A new enzyme catalysis: 3,4-dioxidation of some aryl β-D- glycopyranosides by fungal pyranose dehydrogenase

Article abstract of DOI:10.1016/j.tetlet.2004.09.149

Quinone-dependent pyranose dehydrogenase presents a new tool for versatile conversions of numerous carbohydrates to their di- and tricarbonyl derivatives. This enzyme purified from the basidiomycete Agaricus meleagris catalysed dioxidation of several aromatic β-d-glucopyranosides and a β-d-xylopyranoside into the corresponding 3,4-didehydro-β-d- aldopyranosides (β-d-aldopyranosid-3,4-diuloses) in high yields, typically >80% for 4-nitrophenyl glycosides. These new compounds were doubly hydrated in aqueous solution. According to in situ NMR investigations, the reaction intermediates were the corresponding 3- and 4-dehydro compounds. The analogous anomeric α;-glycosides underwent one-step oxidation only at C-3 to 3-dehydro-α-d-aldopyranosides (α-d-pyranosid-3-uloses).

Full text of DOI:10.1016/j.tetlet.2004.09.149

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 8677–8680
A new enzyme catalysis: 3,4-dioxidation of some aryl
b-D-glycopyranosides by fungal pyranose dehydrogenase
a
Petr Sedmera, Petr Halada, Clemens Peterbauer and Jindrich Volc
a
b
a,
*
ˇ
a
´
ˇ
´
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vıdenska 1083, 142 20 Prague 4, Czech Republic
bDepartment of Food Sciences and Technology, University of Natural Resources and Applied Life Sciences Vienna,
Muthgasse 18, A-1190 Vienna, Austria
Received 17 August 2004; revised 17 September 2004; accepted 21 September 2004
Available online 8 October 2004
Abstract—Quinone-dependent pyranose dehydrogenase presents a new tool for versatile conversions of numerous carbohydrates to
their di- and tricarbonyl derivatives. This enzyme purified from the basidiomycete Agaricus meleagris catalysed dioxidation of sev-
eral aromatic b-D-glucopyranosides and a b-D-xylopyranoside into the corresponding 3,4-didehydro-b-D-aldopyranosides (b-D-
aldopyranosid-3,4-diuloses) in high yields, typically >80% for 4-nitrophenyl glycosides. These new compounds were doubly
hydrated in aqueous solution. According to in situ NMR investigations, the reaction intermediates were the corresponding 3-
and 4-dehydro compounds. The analogous anomeric a-glycosides underwent one-step oxidation only at C-3 to 3-dehydro-a-D-aldo-
pyranosides (a-D-pyranosid-3-uloses).
Ó 2004 Elsevier Ltd. All rights reserved.
Redox enzyme catalysis offers great potential for modi-
fication of sugars to reactive derivatives (possible inter-
mediates for chemical synthesis) by regiospecifically
introducing carbonyl function(s).1 Syntheses of surfac-
tants and polymer building blocks may serve as an
example.2 A powerful tool in this context is now pro-
vided by a novel fungal enzyme, the quinone-dependent
pyranose dehydrogenase (PDH, pyranose:acceptor
oxidoreductase, EC 1.1.99.29), acting on a vast array
of mono- and oligosaccharides. Previously we showed
with aromatic glycosides, some of which proved to be
especially good substrates for the unique C-3,4 dioxida-
tive conversions to new glycosides of tricarbonyl sugars.
Using in situ NMR methodology,8 we detected and elu-
cidated the structures of the oxidation products of sali-
cin (2-hydroxymethylphenyl-b-D-glucopyranoside, 1),
arbutin (4-hydroxyphenyl-b-D-glucopyranoside, 2), 4-
nitrophenyl-b-D-glucopyranoside (4NP b-Glcp, 3), 4-
nitrophenyl-b-D-xylopyranoside (4NP b-Xylp, 4) and
iridoid glycoside harpagoside (5, Fig. 1), respectively.
High substrate conversions approaching 95% were
achieved within 2–24h.
that selective monooxidations (at C-1,3 C-2,3,4 C-35,6
)
or dioxidations (at C-2,34,7) of the sugar molecule can
be performed depending on the nature of the sugar,
the reaction conditions and the enzyme source (mainly
some Agaricales). In addition, the primary oxidation
can proceed specifically at one position only (C-3 for
Me a/b-D-Glcp, sucrose, trehalose5 and D-glucose,6 C-2
for D-galactose4) or in parallel at positions C-2 and C-
3 (D-xylose,7 D-glucose4) or C-1 and C-2 (lactose3) in
various proportions with individual PDHs. In this work,
we extended our investigation to the reaction of PDH
Setting the aglycone resonances aside, the 1H NMR
spectrum (Table 1) of typical end-products consisted
of an AB system (H-1, H-2) and an ABC system
Keywords: 3,4-Didehydroglucopyranoside; 3,4-Didehydroxylopyrano-
side; Aldopyranosid-3,4-diuloses; Aryl glycoside oxidation; Sugar 3,4-
dioxidation; Pyranose dehydrogenase; Agaricus.
*
Corresponding author. Tel.: +42 0241062298; fax: +42
0296442384; e-mail: volc@biomed.cas.cz
Figure 1. Structure of harpagoside 5.
0040-4039/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.09.149

8678
P. Sedmera et al. / Tetrahedron Letters 45 (2004) 8677–8680
Table 1. NMR data of 3,4-dioxidation products (399.89 [1H] and
100.55 [13C] MHz, D2O, 30°C); sugar part only
1h
2
h
3h
4h
5h
Proton
dH
1
2
5
6
4.984
3.693
3.712
3.772
3.604
4.846
3.583
3.652
3.756
3.612
5.119
3.267
3.782
3.787
3.603
5.1104.676
3.692
3.687
3.503a
3.386
Figure 2. Diagnostic HMBC contacts in the biotransformation prod-
uct of arbutin 2.
3.618
3.788
3.670
Hi,Hj
J(i,j), Hz
ions at masses of 4 mass units lower than those of the
parent compounds (m/z 305.2, 291.2, 320.3, 290.2 and
513.4 for the final reaction mixtures upon biotransforma-
tion of 1, 2, 3, 4 and 5, respectively). However, no ketone
signals were present in the 13C NMR spectra and chem-
ical shifts both of C-3 and C-4 (Table 1) indicated the
hydration of both keto groups. HMBC was used to dis-
tinguish these quaternary carbons (Fig. 2).
1,2
8.08.08.07.6
8.2
2.8
5,6d
5,6u
6d,6u
2.7
7.2
2.2
5.1
2.3
7.3
7.9
À12.7
À11.9
À11.6
À12.0b
À11.9
Carbon
dC
1
2
3
4
5
6
100.28
72.66
94.88
94.25
76.02
60.94
101.00
72.66
94.86
94.26
75.92
59.74
99.29
75.45
94.81
94.19
76.21
59.63
99.63
72.44
94.39
92.96
67.53
98.5c
72.2c
94.7c
94.1c
76.0c
59.7c
Careful monitoring of the biotransformations revealed
that the reaction intermediates were 3-dehydroglyco-
sides together with their hydrated forms; a hydrate of
a 4-dehydroglycoside was found by NMR with 4 (see
Table 2 for the spectroscopic characterization). Some
low abundance transient intermediates that might be
due to 4b, 4e or 4f (Scheme 1) were also observed. The
NMR data of the 3-oxidation products (1a, 1c, 2a, 2c,
3a, 3c) were similar to those reported for methyl b-D-
glucopyranoside.5,10 The structure of compound 4d is
unambiguous: its 1H NMR spectrum consisted of a con-
For compound identification, see Scheme 1.
a H-5u.
b J(5d,5u).
c HMQC and HMBC readouts.
(comprising H-5 and both H-6Õs) or two AB systems
(with 4). These properties are consistent with 3,4-dioxida-
tion. Positive-ion ESI mass spectra9 showed [M+Na]+
Table 2. NMR data of reaction intermediates (399.89 [1H] and 100.55 [13C] MHz, D2O, 30°C); sugar part only
1a
1c
2a
2c
3a
3c
4c
4d
5a
5c
Proton
dH
1
2
3
4
5
6
5.013
4.436
—
4.964
3.492
4.860
4.315
—
4.828
3.366
—
5.172
4.435
—
5.102
3.479
—
5.107
3.453
—
5.041
3.528
3.485
—
4.708
4.134
—
4.656
3.174
—
—
3.997
4.275
3.449
3.796
3.659
4.274
3.441
3.355
3.462
4.290
3.584
3.401
3.595
3.598
3.752
4.197
3.407
3.848
3.304
3.431
3.739
3.537
3.6903.783
3.5303.643
3.615
3.520a
a
3.679 3.7103.817
3.5203.5403.636
3.467
3.674
3.550
Hi,Hj
J(i,j), Hz
8.2
1,2
2,4
7.9
1.7
8.087.09
—
9.7
78..20
78..01
1.7
10.3
2.2
—
9.8
1.7
10.3
4.9
—
—
9.5b
1.7
10.4
2.2
—
4,5
10.3
2.2
9.8
4.9
9.8c
—
—
—
10.1
2.3
d
5,6d
5,6u
6d,6u
4.8
4.5
2.022..10
5.5
4.8
À12.6
4.8
À12.6
5.5
À12.1
5.05.8
À12.6
À12.2
À12.5
À11.1e
À12.4e
À12.5
À12.5
Carbon
dC
1
2
3
4
5
6
102.02
76.6073.88
100.06
102.85
100.79
100.2
100.1
98.4
76.67
73.98
72.0 72.2
73.5
n.d.
76.1
77.4
60.8
206.20
72.16
76.25
60.72
94.51
70.30
n.d.
94.48
70.42
75.2
92.2
n.d.
70.6
72.25
75.4076.29
60.54
75.43
68.6
76.3
60.81
61.07
61.0
For compound identification, see Scheme 1; n.d., not determined.
a H-5u.
b J(2,3); carbon chemical shifts reported to one decimal point are HMQC and HMBC readouts.
c J(4,5d).
d J(4,5u).
e J(5d,5u).

ses eventual double oxidation of aryl b-D-glycosides 1,
P. Sedmera et al. / Tetrahedron Letters 45 (2004) 8677–8680
8679
Scheme 1. Reaction scheme for double oxidation of aryl glycosides 1, 2, 3, 4 and 5 by pyranose dehydrogenase (PDH). Ar, aryl; 1, Ar = 2-
hydroxymethylphenyl, R = CH2OH; 2, Ar = 4-hydroxyphenyl, R = CH2OH; 3, Ar = 4-nitrophenyl, R = CH2OH; 4, Ar = 4-nitrophenyl, R = H; 5,
Ar = see Figure 1, R = CH2OH; BQ, benzoquinone, HQ, hydroquinone.
Table 3. Oxidation of glycosides 1–5 by PDH
2, 3, 4, 5 to the corresponding aryl b-D-3,4-didehydro-
glycosides (aryl b-D-glycopyranosid-3,4-diuloses) fol-
lowing Scheme 1, preferentially via 3-dehydro
intermediates.
Substrate
Reaction
time (h)
Products identified by NMR
(%)
a
b
c
d
1
2
3
4
5
24
2
5
—
—
—
1
8
81
23
3
6017
4
The double oxidation is obviously related to the pres-
ence of an aromatic moiety in the molecule since both
methyl b-D-Glcp and methyl b-D-Xylp were transformed
into their 3-keto derivatives only, even after long contact
with an excess of the enzyme.5 On the other hand, 4NP
derivatives of a-Glcp, a-Xylp, a-Galp, b-Galp, a-Arap
and b-Arap also yielded 3-oxidised products only (to
be described elsewhere), so that both the anomeric and
sugar configuration are important. The furanose ring
in 4NP a-Xylf and 4NP a-Araf prevented reaction with
PDH. The effect of the aromatic ring may be a kind of
co-operative binding at the enzyme active site facilitat-
ing a better and longer contact. Interestingly, 5 also gave
the 3,4-dioxidised product (5h, Scheme 1) even though
the aromatic part here is the cinnamic acid ester of
one of the aglycone hydroxyls.
2
92
82
24
2058
—
17
—
8
42
Compounds identified by NMR after substantial (>99%) substrate
conversion.
tiguous three-spin system involving H-1 to H-3 and an
AB system for the two methylene protons at C-5 (proved
by HMQC); C-4 resonates at 92.2ppm, that is the corre-
sponding carbonyl is hydrated. Contrary to the com-
monly held belief that double oxidation takes place
only after prolonged reaction time, the signals of 3,4-
dioxidised products were present from early stages of
the reaction. The results of NMR analysis of the reac-
tion mixtures at the time when almost all substrate
(>99%) had been consumed are given in Table 3. At this
stage, however, both hydration/dehydration reactions
and the 3,4-dioxidation (see Scheme 1) were still taking
place so that these results are only illustrative of multiple
product forms to be analysed for each substrate. Never-
theless, all the data provided confirm that PDH cataly-
C-3 oxidation of the glycosyl moiety is known also to be
catalysed by other enzymes: bacterial glycoside-3-dehy-
drogenase (EC 1.1.99.13) from Agrobacterium tumefac-
iens (e.g., sucrose, maltose, isomaltulose, leucrose,
lactose),1 and fungal pyranose oxidase (EC 1.1.3.10)

8680
P. Sedmera et al. / Tetrahedron Letters 45 (2004) 8677–8680
from Peniophora gigantea (Me a/b-D-Glcp).10 Several
examples of naturally occurring iridoid 3-dehydrogluco-
sides are also known.11,12 In addition to PDH, sugar
dioxidation at adjacent positions has earlier been re-
ported for C-2 and C-3 of some monosaccharides using
pyranose oxidase (D-glucose,13,14 D-galactose,15 1,5-an-
hydro-D-glucitol and 1,5-anhydro-D-galactitol10). Only
recently, a unique glycoside of a contiguous tricarbonyl
sugar derivative, 2-(3,4-dihydroxyphenyl)ethyl 2,3-dide-
hydro-b-D-glucopyranoside, was isolated from a natural
plant source.16 Here, we report for the first time enzy-
matic C-3,4 double oxidation of sugars, D-glucose and
D-xylose in their glycosidic form. According to our liter-
ature search, chemical oxidation of sugars to the corre-
sponding 3,4-didehydroderivatives has not yet been
described. On the contrary, both solid state and solution
structures of a related diketose, D-threo-hexo-3,4-diulose
are well understood.17
´
´
6. Volc, J.; Kubatova, E.; Daniel, G.; Sedmera, P.; Haltrich,
D. Arch. Microbiol. 2001, 176, 178–186.
ˇ
´
7. Volc, J.; Sedmera, P.; Halada, P.; Prikrylova, V.; Haltrich,
D. Carbohydr. Res. 2000, 329, 219–225.
8. The experiments were performed in a 5-mm NMR sample
tube (solution volume 0.7mL) containing substrate
(10mM), 1,4-benzoquinone (30mM) and 0.4U PDH in
deuterium oxide (no buffering). The enzyme catalyst was
PDH purified from mycelial cultures of the fungus
Agaricus meleagris to apparent homogeneity (53Umg
proteinÀ1, ferricenium assay; 121UmLÀ1 distilled water;
3lL per reaction mixture) using a procedure described
previously.6 Monitoring the reaction time course by 1H
NMR spectroscopy provided pseudokinetic data. When
the concentration of the products was sufficient for their
detection ÔsnapshotsÕ were taken using 2D NMR tech-
niques (COSY, TOCSY, HOM2DJ, HMQC) or their
selective one-dimensional variants (1D-TOCSY, 1D-
NOE). Upon substantial substrate depletion and reaching
a steady state, the complete analysis (including 13C NMR
and HMBC) was undertaken to determine the structure
and proportions of the products.
9. The spectra were recorded on a LCQ Deca ion trap mass
spectrometer equipped with a nanospray ion source.
Samples, dissolved in 30% aqueous acetonitrile, were
sprayed directly from EconoTipTM emitters. Spray voltage
was 1.2kV and the heated capillary was kept at 150°C.
Only the final reaction mixtures were studied.
Acknowledgements
This work was supported by grant 2004-4 (Program for
Scientific-Technical Cooperation KONTAKT Austria-
Czech Republic) and grant no. P-16836-B11 from the
Austrian Science Foundation (C. Peterbauer).
10. Freimund, S.; Huwig, A.; Giffhorn, F.; Ko¨pper, S. Chem.
Eur. J. 1998, 4, 2442–2455.
11. Kiuchi, F.; Liu, H. M.; Tsuda, Y. Chem. Pharm. Bull.
1990, 38, 2326–2328.
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´