplemented with
D
-psicose exhibit higher antioxidant activity.15 Furthermore,
D-psi-
cose can be used as a precursor in the synthesis of xylosylpsicoses,
which are promising candidates for prebiotics, cosmetics and ther-
apeutic uses.17 However, only two enzymes
D
-tagatose 3-epimer-
-psicose 3-epimerase
from Agrobacterium tumefaciens20 have been reported for
-psicose
production. In addition, due to the fact that the interconversion be-
tween -fructose and -psicose is an equilibrium process, the large
ase from Pseudomonas cichorii18,19 and
D
D
D
D
scale and high yield production of
challenging.
D-psicose remains quite
A potential solution to the above mentioned problems with rare
sugar production lies in the use of aldolase reactions (i.e., DHAP
aldolase reactions), which are capable of generating vicinal diol
diastereomers and are thus an ideal tool for the construction of
rare sugars.21 In fact, the preparation of rare sugars, as well as their
derivatives, represents the most significant applications of DHAP-
dependent aldolases.8,14,22,23 In our previous work, we synthesized
two rare sugars (D-sorbose and D-psicose) simultaneously with
Rhamnulose-1-phosphate aldolase (RhaD) from Escherichia coli in
good overall yields (Scheme 1). However, RhaD showed no ste-
reo-preference for either product (syn/anti ꢀ1:1) when accepting
example, D-psicose, a rare sugar and C-3 epimer of D-fructose, has
the unique property of being an ideal sucrose substitute. Compared
D-glyceraldehyde. Here we describe another enzyme, L-fuculose-
1-phosphate aldolase, from Thermus thermophilus HB8 (FucAT.HB8
capable of stereoselectively synthesizing -psicose from -glycerol
3-phosphate ( -GP) and -glyceraldehyde. Alternatively, the rare
sugars -tagatose and -fructose were synthesized efficiently with
)
⇑
Corresponding author. Tel.: +1 614 292 9884; fax: +1 614 688 3106.
E-mail addresses: pgwang@chemistry.ohio-state.edu, wang.892@osu.edu
(P.G. Wang).
D
L
L
D
L
L
Authors who contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.072

Z. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5084–5087
5085
we expressed and purified FucAT.HB8 in E.coli (see Supplementary
data) and then employed it in the one-pot reaction under the same
conditions (Scheme 2). To our delight, the yield was greatly im-
O
OH
O
HO
H2O3PO
OH
OH
OH
OH OH
proved (Table 1 and 67% vs 12%) with the diastereomer
being detected as a minor product when -glyceraldehyde was
used as the acceptor (Fig. 1 maroon line). The product ratio of
psicose/
-sorbose was ꢀ5:1 as determined by ion exchange HPLC
(Fig. 1 maroon line).
-glycerol 3-phosphate is a reasonably stable starting material
D-sorbose
DHAP
D-sorbose
RhaD
D
O
D-
O
OH
D
OH
HO
OH
L
OH OH
and commercially available, however it is still quite expensive,
and thus not ideal for large-scale synthesis. In contrast, racemic
glycerol 3-phosphate (DL-GP) is much cheaper and it has been re-
D-glyceraldehyde
D-psicose
syn/anti~1/1
L
-isomer.10,27 There-
Scheme 1. Enzymatic synthesis of
D
-sorbose and D-psicose with RhaD.
ported that GPO can exclusively oxidize the
fore, it was envisioned that a racemic mixture could deliver a
50% yield of DHAP and the remaining -glycerol 3-phosphate could
D
FucAT.HB8 using
isomer.
L-glyceraldehyde as the acceptor instead of the D-
be isolated during purification. Most significantly, the synthetic
cost could be greatly decreased by utilizing racemic DL-glycerol
3-phosphate (DL-GP). Thus, under the same reaction conditions,
FucA is a class II DHAP-dependent aldolase that catalyzes the
reversible cleavage of -fuculose 1-phosphate to DHAP and -lactal-
dehyde, a central step for
-fucose metabolism in bacteria.24 Fur-
thermore, FucA from E. coli (FucAE.coli) is a homotetramer of 215
amino acid residues containing one Zn2+ ion per subunit.25 Impor-
tantly, FucA has demonstrated its value in aldol reactions to selec-
tively afford vicinal diols with the anti configuration, which is
advantageous for
Consequently, we first employed FucAE.coli in the synthesis of
psicose via a one-pot four enzyme reaction in which DHAP was
L
L
staring from racemic DL-GP, a total yield of 58% (Table 1) of
cose/ -sorbose was obtained and the ratio was determined by
HPLC (Fig. 1 green line, -psicose/ -sorbose 8.4/1). The only differ-
D-psi-
L
D
D
D
ence for reactions starting with DL-GP is that DL-GP was used in
excess and the reaction yield was calculated using the acceptor
as the limiting reagent (see Supplementary data for reaction stoi-
chiometry). After silica gel and gel filtration chromatography, the
D
-psicose construction.26
D
-
mixture containing D-psicose/D-sorbose could be easily separated
by a cation exchange resin column (Ca2+ form) under elevated tem-
generated from the oxidation of L-glycerol 3-phosphate (L-GP) by
perature (70 °C) (see Supplementary data).
glycerol phosphate oxidase (GPO) (Scheme 2).27 To degrade the
corresponding by-product, hydrogen peroxide, and regenerate
oxygen, catalase was added to the reaction mixture. Subsequently,
Alternatively, when
instead of the -isomer, two additional rare sugars,
-tagatose, could accordingly be synthesized as allowed by the ste-
L-glyceraldehyde was used as the acceptor,
D
L
-fructose and
L
the DHAP generated in situ was coupled with
FucA to give -psicose 1-phosphate. Lastly, the phosphate group
was removed under acidic conditions by acid phosphatase (AP)
to furnish -psicose. Unfortunately, after silica gel and gel filtration
purification, only a 12% yield of -psicose could be obtained (Ta-
ble 1). Compared to the high yield of -psicose 1-phosphate when
D-glyceraldehyde by
reoselectivity of FucA (Scheme 3). L-fructose is a well known non-
nutritive sweetener30 and an inhibitor of several glycosidases31
with its enzymatic synthesis by the aldolase RhaD being greatly
D
exploited by Wong and co-workers.14,23 However,
L-tagatose,
D
which is a functional sweetener32 and a promising starting mate-
rial for the synthesis of high value-added complex compound,33
has not yet been broadly utilized due to its high cost of production.
D
D
DHAP was used directly for aldol addition26, this low yield can
most likely be attributed to the possibility that FucAE.coli may not
be compatible with the one-pot system.
L
-Tagatose can be produced via oxidation of galactitol by Klebsiella
pneumoniae 40b34 or generated via epimerization of
-sorbose by
-tagatose 3-epimerase from Pseudomonas sp. ST-2435, however,
both methods give low yields.
As shown in Scheme 3, we were able to successfully carry out
the synthesis of -fructose and -tagatose in a one-pot fashion using
DL-GP as starting material and -glyceraldehyde as the acceptor. It
L
To improve the overall yield for this synthesis, we turned our
attention towards enzymes from thermophilic bacteria, which
show a great potential for biotechnological applications.28 Upon
investigation, we noticed that the crystal structure of FucA from
T. thermophilus HB8 (FucAT.HB8) was recently reported but the en-
zyme had yet to be used for synthetic purposes.29 Consequently,
D
L
L
L
is interesting to note that the acid phosphatase (AP) we previously
FucAE.coli or FucAT.HB8
rt. 22 h
pH = 7.0
O
OH
2-
2-
OPO3
HO
OPO3
O32-PO
HO
O
DHAP
OH
OH
GPO
OH OH
L-glycerol 3-phosphate
O
L-GP
OH
O2
H2O2
OH
D-glyceraldehyde
catalase
AP
O
OH
37 oC, 22 h
pH = 4.7
HO
OH
OH OH
D-psicose/D-sorbose
Scheme 2. Synthesis of D-psicose with FucA and acid phosphatase (see Table 1 for yields and product ratio).

5086
Z. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5084–5087
Table 1
Summary of FucA catalyzed reactions using different starting materials and acceptors
Enzyme
Starting saterial
Acceptor
Product(s)
Yield (%)
Ratio
FucAE.coli
FucAT.HB8
FucAT.HB8
FucAT.HB8
L-GP
12
67
58
47
1:0
D
D
D
-Glyceraldehyde
-Glyceraldehyde
-Glyceraldehyde
D
D
D
-Psicose
-Psicose
-Psicose
L-GP
5.3:1
8.4:1
1.2:1
D
-Sorbose
-Sorbose
DL-GP
DL-GP
D
L-Glyceraldehyde
L-Fructose
L-Tagatose
Figure 1. HPLC (hydrogen form, sulfonated divinyl benzene–styrene copolymer support and eluted with 5 mM H2SO4) profile of final reaction mixture in Scheme 2 compared
with authentic samples.
FucAT.HB8
rt. 22 h
pH = 7.0
O
OH
2-
2-
OPO3
HO
2-
OPO3
HO
OPO3
OH
O
OH
GPO
DHAP
OH OH
DL-glycerol 3-phosphate
O
DL-GP
O2
H2O2
OH
OH
L-glyceraldehyde
catalase
O
OH
O
OH
YqaB
HO
37 oC, 12h
HO
OH
OH
+
OH OH
L-tagatose
OH OH
H3PO4
L-fructose
Scheme 3. Synthesis of L-tagatose and L-fructose with FucAT.HB8 using L-glyceraldehyde.
used could not dephosphorylate
L
-tagatose 1-phosphate com-
is that FucAT.HB8 seems to lose its stereoselectivity when accepting
-glyceraldehyde–similar to our previous discovery that -rhamnu-
lose-1-phosphate aldoalse (RhaD) produces a single product (
fructose) when using -glyceraldehyde while losing its stereoselec-
tivity when -glyceraldehyde is the acceptor. Nonetheless, this
pletely to give the desired sugar products. However, upon search-
ing relevant literature we found that YqaB phosphatase from E.coli,
belonging to the haloacid dehalogenase (HAD) superfamily, shows
a remarkably broad substrate range, among which the dephospho-
L
L
L-
L
D
rylation activity toward
D
-fructose-1-phosphate is the highest.36
property of aldolases could be well utilized for the synthesis of var-
ious types of rare sugars.
We thus expressed and purified YqaB phosphatase from E.coli
and the dephosphorylation reaction went quite smoothly under
In summary, we developed a one-pot four-enzyme approach for
neutral conditions.
moderate yield (see Table 1, total yield 47%) and the ratio was
determined by HPLC (Fig. 2, -fructose/ -tagatose 1.2/1). After silica
gel and gel filtration chromatography, the mixture containing
L
-Fructose and
L
-tagatose were afforded in
the synthesis of the rare sugars
D
-psicose,
D-sorbose, L-tagatose,
and
L-fructose with aldolase FucA. All synthesized rare sugars were
L
L
characterized by 1H NMR and compared with commercially
authentic samples (see Supplementary data). To the best of our
knowledge, this is the first use of FucA from a thermophilic source
(T. thermophilus HB8), which proved to be more efficient than its
E.coli counterpart. Importantly, the one-pot four-enzyme approach
L
-
fructose/L-tagatose could be easily separated by cation exchange
resin column (Ca2+ form) under elevated temperature (70 °C) (see
Supplementary data). As shown in Table 1, one thing worth noting

Z. Li et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5084–5087
5087
Figure 2. HPLC (calcium form, sulfonated divinyl benzene–styrene copolymer support and eluted with H2O) profile of final reaction mixture in Scheme 3 compared with
authentic samples.
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Acknowledgment
P.G.W. acknowledges NIH (R01 AI083754, R01 HD061935 and
R01 GM085267) for financial support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.072.
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