Crystal structure, solubility, and mutarotation of the rare monosaccharide D-psicose

Article abstract of DOI:10.1246/bcsj.20100148

X-ray crystal analysis for D-psicose (C3-position epimer of D-fructose) crystallized from aqueous solution was successfully performed for the first time. It was confirmed that D-psicose crystallized solely as β-D-pyranose with 1C (¹C₄ (d)) conformation. The crystal system (orthorhombic), space group (#19, P2₁2₁2₁), and number of molecules per unit cell (Z = 4) are the same as those for β-D-fructopyranose, α-L-sorbopyranose, and α-D-tagatopyranose. Solubility of D-psicose at 25 °C was 291 g per 100 g water. Mutarotation was further investigated recording the time course of specific rotation [α] at 589 nm after the dissolution of D-psicose in water. It is thought that [α] for β-D-psicopyranose in water may be ca.-85degdm ^-1g^-1 cm³. The time course of absorbance at 280 nm after the dissolution of D-psicose was also measured to see the development of open-chain carbonyl-form in the solution, and the first-order kinetic behavior with the rate constant k = 4.44 ms^-1 was confirmed. Assuming the equilibrium content of carbonyl-form of D-psicose as 0.2%, the molar absorption coefficient, ε, for the carbonyl-form was estimated to be 160cm^-1 M^-1.

Full text of DOI:10.1246/bcsj.20100148

©
2010 The Chemical Society of Japan
Bull. Chem. Soc. Jpn. Vol. 83, No. 10, 1193–1197 (2010) 1193
Crystal Structure, Solubility, and Mutarotation
of the Rare Monosaccharide D-Psicose
1
2
2
2
Kazuhiro Fukada,* Tomohiko Ishii, Katsushi Tanaka, Masatsugu Yamaji,
2
2
3
Yuya Yamaoka, Ken-ichi Kobashi, and Ken Izumori
1Department of Applied Biological Science, Faculty of Agriculture, Kagawa University,
393 Ikenobe, Kagawa 761-0795
2
2Department of Advanced Materials Science, Faculty of Engineering, Kagawa University, Takamatsu 761-0396
3Rare Sugar Research Center, Kagawa University, 2393 Ikenobe, Kagawa 761-0795
Received May 27, 2010; E-mail: fukada@ag.kagawa-u.ac.jp
X-ray crystal analysis for D-psicose (C3-position epimer of D-fructose) crystallized from aqueous solution was
successfully performed for the first time. It was confirmed that D-psicose crystallized solely as ¢-D-pyranose with 1C
1
(
C4 (D)) conformation. The crystal system (orthorhombic), space group (#19, P212121), and number of molecules per unit
cell (Z = 4) are the same as those for ¢-D-fructopyranose, ¡-L-sorbopyranose, and ¡-D-tagatopyranose. Solubility of
D-psicose at 25 °C was 291 g per 100 g water. Mutarotation was further investigated recording the time course of specific
rotation [¡] at 589 nm after the dissolution of D-psicose in water. It is thought that [¡] for ¢-D-psicopyranose in water may
¹1
¹1
3
be ca. ¹85 deg dm
g
cm . The time course of absorbance at 280 nm after the dissolution of D-psicose was also
measured to see the development of open-chain carbonyl-form in the solution, and the first-order kinetic behavior with the
¹1
rate constant k = 4.44 ms was confirmed. Assuming the equilibrium content of carbonyl-form of D-psicose as 0.2%, the
¹1
¹1
molar absorption coefficient, ¾, for the carbonyl-form was estimated to be 160 cm
M .
Recent biotechnological advances have dramatically increas-
CH OH
CH OH
2
2
ed the possibility of producing rare monosaccharides in an
environmentally friendly way. Izumoring is a biotechnological
strategy, which allows the green production of any of the
isomeric hexoses, pentoses, and tetroses.1 In Izumoring,
epimerization of ketoses at the C3 position is one of the key
processes for the syntheses of various monosaccharides. D-
Tagatose 3-epimerase (D-TE) is employed as a versatile catalyst
for the C3 epimerization of all ketohexoses and ketopentoses.
A catalytic mechanism of D-TE has been proposed, based on
X-ray crystal analysis for the complexes of D-TE and D-
O
O
­3
HO
OH
OH
OH
OH
OH
CH OH
CH OH
2
2
4
tagatose/D-fructose. In this study we utilized immobilized D-
TE to produce a rare monosaccharide D-psicose (Psi) from
inexpensive D-fructose (Fru) (Figure 1).
D-fructose
D-psicose
It has recently been demonstrated that Psi has interesting
functional properties; for example, Psi could be useful in
Figure 1. Conversion of D-fructose (Fru) to D-psicose (Psi)
by D-tagatose 3-epimerase. The equilibrium molar ratio is
3 (Fru) to 1 (Psi) at 25 °C.
5
preventing postprandial hyperglycemia in diabetic patients and
6
be a potential anthelmintic. It is further expected that Psi may
be used as a sweetener for functional foods with high
antioxidant activity and low calorie content.7
It should be stressed that Psi had only existed as a syrup,
solubility and mutarotation of Psi were also investigated using
the crystalline sample. These solution properties are important
for carbohydrate science as well as for quality control in large-
scale Psi production.
8
and no crystalline sample had been made until recently.
Through the success of large-scale production of highly pure
Psi by the epimerization of Fru with the aid of D-TE, however,
crystallization of Psi was finally achieved and single crystals of
Psi suitable for X-ray diffraction analysis have recently been
obtained. The present work reports the single crystal struc-
ture of Psi to complete the data set of crystal structures of
Experimental
Materials. Psi was obtained by the epimerization of Fru
using immobilized D-TE with the equilibrium molar ratio 1
1
4,15
versus 3.
To remove Fru from the mixed sugar solution,
column chromatography was performed using a Dowex
9­13
2+
ketohexoses.
Besides the crystal structure, the aqueous
50W-X2 (Ca -form) or Hitachi GL-C611, and the fractions
Published on the web September 25, 2010; doi:10.1246/bcsj.20100148

1194 Bull. Chem. Soc. Jpn. Vol. 83, No. 10 (2010)
Crystal Structure and Solubility of D-Psicose
containing Psi was concentrated up to ca. 80 wt % by a rotary
evaporator at 40 °C. Crystallization of Psi succeeded at room
temperature within 2­3 days. The melting point of Psi was
confirmed to be 96 °C by means of differential scanning
calorimetry (Rigaku DSC 8240D).
Table 1. Crystallographic and Refinement Data for
¢-D-Psicopyranose
Formula
C6H12O6
180.16
Orthorhombic
P212121 (#19)
25
Formula weight
Crystal system
Space group
T/°C
X-ray Diffraction Analysis. The X-ray diffraction study
was performed with a colorless block crystal (0.20 © 0.20 ©
3
0
.20 mm ) using a Rigaku AFC7S diffractometer with graphite
Wavelength/¡
0.71069
1.44
monochromated Mo K¡ radiation. The crystal structure was
¹1
®
(Mo K¡)/cm
16
solved by the direct methods SIR92 and expanded using
a/¡
b/¡
c/¡
V/¡3
Z
8.693(2)
11.185(4)
7.745(2)
753.1(3)
4
1
7
Fourier techniques. Non-hydrogen atoms were refined aniso-
tropically and hydrogen atoms were refined using the riding
1
8
model. The final cycle of full-matrix least-squares refinement
on F was based on 1105 observed reflections (I > 3.00·(I))
with the empirical absorption corrections, and 122 variable
parameters converged (largest parameter shift was 0.00 times
¹3
Calculated density/g cm
Reflections collected
1.589
6914
Independent reflections (Rint = 0.080)
Observed reflections (I > 3·(I))
1236
1105
its esd) with unweighted (weighted) agreement factor; R =
1
0
.043 (wR = 0.044).
2
R1 (observed data) = ­(«Fobs« ¹ «Fcalc«)/­«Fobs« 0.043
Crystallographic data have been deposited with Cambridge
2
2 1/2
wR2 = [­w(«Fobs« ¹ «Fcalc«) /­wFobs ]
0.044
Crystallographic Data Centre: Deposition number CCDC-
76516. Copies of the data can be obtained free of charge
7
via http://www.ccdc.cam.ac.uk/conts/retrieving.html (or from
the Cambridge Crystallographic Data Centre, 12, Union Road,
Cambridge, CB2 1EZ, U.K.; Fax: +44 1223 336033; e-mail:
deposit@ccdc.cam.ac.uk).
Solubility. To estimate solubility of Psi in the temperature
range 0 to 50 °C, Psi crystal was finely smashed, mixed with
pure water in a glass vial, and stored in a thermostat water bath
(Lauda RMS6) with occasional stirring for at least five days.
After the dissolution equilibrium was attained, Psi content in
supernatant, which corresponded to aqueous solubility, was
estimated by means of densitometry based on the predeter-
mined calibration curve for the relation between Psi content
and solution density. For the accurate density measurements of
aqueous Psi, an oscillating tube density meter (Anton Paar
DMA5000, Gratz) was employed. The thermostat water bath
was then set at a higher temperature and new dissolution
equilibrium was attained within another five days.
Figure 2. Molecular structure and atom labeling of
-D-psicopyranose in single crystal.
¢
Table 2. Geometric Parameters for Non-Hydrogen Atoms in
-D-Psicopyranose
¢
Mutarotation. The time course of the optical rotation at
89 nm (Na D-line) for the Psi solution was monitored after the
Bond length
Bond angle
5
Distance
Angle
/degree
Atom Atom
Atom Atom Atom
dissolution of the sugar powder in water employing a digital
spectropolarimeter (Jasco P-1010, Tokyo). Psi crystals were
finely crushed for prompt dissolution (within 10 s), and the
solution was introduced into a 1-dm path length quartz cell
equipped with circulating water jacket for temperature control-
ling. Measurements were performed at 15, 25, 35, and 45 °C.
The absorbance at 280 nm was also monitored employing an
UV-VIS spectrometer (Hitachi U-1800, Tokyo) with a 1-cm
path length quartz cell at 25 °C to see the development of the
open-chain carbonyl-form after dissolution.
/¡
O(1)
O(2)
O(3)
O(4)
O(5)
O(6)
O(6)
C(1)
C(2)
C(3)
C(4)
C(5)
C(1)
C(2)
C(3)
C(4)
C(5)
C(2)
C(6)
C(2)
C(3)
C(4)
C(5)
C(6)
1.433(4)
1.411(3)
1.432(3)
1.424(4)
1.426(4)
1.423(4)
1.435(4)
1.517(4)
1.542(4)
1.519(4)
1.517(4)
1.511(4)
O(1)
O(2)
O(2)
O(2)
O(3)
O(3)
O(4)
O(4)
O(5)
O(5)
O(6)
O(6)
C(6)
O(6)
C(1)
C(2)
C(3)
C(4)
C(1)
C(2)
C(2)
C(2)
C(3)
C(3)
C(4)
C(4)
C(5)
C(5)
C(2)
C(2)
O(6)
C(6)
C(2)
C(3)
C(4)
C(5)
C(2)
O(6)
C(1)
C(3)
C(2)
C(4)
C(3)
C(5)
C(4)
C(6)
C(1)
C(3)
C(2)
C(5)
C(3)
C(4)
C(5)
C(6)
109.1(2)
107.3(2)
110.8(2)
109.5(2)
111.2(2)
112.0(2)
110.4(2)
113.0(2)
106.4(2)
112.5(2)
104.6(2)
110.1(2)
115.3(2)
111.1(2)
114.2(2)
109.6(2)
111.3(2)
109.6(3)
Results and Discussion
Molecular Geometry. Crystallographic data and refine-
ment parameters of the X-ray analysis are presented in Table 1.
The perspective view of the Psi molecule obtained from the
X-ray analysis is illustrated in Figure 2 and bond lengths
and bond angles for non-hydrogen atoms are summarized
in Table 2. Although Psi in aqueous solution consists of five
tautomers, ¡-furanose, ¢-furanose, ¡-pyranose, ¢-pyranose,

K. Fukada et al.
Table 3. Summary of Single Crystal Structure of Ketohexoses
Bull. Chem. Soc. Jpn. Vol. 83, No. 10 (2010) 1195
Orientation of OH groups
Unit cell
volume/¡3
Sugar
Ring structure
Reference
C2
C3
C4
C5
D-Psicose
D-Fructose
L-Sorbose
D-Tagatose
¢-Pyranose (1C)
¢-Pyranose (1C)
¡-Pyranose (1C)
¡-Pyranose (C1)
axial
axial
axial
axial
axial
equatorial
equatorial
axial
equatorial
equatorial
equatorial
equatorial
axial
axial
equatorial
equatorial
753.1
747.0
745.9
722.8
this work
10, 11
13
12
b
a
o
a
o
c
c
Figure 3. Molecular packing of ¢-D-psicopyranose in the unit cell (left) and the projected image along the b axis on the a­c plane
right).
(
and carbonyl-form, Psi crystallized solely as ¢-D-pyranose with
1
600
500
1
C ( C (D)) conformation. Figure 2 shows the six-membered
4
pyranose ring with three axial OH groups at C2-, C3-, and C5-
positions and one equatorial OH at C4-position. As had been
suggested with ¡-ribopyranose,9 the ring conformation is
stabilized by intramolecular hydrogen bonding between O(3)H
and O(5). The distance between O(3)£O(5) and H(6)£O(5) is
D-Fructose
4
3
2
00
00
00
2
.719 and 2.035 ¡, respectively. The crystal system (ortho-
D-Psicose
rhombic), space group (#19, P2 2 2 ), and number of mole-
1
1 1
cules per unit cell (Z = 4) are the same as those for ¢-D-
fructopyranose, ¡-L-sorbopyranose, and ¡-D-tagatopyranose.
In Table 3, molecular conformation of the four ketohexoses in
the crystal is summarized.
The molecular packing of Psi in the unit cell and the
projected image along the b axis on the a­c plane is shown
in Figure 3. One can see that the pyranose ring planes are
almost parallel to the a­c plane as reported for ¢-D-fructopyra-
nose. The lattice parameters of Psi are close to those of
100
0
0
10
20
30
40
50
temperature / °C
Figure 4. Solubility of D-psicose in water as a function of
temperature. Literature data of aqueous solubility for
anhydrous D-fructose is indicated by the broken line.
¢
-D-fructopyranose (a = 8.088, b = 9.204, c = 10.034 ¡, V =
3
7
46.95 ¡ ), but the b axis for Psi is slightly longer (Table 1).
Steric hindrance by the axial OH at C3-position is a possible
cause for the elongation of the b axis.
From the measurements of the X-ray crystal orientation
using a rod-shaped crystal of Psi, it was confirmed that the b
axis was almost in parallel with the longitudinal direction of the
crystal. The indexes of the crystal side planes were (101) and
was cracked and the resulting prism-shape had indexes of (120)
ꢀ
ꢀ
and (120), respectively.
Aqueous Solubility.
Figure 4 shows the temperature
dependence of the aqueous solubility of Psi and the literature
ꢀ
ꢀ
ꢀ ꢀ
19
(
101), or (101) and (101), respectively. The end of the crystal
data for the solubility of Fru. It can be seen that Psi is highly

1196 Bull. Chem. Soc. Jpn. Vol. 83, No. 10 (2010)
Crystal Structure and Solubility of D-Psicose
0
0
.3
.2
(b)
0
20
40
60
80
-
-
-
-
0
.8
α
0.4
0
(a)
0.1
260 300 340 380 420 460 500
λ
/ nm
0
500
1000
time / s
Figure 5. Time course of optical rotation at 589 nm (a) and absorbance at 280 nm (b) after dissolution of D-psicose in water
1500
0
500
1000
1500
time / s
(
10 wt %). Temperature is 15 ( ), 25 ( ), 35 ( ), and 45 °C ( ), respectively. Nonlinear least-squares fitting parameters for (a) are
¹3 ¹1
summarized in Table 4 and for (b): abs(t) = 0.28­0.19 ꢂ exp(¹4.44 © 10
solution of D-psicose (30 wt %).
s
ꢂ t). Inset in (b) is the UV spectrum for aqueous
Table 4. Nonlinear Least-Squares Fitting Parameters for the
that [¡] for ¢-D-psicopyranose in water may be ca. ¹85
¹
1
¹1
3
Time Course of [¡] after Dissolution of Psi in Water;
deg dm
g
cm . However, caution is necessary for this
a)
[
¡](t) = [¡]¨ + ([¡]0 ¹ [¡]¨)exp(¹k ꢂ t)
evaluation of [¡] since the ¡- and ¢-configurations for a
reducing sugar frequently coexist in the same crystal structure
and X-ray diffraction analysis cannot accurately detect compo-
¹1
[¡]¨/deg dm¹1 g¹1 cm3
Temperature/°C
k/ms
1
2
3
4
5
5
5
5
1.27
3.12
5.92
3.4
4.6
5.7
8.1
9
nents that make up less than 10% of the crystal.
It is interesting to show the time course of absorbance at
2
80 nm after dissolution of D-psicose (Figure 5b) since the
12.2
absorbance at this wavelength can only be attributed to open-
chain carbonyl-form in the five tautomers. The data points in
Figure 5b were well fitted by a single-exponential equation
a) [¡]0 was estimated to be ¹85 deg dm g¹1 cm3.
¹1
¹
1
soluble in water; at 25 °C (50 °C), 291 g (489 g) of Psi can be
dissolved in 100 g water to give a 74 wt % (83 wt %) solution
having a rate constant, 4.44 ms , which was faster than that
¹1
for mutarotation at 25 °C (3.12 ms ). Figure 6 illustrates
the multiple equilibria between the five tautomers of Psi in
solution. The kinetic behavior for the concentration of chain-
form, [chain], can be described by the following equation;
¹
3
with density of 1.35 g cm . This graph provides us the basic
information for the recrystallization recipe of Psi from aqueous
solution. Solubility of the anhydrous crystal of Fru is higher
than Psi as shown in Figure 4. It is known that the dihydrate of
½
chainꢀ ¼ ½chainꢀ1 þ ð½chainꢀ ꢁ ½chainꢀ Þ exp½ꢁðkþ þ kꢁÞtꢀ
0
1
Fru can be formed below 20 °C, which is supposed to be Fru’s
thermodynamically stable state.19 At the present stage, no
hydrated crystal of Psi has been confirmed.
ð1Þ
represent equilibrium and
where subscript
¨
and
0
Mutarotation.
Finally we investigated mutarotation
initial values, respectively, k = k + k + k + k , and k =
+ 1 2 3 4 ¹
recording the time course of specific rotation [¡] at 589 nm
after the dissolution of D-psicose in water (10 wt %). To our
best knowledge, mutarotation of Psi has not been reported
so far. Figure 5a shows the time course of [¡] at various
temperatures and the nonlinear least-squares fitting curves by
single-exponential equations (Table 4). When temperature was
elevated, significant acceleration of mutarotation and an
increase of the equilibrium value of specific rotation, [¡]¨,
k¹1 + k¹2 + k¹3 + k . Referring to the fitting parameters in
¹4
¹
1
Figure 5b, k+ + k¹ should be 4.44 ms at 25 °C. Interpretation
of the rate constant for mutarotation, on the other hand, is not as
simple as UV absorbance kinetics since all five tautomers
contribute to the specific rotation and the molecular rotatory
power of each tautomer is not yet known. Note that the fitting
curve in Figure 5b started from 0.09 at t = 0 suggesting a
contamination with the absorbance at this wavelength in the
crystal. Subtracting the absorption by the contamination and
assuming the equilibrium content of the carbonyl-form at 25 °C
were confirmed while the initial value, [¡] , did not change
0
with temperature. These results suggest ¢-D-psicopyranose in
crystal first dissolves in water without configuration change
and then equilibrates gradually to a mixture of tautomers
at a temperature dependent rate constant. The equilibrium
ratio of tautomers is also temperature dependent. It is thought
2
0
as 0.2%, the molar absorption coefficient at ­max = 280 nm,
¾, for the open-chain carbonyl-form of Psi was estimated to be
¹
1
¹1
160 cm M , which was approximately ten (eight) times
larger than that of ¾ for aqueous acetone with ­max = 265 nm

K. Fukada et al.
Bull. Chem. Soc. Jpn. Vol. 83, No. 10 (2010) 1197
T. B. Granström, G. Takata, M. Tokuda, K. Izumori, J.
2
in crystal
Biosci. Bioeng. 2004, 97, 89.
OH
3
4
K. Izumori, J. Biotechnol. 2006, 124, 717.
H. Yoshida, M. Yamada, T. Nishitani, G. Takada, K.
Izumori, S. Kamitori, J. Mol. Biol. 2007, 374, 443.
T. Matsuo, K. Izumori, Biosci. Biotechnol. Biochem. 2006,
70, 2081.
O
CH OH
2
dissolution in water
OH
OH
HO
5
β-D-psicopyranose (1C)
6
M. Sato, H. Kurose, T. Yamasaki, K. Izumori, J. Nat. Med.
OH
O
2008, 62, 244.
CH OH
2
O
7
Y. Sun, S. Hayakawa, M. Ogawa, K. Fukada, K. Izumori,
CH OH
2
OH
k1
J. Agric. Food Chem. 2008, 56, 4789.
k2
OH
HO
OH
8
Data Book of Biochemistry I, ed. by The Japanese
HO
OH
k-1
OH
Biochemical Society, Tokyo-Kagaku-Dohjin, Tokyo, 1979,
p. 427.
OH
α-D-psicopyranose
22 %)
O
k-2
HO
HO
β-D-psicopyranose
9
G. A. Jeffrey, W. Saenger, Hydrogen Bonding in Biological
Structures, Springer, Berlin, 1991, Chap. 13.
10 J. A. Kanters, G. Roelofsen, B. P. Alblas, I. Meinders, Acta
OH
(
(24 %)
k4
k3
HO
O
Crystallogr., Sect. B 1977, 33, 665.
11 S. Takagi, G. A. Jeffrey, Acta Crystallogr., Sect. B 1977,
O
OH
OH
OH
OH
chain-form
0.2 %)
k-4
k-3
HO
HO
(
3
3, 3510.
S. Takagi, R. D. Rosenstein, Carbohydr. Res. 1969, 11, 156.
13 S. H. Kim, R. D. Rosenstein, Acta Crystallogr. 1967, 22,
648.
HO
β-D-psicofuranose
15 %)
OH
HO
α-D-psicofuranose
39 %)
OH
1
2
(
(
1
01.
4
H. Itoh, T. Sato, K. Izumori, J. Ferment. Bioeng. 1995, 80,
Figure 6. Illustration for molecular structures of D-psicose
in crystal and in aqueous solution. Equilibrium contents
of each tautomer at 27 °C are indicated according to the
literature.20
1
1
5
K. Takeshita, A. Suga, G. Takada, K. Izumori, J. Biosci.
Bioeng. 2000, 90, 453.
A. Altomare, G. Cascarano, C. Giacovazzo, A. Guagliardi,
M. C. Burla, G. Polidori, M. Camalli, J. Appl. Cryst. 1994, 27,
35.
1
6
1,3-dihydroxy-2-propanone with ­max = 270 nm).21 The ob-
(
4
served increase of extinction coefficient and the red-shift for the
n ¼ ³* transition of C=O group may be attributed to the more
extensive ³* molecular orbital in the open-chain Psi molecule
compared to acetone.
1
7
P. T. Beurskens, G. Admiraal, G. Beurskens, W. P. Bosman,
R. de Gelder, R. Israel, J. M. M. Smits, The DIRDIF-99 Program
System, Technical Report of the Crystallography Laboratory,
University of Nijmegen, The Netherlands, 1999.
1
8
Least-squares function minimized: ­w(«Fobs« ¹ «Fcalc«)2
This work was financially supported by the CITY AREA
program by the Ministry of Education, Culture, Sports, Science
and Technology of Japan (MEXT).
where w is the least-squares weight.
F. E. Young, F. T. Jonses, H. J. Lewis, J. Phys. Chem. 1952,
6, 1093.
R. N. Goldberg, Y. B. Tewari, J. Phys. Chem. Ref. Data
989, 18, 809.
G. Avigad, S. Englard, I. Listowsky, Carbohydr. Res. 1970,
14, 365.
1
9
5
2
0
1
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1
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