Glycosyl azides - An alternative way to disaccharides

Article abstract of DOI:10.1002/adsc.200700028

Glycosyl azides are shown to be efficient donors for β-galactosidases, β-glucosidases and α mannosidases. Only α-galactosidases do not cleave the respective glycosyl azide 1 and, moreover, they exhibit competitive inhibition (especially α-galactosidase fr

Full text of DOI:10.1002/adsc.200700028

FULL PAPERS
DOI: 10.1002/adsc.200700028
Glycosyl Azides – An Alternative Way to Disaccharides
Pavla Bojarovµ,a,c Lucie Petrµskovµ,a Erica Elisa Ferrandi,b Daniela Monti,b
a
a
a
a,
ˇ
Helena Pelantovµ, Marek Kuzma, Pavla Simerskµ, and Vladimír Kren *
a
ˇ
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenskµ 1083, 142 20 Praha 4, Czech Republic
Fax : (+420)-296-442-509; e-mail: kren@biomed.cas.cz
Istituto di Chimica del Riconoscimento Molecolare, CNR, Via Mario Bianco 9, 20131 Milano, Italy
Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, 12840 Praha 2, Czech Republic
b
c
Received: January 16, 2007; Revised: April 4, 2007
This paper is dedicated to the memory of Prof. Zoltµn Gyçrgydeàk, who greatly contributed to the chemistry
of glycosyl azides.
Abstract: Glycosyl azides are shown to be efficient scribed for the first time. All the reactions were
donors for b-galactosidases, b-glucosidases and a- highly regioselective, yielding b
A
mannosidases. Only a-galactosidases do not cleave Galactosidase from E. coli proved to have the best
the respective glycosyl azide 1 and, moreover, they synthetic capabilities. The present study shows that
exhibit competitive inhibition (especially a-galactosi- glycosyl azides are a valuable alternative to common
dase from Talaromyces flavus). High water solubility p-nitrophenyl glycoside donors and in many synthet-
and ready synthesis of glycosyl azides enable trans- ic reactions.
glycosylation reactions even with difficult acceptors
like N-acetyl-d-mannosamine in good yields. The
versatility of glycosyl azides was demonstrated in the Keywords: azides; enzyme catalysis; glycosylation;
synthesis of five disaccharides – two of them are de- modified substrate
Introduction
during the cleavage of glycosyl azides. Azides are
cleaved more slowly than the corresponding p-nitro-
Transglycosylation reactions are a well known method phenyl glycosides[7] and glycosyl fluorides[8] (consider-
of carbohydrate synthesis and glycosidases are widely ing kcat, KM), however, they do not inhibit the enzyme
used for this aim.[1] An efficient transfer of the gly- at higher concentrations and afford good transglyco-
cone donor moiety to an acceptor requires a relatively sylation yields, probably due to the reduction of water
high donor concentration, which limits the risk of side activity by the high donor concentration. Their chemi-
hydrolysis. The hydrophobic leaving groups, although cal behaviour was thoroughly described by Gyçrgy-
providing better substrate cleavage, often cause solu- deàk and Thiem.[9]
bility problems. Therefore, new effective and highly
The cleavage of glycosyl azides was probably first
soluble glycosyl donors are still sought after: 3-nitro- studied by Sinnott with b-galactosidase from E.
and 5-nitro-2-pyridyl glycosides,[2] vinyl glycosides,[3] coli,[10] later also with b-glucosidase from Agrobacteri-
and also donors other than O-glycosides, e.g., glycosyl um sp.[11] b-d-Glucopyranosyl azide was used as a
fluorides,[4] which exhibit many common properties to donor in a coupled screening for thioglycoligase activ-
glycosyl azides.[5]
ity.[12] We have used 2-acetamido-2-deoxy-b-d-gluco-
Glycosyl azides combine some advantages of the pyranosyl and -galactopyranosyl azide in transglycosy-
above glycosyl donors, such as the small size of a leav- lation reactions with b-N-acetylhexosaminidases.[7]
ing group, a strong nucleophilic character and delocal-
In this paper we exploit the versatility of glycosyl
ized p-electron density. Additionally, they are perfect- azides as donors in transglycosylation reactions with a
ly water-soluble and, contrary to, e. g., many glycosyl representative sample of various glycosidases: a- and
fluorides,[4] indefinitely stable even in acidic solutions, b-galactosidases, b-glucosidases, and a-mannosidases.
which enables reactions with enzymes like fungal gly- Three enzyme classes, b-galactosidases, b-glucosidases,
cosidases and a-mannosidase from jack beans. De- and a-mannosidases, proved to be good catalysts for
spite the close chemical resemblance to glycosyl fluo- synthesis with glycosyl azides. Five disaccharidic struc-
rides, no “wrong anomer hydrolysis”[6] was observed tures were selectively prepared, two of which are de-
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Glycosyl Azides – An Alternative Way to Disaccharides
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scribed for the first time. The reactions had good to common rates for modified substrates),[19] contrary
yields (up to 37%) and a high regioselectivity [exclu- to a-galactosidases.
sive formation of b
A
To account for the surprising results that none of
dases tested did not cleave a-d-galactopyranosyl the tested a-galactosidases cleaved azide 1, the inhibi-
azide (1). On the contrary, an inhibition effect of the tion of a-galactosidases by this azide was studied. All
azide was observed (Ki =0.25 mM with a-galactosi- examined enzymes were considerably inhibited by
dase from Talaromyces flavus).
azide 1. For example, the residual activity of the most
inhibited a-galactosidase from Talaromyces flavus
CCF 2686 declined to just 35% in the presence of
azide 1 (2 mM). This inhibition was determined to be
competitive, with Ki =0.25ꢀ0.05 mM. For compari-
son, KM =0.54ꢀ0.07 mM for this enzyme with stan-
dard substrate 5. No inhibition was detected with
Results and Discussion
Preparation and Screening of Glycosyl Azides
Glycosyl azides 1–4 (Figure 1) were prepared in good other glycosidase classes and the respective azides.
yields according to the published procedures.[13–18]
Enzymes suitable for the catalysis of transglycosyla-
The potential of azides 1–4 to act as substrates for tion reactions must meet the following criteria: (i)
the respective glycosidases was assayed. Azides 1–4 reasonable reaction rate (and thus low enzyme con-
were subjected to a hydrolytic screening comprising sumption); (ii) efficient product formation (and thus
43 fungal, microbial, animal and plant glycosidases. high yield). As for condition (i), it is noteworthy that
The total hydrolysis rates of glycosyl azides 1–4 were the fastest hydrolyzing enzymes do not always give
compared to those of the respective p-nitrophenyl the highest yield in transglycosylations, however, a
glycosides 5–8. The results for azides 2 and 3 are certain cleavage rate (optimum ca. 5% cleavage and
given in Table 1. Azide 4 was cleaved by both tested more, compared to standard substrate) is necessary in
a-mannosidases (from jack beans and from Aspergil- order to reduce the enzyme consumption and keep
lus oryzae CCF 1066; 5% referred to 8). On the con- the viscosity of the solution at an acceptable level.
trary, azide 1 was cleaved by none of the a-galactosi- Therefore, the best enzymes from the hydrolytic
dases tested (<1% referred to 5). In summary, the re- screening were selected to test their synthetic poten-
spective azides were cleaved by most tested b-galacto- tial with azides 2–4, and to optimize the reaction con-
sidases, b-glucosidases and a-mannosidases (related ditions on an analytical scale. The transglycosylation
screening comprised b-galactosidases from B. circu-
lans, E. coli, and bovine testes, b-glucosidases from A.
oryzae CCF 1602, A. sojae CCF 3060 and P. chrysoge-
num CCF 1269, and both a-mannosidases (from jack
beans and A. oryzae CCF 1066) with various accept-
ors: glucoside 7, GlcNAc (12), and ManNAc (14). As
a result, the following reactions were proposed for
the semi-preparative scale: (i) azide 2 with glucoside 7
or GlcNAc (12) or ManNAc (14) and b-galactosidase
from E. coli (Scheme 1); (ii) azide 3 with GlcNAc
(12) and b-glucosidase from Penicillium chrysogenum
CCF 1269 (Scheme 2); (iii) azide 4 and a-mannosi-
dase from jack beans (Scheme 3; autocondensation
reaction).
Figure 1. Glycosyl azides 1–4 and respective p-nitrophenyl
glycosides 5–8.
Table 1. Cleavage of glycosyl azides 2 and 3 by respective glycosidases.
Azide 2 and b-galactosidases[a]
Cleavage Azide 3 and b-glucosidases[a]
Cleavage
Escherichia coli[b]
A. oryzae CCF 1602, A. sojae CCF 3060, A. ter-
reus CCF 2539, Bacillus circulans,[b] bovine liver,
bovine testes[b]
++
+
A. oryzae CCF 1602,[b] A. sojae CCF 3060[b]
A. terreus CCF 55, A. terreus CCF 58, A. terreus
CCF 2539, P. brasilianum CCF 2155, P. chrysoge-
num CCF 1269[b]
++
+
A. oryzae (Sigma), Kluyveromyces lactis
ꢁ
almonds, A. flavipes CCF 2026, P. daleae CCF 2365
ꢁ
[a]
The ratio of hydrolyses of azides 2 and 3 related to p-nitrophenyl glycosides 6 and 7, respectively, was determined as the
ratio of total hydrolysis times (assayed under the same conditions and analyzed by TLC). The ratio of hydrolyses was 3–
7% (++), 3–1% (+) or lower than 1% (ꢁ).
[b]
Best cleaving enzymes used in analytical transglycosylations.
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Scheme 3. Transglycosylation reaction with azide donor 4.
Scheme 1. Transglycosylation reactions with azide donor 2.
Scheme 2. Transglycosylation reaction with azide donor 3.
The above synthetic reactions were performed on a semi-preparative scale giving good yields (up to 37%)
semi-preparative scale under optimized conditions. and exclusive regioselectivity [b
A
Products 10, 13, 15, 16, and 17 were obtained in good disaccharides, two of which are novel, were synthe-
yields (up to 37%, for details see Schemes 1–3 and sized under the catalysis by b-galactosidase from E.
the Experimental Section) and with a high regioselec- coli, b-glucosidase from P. chrysogenum CCF 1269
tivity (exclusive formation of b
ACHTREUNG
ucts 15 and 17 were synthesized for the first time. All
products were fully characterized (NMR, MS, optical valuable alternative to common p-nitrophenyl glyco-
rotation) and in the case of known compounds, data side donors in many synthetic reactions, even with dif-
were compared to the literature values. b-Galactosi- ficult acceptors like N-acetyl-d-mannosamine. Their
dase from E. coli proved to be a particularly efficient main advantages are high water solubility, indefinite
catalyst in synthetic reactions. No traces of autocon- stability and ready synthesis.
densation products were observed in the reactions
with azides 2 and 3. This can be explained by a huge
molar excess of acceptor (5–6:1) as well as by a too
Experimental Section
low concentration of donor in reaction mixture
(ꢂ100 mM) for autocondensation reactions.[7]
Materials
All reagents and solvents used were commercially available
Conclusions
and of analytical grade. b-Galactosidases from Aspergillus
oryzae, Escherichia coli, Kluyveromyces lactis and bovine
liver, as well as b-glucosidase from almond, a-galactosidase
from Coffea arabica and a-mannosidase from jack beans,
were purchased from Sigma. b-Galactosidase from Bacillus
circulans was purchased from Daiwa Kasei K. K. (Osaka,
Japan) under the trade name Biolacta FN5. The fungal
strains producing a-galactosidases (EC 3.2.1.22), b-galactosi-
dases (EC 3.2.1.23), b-glucosidases (EC 3.2.1.21), and a-
mannosidases (EC 3.2.1.24) originated from the Culture
Collection of Fungi (CCF), Department of Botany, Charles
This work clearly demonstrates the potential of glyco-
syl azides as donors in transglycosylation reactions
with various glycosidases. The screening for hydrolytic
cleavage revealed that the respective glycosyl azides
are fairly well cleaved by b-galactosidases, b-glucosi-
dases and a-mannosidases, contrary to the tested a-
galactosidases, which were substantially inhibited by
the azide 1. Representative transglycosylation reac-
tions were performed with the selected enzymes on a University in Prague, or from the Culture Collection of the
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Glycosyl Azides – An Alternative Way to Disaccharides
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Institute of Microbiology (CCIM), Prague. The strains were
cultivated as described previously.[20] b-Galactosidase from
bovine testes was a crude preparation obtained by the
method of Hedbys et al.;[21] fresh bovine testes came from a
local slaughterhouse and were stored at ꢁ208C.
d=20.47, 20.55, 20.58, 20.68 (4ꢂAc), 62.11 (C-6), 65.62 (C-
4), 68.20 (C-3), 69.13 (C-2), 70.62 (C-5), 87.41 (C-1), 169.52,
[a]
169.61, 169.72, 170.45 (4ꢂCO). Readout from HOM2DJ;
[b] some acetyls were not found with certainty.
a-d-Mannopyranosyl azide (4) was prepared from 4b by
deacetylation according to Zemplen (MeONa/dry MeOH)
as a yellowish honey in 14% overall yield. In our hands,
both 4b and 4 were repeatedly isolated in a mixture with an
unidentified impurity, inert to enzymatic reactions (ca. 10–
30%), different from the b-anomer (as compared to the
published 13C NMR spectrum).[22]
Methods
Thin-layer chromatography was performed on precoated
Merck silica gel DC-Alufolien Kieselgel 60 F254 plates. The
spots were visualized by charring with 5% H2SO4 in EtOH.
Flash chromatography was performed on silica gel 60 (40–
63 mm, Merck). Gel chromatography was performed on Bio-
ꢁ
1
Gel P-2 (Bio-Rad, U.S.A.). H and 13C NMR spectra were
Enzymatic Methods
recorded on
a
Varian INOVA-400 spectrometer
Enzyme activity assay: The reaction mixture containing the
respective substrate (substrate 5 for a-galactosidases, 6 for
b-galactosidases, 7 for b-glucosidases, or 8 for a-mannosi-
dases; starting concentration 2 mM) and the respective
enzyme (0.02–0.03 UmLꢁ1) in citrate/phosphate buffer
(0.05 M, pH 5.0, for fungal enzymes or pH indicated by pro-
ducer for commercial enzymes) or phosphate buffer (0.05M,
pH 7.3, for E. coli b-galactosidase) was incubated with shak-
ing at 358C (or at the temperature indicated by producer
for commercial enzymes) for 10 min. Liberated p-nitrophe-
nol was determined spectrophotometrically (420 nm) under
alkaline conditions (0.1M Na2CO3). One unit of enzymatic
activity released 1 mmol of p-nitrophenol per min under the
above conditions.
(399.87 MHz and 100.55 MHz, respectively) at 308C in the
indicated solvents. Chemical shifts are expressed in d scale
and were measured with digital resolution justifying the re-
ported values to three or two decimal places, respectively.
HMQC and HMBC readouts are accurate at two decimal
1
places for H NMR chemical shifts and at one decimal place
for 13C NMR shifts. Residual signal of solvents was used as
an internal standard (CDCl3: dH =7.265 ppm, dC =
77.00 ppm, D2O: dH =4.508 ppm). Carbon chemical shifts in
D2O were referred to acetone (dC =30.50 ppm). The report-
ed assignment is based on gCOSY, HOM2DJ, HSQC,
HMQC, HMBC, and 1D-TOCSY experiments. The anome-
ric configuration of mannopyranosides was assigned on the
1
basis of direct coupling constant JC-1,H-1, which was mea-
Hydrolytic screening of glycosyl azides 1–4 with glycosi-
dases: The reaction mixture, composed of glycosyl azide 1–4
or standard p-nitrophenyl glycoside 5–8 (20 mM, starting
concentration) and the respective glycosidase (0.54 UmLꢁ1)
in citrate/phosphate buffer (0.05M, pH 5.0, for fungal en-
zymes or the pH indicated by the producer for commercial
enzymes) or phosphate buffer (0.05M, pH 7.3, for E. coli b-
galactosidase), was shaken at 358C and monitored by TLC
(2-propanol:H2O:aqueous NH4OH, 7:2:1). The total hydrol-
ysis time was indicated as the time when no more starting
material was detectable in the reaction mixture. The en-
zymes were compared according to the ratio of total hydrol-
ysis times for 1–4 and 5–8, respectively. a-Galactosidases,
tested with azide 1 and standard 5, were obtained from the
following sources: Aspergillus flavipes CCF 2026, A. flavus
CCF 1129, A. oryzae CCF 1602, A. parasiticus CCF 3058, A.
phoenicis CCF 61, A. sojae CCF 3060, A. tamarii CCF 3058,
A. terreus CCF 55, A. terreus CCF 2539, Circinella muscae
CCF 1568, Chaetomium globosum, coffee beans, Micromu-
cor ramannianus CCF 1022, Penicillium brasilianum CCF
2155, P. chrysogenum CCF 1269, P. commune CCF 2962, P.
daleae CCF 2365, P. multicolor CCF 2244, P. ochrochloron
CCF 2379, Talaromyces flavus CCF 2324, T. flavus CCF
2686, Trichoderma harzianum CCF 2687. b-Galactosidases
and b-glucosidases, tested with azide 2 and standard 6 or
with azide 3 and standard 7, respectively, are listed in
Table 1. a-Mannosidases from Aspergillus oryzae CCF 1066
and from jack beans were tested with azide 4 and standard
8.
sured by a proton-coupled HMQC experiment. Positive-ion
MALDI mass spectra were measured on a Bruker BIFLEX
II reflectron time-of-flight mass spectrometer (Bruker-Fran-
zen, Bremen, Germany) equipped with a nitrogen laser
(337 nm) and a gridless delayed extraction ion source. A so-
lution (10 mgmLꢁ1) of 2,5-dihydroxybenzoic acid (Sigma) in
50% acetonitrile/0.1% trifluoroacetic acid was used as
MALDI matrix. The matrix solution (1 mL) was premixed
with a sample (1 mL), loaded on the target and allowed to
dry at ambient temperature. Spectra were externally cali-
brated using the monoisotopic [M+H]+ signal of the matrix
and angiotensin II (Sigma). Optical rotation was measured
on a Perkin–Elmer 241 polarimeter at 589 nm.
Synthesis of Substrates
a-d-Galactopyranosyl azide (1) was a kind gift from Prof.
Z. Gyçrgydeàk, Debrecen University, Hungary.
b-d-Galactopyranosyl azide (2) was prepared according
to Korytnyk and Mills[13] or Gyçrgydeàk and Szilàgyi.[14]
b-d-Glucopyranosyl azide (3) was prepared according to
1
Soli et al.[15] or Ogawa et al.[16] The H and 13C NMR data
for compounds 1–3 were consistent with the structures (see
the literature,[14,16] respectively).
a-d-Mannopyranosyl azide (4): 1,2,3,4,6-Penta-O-acetyl-
d-mannose (4a) was prepared according to Watt and Wil-
liams.[17] 2,3,4,6-Penta-O-acetyl-a-d-mannopyranosyl azide
(4b) was prepared according to Ponpipom et al[18] as a yel-
1
1
lowish honey. H NMR (CDCl3; JC-1,H-1 =171 Hz): d=1.971,
2.031, 2.085, 2.144 (each s, 3H, 4ꢂAc), 4.13[a] (m, 1H, H-5),
Enzyme inhibition assay: The reaction mixture contain-
ing the standard p-nitrophenyl glycoside 5–8, the respective
azide 1–4 (both 2 mM) and the respective glycosidase (0.02–
0.03 UmLꢁ1) in citrate/phosphate buffer (0.05 M, pH 5.0, for
fungal enzymes or the pH indicated by the producer for
4.144 (m, 1H, H-6u), 4.28[a] (m, 1H, H-6d), 5.130 (dd, J1,2
1.9 Hz, J2,3 =3.0 Hz, 1H, H-2), 5.223 (dd, J2,3 =3.0 Hz, J3,4
=
=
10.4 Hz, 1H, H-3), 5.261 (dd, J3,4 =10.4 Hz, J4,5 =9.7 Hz, 1H,
H-4), 5.369 (d, J1,2 =1.9 Hz, 1H, H-1); 13C NMR (CDCl3):
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Pavla Bojarovµ et al.
FULL PAPERS
commercial enzymes) or phosphate buffer (0.05M, pH 7.3,
for E. coli b-galactosidase) was incubated under shaking at
358C for 10 min and assayed spectrophotometrically as de-
scribed above. Assayed a-galactosidases as listed above, as-
sayed b-galactosidases and b-glucosidases as listed in
Table 1. Azide 4 was tested with a-mannosidases from As-
pergillus oryzae CCF 1066 and from jack beans. The residual
activity was determined as the ratio of hydrolysis rates of
standard substrate 5–8 in the presence of the respective
azide 1–4 and without it. Kinetic constants (KM, Ki, Vmax) for
a-galactosidase from Talaromyces flavus CCF 2686 were de-
termined from initial hydrolysis rates at various starting con-
centrations of the standard substrate 5 (0.4–2 mM) with
azide 1 (0–2 mM) using SigmaPlot 2001 (SPSS Science,
U.S.A.). Initial hydrolysis rates were determined at 308C
and pH 4 in 1 mm path-length cells. The released p-nitro-
4.5 Hz, J5’,6’d =7.6 Hz, 1H, H-5’), 3.425 (m, 2H, H-2, H-3),
3.489 (dd, J5’,6’u =4.5 Hz, J6’u,6’d =11.6 Hz, 1H, H-6’u), 3.533
(dd, J5’,6’d =7.6 Hz, J6’u,6’d =11.6 Hz, 1H, H-6’d), 3.669 (dd,
J
3’,4’ =3.3 Hz, J4’,5’ =1.0 Hz, 1H, H-4’), 3.69[a] (m, 1H, H-5),
3.70[a] (m, 1H, H-6u), 4.010 (dd,
J5,6d =4.7 Hz, J6u,6d =
14.5 Hz, 1H, H-6d), 4.190 (d, J1’,2’ =7.6 Hz, 1H, H-1’), 5.070
(d, J1,2 =7.7 Hz, 1H, H-1), 7.076 (m, 2H, H-ortho), 8.065 (m,
2H, H-meta); 13C NMR (D2O, HMQC and HMBC read-
outs): d=61.1 (C-6’), 68.5 (C-6), 68.8 (C-4’), 69.3 (C-4), 70.7
(C-2’), 72.8 (C-3’), 72.9 (C-2), 75.1 (C-5’), 75.4 (C-3), 75.5
(C-5), 99.5 (C-1), 103.4 (C-1’), 116.8 (C-ortho), 126.2 (C-
meta), 142.8 (C-para), 161.6 (C-ipso).[a] Readout from
HMQC. The spectroscopic data of 10 were in accord with
the literature values.[24]
phenol was continuously monitored at 348 nm (the pH-inde- b-d-Galactopyranosyl-(1!6)-2-acetamido-2-deoxy-d-
pendent isosbestic point of ionized p-nitrophenol). Under
these conditions, the difference of absorption coefficients of
p-nitrophenol and the substrate was 2698Mꢁ1 cmꢁ1.[23]
glucopyranose (13)
b-d-Galactopyranosyl azide (2; 24.8 mg, 0.121 mmol) and 2-
acetamido-2-deoxy-d-glucopyranose
(12;
132.8 mg,
0.600 mmol) were dissolved in phosphate buffer (1.2 mL,
0.05M, pH 7.3). b-Galactosidase from E. coli (6 U) was
added and the reaction mixture was shaken (850 rpm) at
358C and monitored by TLC (2-propanol:H2O:aqueous
NH4OH, 7:2:1). After 2.5 h, another portion (2 U) of
enzyme was added. The reaction was stopped after 4.5 h by
heating (1008C, 5 min) and centrifuged (13,500 rpm,
10 min). Purification by gel chromatography (Bio-Gelꢁ P-2;
mobile phase H2O, flow rate 13.8 mLhꢁ1, ambient tempera-
ture) afforded compound 13 as a white solid; yield: 6.8 mg
(0.018 mmol; 28% related to consumed donor 2; donor 2
was partially recovered: 11.6 mg). According to NMR data,
13 was a mixture of two anomers (a/b=1.5).
Analytical Transglycosylation Reactions
b-Galactosidases: The reaction mixture containing b-galac-
tosidase from B. circulans, E. coli or bovine testes (0.5 U or
0.7 U), azide 2 (50 mM or 100 mM, respectively), the accept-
or [glucoside 7 (300 mM), GlcNAc (500 mM) or ManNAc
(500 mM)] in citrate/phosphate buffer (50 mM; pH 6.5 for
B. circulans enzyme, pH 4.5 for bovine testes enzyme) or
phosphate buffer (0.05M, pH 7.3, for E. coli enzyme) was
incubated with shaking at 358C for 24 h.
b-Glucosidases: The reaction mixture containing b-gluco-
sidase from A. oryzae CCF 1602, A. sojae CCF 3060 or P.
chrysogenum CCF 1269 (0.5 U), azide 3 (100 mM), the ac-
ceptor (500 mM, GlcNAc or ManNAc) in citrate/phosphate
buffer (0.05M, pH 5.0) was incubated with shaking at 358C
for 24 h.
a-Mannosidases: The reaction mixture containing azide 4
(100 mM, 200 mM, 300 mM, or 500 mM) and a-mannosidase
from jack beans (0.75, 0.95, 1.1 or 1.5 U, respectively) in cit-
rate/phosphate buffer (0.05M, pH 4.5) was incubated with
shaking at 358C for 24 h. The course of all analytical reac-
tions was monitored by TLC (2-propanol:H2O:aqueous
NH4OH, 7:2:1).
1
a-Anomer of 13: H NMR (D2O): d=1.823 (s, 3H, 2-Ac),
3.333 (dd, J2’,3’ =9.9 Hz, J1’,2’ =7.8 Hz, 1H, H-2’), 3.359 (dd,
J
J
4,5 =10.1 Hz, J3,4 =8.9 Hz, 1H, H-4), 3.432 (dd, J2’,3’ =9.9 Hz,
3’,4’ =3.4 Hz, 1H, H-3’), 3.470 (ddd, J5’,6’d =7.8 Hz, J5’,6’u
=
1.5 Hz, J4’,5’ =1.0 Hz, 1H, H-5’), 3.520 (dd, J6’d,6’u =11.7 Hz,
5’,6’u =1.5 Hz, 1H, H-6’u), 3.539 (dd, J2,3 =10.6 Hz, J3,4
J
=
8.9 Hz, 1H, H-3), 3.578 (dd, J6’d,6’u =11.7, J5’,6’d =7.8 Hz, 1H,
H-6’d), 3.668 (dd, J2,3 =10.6 Hz, J1,2 =3.5 Hz, 1H, H-2), 3.675
(dd, J6d,6u =11.4 Hz, J5,6u =5.1 Hz, 1H, H-6u), 3.700 (dd,
J
J
3’,4’ =3.4, J4’,5’ =1.0 Hz, 1H, H-4’), 3.794 (ddd, J4,5 =10.1 Hz,
5,6u =5.1 Hz, J5,6d =2.1 Hz, 1H, H-5), 3.952 (dd, J6d,6u
=
11.4 Hz, J5,6d =2.1 Hz, 1H, H-6d), 4.211 (d, J1’,2’ =7.8 Hz, 1H,
H-1’), 4.977 (d, J1,2 =3.5 Hz, 1H, H-1); 13C NMR (D2O,
HMQC readouts): d=21.3 (2-Ac), 54.4 (C-2), 61.4 (C-6’),
69.1 (C-6, C-4’), 70.3 (C-4), 71.1 (C-3, C-5), 71.2 (C-2’), 73.1
(C-3’), 75.5 (C-5’), 91.3 (C-1), 103.7 (C-1’).
p-Nitrophenyl b-d-Galactopyranosyl-(1!6)-b-d-
glucopyranoside (10)
b-d-Galactopyranosyl azide (2; 5 mg, 24.4 mmol) and p-ni-
trophenyl b-d-glucopyranoside (7; 43.4 mg, 144 mmol) were
dissolved in phosphate buffer (480 mL, 0.05M, pH 7.3). b-
Galactosidase from E. coli (3 U) was added and the reaction
mixture was shaken (850 rpm) at 358C and monitored by
TLC (2-propanol: H2O:aqueous NH4OH, 7:2:1). The reac-
tion was stopped after 2 h by heating (1008C, 5 min) and
centrifuged (13,500 rpm, 10 min). Purification by gel chro-
matography (Bio-Gelꢁ P-2; mobile phase H2O, flow rate
13.8 mLhꢁ1, ambient temperature) afforded compound 10 as
white solid; yield: 1.1 mg, (2.4 mmol; 37% related to con-
sumed donor 2; donor 2 was partially recovered: 3.7 mg).
1H NMR (D2O): d=3.316 (dd, J1’,2’ =7.6 Hz, J2’,3’ =9.9 Hz,
1H, H-2’), 3.373 (m, 1H, H-4), 3.375 (dd, J2’,3’ =9.9 Hz,
1
b-Anomer of 13: H NMR (D2O): d=1.821 (s, 3H, Ac),
3.309 (m, 2H, H-3, H-4), 3.338 (dd, J2’,3’ =9.9 Hz, J1’,2’
7.8 Hz, 1H, H-2’), 3.409 (m, 1H; H-5), 3.435 (dd, J2’,3’
=
=
9.9 Hz, J3’,4’ =3.4 Hz, 1H, H-3’), 3.463 (dd, J2,3 =10.3 Hz,
1,2 =8.4 Hz, 1H, H-2), 3.478 (ddd, J5’,6’d =7.8 Hz, J5’,6’u
1.5 Hz, J4’,5’ =1.0 Hz, 1H, H-5’), 3.530 (dd, J6’d,6’u =11.7 Hz,
5’,6’u =1.5 Hz, 1H, H-6’u), 3.578 (dd, J6’d,6’u =11.7 Hz, J5’,6’d
J
=
J
=
7.8 Hz, 1H, H-6’d), 3.637 (dd, J6d,6u =11.5 Hz, J5,6u =5.9 Hz,
1H, H-6u), 3.700 (dd, J3’,4’ =3.4 Hz, J4’,5’ =1.0 Hz, 1H, H-4’),
4.005 (dd, J6d,6u =11.5 Hz, J5,6d =2.1 Hz, 1H, H-6d), 4.228 (d,
J
1’,2’ =7.8 Hz, 1H, H-1’), 4.506 (d, J1,2 =8.4 Hz, 1H, H-1);
13C NMR (D2O, HMQC readouts): d=21.3 (2-Ac), 57.0 (C-
2), 61.4 (C-6’), 69.1 (C-6, C-4’), 70.2 (C-4), 71.2 (C-2’), 73.1
J
3’,4’ =3.3 Hz, 1H, H-3’), 3.384 (ddd, J4’,5’ =1.0 Hz, J5’,6’u =
1518
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Adv. Synth. Catal. 2007, 349, 1514 – 1520

Glycosyl Azides – An Alternative Way to Disaccharides
FULL PAPERS
(C-3’), 74.2 (C-3), 75.2 (C-5), 75.5 (C-5’), 95.4 (C-1), 103.7
(C-1’). The spectroscopic data of 13 were in accord with the
literature values.[25]
1.207 mmol) were dissolved in citrate/phosphate buffer
(2.4 mL, 0.05M, pH 5.0). b-Gluctosidase from Penicillium
chrysogenum CCF 1269 (12 U) was added and the reaction
mixture was shaken (850 rpm) at 358C and monitored by
TLC (2-propanol:H2O:aqueous NH4OH, 7:2:1). The reac-
tion was stopped after 5 h by heating (1008C, 2 min) and
centrifuged (13,500 rpm, 10 min). Purification by gel chro-
matography (Bio-Gelꢁ P-2; mobile phase H2O, flow rate
14.7 mLhꢁ1, ambient temperature) afforded compound 16 as
a white solid; yield: 17 mg (0.044 mmol; 28% related to
consumed donor 3; donor 3 was partially recovered: 19 mg).
For NMR characterization, compound 16 was peracetylated
(Ac2O/dry py, DMAP, 24 h) and purified by silica gel chro-
matography (CH2Cl2/MeOH, 20:1) to afford peracetate 16a.
According to NMR data, 16a was a mixture of two anomers
(a/b=1.22).
b-d-Galactopyranosyl-(1!6)-2-acetamido-2-deoxy-d-
mannopyranose (15)
b-d-Galactopyranosyl azide (2; 51 mg, 0.249 mmol) and 2-
acetamido-2-deoxy-d-mannopyranose
(14;
266 mg,
1.202 mmol) were dissolved in phosphate buffer (1.8 mL,
0.05M, pH 7.3). b-Galactosidase from E. coli (26 U) was
added and the reaction mixture was shaken (850 rpm) at
358C and monitored by TLC (2-propanol: H2O:aqueous
NH4OH, 7:2:1). The reaction was stopped after 4.5 h by
heating (1008C, 2 min) and centrifuged (13,500 rpm,
10 min). Purification by gel chromatography (Bio-Gelꢁ P-2;
mobile phase H2O, flow rate 13.8 mLhꢁ1, ambient tempera-
ture) afforded compound 15 as a white solid; yield: 6.7 mg
(0.017 mmol; 14% related to consumed donor 2; donor 2
was partially recovered: 26 mg). [a]2D3: +10.0 (c 0.12 in
water). According to NMR data, 15 was a mixture of two
anomers (a/b=1.22).
1
a-Anomer of 16a: H NMR (CDCl3): d=1.934 (s, 3H, 2-
NAc), 1.999 (s, 3H, Ac), 2.020 (s, 3H, Ac), 2.034 (s, 3H,
Ac), 2.047 (s, 3H, Ac), 2.056 (s, 3H, Ac), 2.089 (s, 3H, Ac),
2.188 (s, 3H, Ac), 3.508 (dd, J6d,6u =10.9 Hz, J5,6u =6.6 Hz,
1H, H-6u), 3.679 (ddd, J4’,5’ =9.9 Hz, J5’,6’d =4.6 Hz, J5’,6’u
2.4 Hz, 1H, H-5’), 3.895 (dd, J5,6d =10.9 Hz, J5,6d =2.0 Hz,
1H, H-6d), 3.945 (ddd, J4,5 =10.5 Hz, J5,6u =6.6 Hz, J5,6d
=
a-Anomer of 15: 1H NMR (D2O; 1JC-1,H-1 =176 Hz): d=
1.828 (s, 3H, Ac), 3.349 (dd, J1’,2’ =7.8 Hz, J2’,3’ =9.9 Hz, 1H,
H-2’), 3.445 (dd, J2’,3’ =9.9 Hz, J3’,4’ =3.4 Hz, 1H, H-3’), 3.483
(ddd, J4ꢃ,5ꢃ =1.0 Hz, J5ꢃ,6’u =4.2 Hz, J5ꢃ,6’d =7.9 Hz, 1H, H-5’),
3.489 (dd, J3,4 =9.9 Hz, J4,5 =10.0 Hz, 1H, H-4), 3.528 (dd,
=
2.0 Hz, 1H, H-5), 4.113 (dd, J6’d,6’u =12.3 Hz, J5’,6’u =2.4 Hz,
1H, H-6’u), 4.283 (dd, J6’d,6’u =12.3 Hz, J5’,6’d =4.6 Hz, 1H, H-
6’d), 4.426 (ddd, J2,2-NH =11.1 Hz, J2,3 =8.9 Hz, J1,2 =3.7 Hz,
1H, H-2), 4.522 (d, J1’,2’ =8.0 Hz, 1H, H-1’), 4.954 (dd, J2’,3’
9.6 Hz, J1’,2’ =8.0 Hz, 1H, H-2’), 5.013 (dd, J4,5 =10.5 Hz,
3,4 =9.3 Hz, 1H, H-4), 5.063 (dd, J4’,5’ =9.9 Hz, J3’,4’ =9.5 Hz,
=
J
5’,6’u =4.2 Hz, J6’u,6’d =11.6 Hz, 1H, H-6’u), 3.585 (dd, J5’,6’d
7.9 Hz, J6’u,6’d =11.6 Hz, 1H, H-6’d), 3.707 (dd, J3’,4’ =3.4 Hz,
4’,5’ =1.0 Hz, 1H, H-4’), 3.727 (dd, J5,6u =4.8 Hz, J6u,6d
11.3 Hz, 1H, H-6u), 3.794 (ddd, J4,5 =10.0 Hz, J5,6u =4.8 Hz,
5,6d =2.0 Hz, 1H, H-5), 3.834 (dd, J2,3 =4.7 Hz, J3,4 =9.9 Hz,
=
J
1H, H-4’), 5.197 (dd, J2’,3’ =9.6 Hz, J3’,4’ =9.5, 1H, H-3’),
5.212 (dd, J2,3 =11.1 Hz, J3,4 =9.3 Hz, 1H, H-3), 5.546 (d,
J
=
J
2,2-NH =8.9 Hz, 1H, 2-NH), 6.147 (d, J1,2 =3.7 Hz, 1H, H-1);
J
13C NMR (CDCl3, HMQC readouts): d=23.9–24.5 (7ꢂAc),
26.7 (2-NAc), 54.7 (C-2), 65.4 (C-6’), 72.0 (C-4, C-4’), 71.8
(C-6), 74.4 (C-3), 74.6 (C-5, C-2’), 75.6 (C-5’), 76.3 (C-3’),
94.0 (C-1), 104.3 (C-1’).
1H, H-3), 3.946 (dd, J5,6d =2.0 Hz, J6u,6d =11.3 Hz, 1H, H-
6d), 4.101 (dd, J1,2 =1.6 Hz, J2,3 =4.7 Hz, 1H, H-2), 4.235 (d,
J
1’,2’ =7.8 Hz, 1H, H-1’), 4.896 (d, J1,2 =1.6 Hz, 1H, H-1);
13C NMR (D2O, HMQC and HMBC readouts): d=22.0
(Ac), 53.4 (C-2), 61.1 (C-6’), 66.7 (C-4), 68.7 (C-3, C-6), 68.8
(C-4’), 70.8 (C-2’), 71.0 (C-5), 72.8 (C-3’), 75.4 (C-5’), 93.2
(C-1), 103.3 (C-1ꢃ), 174.8 (CO).
1
b-Anomer of 16a: H NMR (CDCl3): d=1.924 (3H, s, 2-
NAc), 2.002 (s, 3H, Ac), 2.020 (s, 3H, Ac), 2.044 (s, 3H,
Ac), 2.056 (s, 6H, 2ꢂAc), 2.096 (s, 3H, Ac), 2.113 (s, 3H,
Ac), 3.592 (dd, J6d,6u =11.2 Hz, J5,6u =6.1 Hz, 1H, H-6u),
3.672 (ddd, J4’,5’ =9.6 Hz, J5’,6’d =4.6 Hz, J5’,6’u =2.2 Hz, 1H, H-
5’), 3.749 (dd, J4,5 =9.6 Hz, J5,6u =6.1 Hz, J5,6d =2.4 Hz; 1H,
H-5), 3.906 (dd, J6d,6u =11.2 Hz, J5,6d =2.4 Hz, 1H, H-6d),
4.128 (dd, J6’d,6’u =12.4 Hz, J5,6’u =2.2 Hz, 1H, H-6’u), 4.232
(ddd, J2,3 =10.3 Hz, J2,2-NH =9.6 Hz, J1,2 =8.8 Hz, 1H, H-2),
4.264 (dd, J6’d,6’u =12.4 Hz, J5’,6’d =4.6 Hz, 1H, H-6’d), 4.551
b-Anomer of 15: 1H NMR (D2O; 1JC-1,H-1 =161 Hz): d=
1.867 (s, 3H, Ac), 3.34[a] (m, 1H, H-5), 3.356 (dd, J1’,2’
=
7.8 Hz, J2’,3’ =9.9 Hz, 1H, H-2’), 3.36[a] (m, 1H, H-4), 3.445
(dd, J2’,3’ =9.9 Hz, J3’,4’ =3.4 Hz, 1H, H-3’), 3.486 (ddd, J4’,5’
1.0 Hz, J5’,6u’ =4.2 Hz, J5’,6d’ =7.9 Hz, 1H, H-5’), 3.530 (dd,
5’,6u’ =4.2 Hz, J6u’,6d’ =11.6 Hz, 1H, H-6u’), 3.585 (dd, J5’,6d’
7.9 Hz, J6u’,6d’ =11.6 Hz, 1H, H-6d’), 3.610 (dd, J2,3 =4.5 Hz,
3,4 =9.7 Hz, 1H, H-3), 3.692 (dd, 5,6u =4.7 Hz, J6u,6d =
=
J
=
(d, J1’,2’ =8.0 Hz, 1H, H-1’), 4.970 (dd, J4,5 =9.6 Hz, J3,4
=
J
J
9.6 Hz, 1H, H-4), 4.976 (dd, J2’,3’ =9.5 Hz, J1’,2’ =8.0 Hz, 1H,
H-2’),5.063 (dd, J4’,5’ =9.6 Hz, J3’,4’ =9.6 Hz, 1H, H-4’), 5.101
11.4 Hz, 1H, H-6u), 3.707 (dd, J3’,4’ =3.4 Hz, J4’,5’ =1.0 Hz,
1H, H-4’), 3.999 (dd, J5,6d =1.5 Hz, J6u,6d =11.4 Hz, 1H, H-
6d), 4.235 (dd, J1,2 =1.7 Hz, J2,3 =4.5 Hz, 1H, H-2), 4.243 (d,
(dd, J2,3 =10.3 Hz, J3,4 =9.6 Hz, 1H, H-3), 5.190 (dd, J3’,4’
=
9.6 Hz, J2’,3’ =9.5 Hz, 1H, H-3’), 5.517 (d, JH,2-NH =9.6 Hz,
1H, 2-NH), 5.664 (d, J1,2 =8.8, 1H, H-1); 13C NMR (CDCl3,
HMQC readouts): d=23.9–24.5 (7ꢂAc), 26.7 (2-NAc), 56.8
(C-2), 65.5 (C-6ꢃ), 71.6 (C-6), 72.0 (C-4, C-4’), 74.6 (C-2ꢃ),
75.6 (C-5’), 76.3 (C-3, C-3’), 77.8 (C-5), 96.2 (C-1), 104.4 (C-
1’). The spectroscopic data of 16a were in accord with the
structure.[26]
J
1’,2’ =7.8 Hz, 1H, H-1’), 4.812 (d, J1,2 =1.7 Hz, 1H, H-1);
13C NMR (D2O, HMQC and HMBC readouts): d=22.1
(Ac), 54.2 (C-2), 61.1 (C-6’), 66.6 (C-4), 68.6 (C-6), 68.8 (C-
4’), 70.8 (C-2’), 72.0 (C-3), 72.8 (C-3’), 75.4 (C-5, C-5’), 93.1
(C-1), 103.3 (C-1’), 175.7 (CO),[a] Readout from HMQC;
MS (MALDI-TOF): m/z=406.2 [M+Na]+.
b-d-Glucopyranosyl-(1!6)-2-acetamido-2-deoxy-d-
glucopyranose (16)
b-d-Glucopyranosyl azide (3; 51 mg, 0.249 mmol) and 2-
acetamido-2-deoxy-d-glucopyranose
(12;
267 mg,
Adv. Synth. Catal. 2007, 349, 1514 – 1520
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
1519

Pavla Bojarovµ et al.
FULL PAPERS
[4] S. J. Williams, S. G. Withers, Carbohydr. Res. 2000, 327,
27–46.
[5] F. Micheel, A. Klemer, Adv. Carbohydr. Chem. 1961,
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[6] E. J. Hehre, C. F. Brewer, D. S. Genghof, J. Biol. Chem.
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[7] P. Fialovµ, A. T. Carmona, I. Robina, R. Ettrich, P. Sed-
a-d-Mannopyranosyl-(1!6)-a-d-mannopyranosyl
azide (17)
a-d-Mannopyranosyl azide (4; 170 mg, 0.829 mmol) was dis-
solved in citrate/phosphate buffer (1.2 mL, 0.05 M, pH 4.5).
a-Mannosidase from jack beans (24 U) was added and the
reaction mixture was shaken (850 rpm) at 358C and moni-
tored by TLC (2-propanol:H2O:aqueous NH4OH, 7:2:1).
The reaction was stopped after 4.5 h by heating (1008C,
2 min) and centrifuged (13,500 rpm, 10 min). Purification by
gel chromatography (Bio-Gelꢁ P-2; mobile phase H2O, flow
rate 7.6 mLhꢁ1, ambient temperature) afforded compound
17 as a white hygroscopic solid; yield: 6.5 mg, 0.018 mmol;
6% related to consumed donor 4; donor 4 was partially re-
ˇ
ˇ
ˇ
mera, V. Prikrylovµ, L. Petrµskovµ-Husµkovµ, V. Kren,
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235–241.
1
covered: 108 mg). [a]2D3: +120.0 (c 0.1 in water); H NMR
1
1
(D2O; JC-1,H-1 =172 Hz, JC-1’,H-1’ =172 Hz): d=3.424 (dd, J=
9.1 Hz, J=9.6 Hz, 1H, H-4’), 3.461 (m, 1H, H-5’), 3.51[a] (m,
2H, H-3, H-4), 3.53[a] (m, 1H, H-6’u), 3.59[a] (m, 1H, H-6u),
3.61[a] (m, 1H, H-3’), 3.65[a] (m, 1H, H-6’d), 3.651 (dd, J1,2
=
1.8 Hz, J2,3 =2.8 Hz, H-2), 3.707 (m, 1H, H-5), 3.74[a] (m,
1H, H-6d), 3.75[a] (m, 1H, H-2’), 4.691 (d, J1’,2’ =1.7 Hz, 1H,
H-1’), 5.220 (d, J1,2 =1.8 Hz, 1H, H-1); 13C NMR (D2O,
HMQC readouts): d=61.15 (C-6ꢃ), 65.77 (C-6), 66.50 (C-4),
66.94 (C-4’), 69.97 (C-2), 70.17 (C-2’), 70.28 (C-3), 70.75 (C-
[15] E. D. Soli, A. S. Manoso, M. C. Patterson, P. DeShong,
D. A. Favor, R. Hirschmann, A. B. Smith, J. Org.
Chem. 1999, 64, 3171–3177.
[a]
3’), 72.97 (C-5’), 73.05 (C-5), 90.05 (C-1), 99.63 (C-1’),
[16] T. Ogawa, S. Nakabayashi, S. Shibata, Agric. Biol.
Readout from HSQC; MS (MALDI-TOF): m/z=390.1
[M+Na]+, 406.2 [M+K]+.
Chem. 1983, 47, 281–285.
[17] J. A. Watt, S. J. Williams, Org. Biomol. Chem. 2005, 3,
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Acknowledgements
ˇ
[19] P. Fialovµ, L. Weignerovµ, J. Rauvolfovµ, V. Prikrylovµ,
This work was supported by Czech National Science Founda-
tion No. 203/05/0172, grant No. IAA400200503 from Grant
Agency of Academy of Sciences of the Czech Republic, res.
concept no. MSM21620808, ESF project COST Chemistry
D25/0001/01 and D34/001/05, and Czech Ministry of Educa-
tion (projects OC170 and OC136). Czech-Italian interaca-
ˇ
A. Pisvejcovµ, R. Ettrich, M. Kuzma, P. Sedmera, V.
ˇ
Kren, Tetrahedron 2004, 60, 693–701.
[20] a) Z. Hunkovµ, V. Kren, M. Scigelovµ, L. Weignerovµ,
ˇ
ˇ
ˇ
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O. Scheel, J. Thiem, Biotechnol. Lett. 1996, 18, 725–
730; b) Z. Hunkovµ, A. Kubµtovµ, L. Weignerovµ, V.
Kren, Czech Mycol. 1999, 51, 71–88.
ˇ
ˇ
ˇ
demic project between AV CR and CNR (V.K. & D.M.) is
acknowledged for the mobility resources.
[21] L. Hedbys, E. Johansson, K. Mosbach, P. O. Larsson,
A. Gunnarsson, S. Svensson, Carbohydr. Res. 1989, 186,
217–223.
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1520
asc.wiley-vch.de
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