Cyanodeoxy-glycosyl derivatives as substrates for enzymatic reactions

Article abstract of DOI:10.1002/ejoc.200500755

Synthetic routes for the preparation of new sugar nitriles 8-10 derived from 2-acetamido-2-deoxy-β-D-glucopyranosides bearing a cyano group at the C-5 or C-6 position are presented. In an attempt to prepare the glycosyl azide 10 by treatment of tosylate 23 with KCN/DMF at 60°C, an intramolecular 1,3-dipolar cycloaddition reaction occurred to give the highly constrained nonisolable tetrazole 24, which was readily converted into the imino-azido compound 25 through an azido-tetrazole tautomerism. Compounds 8 and 10 were found to be poorer substrates of fungal β-N-acetylhexosaminidases than compound 9 and none of these compounds was accepted as substrates of the nitrilase or nitrile hydratase. Docking of the nitriles 8-10 in the active site of the β-N-acetylhexosaminidase from Aspergillus oryzae gave interaction energies comparable with the natural substrate. Based on these data, which indicate strong binding of these compounds (8 > 9 > 10) to the active site, it has been proposed that some cyano derivatives may act as competitive inhibitors of β-N-acetylhexosaminidases. This hypothesis is consistent with enzyme inhibition experiments which showed strong inhibitory properties of compound 9 (K_I = 0.37 mM) and in particular of compound 8 (K_I = 7.6 μM). Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejoc.200500755

FULL PAPER
DOI: 10.1002/ejoc.200500755
Cyanodeoxy-Glycosyl Derivatives as Substrates for Enzymatic Reactions
[a]
[b]
[b]
[c]
ˇ
Ana T. Carmona, Pavla Fialová, Vladimir Kren,* Rudiger Ettrich,
Ludmila Martínková,[b] Antonio J. Moreno-Vargas,[a] Cristina González,[a] and
Inmaculada Robina*[a]
Keywords: Sugar nitriles / Azido-tetrazole tautomerism / Glycosidase / Inhibitors / Enzymes / Carbohydrates
Synthetic routes for the preparation of new sugar nitriles 8–
10 derived from 2-acetamido-2-deoxy-β- -glucopyranosides
Docking of the nitriles 8–10 in the active site of the β-N-ace-
tylhexosaminidase from Aspergillus oryzae gave interaction
energies comparable with the natural substrate. Based on
these data, which indicate strong binding of these com-
pounds (8 Ͼ 9 Ͼ 10) to the active site, it has been proposed
that some cyano derivatives may act as competitive inhibitors
of β-N-acetylhexosaminidases. This hypothesis is consistent
with enzyme inhibition experiments which showed strong in-
D
bearing a cyano group at the C-5 or C-6 position are pre-
sented. In an attempt to prepare the glycosyl azide 10 by
treatment of tosylate 23 with KCN/DMF at 60 °C, an intra-
molecular 1,3-dipolar cycloaddition reaction occurred to give
the highly constrained nonisolable tetrazole 24, which was
readily converted into the imino-azido compound 25 through
an azido-tetrazole tautomerism. Compounds 8 and 10 were
found to be poorer substrates of fungal β-N-acetylhexosamin-
idases than compound 9 and none of these compounds was
accepted as substrates of the nitrilase or nitrile hydratase.
hibitory properties of compound 9 (KI = 0.37 m
ticular of compound 8 (KI = 7.6 µ ).
M) and in par-
M
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
the traditional nitrophenyl glycosides,[3] glycosyl azides[4]
have been demonstrated as efficient substrates.
Introduction
Glycoscience covers a multifaceted range of activities and
applications of carbohydrate research. Glycostructures
themselves play a highly diverse and crucial role in a myriad
of organisms and in important systems in biology, physiol-
ogy, medicine, bioengineering and technology. Yet it is only
in recent years that the tools have been developed that lead
to an understanding of their highly complex functions and
chemical background.
Glycosidases are known to accept structurally modified
substrates, which enables researchers to investigate various
aspects of their substrate specificity. As a result, new com-
petitive inhibitors are being found that can be used against
glycosidase-induced infections in human and veterinary
medicine and in agriculture.[5] Furthermore, structure–func-
tion-relationship studies form the basis for targeted muta-
genesis.[6]
In the synthesis of complex carbohydrate structures, en-
zymatic methods, which are simple, selective and mild alter-
natives to synthetic chemistry, are often applied.[1] Glyco-
sidases (EC, 3.2) are widely used for this purpose owing to
their stability, low cost and virtually absolute stereoselectiv-
ity.[2] The main requirement of their substrates is the pres-
ence of a good leaving group, and recently, in addition to
A large group of well-accepted structural modifications
in the substrate pyranose ring are those at the C-6 posi-
tion.[7] The introduction of a highly versatile functionality
such as a nitrile group brings about the possibility of fur-
ther modifications,[8] for example, reduction to an amino
group possibly followed by conjugation with aldehydes or
isothiocyanates or hydrolysis (chemical or enzymatic) to the
respective carboxylic acid.
[a] Departamento de Química Orgánica, Facultad de Química,
Universidad de Sevilla, Apartado 553,
Several procedures for attaching a cyano group to dif-
ferent carbon atoms of the sugar backbone have been re-
ported,[9] yielding, for example, methyl 3-cyano-3-deoxyal-
tropyranoside (1),[10] methyl 2-cyano-2-deoxy-α--altropyr-
anoside (2),[11] and isopropyl 2-cyano-2-deoxy-α--glucopy-
ranoside (3) (Figure 1).[12]
As for compounds carrying a cyano group at the C-5
position, several methods have been described that yield, for
example, methyl α--galactopyranuronitrile (4) or methyl α-
-glucopyranuronitrile (5) (Figure 1).[13–15] 2,3,6-Trideoxy-
6-cyano derivative 6 and the unsaturated 6-deoxy-6-cyano
41071 Sevilla, Spain
Fax: +34-954624960
E-mail: robina@us.es
[b] Institute of Microbiology, Laboratory of Biotransformation,
Academy of Sciences of the Czech Republic,
Vídenˇská 1083, 14220 Prague 4, Czech Republic,
Fax: +420-296442509
E-mail: kren@biomed.cas.cz
[c] Laboratory of High Performance Computing, Institute of Phys-
ical Biology USB and Institute of Landscape Ecology AS CR,
Zámek 136, 37333 Nové Hrady, Czech Republic
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
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Cyanodeoxy-Glycosyl Derivatives as Substrates for Enzymatic Reactions
FULL PAPER
and other cyanides in DMF did not give the desired com-
pound. Instead the basic medium promoted an internal dis-
placement of the tosylate by the OH group at the C-3 posi-
1
tion to give the bicyclic compound 14. In the H NMR
spectra of 13 and 14, a remarkable change in the signals of
1-H (δ =5.13 ppm, d, J1,2 = 8.4 Hz for 13 to 5.60 ppm, s for
14) and of 2-H (δ = 3.86 ppm, dd, J2,3 = 10.3 Hz, J1,2
=
8.4 Hz for 13 to 4.43 ppm, d, J2,3 = 3.0 Hz for 14) was ob-
served, indicating a semi-boat conformation of the pyra-
nose ring. Protection of the hydroxy groups with acetates
or TBS groups (compounds 15 and 16, respectively) also
failed to give the expected results after treatment with KCN.
In the case of 15, the trans-esterified peracetylated com-
pound 17 was obtained and in the case of 16, decomposi-
tion took place. The use of other reagents containing cya-
nide anion (TMSCN/TBAF, Bu4NCN) were also unsuc-
cessful.
Figure 1. Cyanopyranosyl derivatives 1–7.
To avoid internal displacements, we carried out another
synthetic strategy that implies the protection of the O-3-H
and O-4-H atoms in one step using a butanediacetal (BDA)
protecting group. This synthetic scheme was applied to p-
nitrophenyl and azido derivatives 12a and 12b (Scheme 2).
The bis-acetal 18 was obtained in 78–80% yield by reaction
with butanedione and methyl orthoformate applying Ley’s
methodology.[18] Compounds 18a and 18b were obtained as
a mixture of diastereoisomers at the acetalic carbon atoms
and were used directly in the following steps. Singlets due
to 3 H atoms at δ = 3.29 and 3.22 ppm and to 6 H atoms
at δ = 1.32 ppm corresponding to the OMe and Me groups,
respectively, indicated the presence of the BDA protecting
group.
derivative 7 were prepared by displacement of the corre-
sponding 6-O-triflate with tetrabutylammonium cyanide.[16]
This paper presents the synthesis of nitriles derived from
N-acetyl--glucosamine, that is, p-nitrophenyl 2-acetamido-
2-deoxy-β--glucopyranosiduronitrile (8), p-nitrophenyl
2-acetamido-2,6-dideoxy-β--gluco-heptopyranosyluronon-
itrile (9) and 2-acetamido-2,6-dideoxy-β--gluco-heptopyr-
anosyluronitrile azide (10) (Figure 2). Furthermore, the po-
tential of these compounds as substrates of glycosidases
and nitrilases and as inhibitors of glycosidases has been
studied.
Tosylation of 18a at the C-6 position followed by dis-
placement with a cyanide anion led to decomposition prod-
ucts. However, iodination (I2/PPh3, 65% yield) of the mix-
ture of p-nitrophenyl derivatives 18a gave 19, which was iso-
lated as a single diastereoisomer. The iodo substituent in 19
was confirmed by the resonances at δ = 3.59 and 3.18 ppm
for 6-H and 6Ј-H (J6,5 = 2.4 Hz, J6Ј,5 = 8.1 Hz, J6,6
=
1
10.8 Hz) in its H NMR spectrum. Displacement with tet-
rabutylammonium cyanide (Bu4NCN/THF) afforded the
cyano compound 20 in 43% yield, which was finally depro-
tected in aqueous TFA to give 9. In the 1H NMR spectrum
of 20, deshielding of the 6-H and 6Ј-H signals to δ = 2.85
and 2.69 ppm with respect to the same protons as in 19
confirmed the substitution. The signal at δ = 116.2 ppm in
the 13C NMR spectrum of 20 verified the presence of the
cyano group.
Figure 2. Glucopyranosiduronitrile 8 and gluco-heptopyranosyl-
urononitriles 9 and 10.
Results and Discussion
The synthesis of the p-nitrophenyl derivatives starts from
The cyano derivative 10 was obtained by 6-O-triflation
p-nitrophenyl
2-acetamido-2-deoxy-β--glucopyranoside of the bis-acetal 18b and subsequent displacement at room
(12a),[17] which was prepared from the readily available 2- temperature with tetrabutylammonium cyanide followed by
acetamido-2-deoxy-α--glucopyranosyl chloride (11) by nu- acidic deprotection (15% overall yield from 12b). Com-
cleophilic displacement of the chloride with sodium p-nitro- pounds 18b were obtained from the known 2-acetamido-2-
phenolate in DMF followed by methanolysis. The simplest deoxy-β--glucopyranosyl azide 12b[19] under BDA condi-
route for the introduction of the cyano group at the C-6 tions.
position, which implies the regioselective introduction of a
With the idea of improving the yield of the preparation
bulky leaving group at C-6, such as tosylate, followed by of the azido-derivative 10, we changed the starting material
displacement was unsuccessful (Scheme 1). Although com- from the highly reactive triflate 21 to the isolable tosylate
pound 13 was obtained in 60% yield, treatment with KCN 23 (Scheme 3), which was readily obtained from 18b in 80%
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Scheme 1.
Scheme 2.
yield. At room temperature, only a partial reaction of 23 derivative 24, followed by a tetrazole ring-opening[21] pro-
with cyanide anions occurred. cess that involves breakage of the N-1–N-2 bond in the
However, when the reaction was carried out with KCN/ tetrazole moiety. This process is highly favourable as a re-
DMF at 60 °C, derivative 25 was obtained. Its formation sult of the high conformational strain of 24.
can be explained by a 1,3-dipolar cycloaddition[20] between
Compound 25 exhibits a UV absorption at 274 nm, a
the 6-cyano and the 1-azido groups of 22, which affords strong band in its IR spectrum at 2191 cm–1 and a signal in
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Cyanodeoxy-Glycosyl Derivatives as Substrates for Enzymatic Reactions
FULL PAPER
Scheme 3.
its 13C NMR spectrum at δ = 115.7 ppm which has been no hydrolysis was observed (lower than 1% relative to 12a).
assigned to the C-7 atom based upon its HSQC spectrum. With such a low hydrolytic potential, transglycosylation re-
The fact that the tetrazole carbon atom resonates at δ = actions are not feasible due to very high enzyme consump-
143–154 ppm[22] and that the tetrazole ring exhibits no IR tion. None of the compounds were accepted by nitrile-con-
absorption at around 2000–2200 cm–1 [23] confirms the verting enzymes, a nitrile hydratase from Rhododoccus equi
tetrazole ring-opening. Additionally, hydrogenation (H2/Pd/ A4[25] and a nitrilase from Aspergillus niger K10.[26] This is
C, 1 atm) of 25 provokes the disappearance of the band at not surprising in view of the previous observations that
2191 cm–1 from the IR spectrum and the UV absorption. bulky nitriles are hardly accepted by these enzymes (see
This is further evidence of tetrazole ring-opening after the ref.[27] for a review).
cycloaddition reaction. The 1H NMR spectroscopic data of
However, compounds 8–10 could dock into the active
25 also indicate that the sugar moiety adopts a boat confor- site of the β-N-acetylhexosaminidase derived from Aspergil-
mation, which was unequivocally assigned by the change in lus oryzae CCF 1066, one of the most frequently studied
J1,2 and J4,5 from 8.9 and 10.0 Hz, respectively, in 23 to J1,2 representatives of the eukaryotic β-N-acetylhexosaminid-
= 0 Hz and J4,5 = 2.3 Hz. The other coupling constants ases (Figure 3 and Figure 4).
remain practically unaltered as a result of the torsional
requirements of the bis-acetal.
The interaction energies of these compounds with the en-
zyme (steric and electrostatic contributions, Table 1) were
Nitrile 8 was synthesized by oxidation of the 6-OH group compared with the corresponding substrates without the
in 18a under Swern conditions to afford carbaldehyde 26 in cyano group which are hydrolyzed well by the enzyme[4]
an almost quantitative yield. The resonance at δ = 9.6 ppm (that is 8 and 9 are compared with 12a and 10 with 12b).
in its 1H NMR spectrum confirmed the presence of the for-
With respect to the above results from hydrolytic screen-
myl group. The nitrile group was formed under very mild ing, the results of molecular modeling were rather surpris-
conditions with iodine and ammonia in THF at room tem- ing. The interaction energy of a poor substrate with this
perature.[24] This transformation took place through the im- enzyme is typically lower than –150 kJ/mol.[7d] Thus, our
ine and diiodo derivative to give the triple bond after dehy- results clearly indicate that the substrates bind well to the
drohalogenation. Finally, deprotection with aqueous TFA active site, although the sum of the steric and electrostatic
yielded 8 in 93% yield (Scheme 4).
energy contributions are smaller than those of the standard
Compounds 8–10 were then subjected to an enzymatic substrate. Azido derivatives 12b and 10 show similar but
hydrolysis screening comprising 33 fungal β-N-acetylhexos- slightly weaker binding than the nitrophenyl glycosides,
aminidases mainly from the Aspergillus, Penicillium and Ta- which agrees with the experimental results for the hydrolysis
laromyces genera. Owing to the lack of a chromophore moi- of azides.[4] As a result, we have proposed a hypothesis that
ety, the hydrolysis of compound 10 was analyzed by TLC. compounds 8–10 are competitive inhibitors of the β-N-ace-
The hydrolytic rates were measured relative to the hydroly- tylhexosaminidase from A. oryzae.
sis of the standard substrate 12a. None of the compounds
To verify this assumption, we determined the residual ac-
proved to be a good substrate for the enzymes tested − the tivities of this enzyme towards the standard substrate 12a
best results were obtained with compound 9 and the β-N- in the presence of different concentrations of compounds
acetylhexosaminidases derived from Talaromyces flavus 8–10. The results summarized in Table 2 as well as the inhi-
CCF 2686 (3.3% relative to 12a), Penicillium pittii CCF bition studies show that compound 8 is a strong competi-
2277 (3.0%) and Hamigera avellanea CCF 2923 (2.9%). tive inhibitor of the β-N-acetylhexosaminidase from A. ory-
With compound 8, Fusarium oxysporum CCF 377 (3.4%) zae (KI = 7.6 µ with KM for standard substrate 12a being
proved to be the best source. In other cases, negligible or 0.75 m).[4] Inhibitory properties were also demonstrated
Eur. J. Org. Chem. 2006, 1876–1885
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V. K rˇen, I. Robina et al.
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Scheme 4.
Figure 3. Substrates A) 12a, B) 9 and C) 8 docked into the active site of the β-N-acetylhexosaminidase from A. oryzae CCF 1066.
Hydrogen bonding (in green) to glutamic acid 519, arginine 193 and tryptophan 482 fixes the glycosides. The π-electron system of the
aromatic ring of the p-nitrophenyl group is stabilized by aromatic stacking with tryptophan 482.
Figure 4. Substrates A) 12b and B) 10 docked into the active site of the β-N-acetylhexosaminidase from A. oryzae CCF 1066. Hydrogen
bonding (in green) to glutamic acid 519, arginine 193 and tryptophan 482 fixes the glycosides. The azido group is depicted as a blue
stick.
Table 1. Interaction energies of substrates 8–10, 12a and 12b with compared with that of 12a and 9 (the distance between the
the β-N-acetylhexosaminidase isolated from A. oryzae.
glycosidic oxygen atom and glutamic acid 519 is 2.8 Å for
Interaction energy [kJ/mol][a]
the latter, however, 2.9 Å for 8; Figure 3). These results im-
ply that modifications analogous to compound 8, that is,
conversion of C-6-OH into the CN group, could be a prom-
ising route to other glycosidase inhibitors. Compound 10 is
not an inhibitor, which is probably due to weaker binding
to the enzymatic active site relative to the nitrophenyl deriv-
atives (Table 1).
Substrate
Total
Steric
Electrostatic
12a
12b
8
–300
–257
–249
–238
–208
–84
–57
–86
–96
–65
–216
–200
–163
–142
–143
9
10
[a] Drop in the enzyme–substrate complex interaction energy re-
flects better binding of the substrate to the enzymatic active site.
Conclusions
by compound 9 (KI = 0.37 m). Thus, compound 8, though
a structural analogue of 9, exhibits a 50-fold stronger inhi-
New nitrile-substituted derivatives of 2-acetamido-2-de-
bition effect, which can be explained by comparison of in- oxy-β--glucopyranosides 8–10 were prepared for enzy-
teraction energies at the enzymatic active site (Figure 1, matic studies with respective glycosidases, both as potential
Table 1). Additionally, the better cleavage of compound 9 substrates and inhibitors. A panel of 33 fungal β-N-acetyl-
in comparison with compound 8 may be influenced by a hexosaminidases was tested and slight cleavage by several
slight shift in the position of compound 8 in the active site enzymes was observed (ca. 3% hydrolytic rate relative to
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Cyanodeoxy-Glycosyl Derivatives as Substrates for Enzymatic Reactions
FULL PAPER
Table 2. Residual activities of the β-N-acetylhexosaminidase from A. oryzae in the presence of 8–10.
Inhibitor concentration
[m]
Substrate/inhibitor ratio
Residual activity towards 12a (2 m) [%]
Compound 8
Compound 9
Compound 10
0
–
10
5
2
1
100
16
10
3
100
88
82
60
44
100
100
100
100
100
0.2
0.4
1
2
1
CCF 2171, P. chrysogenum CCF 1269, P. funiculosum CCF 2985,
P. multicolor CCF 2244, P. oxalicum CCF 1959, P. oxalicum CCF
2315, P. oxalicum CCF 2430, P. pittii CCF 2277, P. spinulosum CCF
2159, Talaromyces flavus CCF 2573, T. flavus CCF 2686, T. ohiensis
CCF 2229, Trichoderma harzianum CCF 2687.
the standard substrate 12a). Compound 9 proved to be best
accepted whereas glycosyl azide 10 was cleaved by none of
the enzymes tested. Neither was hydrolysis of any of the
compounds by a nitrile hydratase or a nitrilase observed,
probably due to steric factors.
All assays with the nitrile hydratase from Rhododoccus equi A4
were performed in Na/K phosphate buffer (0.05 , pH 7.5) with
0.5 m substrate. The enzyme was prepared and purified as de-
scribed previously.[28]
Molecular modeling and docking of the respective com-
pounds into the active site of the β-N-acetylhexosaminidase
from A. oryzae revealed interaction energies that suggest
strong binding to the active site. Correspondingly, experi-
ments showed competitive inhibition by compound 9 (KI =
0.37 m) and in particular by compound 8 (KI = 7.6 µ).
As a result, compound 8 appears to be a good lead struc-
ture for the further design of efficient glycosidase inhibitors.
An intramolecular 1,3-dipolar cycloaddition reaction
was observed during the preparation of cyano-glycosyl
azide 22, giving the nonisolable tetrazole 24, which, owing
to the high conformational strain of its structure, was
converted into the imino-azido compound 25 through a
tetrazole/azido tautomerism.
Activity Assay for β-N-Acetylhexosaminidases:[29] The reaction mix-
ture containing compound 8, 9 or the standard substrate 12a
(2 m, starting concentration) and β-N-acetylhexosaminidase
(0.15–0.3 U/mL for 8 and 9; 0.01–0.02 U/mL for 12a) in buffer (as-
say volume 50 µL) was incubated in microplates at 35 °C for
10 min. The reaction was stopped by adding aq. Na2CO3 (0.1 ,
150 µL). Liberated p-nitrophenol was determined spectrophoto-
metrically (414 nm) on Titertek Multiscan® MCC/340 (Flow
Laboratories, McLean, U.S.A.) microplate reader. One unit of en-
zymatic activity is defined as the amount of enzyme that releases
1 µmol of p-nitrophenol per minute under the above conditions.
Enzymes were classified according to the ratio of hydrolysis rates
of compound 8 or 9 and of the standard substrate 12a, extrapolated
to the same amount of enzyme. The activity towards compound 10
was estimated as follows. Compound 10 or the standard substrate
12a (10 m, starting concentration) and β-N-acetylhexosaminidase
(1.79 U/mL for 10, 0.09 U/mL for the standard substrate) in buffer
were incubated at 35 °C under shaking. Samples were taken at reg-
ular intervals and analyzed by TLC (2-propanol/H2O/aq. NH3,
7:2:1). The spots were visualized by UV light and by charring with
5% sulfuric acid in ethanol. Enzymes were classified according to
the ratio of total hydrolysis times for compound 10 and the stan-
dard substrate 12a, extrapolated to the same amount of enzyme.
As far as we are aware, this is the first example of this
type of tautomerism in nonheteroaromatic compounds.
Experimental Section
General: Optical rotations were measured in a 1.0 cm tube with a
Perkin–Elmer 241 MC spectropolarimeter. 1H and 13C NMR spec-
tra were obtained for solutions in CDCl3, [D6]DMSO, CD3OD and
D2O; J values are given in Hz and δ in ppm. All the assignments
were confirmed by two-dimensional NMR experiments. The FAB
mass spectra were obtained using glycerol or 3-nitrobenzyl alcohol
as the matrix. TLC was performed on silica gel HF254 (Merck),
with detection by UV light and Pancaldi reagent [(NH4)6MoO4,
Ce(SO4)2, H2SO4, H2O]. Silica gel 60 (Merck, 230 mesh) was used
for preparative chromatography. Anhydrous solvents and reagents
were freshly distilled under N2 prior to use.
Inhibition Assay for β-N-Acetylhexosaminidases: Residual enzy-
matic activities in the presence of compounds 8–10 were deter-
mined as follows. The reaction mixture containing the standard
substrate 12a (2 m), compound 8, 9 or 10 (0–3 m) and the β-N-
acetylhexosaminidase from A. oryzae CCF 1066 (0.11 U/mL) was
incubated at 35 °C under shaking. Samples were taken, quenched
by adding aq. Na2CO3 (0.1 ) and analyzed spectrophotometrically
as above. The residual activity is defined as the ratio of initial rates
of standard substrate hydrolysis in the presence and absence of in-
hibitor 8, 9 or 10. Kinetic constants (KM, KI, Vmax) were deter-
mined from initial reaction rates at different starting concentrations
of the standard substrate 12a (0.5–2.0 m) in the presence of inhib-
itor 8 or 10 (0–3 m) using SigmaPlot 2001 (SPSS Science, U.S.A.).
Hydrolysis rates were determined as described above.
Enzymes: All assays with β-N-acetylhexosaminidases were per-
formed in citrate/phosphate buffer (0.05 , pH 5.0). The fungal
strains producing β-N-acetylhexosaminidases (EC, 3.2.1.52) origi-
nated from the Culture Collection of Fungi (CCF), Department of
Botany, Charles University Prague, or from the Culture Collection
of the Institute of Microbiology (CCIM), Prague, and were culti-
vated as described previously.[28] The screening comprised 33 en-
zymes: Acremonium persicinum CCF 1850, Aspergillus awamori
CCF 763, A. flavipes CCF 1895, A. flavipes CCF 3067, A. flavofur-
catis CCF 3061, A. flavus CCF 3056, A. niger CCIM K2, A. niveus
CCF 3057, A. nomius CCF 3086, A. oryzae CCF 147, A. oryzae
Activity Assay for Nitrile Hydratase: The reaction mixture contain-
ing 0.5 m of compounds 8–10 and the purified nitrile hydratase
CCF 1066, A. parasiticus CCF 1298, A. sojae CCF 3060, A. tamarii from Rhododoccus equi A4[25] (0.06 mg of protein/mL, that is, ap-
CCF 3085, A. terreus CCF 2539, A. terreus USA, Fusarium oxyspo-
rum CCF 377, Hamigera avellanea CCF 2923, Chaetomium glo-
bosum CCF 430, Penicillium brasilianum CCF 2155, P. brasilianum
prox. 3 U/mL, as assayed with benzonitrile) in 50 m Na/K phos-
phate buffer, pH 7.5, (assay volume 0.5 mL) was incubated at 28 °C
with shaking. At intervals (1, 3, 5 and 20 h) 0.05 mL of the reaction
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V. K rˇen, I. Robina et al.
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mixture was withdrawn, mixed with 0.05 mL methanol and centri-
fuged. The supernatants were analyzed by HPLC using a system
which consisted of the solvent delivery system 600 and the PDA
detector 996 (Waters) and the Nova-Pak C18 column (5 µm,
3.9×150 mm; Waters). Nitriles 8 and 9 were eluted with a mobile
phase consisting of 25% (v/v) acetonitrile and 0.1% (v/v) H3PO4
at a flow rate of 0.9 mL/min and detected at 296 nm. Nitrile 9 was
eluted with 10% (v/v) acetonitrile and 0.1% (v/v) H3PO4 at the
same flow rate and detected at 210 nm. Nitriles 8–10 remained un-
reacted.
which was used directly in the next step. A mixture of 18a (270 mg,
0.592 mmol), triphenylphosphane (435 mg, 1.660 mmol), imidazole
(113 mg, 1.660 mmol) and iodine (281 mg, 1.106 mmol) in toluene
(20 mL) was stirred under reflux for 2 h. The reaction mixture was
cooled, an equal volume of saturated aqueous sodium hydrogencar-
bonate was added and the mixture was stirred for 5 min. Iodine
was added in portions until the toluene phase remained iodine-
colored. It was then stirred for an additional 10 min and then the
excess of iodine was removed by addition of aqueous thiosulfate.
The mixture was transferred to a separating funnel; the organic
layer was diluted with toluene, and, after separation, the toluene
layer was washed with water, dried, filtered and concentrated. Tri-
phenylphosphane oxide was then precipitated in diethyl ether and
the filtrate concentrated. The residue was purified by column
chromatography on silica gel (diethyl ether/petroleum ether, 5:1) to
Activity Assay for Nitrilase: The reaction mixture containing
0.5 m of compounds 8–10 and the cell-free extract from Aspergil-
[26]
lus niger A4 possessing nitrilase activity
(1 mg of protein/mL,
that is, approx. 2.8 U/mL, as assayed with benzonitrile) in 50 m
Tris/HCl buffer, pH 8.0 (assay volume 0.5 mL), was incubated at
35 °C with shaking. Work up of the samples and HPLC analysis
were performed as described for the nitrile hydratase assay. The
unreacted nitriles 8, 9 and 10 but no reaction products were de-
tected.
give 19 (218 mg, 65%). [α]2D8 = +28 (c = 0.75, CH Cl ). IR: ν
=
˜
max
2
2
3264, 3086, 165, 1524, 1344, 1111, 1071, 1036, 887, 745, 596 cm–1.
1H NMR (300 MHz, CDCl3): δ = 8.21–8.17 (m, 2 H, Ph), 7.21–
7.16 (m, 2 H, Ph), 5.88 (d, J1,2 = 8.1 Hz, 1 H, 1-H), 5.70 (d, JNH,2
= 6.9 Hz, 1 H, NH), 4.55 (dd, J3,2 = 10.8, J3,4 = 9.6 Hz, 1 H, 3-
2
H), 3.71 (m, 1 H, 5-H), 3.59 (dd, J6,5 = 2.4, J6,6Ј = 10.8 Hz, 1 H,
Molecular Modeling: The primary sequence of the β-N-acetylhexos-
aminidase from A. oryzae CCF 1066 was aligned with the known
X-ray structures of the β-N-acetylhexosaminidases from Serratia
marcescens and Streptomyces plicatus, extracted from the Brook-
haven Protein Database (PDB entry: 1QBA and 1HP4, respec-
tively). The sequence data are available from the DDBJ/EMBL/
GeneBank databases (http://www.ncbi.nlm.nih.gov/) under the ac-
cess number AY091636. Three-dimensional models were generated
using the Modeller6 package.[30] For model refinement and minim-
ization, the SYBYL package with the TRIPOS force field (TRIPOS
Associates Inc.) was used. The complete modeling, including the
alignment and energy minimization, was performed exactly as de-
scribed previously.[3b] The docking of ligands was performed as de-
scribed earlier.[3b] The positioning of the ligands in the arbitrary
site was carried out using the DOCK module included in SYBYL/
MAXIMIN2, which calculates interaction energies based on steric
contributions from the TRIPOS force field and on electrostatic
contributions from any atomic charges present in the ligand. Exact
positioning of the ligand was achieved by a two-step procedure,
that is, energy minimization followed by molecular dynamics, in
exactly the same manner for all ligands described. The ligand–pro-
tein system was minimized by 1000 interactions with the Powell
minimizer and the TRIPOS force field including electrostatic inter-
actions based on Gasteiger–Hückel partial charge distributions
using a dielectric constant with a distance-dependent function ε =
4r and a nonbonded interaction cut off of 8 Å. A molecular dy-
namics simulation at 290 K followed the minimization with the
NTV ensemble over 15 ps. The resulting structure was then mini-
mized with the same parameters as above to a convergence of the
energy gradient less than 0.04 kJ/mol. The nonbinding interaction
energy between the model and the ligands within the optimized
complex was calculated using the TRIPOS force field. This estima-
tion of real interaction energy neglects solvation and desolvation
effects.
6-H), 3.50 (t, J4,5 = 9.6 Hz, 1 H, 4-H), 3.48 (m, 1 H, 2-H), 3.18
(dd, J6Ј,5 = 8.1 Hz, J6,6Ј = 10.8 Hz, 1 H, 6Ј-H), 3.29 and 3.22 (2s,
3 H each, OMe), 1.98 (s, 3 H, CH3CO), 1.32 (s, 6 H, CH3) ppm.
13C NMR (75.4 MHz, CDCl3): δ = 170.5 (C=O), 161.4 (C-1 Ph),
142.8 (C-4 Ph), 125.6, 116.8 (Ph), 99.9, 99.8 (Cq BDA), 96.9 (C-1),
70.0 (C-5), 69.7 (C-4), 66.2 (C-3), 56.2 (C-2), 48.3, 47.8 (2 OMe),
20.2 (C-6), 23.4 (NHCOCH3), 17.5, 17.4 (2 CH3) ppm. FABMS:
m/z (%) = 589 (100) [M + Na]+, 535 (70) [M – OMe]+. CIMSHR:
calcd. for C20H27N2O9I + H: 567.0840; found 567.0795.
p-Nitrophenyl 2-Acetamido-2,6-dideoxy-3,4-O-(2Ј,3Ј-dimethoxybu-
tane-2Ј,3Ј-diyl)-β-D-gluco-heptopyranosylurononitrile (20): Tetrabu-
tylammonium cyanide (135 mg, 0.504 mmol) dissolved in THF
(1 mL) was added to a stirred solution of 19 (190 mg, 0.336 mmol)
in dry THF (3 mL). The mixture was stirred at room temp. for
16 h and then concentrated. The residue was dissolved in CH2Cl2,
washed with sodium hypochlorite and water, dried (Na2SO4) and
the solvents evaporated. The residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH, 25:1Ǟ18:1) to give
20 (67 mg, 43%). [α]2D8 = +25 (c = 0.8, CH Cl ). IR: ν
= 3268,
˜
2
2
max
1
3082, 1661, 1593, 1346, 1113, 1071, 1042, 889, 746 cm–1. H NMR
(300 MHz, CDCl3): δ = 8.22–8.17 (m, 2 H, Ph), 7.20–7.07 (m, 2 H,
Ph), 5.96 (d, J1,2 = 8.0 Hz, 1 H, 1-H), 5.82 (d, JNH,2 = 7.1 Hz, 1
H, NH), 4.58 (dd, J3,4 = 9.7, J3,2 = 11.0 Hz, 1 H, 3-H), 3.94 (ddd,
J5,6 = 3.4 Hz, J5,6Ј = 6.5 Hz, J5,4 = 9.7 Hz, 1 H, 5-H), 3.62 (t, J4,5
= 9.7 Hz, 1 H, 4-H), 3.47 (ddd, J2,NH = 7.1 Hz, J2,1 = 8.0 Hz, J2,3
= 11.0 Hz, 1 H, 2-H), 3.29 and 3.22 (2s, 3 H each, OMe), 2.85 (dd,
2J6,6Ј = 16.9, J6,5 = 3.4 Hz, 1 H, 6-H), 2.69 (dd, J6Ј,5 = 6.5 Hz, J6Ј,6
= 16.9 Hz, 1 H, 6Ј-H), 1.98 (s, 3 H, MeCO), 1.34 and 1.33 (2s, 3
H each, Me) ppm. 13C NMR (75.4 MHz, CDCl3): δ = 170.9 (C=O),
161.6 (C-1 Ph), 143.1 (C-4 Ph), 125.9, 116.8 (Ph), 116.2 (CN),
100.2, 100.1 (Cq BDA), 97.0 (C-1), 70.0 (C-5), 69.7 (C-4), 66.2 (C-
3), 56.2 (C-2), 48.4, 48.1 (2 OMe), 23.6 (NHCOCH3), 20.2 (C-6),
17.7, 17.6 (2 CH3) ppm. FABMS: m/z = 488 (30) [M + Na]+.
CIMSHR: calcd. for C21H27N3O9 + H: 466.1826; found 466.1802.
p-Nitrophenyl 2-Acetamido-2,6-dideoxy-6-iodo-3,4-O-(2Ј,3Ј-dimeth-
oxybutane-2Ј,3Ј-diyl)-β-
D
-glucopyranoside (19): p-Nitrophenyl β--
p-Nitrophenyl 2-Acetamido-2,6-dideoxy-β-D-gluco-heptopyranosylu-
rononitrile (9): Compound 20 (50 mg, 0.108 mmol) was stirred in a
glucopyranoside 12a[17] (1.5 g, 4.38 mmol), butane-2,3-dione
(1.91 mL, 21.9 mmol), CH(OMe)3 (7.2 mL, 65.7 mmol) and CSA solution of aqueous TFA (2:1) (2 mL) for 2 h at 40 °C. The mixture
(274 mg, 1.18 mmol) were stirred under reflux in MeOH (50 mL)
for 6 h. Et3N was added and the solution concentrated. Column
chromatography on silica gel (CH2Cl2/MeOH, 35:1) afforded p-ni-
trophenyl 2-acetamido-2-deoxy-3,4-O-(2Ј,3Ј-dimethoxybutane-
2Ј,3Ј-diyl)-β--glucopyranoside (18a) (1.58 g, 79%) as a solid,
was concentrated and the residue purified by column chromatog-
raphy on silica gel (CH2Cl2/MeOH, 12:1) to give 9 (32 mg, 84%)
as a white solid. [α]2D8 = –57 (c = 0.5, MeOH). 1H NMR (300 MHz,
MeOD): δ = 8.24–8.18 (m, 2 H, Ph), 7.24–7.17 (m, 2 H, Ph), 5.28
(d, J1,2 = 8.5 Hz, 1 H, 1-H), 3.95 (dd, J2,3 = 10.4 Hz, J2,1 = 8.5 Hz,
1882
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 1876–1885

Cyanodeoxy-Glycosyl Derivatives as Substrates for Enzymatic Reactions
FULL PAPER
1 H, 2-H), 3.76 (ddd, J5,6 = 3.3 Hz, J5,6Ј = 7.4 Hz, J5,4 = 9.7 Hz, 1
H, 5-H), 3.61 (dd, J3,4 = 8.7 Hz, J3,2 = 10.4 Hz, 1 H, 3-H), 3.33
(dd, J4,3 = 8.7 Hz, J4,5 = 9.7 Hz, 1 H, 4-H), 3.01 (dd, J6,5 = 3.3,
1.46 mmol) in dry pyridine (3 mL) was added dropwise. The mix-
ture was stirred at room temp. for 2 h. Then H2O (0.5 mL) was
added, the solution stirred for 15 min and the mixture concentrated
2J6,6Ј = 17.1 Hz, 1 H, 6-H), 2.80 (dd, J6Ј,5 = 7.4 Hz, 1 H, 6Ј-H), under vacuum. The corresponding residue was dissolved in dichlo-
1.97 (s, 3 H, Me-CO) ppm. 13C NMR (75.4 MHz, MeOD): δ =
romethane and washed with HCl (1 ), sat. aq. NaHCO3 and brine.
173.9 (C=O), 163.4 (C-1 Ph), 144.2 (C-4 Ph), 126.7, 117.7 (Ph),
The organic layer was dried with Na2SO4, filtered, and concen-
118.5 (CN), 99.6 (C-1), 74.9 (C-3), 74.5 (C-4), 73.3 (C-5), 57.1 (C- trated. The corresponding residue was purified by column
2), 22.9 (NHCOCH3), 21.4 (C-6) ppm. CIHRMS: calcd. for
C15H17N3O7 + H: 352.1145; found 352.1173.
chromatography (diethyl ether) to give 23 (399 mg, 80%) as a white
foam. [α]2D8 = +25 (c = = 0.8, CH Cl ). IR: νmax = 3268, 3082, 1661,
˜
2
2
1
1593, 1346, 1113, 1071, 1042, 889, 746 cm–1. H NMR (300 MHz,
CDCl3): δ = 7.82 (m, 2 H, Ph), 7.36 (m, 2 H, Ph), 5.59 (br. d, JNH,2
= 7.5 Hz, 1 H, NH), 5.18 (d, J1,2 = 8.9 Hz, 1 H, 1-H), 4.32 (dd,
J3,4 = 9.7, J3,2 = 10.9 Hz, 1 H, 3-H), 4.27 (d, J6,5 = 3.1 Hz, 1 H, 6-
H), 3.76 (dt, J4,5 = 10.0 Hz, 1 H, 5-H), 3.62 (t, 1 H, 4-H), 3.26 and
3.18 (2s, 3 H each, OMe), 3.15 (ddd, 1 H, 2-H), 2.46 (s, 3 H, CH3
of tosyl), 2.00 (s, 3 H, Me-CO), 1.29 (2s, 3 H each, Me) ppm. 13C
NMR (75.4 MHz, CDCl3): δ = 170.7 (C=O), 145.1 (C-1 of Ph),
132.5 (C-4 of Ph), 129.8, 128.0 (Ph), 100.0, 99.8 (Cq BDA), 87.4
(C-1), 73.4 (C-5), 67.2, 67.1 (C-3, C-6), 65.9 (C-4), 55.2 (C-2), 48.3,
48.0 (2 OMe), 23.5 (NHCOCH3), 21.7 (CH3 of tosyl), 17.6, 17.5 (2
CH3) ppm.
2-Acetamido-2,6-dideoxy-β-D-gluco-heptopyranosylurononitrile
Azide (10): Compound 18b (762 mg, 2.12 mmol) was suspended in
dry CH2Cl2 (40 mL) under Ar. The reaction mixture was cooled
to –20 °C and 2,6-lutidine (0.626 mL, 5.29 mmol) was added drop-
wise, followed by triflic acid anhydride (1.071 mL, 6.35 mmol). The
reaction mixture was stirred at –15 °C and monitored by TLC
(CH2Cl2/MeOH, 12:1). After 4 h, the reaction was quenched by
adding sat. aq. NaHCO3 (40 mL), then CH2Cl2 (50 mL) was added
and the organic phase was separated. The aqueous phase was ex-
tracted with CH2Cl2 and the combined organic phases were washed
with water. All washing procedures were completed within 15 min
at 0 °C. The organic phase was dried with Na2SO4 and the solvents
evaporated under vacuum. The crude 2-acetamido-2-deoxy-3,4-O-
(2Ј,3Ј-dimethoxybutane-2Ј,3Ј-diyl)-6-O-trifluoromethylsulfonyl-β-
-glucopyranosyl azide (21) was immediately used in the next step
without further purification.
(1R,2R,4R,5R,7R,8R,9R)-N-(11-Azido-4,5-dimethoxy-4,5-dimethyl-
3,6,13-trioxa-10-azatricyclo[7.3.1.02,7]tridec-10-en-8-yl)acetamide
(25): KCN (160 mg, 2.46 mmol) was added in one portion to a
stirred solution of 23 (350 mg, 0.681 mmol) in DMF (10 mL). The
mixture was stirred at 60 °C for 2 h. Then the solution was concen-
trated under vacuum and the resulting residue was purified by col-
umn chromatography (diethyl ether/acetone, 20:1) to give 25
(145 mg, 58%) as a white solid. [α]2D8 = +25 (c = 0.8, CH2Cl2). IR:
The crude compound 21 was dissolved in dry CH2Cl2 (10 mL) un-
der argon and a solution of Bu4NCN (1.7 g, 6.33 mmol) in dry
CH2Cl2 (9 mL) was added at 0 °C whilst stirring. The reaction mix-
ture was allowed to react for 4 h at room temp. Then the reaction
mixture was diluted with CH2Cl2 (80 mL) and the organic phase
was washed with NaClO4 (2×40 mL) and water, dried (Na2SO4)
and the solvent evaporated under vacuum to dryness to give 2-
acetamido-6-cyano-2,6-dideoxy-3,4-O-(2Ј,3Ј-dimethoxybutane-
2Ј,3Ј-diyl)-β--glucopyranosyl azide (22) which was used without
purification in the next step.
ν
= 3289, 2949, 2191, 1665, 1549, 1425, 1140 cm–1. 1H NMR
˜
max
(300 MHz, CDCl3): δ = 5.77 (br. d, JNH,2 = 5.1 Hz, 1 H, NH), 5.71
(s, 1 H, 1-H), 4.73 (dd, J5,4 = 2.3, J5,6b = 5.2 Hz, 1 H, 5-H), 3.98
(dd, J3,4 = 9.0, J3,2 = 9.9 Hz, 1 H, 3-H), 3.92 (dd, 1 H, 2-H), 3.74
2
(d, 1 H, 6a-H), 3.64 (dd, 1 H, 4-H), 3.54 (dd, J6b,5 = 12.6, 1 H,
6b-H), 3.30 and 3.25 (2s, 3 H each, OMe), 2.10 (s, 3 H, Me-CO),
1.33 and 1.30 (2s, 3 H each, Me) ppm. 13C NMR (75.4 MHz,
CDCl3): δ = 170.4 (C=O), 100.7, (Cq BDA), 93.2 (C-1), 76.4 (C-5),
71.6 (C-4), 66.4 (C-3), 54.6 (C-2), 52.8 (C-6), 48.1 (2 OMe), 23.2
(NHCOCH3), 17.6 (2 CH3) ppm. FABMS: m/z = 488 (30) [M +
Na]+. CIMSHR: calcd. for C21H27N3O9 + H: 466.1826; found
466.1802.
Compound 22 (506 mg, 1.37 mmol) was dissolved in TFA/H2O, 2:1
(20 mL) at 0 °C and stirred at room temp. for 5 h (TLC: CH2Cl2/
MeOH, 8:1). The solvent was evaporated under vacuum. Purifica-
tion by flash chromatography on silica gel (CH2Cl2/MeOH, 10:1)
afforded 10 (75 mg, 0.294 mmol; 16% overall from 18b). [α]2D8
=
–17.9 (c = 0.67, MeOH). IR: ν
= 3407, 2122, 1649, 1559, 1377,
˜
max
1202, 1130, 1071, 1020, 721 cm–1. H NMR (300 MHz, MeOD): δ
= 4.62 (d, J1,2 = 9.3 Hz, 1 H, 1-H), 3.72 (dd, J2,3 = 10.2 Hz, 1 H,
2-H), 3.62 (ddd, J5,4 = 9.0, J5,6 = 3.6, J5,6Ј = 6.3 Hz, 1 H, 5-H),
3.50 (dd, J3,4 = 8.7 Hz, 1 H, 3-H), 3.61 (t, 1 H, 4-H), 3.02 (dd,
2J6,6Ј = 17.1 Hz, 1 H, 6-H), 2.86 (dd, 1 H, 6Ј-H), 2.02 (s, 3 H, Me-
CO) ppm. 13C NMR (75.4 MHz, MeOD): δ = 174.1 (C=O), 118.4
(CN), 90.2 (C-1), 75.4 (C-3), 75.1 (C-5), 74.6 (C-4), 56.9 (C-2), 23.1
(NHCOCH3), 21.6 (C-6) ppm. CIHRMS: calcd. for C9H13N5O4 +
H: 256.1046; found 256.1060.
1
p-Nitrophenyl 2-Acetamido-2-deoxy-3,4-O-(2Ј,3Ј-dimethoxybutane-
2Ј,3Ј-diyl)-β-D-glucopyranosiduronitrile (27): A solution of DMSO
(121 µL, 1.71 mmol) in anhydrous CH2Cl2 (0.5 mL) was added
dropwise to a stirred solution of oxalyl chloride (75 µL,
0.855 mmol) in anhydrous CH2Cl2 (2 mL) at –70 °C. The mixture
was stirred at –70 °C for 15 min and then a solution of alcohol
18a (300 mg, 0.658 mmol) in anhydrous CH2Cl2 (2 mL) was added
dropwise. The mixture was stirred for 1 h at –70 °C and then Et3N
(0.5 mL, 3.29 mmol) was added. The mixture was allowed to reach
room temp. and then was diluted with CH2Cl2 and washed with
water and brine. The organic phase was dried (Na2SO4) and evapo-
rated to give crude p-nitrophenyl 2-acetamido-2-deoxy-3,4-O-
(2Ј,3Ј-dimethoxybutane-2Ј,3Ј-diyl)-β--gluco-hexodialdo-1,5-
pyranoside (26), which was used in the following step without fur-
ther purification. CIMSHR: m/z = 455.1688 (calcd. for
C20H26N2O10 + H: 455.1666).
2-Acetamido-2-deoxy-3,4-O-(2Ј,3Ј-dimethoxybutane-2Ј,3Ј-diyl)-6-O-
tosyl-β-D-glucopyranosyl Azide (23): 2-Acetamido-2-deoxy-β--glu-
copyranosyl azide[19] (12b) (1 g, 4.06 mmol) was dissolved in dry
methanol (46 mL) and butane-2,3-dione (1.77 mL; 20.3 mmol),
CH(OMe)3 (6.67 mL, 60.9 mmol) and CSA (255 mg; 1.096 mmol)
were added under argon. The reaction mixture was refluxed for 8 h
and then concentrated under vacuum to dryness. The crude prod-
uct, 2-acetamido-2-deoxy-3,4-O-(2Ј,3Ј-dimethoxybutane-2Ј,3Ј-
diyl)-β--glucopyranosyl azide (18b), was used without purification
in the next step.
Iodine (31 mg, 0.121 mmol) was added to a solution of aldehyde
26 (50 mg, 0.110 mmol) in a solution of ammonia (1.1 mL) and
THF (0.15 mL). The mixture was stirred at room temp. for 5 h and
then a 5% aqueous solution of Na2S2O3 was added and the mixture
was stirred for 10 min. The mixture was extracted with CH2Cl2
A solution of alcohol 18b (350 mg, 0.97 mmol) in dry pyridine
(3 mL) was cooled to 0 °C and a solution of TsCl (277 mg,
Eur. J. Org. Chem. 2006, 1876–1885
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
1883

V. K rˇen, I. Robina et al.
FULL PAPER
[7]
a) T. Kimura, S. Takayama, H. Huang, C.-H. Wong, Angew.
Chem. Int. Ed. Engl. 1996, 35, 2348–2351; b) A. MacManus,
U. Grabowska, K. Biggadike, M. I. Bird, S. Davies, E. N. Vulf-
son, T. Gallagher, J. Chem. Soc., Perkin Trans. 1 1999, 295–305;
c) L. Husˇáková, S. Riva, M. Casali, S. Nicotra, M. Kuzma, Z.
Hunˇková, V. Krˇen, Carbohydr. Res. 2001, 331, 143–148; d) P.
Fialová, D.-J. Namdjou, R. Ettrich, V. Prˇikrylová, J. Rauvol-
fová, K. Krˇenek, M. Kuzma, L. Elling, K. Bezousˇka, V. Krˇen,
Adv. Synth. Catal. 2005, 347, 997–1006.
(3×15 mL) and the combined organic phases were then dried and
the solvents evaporated. The residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH, 50:1) to give 27
(35 mg, 71%). [α]2D5 = +25 (c = 1, CH Cl ). IR: ν
= 3283, 3082,
˜
max
2
2
1667, 1593, 1346, 1111, 1067, 1058, 887, 745 cm–1 1H NMR
.
(300 MHz, CDCl3): δ = 8.22–8.20 (m, 2 H, Ph), 7.12–7.10 (m, 2 H,
Ph), 5.96 (d, J1,2 = 6.9 Hz, 1 H, 1-H), 5.76 (d, JNH,2 = 6.9 Hz, 1
H, NH), 4.51 (d, J5,4 = 10.2 Hz, 1 H, 5-H), 4.48 (t, J4,3 = 10.3 Hz,
1 H, 4-H), 3.97 (t, J3,2 = 9.9 Hz,1 H, 3-H), 3.48 (dt, 1 H, 2-H),
3.35 and 3.23 (2s, 3 H each, OMe), 2.00 (s, 3 H, Me-CO), 1.35 and
1.34 (2s, 3 H each, Me) ppm. 13C NMR (125.7 MHz, CDCl3): δ =
171.1 (C=O), 161.2 (C-1 Ph), 143.7 (C-4 Ph), 126.0, 117.1 (Ph),
115.2 (CN), 100.6, 100.5 (Cq BDA), 98.3 (C-1), 68.9 (C-3), 66.0 (C-
4), 63.5 (C-5), 56.2 (C-2), 48.5, 48.3 (2 OMe), 23.6 (NHCOCH3),
17.7, 17.5 (2 CH3) ppm. FABMS: m/z = 474 (40) [M + Na]+, 420
(80) [M – OMe]+. CIMSHR: calcd. for C20H25N3O9 + H: 452.1669;
found 452.1660.
[8]
[9]
S. Murahashi, “Three Carbon-Heteroatom Bonds: Nitriles,
Isocyanides, and Derivatives”, in Science of Synthesis, Thieme,
Stuttgart, 2004, vol. 19.
a) L. Hough, A. C. Richardson in The Carbohydrates. Chemis-
try and Biochemistry (Eds.: W. Pigman, D. Horton), Academic
Press, San Diego, 1972, vol. IA, pp. 118–120; b) G. D.
McGinnis, Carbohydr. Res. 1982, 108, 284–292; c) P. Ermert,
A. Vasella, Helv. Chim. Acta 1991, 74, 2043–2053; d) A. D.
Doresey, J. E. Barbarow, D. Trauner, Org. Lett. 2003, 5, 3237–
3239; e) O. Attanasi, P. Palma, F. Serra-Zanetti, Synthesis 1983,
741–742; f) A. Fürstner, J.-P. Praly, Angew. Chem. Int. Ed. Engl.
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of TFA/H2O, 2:1 (1.5 mL) for 3 h at 40 °C. The mixture was con-
centrated to give pure 8 (23 mg, 93%) as a white solid. [α]2D5 = –64
(c = 0.45, MeOH). 1H NMR (300 MHz, MeOD): δ = 8.25–8.20
(m, 2 H, Ph), 7.25–7.16 (m, 2 H, Ph), 4.58 (d, J1,2 = 8.1 Hz, 1 H,
1-H), 4.58 (d, J5,4 = 8.7 Hz, 1 H, 5-H), 3.94 (dd, J2,3 = 9.6 Hz, 1
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173.9 (C=O), 163.0 (C-1 Ph), 144.7 (C-4 Ph), 126.7, 117.9 (Ph),
117.5 (CN), 100.0 (C-1), 74.0, 73.4 (C-3, C-4), 67.0 (C-5), 56.9 (C-
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