Towards the biotechnological isomerization of branched sugars: d-tagatose-3-epimerase equilibrates both enantiomers of 4-C-methyl-ribulose with both enantiomers of 4-C-methyl-xylulose

Article abstract of DOI:10.1016/j.tetlet.2008.03.047

Microbial oxidation of 2-C-methyl-d-ribitol and 2-C-methyl-d-arabinitol by Gluconobacter thailandicus NBRC 3254 produces 4-C-methyl-l-ribulose and 4-C-methyl-d-ribulose, respectively. Further, 4-C-methyl-l-ribulose and 4-C-methyl-d-ribulose were equilibra

Full text of DOI:10.1016/j.tetlet.2008.03.047

Available online at www.sciencedirect.com
Tetrahedron Letters 49 (2008) 3316–3321
Towards the biotechnological isomerization of branched sugars:
D-tagatose-3-epimerase equilibrates both enantiomers of
4-C-methyl-ribulose with both enantiomers of 4-C-methyl-xylulose
Devendar Rao a, Akihide Yoshihara a, Pushpakiran Gullapalli a, Kenji Morimoto a,
Goro Takata a, Filipa P. da Cruz b, Sarah F. Jenkinson b, Mark R. Wormald c,
Raymond A. Dwek c, George W. J. Fleet b,*, Ken Izumori a,*
a Rare Sugar Research Center, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Mik-choi, Kita-gun, Kagawa 761-0795, Japan
b Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road OX1 3TA, UK
c Glycobiology Institute, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK
Received 4 February 2008; revised 26 February 2008; accepted 7 March 2008
Available online 12 March 2008
Abstract
Microbial oxidation of 2-C-methyl-D-ribitol and 2-C-methyl-D-arabinitol by Gluconobacter thailandicus NBRC 3254 produces
4-C-methyl-L-ribulose and 4-C-methyl-D-ribulose, respectively. Further, 4-C-methyl-L-ribulose and 4-C-methyl-D-ribulose were equili-
brated by D-tagatose-3-epimerase (DTE) with 4-C-methyl-L-xylulose and 4-C-methyl-D-xylulose, respectively. These transformations
demonstrate that polyol dehydrogenase and DTE act on branched synthetic sugars. The green preparation of all of the stereoisomers
of 4-C-methyl pentuloses illustrates the ability of biotechnology to generate novel branched monosaccharides.
Ó 2008 Elsevier Ltd. All rights reserved.
Although the driving force for the initial preparation of
rare sugars has been their potential as alternative food-
stuffs,1 it has become clear that rare and new monosaccha-
rides interact with a number of biological receptors and
have a wide range of potential chemotherapeutic uses.2
Application of enzymes to organic synthesis3 to perform
specific transformations is generally limited by their
substrate specificity but the directed evolution and the dis-
covery of new enzymes have shown the potential of such
biotechnological procedures.4 The technique of Izumoring5
has been developed under environmentally friendly condi-
tions to allow the isomerization of all of the 16 aldohexoses
and 8 ketohexoses in large amounts in water; this depends
on (i) polyol dehydrogenases which allow the specific oxi-
dation of alditols to ketoses,6 (ii) D-tagatose-3-epimerase
(DTE) which equilibrates C-3 in each of the four pairs of
ketoses,7 and (iii) aldose isomerases which equilibrate
ketoses and aldoses; the isomerism on a multi-gram scale
of L-rhamnose to 1-deoxy-L-fructose indicates that the
technology may be extended to deoxy sugars.8
It may be that the concept of Izumoring can be applied
generally to the synthesis of unnatural carbohydrates. As
part of a project to investigate the synthesis of new
branched sugars by green procedures (Scheme 1), this Let-
ter establishes that 2-C-methyl-alditols 1 and 2 [in red] are
selectively oxidized at C-2 to afford the corresponding
ketoses 3L and 4D [in blue], respectively; DTE equilibrates
3L with 4L, and 4D with 3D—allowing for the first time,
the preparation of all of the 4-C-methyl-pentuloses. There
have been no reports of such C-methyl branched pentu-
loses although both enantiomers of ribulose and xylulose
have wide ranging biological roles. L-Xylulose may be used
as an inhibitor of glycosidases and also as an indicator of
patients suffering from acute or chronic hepatitis and liver
*
Corresponding authors.
E-mail addresses: george.fleet@chem.ox.ac.uk (G. W. J. Fleet), izumori@
kagawa-u.ac.jp (K. Izumori).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.03.047

D. Rao et al. / Tetrahedron Letters 49 (2008) 3316–3321
3317
L-sugars
OH
OH
HO Me
HOH2C
HO Me
OH
OH
HO Me
HOH2C
HO Me
CHO
HOH2C
CHO
CHO
HOH2C
CHO
OH
OH
OH
OH
4-C-methyl-L-ribose
4-C-methyl-L-arabinose
4-C-methyl-L-xylose
4-C-methyl-L-lyxose
(iii)
(iii)
(iii)
(iii)
O
O
HO
(i)
(ii)
(ii)
HO Me
HO Me
HO Me
OH
HO Me
HOH2C
HOH2C
CH2OH
HOH2C
CH2OH
HOH2C
CH2OH
CH2OH
OH
OH
OH
OH
4-C-methyl-L-ribulose 3L
4-C-methyl-L-xylulose 4L
2-C-methyl-D-arabinitol
1
180o
180o
2
O
O
OH
(i)
HO Me
HO Me
Me OH
HOH2C
HO
Me OH
CH2OH
OH
HOH2C
CH2OH
HOH2C
CH2OH
CH2OH
HOH2C
OH
OH
OH
2-C-methyl-D-ribitol
4-C-methyl-D-ribulose 3D
4-C-methyl-D-xylulose 4D
(iii)
(iii)
(iii)
(iii)
OH
OH
OH
OH
HO Me
HOH2C
HO Me
HOH2C
HO Me
HO Me
CHO
CHO
HOH2C
CHO
HOH2C
CHO
OH
OH
OH
OH
4-C-methyl-D-ribose
4-C-methyl-D-lyxose
4-C-methyl-D-arabinose
4-C-methyl-D-xylose
D-sugars
Scheme 1. Reagents: (i) Polyol dehydrogenases; (ii) D-tagatose-3-epimerase (DTE); (iii) aldose isomerases.
HO
HOH2C
OH
HO
HOH2C
OH
HO
HOH2C
Me OH
CH2OH
O
(iii)
(i)
(ii)
O
Me
CH2NMe2
CHO
HOH2C
OH
O
OH OH
OH
HO OH
2-C-methyl-D-ribitol
6
7
5
180o
1
O
OH
HO Me
HOH2C
HO Me
HOH2C
O
HO Me
HOH2C
(v)
(iv)
CH2OH
CH2OH
CH2OH
OH
OH
OH
4L
4-C-methyl-L-ribitol
4-C-methyl-L-xylulose
3L
4-
C-methyl-L-ribulose
Scheme 2. Reagents and conditions: (i) Me2NH, AcOH, EtOH, 80 °C; (ii) Ca(OH)2, H2O, 70 °C ꢀ20% over two steps; (iii) NaBH4, MeOH, 100%; (iv) G.
thailandicus NBRC 3254, H2O, 65%; (v) DTE, H2O, ratio of 68:32, isolated 25% yield.
Fig. 1. HPLC profiles of purified products (a) 4-C-methyl-L-ribulose 3L, (b) 4-C-methyl-L-xylulose 4L.

3318
D. Rao et al. / Tetrahedron Letters 49 (2008) 3316–3321
(ii)
O
O
HOH2C
O
O
HO
(i)
HO Me
CH2OH
O
OH
OH
O
(iii)
+
HOH2C
O
Me
Me
HO
O
O
O
OH
2-
C-methyl-D-arabinitol
10
9
8
180o
2
OH
O
O
Me OH
(v)
(iv)
HO Me
HOH2C
HO Me
HOH2C
HOH2C
CH2OH
CH2OH
CH2OH
OH
OH
OH
4-C-methyl-D-ribulose
4-C-methyl-D-xylulose
4D
3D
Scheme 3. Reagents and conditions: (i) MeMgBr, THF; then NaCN, H2O, ꢀ65%; (ii) CF3COOH, H2O; (iii) NaBH4, MeOH, 99%; (iv) G. thailandicus
NBRC 3254, H2O, 70%; (v) DTE, H2O, ratio of 70:30, isolated 50% yield.
Fig. 2. HPLC profiles of purified products (a) 4-C-methyl-D-ribulose 3D, (b) 4-C-methyl-D-xylulose 4D.
cirrhosis. D-Xylulose can induce depletion of ATP and Pi in
isolated rat hepatocytes. L-Ribulose is a promising precur-
sor for the production of various biologically important
compounds that show antiviral properties against HIV
and the hepatitis virus and also plays an important role
in human body sugar metabolism. D-Ribulose derivatives
occur extensively in mammals such as in D-ribulose-
peptides in human semen;9 the syntheses described in this
Letter will allow the evaluation of the effect of carbon
branching on such sugars.
No 4-C-methyl or 2-C-methyl sugars occur as natural
products, so the chemical synthesis of the 2-C-methyl pent-
itols is necessary. For the synthesis of the L-sugars 3L and
4L, 2-C-methyl-D-ribitol 1 [which is related to 4-C-methyl-
L-ribitol by a 180° rotation] was prepared from D-glucose 5
(Scheme 2).
Treatment of glucose 5 with dimethylamine and acetic
acid in ethanol afforded the Amadori ketose 6, which on
treatment with calcium hydroxide in water gave the
branched ribonolactone 7 in around 20% yield on a large
scale; lactone 7 is the most readily available C-branched
carbohydrate chiron and may be prepared in substantial
amounts by this method.10 Reaction of lactone 7 with
sodium borohydride in methanol afforded the correspond-
ing C-methyl ribitol 111 (quantitative yield).
Table 1
1H and 13C assignments in 2H2O, pH 6.6, referenced to acetone at
2.220 ppm (1H) and 30.90 ppm (13C), were made based on the 1D, 2D
COSY and 2D HSQC spectra
4-C-Methyl-ribulose 3
4-C-Methyl-xylulose 4
3af
3bf
4af
4bf
78%
22%
24%
76%
1H d (ppm)
C-1 H
C-1H0
C-3H
C-5H
C-5H0
3.584
3.701
3.774
3.642
3.542
3.883
3.981
3.768
1.334
3.610
3.953
3.952
3.831
1.326
3.613
3.913
4.018
3.858
1.337
3.611
3.929
3.862
3.734
1.327
C-4CH3
JHH (Hz)
H-1-H-10
H-5-H-50
ꢁ12.1
ꢁ11.8
ꢁ11.9
ꢁ12.0
ꢁ9.9
ꢁ10.1
ꢁ9.8
ꢁ9.7
13C d (ppm)
C-1
C-2
C-3
C-4
C-5
C-4CH3
63.33
103.68
74.62
76.91
76.72
21.65
63.33
106.04
83.22
77.60
76.10
20.81
63.21
107.80
81.77
80.43
78.07
18.99
64.87
104.73
78.55
79.55
76.30
19.65
The ring forms were confirmed from the 2D HMBC spectra. Proportions
of the anomers were determined from the peak intensities in the 1D 1H
spectra.

D. Rao et al. / Tetrahedron Letters 49 (2008) 3316–3321
3319
CH2OH
OH
OH
CH2OH
OH
OH
CH2OH
OH
O
O
O
O
CH2OH
OH
H3C
HO
H3C
HO
H3C
HO
H3C
HO
OH
OH
3L-β
f
3L-α
f
4L-β
f
4L-αf
Scheme 4. Furanose forms of 4-C-methyl-L-ribulose 3 and 4-C-methyl-L-xylulose 4.
Gluconobacter thailandicus NBRC 3254 has been shown
to oxidize meso-ribitol to L-ribulose with no oxidation to
the enantiomer;12 similarly, the branched pentitol 4-C-
methyl-L-ribitol 1 was oxidized at C-2 by using the resting
cell of Gluconobacter thailandicus NBRC 3254 to afford 4-
2-C-methyl-D-arabinitol 2 required several steps for the
synthesis (Scheme 3). Thus, the protected D-erythrono-
lactone 8 was treated first with methyl magnesium bromide
followed by a Kiliani reaction to give the protected d-lac-
tone 9 and the c-lactone 10 in a combined yield of approx-
20
C-methyl-L-ribulose 3L [oil, ½aꢂD ꢁ2.9 (c 1.0, water)] in
imately 65%;14
a
small amount of the epimeric
65% yield.
ribonolactone 7 [<10%] was formed during the reaction.
Acetonide 9 was removed by the treatment with aqueous
trifluoroacetic acid to form 10, which on treatment with
sodium borohydride in methanol afforded 2-C-methyl-D-
arabinitol 215 in 99% yield as a substrate for the microbial
oxidation.
DTE epimerizes the C-3 position of many ketoses and,
in particular, equilibrates L-ribulose with L-xylulose.13 Epi-
merization of 4-C-methyl-L-ribulose 3L by DTE gave a
mixture of 3L and 4L in a ratio of 68:32 and allowed the
20
isolation of 4-C-methyl-L-xylulose 4L [oil, ½aꢂD +7.7 (c
1.0, water)] in 25% yield. The HPLC profiles of purified
3L and 4L are shown in Figure 1.
Gluconobacter thailandicus NBRC 3254 also oxidizes D-
arabinitol at C-4 to produce D-xylulose with no other rele-
vant by-products;12 under the same conditions, the resting
cells of Gluconobacter thailandicus NBRC 3254 were used
to oxidize 2-C-methyl-D-arabinitol 2 at C-4 to produce 4-
For the synthesis of the D-pentuloses, 2-C-methyl-D-ara-
binitol 2, again related to 4-C-methyl-D-lyxitol by a 180°
rotation was required. In comparison, with the short envi-
ronmentally friendly preparation of ribitol 1, from glucose,
20
C-methyl-D-xylulose, 4D [oil, ½aꢂD ꢁ7.2 (c 1.0, water)] in
HDO
4-methyl-ribulose 3D and 3L
Acetone
1αH/H’
6αH
6βH
3αH
3βH
5βH
5αH’
5αH
1βH’
5βH’
1βH
4.5
4.0
3.5
3.0
2.5
2.0
1.5
Chemical Shift (ppm)
Fig. 3. 1H NMR of 4-C-methyl-ribulose 3 L-enantiomer in black, D-enantiomer in red.

3320
D. Rao et al. / Tetrahedron Letters 49 (2008) 3316–3321
70% yield. DTE also epimerizes D-xylulose at C-3 to pro-
duce D-ribulose.13 Reaction of 4-C-methyl-D-xylulose 4D
with DTE afforded a mixture of the two epimers 4D and
3D in a ratio of 30:70, allowing the isolation of 4-C-
3, and less certainly in 4, the major isomer shows an
NOE between C-1H/H0 and C-3H, indicating that the a
anomer is the major isomer present in 3 and the b anomer
is the major isomer present in 4.
20
methyl-D-ribulose 3D [oil, ½aꢂD +2.7 (c 1.0, water)] in
NMR studies of D-ribulose and D-xylulose have been
previously reported.16 The general pattern of 13C chemical
shifts is similar for the pentuloses and the 4-C-methyl
pentuloses. The major anomers reported for the pentuloses
are a-ribulose (61%) and b-xylulose (62%), as is found for
the 4-C-methyl pentuloses. However, a significant propor-
tion of the open-chain keto-form was observed for both
pentuloses (ꢀ19%), whereas these forms are below the
detection limit for the 4-C-methyl pentuloses (Figs. 3
and 4).
In summary, this Letter reports the first syntheses of
both enantiomers of the hitherto unknown 4-C-methyl
pentuloses. Although the isomerization of the ketoses with
the corresponding aldoses [in purple in Scheme 1] by aldose
isomerases has yet to be confirmed, this work indicates that
the technique of Izumoring may be applied to a wide range
of both naturally occurring and synthetic carbohydrates
and provides practical amounts of the new monosaccha-
rides to evaluate their biological potential. The combina-
tion of chemistry with biotechnology in this Letter
illustrates the synergistic potential in the synthesis of novel
sugars.
50% yield. The purity of the two epimers was established
by HPLC (Fig. 2).
A detailed NMR study of pentuloses 3 and 4 was under-
taken in order to establish their structures in solution. The
1H NMR spectra of 3L and 3D are identical, as are the
spectra of 4L and 4D. All the four samples are greater than
95% pure as judged by the NMR spectra.
Full 1H and 13C NMR assignments for 4-C-methyl ribu-
lose 3 and the 4-C-methyl xylulose 4 are given in Table 1.
Both 3 and 4 are present in solution in the two anomeric
furanose forms (Scheme 4). If the keto-form is present in
either, it is below the detection limit. In 3, the C-4CH3 res-
onances give an NOE to the C-3H resonances of similar
magnitude to the stronger of the two NOEs to the C-
5Hs, indicating that the methyl group and C-3H are on
the same side of the ring (consistent with a ribulose config-
uration). In the major anomer of 4, the C-4CH3 resonance
gives an NOE to the C-3H resonance of similar magnitude
to the weaker of the two NOEs to the C-5Hs, indicating
that the methyl group and C3H are on the opposite sides
of the ring (consistent with a xylulose configuration). In
HDO
4-C-methyl-xylulose 4D and 4L
Acetone
1βH/H’
X
6βH
5αH’
5βH
6αH
3αH
3βH
5βH’
1aH’
1αH
5αH
4.5
4.0
3.5
3.0
2.5
2.0
1.5
Chemical Shift (ppm)
Fig. 4. 1H NMR of 4-C-methyl-xylulose 4 L-enantiomer in black, D-enantiomer in red.

D. Rao et al. / Tetrahedron Letters 49 (2008) 3316–3321
3321
Rao, D.; Morimoto, K.; Takata, G.; Jones, N. A.; Jenkinson, S. F.;
Wormald, M. R.; Dwek, R. A.; Fleet, G. W. J.; Izumori, K.
Tetrahedron: Asymmetry 2008, 19.
Acknowledgement
This work was supported in part by the Program for
Promotion of Basic Research Activities for Innovative Bio-
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22
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T.; Takuchi, T.; Khan, A. R.; Izumori, K. . J. Ferment. Bioeng. 1995,
79, 184–185; (e) Ishida, Y.; Kamiya, T.; Izumori, K. J. Ferment.
Bioeng. 1997, 84, 348–350.
14. The ratio of the protected lactone to the unprotected lactone varies in
regard to the work-up of the Kiliani reaction. See Ref. 10 and:
Jenkinson, S. F.; Jones, N. A.; Moussa, A.; Stewart, A. J.; Heinz, T.;
Fleet, G. W. J. Tetrahedron Lett. 2007, 48, 4441–4445.
5. (a) Izumori, K. J. Biotechnol. 2006, 124, 717–722; (b) Izumori, K.
Naturwissenschaften 2002, 89, 120–124.
6. (a) Rahman, A. K.; Tokunaga, H.; Yoshida, K.; Izumori, K. J.
Ferment. Bioeng. 1991, 72, 488–490; (b) Muniruzzaman, S.; Toku-
naga, H.; Izumori, K. J. Ferment. Bioeng. 1995, 9, 323–327; (c)
Bhuiyan, S. H.; Ahmed, Z.; Utamura, M.; Izumori, K. J. Ferment.
Bioeng. 1998, 86, 513–516.
7. (a) Yoshida, H.; Yamada, M.; Nishitani, T.; Takada, G.; Izumori, K.;
Karnitori, S. J. Mol. Biol. 2007, 374, 443–453; (b) Ishida, Y.; Kamiya,
T.; Izumori, K. J. Ferment. Bioeng. 1997, 84, 348–350.
15. Data for 2-C-methyl-D-arabinitol 2: HRMS (ESI+ve): 189.0734
21
(M+Na+); C6H14NaO5 requires: 189.0733; ½aꢂD +13.5 (c 1.0 in
MeOH); mmax (thin film): 3356 (s, br, OH); dH (D2O, 400 MHz): 1.20
(3H, s, Me), 3.58–3.67 (4H, m, 3 ꢃ CH2, 1 ꢃ CH), 3.80–3.85 (2H, m,
1 ꢃ CH2, 1 ꢃ CH); dH (MeOD, 400 MHz): 1.23 (3H, s, Me), 3.52 (1H,
d, H-3, J 7.1), 3.56–3.66 (3H, m, 3 ꢃ CH2), 3.75–3.84 (2H, m, H-4,
CH2); dC (D2O, 100.6 MHz): 20.1 (Me), 63.7 (CH2), 67.0 (CH2), 72.2
(CH), 74.8 (CH), 75.4 (C-2); dC (MeOD, 100.6): 21.7 (Me), 65.0
(CH2), 68.2 (CH2), 73.7 (C-4), 75.6 (C-2), 76.2 (C-3); m/z (ESIꢁve):
165 (M–H+, 100%).
8. (a) Gullapalli, P.; Shiji, T.; Rao, D.; Yoshihara, A.; Morimoto, K.;
Takata, G.; Fleet, G. W. J.; Izumori, K. Tetrahedron: Asymmetry
2007, 18, 1995–2000; (b) Yoshihara, A.; Haraguchi, S.; Gullapalli, P.;
16. Vuorinen, T.; Serianni, A. S. Carbohydr. Res. 1991, 209, 13–31.