Studies on the synthesis and the antimicrobial and antioxidant activities of a novel class of fluorescein-based glycosides

Article abstract of DOI:10.1016/j.carres.2011.08.005

Facile glycosylation of a fluorescein diol derivative with per-O-acetyl/benzoyl sugar derivatives using BF₃·Et ₂O catalyst resulted in the formation of the expected glycosides in 54-66% yield. The biological screening of the glycosid

Full text of DOI:10.1016/j.carres.2011.08.005

Carbohydrate Research 346 (2011) 2362–2367
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Studies on the synthesis and the antimicrobial and antioxidant activities
of a novel class of fluorescein-based glycosides
Mani Rajasekar a, Raman Jegadeesh b, Nanjian Raaman b, Thangamuthu Mohan Das a,
⇑
a Department of Organic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, India
b Department of Botany, CAS, 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:
Received 4 May 2011
Received in revised form 27 July 2011
Accepted 3 August 2011
Available online 16 August 2011
Facile glycosylation of a fluorescein diol derivative with per-O-acetyl/benzoyl sugar derivatives using
BF3ꢀEt2O catalyst resulted in the formation of the expected glycosides in 54–66% yield. The biological
screening of the glycosides against different microbes shows good inhibitory activity. The antioxidant
activity of the fluorescein-based glycosides shows remarkable inhibition (IC50 ꢁ80%).
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Partially protected sugars
Glycosides
Fluorescein diol
Antimicrobial activity
Antioxidant activity
1. Introduction
comes one of the important fields of current research, which is
mainly due to their inhibitory activity against several enzymes22
Due to the intrinsic properties of the fluorescein moiety re-
ported for several fluorescein derivatives in the recent literature,1
these compounds find a wide range of applications, especially in
the fields of drug discovery, gene delivery systems, cancer therapy,
neurodegenerative diseases,2 biosensors,3–9 bioimaging10–12 and
absorption studies of protein-based indicators.13,14 Although the
first report on a fluorescein derivative appeared during the 19th
century, fluorescein chemistry is one of the underdeveloped fields
in synthetic organic chemistry. These fluorescent molecules emit
longer wavelength light upon excitation at around 500 nm in aque-
ous media. Recently, several research groups have used these
derivatives as fluorescent tags for several biological molecules,
such as, protein, DNA, etc., as well as a reliable platform for many
kinds of fluorescence probes.15 Thus, by making use of the fluores-
cent nature of the fluorescein-based sugar derivatives reported in
the present work, one can presume that these derivatives are ex-
pected to find potential applications in the field of biology.
and their serving as inducers of a
-glycosidase.23 In recent years,
several organic derivatives that are reported to have good-to-
excellent antioxidant activity belong to the family of flavonoids
and fluorescein derivatives.24,25 However, these classes of com-
pounds are either defensive molecules used in the prevention of
different pathological disorders or are used in industry for the pre-
vention of oxidative damage to polymers and natural pigments.26
Though several reports27,28 using BF3ꢀEt2O, TMSOTf, AgClO4,
among others, have appeared on the synthesis of glycosides,29–33
to the best of our knowledge, this is the first report on the reaction
of per-O-acetylated/benzoyl monosaccharides with fluorescein
diol derivatives in the presence of BF3ꢀEt2O as a Lewis acid catalyst.
Since fluorescein reacts with lipid radicals34 to form phenoxy
radicals that act as effective chain-breaking antioxidants, we also
expect fluorescein diol derivatives to have characteristic antioxi-
dant properties. Moreover, alkyl-substituted fluorescein, which in-
creases the electron density on the hydroxyl group by an inductive
effect, increases its activity toward the generation of lipid radi-
cals.35 Thus it has been proposed that these two potentially useful
properties could be combined in a properly functionalized aro-
matic alcohol that might act as an efficient drug.36 In addition to
our ongoing research37,38 in the area of sugar chemistry, here we
investigate the influence of the sugar skeleton and glycon motifs
on antimicrobial activity by the well diffusion method and deter-
mine the antioxidant properties using the 1,1-diphenyl-2-pic-
rylhydrazide (DPPH) radical-scavenging activity assay.
Development of several of our existing front-line drugs16,17 and
organic materials18,19 are essential and reliable platforms, which
are, for the most part, dependent on glycosylated products. More-
over, understanding of the relationship that exists between the
sugars and bioactive entities shed light on the biological signifi-
cance20,21 of these derivatives. Thus, the synthesis of glycosides be-
⇑
Corresponding author. Tel.: +91 44 22202814; fax: +91 44 22352494.
E-mail address: tmdas_72@yahoo.com (T. Mohan Das).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.08.005

M. Rajasekar et al. / Carbohydrate Research 346 (2011) 2362–2367
2363
Staphylococcus aureus (ATCC 12600), Bacillus subtilis (ATCC 6633),
Klebsiella pneumoniae (ATCC 13883), Escherichia coil (ATCC 11775)
and a yeast (Candida albicans). The screening results are shown in
Figure 3.
The results of the antibacterial and antifungal screening of fluo-
rescein diol (2) show poor biological activities against pathogens.
The glycosylated fluorescein derivatives 4(a–g) containing elec-
tron-donating groups (4a and 4b) show higher activity, which indi-
cates an altered biological response upon appending the glycosyl
moiety. We have also evaluated the antifungal activity of the fluo-
rescein-based glycoside derivatives against C. albicans. The screen-
ing data are given in Figure 3. However, the antifungal activity of
4(c–g) is low in comparison with 4a and 4b, which also affirms
2. Results and discussion
2.1. Synthesis of fluorescein-based glycosides 4(a–g)
40,50-Bis(bromomethyl)fluorescein dibenzoate (1) was synthe-
sized from the corresponding methyl fluorescein derivative, that
is, 40,50-dimethylfluorescein dibenzoate, by adopting the literature
procedure.39 1,2,3-Tri-O-acetyl-4,6-O-butylidene-b-
D
-glucopyran-
-glucopyranose, per-
-xylose, -galactose, -glu-
-glucose41,42 were synthesized by
ose, 1,2,3-tri-O-benzoyl-4,6-O-butylidene-b-
O-acetylated -sugars of -mannose,
cose40 and per-O-benzoylated-
D
D
D
D
D
D
D
adopting the literature procedures and characterized using spec-
tral techniques. The 40,50-bis(bromomethyl)fluorescein dibenzoate
(1) was reacted with NaHCO3 in a CH3CN–H2O solvent mixture to
give the corresponding hydrolyzed fluorescein diol derivatives (2)
in 68% of yield. Fluorescein diol (2) was then reacted with per-O-
acetyl/benzoyl sugar derivatives 3(a–g) using the Lewis acid
BF3ꢀEt2O as a catalyst in a TEA–CH2Cl2 mixture to give the corre-
sponding fluorescein-based glycoside products 4(a–g) in 54–68%
the higher activities of protected saccharide derivatives of D-glu-
cose (4a and 4b).
In conclusion, this report deals with a facile synthesis of fluores-
cein-based glycosides in moderate-to-good yield. Glycosylation of
fluorescein diol derivatives with per-O-acetyl/benzoyl
D-sugar
derivatives using BF3ꢀEt2O as Lewis acid catalyst resulted in the for-
mation of the corresponding glycosides in high yield with good
selectivities. The anomeric configuration assignment of the glyco-
sides was determined by 1H NMR spectroscopy. The preliminary
biological evaluation of these synthesized compounds showed
weak-to-moderate antimicrobial and antioxidant activities.
yield, respectively (Table 1). The
a:b ratios have been calculated
from the reaction mixtures using 1H NMR spectroscopy. The peaks
observed for compound 4b at around d 5.70 and 8.4 ppm corre-
spond to an
the peaks were observed at around d 5.72 and 8.4 ppm with an
:b ratio of 14:86.
a:b ratio of 49:51, whereas, in case of compound 4c
a
3. Experimental section
3.1. General methods
2.2. Biological studies
2.2.1. Antioxidant studies
Fluorescein-based glycoside derivatives 4(a–g) were assessed
for their capacity to prevent the formation of DPPH peroxide radi-
cals in a peroxy-generating system as described in Section 3.2.2.
The antioxidant activity of fluorescein diol (2) tested shows poor
activity. All the compounds are shown in Figure 1. Except for the
D
-Glucose, D-galactose, D-mannose, D-xylose, butyraldehyde, 2-
methylresorcinol, BF3ꢀEt2O, and other alcohols were obtained from
SRL, Chennai, Tamil Nadu, India. Ac2O was purchased from Fisher
Chemicals Pvt. Ltd, Chennai, Tamil Nadu India. Column chromatog-
raphy was performed on Silica Gel 60 (100–200 mesh). Melting
points were measured on a Sigma micro melting point apparatus
and are uncorrected. NMR spectra were recorded on a Bruker
DRX 300 in CDCl3 or DMSO-d6 at the University of Madras (Chen-
nai, Tamil Nadu, India). Me4Si was used as the internal standard
(d = 0.00 ppm), and all J values are given in hertz. Elemental analy-
ses were performed using a Perkin–Elmer 2400 elemental analyzer,
and optical rotations were determined using a Rudolph Autopol II
digital polarimeter.
4,6-O-protected acetyl/benzoyl D-sugar derivatives 4a and 4f, all
other derivatives, exhibit very low antioxidant activities.
However, when the sugar moieties are coupled with the aglycon
motifs such as fluorescein diol (2), the glycosides 4(a–g) show
moderate activities, when compared to standard
shows an activity of 70% at minimum concentration (10
a
-tocopherol that
g/mL).
l
The change in color intensity in 96-well microplates was observed
initially from pink to yellow (Fig. 2), which indicates the antioxi-
dant activities of the tested compounds. It has been observed that
the activity increases proportionately with the increase in concen-
3.2. Antimicrobial activities
tration. Thus the compounds 4a (IC50 33.6
lg/mL) and 4f (IC50
12.7 g/mL) show more activity compared to the other derivatives.
l
Antifungal and antibacterial activities of the fluorescein-based
glycoside derivatives 4(a–g) were determined by the well-diffu-
sion method using Sabouraud dextrose agar and Müller–Hinton
agar.44 The zone of inhibition was recorded (in mm) on the com-
pletion of the incubation, and the mean diameter for each com-
pound at 2 mg mLꢂ1 was determined. Stock solutions of the test
compounds were prepared in DMSO. The diameters of the zone
of inhibition produced by the compounds were compared with
The influence of fluorescein diol (2) coupled with a 4,6-O-protected
glycosyl moiety (4a and 4f) is greater when compared to the corre-
sponding per-O-acetyl/benzoyl sugar derivatives (4b–e and 4g),
which may be due to the facile release of hydride radicals from
the former substrate.
The glycosyl derivatives 4a and 4f with a protected group as the
aglycon shows comparatively higher activities among the other
compounds evaluated. The fluorescein derivative that has a longer
alkyl chain shows a maximum inhibitory activity (80.1%), whereas
the derivatives without an alkyl chain show only moderate activity
(19.3%). From these results it is evident that the antioxidant activ-
ity of 4,6-O-protected-D-glucopyranose coupled with a fluorescein
unit possessing a hydrophobic alkyl chain increases the antioxi-
dant activity.
the standard antibiotics tetracycline at 30 lg per well. Each exper-
iment was repeated three times to minimize error.
3.2.1. Inoculum preparation
Fresh bacterial cultures were used for the antibacterial suscep-
tibility tests. Five MTCC colonies of the strains were inoculated into
tryptic soy or brain–heart infusion broths and incubated at 37 °C
for 22–24 h. The turbidity was adjusted with sterile broth to corre-
spond to the 0.5 McFarland standard; standard inoculum of the
microorganism of 1.5 ꢃ 106 colony-forming units CFU mLꢂ1 di-
luted 1:100 gives suspension of turbidity equal to a McFarland
standard of 0.5. The inoculum used was prepared from a 24-h-
2.2.2. Antibacterial and antifungal studies
The fluorescein-based glycoside derivatives 4(a–g) were sub-
jected to assays for antibacterial activity against five human path-
ogenic43 bacteria using Pseudomonas aeruginosa (ATCC 10145),

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M. Rajasekar et al. / Carbohydrate Research 346 (2011) 2362–2367
Table 1
Synthesis of fluorescein-based glycoside derivatives 4(a–g)
OR
RO
O
Br
OH
O
Br
HO
BzO
O
O
O
O
BzO
O
OBz
O
OBz
BF3.Et2O,
TEA, DCM
BzO
O
OBz
NaHCO3
O
O
O
CH3CN-H2O,
reflux
O
RO
O
OR
R = Ac, Bz
3 (a-g)
2
1
4 (a-g)
Entry
Sugar part (3/4)
Time (h)
Selectivity ratioa (%)
d (H-1) (3J H1,H2/Hz)
Yield (%)
H3C
O
1
5
b only
5.73, 8.1
61
O
O
AcO
OAc
4a
OAc
O
AcO
AcO
2
3
7
4
49:51
5.70, 8.4
5.72, 8.4
56
66
OAc
4b
OAc
OAc
O
14:86
AcO
OAc
O
4c
AcO
AcO
4
5
5
8
b only
5.73, 6.9
5.72, 6.9
54
61
OAc
4d
OAc
OAc
O
AcO
AcO
a
only
4e
H3C
O
6
4
b only
6.17, 7.5
6.30, 7.8
60
59
O
O
BzO
OBz
4f
OBz
O
BzO
BzO
7
6
b only
OBz
4g
a
The a
:b ratio was determined by 1H NMR spectroscopy before column chromatographic purification.
old culture on brain–heart infusion agar (BHIA). With a sterile loop,
the tops of 2–4 colonies were transferred to a tube containing 5 mL
of Müller–Hinton broth or brain–heart infusion broth. The tube
was incubated at 35 °C for 24 h. The turbidity of the culture sus-
pension was adjusted with broth or a sterile saline solution
(0.85–0.90%). The culture density was adjusted with a spectropho-
tometer (kmax at 530 nm) to obtain a final concentration to match
that of a 0.5 McFarland standard. The final inoculum size was
approximately 0.5–2.5 ꢃ 103 CFU/mL.
hydrazine when it reacts with hydrogen donors, and its stability is
evaluated by the decoloration assay, which evaluates the decrease
in absorbanceat 517 nm produced by the addition of the antioxidant
to a DPPH solution in ethanol. Assays were performed in 1.5-mL
reaction mixtures containing 1 mL of 0.1 mM DPPH ethanol solution
and 1 mL of different concentrations of fluorescein-based glycoside
derivatives 4(a–g) (10–100 lg/mL)/0.5 mL of ethanol (as control).
After 30 min of incubation at 37 °C in the dark, the absorbances of
the reaction mixtures were measured at 517 nm. The IC50 is the con-
centration of the sample required to scavenge 50% of the DPPH free
radicals. The percentage of inhibition is calculated by subtracting
the absorbance of the sample from the absorbance of the control di-
vided by absorbance of the control. The lower the IC50, the higher is
the antioxidant activity of the examined compound. From the differ-
ence in the absorbance of DPPH, the percentage of inhibition was
calculated as a function of antioxidant activity.
3.2.2. In vitro antioxidant activity
In vitro antioxidant activity for fluorescein-based glycoside
derivatives 4(a–g) were evaluated by the DPPH radical-scavenging
method.45 The principle of this assay is based on the measurement
of the scavenging ability of the antioxidant toward the stable
radical. The free radical of DPPH is reduced to the corresponding

M. Rajasekar et al. / Carbohydrate Research 346 (2011) 2362–2367
2365
100
90
80
70
60
50
40
30
20
10
0
10
20
30
40
50
60
70
80
90
100
ug/mL
α-tocopherol
4a
4b
4c
4d
4e
4f
4g
Figure 1. Antioxidant activities of fluorescein-based glycosides 4(a–g) as measured by the DPPH method.
nitrogen atmosphere was added Et3N (0.15 mmol) in CH2Cl2
(20 mL), followed by BF3ꢀEt2O (23 mmol, 3 mL) in CH2Cl2 (5 mL)
over 30 min. The reaction mixture was then kept stirring until
TLC showed completion of the reaction. Satd aq NaHCO3 was
added, and the reaction mixture was then extracted with EtOAc
(30 mL). The organic layer was dried over anhyd MgSO4, followed
by column chromatographic purification using CHCl3–MeOH on
silica gel to give the corresponding glycosides 4(a–g).
3.4.1. Synthesis of 40,50-dimethylfluorescein dibenzoate 2,3-di-
O-acetyl-4,6-O-butylidene-b-
D-glucopyranoside (4a)
From 1,2,3-tri-O-acetyl-4,6-O-butylidene-b-
D-glucopyranose
(3a) (0.24 g, 0.60 mmol), fluorescein diol (2) (0.18 g, 0.30 mmol)
and BF3ꢀEt2O (23 mmol, 3 mL). Yellow solid: (0.22 g, 61%); mp
158–160 °C; ½a 2D0
ꢄ
+50.2 (c 0.2, CHCl3). 1H NMR (300 MHz, CDCl3):
d 0.90 (t, J = 7.2 Hz, 6H), 1.31–1.43 (m, 4H), 1.56–1.63 (m, 4H),
2.04 (s, 3H), 2.06 (s, 3H), 2.09 (s, 3H), 2.10 (s, 3H), 3.50 (t,
J = 8.7 Hz, 5H), 4.22 (d, J = 6.0 Hz, 3H), 4.50 (t, J = 5.1 Hz, 2H), 4.79
(d, J = 4.8 Hz, 2H), 5.07 (t, J = 8.5 Hz, 4H), 5.26 (t, J = 9.0 Hz, 2H),
5.73 (d, J = 8.1 Hz, 2H), 6.73 (d, J = 3.6 Hz, 1H), 6.76 (d, J = 3.6 Hz,
1H), 6.88–6.96 (m, 2H), 7.06 (t, J = 9.0 Hz, 1H), 7.30 (t, J = 9.0 Hz,
2H), 7.55 (q, J = 15.0 Hz, 5H), 7.65–7.76 (m, 3H), 8.05 (q,
J = 7.2 Hz, 1H), 8.25 (t, J = 8.1 Hz, 2H). 13C NMR (75 MHz, CDCl3):
d 14.0, 17.7, 20.7, 20.9, 21.0, 21.1, 29.8, 36.2, 67.4, 68.0, 71.4,
72.0, 77.9, 92.4, 103.0, 110.2, 116.6, 118.1, 118.7, 124.5, 125.6,
125.8, 128.9, 129.1, 130.4, 130.5, 130.7, 134.1, 134.5, 169.1,
169.8, 170.3. Anal. Calcd for C64H64O23: C, 63.99; H, 5.37. Found:
C, 63.64; H, 5.01.
Concentration in µg /mL
Figure 2. Antioxidant activities of fluorescein-based glycosides 4(a–g) in 96-well
microplates: (1) control; (2) 10
60 g; (8) 70 g; (9) 80 g; (10) 90
4c, (d) 4d, (e) 4e, (f) 4f, and (g) 4g.
l
g; (3) 20
l
g; (4) 30
lg; (5) 40 lg; (6) 50 lg; (7)
l
l
l
lg; (11) 100 l
g; (12) Control; (a) 4a, (b) 4b, (c)
3.3. Synthesis of 40,50-bis(hydroxymethyl)fluorescein dibenzoate
(2)
To a solution of 40,50-bis(bromomethyl)fluorescein dibenzoate
(1) (0.72 g, 1.0 mmol) in a mixture of CH3CN (50 mL) and water
(20 mL), NaHCO3 (0.84 g, 1.0 mmol) was added and refluxed for
2 h. The crude mixture was extracted with CH2Cl2, dried over
anhyd Na2SO4, the mixture was filtered, and the solvent was evap-
orated to give fluorescein diol (2) as a yellow solid. Yield: 0.41 g
(68%); mp 125–127 °C. 1H NMR (300 MHz, CDCl3): d 4.10 (s, 2H),
4.89 (s, 4H), 6.92 (d, J = 9.0 Hz, 3H), 7.01 (d, J = 9.0 Hz, 1H), 7.32
(d, J = 7.5 Hz, 2H), 7.46–7.80 (m, 7H), 8.10 (t, J = 9.0 Hz, 2H), 8.28
(d, J = 7.8 Hz, 3H). 13C NMR d: 20.4, 81.4, 116.5, 117.0, 119.0,
119.2, 124.2, 125.4, 126.1, 128.4, 128.7, 128.9, 129.2, 130.1,
130.3, 130.4, 133.7, 134.3, 135.5, 149.4, 150.6, 152.3, 164.2,
168.9. Anal. Calcd for C36H24O9: C, 72.00; H, 4.03. Found: C,
72.32; H, 3.79.
3.4.2. Synthesis of 40,50-dimethylfluorescein dibenzoate 2,3,4,6-
tetra-O-acetyl-b-
D
-glucopyranoside (4b)
-glucopyranose (3b) (0.23 g, 0.60 mmol),
From per-O-acetyl-b-
D
fluorescein diol (2) (0.18 g, 0.30 mmol) and BF3ꢀEt2O (23 mmol,
3 mL). Yellow solid (0.21 g, 56%); mp 190–192 °C; ½a D20
ꢄ
+39.6 (c
0.2, CHCl3). 1H NMR (300 MHz, CDCl3): d 2.00 (s, 6H), 2.05 (s, 6H),
2.12 (s, 6H), 2.17 (s, 6H), 4.06–4.20 (m, 6H), 4.80 (d, J = 4.5 Hz,
1H), 5.06 (d, J = 3.6 Hz, 1H), 5.11 (d, J = 3.3 Hz, 2H), 5.33 (q,
J = 5.4 Hz, 4H), 5.43 (d, J = 2.7 Hz, 2H), 5.70 (d, J = 8.4 Hz, 2H), 6.77
(q, J = 7.1 Hz, 2H), 6.88–6.96 (m, 3H), 7.02–7.31 (m, 2H), 7.52–
7.72 (m, 6H), 8.03–8.06 (m, 1H), 8.31 (m, 4H). 13C NMR (75 MHz,
CDCl3): d 18.7, 18.8, 19.0, 29.1, 59.2, 65.0, 66.0, 69.0, 69.9, 74.8,
75.2, 75.7, 79.5, 90.3, 108.6, 115.3, 116.0, 122.0, 122.2, 123.5,
124.2, 127.4, 128.0, 128.4, 129.6, 132.5, 133.6, 143.0, 145.8, 149.2,
149.8, 150.0, 150.9, 161.0, 167.1, 167.5, 168.1, 168.3, 168.5. Anal.
Calcd for C64H60O27: C, 60.95; H, 4.80. Found: C, 61.26; H, 4.48.
3.4. General method for synthesis of fluorescein-based
glycosides 4(a–g)
To a mixture of per-O-acetyl/benzoyl
D-sugar derivatives 3(a–
g), (0.60 mmol) and the fluorescein diol (2), (0.30 mmol) under

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M. Rajasekar et al. / Carbohydrate Research 346 (2011) 2362–2367
40
35
30
25
20
15
10
5
0
Concentration in µg /mL
S.a
B.s
E.c
K.p
P.a
C.a
Figure 3. Antimicrobial activities of fluorescein-based glycosides 4(a–g).
3.4.3. Synthesis of 40,50-dimethylfluorescein dibenzoate 2,3,4,6-
tetra-O-acetyl-b- -galactopyranoside (4c)
From per-O-acetyl-b- -galatopyranose (3c) (0.25 g, 0.60 mmol),
(23 mmol, 3 mL). Yellow solid (0.23 g, 61%): mp 176–178 °C; ½a D20
+50.1 (c 0.2, CHCl3). 1H NMR (300 MHz, CDCl3): d 2.06 (s, 6H),
2.08 (s, 6H), 2.11 (s, 6H), 2.40 (s, 6H), 3.53 (d, J = 3.6 Hz, 2H), 3.56
(s, 1H), 4.15 (dd, J = 5.1 Hz, J = 5.1 Hz, 2H), 4.79 (d, J = 4.5 Hz, 4H),
4.97–5.06 (m, 3H), 5.20 (t, J = 8.1 Hz, 3H), 5.72 (d, J = 6.9 Hz, 3H),
6.73–6.77 (m, 2H), 6.88–6.96 (m, 3H), 7.04 (d, J = 8.7 Hz, 3H),
7.28 (t, J = 8.5 Hz, 4H), 7.51–7.73 (m, 1H), 8.04 (t, J = 8.6 Hz, 2H),
8.22 (t, J = 7.6 Hz, 3H). 13C NMR (75 MHz, CDCl3): d 20.8, 62.8,
68.3, 69.5, 71.0, 92.0, 116.4, 117.9, 118.4, 119.4, 124.2, 125.2,
125.6, 126.4, 128.7, 128.8, 129.0, 129.9, 130.3, 130.4, 133.9,
135.1, 150.2, 150.2, 152.8, 164.5, 169.0, 169.8. Anal. Calcd for.
ꢄ
D
D
fluorescein diol (2) (0.18 g, 0.30 mmol) and BF3ꢀEt2O (23 mmol,
3 mL). Yellow solid (0.31 g, 66%); mp 186–188 °C; ½a D20
ꢄ
+80.2
(c 0.2, CHCl3). 1H NMR (300 MHz, CDCl3): d 2.02 (s, 6H), 2.03
(s, 6H), 2.05 (s, 6H), 2.10 (s, 6H), 4.07 (d, J = 3.5 Hz, 1H), 4.13
(t, J = 6.0 Hz, 2H), 4.25 (d, J = 4.2 Hz, 1H), 4.29 (d, J = 4.2 Hz, 1H),
4.79 (d, J = 4.8 Hz, 1H), 5.08 (d, J = 3.6 Hz, 1H), 5.12 (d, J = 3.6 Hz,
2H), 5.14 (s, 3H), 5.18 (s, 2H), 5.47 (t, J = 9.0 Hz, 2H), 5.72 (d,
J = 8.4 Hz, 2H), 6.33 (d, J = 3.6 Hz, 2H), 6.73–6.77 (dd, J = 7.5 Hz,
J = 7.5 Hz, 2H), 6.88–7.05 (m, 3H), 7.28 (t, J = 9.0 Hz, 1H), 7.52–
7.59 (m, 4H), 7.63–7.77 (m, 3H), 8.06 (m, 2H), 8.26 (t, J = 7.2 Hz,
1H). 13C NMR (75 MHz, CDCl3): d 20.6, 69.5, 70.6, 71.6, 72.6, 80.2,
92.8, 109.5, 116.0, 117.1, 124.2, 126.0, 126.4, 128.2, 128.8, 129.5,
133.3, 134.8, 149.9, 150.3, 150.9, 161.7, 167.5. Anal. Calcd for
C64H60O27: C, 60.95; H, 4.80. Found: C, 61.24; H, 4.25.
3.4.6. Synthesis of 40,50-dimethylfluorescein dibenzoate 2,3-di-
O-benzoyl-4,6-O-butylidene-b- -glucopyranoside (4f)
From 1,2,3-tri-O-benzoyl-4,6-O-butylidene-b- -glucopyranose
D
D
C64H60O27: C, 60.95; H, 4.80. Found: C, 61.03; H, 5.24.
(3f) (0.32 g, 0.60 mmol), fluorescein diol (2) (0.18 g, 0.30 mmol)
and BF3ꢀEt2O (23 mmol, 3 mL). Yellow solid (0.26 g, 60%); mp
3.4.4. Synthesis of 40,50-dimethylfluorescein dibenzoate 2,3,4-
tri-O-acetyl-b- -xylopyranoside (4d)
From per-O-acetyl-b- -xylopyranose (3d) (0.19 g, 0.60 mmol),
162–164 °C; ½a 2D0
ꢄ
+49.6 (c 0.2, CHCl3). 1H NMR (300 MHz, CDCl3):
0.84 (t, 6H), 1.32–1.59 (q, 4H), 1.59–1.62 (q, 4H), 3.62
D
d
D
(t, J = 8.2 Hz, 3H), 3.78 (d, J = 6.9 Hz, 4H), 4.31 (t, J = 5.4 Hz, 2H),
4.56 (t, J = 5.1 Hz, 2H), 4.79 (d, J = 4.8 Hz, 2H), 4.87 (s, 1H), 5.74–
5.80 (m, 4H), 6.17 (d, J = 7.5 Hz, 2H), 6.77 (d, J = 7.2 Hz, 2H), 6.82
(d, J = 7.2 Hz, 2H), 6.88–7.08 (m, 5H), 7.28–7.43 (m, 7H), 7.52–
7.59 (q, 6H), 7.65–7.73 (m, 6H), 7.90 (d, J = 7.2 Hz, 2H), 8.01 (t,
J = 7.2 Hz, 2H), 8.25 (t, J = 8.1 Hz, 6H). 13C NMR (125 MHz, CDCl3):
d 13.8, 17.4, 20.5, 28.1, 29.6, 35.4, 36.0, 45.1, 47.8, 67.5, 68.0,
71.4, 77.1, 81.5, 82.2 92.1, 93.1, 95.9, 102.9, 108.8, 109.6, 116.4,
117.9, 128.4, 128.5, 128.6, 128.8, 129.9, 130.3, 145.6, 149.9,
154.0, 164.6, 165.3, 165.5, 169.6, 167.7. Anal. Calcd for
fluorescein diol (2) (0.18 g, 0.30 mmol) and BF3ꢀEt2O (23 mmol,
3 mL). Yellow solid (0.18 g, 54%); mp 168–170 °C; ½a D20
ꢄ
+49.3
(c 0.2, CHCl3). 1H NMR (300 MHz, CDCl3): d 2.05 (s, 6H), 2.06
(s, 6H), 2.11 (s, 6H), 3.49–3.56 (q, 2H), 4.13 (d, J = 4.8 Hz, 2H),
4.17 (d, J = 4.8 Hz, 3H), 4.78 (d, J = 4.5 Hz, 1H), 4.94–5.06 (m, 5H),
5.21 (t, J = 6.0 Hz, 2H), 5.72 (d, J = 6.9 Hz, 1H), 6.73 (s, 2H), 6.75
(d, J = 8.7 Hz, 3H), 6.91 (t, J = 6.0 Hz, 2H), 7.24–7.31 (m, 2H), 7.57
(q, J = 9.0 Hz, 3H), 7.65–7.74 (m, 2H), 8.05 (m, 2H), 8.25 (t,
J = 6.0 Hz, 2H). 13C NMR (75 MHz, CDCl3): d 20.4, 20.6, 20.7, 20.8,
62.8, 68.3, 69.5, 71.0, 76.6, 77.0, 77.5, 92.0, 116.4, 117.9, 118.5,
119.4, 124.2, 125.2, 125.6, 126.4, 128.7, 129.0, 130.3, 133.9,
150.3, 150.6, 164.5, 169.0, 169.3, 169.8. Anal. Calcd for
C84H72O23: C, 69.61; H, 5.01. Found: C, 69.32; H, 5.29.
3.4.7. Synthesis of 40,50-dimethylfluorescein dibenzoate 2,3,4,6-
tetra-O-benzoyl-b- -glucopyranoside (4g)
From per-O-benzoyl-b- -glucopyranose (3g) (0.42 g, 0.60
C58H52O23: C, 62.36; H, 4.69. Found: C, 62.19; H, 4.88.
D
3.4.5. Synthesis of 40,50-dimethylfluorescein dibenzoate 2,3,4,6-
tetra-O-acetyl- -mannopyranoside (4e)
From per-O-acetyl- -mannopyranose (4e) (0.23 g, 0.60
mmol), fluorescein diol (2) (0.18 g, 0.30 mmol), and BF3ꢀEt2O
D
mmol), fluorescein diol (2) (0.18 g, 0.30 mmol), and BF3ꢀEt2O
a-D
(23 mmol, 3 mL). Yellow solid (0.31 g, 59%); mp 158–160 °C; ½a D20
ꢄ
a-D
+50.2 (c 0.2, CHCl3). 1H NMR (300 MHz, CDCl3): d 4.53 (d,

M. Rajasekar et al. / Carbohydrate Research 346 (2011) 2362–2367
14. Lyons, A. B. J. Immunol. Methods 2000, 243, 147–154.
2367
J = 4.8 Hz, 2H), 4.64 (d, J = 2.1 Hz, 6H), 4.78 (d, J = 4.2 Hz, 2H), 5.86
(q, J = 9.1 Hz, 4H), 6.04 (t, J = 9.1 Hz, 2H), 6.30 (d, J = 7.8 Hz, 2H),
6.75 (q, J = 7.5 Hz, 2H), 6.91 (m, 4H), 7.04 (d, J = 9.0 Hz, 1H), 7.26–
7.48 (m, 9H), 7.54 (t, J = 7.5 Hz, 10H), 7.68 (q, J = 6.75 Hz, 5H), 7.90
(q, J = 7.8 Hz, 9H), 8.05 (t, J = 7.3 Hz, 10H), 8.25 (t, J = 8.1 Hz, 8H). 13
C NMR (75 MHz, CDCl3): d 62.7, 69.1, 70.5, 70.9, 72.09, 73.2, 92.7,
128.4, 128.4, 128.5, 128.5, 128.7, 128.8, 129.0, 129.6, 129.8,
129.8, 130.0. 130.2, 130.3, 130.4, 133.1, 133.4, 133.5, 133.5,
133.8, 133.9, 164.6, 165.2, 165.7, 166.1. Anal. Calcd for
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Acknowledgment
T.M. acknowledges financial support from the UGC, New Delhi.
We also thank DST, New Delhi for 300 MHz NMR spectrometer un-
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