Facile enzymatic synthesis of ketoses

Article abstract of DOI:10.1002/anie.201505714

Studies of rare ketoses have been hampered by a lack of efficient preparation methods. A convenient, efficient, and cost-effective platform for the facile synthesis of ketoses is described. This method enables the preparation of difficult-to-access ketopentoses and ketohexoses from common and inexpensive starting materials with high yield and purity and without the need for a tedious isomer separation step. A spoonful of sugar: A convenient, efficient, and cost-effective platform for the facile synthesis of ketoses is described. This method, which involves a one-pot mulitenzyme (OPME) reaction, enables the preparation of rare ketopentoses and ketohexoses from common and inexpensive starting materials with high yield and purity and without the need for a tedious isomer separation step.

Full text of DOI:10.1002/anie.201505714

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Angewandte
Communications
International Edition: DOI: 10.1002/anie.201505714
German Edition: DOI: 10.1002/ange.201505714
Monosaccharides
Facile Enzymatic Synthesis of Ketoses
Liuqing Wen, Kenneth Huang, Mohui Wei, Jeffrey Meisner, Yunpeng Liu, Kristina Garner,
Lanlan Zang, Xuan Wang, Xu Li, Junqiang Fang, Houcheng Zhang, and Peng George Wang*
Abstract: Studies of rare ketoses have been hampered by a lack
of efficient preparation methods. A convenient, efficient, and
cost-effective platform for the facile synthesis of ketoses is
described. This method enables the preparation of difficult-to-
access ketopentoses and ketohexoses from common and
inexpensive starting materials with high yield and purity and
without the need for a tedious isomer separation step.
Aldose–ketose isomerization is the most important
[4]
method for ketose preparation, even though the reaction
[5]
equilibrium is very unfavorable for ketose formation.
Significant progress has been achieved by using both chemical
and enzymatic isomerization schemes over the last two
[6]
decades. Approaches to improve aldose–ketose conversion,
including the addition of borate to break the aldose–ketose
[
7]
[8]
reaction equilibrium, directed evolution, and the discov-
[9]
T
he only seven monosaccharides that occur naturally in
ery of novel enzymes in nature, have also been suggested.
Nevertheless, these methods require a complicated isomer
separation step to obtain the ketose in pure form. Further-
more, only four rare ketoses (d-xylulose, d-ribulose, l-
ribulose, and d-tagatose) can be directly isomerized from
the most common aldoses, and the synthesis of other rare
ketoses has thus been more challenging. Chemical methods
for the synthesis of these rare ketoses require tedious
substantial quantities are d-xylose, d-ribose, l-arabinose, d-
[1]
galactose, d-glucose, d-mannose, and d-fructose. Of all
possible structures, the twelve ketopentoses and ketohexo-
[2]
ses can be divided into two types based on their C-3
configuration: (3S)-ketoses and (3R)-ketoses (Figure 1). With
the exception of d-fructose, all of these ketoses are defined as
[
2]
“
rare sugars”. Despite their lower accessibility, rare ketoses
[10]
offer enormous potential for applications in food, pharma-
protection/deprotection manipulations.
Alternatively,
[
3]
ceutical, medicinal, and synthetic chemistry.
enzymatic preparation by epimerizing ketoses at the C-3
[
11]
[12]
position, oxidizing polyols, or relying on aldol condensa-
[13]
tion proceeds regio- and stereoselectively without protec-
tion. However, these processes suffer from low yields,
expensive starting materials, or complicated isomer separa-
tion. Therefore, most of the rare ketoses are still not readily
available, which in turn has hindered studies of this funda-
mental class of carbohydrates.
Herein, a convenient, efficient, and cost-effective plat-
form for ketose syntheses is described. All of the rare
ketopentoses and ketohexoses were produced by utilizing this
platform, with the exception of l-sorbose. l-sorbose, a starting
material for the production of vitamin C, has been produced
[14]
in industrial settings and is consequently inexpensive.
Our strategies are based on a “phosphorylation!de-
phosphorylation” cascade reaction. In the first reaction step
(
Figure 1. Twelve naturally occurring ketopentoses and ketohexoses
including six (3S)-ketoses and six corresponding (3R)-ketoses.
Scheme 1), we combined thermodynamically unfavorable
bioconversions of common (3S)-sugars to the desired (3S)-
ketoses (route A) or (3R)-ketoses (route B) with phosphor-
ylation reactions by substrate-specific kinases ( fructokinase
[
*] L. Wen, K. Huang, M. Wei, Dr. J. Meisner, Dr. Y. Liu, K. Garner,
L. Zang, X. Wang, X. Li, Prof. Dr. P. G. Wang
Department of Chemistry and
(
(
HK) from humans in route A and l-rhamnulose kinase
RhaB) from Thermotoga maritima MSB8 in route B). In the
Center for Therapeutics and Diagnostics
Georgia State University, Atlanta, GA 30303 (USA)
E-mail: pwang11@gsu.edu
second reaction step, adenosine phosphates (ATP and ADP)
were selectively removed by a convenient method called
silver nitrate precipitation. Then, acid phosphatase (AphA)
was added to hydrolyze the phosphate groups and produce
the desired ketoses. Notably, both reaction steps can be
performed in a one-pot fashion. The preparation of l-fructose
from dl-glycerol 3-phosphate and glycerol by relying on aldol
condensation using rhamnulose bisphosphate aldolase
Dr. J. Fang, Dr. H. Zhang, Prof. Dr. P. G. Wang
National Glycoengineering Research Center, Shandong University
Jinan 250100 (China)
Dr. J. Meisner
Current Address: Division of Pulmonary, Allergy and Immunology,
Cystic Fibrosis, and Sleep, Department of Pediatrics
Emory University School of Medicine
Atlanta, GA 30322 (USA)
(RhaD) is illustrated in Scheme 2. l-Psicose was further
prepared from l-fructose by using the targeted phosphoryla-
tion strategy shown in Scheme 3.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201505714.
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Scheme 3. One-pot multienzyme reaction for the synthesis of l-psi-
cose.
(pentose) or C-6 (hexose) position is used. To avoid such
unwanted side reactions, kinases that phosphorylate ketoses
[16]
at the C-1 position were utilized. In route A (Scheme 1),
a kinase that can phosphorylate (3S)-ketoses (d-xylulose, l-
ribulose, and d-tagatose) but not (3S)-aldoses (d-xylose, l-
arabinose and d-galactose) is necessary. In route B, a kinase
that specifically recognizes (3R)-ketoses but not (3S)-ketoses
and (3S)-aldoses is required. By screening the substrate
specificity of many kinases (data not shown), we discovered
Scheme 1. One-pot two-step enzymatic synthesis of l-ribulose, d-
xylulose, and d-tagatose (Route A), and l-xylulose, d-ribulose, d-
sorbose, d-psicose, and l-tagatose (Route B).
[17]
that fructokinase (HK) from humans accords well with the
requirements of route A, and l-rhamnulose kinase (RhaB)
from Thermotoga maritima MSB8, a novel enzyme that
absolutely requires ketoses with (3R)-configuration, accords
well with the requirements of route B (see the Supporting
Information). HK can also phosphorylate l-psicose effi-
ciently, while no activity towards its corresponding (3R)-
ketose (l-fructose) was detected, thus making it possible to
establish the reaction for the preparation of l-psicose from l-
fructose (Scheme 3). Another requirement for the establish-
ment of the transformations shown in (Schemes 1, route B
and Scheme 3) is that DTE, which catalyzes the interconver-
[11]
sion of (3S)- and (3R)-ketoses, does not epimerize ketose 1-
phosphates, otherwise the products obtained will be a mixture
containing both (3S)-ketoses and (3R)-ketoses. DTE does not
recognize d-fructose 6-phosphate or d-ribulose 5-phos-
Scheme 2. One-pot multienzyme reaction for the synthesis of l-fruc-
tose.
[11]
phate.
We therefore hypothesized that it would not
recognize other ketose phosphates such as ketose 1-phos-
phates. This hypothesis was supported by purity analysis of
the final products (Table 1).
To achieve the designed outcomes, there are three
challenges: 1) the identification of kinases that can phosphor-
ylate the desired ketoses at the C-1 position but not starting
sugars or intermediates, 2) the availability of a d-tagatose 3-
epimerase (DTE) that specifically epimerizes ketoses at C-3
but not ketose 1-phosphates, and 3) an efficient method to
separate sugar phosphates from the adenosine phosphates
In the second reaction step, acid phosphatase was used to
hydrolyze the phosphate group of ketose 1-phosphates to
produce ketoses under acidic conditions (pH 5.5) in which
monosaccharides are stable. However, the presence of
adenosine phosphates (ATP and ADP) inhibits the hydrolytic
[18]
activity of the acid phosphatase. In this work, a convenient
method referred to as silver nitrate precipitation was used to
selectively remove the adenosine phosphates. Silver phos-
phate is insoluble and silver ions can thus precipitate ADP
(
ATP and ADP).
The first reaction step for the approaches shown in
Schemes 1 and 3 was carried out under slightly basic
conditions, in which all of the enzymes were quite active.
Nevertheless, monosaccharides are unstable in alkaline
[
19]
and ATP. Interestingly, we noticed that silver ions cannot
precipitate monophosphate sugars when the sugars are
composed of four or more carbons (Table S2 in the Support-
ing Information). It appears that binding of the sugar group to
phosphate prevents silver phosphate precipitation. By apply-
[15]
media,
and the isomerases in the reaction system may
also isomerize the intermediate ketose phosphates to a certain
extent when the kinase that phosphorylates ketoses at the C-5
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Table 1: Synthesis of rare ketoses using the two-step strategy shown in Schemes 1 to 3.
[a]
[b]
Entry
Starting material
Enzymes
Scheme
Intermediate
Ketose
Yield [%]
Purity [%]
AraA
HK
Route A
Scheme 1
1
93
>99
l-arabinose
l-ribulose 1-phosphate
l-ribulose
XylA
HK
Route A
Scheme 1
2
3
4
5
6
7
8
92
92
91
91
94
93
95
>99
>99
>99
98.2
>99
>99
>99
d-xylose
d-xylulose 1-phosphate
d-tagatose 1-phosphate
l-xylulose 1-phosphate
d-ribulose 1-phosphate
d-sorbose 1-phosphate
d-psicose 1-phosphate
l-tagatose 1-phosphate
d-xylulose
d-tagatose
l-xylulose
d-ribulose
d-sorbose
d-psicose
l-tagatose
AraA
HK
Route A
Scheme 1
d-galactose
l-arabinose
d-xylose
AraA
DTE
RhaB
Route B
Scheme 1
XylA
DTE
RhaB
Route B
Scheme 1
AraA
DTE
RhaB
Route B
Scheme 1
d-galactose
d-fructose
l-sorbose
DTE
RhaB
Route B
Scheme 1
DTE
RhaB
Route B
Scheme 1
GPO
GO
catalase
RhaD
DL-glycerol 3-phosphate
glycerol
9
Scheme 2
70
99
l-fructose 1-phosphate
l-psicose 1-phosphate
l-fructose
l-psicose
DTE
HK
1
0
Scheme 3
90
>99
l-fructose
[
a] All products were prepared on a preparative (408 mg–628 mg) scale. [b] Defined as the percentage of desired ketose out of the sum of all possible
sugars.
ing this selective precipitation ability, sugar phosphates can be
easily and cleanly separated from adenosine phosphates
precipitation, another sugar phosphate purification method
in which sugar phosphate was isolated as a barium salt by
[20]
(
ATP and ADP). The method was tested in the synthesis of
l-rhamnulose 1-phosphate from l-rhamnulose by using RhaB
see the Supporting Information). Once no l-rhamnulose
using barium, no additional steps to remove toxic ions and
no accurate pH control are necessary. Moreover, silver is safer
than barium and other metal ions that can be used to
(
[
21]
could be detected in the reaction by HPLC, ATP and ADP
were removed by adding a small excess of silver nitrate, which
led to the removal of more than 99% of the ATP and ADP
precipitate adenosine phosphates, such as mercury. These
advantages make this method highly attractive for use in
rapidly purifying sugar phosphate.
(
Figure S2). Then, excess sodium chloride was added to
Having overcome these problems, other conversion-
[22]
remove the redundant silver ions. The total separation process
can be completed in less than 15 min. After desalting by using
Bio-Gel P-2 column, l-rhamnulose 1-phosphate sodium was
isolated in 94% yield, which is comparable to the yield when
using ion-exchange purification methods but it is more
convenient. The precipitate can be redissolved in ammonium
hydroxide, and the adenosine phosphates (ATP and ADP) or
silver ions can be then recycled. Compared to barium
related enzymes including d-xylose isomerase (XylA),
[23]
[18]
RhaD,
and AphA
from Escherichia coli, l-arabinose
and d-galactose isomerase (AraA) from Thermotoga mar-
[
24]
[11]
itima MSB8, and DTE from Pseudomonas Sp. St-24 were
prepared as described in Supporting Information.
In route A (Scheme 1), l-arabinose, d-xylose, and d-
galactose were incubated with their corresponding isomerases
and HK to prepare l-ribulose, d-xylulose and d-tagatose,
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respectively (Table 1, entries 1–3). The reactions were
allowed to proceed until no detectable starting aldoses were
found by HPLC (conversion ratios exceeding 99%), thus
making isomer separation unnecessary. Silver nitrate precip-
itation was used to remove ATP and ADP. Proteins were also
removed during this process (as confirmed by Bradford
assay). Consequently, two steps of the method could be
carried out in one pot. After the hydrolysis of phosphate
groups in step 2, the solution from each reaction was desalted
by using a Bio-Gel P-2 column. l-ribulose, d-xylulose, and d-
tagatose were finally obtained in more than 90% yield
unfavorable for l-psicose formation, for which the conversion
[25]
ratio is only 24%. Applying the discovery that HK from
humans could efficiently phosphorylate l-psicose but not l-
fructose, l-psicose was prepared from l-fructose by using the
described targeted phosphorylation strategy (Scheme 3) in
90% yield with a product purity exceeding 99%.
In summary, a novel method for the facile synthesis of
ketoses was established. This method relies on substrate-
specific kinases and the improved aldol condensation reac-
tion, and makes it possible to use one-pot multienzyme
[27]
(OPME) reactions to prepare difficult-to-access ketopen-
toses and ketohexoses from common and inexpensive mate-
rials. The described two-step strategy not only provides
unprecedentedly high yields but also avoids the need for
a complicated isomer separation step. ATP is commercially
cheap owing to increased industrial production over the past
decade and an ATP-regeneration system has also been
(
Table 1). The products were confirmed by NMR, HPLC and
MS analysis (see the Supporting Information). HPLC and
NMR analysis indicated product purity exceeding 99%.
In route B (Scheme 1), five (3R)-ketoses (l-xylulose, d-
ribulose, d-sorbose, d-psicose, and l-tagatose) were prepared
from five common (3S)-sugars (l-arabinose, d-xylose, d-
galactose, d-fructose, and l-sorbose, respectively). The dis-
covery of DTE made it possible to achieve the interconver-
sion between (3S)-ketoses and (3R)-ketoses, which is espe-
[28]
suggested,
thus making the transformation reaction de-
scribed herein of particular interest for large-scale produc-
tion.
[
11]
cially important for the preparation of (3R)-ketoses.
This study represents a highly convenient and efficient
strategy for ketose synthesis. We anticipate that this method
will accelerate progress in understanding the biological roles
and synthetic applications of rare ketoses, as well as advanc-
ing the synthesis of rare aldoses since the aldose–ketose
isomerization reaction is very favorable for aldose formation.
Future studies will enable the identification of new kinases for
use in sugar syntheses, thereby providing a powerful set of
tools for carbohydrate research.
Nevertheless, with the exceptions of two (3S)-ketoses (d-
fructose and l-sorbose), ketoses are not readily available, and
the conversions catalyzed by DTE are an equilibrium
reaction. For example, the conversion ratio is only 20% for
d-fructose to d-psicose and 27% for l-sorbose to l-taga-
[25]
tose. The separation of (3S)-ketoses and (3R)-ketoses is
difficult owing to their similar properties. In this work, we
combined DTE-catalyzed epimerization with targeted phos-
phorylation of (3R)-ketose by RhaB (entries 7 and 8). To
avoid using ketoses that are not readily available, we included
enzymatic isomerization when starting with (3S)-aldose Acknowledgements
entries 4–6). All reactions were allowed to proceed until no
(
detectable starting sugars were found by HPLC (conversion
ratios exceeding 99%). After the hydrolysis of phosphate
groups in step 2, all five (3R)-ketoses were obtained in more
than 90% yield (Table 1). Given the high substrate specificity
of RhaB, the products l-xylulose, d-sorbose, d-psicose, and l-
tagatose were obtained in more than 99% purity. d-ribulose
was obtained in 98.2% purity while 0.6% of d-xylulose and
Prof. Dr. P. Wang thanks National Institute of Health
(R01GM085267), National Basic Research(973) Program of
China (2012CB822102), and the Key Grant Project of
Chinese Ministry of Education (313033) for financial support.
Keywords: enzymatic synthesis · ketoses · one-pot reactions ·
phosphorylation · synthetic methods
1
.2% of d-xylose were observed because d-xylose and d-
How to cite: Angew. Chem. Int. Ed. 2015, 54, 12654–12658
Angew. Chem. 2015, 127, 12845–12849
xylulose could be phosphorylated by RhaB to a certain extent
(
Table S1 in the Supporting Information).
l-fructose was synthesized by using RhaD because there
is a lack of common corresponding sugars. RhaD exclusively
produces l-fructose from dihydroxyacetone phosphate
[1] K. Izumori, J. Biotechnol. 2005, 118, S89 – S90.
[23]
[
2] T. B. Granstrçm, G. Takata, M. Tokuda, K. Izumori, J. Biosci.
Bioeng. 2004, 97, 89 – 94.
(
DHAP) and l-glyceraldehyde. However, DHAP and l-
glyceraldehyde are costly and unstable. To increase the
practicality of the process, two previously reported strat-
[
3] a) Z. Ahmed, Electron. J. Biotechnol. 2001, 4, 13 – 14; b) B. Li, S.
Varanasi, P. Relue, Green Chem. 2013, 15, 2149 – 2157; c) Y.
Ishida, K. Kakibuchi, R. Kudo, K. Izumori, S. Tajima, K.
Akimitsu, K. Tanaka, Acta Hortic. 2012, 927, 929 – 934.
[23,26]
egies
were combined to allow the use of the inexpensive
materials glycerol and dl-glycerol 3-phosphate to produce l-
fructose 1-phosphate in a one-pot reaction (Scheme 2) in
which l-glyceraldehyde is produced from glycerol by galac-
tose oxidase (GO) and DHAP is produced from dl-glycerol
[4] Z. Li, Y. Gao, H. Nakanishi, X. Gao, L. Cai, Beilstein J. Org.
Chem. 2013, 9, 2434 – 2445.
[
5] D. Ekeberg, S. Morgenlie, Y. Stenstrom, Carbohydr. Res. 2007,
42, 1992 – 1997.
3
3
-phosphate by glycerol phosphate oxidase (GPO). After the
[
6] a) Y. Romµn-Leshkov, M. Moliner, J. A. Labinger, M. E. Davis,
hydrolysis of the phosphate group in step 2, l-fructose was
finally obtained in 70% yield with a purity of 99%. l-psicose
was then prepared from l-fructose by using DTE. However,
the conversion of l-fructose into l-psicose by DTE is
Angew. Chem. Int. Ed. 2010, 49, 8954 – 8957; Angew. Chem. 2010,
1
22, 9138 – 9141; b) K. Beerens, T. Desmet, W. Soetaert, J Ind
Microbiol. Biotechnol. 2012, 39, 823 – 834; c) D. Ekeberg, S.
Morgenlie, Y. Stenstrom, Carbohydr. Res. 2005, 340, 373 – 377;
Angew. Chem. Int. Ed. 2015, 54, 12654 –12658
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 12657

.
Angewandte
Communications
d) S. Saravanamurugan, M. Paniagua, J. A. Melero, A. Riisager,
J. Am. Chem. Soc. 2013, 135, 5246 – 5249.
[19] G. M. Whitesides, P. E. Garrett, M. G. Siegel, Patents
(US4164444 A), 1979.
[
[
7] J. F. Mendicino, J. Am. Chem. Soc. 1960, 82, 4975 – 4979.
8] H. Leemhuis, R. M. Kelly, L. Dijkhuizen, IUBMB Life 2009, 61,
[20] C.-H. Wong, S. D. McCurry, G. M. Whitesides, J. Am. Chem. Soc.
1980, 102, 7938 – 7939.
2
22 – 228.
9] J. K. Vester, M. A. Glaring, P. Stougaard, Microb. Cell Fact. 2014,
3, 72.
10] a) T. Mukaiyama, Y. Yuki, K. Suzuki, Chem. Lett. 1982, 1169 –
170; b) T. Matsumoto, T. Enomoto, T. Kurosaki, J. Chem. Soc.
[21] K. Tanaka, M. Honjo, Patents (US3079379 A), 1963.
[22] A. S. Rozanov, S. N. Zagrebelny, A. B. Beklemishev, Appl.
Biochem. Microbiol. 2009, 45, 31 – 37.
[23] D. Franke, T. Machajewski, C. C. Hsu, C. H. Wong, J. Org. Chem.
2003, 68, 6828 – 6831.
[
1
[
1
Chem. Commun. 1992, 610 – 611.
[24] D. W. Lee, H. J. Jang, E. A. Choe, B. C. Kim, S. J. Lee, S. B. Kim,
Y. H. Hong, Y. R. Pyun, Appl. Environ. Microbiol. 2004, 70,
1397 – 1404.
[25] H. Itoh, K. Izumori, J. Ferment. Bioeng. 1996, 81, 351 – 353.
[26] Z. Li, L. Cai, Q. Qi, P. G. Wang, Bioorg. Med. Chem. Lett. 2011,
21, 7081 – 7084.
[
[
[
[
11] H. Itoh, H. Okaya, A. R. Khan, S. Tajima, S. Hayakawa, K.
Izumori, Biosci. Biotechnol. Biochem. 1994, 58, 2168 – 2171.
12] W. Poonperm, G. Takata, K. Morimoto, T. B. Granstrom, K.
Izumori, Enzyme Microb. Technol. 2007, 40, 1206 – 1212.
13] T. D. Machajewski, C. H. Wong, Angew. Chem. Int. Ed. 2000, 39,
1
352 – 1375; Angew. Chem. 2000, 112, 1406 – 1430.
14] R. Giridhar, A. K. Srivastava, World J. Microbiol. Biotechnol.
001, 17, 185 – 189.
[27] H. Yu, K. Lau, V. Thon, C. A. Autran, E. Jantscher-Krenn, M.
Xue, Y. Li, G. Sugiarto, J. Qu, S. Mu, L. Ding, L. Bode, X. Chen,
Angew. Chem. Int. Ed. 2014, 53, 6687 – 6691; Angew. Chem. 2014,
126, 6805 – 6809.
[28] Z. Liu, J. Zhang, X. Chen, P. G. Wang, ChemBioChem 2002, 3,
348 – 355.
2
[
[
15] J. C. Speck, Jr., Adv. Carbohydr. Chem. 1958, 13, 63 – 103.
16] R. W. Nagorski, J. P. Richard, J. Am. Chem. Soc. 1996, 118,
7
432 – 7433.
[
17] a) A. Asipu, B. E. Hayward, J. OꢀReilly, D. T. Bonthron,
Diabetes 2003, 52, 2426 – 2432; b) R. Bais, H. M. James, A. M.
Rofe, R. A. Conyers, Biochem. J. 1985, 230, 53 – 60.
[
18] G. M. Rossolini, M. C. Thaller, R. Pezzi, G. Satta, FEMS
Microbiol. Lett. 1994, 118, 167 – 173.
Received: July 3, 2015
Published online: August 14, 2015
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