Efficient enzymatic synthesis of l-rhamnulose and l-fuculose

Article abstract of DOI:10.1016/j.bmcl.2015.12.051

l-Rhamnulose (6-deoxy-l-arabino-2-hexulose) and l-fuculose (6-deoxy-l-lyxo-2-hexulose) were prepared from l-rhamnose and l-fucose by a two-step strategy. In the first reaction step, isomerization of l-rhamnose to l-rhamnulose, or l-fucose to l-fuculose was combined with a targeted phosphorylation reaction catalyzed by l-rhamnulose kinase (RhaB). The by-products (ATP and ADP) were selectively removed by silver nitrate precipitation method. In the second step, the phosphate group was hydrolyzed to produce l-rhamnulose or l-fuculose with purity exceeding 99% in more than 80% yield (gram scale).

Full text of DOI:10.1016/j.bmcl.2015.12.051

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient enzymatic synthesis of L-rhamnulose and L-fuculose
a,
Liuqing Wen a,y, Lanlan Zang b,y, Kenneth Huang a, Shanshan Li a, Runling Wang b, , Peng George Wang
⇑
⇑
a Department of Chemistry and Center for Therapeutics and Diagnostics, Georgia State University, Atlanta, GA 30303, USA
b Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University,
Tianjin 300070, China
a r t i c l e i n f o
a b s t r a c t
Article history:
L
-Rhamnulose (6-deoxy-
from -rhamnose and -fucose by a two-step strategy. In the first reaction step, isomerization of
nose to -rhamnulose, or -fucose to -fuculose was combined with a targeted phosphorylation reaction
catalyzed by -rhamnulose kinase (RhaB). The by-products (ATP and ADP) were selectively removed by
silver nitrate precipitation method. In the second step, the phosphate group was hydrolyzed to produce
-rhamnulose or -fuculose with purity exceeding 99% in more than 80% yield (gram scale).
Ó 2015 Elsevier Ltd. All rights reserved.
L
-arabino-2-hexulose) and
L
-fuculose (6-deoxy-
L
-lyxo-2-hexulose) were prepared
Received 2 November 2015
Revised 12 December 2015
Accepted 15 December 2015
Available online xxxx
L
L
L
-rham-
L
L
L
L
L
L
Keywords:
L
-Rhamnulose
-Fuculose
L
Enzymatic synthesis
Phosphorylation
De-phosphorylation
Among 36 hexoses and pentoses (12 ketoses and 24 corre-
sponding aldoses), only seven monosaccharides are naturally
(RhaD) or L-fuculose 1-phosphate aldolase (FucA) to facilitate fur-
ther metabolic function.11 RhaD and FucA are two powerful biocat-
alysts and have been widely used in synthetic chemistry to
present in substantial quantities (
D
-xylose,
D-ribose, L-arabinose,
D
-galactose,
D
-glucose,
D
-mannose, and
D
-fructose).1 With the
produce rare ketoses or their derivatives.12 Moreover,
lose and L
L
-rhamnu-
-fuculose can also be directly isomerized or epimerized
into other rare sugars.13 Therefore,
-rhamnulose and -fuculose
exception of
D
-fructose, all other ketoses are defined as ‘rare sug-
ars’.2 Despite their limited accessibility, rare ketoses offer a lot of
potential for applications in pharmaceutical, medicinal, food, and
L
L
are not only primary targets for investigating the mechanistic
and regulatory aspects of sugar metabolism, but also important
starting materials in synthetic chemistry. An efficient system to
readily provide both ketoses in considerable amounts is highly
attractive in enabling the studies of both deoxy ketoses.
synthetic chemistry.3 For example,
D-psicose has about 70% of
the sweetness but only 0.3% of the energy calories of sucrose.4 It
can also inhibit hepatic lipogenous enzyme activity, helping reduce
abdominal fat accumulation.5
L-xylulose was reported to be an
inhibitor of glycosidase6, while also serving as an indicator for
acute or chronic hepatitis and liver cirrhosis.7 The investigations
of the new properties of rare ketoses and their applications have
drawn much attention recently.8
There are two methods that could be used to produce
L
-rhamnulose and
L
-fuculose. The most common method is isomer-
izing -rhamnose to
L
L
-rhamnulose or -fucose to
L
L
-fuculose.13,14
However, aldose–ketose isomerization mediated by either chemi-
cal or enzymatic method (isomerase) is reversible, with reaction
equilibrium being very unfavorable for ketose formation.15 For
L
-Rhamnulose and
that offer many potential applications. For example,
is a precursor of furaneol that has been used in the flavor industry
for its sweet strawberry aroma.9 In addition,
-rhamnulose and
-fuculose play important roles in sugar metabolism.10 In bacteria,
-rhamnose and -fucose must be converted to their ketose 1-phos-
L
-fuculose are two crucial rare deoxy ketoses
L
-rhamnulose
example, only 11% of
L
L-fucose can be isomerized to L-fuculose by
L
-fucose isomerase (FucI) in the final reaction equilibrium.14 More-
L
L
over, an extensive isomer separation step is still necessary to
obtain a ketose in pure form. Ion-exchange chromatography
(Ca2+ form) is the main method for sugar isomer separation.16
L
phate forms, which are later split into dihydroxyacetone phos-
phate and
L
-lactaldehyde by
L
-rhamnulose 1-phosphate aldolase
Nevertheless, it was reported that
separated from
L-rhamnulose is hard to be
L
-rhamnose using ion-exchange chromatography
(Ca2+ form) column.13b They even can’t be separated well by
HPLC. Commercially available product only has 80% purity
(Sigma–Aldrich). Selective degradation of unwanted isomer by
⇑
Corresponding authors. Tel.: +86 022 83336658 (R.W.), +1 404 413 3009
(P.G.W.).
E-mail addresses: wangrunling@tmu.edu.cn (R. Wang), pwang11@gsu.edu
(P.G. Wang).
bacteria to isolate the desired ketose has also been explored17
,
y
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2015.12.051
0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wen, L.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2015.12.051

2
L. Wen et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
but this method is time-consuming and suffer from low efficiency.
The addition of borate into reaction system has been suggested to
improve aldose–ketose isomerization because borate can bind
ketose stronger than aldose.18 Such strategy has been applied on
Substrate specificity of RhaB towards several deoxy sugars
Substrate
RhaB activity (%)
100
81.3
ND
L-Rhamnulose
L-Fuculose
L-Rhamnose
L-Fucose
the conversion of L-fucose to L-fuculose, in which a 85% conversion
ratio was observed.14 However, the purification steps require the
separation of ketose-borate complex and the splitting of the
desired ketoses from ketose-borate complex.19 These tedious
manipulations place a limit on the applications of this strategy.
ND
ND: no detectable activity was observed.
The second method for the production of
L
L
-rhamnulose and
-fuculose is based on aldol condensation reaction.20 In this strat-
substrate specificity towards (3R)-ketoses as compared to (3S)-
ketoses or (3S)-aldoses.22 In this work, the substrate specificity of
RhaB towards several deoxy sugars was studied (see Supplemen-
egy, RhaD or FucA were used to produce L-rhamnulose 1-phos-
phate or
phosphate (DHAP) and
group was hydrolyzed to afford
ever, DHAP and -glyceraldehyde are costly and unstable, reducing
L
-fuculose 1-phosphate from dihydroxyacetone
-lactaldehyde, and then the phosphate
-rhamnulose or -fuculose. How-
L
tary data). RhaB failed to recognize
had high activity towards -rhamnulose and
indicating its potential for one-pot multienzyme (OPME) reac-
tions24 to produce
-rhamnulose and -fuculose.
Having met the prerequisite, other conversion-related enzymes
L-rhamnose or
L-fucose but
L
L
L
L
-fuculose (Table 1),
L
the synthetic practicality. Although DL-glycerol 3-phosphate, an
inexpensive starting material, has been used to produce DHAP in
a one-pot reaction fashion21, ketose production mediated by aldo-
lase still suffers from low yields and tedious purification manipu-
L
L
including
L , L-fucose isomerase
-rhamnose isomerase (RhaA)25
(FucI)26, and acid phosphatase (AphA)27 from Escherichia coli were
prepared as described in Supplementary data. To test the potential
of RhaB in OPME reactions, analytical scale reactions (Table 2) were
lations.20b Therefore, while
L
-rhamnulose and
commercially available, they are cost prohibitive (
$178/10 mg, Sigma–Aldrich; -fuculose, $199/10 mg, Carbosynth).
The study of -rhamnulose and -fuculose has been hindered due
to their limit availability. Herein an enzymatic method for the
efficient and convenient preparation of rare ketoses -rhamnulose
and -fuculose from readily available aldoses is reported.
L
-fuculose are
L-rhamnulose,
L
performed in one-pot (164 lg scale), and tested by TLC. Reactions
L
L
without isomerases were done as negative controls. Once the for-
mation of sugar phosphates was observed on TLC while no reac-
tions were observed on the control reactions, preparative
reactions (gram scale) were performed.
L
L
Recently, we developed a convenient, efficient and cost-effec-
tive platform for the facile synthesis of ketoses, by which 10
In the first reaction step, L-rhamnose was incubated with RhaA
and RhaB in one-pot in the presence of ATP as the phosphate donor
(OPME 1, Scheme 1). No buffer was used in consideration to purifi-
cation. The reaction pH was held near 7.5, where all enzymes are
quite active, using sodium hydroxide as the reaction occurred. In
non-readily available ketopentoses
(
L
-ribulose,
-xylulose) and ketohexoses ( -tagatose,
-tagatose, -fructose and -psicose) were prepared from
D
-xylulose,
D
-ribulose, and
-psicose,
L
D
D-sorbose,
D
L
L
L
common and inexpensive starting materials with both high yield
and purity without having to undergo a tedious isomer separation
step.22 The basic concept of this strategy is based on ‘phosphoryla-
tion?dephosphorylation’ cascade reaction. In this work, this
this one-pot two-enzyme system, RhaA isomerized
L-rhamnose to
L-rhamnulose, which was immediately phosphorylated by RhaB.
It seems that L-rhamnulose was taken out of the reaction balance,
and thus the reaction was driven towards the formation of
-rhamnulose in its ketose 1-phosphate form -rhamnulose
strategy was applied to produce
L-rhamnulose from L-rhamnose
L
(L
and -fuculose from -fucose, respectively. Thermodynamically
L
L
1-phosphate). The reaction was monitored by TLC and HPLC
equipped with ELSD detector (HPX-87H column). Once the reac-
tion finished (conversion ratio exceeding 99%), silver nitrate pre-
cipitation was used to precipitate ATP and ADP.22 In detail, silver
nitrate was added into reaction system until no new precipitate
formed, and the precipitate was removed by centrifugation.
Sodium chloride was added to precipitate the remaining silver
ions, and the precipitate was removed by centrifugation. After
unfavorable aldose–ketose conversions were combined with
phosphorylation reactions by substrate-specific kinases to increase
conversion ratio in step 1. Sugar phosphates were purified by silver
nitrate precipitation. The phosphate group was hydrolyzed to
produce ketoses in step 2 (Scheme 1).
To apply the described scheme on
production in this work, the prerequisite is the availability of a
kinase that specifically recognizes -rhamnulose and -fuculose
but not -rhamnose or -fucose. Otherwise, the products obtained
finally will be a mixture containing both aldose and ketose.
-rhamnulose kinase is the enzyme that prefers ketoses with
(3R)-configuration.23 Recently, we identified an
-rhamnulose
kinase from Thermotoga maritima MSB8, which show high
L-rhamnulose and L-fuculose
L
L
desalting by using P-2 column, L-rhamnulose 1-phosphate was iso-
L
L
lated in 91% yield. The product was analyzed by NMR and MS (see
Supplementary data). The NMR spectra and MS data are well in
L
accord with previously reported data20b,28
, confirming the
L
isolated product is
L-rhamnulose 1-phosphate. In the second
OH OH
OH
OH OH
OH OH
OH OH
FucI
OH OH
OH
OH OH
O
P
O
P
RhaB
RhaB
RhaA
HO
O
HO
O
O
O
HO
HO
O
O
OH
OH OH
-rhamnose
OH
OH OH
OH
O
O
OH
-fuculose
OPME 2 step1 (pH 7.5)
ATP
ADP
ATP
ADP
L
L
-fucose
L
-rhamnulose
L
L
-rhamnulose 1-phosphate
L
-fuculose 1-phosphate
OPME 1 step 1 (pH 7.5)
1) AgNO3
1) AgNO3
step 2
step 2
2) NaCl
2) NaCl
3) AphA (pH5.5)
Pi
Pi
3) AphA (pH 5.5)
OH OH
OH OH
RhaA: L-rhamnose isomerase
RhaB: L-rhamnulose kinase
FucI: L-fucose isomerase
AphA: acid phosphatase
HO
HO
O
OH
O
OH
L
-fuculose
L
-rhamnulose
Scheme 1. Two-step strategy for the enzymatic synthesis of L-rhamnulose and L-fuculose from L-rhamnose and L-fucose.
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L. Wen et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 2
Enzymatic synthesis of L-rhamnulose and L-fuculose from L-rhamnose or L-fucose using the strategy shown in Scheme 1
Entry
Starting material
Enzymes
Intermediate
OH OH
Step 1 yield (%)
Product
OH OH
Total yield (%)
Scale (mg)
1080
Puritya (%)
>99
OH OH
RhaA
RhaB
AphA
O
P
HO
HO
HO
O
O
1
91
82
O
OH
-rhamnulose
OH OH
OH OH
O
OH
OH
L
L-rhamnose
L-rhamnulose 1-phosphate
OH OH
OH OH
Fucl
RhaB
AphA
O
P
HO
O
O
2
93
84
1099
>99
OH OH
-fucose
O
OH
O
OH
OH
L
L-fuculose
L
-fuculose 1-phosphate
a
Defined as the percentage of desired ketose out of the sum of starting aldose and product.
L-rhamnose
L-fucose
L-fuculose
L-rhamnulose
9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0
Time
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23
Time
Figure 1. HPLC profiles of L-rhamnulose (HPX-87H column) and L-fuculose (Sugar-Pak 1 column) compared with starting aldoses.
L
-fuculose was obtained in 84% yield with regard to
L-fucose. The
reaction step, the phosphate group of
was hydrolyzed by AphA in pH 5.5 to produce
phosphate sugar was no longer observed on TLC, the solution was
concentrated and purified by P-2 column to afford final product in
L
-rhamnulose 1-phosphate
product was analyzed by HPLC, NMR and MS (see Supplementary
data). HPLC and NMR analysis indicates
exceeding 99%.
L-rhamnulose. Once
a product purity
In summary, based on the ‘phosphorylation?de-phosphoryla-
82% yield with regard to
HPLC, NMR and MS (See Supplementary data). No obvious peak of
-rhamnose can be found by HPLC (Fig. 1) and no characteristic
L-rhamnose. The product was analyzed by
tion’ cascade reaction and silver nitrate precipitation purification
method,
niently prepared from readily available starting aldoses
-rhamnose and -fucose) in more than 80% yield in gram scale.
L-rhamnulose and L-fuculose were efficiently and conve-
L
peak of aldose can be found in NMR spectra (See Supplementary
data) indicating product purity exceeding 99%.
(
L
L
Using a single desalting column (P-2 column), product with high
purity was obtained. ATP is also commercially inexpensive owing
to increased industrial production over the past decade and an
ATP-regeneration system has also been suggested29, thus making
the preparation process described herein of particular interest for
large-scale production. We anticipate that this work will accelerate
the progress in understanding the biological roles and synthetic
In our previously reported strategy, phosphorylation and
dephosphorylation reactions were carried in a one-pot fashion.
Although a total isolated yield exceeding 90% was reached, the
hydrolyzing reaction required a long reaction time and a large
amount of acid phosphatase due to unfavorable reaction condi-
tions. In this work, the intermediate of phosphate sugar was puri-
fied by P-2 column to remove most of impurities (mainly salt)
which affect the activity of acid phosphatase. Although a slightly
lower isolated yield was obtained, this two-step strategy reduced
reaction time and usage of acid phosphatase.
applications of L-rhamnulose and L-fuculose.
Acknowledgments
Similarly, preparative scale synthesis of
was also carried in gram scale. In the first reaction step,
L
-fuculose from
L
-fucose
-fucose
L
This work was financially supported National Institute of Health
(R01GM085267 to P.G.W.), and National Natural Science Founda-
tion of China (21202120 to R.L.W.).
was incubated with FucI and RhaB in the presence of ATP as phos-
phate donor (OPME 2, Scheme 1). Conversion ratio exceeding 99%
can be reached. Following the same manipulation as described
above,
and MS data are well in accord with the previously reported
data,20b,28 confirming the isolated product is
-fuculose
1-phosphate (see Supplementary data). After hydrolyzing the
phosphate group of -fuculose 1-phosphate by AphA in step 2,
L-fuculose 1-phosphate was isolated in 93% yield. NMR
Supplementary data
L
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.12.051.
L
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4
L. Wen et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
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