Conversion of D-galactose to D-threo-hexos-2,3-diulose by fungal pyranose oxidase

Article abstract of DOI:10.1081/CAR-120021701

Pyranose oxidase (pyranose:O₂ 2-oxidoreductase, EC 1.1.3.10) purified from mycelia of the basidiomycete fungi Trametes versicolor and Oudemansiella mucida catalyzed oxidation of D-galactose successively at C-2 and C-3 to D-threo-hexos-2,3-diulo

Full text of DOI:10.1081/CAR-120021701

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Conversion of d‐Galactose to
d‐threo‐Hexos‐2,3‐diulose by Fungal Pyranose
Oxidase
Jind[rcheck]ich Volc , Petr Sedmera , Petr Halada , Padmanabh Dwivedi &
Maria Costa‐Ferreira
To cite this article: Jind[rcheck]ich Volc , Petr Sedmera , Petr Halada , Padmanabh Dwivedi &
Maria Costa‐Ferreira (2003) Conversion of d‐Galactose to d‐threo‐Hexos‐2,3‐diulose by
Fungal Pyranose Oxidase, Journal of Carbohydrate Chemistry, 22:3-4, 207-216, DOI: 10.1081/
CAR-120021701
To link to this article: http://dx.doi.org/10.1081/CAR-120021701
Published online: 28 Oct 2011.
Submit your article to this journal
Article views: 12
View related articles
Citing articles: 8 View citing articles
Full Terms & Conditions of access and use can be found at
http://www.tandfonline.com/action/journalInformation?journalCode=lcar20
Download by: [Central Michigan University]
Date: 15 October 2015, At: 09:16

JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, Nos. 3 & 4, pp. 207–216, 2003
Conversion of D-Galactose to D-threo-Hexos-2,3-diulose
by Fungal Pyranose Oxidase
1,*
1
Jind rˇ ich Volc, Petr Sedmera, Petr Halada, Padmanabh Dwivedi,
1
1,
#
2
and Maria Costa-Ferreira
1
Institute of Microbiology, Academy of Sciences of the Czech Republic,
Czech Republic
2
Departamento de Biotecnologia, Instituto Nacional de Engenharia e Tecnologia
Industrial (INETI), Lisboa Codex, Portugal
ABSTRACT
Pyranose oxidase (pyranose:O 2-oxidoreductase, EC 1.1.3.10) purified from mycelia
2
of the basidiomycete fungi Trametes versicolor and Oudemansiella mucida catalyzed
oxidation of D-galactose successively at C-2 and C-3 to D-threo-hexos-2,3-diulose
(
2,3-dehydro-D-galactose, 2,3-diketo-D-galactose) in the yields up to 80%. The sites of
oxidation were deduced from structures of the N,N-diphenylhydrazone derivatives of
the reaction products. Under the reaction conditions used, the diulose was susceptible
to non-enzymatic oxidative decarboxylation to D-threo-pentos-2-ulose (2-dehydro-D-
xylose, 2-keto-D-xylose) in yields of 5–10%.
Key Words: D-Galactose; D-lyxo-Hexos-2-ulose; D-threo-Hexos-2,3-diulose;
Pyranose dehydrogenase; Pyranose oxidase; Trametes versicolor; Oudemansiella
mucida.
*
Correspondence: Jind rˇ ich Volc, Institute of Microbiology, Academy of Sciences of the Czech
Republic, Videnska 1083, 142 20 Prague 4, Czech Republic; Fax: +420-2-4106-2384; E-mail:
volc@biomed.cas.cz.
#
Current address: Department of Botany, Arunachal University, Rono Hills, Itanagar, India.
2
07
DOI: 10.1081/CAR-120021701
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

208
Volc et al.
INTRODUCTION
Highly reactive carbonyl sugar derivatives are gaining in importance in car-
bohydrate chemistry as intermediates or synthons in procedures leading to useful
[
1]
products and rare sugars. This is now also due to their easier accessibility through
enzymatic conversions of common sugars by employing stereoselective sugar oxi-
doreductases, such as fungal pyranose oxidase (P2O, pyranose:O 2-oxidoreductase, EC
2
[
2]
[3]
1
.1.3.10)
and pyranose dehydrogenase.
These compounds have high potential
applicability as intermediates in the isomerizations of aldoses to ketoses. P2O shows
a broad substrate tolerance, acting on C-2 equatorial hydroxyls of some aldopyra-
noses. Its activity at C-3 of several substrates has also been recognized.
far characterized from different sources, oxidize D-galactose with activities ranging
[
2]
[
2,4]
[5,6]
P2Os, so
[7–9]
from 0.9 to 8.8% relative to preferred D-glucose (100%). The corresponding re-
action product was identified as D-lyxo-hexos-2-ulose (Scheme 1, 1; 2-dehydro-D-ga-
lactose). This compound is attractive due to its intermediate role in the proposed
(chemo)enzymatic processes for production of D-tagatose from D-galactose. How-
[
4,10]
[
10,11]
ever, our previous observations indicated that P2O of some fungi may exhibit modified
catalytic properties towards D-galactose, resulting in its further transformation.
In this paper we demonstrate that P2O purified from Trametes versicolor and
Oudemansiella mucida converts D-galactose, upon prolonged incubations, to D-threo-
hexos-2,3-diulose (Scheme 1, 2; 2,3-didehydro-D-galactose), a new sugar derivative with
three adjacent carbonyl groups. Its structure was determined spectroscopically using the
corresponding diphenylhydrazone derivatives.
RESULTS AND DISCUSSION
Time Course of D-Galactose Oxidation
The HPLC analyses of sugar components during the course of D-galactose trans-
formation carried out on an analytical scale using homogeneous P2O from T. versicolor
revealed that 1 (Figure 1, peak III, T= 15.85 min) accumulated in the reaction mixture
only transiently being in turn converted to a final product (peak I, T =7.95 min). The
latter compound was eluted near the column void volume as observed earlier for the
[12]
end product of D-glucose double oxidation. The similar chromatographic pattern was
also obtained for products of D-galactose transformation by P2O partially purified from
mycelia of O. mucida (not shown). These enzyme activities were in contrast with high
[
11]
C-2 regioselectivity of P2O from T. multicolor
quinone-dependent flavoglycoprotein from Agaricus bisporus,
and pyranose dehydrogenase, a
[12]
which oxidized
D-galactose almost quantitatively to 1. The enzyme source-dependent variability in
catalytic regioselectivity has already been demonstrated before with pyranose dehy-
[
drogenase and D-glucose.
13]
Structure Elucidation
To determine spectroscopically the site of the second oxidation of D-galactose
by P2O, the products in the semipreparative reaction mixture were treated with

Conversion of D-Galactose
209
Figure 1. (A)–(E) HPLC monitoring of D-galactose oxidation by pyranose 2-oxidase from
T. versicolor at incubation times of 0, 1.5, 4 and 20 h, respectively. Peaks: I, D-threo-hexos-2,3-
diulose; II, D-galactose; III, D-lyxo-hexos-2-ulose; the peak I at (A) corresponds to the salt
originating from the enzyme preparations added to the reaction mixture.
Figure 2. Structures and FAB-MS fragmentation of N,N-diphenylhydrazone derivatives 4–9 used
for identification of the oxidation products of D-galactose by pyranose oxidase.

210
Volc et al.
N,N-diphenylhydrazine and the diphenylhydrazone (DPH) compounds formed were
isolated using semipreparative TLC. This step substantially simplified the NMR
measurements by converting multiple (bi)cyclic isomers of the individual free
oxidation products to the mostly ‘linear’ structures, the only source of diversity
being then the extent of derivatization of carbonyl groups.
+
The compound displaying [M +H] ion at m/z 345, containing one DPH moiety,
one carbonyl group, and a contiguous five-spin system -[CH(OH)] CH OH was as-
signed the structure of mono-DPH derivative (4, Figure 2) of D-lyxo-hexos-2-ulose. The
3
2
+
next compound with [M +H] at m/z 511, contained two DPH units and a similar five-
spin system. It was therefore assigned the structure of bis-DPH derivative (5) of D-lyxo-
+
hexos-2-ulose. Both compounds 6 and 7 exhibited [M +H] ions at m/z 509, contained
two DPH units each, one free carbonyl per molecule, and a contiguous four-spin
1
system in their H NMR spectra (Table 1). The localization of the free carbonyl group
(Table 2) was determined by HMBC: at C-3 in 6 and at C-2 in 7. These two compounds
were DPH derivatives of the parent D-threo-hexos-2,3-diulose (2, Scheme 1). The
+
compound exhibiting [M +H] ion at m/z 675 contained three DPH units, no free
carbonyl, and a similar proton spin system to those described above, clearly consistent
1
Table 1. H NMR data of compounds 4–9 (399.91 MHz, CD
3
OD, 30°C).
Proton/J(i,j)
4
5
6
7
8
9
H-1
H-3
6.681
5.406
7.2
7.095
5.173
8.0
6.450
–
6.030
–
6.107
–
7.038
5.244
2.9
J
H-4
3,4
4.018
1.8
4.306
1.7
5.440
2.1
4.698
4.1
4.966
2.7
4.181
6.6
J4,5
6
.0*
3.736
.847
11.0**
H-5
3.908
4.114
4.266
4.077
4.178
3
J
J
5,6d
5,6u
6.0
6.6
6.4
6.4
6.4
6.7
5.5
6.4
6.2
6.2
H-6u
H-6d
3.634
3.667
3.728
3.728
–
3.679
3.774
11.0
3.700
3.824
11.1
3.795
3.885
11.0
J
6d,6u
11.0
Additional signals - 4: 7.245 (4 H, m, ortho-Ph), 7.340 (2 H, m, para-Ph), 7.499 (4 H, m, meta-Ph);
: 6.896 (4 H, m, ortho-Ph ), 6.915 (4 H, m, ortho-Ph ), 7.037 (2 H, m, para-Ph ), 7.220 (4 H, m,
meta-Ph ), 7.237 (4 H, m, meta-Ph ),; 6: 6.823 (4 H, m, ortho-Ph ), 7.086 (4 H, m, ortho-Ph ),
5
2
1
1
1
2
1
2
7
.169 (2 H, m, para-Ph
1
), 7.194 (2 H, m, para-Ph
2
), 7.312 (4 H, m, meta-Ph ), 7.333 (4 H, m, meta-
2
1 1 1 2
Ph ); 7: 7.069 (4 H, m, ortho-Ph ),7.086 (2 H, m, para-Ph ), 7.165 (4 H, m, ortho-Ph ), 7.249 (2 H,
m, para-Ph ), 7.266 (4 H, m, meta-Ph ), 7.381 (4 H, m, meta-Ph ); 8: 6.696 (4 H, m, ortho-Ph ),
2
2
1
2
6.728 (4 H, m, ortho-Ph
ortho-Ph
1
), 7.022 (2 H, m, para-Ph ), 7.037 (2 H, m, para-Ph
3
2
), 7.054 (4 H, m,
3
), 7.166 (2 H, m, para-Ph
1
), 7.169 (4 H, m, meta-Ph ), 7.204 (4 H, m, meta-Ph
2
3
), 7.281
(
Ph ), 7.237 (2 H, m, para-Ph ), 7.243 (4 H, m, meta-Ph ), 7.390 (4 H, m, meta-Ph ).
4 H, m, meta-Ph
1
); 9: 6.887 (4 H, m, ortho-Ph
2
1
), 6.915 (4 H, m, ortho-Ph ), 7.048 (2 H, m, para-
2
1
7
1
*J
4,5u
.
**J5d,5u; d, downfield; u, upfield.

Conversion of D-Galactose
211
1
3
Table 2.
C NMR data of compounds 4–9 (100.57 MHz, CD OD, 30°C).
3
Carbon
4
5
6
7
8
9
1
2
3
4
5
6
133.46
201.05
73.52
73.57
72.40
64.68
130.19
162.91
73.05
74.08
72.37
65.14
128.31
141.82
201.04
75.41
134.42
194.46
155.45
75.04
129.28
143.90
161.54
75.91
128.76
161.01
71.58
75.47
64.95
74.87
64.60
74.49
64.45
74.46
64.57
Chemical shifts of free keto group are given in bold.
Additional signals -4: 123.86 d (4 C, ortho-Ph), 128.09 d (2 C, para-Ph), 131.44 d (4 C, meta-Ph),
43.82 s (2 C, ipso-Ph); 5: 123.09 d (4 C, ortho-Ph ), 123.88 d (4 C, ortho-Ph ),124.76 d (2 C,
ortho-Ph ), 126.91 d (2 C, para-Ph ), 130.49 d (4 C, meta-Ph ), 131.34 d (4 C, meta-Ph ), 144.06 s
2 C, ipso-Ph ), 150.38 s (2 C, ipso-Ph ); 6: 123.95 d (4 C, ortho-Ph ), 124.19 d (4 C, ortho-Ph ),
1
2
1
2
1
2
1
(
1
2
1
2
1
Ph
26.49 d (2 C, para-Ph ), 127.21 d (2 C, para-Ph ), 130.86 d (4 C, meta-Ph ), 131.01 d (4 C, meta-
1
2
2
1
), 144.06 s (2 C, ipso-Ph
1
), 147.71 s (2 C, ipso-Ph ); 7: 124.31 d (4 C, ortho-Ph ), 124.54 d (4 C,
2 2
ortho-Ph ), 125.82 d (2 C, para-Ph ), 127.82 d (2 C, para-Ph ), 130.34 d (4 C, meta-Ph ), 131.47 d
1 1 2 1
(
4 C, meta-Ph ), 148.37 s (4 C, ipso-Ph); 8: 123.50 d (4 C, ortho-Ph ), 123.66 d (4 C, ortho-Ph ),
3
2
2
1
1 2
23.99 d (4 C, ortho-Ph ), 125.02 d (2 C, para-Ph3), 125.91 d (2 C, para-Ph ), 126.72 d (2 C, para-
1 3 2 1
Ph ), 130.22 d (4 C, meta-Ph ), 130.64 d (4 C, meta-Ph ), 131.13 d (4 C, meta-Ph ), 148.20 s (2 C,
ipso-Ph ), 148.78 s (2 C, ipso-Ph ), 148.92 s (2 C, ipso-Ph ); 9: 122.97 d (4 C, ortho-Ph ), 123.74 d
3
1
2
2
(
4 C, ortho-Ph
1
), 124.87 d (2 C, para-Ph
2 1 2
), 127.03 d (2 C, para-Ph ), 130.56 d (4 C, meta-Ph ),
1
1 1 2
31.41 d (4 C, meta-Ph ), 144.80 s (2 C, ipso-Ph ), 150.42 s (2 C, ipso-Ph ).
with structure 8 (Figure 2), a tris-DPH derivative of 2. The last reaction product of
[
+
M +H] ion m/z 481 contained two DPH units, had only five carbon atoms derived
from the parent sugar and its contiguous spin system contained also four protons.
Therefore, this compound was a bis-DPH derivative (9, Figure 2) of a product of 2
oxidative decarboxylation.
P2O Catalysis with D-Galactose and Related P2O Activities
Based on the structures of free di- and tricarbonyl sugars deduced from the above
identified hydrazones and HPLC monitoring of the time course of P2O catalysis, we
Scheme 1. Reaction scheme for double oxidation of D-galactose by pyranose oxidase from
Trametes versicolor and Oudemansiella mucida. D-Galactose is successively oxidized at C-2 and
C-3 to diketoaldose 2 (D-erythro-hexos-2,3-diulose), which is apparently susceptible to non-
enzymatic oxidative decarboxylation giving 3 (D-threo-pentos-2-ulose) via a keto/enol intermediate
(not shown). 1, intermediate D-lyxo-hexos-2-ulose; for simplicity, only one of the possible equi-
librium cyclic forms is shown for 1 and 2, carbonyl hydration also takes place.

212
Volc et al.
conclude that D-galactose is transformed by P2O and molecular oxygen to ketoaldose
1
2
, which is subsequently oxidized by the same enzyme at C-3 to give the diketoaldose
(Scheme 1). It is assumed that prior to the second P2O-catalyzed step, 1 is hydrated
at C-2 carbonyl or the 1,5-pyranose isoform of 1 is spontaneously recycled into 2,6-
[
10]
pyranose hydrated at the C-1 carbonyl.
The ability of fungal sugar oxidoreductases to catalyse C-2,3 double oxidation
[6]
of aldoses was first demonstrated for P2O from O. mucida and pyranose dehydro-
12]
[
genase of Agaricus bisporus
meric to 2. Pyranose dehydrogenase of A. meleagris was shown to catalyse C-2,3
with D-glucose giving D-erythro-hexos-2,3-diulose epi-
[14]
oxidation of D-xylose to another tricarbonyl derivative, D-erythro-pentos-2,3-diulose.
To our knowledge, none of the aldos-2,3-diuloses exhibiting three adjacent carbonyl
groups have been prepared to date by means of carbohydrate chemistry and this is the
first report describing enzymatic formation of 2. The search of Chemical Abstracts
database for double oxidation products of D-galactose revealed one item, D-lyxo-hex-
2-ulosonic acid. However, its formation involves an oxidation at C-1 and C-2. Similar
C-2,3 dioxidation by P2O (Peniophora gigantea) was also observed with 1,5-anhydro-
D-glucitol and 1,5-anhydro-D-galactitol yielding the corresponding 1,5-anhydro-hex-2,3-
[
diuloses.
5]
Oxidative decarboxylation of 2 to D-threo-pentos-2-ulose (3, Scheme 1, 2-dehydro-
D-xylose) was inferred from the structure of the isolated hydrazone 9. Formation of this
by-product in yields of 5–10% was dependent on intensity of aeration of the reaction
mixture. Analogous degradative reaction was earlier shown for C-3 carbonyl deriva-
[
5]
[14]
tives of 2-deoxy-D-glucose and D-glycero-pentos-2,3-diulose.
In the semipreparative transformations described, D-galactose was converted by
T. versicolor or O. mucida P2O to 2 in yields approaching 80% (HPLC analysis). In
contrast with D-erythro-hexos-2,3-diulose previously isolated also in a crystalline
[
15]
form,
reaction mixture. The present study extends our earlier findings
we were unsuccessful in crystallizing 2 from the ultrafiltrate of a completed
demonstrating
[6,12,16]
high potential of pyranose oxidoreductases for conversions of various sugars to their
reactive di- and tricarbonyl derivatives, possible building blocks in synthetic carbo-
hydrate chemistry. In view of the earlier designed enzymatic processes for isome-
[10,11]
rization of D-galactose to D-tagatose,
our present findings point to the importance
of the fungal source of P2O, i.e., the enzyme of choice should have no or minimum
activity for the C-3 oxidation.
EXPERIMENTAL
Enzyme Source, Purification, and Assay
Fungi Trametes versicolor, CCBAS 614 and Oudemansiella mucida, CCBAS 428
were obtained from the Culture Collection of Basidiomycetes at the Institute of Mic-
robiology, Prague. The fungi were grown under submerged conditions on a complex
[
17]
[6]
medium
mucida). T. versicolor P2O was purified from mycelial extracts to apparent homo-
for three days (T. versicolor) or a synthetic medium for four days (O.
geneity (39 U/mg protein) following substantially the three-step procedure described
[
17]
previously.
hydrophobic interaction chromatography.
P2O from O. mucida was partially purified by applying the first step,

Conversion of D-Galactose
213
The enzyme activities were assayed spectrophotometrically by measuring at 590
nm the production of H O in a coupled peroxidase/chromogen (3-methyl-2-benzo-
2
2
[
17]
thiazolinone hydrazone-3-dimethylaminobenzoic acid) system.
1
enzyme activity oxidizes D-glucose at 1 mmol min
One unit (U) of the
under the assay conditions.
ꢀ
P2O Catalyzed Transformation of D-Galactose
The analytical transformations of D-galactose were performed for 20 h at 30°C
under stirring with 13.5 mg (50 mM) D-galactose, 6.2 U purified P2O (T. versicolor
or O. mucida) and 350 Sigma Units of bovine liver catalase (Reanal, Budapest, Hun-
gary) in 10 mM bisTris HCl buffer, pH 7.2 (1.5 mL). Excess catalase was added in
order to prevent P2O deactivation and product decomposition by co-produced H O .
2
2
Time course of the reaction was followed by HPLC analysis of the samples (60 mL)
withdrawn at time intervals, passed through Ultrafree-MC 30,000 NMWL filter units
(
Millipore, Bedford, MA) and diluted 10 times with water. HPLC monitoring was
performed on an SP 8800 liquid chromatograph (Spectra Physics, San Jose, CA, USA)
2
+
fitted with a refractive index detector, using Ostion LG KS 0800 Ca column 250 x
8
mm (Watrex, Prague, Czech Republic), eluted at 80°C with deionized water (0.5 mL
ꢀ
1
min ). The reaction mixture (25 mL) for semipreparative transformations (for 28 h at
5°C under stirring) contained 225 mg D-galactose, 60 U P2O (T. versicolor), 2000
Sigma Units catalase added in three aliquots at 0, 6, and 14 h, and 0.03% ProClin 300
Supelco, Bellefonte, PA) antimicrobial preservative in 50 mM 2(N-morpholino)etha-
nesulfonic acid (MES), pH 7.0.
2
(
Derivatization of D-Galactose Oxidation Products
The semipreparative reaction mixture was vacuum concentrated to 15 mL, passed
through a YM 30 membrane (Amicon, Danvers, MA), and the filtrate was sup-
plemented with the same volume of ethanol and 366 mL of freshly distilled N,N-
diphenylhydrazine (Koch-Light Lab., Colnbrook, UK). After acidification (0.3 mL 99 %
acetic acid) and vigorous stirring for 2 h, the mixture was left standing overnight at
22°C and then the solvent removed by vacuum evaporation. The solid obtained was
extracted into 2 mL of methanol, most of crystalline MES being separated. The
hydrazones contained in the methanol fraction were isolated using TLC on Silica Gel
60 F254 aluminium sheets (Merck, Darmstadt, Germany) in 50:3 CHCl –MeOH and
3
UV/VIS detection. Pale-yellow to orange streaks of six major hydrazones were excised,
combined separately, and extracted into methanol. The individual hydrazones were
further purified by rechromatography and spectroscopically identified.
Spectral Analysis
1
13
H and C NMR spectra of the isolated N,N-diphenylhydrazones were measured
on a Varian INOVA-400 spectrometer in CD OD at 30°C. The residual solvent signals
3
were used as an internal standard (dH 3.33, dC 49.3). Carbon signal multiplicity was
determined by APT (Attached Proton Test). 2D NMR experiments (gCOSY, TOCSY,
HMQC, and HMBC) were performed using manufacturer’s software. Positive ion FAB
mass spectra were recorded on a Finnigan MAT 95 double focusing instrument using

214
Volc et al.
m-nitrobenzyl alcohol as a matrix. The saddle field FAB gun (Ion Tech, Teddington,
ꢀ
5
UK) was operated at 2 mA current and 6 kV energy; xenon (1 ꢁ 10
bar) was used as
a bombarding gas.
D-lyxo-Hexos-2-ulose 1-(N,N-diphenylhydrazone) (4). Compound of R 0.11,
f
rechromatographed using 50:1 CHCl –MeOH, and crystallized from EtOH, pale yellow
3
needles (27 mg), mp 148–149°C, UV/VIS: l
m/z (% rel. int.): 367 [M + Na] (72), 345 [M +H] (33), 327 (58), 254 (12), 223 (18),
(MeOH) 229, 247, 412 nm. FAB-MS,
+
max
+
1
95 (5), 183 (9), 168 (100), 121 (3).
D-lyxo-Hexos-2-ulose 1,2-bis(N,N-diphenylhydrazone) (5). R 0.28, rechroma-
f
tographed in 50:1 CHCl -MeOH, dark yellow syrup (45 mg). UV/VIS: l
(MeOH)
max
3
+
12, 293, 300, 344, 389 nm. FAB-MS, m/z (% rel. int.): 533 [M +Na] (29), 511
2
+
M +H] (26), 493 (1), 449 (2), 419 (5), 389 (1), 342 (4), 327 (6), 252 (7), 222 (14)
[
1
95 (6), 183 (7), 168 (100).
D-threo-Hexos-2,3-diulose 1,2-bis(N,N-diphenylhydrazone) (6). R 0.45, rechro-
f
matographed in 100:1 CHCl -MeOH, silica gel 60 foils, orange syrup (36 mg). UV/
3
+
VIS: lmax (MeOH) 210, 240, 288, 394 nm. FAB-MS, m/z (% rel. int.): 531 [M +Na]
(
+
48), 509 [M +H] (10), 508 (24), 491 (1), 389 (2), 340 (3), 325 (4), 222 (32), 195 (9),
1
83 (8), 168 (100).
D-threo-Hexos-2,3-diulose 1,3-bis(N,N-diphenylhydrazone) (7). R 0.39, rechro-
f
matographed using 100:1 CHCl -MeOH and aluminium foils with concentration zone,
3
deep yellow syrup (63 mg). UV/VIS: l
m/z (% rel. int.): 531 [M +Na] (62), 509 [M +H] (26), 508 (18), 491 (6), 447 (2),
(MeOH) 212, 239, 278, 357 nm. FAB-MS,
+
max
+
3
40 (2), 325 (3), 285 (1), 250 (3), 223 (47), 195 (6), 183 (7), 168 (100).
D-threo-Hexos-2,3-diulose 1,2,3-tris(N,N-diphenylhydrazone) (8). Rf 0.74,
(MeOH) 215,
max
rechromatographed in CHCl , orange solid (99 mg). UV/VIS: l
2
3
+
42, 289, 390 nm. FAB-MS, m/z (% rel. int.): 697 [M +Na] (12), 675 [M + H] (3),
+
6
13 (2), 506 (3), 491 (4), 446 (1), 415 (2), 323 (1), 222 (4), 195 (4), 168 (100).
D-threo-Pentos-2-ulose 1,2-bis(N,N-diphenylhydrazone) (9). R 0.54, rechroma-
f
tographed in 100:1 CHCl -MeOH, yellow syrup (15 mg). UV/VIS: l
(MeOH) 239,
max
3
+
94, 344, 388 nm. FAB-MS, m/z (% rel. int.): 503 [M +Na] (12), 481 [M + H] (10),
+
2
4
1
80 (15), 463 (2), 419 (3), 389 (2), 312 (5), 297 (7), 252 (5), 222 (11), 195 (7), 183 (6),
68 (100).
ACKNOWLEDGMENTS
This research was supported by grant AV0Z5020903 (Institutional Research Con-
cept). Financial support from UNESCO to Padmanabh Dwivedi during his International
Postgraduate Training Course at the Institute of Microbiology, Prague, is acknowledged.

Conversion of D-Galactose
215
REFERENCES
1.
2.
3.
4.
5.
6.
Giffhorn, F.; K o¨ pper, S.; Huwig, A.; Freimund, S. Rare sugars and sugar-based
synthons by chemo-enzymatic synthesis. Enzyme Microb. Technol. 2000, 27, 734–
42.
Giffhorn, F. Fungal pyranose oxidases: occurrence, properties and biotechnical
applications in carbohydrate chemistry. Appl. Microbiol. Biotechnol. 2000, 54,
7
727–740.
Volc, J.; Kub a´ tov a´ , E.; Wood, D.; Daniel, G. Pyranose 2-dehydrogenase, a novel
sugar oxidoreductase from the basidiomycete fungus Agaricus bisporus. Arch.
Microbiol. 1997, 167, 119–125.
Ruelius, H.W.; Kerwin, R.M.; Janssen, F.W. Carbohydrate oxidase, a novel enzyme
from Polyporus obtusus II. Specificity and characterization of reaction products.
Biochim. Biophys. Acta 1968, 167, 501–510.
Freimund, S.; Huwig, A.; Giffhorn, F.; K o¨ pper, S. Rare keto-aldoses from enzy-
matic oxidation: substrates and oxidation products of pyranose 2-oxidase. Chem.
Eur. J. 1998, 4 (12), 2442–2455.
Volc, J.; Sedmera, P.; Havl ´ı cˇ ek, V.; P rˇ ikrylov a´ , V.; Daniel, G. Conversion of
D-glucose to D-erythro-hexos-2,3-diulose (2,3-diketo-D-glucose) by enzyme pre-
parations from the basidiomycete Oudemansiella mucida. Carbohydr. Res. 1995,
278, 59–70.
7.
Danneel, H.J.; Rossner, E.; Zeeck, A.; Giffhorn, F. Purification and character-
ization of pyranose oxidase from the basidiomycete Peniophora gigantea and
chemical analyses of its reaction products. Eur. J. Biochem. 1993, 214, 795–
802.
8
.
.
Masuda-Nishimura, I.; Minamihara, T.; Koyama, Y. Improvement in thermal
stability and reactivity of pyranose oxidase from Coriolus versicolor by random
mutagenesis. Biotechnol. Lett. 1999, 21, 203–207.
Machida, Y.; Nakanishi, T. Purification and properties of pyranose oxidase from
Coriolus versicolor. Agr. Biol. Chem. 1984, 48, 2463–2470.
9
1
0. Freimund, S.; Huwig, A.; Giffhorn, F.; K o¨ pper, S. Convenient chemo-enzymatic
synthesis of D-tagatose. J. Carbohydr. Chem. 1996, 15 (1), 115–120.
1. Haltrich, D.; Leitner, C.; Neuhauser, W.; Nidetzky, B.; Kulbe, K.D.; Volc, J. A
convenient enzymatic procedure for the production of aldose free D-tagatose. Ann.
N.Y. Acad. Sci. 1998, 864, 296–313.
1
1
1
2. Volc, J.; Sedmera, P.; Halada, P.; P rˇ ikrylov a´ , V.; Daniel, G. C-2 and C-3 oxidation
of D-Glc, and C-2 oxidation of D-Gal by pyranose dehydrogenase from Agaricus
bisporus. Carbohydr. Res. 1998, 310, 151–156.
3. Volc, J.; Kub a´ tov a´ , E.; Daniel, G.; Sedmera, P.; Haltrich, D. Screening of basi-
diomycete fungi for the quinone-dependent sugar C-2/C-3 oxidoreductase, pyranose
dehydrogenase, and properties of the enzyme from Macrolepiota rhacodes. Arch.
Microbiol. 2001, 176, 178–186.
1
4. Volc, J.; Sedmera, P.; Halada, P.; P rˇ ikrylov a´ , V.; Haltrich, D. Double oxidation of
D-xylose to D-glycero-pentos-2,3-diulose (2,3-diketo-D-xylose) by pyranose dehy-
drogenase from the mushroom Agaricus bisporus. Carbohydr. Res. 2000, 329, 219–
225.
15. Sedmera, P.; Volc, J.; Havl ´ı cˇ ek, V.; Pakhomova, S.; Jegorov, A. Structural analysis

216
Volc et al.
of crystalline D-erythro-hexos-2,3-diulose (2,3-diketo-D-glucose) prepared enzy-
matically from D-glucose. Carbohydr. Res. 1997, 297, 375–378.
6. Volc, J.; Leitner, C.; Sedmera, P.; Halada, P.; Haltrich, D. Enzymatic formation of
1
1
dicarbonyl sugars: C-2 Oxidation of 1 ! 6 disaccharides gentiobiose, isomaltose
and melibiose by pyranose 2-oxidase from Trametes multicolor. J. Carbohydr.
Chem. 1999, 18, 999–1007.
7. Volc, J.; Kub a´ tov a´ , E.; Sedmera, P.; Daniel, G.; Gabriel, J. Pyranose oxidase and
pyranosone dehydratase: enzymes responsible for conversion of D-glucose to
cortalcerone by the basidiomycete Phanerochaete chrysosporium. Arch. Microbiol.
1991, 156, 297–301.
Received August 12, 2002
Accepted February 6, 2003