Design, synthesis, and biological evaluation of a novel class of fluorescein-based N-glycosylamines

Article abstract of DOI:10.1016/j.carres.2011.06.001

A series of fluorescein-based N-glycosylamines was synthesized from the corresponding fluorescein amine and a partially protected D-glucose. The physiochemical investigation of these compounds by spectral and morphological studies reveals their gelation p

Full text of DOI:10.1016/j.carres.2011.06.001

Carbohydrate Research 346 (2011) 1776–1785
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Design, synthesis, and biological evaluation of a novel class of fluorescein-based
N-glycosylamines
Mani Rajasekar a, Sulaiman Mahaboob Khan b, Sivasithamparam Niranjali Devaraj b,
Thangamuthu Mohan Das a,
⇑
a Department of Organic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, India
b Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600 025, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fluorescein-based N-glycosylamines was synthesized from the corresponding fluorescein
Received 11 April 2011
Received in revised form 19 May 2011
Accepted 1 June 2011
amine and a partially protected D-glucose. The physiochemical investigation of these compounds by spec-
tral and morphological studies reveals their gelation potential. The exclusive localization of fluorescence
in the cytoplasm through cell imaging studies reveals the anti-cancer potentials of N-glycosylamines.
Ó 2011 Elsevier Ltd. All rights reserved.
Available online 13 June 2011
Keywords:
Fluorescein derivatives
N-Glycosylamines
Organogelators
Morphological studies
Cell imaging studies
1. Introduction
characterization, gelation, and cell imaging studies of novel class of
fluorescein-based N-glycosylamines. Generally, fluorescein-based
In recent years, fluorescein derivatives have played an impor-
tant role in the field of drug discovery and gene delivery systems,
cancer,1 neurodegenerative diseases,2 biosensors,3–9 bioimag-
ing,10–12 and absorption studies of protein-based indicators.13,14
Fluorescein was first developed in 19th century and has become
widely known as a highly fluorescent molecule that emits longer
wavelength light upon excitation at around 500 nm in aqueous
media. These derivatives have been used as fluorescent tags for
many biological molecules, such as proteins and DNA, as well as
serving as a platform for many kinds of fluorescence probes.15 In
addition, disulfide-linked bioconjugate16 and aldehyde-based cy-
clized flourescein12 have been shown to specifically target intracel-
lular glutathione by undergoing chemical modification to end up
with the original unmodified drug as the product. Before binding,
all these probes are almost non-fluorescent, but upon binding with
target molecules, tagging or binding, these compounds become
highly fluorescent. Thus, the current research is mainly focused
on the identification of nonspecific but equally effective water-
soluble, thiol-targeting probes that are membrane-permeable and
form less toxic end products. Herein, we report the synthesis,
organic molecules are synthesized in multistep reaction sequences
that result in only poor yields,17 whereas the current study reports,
a facile synthesis of fluorescein-based N-glycoslamine derivatives
carried out using fluorescein-based amines and partially protected
D-glucose that results in good yields of fluorescent products.
2. Results and discussion
2.1. Synthesis of fluorescein-based N-glycosylamines
Fluorescein (1a),18 40,50-dimethylfluorescein (1b),19 4,6-O-butyl-
idene-
D
-glucopyranose (BGP),20,21 4,6-O-ethylidene-
D
-glucopyran
ose (EGP),22 and 4,6-O-benzylidene-
D
-glucopyranose (BzGP)23 were
synthesized by adopting literature procedures. The products were
characterized by spectral techniques. Fluorescein (1a), and 40,50-
dimethylfluorescein (1b) were reacted with p-nitrobenzoyl chloride
and p-nitrobenzyl bromide to give the corresponding etherified 2a
and 2b, and esterified 3a and 3b fluorescein nitro derivatives in
48–60% yields, respectively.24–27 The nitro group of etherified and
esterified fluorescein derivatives was reduced using Pd/C (10% acti-
vated) in H2(g) to give the corresponding fluorescein-based etheri-
fied 4a and 4b, and esterified amine derivatives 5a and 5b in 60–80%
yields, respectively. BGP, EGP, and BzGP were further reacted
with the fluorescein amine derivatives to give the corresponding
⇑
Corresponding author. Tel.: +91 44 22202814; fax: +91 44 22352494.
E-mail address: tmdas_72@yahoo.com (T.M. Das).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.06.001

M. Rajasekar et al. / Carbohydrate Research 346 (2011) 1776–1785
1777
fluorescein-based N-glycosylated products 6(a–c), 7(a–c), 8(a–c),
2.2. Absorption and emission studies
and 9(a–c) in 44–59%yield (Scheme1). The 1H NMRspectraof N-gly-
cosylated products 6(a–c), 7(a–c), 8(a–c), and 9(a–c) showed the
glycosylic-NH resonance at ꢀ4.5–6.0 ppm, and the anomeric proton
at ꢀ4.5 ppm. Thechemicalshiftpositionoftheacetalprotonschange
with respect to the protecting group (BGP: d 4.62–4.71, EGP: d 4.48–
4.51, BzGP: d 5.52–5.57 ppm), and the existence of the b-anomeric
proton at ꢀ4.5 ppm in both the esterified and etherified fluores-
cein-based N-glycosylamine derivatives were identified from the
corresponding coupling constant values (J = 4.9–9.1 Hz) (Table 1).
Fluorescein-based N-glycosylamines 8(a–c)–9(a–c) show char-
acteristic absorption and emission bands at around ꢀ490 nm and
ꢀ550 nm, respectively. The band that appeared between 440 and
490 nm in these N-glycosylated fluorescein derivatives is due to
a
p–
p⁄ transition. Upon irradiation of the N-glycosylated product
at 490 nm, an emission band was observed at ꢀ550 nm. Moreover,
a marginal shift of ꢀ5 nm has been observed among the different
N-glycosylated fluorescein derivatives 6(a–c)–9(a–c). However,
the methyl-substituted fluorescein-based N-glycosylamines
9
NO2
NO2
NO2
NO2
NO2
NO2
R
R
HO
O
O
R
R
R
R
O
O
O
O
Cl
O
O
O
O
O
Br
OH
CHCl3,
DMF,
O
Pyridine
O
O
K2CO3
O
O
1 (a, R = H; b, R = CH3)
2 (a, R = H; b, R = CH3)
3 (a , R = H; b , R = CH3)
THF, Pd/C, H2 (g)
THF, Pd/C, H2 (g)
NH2
NH2
NH2
NH2
R
R
R
R
O
O
O
O
O
O
O
O
O
O
O
O
4 (a, R = H; b, R = CH3)
5 (a, R = H; b, R = CH3)
R'
O
R'
O
O
MeOH,
reflux
O
O
MeOH,
reflux
HO
OH
O
OH
HO
HO
OH
O
R'
O
R'
O
O
O
NH
HO
O
NH
NH
HO
HO
NH
HO
OH
O
OH
OH
O
OH
O
O
R'
O
R'
O
R
R
R
R
O
O
O
O
O
O
O
O
O
O
O
O
6, 7 (R = H, CH3)
6a R = - H, R' = - CH3
8, 9 (R = H, CH3)
8a R = - H,
8b R = - H,
8c R = - H,
R' = - CH3
6b R = - H, R' = - C3H7
6c R = - H, R' = - C6H5
7a R = - CH3, R' = - CH3
7b R = - CH3, R' = - C3H7
7c R = - CH3, R' = - C6H5
R' = - C3H7
R' = - C6H5
9a R = - CH3, R' = - CH3
9b R = - CH3, R' = - C3H7
9c R = - CH3, R' = - C6H5
Scheme 1. Synthesis of fluorescein-based N-glycosylamines.

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M. Rajasekar et al. / Carbohydrate Research 346 (2011) 1776–1785
Table 1
2.3. Gelation studies
Synthesis and spectral data of fluorescein-based N-glycosylamines 6(a–c)–9(a–c)
Compounds t (h) NMR data d H-1, 3JH1,H2/Hz Yield
CGC (%) g mLꢂ1
Recently, we have reported the gelating abilities of different
classes of sugar derivatives28 and have observed that N-glycosyl-
amines are better candidates for gel formation (CGC: 1%). These
findings prompted us to investigate the gelating ability of fluores-
cein-based N-glycosylamines. The presence of more aromatic moi-
(%)
6a
6b
6c
7a
7b
7c
8a
8b
8c
9a
9b
9c
8
8
6
8
10
8
8
8
6
8
4.66, 7.8
4.55, 5.8
4.61, 7.8
4.61, 6.9
4.56, 5.0
4.68, 6.9
4.71, 4.5
4.57, 4.6
4.65, 9.1
4.76, 5.1
4.55, 4.9
4.62, 6.8
59
53
52
44
54
46
56
55
46
53
50
58
1.5
1.0
1.0
1.0
1.0
1.5
-
-
-
-
-
ety favors
p–p stacking of the fluorescein-based N-glycosylated
molecules, which is a pre-requisite for gelating ability. All gel sam-
ples were prepared by dissolving the gelator in a solvent in such a
way that it forms a homogenous solution. The solution was al-
lowed to cool down to room temperature, whereby the gel is
formed. The gelation ability of fluorescein-based N-glycosylamines
6(a–c)–9(a–c) has been assessed by using ‘stable to inversion of
the container’ method.29 The study includes twelve different
ether-6(a–c) and 7(a–c) and ester-based 8(a–c) and 9(a–c) fluores-
cein N-glycosylamines in 21 different solvents [see Supplementary
data for more details], and the results of gelation are summarized
in Table 2.
10
8
-
(a–c) show a bathochromic shift of ꢀ15 nm, which may be due to
the effect of substituent. Among the six different fluorescein-based
N-glycosylamine derivatives studied, methyl substituted 4,6-O-
benzylidene derivative (9c) showed a maximum shift of ꢀ15 nm,
which may be due to the presence of substituent on the aromatic
fluorescein ring (Fig. 1).
However, formation of gel has been observed with ether-based
fluorescein N-glycosylamines 6(a–c) and 7(a–c). In general, protect-
ing groups, such as, ethylidene, butylidene, and benzylidene present
A
600000
B
a
b
400000
200000
c
d
e
f
0
500
600
Wavelength (nm)
700
Figure 1. (A) Absorption spectra of fluorescein-based N-glycosylamines: (a) 8a; (b) 9a; (c) 8b; (d) 9c; (e) 9b, and (f) 8c; (B) emission spectra of fluorescein-based N-
glycosylamines, (a) 8a; (b) 9c; (c) 8c; (d) 9a; (e) 8b, and (f) 9b recorded at 2.5 ꢁ 10ꢂ4 M concentration in DMSO at 298 K (kex = 490 nm).

M. Rajasekar et al. / Carbohydrate Research 346 (2011) 1776–1785
1779
onthe
D
-glucosemoietyand alsothesubstituentspresent onthearo-
protecting group, and also the substituents present on the aromatic
ring as we have reported in the case of sugar–heterocyclic deriva-
matic ring showed remarkable changes in the gelating ability.
Among the various polar and nonpolar solvents used for gelation,
aromatic solvents, such as 1,2-dichlorobenzene (1,2-DCB), o-xylene,
m-xylene, benzene, and toluene were found to be the best solvents,
which may be attributed to a strong solute–solvent interaction and
the corresponding critical gelation concentrations (CGC) of the gela-
tors 6(a–c)–9(a–c) are given in Table 1. Among the six fluorescein-
based N-glycosylamine derivatives studied, N-glycosylamines 6b,
6c, 7a, and 7b act as efficient organogelators and gelate at lower con-
centrations (CGC = 1%). Thus, the gelation ability of the N-glycosyl-
amine-based organogelator has a significant dependence on the
tives.30–34 The greater gelation ability of 4,6-O-butylidene-
D-gluco-
pyranose (BGP), and 4,6-O-ethylidene-D-glucopyranose (EGP)
derivatives are due to higher London dispersion forces that exist be-
tween the alkyl chain groups. However, in case of 4,6-O-benzyli-
dene-
seem to be largely responsible for the gelation properties. In addi-
tion, anisotropic interactions through H-bonding, CH– stack-
D-glucopyranose (BzGP) derivatives, the p–p interactions
p, p–p
ing, and dipole–dipole interactions are also responsible for the linear
dipole–dipole interactions of low molecular weight organogelators
that allow gel formation.
2.4. Morphological studies
Table 2
Gelation studies of ether-based fluorescein N-glycosylamines 6(a–c)–7(a–c)
Scanning electron microscopy (SEM) images show the presence
of elongated nanofibers, which give visual insight into the aggrega-
tion modes. SEM images of etherified fluorescein-based N-glyco-
sylamines 6(a–c) and 7(a–c) show well-defined 3D fibrous
networks as well as bundles of thin flakes (Fig. 2a–f). Morphologi-
cal images of gels derived from N-glycosylamines 6(a–c), 7a, and
7b appeared as smooth sheets or ribbons (Fig. 2a–e). The fibrous
assemblies also been observed, along with the unique flower-
shaped morphologies in case of N-glycosylamine 6c and 7a
Solvent/compounds
6a
6b
6c
7a
7b
7c
C6H6
G
G
G
G
PG
G
G
S
S
S
G
G
PG
PG
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
NO2C6H5
CH3C6H5
(Cl)2C6H4
p-Xylene
m-Xylene
o-Xylene
Note: G = good gelators; PG = partial gelators; S = solution.
Figure 2. SEM images of organogel (pore labeled) formed from, (a) 6a in 1,2-DCB; (b) 6b in 1,2-DCB (flower); (c) 6c in 1,2-DCB; (d) 7a in benzene; (e) 7b in benzene (fiber),
and (f) 7c in benzene.

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M. Rajasekar et al. / Carbohydrate Research 346 (2011) 1776–1785
Table 3
(Fig. 2c and d). The formation of more flat sheets or ribbon-type
features has been observed with N-glycosylamine 6b. However,
all of these are composed of fibrous strands that are longer than
Thermodynamic parameters for sol–gel transition of fluorescein-based N-glycosyl-
amines 6a and 6c
Compounds
Tgs (°C)
D )
H (J gꢂ1
5 lm length and with diameter in the range of 20–60 nm
(Fig. 2a–e). From the SEM images, the pore size of 7c (CGC = 1%)
is greater than that of 6a–c, 7a, and 7b (CGC = 1.5%); hence, com-
pounds 6c and 7(a–c) act as good gelators, compared to the other
derivatives. Three-dimensional fibrous networks hold the solvent
molecules together, which is due to the existence of surface ten-
sion in the gels. The SEM images of all the compounds are shown
in Figure 2.
6a
6c
110
280
190
95
gel phase, the molecules are loosely packed and the system moves
from the most ordered state to a less ordered state.
The phase-transition temperature of compounds 6a, and 6c and
their corresponding gels are given in Table 3. The melting temper-
atures of the gelator and gel are obtained from DSC experiments.
2.5. Thermal analysis
To study the thermal properties of the organogelators, and gels,
Differential Scanning calorimetry (DSC) experiments for two differ-
ent etherified fluorescein-based N-glycosylamines 6a and 6c were
performed. The melting point and enthalpy of organogelator 6a in
2.6. Cell imaging studies
Incubation of HT-29 cells with 40
weak fluorescence at the emission wavelength of 520 nm. In con-
trast, 40 M of esterified fluorescein-based N-glycosylamine 8b
lM fluorescein (1a) showed
the solid phase are
values are
H = 190 J gꢂ1 and 110 °C. The organogelator 6c shows
enthalpy values and the melting point peaks for
H = 7.5 J gꢂ1
H = 280 J gꢂ1 and 95 °C in the
D
H = 9.5 J gꢂ1 and 125 °C, and in gel phase the
l
D
showed a bright fluorescence at its emission wavelength of
550 nm. The bright fluorescence localized exclusively in cytoplas-
mic region of cells but not in the cell membrane. The background
fluorescence was remarkably lower, and staining was seen pro-
fusely in cancer cells. This observation reveals that these molecules
have strong potential to act as anti-cancer agents. In addition to
fluorescein, sugar-based compounds have also been used in specif-
ically targeting cancer cells.35–37 2-Deoxy- or 2-substituted amino
sugar derivatives are precursors for synthesis of pharmaceutically
and biologically important 2-deoxy-N-glycopeptides, which are
normally used in the treatment of cancer and acquired immune
deficiency syndrome (AIDS).
D
and 120 °C in the solid phase, and
gel phase, respectively (Fig. 3).
The peak appearing at ꢀ112 °C (
D
D
H = 138 J gꢂ1) is due to the lib-
eration of moisture. These results indicate the thermal stabilities of
both gel and organogelator. DSC studies further reveal that in the
10
a
Gel
Solid
Recently, Rawal et al.38 reported that 2-deoxy-N-glycopeptides
have strong anticancer activity (IC50 22 lM) against U-87 malig-
8
nant glioblastoma (brain tumor) when compared to the normal hu-
man embryonic kidney (Hek) cell line. The preferential staining of
the cytoplasm may most likely reflect binding to the cellular thiol-
containing molecules like cysteine (Cys), homocysteine (Hcy), and
glutathione (GSH), which are concentrated more in the cytoplasm
of cells. This notion is supported by the fact that fluorescein also
shows a similar staining pattern, although at very low intensity.
Rhodamine,16 derived from the same parent fluorescein, has been
shown to preferentially bind glutathione. It is possible to perturb
the amount of cellular glutathione (GSH) by pre-incubating the
cells with the known thiol-blocking agent, diethyl maleate
(DEM). To prove the hypothesis that esterified fluorescein-based
N-glycosylamine 8b targets cellular glutathione, we incubated
HT-29 cells with 0.5 mM DEM, a concentration effective to block
GSH in hepatocytes,39 followed by esterified fluorescein-based N-
glycosylamine 8b, and measured the cellular fluorescence (Fig. 4).
The level of cellular fluorescence was dramatically lower fol-
lowing pre-treatment with DEM, indicating that esterified fluores-
cein-based N-glycosylamine 8b is responsive to intracellular GSH
concentrations. Hence, the ability of esterified fluorescein-based
N-glycosylamine 8b probes to image endogenous thiols offer new
promise in significant applications to evaluate oxidative stress in
biological systems during which glutathione levels are frequently
altered. Esterified fluorescein-based N-glycosylamine 8b can pro-
duce discernable change, and specific cytoplasmic localization
even though the parent compound fluorescein has relatively weak
fluorescence.
6
4
2
0
0
50
100
150
200
250
Temperature/ 0C
b
4
2
0
Gel
Solid
In summary, a novel class of fluorescein-based N-glycosylam-
ines have been synthesized and characterized using different spec-
tral techniques. The etherified fluorescein-based N-glycosylamines
are found to be good organogelators and are able to gelate even at
CGC 1 w/v %. Morphological and thermal studies show the various
modes of aggregation and stability of gels, respectively, which de-
0
50
100
150
200
250
Temperature/ 0C
Figure 3. DSC spectra of fluorescein-based N-glycosylamines: (a) 6a (gel formed
from benzene); (b) 6c (gel formed from 1,2-DCB).

M. Rajasekar et al. / Carbohydrate Research 346 (2011) 1776–1785
1781
Figure 4. Fluorescence images of live HT-29 cells, (a) phase-contrast image of HT-29 cells; (b) HT-29 cells treated with 40
l
M fluorescein; (c) HT-29 cells treated with 40
lM
esterified fluorescein-based N-glycosylamine (8b); (d) HT-29 cells pre-incubated with 500
(8b).
lM DEM and treated with 40 lM esterified fluorescein based N-glycosylamine
pend on the protecting groups and also on the substituents in the
fluorescein moiety. The exclusive localization of fluorescence in the
cytoplasm through cell imaging studies reveals the anti-cancer po-
tential of N-glycosylamines.
air to 25 °C, left for 12 h at this temperature, and then turned up-
side down. When the gelator formed a clear or slightly opaque
gel by immobilizing the solvent at this stage, it was denoted by
‘G’. Partial gel is denoted as PG, insoluble is as I. Some samples re-
mained as solutions and are denoted as S. Those that precipitated
were denoted as P.
3. Experimental
3.2. Cell preparation
D
-Glucose, resorcinol, 2-methylresorcinol, and phthalic anhy-
dride were purchased from Sd-fine, India. p-Nitrobenzoyl chloride
and p-nitrobenzyl bromide were purchased from Sigma–Aldrich
Chemical Co., USA and were of high purity. Paraldehyde, n-butyral-
dehyde, catalyst Pd/C, pyridine, potassium carbonate, and zinc
chloride were purchased from SRL, India. Chloroform, methanol,
DMF, and THF were used after distillation. Column chromatogra-
phy was performed on silica gel (100–200 mesh). NMR spectra
were recorded on Bruker DRX 300 MHz, and JEOL GSX 400 MHz
spectrometers. Elemental analysis was performed by using Perkin–
Elmer 2400 series CHNS/O analyser. ESI mass spectra were
recorded on JEOL JMS-D-300 spectrometer with an ionization
potential of 70 eV, and ES mass spectra were determined on a
Micromass Quattro-II instrument. The gels were imaged with a
HITACHI-S-3400 W scanning electron microscope. All absorption
spectra were obtained with a UV-1600 UV/vis spectrometer (Shi-
madzu). All fluorescence spectra were obtained with an F4500
fluorescence spectrometer (Hitachi) and optical rotations were
performed using a Rudolph Autopol II digital polarimeter.
HT-29 cells were grown in 25 cm2 UV-sterilized flasks to 70–
80% confluence at 37 °C in Dulbecco’s Modified Eagle Medium
(DMEM, Invitrogen catalog No. 31053-028) supplemented with
10% Fetal Bovine Serum (FBS, Invitrogen). For labeling, the cells
were washed once with DMSO supplemented with 2 g/L
and 20 mM HEPES buffer pH 7.4, then incubated with 40
fluorophore in DMSO at 37 °C for 4 h. One set of cells were pre-
incubated with 500 M DEM, followed by incubation with 40 lM
D-glucose
l
M
l
fluorophore in DMSO taken for imaging studies. Prior to imaging,
the labeling solution was aspirated, and the cells were washed
three times with phosphate-buffered saline (PBS).
3.3. Cell Imaging
Cells were imaged on a Carl Zeiss fluorescent microscope. Exci-
tation light was provided by a 150 W Xe arc lamp transmitted
through a 465–495 nm excitation filter and a 505 nm long-pass di-
chroic mirror, and fluorescence was measured after passage
through a 513–558 nm emission filter.
3.1. Synthesis of gels
The gelation ability of fluorescein-based N-glycosylamines was
determined by the ‘stable to inversion of the container’ method.29
The gelation properties have been tested with 21 different solvents
as follows: gelator (1.5 mg) was mixed in a close-capped test tube
with 1 mL of solvent to result in a concentration of 1.5% (g mLꢂ1),
and the mixture was heated until the solid was dissolved. By this
procedure, the solvent’s boiling point became higher than that un-
der standard atmospheric pressure. The sample vial was cooled in
3.4. General procedure for the synthesis of fluorescein
dinitrobenzyl ether 2a and 2b
To a solution of fluorescein (1 mmol), and p-nitrobenzyl bro-
mide (2 mmol) were added K2CO3 in DMF. The reaction mixture
was stirred at reflux temperature for 18 h and then cooled to room
temperature. The solvent was evaporated under reduced pressure.
The crude product was purified by column chromatography.

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M. Rajasekar et al. / Carbohydrate Research 346 (2011) 1776–1785
3.4.1. Synthesis of fluorescein dinitrobenzyl ether 2a
3.6. General procedure for the synthesis of fluorescein
diaminobenzyl ethers 4a, 4b, 5a and 5b
Compound 2a was obtained by the reaction of fluorescein 1a
(1 mmol, 0.33 g), and p-nitrobenzyl bromide (2 mmol, 0.43 g) as
a brown solid. Yield: 0.31 g (51%); mp 144–145 °C; 1H NMR
(300 MHz, CDCl3): d 5.00 (s, 2H, –CH2), 5.18 (s, 2H, –CH2), 6.59–
6.88 (m, 6H, Ar-H), 7.03–7.31 (m, 3H, Ar-H), 7.58–7.74 (m, 4H,
Ar-H), 8.02 (d, 3H, J = 7.5 Hz, Ar-H), 8.25–8.31 (t, 2H, J = 7.2 Hz,
Ar-H). 13C NMR (75 MHz, CDCl3): d 62.8, 68.6, 82.6, 101.8, 111.7,
111.9, 123.2, 123.6, 123.7, 124.7, 126.3, 126.7, 127.5, 129.0,
129.8, 135.1, 143.6, 146.6, 147.3, 149.9, 152.1, 152.4, 159.6,
169.0. Anal. Calcd for C34H22N2O9: C, 67.77; H, 3.68; N, 4.65. Found:
C, 67.52; H, 3.59; N, 4.56.
A flask containing Pd/C (10% activated 180 mg) was placed in a
flask purged with argon. A portion of 2a and 2b and 3a and 3b
(1 mmol) which was dissolved in 25 mL of dry THF was added,
and a balloon filled with H2 was attached. The reaction mixture
was stirred vigorously under H2 for 25 h, then filtered through Cel-
ite; the solvent was evaporated under reduced pressure. The crude
product was purified by column chromatography.
3.6.1. Synthesis of fluorescein diaminobenzyl ether 4a
Compound 4a was obtained by the reaction of fluorescein dinit-
robenzyl ether 2a (1 mmol, 0.60 g), and Pd/C (10% activated
180 mg) as a yellow solid. Yield: 0.38 g (70%); mp 150–152 °C;
1H NMR (300 MHz, CDCl3): d 2.96 (s, 4H, –NH2), 4.96–5.25 (m,
4H, –CH2), 6.60–7.00 (m, 6H, Ar-H), 7.09–7.17 (q, 2H, J = 7.2 Hz,
Ar-H), 7.28–7.78 (m, 4H, Ar-H), 8.02–8.31 (m, 4H, Ar-H), 8.25–
8.31 (t, 2H, J = 8.4 Hz, Ar-H). 13C NMR (75 MHz, CDCl3): d 80.9,
102.3, 109.5, 110.0, 112.3, 116.5, 117.6, 122.9, 123.4, 123.6,
124.8, 125.4, 128.5, 128.7, 130.1, 130.4,131.0, 133.8, 135.4, 150.4,
150.9, 151.5, 151.8, 152.1, 162.2, 168.3. Anal. Calcd for
3.4.2. Synthesis of 40,50-dimethylfluorescein dinitrobenzyl
ether 2b
Compound 2b was obtained by the reaction of 40,50-dimethyl-
fluorescein 1b (1 mmol, 0.36 g), and p-nitrobenzyl bromide
(2 mmol, 0.43 g) as a brown solid. Yield: 0.33 g (53%); mp 158–
160 °C; 1H NMR (300 MHz, CDCl3): d 2.28 (s, 6H, –CH3), 5.01–
5.26 (m, 4H, –CH2), 6.61–6.78 (m, 5H, Ar-H), 6.89–7.17 (m, 4H,
Ar-H), 7.30 (d, 1H, J = 7.2 Hz, Ar-H), 7.57–7.75 (m, 2H, Ar-H), 8.03
(d, 2H, J = 8.4 Hz, Ar-H), 8.21–8.30 (m, 2H, Ar-H). 13C NMR
(75 MHz, CDCl3): d 81.3, 96.0, 109.1, 110.1, 110.4, 113.1, 113.5,
117.0, 117.7, 123.7, 123.9, 125.7, 126.4, 128.7, 129.1, 129.4,
130.0, 130.7, 131.3, 132.6, 134.2, 134.3, 135.7, 150.9, 151.5,
151.7, 152.0, 154.9, 157.4, 159.8, 162.6, 168.8, 169.1, 171.1. Anal.
Calcd for C36H26N2O9: C, 68.57; H, 4.16; N, 4.44. Found: C, 68.81;
H, 4.07; N, 4.73.
C34H26N2O5: C, 75.26; H, 4.83; N, 5.16. Found: C, 74.94; H, 4.98;
N, 4.94.
3.6.2. Synthesis of 40,50-dimethylfluorescein diaminobenzyl
ether 4b
Compound 4b was obtained by the reaction of 40,50-dimethyl-
fluorescein dinitrobenzyl ether 2b (1 mmol, 0.63 g), and Pd/C
(10% activated 180 mg) as a yellow solid. Yield: 0.29 g (50%); mp
144–146 °C; 1H NMR (300 MHz, CDCl3): d 1.43 (s, 3H, –CH3),
1.84–1.88 (t, 3H, –CH3), 3.82 (s, 4H, –NH2), 5.02–5.25 (m, 4H, –
CH2), 6.73–7.33 (m, 8H, Ar-H), 7.64–7.74 (m, 3H, Ar-H), 8.04 (d,
3H, J = 7.8 Hz, Ar-H), 8.29 (d, 2H, J = 7.8 Hz, Ar-H). 13C NMR
(75 MHz, CDCl3): d 25.6, 30.3, 65.9, 67.9, 103.7, 114.8, 122.1,
123.8, 123.9, 124.0, 127.6, 127.9, 128.7, 128.9, 129.7, 129.9,
130.4, 130.5, 131.4, 133.0, 134.1, 141.3, 142.5, 147.8, 154.8,
157.4, 165.0, 175.4. Anal. Calcd for C36H30N2O5: C, 75.77; H, 5.30;
N, 4.91. Found: C, 75.92; H, 5.48; N, 4.67.
3.5. General procedure for the synthesis of fluorescein
dinitrobenzoyl ester 3a and 3b
To a solution of fluorescein (1 mmol), and p-nitrobenzoyl chlo-
ride (2 mmol) were added pyridine in chloroform. The reaction
mixture was stirred at reflux temperature for 12 h and then cooled
to room temperature. The solvent was evaporated under reduced
pressure. The crude product was purified by column
chromatography.
3.5.1. Synthesis of fluorescein dinitrobenzoyl ester 3a
Compound 3a was obtained by the reaction of fluorescein 1a
(1 mmol, 0.33 g), and p-nitrobenzoyl chloride (2 mmol, 0.35 g) as
a colorless solid. Yield: 0.38 g (60%); mp 136–138 °C; (lit.27 140–
142 °C); 1H NMR (300 MHz, CDCl3): d 6.92–7.01 (m, 3H, Ar-H),
7.26–7.28 (m. 3H, Ar-H), 7.69–7.75 (m, 2H, Ar-H), 8.08 (d, 1H,
J = 7.5 Hz, Ar-H), 8.27–8.31 (d, 1H, J = 1.5 Hz, Ar-H), 8.38 (s, 8H,
Ar-H). 13C NMR (75 MHz, CDCl3): d 81.5, 103.1, 110.3, 110.5,
117.0, 117.8, 123.6, 123.8, 124.0, 125.4, 126.0, 129.3, 130.0,
130.3, 130.7, 131.1, 131.4, 134.3, 135.3, 135.5, 151.0, 151.6,
151.8, 152.1, 152.7, 162.8, 169.2. Anal. Calcd for C34H18N2O11: C,
64.77; H, 2.88; N, 4.44. Found: C, 64.90; H, 2.79; N, 4.59.
3.6.3. Synthesis of fluorescein diaminobenzoyl ester 5a
Compound 5a was obtained by the reaction of fluorescein dini-
trobenzoyl ester 3a (1 mmol, 0.63 g), and Pd/C (10% activated
180 mg) as a yellow solid. Yield: 0.28 g (60%); mp 134–136 °C;
1H NMR (300 MHz, CDCl3): d 4.73 (s, 4H, –NH2), 6.85–7.01 (m,
4H, Ar-H), 7.26–7.28 (t, 3H, J = 7.2 Hz, Ar-H), 7.64–7.77 (m, 2H,
Ar-H), 8.03–8.09 (m, 1H, Ar-H), 8.38 (s, 8H, Ar-H). 13C NMR
(75 MHz, CDCl3):
d 81.5, 110.5, 117.1, 117.7, 123.8, 124.1,
125.4,126.1, 129.3, 130.3, 131.4, 134.3, 135.3, 135.5, 151.0, 151.6,
151.8, 152.8, 162.8, 169.6. Anal. Calcd for C34H22N2O7: C, 71.57;
H, 3.89; N, 4.91. Found: C, 71.82; H, 3.96; N, 5.08.
3.5.2. Synthesis of 40,50-dimethylfluorescein dinitrobenzoyl
ester 3b
3.6.4. Synthesis of 40,50-dimethylfluorescein diaminobenzoyl
ester (5b)
Compound 3b was obtained by the reaction of 40,50-dimethyl-
fluorescein 1b (1 mmol, 0.36 g) and p-nitrobenzoyl chloride
(2 mmol, 0.33 g) as a colorless solid. Yield: 0.31 g (48%); mp 122–
124 °C; 1H NMR (300 MHz, CDCl3): d 2.41 (s, 6H, –CH3), 6.78 (d,
2H, J = 8.7 Hz, Ar-H), 6.93 (d, 2H, J = 8.7 Hz, Ar-H), 7.26 (s, 1H, Ar-
H), 7.65–7.77 (m, 2H, Ar-H), 8.07 (d, 1H, J = 7.2 Hz, Ar-H), 8.41 (d,
8H, J = 7.6 Hz, Ar-H). 13C NMR (75 MHz, CDCl3): d 24.7, 106.1,
111.7, 115.6, 118.6, 122.8, 123.3, 125.2, 128.9, 130.2, 130.9,
131.0, 136.2, 149.7, 155.7, 156.0, 165.9, 168.5. Anal. Calcd for
Compound 5b was obtained by the reaction of 40,50-dimethyl-
fluorescein dinitrobenzoyl ester 3b (1 mmol, 0.64 g), and Pd/C
(10% activated 180 mg) as a yellow solid. Yield: 0.47 g (77%); mp
138–140 °C; 1H NMR (300 MHz, CDCl3): d 2.41 (s, 6H, –CH3), 3.75
(s, 2H, –NH2), 6.78 (d, 2H, J = 8.4 Hz, Ar-H), 6.93 (d, 2H, J = 8.7 Hz,
Ar-H), 7.29 (s, 2H, Ar-H), 7.67–7.74 (q, 2H, J = 8.4 Hz, Ar-H), 8.06
(d, 2H, J = 7.2 Hz, Ar-H), 8.40 (s, 8H, Ar-H). 13C NMR (75 MHz,
CDCl3): d 29.7, 82.5, 117.0, 117.7, 119.3, 120.1, 123.9, 124.1,
125.4, 125.8, 126.3, 127.0, 130.2, 131.4, 134.3 135.3, 150.2, 151.1,
152.6, 162.7, 169.3. Anal. Calcd for C36H26N2O7: C, 72.23; H, 4.38;
N, 4.68. Found: C, 72.47; H, 4.51; N, 4.81.
C36H22N2O11: C, 65.66; H, 3.37; N, 4.25. Found: C, 65.42; H, 3.62;
N, 4.08.

M. Rajasekar et al. / Carbohydrate Research 346 (2011) 1776–1785
1783
3.7. General procedure for the synthesis of fluorescein-based
N-glycosylamines
CDCl3 + DMSO-d6): d 27.0, 67.0, 71.1, 73.5, 73.9, 75.3, 78.0, 78.6,
80.7, 98.0, 102.4, 106.4, 115.4, 117.9, 128.5, 128.8, 130.0, 131.3,
132.9, 133.8, 135.6, 135.8, 136.2, 136.9, 140.4, 142.3, 156.4,
157.6, 167.4, 169.9, 173.8. ESIMS: calcd for 333.3 (100%). Anal.
Calcd for C60H54N2O15: C, 69.09; H, 5.22; N, 2.69. Found: C,
68.91; H, 5.35; N, 2.61.
To a solution of diamine 4a, 4b and 5a, 5b (1 mmol) in dry
MeOH and 4,6-O-protected-D-glucopyranose (BGP, EGP, BzGP)
(2 mmol) were added. After stirring at room temperature for given
period of time, the reaction mixture was evaporated under reduced
pressure. The crude product was slurried with silica gel and puri-
fied by column chromatography. For details (reaction time and
yields of products) see Table 1.
3.7.4. Synthesis of 40,50-dimethylfluorescein-di-(4,6-O-
ethylidene-b-
Compound 7a was obtained by the reaction of 4,6-O-ethyl
idene-b- -glucopyranose (2 mmol, 0.54 g) and diamine 4b
D-glucopyranosylamine) benzyl ether (7a)
D
3.7.1. Synthesis of fluorescein-di-(4,6-O-ethylidene-b-
glucopyranosylamine) benzyl ether (6a)
D
-
(1 mmol, 0.57 g) as a yellow solid. Yield: 0.48 g (44%); mp 167–
169 °C; ½a 2D3
ꢃ
-9.1 (c 1.0, EtOH); 1H (300 MHz, CDCl3 + DMSO-d6):
Compound 6a was obtained by the reaction of 4,6-O-ethylidene-
D-glucopyranose (2 mmol, 0.41 g) and diamine 4a (1 mmol,
d 1.36 (t, 6H, J = 4.8 Hz, –CH3), 2.17 (s, 1H, Sac-H), 2.26 (s, 1H,
Sac-H), 3.34–3.39 (m, 6H, Sac-H), 3.46–3.55 (q, 3H, Sac-H), 3.73
(t, 5H, J = 6.0 Hz, Sac-H), 3.85–3.94 (m, 3H, Sac-H), 4.04–4.13 (m,
2H, Sac-H), 4.61 (d, 2H, J = 6.9 Hz, Ano-H), 4.70–4.46 (q, 2H, Sac-
H), 5.04 (s, 2H, –NH), 5.23 (t, 2H, J = 13.2 Hz, Sac-H), 6.56 (t, 3H,
J = 9.0 Hz, Ar-H), 6.68–6.82 (m, 2H, Ar-H), 7.09 (d, 1H, J = 8.1 Hz
Ar-H), 7.16 (t, 1H, J = 6.0 Hz, Ar-H), 7.31 (d, 3H, J = 6.9 Hz, Ar-H),
7.62–7.78 (m, 4H, Ar-H), 8.04–8.31 (m, 5H, Ar-H). 13C NMR
(75 MHz, CDCl3 + DMSO-d6): d 20.2, 29.5, 30.2, 62.0, 65.7, 66.3,
68.1, 68.5, 70.6, 73.2, 73.3, 75.9, 80.1, 80.7, 92.9, 97.3, 99.3,
103.8, 106.7, 123.6, 123.7, 123.8, 128.7, 129.6, 130.4, 132.9,
134.1, 141.4, 159.4, 161.5, 165.2. Anal. Calcd for C52H54N2O15: C,
65.95; H, 5.75; N, 2.96. Found: C, 66.29; H, 5.53; N, 2.81.
b-
0.54 g) as a yellow solid. Yield: 0.63 g (59%); mp 168–170 °C;
½
a 2D3
ꢃ
ꢂ30.0 (c 1.0, EtOH); 1H NMR (300 MHz, CDCl3 + DMSO-d6): d
1.35 (d, 6H, J 5.1 Hz, –CH3), 3.20–3.34 (m, 3H, Sac-H), 3.43–3.49
(m, 2H, Sac-H), 3.55–3.61 (m, 2H, Sac-H), 3.79–3.86 (m, 2H, Sac-
H), 4.01–4.06 (m, 2H, Sac-H), 4.09 (d, 1H, J = 4.2 Hz, Sac-H), 4.12
(d, 1H, J = 3.9 Hz, Sac-H), 4.66 (d, 2H, J = 7.8 Hz, Ano-H), 4.65 (s,
2H, Sac-OH), 4.71–4.76 (q, 5H, Sac-H), 5.10 (s, 1H, Sac-H), 5.14 (t,
1H, J = 3.3 Hz, Sac-H), 5.30 (s, 1H, –NH), 5.36 (s, 1H, –NH), 6.49–
6.99 (m, 4H, Ar-H), 7.22 (t, 3H, Ar-H), 7.37 (t, 2H, Ar-H), 7.68–
7.84 (m, 4H, Ar-H), 7.99–8.09 (m, 3H, Ar-H), 8.29–8.33 (m, 2H,
Ar-H). 13C NMR (75 MHz, CDCl3 + DMSO-d6): d 12.9, 16.3, 35.1,
61.2, 65.4, 67.1, 67.5, 69.3, 72.2, 75.0, 79.4, 80.1, 92.0, 96.5, 101.0,
109.4, 116.0, 117.0, 122.5, 122.7, 127.8, 128.1, 128.7, 129.4,
130.4, 133.2, 149.8, 150.4, 150.8, 161.7. ESIMS: calcd for 608.1
(100%), 333.3 (50%). Anal. Calcd for C50H50N2O15: C, 65.35; H,
5.48; N, 3.05. Found: C, 65.11; H, 5.35; N, 3.17.
3.7.5. Synthesis of 40,50-dimethylfluorescein-di-(4,6-O-
butylidene-b-D-glucopyranosylamine) benzyl ether (7b)
Compound 7b was obtained by the reaction of 4,6-O-butyli-
dene-b-D-glucopyranose (2 mmol, 0.46 g) and diamine 4b (1 mmol,
0.57 g) as a yellow solid. Yield: 0.54 g (54%); mp 171–173 °C; ½a D23
ꢃ
3.7.2. Synthesis of fluorescein-di-(4,6-O-butylidene-b-
glucopyranosylamine) benzyl ether (6b)
D
-
ꢂ20.5 (c 1.0, EtOH); 1H NMR (300 MHz, CDCl3 + DMSO-d6): d 0.93
(t, 6H, –CH3), 1.25 (m, 4H, –CH2), 1.43–1.46 (m, 4H, –CH2), 1.59–
1.63 (m, 6H, –CH3), 3.24–3.53 (m, 6H, Sac-H), 3.63–4.15 (m, 8H,
Sac-H), 4.56 (t, 2H, J = 5.0 Hz, Ano-H), 4.62 (d, 2H, J = 7.2 Hz, Sac-
H), 5.04 (s, 1H, Sac-H), 5.19 (d, 2H, J = 3.6 Hz, Sac-H), 5.23 (s, 1H,
–NH), 5.28 (s, 1H, –NH), 6.54–6.87 (m, 6H, Ar-H), 7.07–7.19 (m,
3H, Ar-H), 7.30 (d, 3H, J = 6.3 Hz, Ar-H), 7.62–7.78 (m, 3H, Ar-H),
8.02–8.31 (m, 4H, Ar-H). 13C NMR (75 MHz, CDCl3 + DMSO-d6): d
17.2, 29.5, 30.2, 36.2, 62.2, 65.7, 66.5, 68.2, 68.6, 68.7, 70.8, 73.3,
76.0, 80.2, 80.7, 92.9, 97.4, 101.8, 102.2, 102.8, 103.8, 111.8,
113.0, 123.5, 123.7, 123.8, 123.9, 124.7, 127.6, 127.8, 128.6,
128.7, 128.9, 129.0, 129.1, 129.6, 129.8, 130.4, 131.2, 132.9,
134.1, 134.9, 142.7, 143.8, 147.4, 147.6, 152.5, 159.6, 169.3. ESIMS:
calcd for 597.1 (100%), 668.0 (98%). Anal. Calcd for C56H62N2O15: C,
67.05; H, 6.23; N, 2.79. Found: C, 66.75; H, 6.44; N, 2.96.
Compound 6b was obtained by the reaction of 4,6-O-butyli-
dene-b- -glucopyranose (2 mmol, 0.46 g), and diamine 4a
D
(1 mmol, 0.54 g) as a yellow solid. Yield: 0.53 g (53%); mp 162–
164 °C;
½
a 2D3
ꢃ
ꢂ18.2 (c 1.0, EtOH); 1H NMR (300 MHz,
CDCl3 + DMSO-d6): d 0.93 (t, 6H, –CH3), 1.38–1.47 (m, 4H, –CH2),
1.56–1.64 (m, 4H, –CH2), 3.16–3.34 (m, 6H, Sac-H), 3.73–3.87 (m,
3H, Sac-H), 3.98–4.13 (m, 1H, Sac-H), 4.55 (t, 5H, J = 5.8 Hz, Ano-
H), 5.09 (s, 2H, Sac-H), 5.12 (d, 2H, J = 3.9 Hz, Sac-H), 5.28 (s, 1H,
–NH), 5.35 (s, 1H, –NH), 6.55–7.35 (m, 9H, Ar-H), 7.63–7.85 (m,
4H, Ar-H), 7.98–8.11 (m, 2H, Ar-H), 8.21–8.31 (m, 2H, Ar-H), 8.41
(s, 6H, Ar-H). 13C NMR (75 MHz, CDCl3 + DMSO-d6): d 18.9, 22.2,
34.5, 35.7, 41.1, 67.1, 71.2, 73.0, 73.4, 75.1, 78.1, 80.8, 98.0,
102.5, 106.8, 115.4, 123.0, 128.7, 133.7, 134.1, 134.9, 135.1,
136.4, 139.1, 140.8, 155.7, 156.2, 156.8, 167.6, 173.5. ESIMS: calcd
for 333.3 (100%), 409.1 (60%). Anal. Calcd for C54H58N2O15: C,
66.52; H, 6.00; N, 2.87. Found: C, 66.30; H, 6.20; N, 2.99.
3.7.6. Synthesis of 40,50-dimethylfluorescein-di-(4,6-O-
benzylidene-b-
Compound 7c was obtained by the reaction of 4,6-O-benzyl
idene-b- -glucopyranose (2 mmol, 0.53 g), and diamine 4b
D-glucopyranosylamine) benzyl ether (7c)
3.7.3. Synthesis of fluorescein-di-(4,6-O-benzylidene-b-
D
-
D
glucopyranosylamine) benzyl ether (6c)
(1 mmol, 0.57 g) as a yellow solid. Yield: 0.51 g (46%); mp 167–
Compound 6c was obtained by the reaction of 4,6-O-benzyl
idene-b- -glucopyranose (2 mmol, 0.53 g), and diamine 4a
169 °C;
½
a 2D3
ꢃ
ꢂ25.6 (c 1.0, EtOH); 1H NMR (300 MHz,
D
CDCl3 + DMSO-d6): d 2.17 (s, 6H, –CH3), 3.39–3.58 (m, 3H, Sac-H),
3.68–3.80 (m, 3H, Sac-H), 3.92–4.10 (m, 2H, Sac-H), 4.21–4.38
(m, 3H, Sac-H), 4.53 (s, 1H, Sac-H), 4.68 (d, 2H, J = 6.9 Hz, Ano-H),
5.01 (s, 2H, Sac-OH), 5.05 (s, 2H, –CH2), 5.11 (s, 1H, Sac-OH), 5.16
(s, 1H, Sac-OH), 5.23 (s, 2H, –CH2), 5.28 (s, 1H, Sac-H), 5.53 (s,
2H, Ace-H), 6.10 (s, 2H, –NH), 6.53–6.87 (m, 8H, Ar-H), 7.08–7.50
(m, 9H, Ar-H), 7.62–7.79 (m, 4H, Ar-H), 8.04–8.31 (m, 4H, Ar-H).
13C NMR (75 MHz, CDCl3 + DMSO-d6): d 29.4, 30.2, 30.8, 62.0,
65.7, 66.3, 68.6, 69.0, 70.6, 73.2, 75.8, 77.6, 80.8, 81.4, 93.0, 97.4,
101.5, 101.6, 111.7, 123.5, 123.6, 123.7, 124.6, 126.3, 127.6,
127.8, 127.9, 128.6, 128.7, 128.8, 129.1, 129.6, 130.4, 131.2,
132.8, 137.3, 137.4 158.4, 164.8, 165.0. ESIMS: calcd for 333.3
(1 mmol, 0.57 g) as a yellow solid. Yield: 0.57 g (52%); mp 179–
181 °C;
½
a 2D3
ꢃ
ꢂ37.1 (c 1.0, EtOH); 1H NMR (300 MHz,
CDCl3 + DMSO-d6): d 3.30 (t, 2H, J = 8.5 Hz, Sac-H), 3.41–3.46 (m,
2H, Sac-H), 3.51–3.56 (m, 3H, Sac-H), 3.61–3.77 (m, 5H, Sac-H),
3.88 (t, 1H, J = 9.3 Hz, Sac-H), 3.96–4.04 (ddd, 1H, J = 4.8 Hz,
J = 9.5 Hz, J = 12.0 Hz, Sac-H), 4.17–4.29 (m, 1H, Sac-H), 4.61 (d,
2H, J = 7.8 Hz, Ano-H), 5.09 (d, 2H, J = 8.4 Hz, Sac-H), 5.17 (d, 2H,
J = 3.6 Hz, Sac-H), 5.26 (s, 1H, –NH), 5.33 (s, 1H, –NH), 5.52 (s,
3H, Ace-H), 6.55–6.67 (m, 4H, Ar-H), 6.82–7.19 (m, 6H, Ar-H),
7.32–7.48 (m, 10H, Ar-H), 7.67–7.80 (m, 2H, Ar-H), 8.01–8.06 (m,
3H, Ar-H), 8.23–8.31 (m, 3H, Ar-H). 13C NMR (75 MHz,

1784
M. Rajasekar et al. / Carbohydrate Research 346 (2011) 1776–1785
(100%). Anal. Calcd for C62H58N2O15: C, 69.52; H, 5.46; N, 2.62.
Found: C, 69.70; H, 5.36; N, 2.49.
3.7.10. Synthesis of 40,50-dimethylfluorescein-di-(4,6-O-
ethylidene-b- -glucopyranosylamine) benzoyl ester (9a)
Compound 9a was obtained by the reaction of 4,6-O-ethyli-
dene-b- -glucopyranose (2 mmol, 0.54 g), and diamine 5b
D
3.7.7. Synthesis of fluorescein-di-(4,6-O-ethylidene-b-
D
-
D
glucopyranosylamine) benzoyl ester (8a)
(1 mmol, 0.59 g) as a yellow solid. Yield: 0.61 g (53%); mp 156–
Compound 8a was obtained by the reaction of 4,6-O-ethyli-
160 °C;
½
a 2D3
ꢃ
ꢂ30.5 (c 1.0, EtOH); 1H NMR (300 MHz,
dene-b-
D
-glucopyranose (2 mmol, 0.41 g) and diamine 5a (1 mmol,
CDCl3 + DMSO-d6): d 1.35 (d, 6H, J = 5.1 Hz, –CH3), 2.42 (s, 6H, –
CH3), 3.24–3.88 (m, 9H, Sac-H), 4.02–4.13 (m, 5H, Sac-H), 4.55 (d,
1H, J = 3.0 Hz, Sac-H), 4.60 (d, 1H, J = 6.9 Hz, Sac-H), 4.68 (d, 1H,
J = 3.3 Hz, Sac-H), 4.76 (q, 2H, J = 5.1 Hz, J = 10.2 Hz, Ano-H), 4.79
(d, 1H, J = 3.3 Hz, Sac-H), 5.16 (s, 2H, –NH), 6.23 (d, 1H, J = 3.6 Hz,
Ar-H), 6.77 (d, 2H, J = 8.7 Hz, Ar-H), 6.97 (d, 2H, J = 8.7 Hz, Ar-H),
7.30 (d, 1H, J = 7.5 Hz, Ar-H), 7.69–7.81 (m, 2H, Ar-H), 8.05 (d,
1H, J = 7.5 Hz, Ar-H), 8.38–8.45 (m, 8H, Ar-H). 13C NMR (75 MHz,
CDCl3 + DMSO-d6): d 14.3, 25.6, 66.9, 71.2, 73.6, 73.9, 75.3, 78.0,
80.7, 85.8, 86.4, 98.0, 102.4, 106.4, 120.9, 122.6, 124.0, 129.8,
130.1, 131.2, 132.9, 133.8, 140.1, 155.2, 173.6. Anal. Calcd for
0.57 g) as a yellow solid. Yield: 0.51 g (56%); mp 178–180 °C; ½a D23
ꢃ
ꢂ12.5 (c 1.0, EtOH); 1H NMR (300 MHz, CDCl3 + DMSO-d6): d 1.35–
1.37 (q, 6H, –CH3), 3.23–3.36 (m, 4H, Sac-H), 3.49–3.52 (m, 2H, Sac-
H), 3.63–3.69 (t, 1H, Sac-H), 3.85–3.90 (m, 3H, Sac-H), 4.04–4.11
(m, 5H, Sac-H), 4.26 (s, 1H, Sac-OH), 4.45 (s, 1H, Sac-OH), 4.62 (d,
1H, J = 6.9 Hz, –NH), 4.71–4.76 (q, 2H, J = 4.5 Hz, J = 9.9 Hz, Ano-
H), 5.19 (s, 1H, –NH), 5.97 (s, 2H, Sac-OH), 6.52–7.03 (m, 3H, Ar-
H), 7.22–7.30 (m, 3H, Ar-H), 7.66–7.80 (m, 2H, Ar-H), 8.01–8.08
(q, 1H, Ar-H), 8.39 (s, 8H, Ar-H). 13C NMR (75 MHz,
CDCl3 + DMSO-d6): d 20.3, 62.1, 66.4, 68.2, 68.6, 70.8, 73.2, 73.3,
80.1, 80.7, 81.4, 93.0, 97.4, 99.4, 102.9, 109.4, 110.2, 110.4, 113.1,
117.0, 117.4, 117.7, 123.7, 124.0, 124.9, 125.3, 126.0, 126.5,
129.0, 129.2 129.9, 130.3, 131.4, 134.2, 135.2, 135.5, 150.9, 151.5,
151.8, 152.0, 152.1 152.5, 152.7, 159.7, 162.7, 168.9. ESIMS: calcd
for 640.0 (100%), 333.2 (98%), 493.2 (92%). Anal. Calcd for
C52H54N2O15: C, 65.95; H, 5.75; N, 2.96. Found: C, 66.29; H, 5.53;
N, 2.81.
3.7.11. Synthesis of 40,50-dimethylfluorescein-di-(4,6-O-
butylidene-b-
D-glucopyranosylamine) benzoyl ester (9b)
C50H46N2O17: C, 65.42; H, 4.90; N, 2.96. Found: C, 63.70; H, 5.09;
Compound 9b was obtained by the reaction of 4,6-O-butyli-
N, 2.85.
dene-b-D-glucopyranose (2 mmol, 0.46 g) and diamine 5b (1 mmol,
0.59 g) as a yellow solid. Yield: 0.53 g (50%); mp 165–167 °C; ½a D23
ꢃ
3.7.8. Synthesis of fluorescein-di-(4,6-O-butylidene-b-
glucopyranosylamine) benzoyl ester (8b)
Compound 8b was obtained by the reaction of 4,6-O-butyli-
dene-b- -glucopyranose (2 mmol, 0.46 g), and diamine 5a
(1 mmol, 0.57 g) as a yellow solid. Yield: 0.57 g (55%); mp 192–
D
-
ꢂ19.5 (c 1.0, EtOH); 1H NMR (300 MHz, CDCl3 + DMSO-d6): d 0.88–
0.93 (t, 6H, –CH3), 1.39–1.46 (q, 4H, –CH2), 1.58–1.64 (q, 4H, –CH2),
1.83–1.87 (q, 6H, –CH3), 3.19–3.30 (m, 4H, Sac-H), 3.43–3.50 (t, 3H,
Sac-H), 3.84–4.10 (m, 5H, Sac-H), 4.36 (d, 1H, J = 3.6 Hz, Sac-H),
4.52 (d, 1H, J = 3.0 Hz, Sac-H), 4.55 (t, 2H, J = 4.9 Hz, Ano-H), 4.61
(d, 1H, J = 5.1 Hz, Sac-H), 5.15 (d, 1H, J = 3.9 Hz, Sac-H), 6.19 (d,
1H, J = 4.2 Hz, Sac-H), 6.77 (d, 2H, J = 8.7 Hz, Ar-H), 6.97 (d, 3H,
J = 8.7 Hz, Ar-H), 7.30 (d, 1H, J = 7.2 Hz, Ar-H), 7.69–7.81 (m, 3H,
Ar-H), 8.05 (d, 2H, J = 7.2 Hz, Ar-H), 8.42 (s, 8H, Ar-H). 13C NMR
(75 MHz, CDCl3 + DMSO-d6): d 12.8, 16.0, 29.6, 34.9, 38.1, 61.1,
65.1, 66.8, 67.3, 68.9, 72.1, 74.6, 76.9, 79.2, 80.1, 91.9, 96.3,
100.6, 109.2, 116.8, 122.6, 127.6, 127.9, 128.8, 129.9, 130.2,
132.9, 134.6, 149.6, 150.1, 150.6, 161.4, 167.4. Anal. Calcd for
D
194 °C;
½
a 2D3
ꢃ
ꢂ17.1 (c 1.0, EtOH); 1H NMR (300 MHz,
CDCl3 + DMSO-d6): d 0.93 (t, 6H, –CH3), 1.38–1.50 (m, 4H, –CH2),
1.60–1.68 (m, 4H, –CH2), 3.21–3.35 (m, 4H, Sac-H), 3.49–3.89 (m,
5H, Sac-H), 4.06–4.16 (m, 3H, Sac-H), 4.44 (s, 1H, Sac-OH), 4.57
(t, 2H, J = 4.6 Hz, Ano-H), 4.63 (s, 2H, Sac-OH), 5.18 (s, 1H, Sac-H),
6.22 (s, 2H, –NH), 6.62–7.06 (m, 5H, Ar-H), 7.25–7.33 (m, 3H, Ar-
H), 7.67–7.84 (m, 2H, Ar-H), 8.03–8.10 (q, 2H, Ar-H), 8.42 (s, 8H,
Ar-H). 13C NMR (75 MHz, CDCl3 + DMSO-d6): d 18.7, 22.1, 41.0,
67.0, 71.3, 73.0, 73.4, 75.4, 78.1, 80.7, 85.1, 85.7, 86.2, 97.8,
102.2, 106.9, 107.7, 115.0, 115.2, 117.9, 121.7, 122.5, 128.5,
128.8, 129.7, 130.1, 130.7, 131.3, 133.7, 133.9, 134.8, 135.1,
136.2, 139.0, 140.1, 140.4, 155.7, 156.3, 156.6, 156.8, 157.1,
167.5, 173.7. ESIMS: calcd for 668.2 (100%), 333.2 (60%), 817.0
(40%), 1003.2 (30%). Anal. Calcd for C54H54N2O17: C, 64.66; H,
5.43; N, 2.79. Found: C, 64.90; H, 5.19; N, 2.65.
C56H62N2O15: C, 67.05; H, 6.23; N, 2.79. Found: C, 66.75; H, 6.44;
N, 2.96.
3.7.12. Synthesis of 40,50-dimethylfluorescein-di-(4,6-O-
benzylidene-b-
Compound 9c was obtained by the reaction of 4,6-O-benzyli-
dene-b- -glucopyranose (2 mmol, 0.53 g), and diamine 5b
D-glucopyranosylamine) benzoyl ester (9c)
D
(1 mmol, 0.59 g) as a yellow solid. Yield: 0.61 g (58%); mp 188–
3.7.9. Synthesis of fluorescein-di-(4,6-O-benzylidene-b-
glucopyranosylamine) benzoyl ester (8c)
D
-
190 °C;
½
a 2D3
ꢃ
ꢂ12.5 (c 1.0, EtOH); 1H NMR (500 MHz,
CDCl3 + DMSO-d6): d 2.41 (d, 6H, J = 12.1 Hz, –CH3), 3.22–3.24 (m,
4H, Sac-H), 3.43–3.49 (m, 2H, Sac-H), 3.69–3.70 (m, 5H, Sac-H),
3.89–4.02 (m, 3H, Sac-H), 4.22–4.26 (m, 2H, Sac-H), 4.62 (t, 1H,
J = 6.8 Hz, Ano-H), 4.85 (d, 1H, J = 3.8 Hz, Sac-H), 4.91 (s, 1H, Sac-
OH), 5.05 (s, 1H, Sac-OH), 5.17 (s, 1H, –NH), 5.52 (s, 1H, –NH),
6.43 (d, 1H, J = 4.6 Hz, Ar-H), 6.77 (d, 2H, J = 8.4 Hz, Ar-H), 6.93
(d, 1H, J = 6.8 Hz, Ar-H), 6.98 (d, 2H, J = 9.1 Hz, Ar-H), 7.34 (t, 4H,
J = 2.6 Hz, Ar-H), 7.48 (d, 2H, J = 4.8 Hz, Ar-H), 7.70–7.87 (m, 3H,
Ar-H), 8.05 (d, 1H, J = 7.6 Hz, Ar-H), 8.40–8.45 (q, 10H, Ar-H). 13C
NMR (125 MHz, CDCl3 + DMSO-d6): d 10.7, 60.2, 67.6, 68.2, 73.4,
79.3, 79.5, 92.9, 102.1, 130.2, 105.1, 111.4, 115.0, 117.9, 124. 4,
125.4, 128.0, 130.9, 140.0, 145.2, 163.2, 165.1. Anal. Calcd for
Compound 8c was obtained by the reaction of 4,6-O-benzyli-
dene-b-D-glucopyranose (2 mmol, 0.53 g) and diamine 5a (1 mmol,
0.57 g) as a yellow solid. Yield: 0.47 g (46%); mp 148–150 °C; ½a D23
ꢃ
ꢂ40.5 (c 1.0, EtOH); 1H NMR (300 MHz, CDCl3 + DMSO-d6): d 3.35–
3.57 (m, 4H, Sac-H), 3.71–3.76 (q, 2H, Sac-H), 3.92–4.09 (m, 3H,
Sac-H), 4.21–4.32 (m, 2H, Sac-H), 4.61 (d, 1H, J = 9.0 Hz, Sac-H),
4.65–4.71 (t, 3H, J = 9.1 Hz, Ano-H), 5.23 (s, 1H,–NH), 5.53 (s, 4H,
Sac-OH), 6.21 (s, 1H, –NH), 6.61–7.04 (m, 5H, Ar-H), 7.22–7.35
(m, 7H, Ar-H), 7.47–7.51 (m, 5H, Ar-H), 7.64–7.81 (m, 3H, Ar-H),
8.01–8.09 (q, 1H, Ar-H), 8.39 (s, 8H, Ar-H). 13C NMR (75 MHz,
CDCl3 + DMSO-d6): d 67.0, 71.1, 73.5, 73.9, 75.4, 78.0, 78.0, 80.7,
85.7, 86.2, 86.3, 97.9, 102.3, 106.4, 106.5, 115.0, 115.2, 121.7,
122.6, 128.6, 128.8, 129.7, 130.1, 130.7, 131.2, 131.2, 132.9,
133.7, 133.9, 134.7, 135.1, 136.1, 139.0, 139.1, 140.0, 140.4,
142.2, 142.3, 155.7, 156.3, 156.6, 156.9, 157.7, 167.5. ESIMS: calcd
for 702.2 (100%), 493.3 (98%), 851.0 (85%). Anal. Calcd for
C62H54N2O17: C, 67.75; H, 4.95; N, 2.55. Found: C, 67.61; H, 5.07;
N, 2.41.
Acknowledgments
C
60H50N2O17: C, 67.28; H, 4.71; N, 2.62. Found: C, 67.70; H, 4.19;
T. M. acknowledges financial support from the UGC, New Delhi,
India. We thank DST, New Delhi for NMR facilities under the DST-
N, 2.35.

M. Rajasekar et al. / Carbohydrate Research 346 (2011) 1776–1785
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