Isomerization of deoxyhexoses: green bioproduction of 1-deoxy-d-tagatose from l-fucose and of 6-deoxy-d-tagatose from d-fucose using Enterobacter agglomerans strain 221e

Article abstract of DOI:10.1016/j.tetasy.2008.02.013

1-Deoxy-d-tagatose was produced by the hydrogenation of 6-deoxy-l-galactose (l-fucose) to l-fucitol followed by oxidation with Enterobacter agglomerans 221e; a similar sequence on d-fucose afforded 6-deoxy-d-tagatose. Thus, the polylol dehydrogenase recognizes the d-galacto-configuration of both d-fucitol and l-fucitol. The procedures were conducted in water and show the power of green, environmentally friendly biotechnology in the preparation of new monosaccharides with a potential for novel bioactive properties. 6-Deoxy-d-tagatose was also synthesized from d-tagatose via the efficient formation of 1,2:3,4-di-O-isopropylidene-α-d-tagatofuranose; a difficult final removal of protecting groups by acid makes the biotechnological route more attractive.

Full text of DOI:10.1016/j.tetasy.2008.02.013

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 19 (2008) 739–745
Isomerization of deoxyhexoses: green bioproduction of 1-deoxy-
D-tagatose from L-fucose and of 6-deoxy-D-tagatose from
D-fucose using Enterobacter agglomerans strain 221e
Akihide Yoshihara,a Satoshi Haraguchi,a Pushpakiran Gullapalli,a Davendar Rao,a
Kenji Morimoto,a Goro Takata,a Nigel Jones,b Sarah F. Jenkinson,b Mark R. Wormald,c
Raymond A. Dwek,c George W. J. Fleetb and Ken Izumoria,*
aRare Sugar Research Center, Faculty of Agriculture, Kagawa University, 2393 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0795, Japan
bDepartment of Chemistry, Chemistry Research Laboratory, University of Oxford, Mansfield Road, OX1 3TA, UK
cGlycobiology Institute, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, UK
Received 29 January 2008; accepted 8 February 2008
Abstract—1-Deoxy-D-tagatose was produced by the hydrogenation of 6-deoxy-L-galactose (L-fucose) to L-fucitol followed by oxidation
with Enterobacter agglomerans 221e; a similar sequence on D-fucose afforded 6-deoxy-D-tagatose. Thus, the polylol dehydrogenase rec-
ognizes the D-galacto-configuration of both D-fucitol and L-fucitol. The procedures were conducted in water and show the power of
green, environmentally friendly biotechnology in the preparation of new monosaccharides with a potential for novel bioactive properties.
6-Deoxy-D-tagatose was also synthesized from D-tagatose via the efficient formation of 1,2:3,4-di-O-isopropylidene-a-D-tagatofuranose;
a difficult final removal of protecting groups by acid makes the biotechnological route more attractive.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
microbe selectively recognizes the D-galacto—rather than
the L-galacto—structural motif. This is consistent with
the selectivity in the oxidation of achiral galactitol 6 by
E. agglomerans 221e, which exclusively affords D-tagatose
5D as the sole product with none of the enantiomeric
L-tagatose 5L being formed (Scheme 2).4 An authentic
sample of 6-deoxy-D-tagatose 4 was prepared from D-taga-
tose 5D via the efficient formation of diacetone tagatose 7.
Although Izumoring has provided the biotechnology for
the large scale production of any hexose,1 very few reports
of the isomerization of deoxy hexoses have appeared.2
Recently, the efficient isomerization of L-rhamnose (the
only cheaply available deoxy hexose) to 1-deoxy-L-fructose
has been reported on a multigram scale.3
Herein, we extend the isomerization of deoxyhexoses by
the conversion of L-fucose 1L to 1-deoxy-D-tagatose 3 by
initial hydrogenation to L-fucitol 2L followed by microbial
oxidation using Enterobacter agglomerans 221e; a similar
isomerization of D-fucose 1D involved sequential hydro-
genation to D-fucitol 2D and oxidation by the same
microbe to afford 6-deoxy-D-tagatose 4 (Scheme 1).
L-Fucitol 1L can be viewed as either 6-deoxy-L-galactitol
or as 1-deoxy-D-galactitol; both enantiomers of fucitol
are oxidized to form D-tagatoses 3 and 4 so that the
2. Results and discussion
2.1. Synthesis of 1-deoxy-D-tagatose 3 and of 6-deoxy-D-
tagatose 4
1-Deoxy-D-tagatose 3 was prepared by using a combined
biotechnological and chemical route from L-fucose 1L
(Scheme 3). The hydrogenation of L-fucose 1L in water
in the presence of a nickel catalyst at 1.2 MPa, 50 °C gave
L-fucitol 2L in virtually quantitative yield when 1.0% (w/v)
of the substrate was used. The NMR of L-fucitol 2L (Table
1) is unusual in that there is no averaging of the coupling
constants for C2H–C3H–C4H–C5H.
*
Corresponding author. Tel.: +81 87 891 3290; fax: +81 87 891 3289;
e-mail: izumori@ag.kagawa-u.ac.jp
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.02.013

740
A. Yoshihara et al. / Tetrahedron: Asymmetry 19 (2008) 739–745
Me
Me
CHO
CH2OH
OH
OH
O
HO
HO
HO
HO
reduction
(i)
oxidation
(ii)
OH
OH
OH
OH
HO
HO
HO
HO
180o
OH
CH2OH
CH2OH
Me
Me
L-Fucose 1L
L-Fucitol 2L
1-Deoxy-D-tagatose 3
6-Deoxy-L-galactose
OH OH
6-Deoxy-L-galactitol
1-Deoxy-D-galactitol
OH OH
OH OH
CH2OH
OH OH
HOH2C
(i)
(ii)
Me
CHO
Me
Me
180o
HOH2C
Me
OH
O
OH OH
OH OH
OH OH
CH2OH
O
CH2OH
O
CHO
OH
CH2OH
OH
oxidation
(ii)
reduction
(i)
HO
HO
HO
HO
HO
HO
HO
HO
OH
Me
(iii)
OH
CH2OH
OH
Me
OH
Me
D-Tagatose 5D
6-Deoxy-D-tagatose 4
D-Fucitol 2D
6-Deoxy-D-galactitol
D-Fucose 1D
6-Deoxy-D-galactose
OH OH
(iii)
OH OH
OH OH
OH OH
CH2OH
(i)
(ii)
CH2OH
CH2OH
CHO
Me
HOH2C
Me
Me
OH
O
OH
O
OH OH
OH OH
Scheme 1. Reagents: (i) H2, Ni, H2O; (ii) Enterobacter agglomerans 221e, H2O; (iii) see text.
CH2OH
CH2OH
O
CH2OH
O
CH2OH
OH
HO
HO
OH
OH
OH
HO
HO
HO
HO
(i)
OH
(i)
180o
HO
OH
OH
CH2OH
CH2OH
CH2OH
CH2OH
L-galacto
D-galacto
meso-Galactitol 6
L-Tagatose 5L
D-Tagatose 5D
(i)
OH OH
OH OH
(i)
OH OH
OH OH
CH2OH
CH2OH
CH2OH
CH2OH
HOH2C
180o
HOH2C
HOH2C
HOH2C
OH OH
OH
O
OH
O
OH OH
Scheme 2. Reagents: (i) Enterobacter agglomerans 221e, H2O.
OH
OH
OH
OH
OH
OH
OH
OH
(i)
(ii)
CHO
CH2OH
Me
Me
180o
HOH2C
HOH2C
Me
Me
OH
OH
OH
OH
OH
OH
OH
O
6-Deoxy-L-galactitol 1-Deoxy-D-galactitol
L-Fucitol 2L
6-Deoxy-L-galactose 1L
1-Deoxy-D-tagatose 3
L-Fucose
Scheme 3. Reagents and conditions: (i) Nickel, H2, H2O, 1.2 MPa pressure, 50 °C, 96%; (ii) Enterobacter agglomerans strain 221e, 86%.

isolated from L
-fucitol 2L in 86% yield; a very small quan-
A. Yoshihara et al. / Tetrahedron: Asymmetry 19 (2008) 739–745
741
Table 1. NMR analysis of 1D (1H and 13C) and HSQC of L-fucitol 2L
(chemical shift referenced to acetone at 2.22 ppm for 1H and 30.9 ppm for
13C), pH 5.0, 2H2O
tity of a by-product was also formed but was removed by
crystallization. The purity of both substrate 2L and of
product 3 was established by HPLC (Fig. 1).
Label
1H
13C
d (ppm) Mult 3JHH (Hz) d (ppm)
1-Deoxy-D-tagatose 3 crystallizes as two different poly-
morphs of 1-deoxy-a-D-tagatopyranose,6 which is also the
major form present in the solution; the b-pyranose form
is present at about one quarter the concentration together
with minor amounts of three other forms, giving a complex
NMR spectrum in solution. The 13C NMR of 1-deoxy-D-
tagatose 3 in water was identical to that of a synthetic sam-
ple.7 Although 3 has been synthesized in small amounts by
chemical reactions before,8 the present biotechnological
procedure allows sufficient quantities to be readily pre-
pared for full biological evaluation.
HOH2C1
HO
1
2
3
4
5
6
3.676
3.955
3.633
3.475
4.082
1.237
m
m
dd
dd
dq
d
63.9
71.2
70.5
73.6
66.7
19.3
OH
OH
1.5/9.0
9.0/1.8
1.8/6.6
6.6
HO
6CH3
Microbial oxidation of L-fucitol 2L to afford 1-deoxy-D-
tagatose 3 was carried out using E. agglomerans 221e, a
well-established catalyst for oxidation–reduction reactions
between various polyols and ketoses.5 The highest activity
for the oxidation of L-fucitol 2L was observed when this
microbe was grown on a medium containing 0.5% poly-
pepton, 0.5% yeast extract, 0.5% NaCl, 1.0% erythritol
and 1.0% glycerol. A study of a range of different buffers
between pH 4.0 and 11.0 (50 mM sodium acetate pH
4.0–6.0, 50 mM sodium–phosphate pH 6.0–8.0, 50 mM
Tris–HCl pH 7.0–9.0 and 50 mM glycine–NaOH pH 9.0–
11.0) showed that the optimum conditions for the oxida-
tion occurred at a pH of 8.0 in a Tris–HCl buffer. Under
these optimum conditions, 1-deoxy-D-tagatose 3 was
A similar procedure was used for the conversion of D-fu-
cose 1D to 6-deoxy-D-tagatose 4 (Scheme 4). Hydrogena-
tion of D-fucose 1D in water in the presence of a nickel
catalyst proceeded to give D-fucitol 2D in nearly quantita-
tive yield. The optimum conditions for the oxidation of
D-fucitol 2D by E. agglomerans 221e to 6-deoxy-D-tagatose
4 were the same as those for the conversion of L-fucitol 2D
to 1-deoxy-D-tagatose 2 (80% yield). Separation and purifi-
cation of 6-deoxy-D-tagatose 4 was achieved by column
chromatography on Dowex 50W-X2 (Ca2+) at 60 °C.
Figure 1. HPLC analysis of (a) L-fucitol 2L and (b) purified 1-deoxy-D tagatose 3.
OH OH
OH OH
OH OH
O
(ii)
(i)
(vii)
O
Me
CH2OH
CHO
CH2OH
O
Me
Me
Me
OH
O
OH OH
OH OH
O
O
6-Deoxy-D-galactose 1D
6-Deoxy-D-galactitol
6-Deoxy-D-tagatose 4
10
(vi)
D-Fucose
D-Fucitol 2D
OH OH
CH2OH
O
O
O
(iv)
(v)
O
O
O
(iii)
F3CO2SOH2C
IH2C
HOH2C
O
O
O
HOH2C
OH
O
O
O
O
O
O
O
D-Tagatose 5D
7
8
9
Scheme 4. Reagents and conditions: (i) Nickel, H2, H2O, 1.2 MPa pressure, 50 °C, 86%; (ii) Enterobacter agglomerans strain 221e, 74%; (iii) Me2CO,
CuSO4, concd H2SO4, 82%; (iv) (CF3SO2)2O, CH2Cl2, pyridine, 98%; (v) Bu4N+Iꢀ, THF, 96%; (vi) H2, 10% Pd/C, Et3N, EtOH, 96%—see text; (vii)
Dowex (50W-X8, H+), H2O, 90%.

742
A. Yoshihara et al. / Tetrahedron: Asymmetry 19 (2008) 739–745
Figure 2. HPLC analysis of (a) D-fucitol 2D and (b) purified 6-deoxy-D tagatose 4.
6-Deoxy-D-tagatose 4, was identified by HPLC (Fig. 2).
This is the first report of the bioproduction of 6-deoxy-
D-tagatose 4 from 6-deoxy-D-galactose (D-fucose) 1D.
constants. There are very few useful NOEs; a medium
enhancement of 4b-1 to 4b-3 is consistent with the b-fura-
nose form and the weak enhancement of 4a-1 to 4a-3 is
consistent with the a-furanose For both tagatose 5 and 6-
deoxy-tagatose 4, the a-furanose forms have a C3H–C4H
coupling of 5.0 and 5.3 Hz, respectively, and the b-furanose
a coupling of 4.9 and 4.5 Hz, consistent with spin-system 2
being the a-anomer. In the furanose forms of tagatose 5
and 1-deoxy-tagatose 3, the ratios of a/b anomer are
0.35/1 and 1/1, respectively, the former again being consis-
tent with spin system 2 being the a-anomer.
An authentic sample of 6-deoxy-D-tagatose 4 was also pre-
pared by a chemical route from D-tagatose 5D. D-Tagatose
5D, a possible food substitute,9 has only recently become
readily available at a reasonable cost10 for the use as a
member of the chiral pool.11 In the past, the acetonides
of tagatose have usually been made by a multi-step pro-
cesses from fructose12 or sorbose.13 However, treatment
of D-tagatose 5D with acetone in the presence of copper
sulfate and sulfuric acid gave the easily crystallized diaceto-
nide 714 in 82% yield. The free primary alcohol in 7 was
esterified with trifluoromethanesulfonic (triflic) anhydride
in dichloromethane in the presence of pyridine to afford tri-
flate 8 (98% yield), which on reaction with tetrabutylam-
monium iodide in THF gave the corresponding iodide 9
in 96% yield. Hydrogenolysis of iodide 9 with 10% palla-
dium on carbon in ethanol in the presence of triethylamine
formed the protected deoxy tagatose 10 in 96% yield; the
diacetonide 10 is sensitive to acid and partially decomposed
on silica gel chromatography. Careful hydrolysis of 10 to
remove the isopropylidene protecting groups by Dowex
ion exchange resin (50W-X8, H+) gave an authentic sample
of 6-deoxy-D-tagatose 4 in 90% yield; the hydrolysis of
ketose diacetonides is frequently experimentally difficult,
probably due to the sensitivity of the monoacetonides to
acid.15 Although the synthetic sequence is efficient (70%
overall yield from D-tagatose 5D), the difficulties of acid
hydrolysis in the final step make this an unattractive
method in comparison to the biotechnological route.
3. Conclusion
In conclusion, we have reported the overall isomerizations
of the enantiomers of fucose 2L and 2D to 1-deoxy-D-tag-
atose 3 and 6-deoxy-D-tagatose 4, respectively. These
Table 2. NMR analysis of 1D (1H and 13C) and HSQC of 6-deoxy-D-
tagatose 4 (chemical shift referenced to acetone at 2.22 ppm for 1H and
30.9 ppm for 13C), pH 6.9, 2H2O
Label
1H
13C
d
Mult 3JHH
d
(ppm)
(Hz)
(ppm)
4b-1
3.570
3.504
—
4.233
4.122
4.115
1.277
d
d
ꢀ12.0
ꢀ12.0
—
63.39
CH2OH
OH
O
Me
4b-2
4b-3
4b-4
4b-5
4b-6
—
d
dd
dq
d
102.86
71.80
73.17
77.09
15.21
4.7
O
O
4.7/?
?/6.2
6.2
4β
4a-1
3.650
3.576
—
4.287
4.13
d
d
—
d
dd
ꢀ11.8
ꢀ11.8
—
5.0
5.0/3.5
63.10
The NMR of 6-deoxy-D-tagatose 4 in solution is complex
but it mainly exists as the two furanoses, together with
other minor forms. The 13C NMR of the sample of 6-
deoxy-D-tagatose 4 prepared from D-fucitol 2D was identi-
cal to that of the sample prepared from D-tagatose 5D.
4a-2
4a-3
4a-4
105.24
79.21
73.54
OH OH
CH2OH
Me
OH
O
4a-5
4a-6
4.338
1.216
dq
d
3.5/6.5
6.5
76.10
14.22
4
The detailed NMR analysis of the solution spectrum of 6-
deoxy-D-tagatose 4 is shown in Table 2. There are two ma-
jor spin systems, with relative intensities of approximately
2 to 1 which probably correspond to the b 4b and a 4a ano-
mers of the furanose form, respectively; both tagatose 5
and 1-deoxy-D-tagatose 3 exist mainly as pyranose forms
with only minor amounts of the respective furanoses pres-
ent. As both spin-systems are in the furanose form, it is dif-
ficult to assign relative configurations based on coupling
OH
O
Me
CH2OH
O
O
4α

A. Yoshihara et al. / Tetrahedron: Asymmetry 19 (2008) 739–745
743
syntheses are further examples of environmentally friendly
access to very rare deoxy sugars to allow evaluation of their
biological properties; the biotechnological synthesis of such
sugars gives much cleaner materials due to the sensitivity of
the product ketoses to the acid conditions needed at the
final deprotection in chemical synthesis.
(NMR) spectra were recorded on Bruker AMX 500 (1H:
500 MHz and 13C: 125.7 MHz) and Bruker DPX 400 and
DQX 400 spectrometers (1H: 400 MHz and 13C:
100.6 MHz) in the deuterated solvent stated. For spectra
recorded in D2O methanol was used as an internal refer-
ence. Residual signals from other solvents were used as
an internal reference. NMR spectra for 2L and 4 were
recorded on a Varian Unity INOVA 500 (1H: 500 MHz
and 13C: 125 MHz) spectrometer, in D2O, with a probe
temperature of 30 °C. Chemical shifts were measured
relative to internal standards (1H: acetone at 2.220 ppm
and 13C: acetone at 30.9 ppm). Two-dimensional gradient
COSY, HSQC, HMBC and HSQC-TOCSY spectra were
4. Experimental
L-Fucose and D-fucose and all other biochemicals were
purchased from Sigma Chemical Co. (MO, USA) and
Wako Pure Chemicals (Osaka, Japan). D-Tagatose was
obtained as a generous gift from Arla Foods. 50 mM
Tris–HCl buffer, used for L- and D-fucitol oxidations,
was prepared from tris(hydroxymethy)aminoethane
H2NC(CH2OH)3 by adjusting the pH to 8.0 with 1.0 N
HCl. D- and L-Fucose hydrogenation was performed in
TEM-1000M hydrogenation apparatus (Taiatsu Techno
Co. Ltd, Japan). Microbe growth and oxidation reactions
were carried in bioreactors (TS-M-15L fermentor and
TS-M-5L fermentor) from Takasugi Seisakusho Co. Ltd
and oxidation reactions were carried out in Erlenmeyer
flasks (500 mL). Polyol oxidation and ketose accumulation
in the reaction mixture was determined by Nelson–Somo-
gyi method16 by UV by a visible spectrophotometer (UV-
1700 pharmaspec, Shimadzu, Kyoto). 13C NMR spectra
(Bruker AMX 500, 126 MHz) were recorded in D2O using
acetone as internal standard. Optical rotations were
recorded on a Jasco R1030 polarimeter, Na+ lamp, (Jasco,
Tokyo, Japan) at 20 °C in deionized H2O polarimeter with
a path length of 1 dm. Concentrations are quoted in
g 100 mLꢀ1. The product was analyzed by high-perfor-
mance liquid chromatography (Hitachi GL-611 column,
Tokyo, Japan and Shimadzu RID-6A refractive index
detector, Kyoto, Japan) at 60 °C, eluted with 10ꢀ4 M
NaOH at a flow rate of 1.0 mL/min. Tetrahydrofuran
was purchased dry from the Aldrich chemical company
in sure-seal bottles. Pyridine was purchased dry from the
Fluka chemical company in sure-seal bottles over molecu-
lar sieves. All other solvents were used as supplied (Analyt-
ical or HPLC grade) without prior purification. Reactions
were performed under an atmosphere of nitrogen or argon,
unless stated otherwise. Thin layer chromatography (TLC)
was performed on aluminum sheets coated with 60 F254
silica. Sheets were visualized using a spray of 0.2% w/v
cerium(IV) sulfate and 5% ammonium molybdate in 2 M
sulfuric acid. Flash chromatography was performed on
Sorbsil C60 40/60 silica. Melting points were recorded on
a Kofler hot block and are uncorrected. Optical rotations
were recorded on a Perkin–Elmer 241 polarimeter with a
path length of 1 dm. Concentrations are quoted in
g 100 mLꢀ1. Infrared spectra were recorded on a Perkin–
Elmer 1750 IR Fourier Transform spectrophotometer
using thin films on NaCl plates (thin film). Only the char-
acteristic peaks are quoted. Low resolution mass spectra
(m/z) were recorded on VG MassLab 20–250, Micromass
BIOQ-II, Micromass Platform 1, Micromass TofSpec 2E,
or Micromass Autospec 500 OAT spectrometers and high
resolution mass spectra (HRMS m/z) on a Micromass
Autospec 500 OAT spectrometer. The technique used was
electrospray ionization (ESI). Nuclear magnetic resonance
1
used to aid assignment of H and 13C spectra. NOESY
1
spectra were recorded with a 400 ms mixing time. 1D H
NMR spectral simulations were performed using the
program gNMR (Cherwell Scientific Publishing). All
chemical shifts (d) are quoted in ppm and coupling
constants (J) in Hertz.
4.1. L-Fucitol 2L and D-fucitol 2D
4.1.1. Preparation of catalyst. Raney nickel (30 g) was dis-
solved in 300 mL of 20% aqueous sodium hydroxide
(300 mL). The catalyst was heated at 80 °C for 6 h and then
washed several times with distilled water to obtain pH 9.2.
The resultant precipitate was used for hydrogenation of
both enantiomers of fucose.
4.1.2. L-Fucitol 2L. The prepared catalyst was added to
1% solution of L-fucose 1L (5 g) in water; the hydrogena-
tion reaction mixture was adjusted to a total volume of
500 mL. The reaction was carried out at 1.2 MPa hydrogen
pressure at 50 °C for 24 h to afford L-fucitol 2L, (4.8 g,
20
96%), as an oil, ½aꢁD ¼ þ1:9 (c 1.0, water).
D-Fucitol 2D was prepared in an identical procedure from
D-fucose 1D (5 g) in water to give D-fucitol 2D (4.8 g, 96%),
20
oil, ½aꢁD ¼ ꢀ1:9 (c 1.0, water).
4.2. 1-Deoxy-D-tagatose 3 and 6 by microbial oxidation of
2L and 2D
4.2.1. Optimization of conditions. E. agglomerans 221e,
isolated in our laboratory,17 was used for the oxidations
of L-fucitol 2L to 1-deoxy-D-tagatose 3 and of D-fucitol 2L
to 6-deoxy-D-tagatose 4. The strain was maintained at
4 °C on 2% TSB agar slant. This bacteria was grown aerobi-
cally in Erlenmeyer flasks (500 mL) containing 200 mL med-
ium (0.5% polypepton, 0.5% yeast extract, 0.5% NaCl, 1.0%
erythritol and 1.0% glycerol) at 30 °C for 24 h. The cells
were cultivated using the above medium for 24 h and har-
vested by centrifugation at 9000 rpm for 10 min. The col-
lected cells were washed twice with deionized water and
centrifuged at 9000 rpm for 10 min. The washed cells were
suspended in various buffers for carrying out the biotrans-
formations. The effect of pH from 6.0 to 11.0 on both oxida-
tions was studied using buffers (50 mM sodium acetate pH
4.0–6.0, 50 mM sodium phosphate pH 6.0–8.0, 50 mM
Tris–HCl pH 7.5–9.0, 50 mM glycine–NaOH pH 9.0–
11.0). The reaction mixtures were analyzed by HPLC after

744
A. Yoshihara et al. / Tetrahedron: Asymmetry 19 (2008) 739–745
2 h for the transformations of L-fucitol 2L to 1-deoxy-D-tag-
atose 3 and of D-fucitol 2D to 6-deoxy-D-tagatose 4.
66 °C; [a]D = ꢀ63 (c 1.0, CHCl3)}; mmax (thin film): 3491
(m, br, OH); dH (CDCl3, 400 MHz): 1.31 (3H, s, Me),
1.40 (3H, s, Me), 1.43 (3H, s, Me), 1.47 (3H, s, Me), 2.21–
2.24 (1H, dd, OH, J 4.8, 7.7), 3.85–3.97 (2H, m, H6, H60),
4.04–4.08 (1H, m, H5), 4.08 (1H, a-d, H1, J 9.7), 4.27
(1H, d, H10, J 9.7), 4.62 (1H, d, H3, J 5.9), 4.83–4.85 (1H,
dd, H4, J 5.8, 3.9); dC (CDCl3, 100.6 MHz): 24.6, 25.9,
26.4 (4 ꢂ CH3CO), 61.1 (C6), 69.2 (C1), 78.8 (C5), 80.5
(C4), 85.4 (C3), 111.7 (ꢂ2), 112.9 (2 ꢂ CH3CO and C2).
4.2.2. 1-Deoxy-D-tagatose 3. The E. agglomerans 221e
cells were cultivated, harvested and washed as described
above. The collected cells were suspended in 50 mM
Tris–HCl buffer (pH 8.0) and the transformation reaction
was carried out in a 500 mL Erlenmeyer flasks containing
1% L-fucitol 2L (4.8 g, 0.03 mol) at 30 °C with continuous
shaking at 180 rpm. The formation of 1-deoxy-D-tagatose 3
was monitored by HPLC. At the end of the reaction, the
cells were removed by centrifugation at 10,000 rpm for
10 min. The supernatant was decolorized using activated
charcoal which was then removed using centrifugation
and filtration. The decolorized supernatant was deionized
using ion exchange resin diaion (SKIB, H+) and amberlite
(IRA-411, CO32ꢀ). This deionized solution was concen-
trated using a rotary evaporator; the product 3 (3.8 g,
4.3.2. 1,2:3,4-Di-O-isopropylidene-6-trifluoromethanesulfon-
yl-a-D-tagatofuranose 8. A solution of the diacetonide
7 (1.56 g, 6.0 mmol) in dichloromethane (8 mL) and pyri-
dine (1.45 mL, 18 mmol) was cooled to ꢀ30 °C and treated
with triflic anhydride (1.61 mL, 9.6 mmol). The reaction
mixture was stirred at ꢀ30 to ꢀ10 °C for 3 h after which
time TLC (EtOAc/cyclohexane, 1:1) showed the complete
consumption of the starting material (Rf 0.55) and the for-
mation of one major product (Rf 0.83). The reaction mix-
ture was diluted with dichloromethane (30 mL) and
washed with aqueous hydrochloric acid (2 M, 20 mL),
and the aqueous layer was extracted with dichloromethane
(3 ꢂ 20 mL). The combined organic layers were washed
with brine (30 mL), dried over magnesium sulfate and con-
centrated under reduced pressure to give the crude triflate 8
0.02 mol 80%) purified by crystallization mp 132 °C
20
½aꢁD ¼ ꢀ14:7 (c 1.0, water) with identical 1H and 13C
NMR to an authentic sample. 1-Deoxy-D-tagatose 4 was
recrystallized from a mixture of EtOAc and MeOH to give
colorless crystals as two polymorphs;17 mp 136–138 °C and
143–145 °C (lit.23a mp 121–123 °C, lit.23b mp 130 °C);
22
23
½aꢁD ¼ ꢀ13 (c 2.0, H2O) {lit.23a ½aꢁD ¼ ꢀ14 (c 2.0, H2O)}.
(2.307 g, 98%) as a crystalline solid which was used without
22
4.2.3. 6-Deoxy-D-tagatose 4. The oxidation of D-fucitol
2D (4.8 g, 0.03 mol) by E. agglomerans 221 to 6-deoxy-D-
tagatose 4 was performed in an identical manner to that
of L-fucitol 2D above. After removal of the animal char-
coal, the supernatant was deionized using ion exchange
resin diaion (SKIB, H+) and amberlite (IRA-411, CO32ꢀ).
The deionized supernatant was concentrated and the prod-
uct separated using Dowex 50W-X2 resin (Ca2+). The pur-
ity of the product 4 was established by HPLC (Fig. 2); 6-
further purification, mp 44–46 °C; ½aꢁD ¼ þ43:6 (c 0.89,
CHCl3); dH (CDCl3, 400 MHz): 1.30 (3H, s, Me), 1.41
(3H, s, Me), 1.42 (3H, s, Me), 1.47 (3H, s, Me), 4.08 (1H,
d, H1, J 9.9), 4.24–4.28 (1H, a-dt, H5, J 3.8, 7.5), 4.29
(1H, d, H10, J 9.8), 4.60–4.64 (1H, dd, H6, J 7.5, 10.9),
4.64 (1H, d, H3, J 5.7), 4.75–4.78 (1H, dd, H60, J 3.9,
10.9), 4.83–4.85 (1H, dd, H4, J 3.8, 5.7); dC (CDCl3,
100.6 MHz): 24.7, 25.8, 26.1, 26.4 (4 ꢂ CH3CO), 69.2
(C1), 74.1 (C6), 76.2 (C5), 79.5 (C4), 85.0 (C3), 112.1,
112.2, 113.5 (2 ꢂ CH3CO and C2), 118.6 (CF3, d, J 320).
deoxy-D-tagatose 4 (3.5 g, 0.02 mol 73%) was isolated as
20
an oil, ½aꢁD ¼ ꢀ2:2 (c 1.0, water) {lit. an oil, except for
20
enantiomer mp 68–69 °C21b}; ½aꢁD ¼ ꢀ1:6 (c 1.4, water)
4.3.3. 6-Deoxy-1,2:3,4-di-O-isopropylidene-6-iodo-a-D-taga-
tofuranose 9. Tetrabutylammonium iodide (1.4 g,
3.78 mmol) was added to a solution of triflate 8 (987 mg,
2.52 mmol) in THF (10 mL). The reaction mixture was stir-
red in the dark at room temperature for 18 h after which
time TLC analysis (EtOAc/cyclohexane, 1:1) showed the
consumption of the starting material (Rf 0.83) and the for-
mation of one major product (Rf 0.88). The reaction was
concentrated in vacuo and the residue dissolved in dichloro-
methane (50 mL) after which it was washed with sodium
thiosulfate (satd, aq, 30 mL). The aqueous layer was re-ex-
tracted with dichloromethane (2 ꢂ 15 mL) and the com-
bined organics were dried over magnesium sulfate and
concentrated in vacuo. The resulting residue was purified
by column chromatography (EtOAc/cyclohexane, 1:6) to
give the iodide 9 (896 mg, 96%) as a pale yellow oil which
crystallized on standing, HRMS (ESI +ve) found:
20
18
23
{½aꢁD ¼ ꢀ3 (c 2, water),18 ½aꢁD ¼ ꢀ2 (c 2, water),19 ½aꢁD
¼
18
0 (c 1.9, water),20 for L enantiomer ½aꢁD ¼ þ2:7 (c 12.8,
18
water) ½aꢁD ¼ ꢀ2 (c 2, water).21 HRMS (ESI ꢀve) found:
163.0608 (MꢀH+); C6H11O5 requires: 163.0601; m/z (ESI
ꢀve): 163 (MꢀH+, 100%). For NMR data, which is the
same as the chemical sample prepared below, see text
above and Table 2.
4.3. 6-Deoxy-D-tagatofuranose 4 from D-tagatose 5D
4.3.1. 1,2:3,4-Di-O-isopropylidene-a-D-tagatofuranose 7.
Anhydrous copper sulfate (35.8 g, 0.22 mol) and concen-
trated sulfuric acid (0.4 mL) were added to a solution of
D-tagatose 5D (10.1 g, 0.06 mol) in acetone (200 mL). The
reaction mixture was stirred at room temperature for
18 h, after which time TLC (EtOAc/cyclohexane, 1:1)
showed the formation of one major product (Rf 0.55).
The reaction mixture was neutralized with sodium carbon-
ate (solid), filtered through Celite and concentrated in
vacuo. The crude residue was purified by column chroma-
tography (EtOAc/cyclohexane, 1:4) to give the diacetonide
393.0168 (M+Na+); C12H19INaO5 requires: 393.0169; mp
22
37–39 °C; ½aꢁD ¼ þ44:5 (c 0.92, CHCl3); dH (CDCl3,
400 MHz): 1.33 (3H, s, Me), 1.40 (3H, s, Me), 1.42 (3H, s,
Me), 1.48 (3H, s, Me), 3.24–3.28 (1H, dd, H6, J 6.1, 9.6),
3.31–3.35 (1H, dd, H60, J 8.2, 9.5), 3.89 (1H, d, H1, J
9.8), 4.18–4.23 (1H, ddd, H5, J 3.5, 6.1, 8.2), 4.25 (1H, d,
H10, J 9.8), 4.64 (1H, d, H3, J 5.8), 4.82–4.85 (1H, dd,
H4, J 3.5, 5.8); dC (CDCl3, 100.6 MHz): ꢀ1.0 (C6), 25.0
7 (12 g, 82%) as a white crystalline solid, mp 55–58 °C
22
½aꢁD ¼ þ66:3 (c 1.04, CHCl3), {lit.22 mp 63–65 °C;
[a]D = +81.5 (c 1.7, Me2CO)} for enantiomer:13 mp 65–

A. Yoshihara et al. / Tetrahedron: Asymmetry 19 (2008) 739–745
745
3. Gullapalli, P.; Shiji, T.; Rao, D.; Yoshihara, A.; Morimoto,
K.; Takata, G.; Fleet, G. W. J.; Izumori, K. Tetrahedron:
Asymmetry 2007, 18, 1995–2000.
4. Muniruzzaman, S.; Tokunaga, H.; Izumori, K. J. Ferment.
Bioeng. 1994, 78, 145–148.
(Me), 26.1 (Me), 26.4 (ꢂ2, Me), 69.4 (C1), 79.8 (ꢂ2, C4,
C5), 85.4 (C3), 111.9 (ꢂ2), 112.9 (C2, 2 ꢂ CH3CO); m/z
(ESI +ve): 393 (M+H+, 100%).
4.3.4. 6-Deoxy-1,2:3,4-di-O-isopropylidene-a-D-tagatofura-
nose 10. A solution of the iodide (850 mg, 2.3 mmol)
and triethylamine (0.96 mL, 6.9 mmol) in ethanol (23 mL)
in the presence of 10% Pd/C (85 mg, 10 wt %.) was flushed
with argon and then hydrogen and stirred in the dark under
hydrogen for 18 h. After this time TLC showed the conver-
sion of the starting material (Rf 0.88) to one major product
(Rf 0.80). The reaction was flushed with argon, filtered
through Celite and concentrated in vacuo. The residue
was dissolved in dichloromethane (50 mL) and washed with
sodium thiosulfate (satd, aq, 30 mL), dried over magnesium
sulfate and evaporated under reduced pressure. The crude
product was purified by column chromatography (EtOAc/
cyclohexane, 1:6) to give the 6-deoxy compound 10 as a col-
orless oil (538 mg, 96%). (The compound appeared to be
unstable on silica as on re-columning only 109 mg,
5. (a) Izumori, K.; Tsuzaki, K. J. Ferment. Technol. 1988, 66,
225–227; (b) Izumori, K.; Yamakita, M.; Tsumura, T.;
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Bioeng. 1995, 79, 620–622; (f) Takeshita, K.; Shimonishi, T.;
Izumori, K. J. Ferment. Bioeng. 1996, 81, 212–215; (g)
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1998, 85, 84–88; (h) Bhuiyan, S. H.; Ahmed, Z.; Utamura, M.;
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19%, was isolated). HRMS (ESI +ve) found: 267.1201
22
(M+Na+); C12H20NaO5 requires: 267.1203; ½aꢁ ¼ þ64:3
D
(c 0.96, CHCl3); dH (CDCl3, 400 MHz): 1.31 (3H, d, Me,
J 6.4), 1.33 (3H, s, CH3CO), 1.40 (3H, s, CH3CO), 1.43
(3H, s, CH3CO), 1.47 (3H, s, CH3CO), 4.03 (1H, d, H1, J
9.7), 4.06–4.11 (1H, dq, H5, J 3.5, 6.4), 4.26 (1H, d, H10,
J 9.7), 4.61 (1H, d, H3, J 5.9), 4.63–4.66 (1H, dd, H4 J
3.5, 5.8); dC (CDCl3, 100.6 MHz): 13.5 (Me), 25.1 (CH3CO),
26.1 (CH3CO), 26.5 (ꢂ2) (CH3CO), 69.2 (C1), 75.2 (C5),
81.3 (C4), 85.8 (C3), 111.4, 111.5, 112.5 (C2, 2 ꢂ CH3CO);
m/z (ESI +ve): 267 (M+H+, 100%).
4.3.5. 6-Deoxy-D-tagatofuranose 4. Dowex (50W-X8, H+)
was added to a suspension of diacetonide 4 (76 mg,
0.31 mmol) in water (1 mL). The reaction was stirred at
room temperature for 6 days after which time TLC analysis
(EtOAc) showed almost complete conversion of the start-
ing material to one major product (Rf 0.0). The reaction
was filtered and diluted with water (20 mL) and washed
with EtOAc (2 ꢂ 20 mL). The aqueous layer was concen-
trated under reduced pressure to give 6-deoxy-D-tagato-
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furanose
4 (46 mg, 90%) as a colorless oil and
approximately 5:2 mixture of anomers; for data for 4, see
bioproduction of 4 above.
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Acknowledgments
This work was supported in part by the Program for Pro-
motion of Basic Research Activities for Innovative Biosci-
ences (PROBRAIN) and by Summit plc.
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