Synthesis and characterization of novel fluorescent N-glycoconjugates

Article abstract of DOI:10.1016/j.tet.2005.07.006

Novel fluorescent N-glycoconjugates containing d-glucose, glycine and coumarin or naphthalenetriazole derivatives were prepared by peptide synthesis type methods. The fluorescence properties (spectra, quantum yields) of the compounds were evaluated.

Full text of DOI:10.1016/j.tet.2005.07.006

Tetrahedron 61 (2005) 8625–8632
Synthesis and characterization of novel fluorescent
N-glycoconjugates
a
a
a
Ana P. Esteves,a, Lıgia M. Rodrigues, Marılia E. Silva, Sanjay Gupta,
*
´
Ana M. F. Oliveira-Campos, Oldrich Machalicky and Antonio J. Mendonc¸a
´
a
b
c
´
aDepartment of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal
bDepartment of Organic Technology, Faculty of Chemical Technology, University of Pardubice, Czech Republic
cDepartment of Chemistry, University of Beira Interior, Covilha˜, Portugal
Received 29 March 2005; revised 30 June 2005; accepted 5 July 2005
Available online 26 July 2005
Abstract—Novel fluorescent N-glycoconjugates containing D-glucose, glycine and coumarin or naphthalenetriazole derivatives were
prepared by peptide synthesis type methods. The fluorescence properties (spectra, quantum yields) of the compounds were evaluated.
q 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Sugars have also been used in the development of
fluorescent reagents because they confer water solubility
to organic fluorophores with no significant change in
absorption and fluorescence properties.20
The glycoconjugates have an enormous potential in drug
design.1 Glycoproteins are widely distributed in nature and
the sugar fragment influences their conformation and
folding,2 their properties such as solubility, bioavailability,
thermal and proteolytical stability,3 and enhances the
transport through cell membranes.2,4–6
The need for homogeneous samples of the desired
glycopeptides leads to significant development of several
synthetic strategies.7,8 The glycoamino acids are the
building blocks for glycopeptide synthesis and several
routes for their preparation have been reported.21–25 The
most commonly employed methods for the preparation of
N-glycopeptides proceed through reduction of glycosyl
azide to a labile intermediate glycosylamine that is
subsequently condensed with the appropriately protected
amino acid derivative.15,21,26
Glycopeptides, (which retain the carbohydrate–peptide
linkage of a glycoprotein but lack its size and complexity)
can mimic natural fragments of glycoproteins and have been
largely used as targets for therapeutic agents and as models
for biologically relevant systems.7–11
Another important field that has registered development is
the application of fluorescent labels for several compounds
with potential biological activity.12,13 Among them, the
fluorescent peptides7,14–17 have a large number of appli-
cations in biochemistry and biology, namely in studies of
protein interactions and conformational analysis.
The work presented in this paper, is part of an ongoing
project towards of the synthesis of fluorescent N-glycopep-
tides. Model compounds with a fluorescent aminoacid (the
fluorescent labels were introduced at the C-terminus of
glycine through an amide bond) were prepared to test the
methodologies of synthesis and evaluate the influence either
of the amino acid or/and the sugar in the fluorescent
properties of the fluorophore (coumarin-3-carboxilic acid
and 4-(naphtho[1,2-d][1,2,3]triazol-2-yl)benzoic acid). The
labeled amino acid will be used in further work as building
block for sequence analogues of RGD (arginine-glycine-
aspartic acid) motif for binding essays. The absorption
and fluorescence properties of these compounds were
determined, in acetonitrile, and compared.
Fluorescent markers are also being investigated for in vivo
imaging studies, for example, in Alzheimer disease.18 The
most used fluorescent markers for peptides are rhodamine,
fluorescein, coumarin and their derivatives.16,19
Keywords: N-Glycoconjugates; Fluorescent; Coumarin; Naphthalenetriazole.
*
Corresponding author. Tel.: C351253604373; fax: C351253678983;
e-mail: aesteves@quimica.uminho.pt
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.07.006

8626
A. P. Esteves et al. / Tetrahedron 61 (2005) 8625–8632
2. Results and discussion
The steady-state fluorescence spectrum of 2 shows an
emission band also with vibrational structure (maxima at
370 and 380 nm). The transformation of the carboxyl group
of 2 to amido groups (4, 6 and 10) does not affect either the
shape or position of the fluorescence band but it has
considerable effect on fluorescence quantum yield (53% for
2 and close to 100% for the other compounds, standard
deviation is %2%). The relatively high absorption
coefficient of the first absorption band of 2 and high
fluorescence quantum yield lead to the conclusion that the
first absorption band of the studied triazole derivatives has
the character of S0–S1(pp*) transition. Moreover, the high
quantum yield shows that none of possible triplet np* states
(nonbonding electrons on two heterocyclic nitrogens and
nonbonding electrons on carbonyl group) is situated below
S1(pp*) state.
In this work, the preparation of new fluorescent N-glycoconju-
gates based on acetylated D-glucose, glycine and coumarin
or naphthalenetriazole derivatives was studied (Scheme 1).
Evaluation of the fluorescence properties shown by different
substrates was also carried out.
Treatment of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl
bromide with sodium azide27 gave the corresponding b-D-
glucopyranosyl azide in 86% yield after recrystallisation.
Catalytic hydrogenation of the azide28 with Pd/C afforded
1
the glucosylamine as the b anomer (based on H NMR;
JH1H2Z8.7 Hz) in almost quantitative yield (94%) and it
was used with no further purification. Compounds 4 and 5
were obtained by the mixed anhydride method in 13 and
42% yield, respectively, but the same method was
unsuccessful for the synthesis of compounds 6 and 7.
Therefore, glycine was coupled to the fluorescent0dye by the
HBTU method (O-(benzotriazol-1-yl)-N,N,N ,N0,-tetra-
methyl-uroniumhexa-fluorophosphate) for compound 6
and by the DCC/HOBt (N,N0-dicyclohexylcarbodiimide/1-
hydroxy-benzotriazole) method for 7 with yields of 45%.
Hydrolysis of compounds 6 and 7 afforded the correspond-
ing acids 8 and 9 in yields over 80%. Glycoconjugates 10
and 11 were synthesised by DCC/HOBt method in moderate
yields, 45 and 46%, respectively. The synthesis of 10 was
attempted by the Staudinger reaction between glycosyl
azide and amino acid-dye substrate 8 in the presence of
tributyl phosphine.29 This method afforded the a-anomer, as
deduced by 1H NMR spectrum (JZ3.6 Hz for the anomeric
proton), instead of the b-anomer obtained by the DCC/
HOBt method.
The steady-state fluorescence spectrum of 3 displays a broad
emission band with maximum at 400 nm (shoulder at
370 nm) similar to the emission spectra of derivatives 5, 7
and 11. Fluorescence quantum yields were 1.8% (standard
deviation is 0.1%) for 3 and 2.1–2.4% for compounds 5, 7
and 11.
As the spectra of compounds 2, 4, 6 and 10 and 3, 5,7 and 11
show the same patterns, it is possible to conclude that the
substitution on carboxylic groups of both tested fluor-
escence probes does not affect the p-electronic structure of
fluorophores. Therefore, character, energy and sequence of
excited states do not change.
3. Conclusions
The fluorescent probes studied show that the transformation
of the carboxylic group into amido group in the different
N-conjugates does not affect either the shape or the position
of their fluorescence bands. On the other hand, in case of
naphthalenetriazole derivatives, it has considerable effect on
fluorescence quantum yield w50% and almost 100% for the
original carboxylic acid and amides, respectively. No
appreciable changes in low quantum fluorescence yields
(2%) were detected among original coumarin-3-carboxylic
acid and its amido N-conjugates.
The compounds were isolated and characterized by NMR
spectroscopy (1H, 13C, HMQC, HMBC) and elemental
analysis or HRMS.
As the compounds prepared differ markedly in water
solubility, acetonitrile, a common polar aprotic solvent,
was chosen to measure their spectral properties. Besides,
acetonitrile avoids changes in spectral curves resulting from
dissociation of carboxylic hydrogen in non conjugated
probes. Then it was possible to compare a substitution effect
on fluorescence properties of starting dyes and their
conjugates.
Our findings showed that the usual methods used in peptide
synthesis gave acceptable results for the coupling of the
glycosylamine to a fluorescent carboxylic dye or to
fluorescent amino acid.
The spectra for compounds 2 and 10 and 3 and 11 are shown
on Figures 1 and 2, respectively.
This methodology may provide a useful contribution for the
design of new fluorescent glycopeptides.
UV–vis absorption spectrum of 2 shows vibrational
structure at 340 (3maxz24,500 dm3 molK1 cmK1) and
360 nm, while for compound 3, only a broad band at
300 nm (3maxz11,400 dm3 molK1 cmK1) and a shoulder
around 330 nm are observed. Replacement of the carboxyl
group by amido groups (such as in 4, 6 and 10) has no
significant effect on the position or molar absorptivity of the
long-wavelength absorption bands (Fig. 1). A similar
observation may be made for the position of the long-
wavelength absorption bands of 3, 5, 7 and 11 (Fig. 2).
However, the carboxylic acid 3 has a lower molar
absorptivity (3maxz11,400 dm3 molK1 cmK1) than the
corresponding amides (3maxz13,200 dm3 molK1 cmK1).
4. Experimental
4.1. General
Dry solvents, referred to in the ensuing experiments, were
prepared as follows: Acetone was refluxed over magnesium
sulphate, decanted, stirred overnight with calcium
chloride, decanted, refluxed over fresh magnesium sulphate,
˚
distilled from it and stored over 4 A molecular sieves;

A. P. Esteves et al. / Tetrahedron 61 (2005) 8625–8632
8627
Scheme 1. Reagents and conditions: (i) ClCO2Et, Et3N, DMF, rt, 48 h; (ii) ClCO2Et, Et3N, acetone, reflux, 3 h; (iii) HBTU, DIPEA, DMF, rt, 12 h; (iv) DCC/
HOBt, Et3N, EtOAc, rt, 12 h; (v) (1) NaOH, MeOH, rt, 4 h; (2) HCl; (vi) DCC/HOBt, DMF, rt, 12 h; (vii) DCC/HOBt, DMF, rt, 48 h.

8628
A. P. Esteves et al. / Tetrahedron 61 (2005) 8625–8632
Figure 1. Normalised absorption (curve 1) and fluorescence emission (curve 2) spectra of naphthalenetriazole derivative 2 (a) and 10 (b).
dichloromethane was refluxed over phosphorus pentoxide,
˚
distilled from it and stored over 4 A molecular sieves.
Light petroleum refers to the fraction boiling in the range
40–60 8C. Sodium azide was dried in vacuo overnight.
spectrofluorometer. Quantum yields were determined in
acetonitrile using quinine sulphate as the fluorescence
standard. The path length was 1 cm in quartz cells.
4.1.1. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylazide.26
Dry sodium azide (0.163 g, 2.5 mmol) was added to a
solution of a-acetobromoglucose (0.411 g, 1.0 mmol) in dry
acetone (40 mL) and the mixture refluxed for 7 h. Then it
was portioned between dichloromethane (40 mL) and cold
water (30 mL). The aqueous phase was extracted with
dichloromethane, dried (MgSO4) and evaporated affording
the impure azide as a light yellow solid in quantitative yield.
Recrystallisation from dichloromethane/diethyl ether/light
petroleum afforded the azide as a crystalline white solid in
86% yield, mp 125.6–126.5 8C (lit.30 126–127 8C). [a]DK
29 (c 2.0, CHCl3); nmax (Nujol) 2115; 1746; 1213; 1105–
1042 cmK1. 1H NMR (CDCl3) d (ppm): 2.02, 2.04, 2.09 and
2.11 (4s, 12H, 4!OCH3), 3.80 (ddd, JZ10.0, 4.8, 2.4 Hz,
1H, H-5), 4.18 (dd, JZ12.6, 2.4 Hz, 1H, Ha-6), 4.29 (dd,
JZ12.6, 4.8 Hz, 1H, Hb-6), 4.66 (d, JZ9.0 Hz, 1H, H-1),
4.98 (app t, JZ9.3, 9.6 Hz, 1H, H-2), 5.11 (app t, JZ9.6 Hz,
1H, H-4), 5.23 (app t, JZ9.3, 9.6 Hz, 1H, H-3).
The reactions were followed by thin layer chromatography,
which was carried out on pre-coated plates (Merck
Kieselgel 60F254) and compounds were visualized by
UV-light and exposure to vaporized iodine. Merck
Kieselgel 60 (230–400 mesh) was used in column
chromatography. Melting points were determined on a
Stuart SMP3 melting point apparatus and are uncorrected.
Specific optical rotations were calculated from measure-
ments carried out with an Optical Activity-AA-1000
Polarimeter at 27 8C. IR spectra were recorded on a FTIR/
1
Diffus Bomem MB spectrometer. H and 13C NMR data
were recorded on a Varian Unity Plus 300 Spectrometer in
CDCl3 and DMSO-d6; d ppm were measured versus residual
peak of the solvent and J values are given in Hz. HMQC
and HMBC experiments were carried out for complete
assignment of proton and carbon signals in the NMR spectra
(in descripton of 1H signals app means apparent). Elemental
analyses were carried out on a Leco CHNS 932 instrument.
MS spectra were obtained in a VG Autospec mass
spectrometer. Absorption spectra were recorded using a
Perkin-Elmer Lambda 35 spectrophotometer. Steady-state
fluorescence spectra were measured on a Perkin-Elmer LS-5
4.1.2. 2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosylamine28
(1). To a solution of the 2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl azide (0.336 g, 0.9 mmol) in ethyl acetate/
methanol 1:1 (30 mL), Pd/C 5% (0.119 g) was added. The
Figure 2. Normalised absorption (curve 1) and fluorescence emission (curve 2) spectra of coumarin derivatives 3 (a) and 11 (b).

A. P. Esteves et al. / Tetrahedron 61 (2005) 8625–8632
8629
resulting mixture was deaerated for 30 min under a nitrogen
stream. Then it was submitted to a hydrogen atmosphere, at
normal pressure, for 3 h. The final mixture was filtered
through a pad of Celite and concentrated affording the
product as a yellowish solid in 94% that was used with no
further purification, mp 105–106 8C, mp 115–116 8C after
crystallization from methanol/light petroleum (lit.,31 mp not
reported, unstable, mp32 138–140 8C). [a]DC16 (c 2.0,
CHCl3); nmax (Nujol) 3410; 3337; 2725; 2668; 2257; 1754;
amine (1) (0.251 g, 0.723 mmol) and the resulting mixture
was refluxed for 3 h and evaporated. The crude residue
(0.357 g) was purified by flash column chromatography
using ethyl acetate/diethyl ether 1:5 as eluent. The isolated
fraction (0.158 g, 42% yield) was recrystallised from
dichloromethane/diethyl ether/light petroleum affording
the amide 5 as a white solid, mp 160.0–162.3 8C. [a]DK
26 (c 2.0, CHCl3); nmax (Nujol) 3306; 2725; 2670; 1748;
1722; 1672; 1610; 1376; 1214; 1159–1034 cmK1. 1H NMR
(CDCl3) d (ppm): 2.01, 2.04, 2.05 and 2.10 (4s, 12H, 4!
OCH3), 3.90 (ddd, JZ10.0, 4.0, 2.1 Hz, 1H, H-50), 4.14 (dd,
JZ12.6, 2.1 Hz, 1H, Ha-60), 4.30 (dd, JZ12.6, 4.0 Hz, 1H,
Hb-60), 5.17 (app q, JZ9.6, 9.9, 9.6 Hz, 2H, H-20 and H-40),
5.36 (app t, JZ9.3, 9.6 Hz, 1H, H-30), 5.50 (app t, JZ9.0,
9.3 Hz, 1H, H-10), 7.38–7.44 (m, 2H, H-8 and H-7), 7.68–
7.73 (m, 2H, H-6 and H-5), 8.90 (s, 1H, 4-H), 9.31 (d, JZ
9.6 Hz, 1H, NH). 13C NMR (CDCl3) d (ppm): 20.47, 20.54,
20.56 and 20.69 (4!CH3), 61.27 (C-60), 68.04 (C-20 or
C-40), 70.30 (C-20 or C-40), 73.01 (C-30), 73.63 (C-50), 78.16
(C-10), 116.75 (C-7), 117.43 (C-3), 118.28 (C-8a), 125.36
(C-8), 129.95 (C-5), 134.65 (C-6), 149.48 (C-4), 154.62
(C-4a), 160.76 (C]O, coumarin), 162.25 (C]O, amide),
169.39, 169.73, 170.03 and 170.65 (4!C]O, acetyl).
Anal. Calcd for C24H25NO12: C, 55.48; H, 4.86; N, 2.70.
Found: C, 55.47; H, 5.08; N, 2.56. FAB (MCC1) 520.22.
1
1639; 1377; 1224; 1095–1062 cmK1. H NMR (CDCl3) d
(ppm): 2.02, 2.04, 2.08 and 2.10 (4s, 12H, 4!OCH3),
2.02–2.10 (br s, 2H, NH2), 3.70 (ddd, JZ10.0, 4.8, 2.4 Hz,
1H, H-5), 4.12 (dd, JZ12.3, 2.4 Hz, 1H, Ha-6), 4.20 (d, JZ
8.7 Hz, 1H, H-1), 4.24 (dd, JZ12.3, 4.8 Hz, 1H, Hb-6), 4.84
(app t, JZ9.3, 9.6 Hz, 1H, H-2), 5.05 (app t, JZ9.6 Hz, 1H,
H-4), 5.25 (app t, JZ9.3, 9.6 Hz, 1H, H-3). Anal. Calcd for
C14H21NO9: C, 48.41; H, 6.10; N, 4.03. Found: C, 48.44; H,
6.09; N, 4.07.
4.1.3. N-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)
naphtho[1,2-d][1,2,3]triazol-2-yl-benzamide (4). To a
solution of compound 2 (0.280 g, 0.969 mmol) in DMF
(200 mL) at K5 8C, triethylamine (0.098 g, 0.134 mL,
0.969 mmol) and ethyl chloroformate (0.108 g, 0.096 mL,
0.969 mmol) were added. The solid precipitated was filtered
off and the filtrate was maintained at K5 8C. Then, it was
added to the amine (1) (0.337 g, 0.969 mmol). The resulting
mixture was stirred at rt for 48 h. After evaporation, the
crude residue (0.404 g) was purified by flash column
chromatography using diethyl ether/light petroleum 2:1–2.5:1
as eluent. The isolated fraction (0.078 g, 13% yield) was
recrystallised from ethyl acetate/light petroleum affording
the amide 4 as a light pink solid, mp 230–233 8C (dec).
[a]DK53 (c 1.0, CHCl3); nmax (Nujol) 3430; 1748; 1680;
4.1.5. 4-Naphtho[1,2-d][1,2,3]triazol-2-yl-benzoylamino
acetic acid methyl ester (6). To a solution of 2 (0.145 g,
0.5 mmol), HGlyOMe, HCl (0.063 g, 0.5 mmol) and
DIPEA (0.170 mL, 1 mmol) in DMF (10 mL), cooled to
C5 8C, HBTU (0.190 g, 0.5 mmol) was added. The reaction
mixture was stirred at rt overnight and diluted with water
(50 mL). After cooling for 3 h the solid precipitated was
filtered and washed with water. The crude solid was purified
by column chromatography using ethyl acetate/dichloro-
methane 5:1 as eluent. Recrystallisation from methanol/
dichloromethane/light petroleum afforded the pure com-
pound as an orange solid in 45% yield, mp 229–231 8C.
[a]DK2 (c 1.0, DMF); nmax (KBr) 3362; 1754; 1643; 1606;
1
1607; 1370; 1291; 1228; 1217 cmK1. H NMR (CDCl3) d
(ppm): 2.07, 2.08, 2.09 and 2.11 (4s, 12H, 4!OCH3), 3.95
(ddd, JZ10.0, 4.0, 1.8 Hz, 1H, H-50), 4.15 (dd, JZ12.06,
1.8 Hz, 1H, Ha-60), 4.40 (dd, JZ12.6, 4.0 Hz, 1H, Hb-6 ),
5.11 (app t, JZ9.6, 9.9 Hz, 1H, H-20 or H-40), 5.15 (app t,
JZ9.3, 10.2 Hz, 1H, H-20 or H-40), 5.45 (app t, JZ9.9,
9.6 Hz, 1H, H-30), 5.48 (t, JZ9.0 Hz, 1H, H-10), 7.16 (d, JZ
9.0 Hz, 1H, NH), 7.62–7.82 (m, 4H, H-5, H-6, H-9, H-8),
7.92 (dd, JZ8.1, 1.8 Hz, 1H, H-4), 7.98 (d, JZ9.0 Hz, 2H,
H-300 and H-500), 8.50 (d, JZ9.0 Hz, 2H, H-200 and H-600),
8.65 (dd, JZ7.5, 1.8 Hz, 1H, H-7). 13C NMR (CDCl3) d
(ppm): 20.77, 20.74, 20.60 (4!CH3), 61.58 (C-60), 68.16
(C-200 or C-40), 70.87 (C-20 or C-40), 72.51 (C-30), 73.65
(C-5 ), 79.03 (C-10), 116.31 (C-8 or C-9), 119.96 (C-200 and
C-600), 123.40 (C-7), 124.77 (C-3b), 127.73 (C-5), 127.92
(C-6), 128.70 (C-300and C-500), 129.00 (C-4), 130.42 (C-8 or
C-9), 131.95 (C-400), 132.51 (C-7a), 143.03 (C-100), 143.42
(C-3a), 144.03 (C-9a), 166.10 (C]O from dye), 169.60,
169.88, 170.64 and 171.74 (4!C]O, acetyl). HRMS
(FAB) calcd for C31H30N4O10 (MCC1) 619.2040, found
619.2051.
1367; 1295; 1206; 1179 cmK1 1H NMR (DMSO-d6) d
.
(ppm): 3.67 (s, 3H, OCH3), 4.06 (d, JZ5.7 Hz, 2H, CH2),
7.69–7.79 (m, 2H, H-5 and H-6), 7.91 (s, 2H, H-8 and H-9),
8.07 (dd, JZ6.9, 2.0 Hz, 1H, H-4), 8.15 (d, JZ8.7 Hz, 2H,
H-200 and H-600), 8.42 (d, JZ8.7 Hz, 2H, H-300and H-500),
8.55 (dd, JZ7.2, 2.0 Hz, 1H, H-7), 9.19 (t, JZ5.7 Hz, 1H,
NH). 13C NMR (DMSO-d6) d (ppm): 41.35 (CH2), 51.87
(OCH3), 116.27 (C-8 or C-9), 119.46 (C-300 and C-500),
122.88 (C-7), 123.8700(C-3b), 128.18 (C-5), 128.30 (C-6),
129.18 (C-200 and C-6 ), 129.39 (C-4), 130.55 (C-8 or C-9),
132.17 (C-7a), 133.38 (C-400), 141.49 (C-100), 142.53 (C-3a),
143.40 (C-9a), 165.66 (C]O, amide), 170.38 (C]O,
ester). Anal. Calcd for C20H16N5O3: C, 66.66; H, 4.48; N,
15.55. Found: C, 66.25; H, 4.57; N, 15.03. FAB (MCC1)
360.15.
4.1.6. [(2-Oxo-2H-chromene-3-carbonyl)-amino]-acetic
acid methyl ester (7). To a solution of 3 (1.90 g,
10 mmol) in ethyl acetate (100 mL) HOBt (1.70 g,
10 mmol) was added. After cooling to 0 8C, DCC (2.16 g,
10.5 mmol) was added followed by addition of the
HGlyOMe, HCl (1.26 g, 10 mmol) and triethylamine
(1.4 mL, 10 mmol). The reaction mixture was stirred at rt
overnight and the insoluble materials were filtered off. The
4.1.4. N-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-(2-
oxo-2H-chromene)-3-carboxamide (5). To a solution of
compound 3 (0.138 g, 0.723 mmol) in dry acetone (20 mL)
at K5 8C, triethylamine (0.146 g, 0.200 mL, 1.45 mmol)
and ethyl chloroformate (0.157 g, 0.143 mL, 1.45 mmol)
were added. The solid precipitated was filtered off and the
filtrate was maintained at K5 8C. Then, it was added to the

8630
A. P. Esteves et al. / Tetrahedron 61 (2005) 8625–8632
organic layer was successively washed with 5% NaHCO3,
water, 5% citric acid, water and dried. The solvent was
removed under reduced pressure and the resulting product
(1.17 g, 45%) crystallized from ethyl acetate/light pet-
roleum, mp 184.8–187.4 8C. [a]DK2 (c 1.0, DMF); nmax
(KBr) 3332; 1748; 1713; 1607; 1372; 1294; 1218 cmK1. 1H
NMR (CDCl3) d (ppm): 3.81 (s, 3H, OCH3), 4.27 (d, JZ
6.0 Hz, 2H, CH2), 7.37–7.44 (m, 2H, H-8 and H-6), 7.67–
7.72 (m, 2H, H-7and H-5), 8.92 (s, 1H, 4-H), 9.26 (br s, 1H,
NH). 13C NMR (CDCl3) d (ppm): 41.62 (CH2), 52.35
(OCH3), 116.64 (C-8), 117.90 (C-3), 118.44 (C-8a), 125.28
(C-6), 129.84 (C-5), 134.23 (C-7), 148.69 (C-4), 154.48
(C-4a), 161.16 (C]O, coumarin), 161.83 (C]O, amide),
169.67 (C]O, ester). Anal. calcd for C13H11NO5: C, 59.76;
H, 4.25; N, 5.36. Found: C, 59.93; H, 4.46; N, 5.18. FAB
(MCC1) 262.05.
(C-8a), 125.19 (C-6), 130.43 (C-5), 134.32 (C-7), 148.12
(C-4), 154.00 (C-4a), 160.39 (C]O, coumarin), 161.18
(C]O, amide), 170.81 (C]O, acid). Anal. Calcd for
C12H9NO5: C, 58.18; H, 3.66; N, 5.65. Found: C, 58.10; H,
3.93; N, 5.60.
4.1.9. N-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-4-
naphtho[1,2-d][1,2,3]triazol-2-yl-benzoylamino aceta-
mide (10). To a solution of 8 (0.258 g, 0.744 mmol) in
DMF (10 mL), glycosylamine 1 (0.258 g, 0.744 mmol) and
HOBt (0.111 g, 0.818 mmol) were added. After cooling to
C5 8C, DCC (0.161 g, 0.781 mmol) was added and the
resulting mixture stirred at rt overnight. Then, water
(50 mL) was added and the mixture cooled for 3 h. The
solid precipitated was filtered, dried (0.414 g, 82%) and
purified by flash column chromatography using ethyl
acetate/dichloromethane 5:1 as eluent. The isolated fraction
(0.226 g, 45%) was recrystallised from dichloromethane/
light petroleum, affording pure 10 as an orange solid, mp
232.2–233.0 8C. [a]DC5 (c 2.0, CHCl3); nmax (neat) 3413;
4.1.7. 4-Naphtho[1,2-d][1,2,3]triazol-2-yl-benzoylamino
acetic acid (8). The ester 6 (0.268 g, 0.744 mmol) was
dissolved in methanol (1 mL) and 1 M NaOH (1.9 mL,
1.861 mmol) added. The mixture was stirred at rt for 4 h and
1 M HCl (0.744 mL, 0.744 mmol) was added. The methanol
was removed under reduced pressure and the residue thus,
obtained cooled in an ice bath and acidified to pHw2–3
with 1 M HCl with vigorous stirring for 1 h. The solid
precipitated was filtered, washed with water and dried.
Compound 8 was isolated in 87% yield and it was used
without further purification, mp 209–213 8C. [a]DK4 (c 1.0,
DMF); nmax (KBr) 3403; 2554; 1941; 1681; 1633; 1605;
1366; 1311; 1289; 1228; 1167 cmK1. 1H NMR (DMSO-d6)
d (ppm): 3.97 (d, JZ5.7 Hz, 2H, CH2), 7.68–7.80 (m, 2H,
H-5 and H-6), 7.92 (s, 2H, H-8 and H-9), 8.08 (br d, JZ
7.5 Hz, 1H, H-4), 8.15 (d, JZ8.4 Hz, 1H, H-200 or H-600),
8.20 (d, 0J0 Z8.7 Hz, 1H, H-200 or H-600), 8.42 (d, JZ8.7 Hz,
2H, H-3 and H-500), 8.55 (br d, JZ7.2 Hz, 1H, H-7), 9.05 (t,
JZ5.7 Hz, 1H, NH), 12.80 (br s, 1H, OH). 13C NMR
(DMSO-d6) d (ppm): 41.34 (CH2), 116.35 (C-8 or C-9),
119.59 (C-300 or C-500), 119.70 (C-300 or C-500), 123.02 (C-7),
1
1745; 1607; 1370; 1227; 1160 cmK1. H NMR (CDCl3) d
(ppm): 2.01, 2.03, 2.07 and 2.09 (4s, 12H, 4!OCH3), 3.90
(ddd, JZ12.6, 4.0, 2.1 Hz, 1H, H-50), 4.12–4.19 (m, 3H,
Ha-60 and CH2Gly), 4.31 (dd, JZ12.6, 4.0 Hz, Hb-60), 5.02
(t, J0Z9.6 Hz, 1H, H-40), 5.10 (app t, JZ9.6, 10.0 Hz, 1H,
H-2 ), 5.34 (app t, JZ9.3, 9.6 Hz, 2H, H-10 and H-30), 7.30
(t, JZ5.1 Hz, 1H, NHGly), 7.51 (d, JZ9.0 Hz, 1H, NH),
7.82 (app d, 10H0 , JZ7.4 Hz, H-4), 8.00 (d, JZ8.7 Hz, 2H,
H-200 and H-6 ), 8.38 (d, JZ8.7 Hz, 2H, H-300 and H-500),
8.53 (app d, 1H, JZ8.0 Hz, H-7), 7.60–7.70 (m, 4H, H-5,
H-6, H-8 and H-9). 13C NMR (CDCl3) d (ppm): 20.72,
20.60, 20.53 (4!CH3), 43.74 (CH2Gly), 61.58 (C-60), 68.05
(C-200 or C-40), 70.34 (C-20 or C-40), 72.73 (C-30), 73.63
(C-5 ), 78.21 (C-10), 116.17 (C-8 or C-9), 119.67 (C-300and
C-500), 123.28 (C-7), 124.65 (C-3b), 127.58 (C-5), 127.74
(C-6), 128.57 (C-200 and C-600), 128.87 (C-4), 130.20 (C-8 or
C-9), 132.36 (C-7a), 132.44 (C-400), 142.55 (C-100), 143.18
(C-3a), 143.80 (C-9a), 166.85 (C]O from dye), 169.52
(C]O, acetyl), 169.91 (C]O, acetyl), 169.97 (C]O,
aminoacid), 170.72 (C]O, acetyl), 170.94 (C]O, acetyl).
Anal. Calcd for C33H33N5O11: C, 58.66; H, 4.92; N, 10.37.
Found: C, 58.84; H, 5.06; N, 10.24.
00
123.98 (C-3b), 128.34 (C-5), 128.44 (C-6), 129.25 (C-2 or
00
00
C-6 ), 129.50 (C-4), 130.68 (C-9 0o0 r C-8), 131.34 (C-2 or
00
C-6 ), 132.29 (C-7a), 133.70 (C-4 ), 141.57 (C-100), 142.66
(C-3a), 143.52 (C-9a), 165.77 (C]O, amide), 171.32
(C]O, acid). HRMS (EI) calcd for C19H14N4O3
346.1066, found 346.1062.
4.1.10. N-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-
[(2-oxo-2H-chromene-3-carbonyl)-amino]-acetamide
(11). To a solution of 9 (0.1236 g, 0.5 mmol) in DMF
(10 mL), glycosylamine 1 (0.1737 g, 0.5 mmol) and HOBt
(0.086 g, 0.55 mmol) were added. After cooling to C5 8C,
DCC (0.108 g, 0.525 mmol) was added and the resulting
mixture stirred at rt for 48 h. Then, water (50 mL) was
added and the mixture cooled for 3 h. The solid precipitated
was filtered, dried (0.131 g, 46%) and purified by flash
column chromatography using ethyl acetate as eluent. The
isolated fraction (0.067 g, 23%) was recrystallised from
dichloromethane/light petroleum, affording pure 11 as a
white solid, mp 245–246 8C. [a]DK11 (c 2.0, CHCl3); nmax
4.1.8. [(2-Oxo-2H-chromene-3-carbonyl)-amino]-acetic
acid (9). The ester 7 (0.769 g, 3 mmol) was dissolved in
methanol (5 mL) and 1 M NaOH (7.5 mL, 7.5 mmol) added.
The mixture was stirred at rt for 4 h and 1 M HCl (3.0 mL,
3 mmol) was added. The methanol was removed under
reduced pressure and the residue thus, obtained cooled in an
ice bath and acidified to pHw2–3 with 1 M HCl with
vigorous stirring for 1 h. The solid precipitated was filtered,
washed with water and dried. Compound 9 was isolated in
86% yield and it was used without further purification, mp
238–239 8C (dec). [a]DK4 (c 1.0, DMF); nmax (KBr) 3315;
2683; 1760; 1712; 1634; 1608; 1372; 1294; 1228;
(neat) 3413; 1746; 1610; 1370; 1229 cmK1 1H NMR
.
1
1162 cmK1. H NMR (DMSO-d6) d (ppm): 4.05 (d, JZ
(CDCl3) d (ppm): 1.99, 2.03 and 2.09 (3s, 12H, 4!OCH3),
3.84 (ddd, JZ12.6, 4.5, 2.4 Hz, 1H, H-50), 4.08–4.15 (m,
1H, Ha-60), 4.14 (d, JZ5.7 Hz, 1H, CH2Gly), 4.30 (dd, JZ
12.6, 4.5 Hz, 1H, Hb-60), 4.90 (t, JZ9.6 Hz, 1H, H-40), 5.05
(app t, JZ9.6, 10.0 Hz, 1H, H-20), 5.24 (app0 t, JZ9.3,
9.6 Hz, 1H, H-10), 5.30 (t, JZ9.6 Hz, 1H, H-3 ), 7.01 (d,
5.7 Hz, 2H, CH2), 7.44 (t, JZ7.4 Hz, 1H, H-6), 7.51 (d, JZ
8.4 Hz, 1H, H-8), 7.75 (dt, JZ1.5, 8.0 Hz, 1H, H-7), 7.99
(dd, JZ1.5, 7.8 Hz, 1H, H-5), 8.90 (s, 1H, H-4), 9.03 (t, JZ
5.7 Hz, 1H, NH), 12.8 (br s, 1H, OH). 13C NMR (DMSO-d6)
d (ppm): 41.52 (CH2), 116.18 (C-8), 118.19 (C-3), 118.43

A. P. Esteves et al. / Tetrahedron 61 (2005) 8625–8632
8631
JZ9.3 Hz, 1H, NH), 7.40 (d, JZ7.8 Hz, 1H, H-8), 7.44 (d,
References and notes
JZ7.8 Hz, 1H, H-5), 7.71 (t, JZ7.8 Hz, 2H, H-6 and H-7),
8.91 (s, 1H, H-4), 9.26 (t, JZ4.8 Hz, 1H, NHGly). 13C
NMR (CDCl3) d (ppm): 20.50, 20.55 and 20.70 (4!CH3),
43.89 (CH2Gly), 61.58 (C-60), 68.09 (C-20 or C-40), 70.26
(C-20 or C-40), 72.55 (C-30), 73.56 (C-50), 78.22 (C-10),
116.76 (C-7), 117.79 (C-3), 118.44 (C-8a), 125.41 (C-8),
129.92 (C-5), 134.44 (C-6), 148.85 (C-4), 154.54 (C-4a),
161.30 (C]O, coumarin), 162.62 (C]O, coumarin amide),
169.28 (C]O, aminoacid), 169.50, 169.80, 170.63 and
171.10 (4!C]O, acetyl). Anal. Calcd for C26H28N2O13:
C, 54.16; H, 4.90; N, 4.86. Found: C, 53.85; H, 5.19; N,
4.72. HRMS (FAB) calcd for C26H29N2O13 (MCC1)
577.1670, found 577.1671.
¨
1. Bottcher, C.; Spengler, J.; Essawy, S. A.; Burger, K.
¨
Monatshefte fur Chemie 2004, 135, 853–863.
2. Tarasenko, O.; Islam, S.; Paquiot, D.; Levon, K. Carbohydr.
Res. 2004, 339, 2859–2870 and references cited therein.
˚
3. Kihlberg, J.; Ahman, J.; Walse, B.; Drakenberg, T.; Nilsson,
¨
A.; Soderberg-Ahlm, C.; Bengtsson, B.; Olsson, H. J. Med.
Chem. 1995, 38, 161–169.
4. Arsequell, G.; Valencia, G. Tetrahedron: Asymmetry 1999, 10,
3045–3094.
5. Dwek, R. A. Chem. Rev. 1996, 96, 683–720.
6. Reuter, G.; Gabius, H. J. Cell Mol. Life Sci. 1999, 55, 368–422.
7. Danishefsky, S. J.; Allen, J. R. Angew. Chem., Int. Ed. 2000,
39, 836–863.
4.1.11. N-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)-4-
naphtho[1,2-d][1,2,3]triazol-2-yl-benzoylamino-aceta-
mide (10) (a-anomer). To a solution of 2,3,4,6-tetra-O-
acetyl-b-D-glucopyranosyl azide (0.195 g, 0.52 mmol), in
anhydrous CH2Cl2 (20 mL), under a stream of argon,
compound 8 (0.20 g, 0.58 mmol) was added. The mixture
was cooled to K78 8C, stirred for 10 min followed by the
addition of tributylphosphine (0.129 mL, 0.522 mmol).
After 10 min, the stream of argon was cut off and the
reaction mixture was stirred at K78 8C for 8 h and at rt
overnight. Then, it was kept in the refrigerator for 3 h. The
precipitate (tributylphosphine oxide) was filtered off and the
solution concentrated affording an oily residue, which was
purified by column chromatography (eluent: methanol/
dichloromethane 3:97). The isolated fraction (0.307 g) was
further purified by preparative layer chromatography, using
as eluents, successively, diethyl ether/dichloromethane 1:1
(two runs) and dichloromethane (three runs). The required
band was removed and extracted with dichloromethane. The
solvent was evaporated yielding a white solid (0.024 g, 7%,
contaminated with traces of the b-anomer), mp 228.7–
230.4 8C; nmax (neat) 3413; 1745; 1607; 1370; 1227;
8. Lohof, E.; Planker, E.; Mang, C.; Burkhart, F.; Dechantsreiter,
¨
M. A.; Haubner, R.; Wester, H.-J.; Schwaiger, M.; Holzemann,
G.; Goodman, S. L.; Kessler, H. Angew. Chem., Int. Ed. 2000,
39, 2761–2764.
9. Coutrot, F.; Grison, C.; Coutrot, P. C.R. Chimie 2004, 7, 3–13.
10. Davis, B. G. Chem. Rev. 2002, 102, 579–602.
11. Meinjohanns, E.; Meldal, M.; Paulsen, H.; Dwek, R. A.; Bock,
K. J. Chem. Soc., Perkin Trans. 1 1998, 549–560.
¨
12. Cuppoletti, A.; Younjin, C.; Jin-Seong, P.; Strassler, C.; Kool,
E. T. Bioconjugate Chem. 2005, 16, 528–534.
13. Wetlz, B.; Gruber, M.; Oswald, B.; Du¨rkop, A.; Weidgans, B.;
Probst, M.; Wolfbeis, O. S. J. Chromatogr. B 2003, 793,
83–92.
14. Fernandez-Carteado, J.; Kogan, M. J.; Castel, S.; Giralt, E.
Angew. Chem., Int. Ed. 2004, 43, 1811–1814.
15. David, R.; Machova, Z.; Beck-Sickinger, A. G. Biol. Chem.
2003, 384, 1619–1639.
16. Faure, M.-P.; Gaudreau, P.; Shaw, I.; Cashman, N. R.;
Beaudet, A. J. Histochem. Cytochem. 1994, 42, 755–763.
17. Fuchs, S.; Otto, H.; Jehle, S.; Henklein, P.; Schu¨ter, A. D.
Chem. Commun. 2005, 1830–1832.
18. Mayo, V. J.; Prabhakaran, J.; Mann, J. J.; Kumar, J. S. D.
Tetrahedron Lett. 2003, 44, 8535–8537.
1
1160 cmK1. H NMR (CDCl3) d (ppm): 2.07, 2.10, 2.11
and 2.12 (4s, 12H, 4!OCH3), 4.21–4.77 (m, 7H, CH2 Gly,
2!H-60, H-50, H-40and H-30), 5.204 (app d, JZ4.2 Hz, 1H,
H-20), 5.57 (d, JZ3.6 Hz, 1H, H-1 ), 6.78 (t, JZ4.8 Hz, 1H,
NHGly), 7.63–7.81 (m, 5H, H-9, H-8, H-6, H-5, NH), 7.91
(dd, JZ7.5, 1.5 Hz, 1H, H-4), 8.03 (d, JZ9.0 Hz, 2H, H-200
and H-600), 8.49 (d, JZ8.7 Hz, 2H, H-300 and H-500), 8.65
(dd, JZ7.8, 1.8 Hz, 1H, H-7). 13C NMR (CDCl3) d (ppm):
20.48, 20.71, 20.75 (4!CH3), 41.99 (CH2Gly), 60.94 (C-30
and C-40), 61.80 (C-60), 66.32 (C-10), 67.46 (C-20), 74.12
(C-50), 116.32 (C-5 or C-6 or C-8 or C-9), 119.88 (C-300 and
C-500), 123.40 (C-7), 124.82 (C-3b), 127.70 (C-5 or C-6 or
C-8 or C-9), 127.86 (C-5 or C-6 or C-8 or C-9), 128.51
(C-200 and C-600), 129.00 (C-4), 130.33 (C-5 or C-6 or C-8 or
C-9), 132.50 (C-7a or C-400), 133.06 (C-7a or C-400), 142.64
(C-100), 143.35 (C-3a), 143.96 (C-9a), 169.48 (C]O from
dye), 169.56, 170.05 and 170.12 (C]O, acetyl), 170.49
(C]O, aminoacid).
19. Bennett, F. A.; Barlow, D. J.; Dodoo, A. N. O.; Hider, R. C.;
Lansley, A. B.; Lawrence, M. J.; Marriott, C.; Bansal, S. S.
Anal. Biochem. 1999, 270, 15–23.
20. Reddington, M. V. J. Chem. Soc., Perkin Trans. 1 1998,
143–147.
21. He, Y.; Hinklin, R. J.; Chang, J.; Kiessling, L. L. Org. Lett.
2004, 6, 4479–4482 and references cited therein.
22. Bejugam, M.; Flitsch, S. L. Org. Lett. 2004, 6, 4001–4004.
23. Huo, C.; Wang, C.; Zhao, M.; Peng, S. Chem. Res. Toxicol.
2004, 17, 1112–1120.
24. Simo, O.; Lee, V. P.; Davies, A. S.; Kreutz, C.; Gross, P. H.;
Jones, P. R.; Michael, K. Carbohydr. Res. 2005, 340, 557–566.
´
25. Monsigny, M.; Quetard, C.; Bourgerie, S.; Delay, D.; Pichon,
C.; Midoux, P.; Mayer, R.; Roche, A. C. Biochemie 1998, 80,
99–108.
26. Boons, G.-J.; Polt, R. L. In Carbohydrate Chemistry;
Boons, G.-J., Ed.; Blackie Academic: London, 1998;
pp 223–242.
27. Michael, F.; Klemer, A. Adv. Carbohydr. Chem. 1961, 16, 95.
28. Paulsen, H.; Pflughaupt, K.-W., 2nd ed. In The Carbohydrates,
Chemistry and Biochemistry, Vol. IB; Academic: New York,
1980; pp 881–927.
Acknowledgements
The authors thank the University of Minho and the
Foundation of Science and Technology (FCT) for financial
support; Lumir Lichy for absorption and fluorescence
measurements, and Dr. Radim Hrdina for a gift of dye 2.
29. (a) Inaku, T.; Kobayashi, K. Synlett 1993, 869–870. (b)
Mizuno, M.; Muramoto, I.; Kobayashi, K.; Yaginuma, H.;
Inazu, T. Synthesis 1999, 162–165.

8632
A. P. Esteves et al. / Tetrahedron 61 (2005) 8625–8632
´
32. Kondratenko, R. M.; Baltina, L. A.; Mustafina, S. R.; Vasileva,
30. Soli, D. E.; Manoso, A. S.; Patterson, M. C.; DeSong, P.
J. Org. Chem. 1999, 64, 3171–3177.
E. V.; Pompei, R.; Deidda, D.; Plyasunova, O. A.; Pokrovskii,
A. G.; Tolstikov, A. Russ. J. Bioorg. Chem. 2004, 30,
308–315.
31. von Roedern, E. G.; Lohof, E.; Hessler, G.; Hoffmann, M.;
Kessler, H. J. Am.Chem. Soc. 1996, 118, 10156–10167.