Monogalactopyranosides of fluorescein and fluorescein methyl ester: Synthesis, enzymatic hydrolysis by biotnylated β-galactosidase, and determination of translational diffusion coefficient

Article abstract of DOI:10.1016/j.carres.2012.05.017

Fluorescein monoglycosides (d-galactopyranoside (FMG) and d-glucopyranoside) and their methyl ester (MFMG) have been prepared from acetobromoglucose/galactose and fluorescein methyl ester in good yields. Enzymatic hydrolysis experiments (using biotinylated β-galactosidase) of the galacto derivatives have been performed and kinetic parameters were calculated. A 15-20 times increase of the fluorescence intensity has been observed during the hydrolysis. A linear increase of fluorescence has been noted at short time and low concentration of substrate, making these compounds useful and sensitive probes for galactosidases. The magnitude of the Michaelis-Menten constant (K_m) value for MFMG is higher than that of FMG suggesting a possible conformational change of the fluorogenic substrate. K_m value for biotinylated β-Gal with FMG is lower than that for the native enzyme. This observation indicates higher substrate affinity of the biotinylated enzyme in comparison to the native enzyme. Translational diffusion coefficients have been measured, for both fluorogenic substrates and both the products, employing fluorescence correlation spectroscopy. Translational diffusion coefficients for fluorogenic substrates and the enzymatic hydrolysis products have been measured to be similar, in the range of 3.5-4.5 × 10^-10 m² s^-1. Thus an enhancement or retardation of the enzymatic kinetics due to difference in translational mobility of substrate and product is not that apparent.

Full text of DOI:10.1016/j.carres.2012.05.017

Carbohydrate Research 358 (2012) 40–46
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Monogalactopyranosides of fluorescein and fluorescein methyl ester: synthesis,
enzymatic hydrolysis by biotnylated b-galactosidase, and determination of
translational diffusion coefficient
Prasun K. Mandal a,b, Laurent Cattiaux c,d,e, David Bensimon a,f, Jean-Maurice Mallet c,d,e,
⇑
a Laboratoire de Physique Statistique, Ecole Normale Supérieure, Département de Physique, UMR CNRS-ENS 8550, 24 rue Lhomond, 75231 Paris, France
b Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) – Kolkata, Mohanpur Campus, Mohanpur 741252, West Bengal, India
c UPMC Paris06, UMR 7203, Laboratoire des BioMolécules, Université P. et M. Curie, 4 Place Jussieu, 75005 Paris, France
d CNRS, UMR 7203, France
e ENS, UMR 7203, Département de Chimie, Ecole Normale Supérieure, 24, Rue Lhomond, 75005 Paris, France
f Department of Chemistry and Biochemistry, University of California, Los Angeles, CA, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorescein monoglycosides (D-galactopyranoside (FMG) and D-glucopyranoside) and their methyl ester
Received 15 March 2012
Received in revised form 11 May 2012
Accepted 15 May 2012
(MFMG) have been prepared from acetobromoglucose/galactose and fluorescein methyl ester in good
yields. Enzymatic hydrolysis experiments (using biotinylated b-galactosidase) of the galacto derivatives
have been performed and kinetic parameters were calculated. A 15–20 times increase of the fluorescence
intensity has been observed during the hydrolysis. A linear increase of fluorescence has been noted at
short time and low concentration of substrate, making these compounds useful and sensitive probes
for galactosidases. The magnitude of the Michaelis–Menten constant (Km) value for MFMG is higher than
that of FMG suggesting a possible conformational change of the fluorogenic substrate. Km value for bio-
tinylated b-Gal with FMG is lower than that for the native enzyme. This observation indicates higher sub-
strate affinity of the biotinylated enzyme in comparison to the native enzyme. Translational diffusion
coefficients have been measured, for both fluorogenic substrates and both the products, employing fluo-
rescence correlation spectroscopy. Translational diffusion coefficients for fluorogenic substrates and the
Available online 23 May 2012
Keywords:
Galactoside
Glucoside
Fluorescein
Biotinylated galactosidase
Fluorogenic probe
Translational diffusion coefficient
enzymatic hydrolysis products have been measured to be similar, in the range of 3.5–4.5 ꢀ 10ꢁ10 m2 sꢁ1
.
Thus an enhancement or retardation of the enzymatic kinetics due to difference in translational mobility
of substrate and product is not that apparent.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
So, the rate of fluorescence increase from FDG was found to be very
slow and not correlated with the b-galactosidase activity as a linear
Fluorogenic glycosides are important probes for reporting glyco-
sidase activities. In such compounds, a sugar blocks a chemical
group necessary for fluorescence through a glycosidic linkage. Fluo-
rescein is a suitable dye for that purpose and fulfills two require-
ments: (a) high fluorescence quantum yield; (b) a free phenol
function that is necessary for fluorescence and can be blocked
through an enzymatically labile glycosidic linkage. In this respect,
fluorescein digalactoside (FDG) is a commercially available com-
pound for galactosidase assays (Scheme 1). However, the fluores-
cence production from FDG needs two enzymatic steps: first step
involves hydrolysis of FDG to give fluorescein mono galactoside
(FMG)—a dark (or weakly-fluorescent) compound- and the second
step to give fluorescein, the final fluorescent product. For Escherichia
coli galactosidase, the first step is 12 times slower than the second:
function at short time and low concentration (<<Km) (Scheme 1).
FMG will therefore be a far better fluorogenic compound than FDG
for kinetic measurements.
In spite of more and more enzymatic assay studies, the struc-
ture–function relationship for enzymes at the single enzyme level
is still not understood completely.2–4 The major hurdle in this direc-
tion is the non availability of suitable fluorogenic probe having neg-
ligible emission and significantly higher signal to noise ratio of the
highly fluorescent enzymatic hydrolysis product. Moreover, photo-
stability of the fluorescent product is also a matter of concern.
We were interested in the enzymatic activity of a single immo-
bilized enzyme, specially b-galactosidase, at the single molecule le-
vel, using a fluorogenic galactosyl reporter. A two step hydrolysis
(of FDG) leading to the final fluorescent product dramatically limits
the performance of this kind of experiment as the first hydrolysis
product will diffuse out of the illuminating volume of the micro-
scope. A single step hydrolysis reaction is needed and FMG could
possibly be a suitable substrate for that purpose. Commercially
(ka = 1.9 l l
mol minꢁ1 mgꢁ1; kb = 22.7 mol minꢁ1 mgꢁ1)(Scheme 1).1
⇑
Corresponding author. Tel.: +33 (0)1 44 32 33 90.
E-mail address: Jean-Maurice.Mallet@ens.fr (J.-M. Mallet).
0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.carres.2012.05.017

P. K. Mandal et al. / Carbohydrate Research 358 (2012) 40–46
41
OH
OH
OH
HO
HO
OH
O
O
O
HO
HO
HO
HO
O
O
O
O
HO
HO
O
O
OH
OH
OH
OH
O
O
O
O
O
O
COOMe
COOH
O
COOH
[Enz] step a
OH
O
CO
HO
HO
O
FMG
MFMG
FMG
OH
OH
O
FDG
HO
O
HO
HO
OH
O
O
O
O
[Enz] step b
COOH
Me
Fluorescein
Tokyo green
Scheme 1. Hydrolysis of FDG by galactosidase.
Figure 2. Chemical structures of MFMG, FMG and Tokyo green.
available alternative probes to FMG, resorufin-b-
side, methylumbelliferyl-galactosides, and DDAO galactoside (9H-
(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) b- -galactopyrano-
D-galactopyrano-
complex (ES) and (b) irreversible conversion of ES to product are
determined partly by the diffusion constants of substrates. Thus
we have measured the translational diffusion coefficients of both
fluorogenic substrates and the products employing fluorescence
correlation spectroscopy.
D
side) have critical drawbacks.5,6 Due to widely separated absorp-
tion profile of fluorogenic substrate and the enzymatic hydrolysis
product two lasers need to be used in order to excite them. Either
it is necessary to excite them in the UV region or significant overlap
of the excitation and emission spectra of the enzymatic hydrolysis
product reduces the sensitivity significantly.5,6 Moreover, these
dyes are not as bright as fluorescein.
2. Results and discussion
2.1. Synthesis of the probes
In this manuscript we report detailed synthesis of Fluorescein
monoglycosides
(
D
-galactopyranoside and
D-glucopyranoside)
We want to describe here the synthesis of fluorescein mono
galactoside (FMG) and of their methyl ester (MFMG) and the kinet-
ics of their enzymatic hydrolysis (Fig. 1). Strangely enough, FMG is
not commercially available and no convenient synthesis of this
compound is described in literature. Alternatively, Urano et al.7
developed Tokyo green(s) (Fig. 2), mono galactosylated closely
related compounds, prepared in several steps from a xanthone
derivative.8 They had shown that the presence of –COOH group
on the pendant benzenic ring causes no change in the fluorescence
(FMG) and their methyl ester (MFMG). Characterization of all prod-
ucts has been made. We have employed biotinylated b-galactosi-
dase instead of native enzyme as the former would be used for
single molecule experiments. Michaelis–Menten constant (Km) va-
lue for FMG and MFMG has been measured in order to verify
whether there is any change because of chemical modification of
the substrate. It is well-known that two steps of enzyme catalyzed
reactions viz. (a) reversible formation of enzyme–substrate
Figure 1. Kinetic profile of FDG ? FMG ? F sequence.

42
P. K. Mandal et al. / Carbohydrate Research 358 (2012) 40–46
OAc
O
OAc
O
AcO
AcO
O
HO
OAc
O
HO
AcO
AcO
AcO
AcO
OAc
Br
OAc
OAc
Br
2
O
O
5
a
O
O
O
6
1
COOMe
O
O
a
b
COOMe
CO
HO
OH
OH
1
O
O
O
OAc
HO
HO
HO
HO
O
AcO
AcO
OH
OH
O
c
O
O
O
O
b
c
4
OAc
d
COOH
COOMe
MFMG
FMG
O
O
8
7
COOMe
3
Scheme 4. Reagents and conditions: (a) Cs2CO3, DMF, 5 h, rt, 72%; (b) MeONa,
MeOH, 2 h, rt, 56%; (c) NaOH, water, 1 h, rt, 69%.
Scheme 2. Synthesis steps of MFMG and FMG. Reagents and conditions: (a) litt:
H2SO4, MeOH reflux; (b) Cs2CO3, DMF, 5 h, rt, 79%; (c) MeONa, MeOH, 2 h, rt, 47%;
(d) NaOH, water, 1 h, rt, 53%.
glucosylation of fluorescein with a plant cell culture (Rauwolfia
serpentina).12
2.2. Enzymatic hydrolysis of galacto derivatives: FMG and MFMG
property of the fluorophore. Rather, it causes the benzene ring to
remain in an orthogonal plane with respect to the xanthenes plane
and thus maintains a high fluorescence quantum yield.7 The
methyl ester of FMG (MFGM) (Fig. 2) is thus expected to have sim-
ilar photo-physical properties but may cause a difference in the
kinetics of the enzymatic hydrolysis
The synthesis of FMG is depicted in Scheme 2. After unsuccess-
ful9 direct galactosylation of fluorescein, we decided to protect the
acid function as a methyl ester and therefore fluorescein methyl
ester (1) was prepared using a method first published in 1901 by
Feuerstein and Dutoit10 and recently improved by Adamczyk
et al.11 by reacting fluorescein with refluxing methanol in the pres-
ence of sulfuric acid. The reaction of this ester with acetobromoga-
lactose 2 in dimethylformamide (DMF) in the presence of cesium
carbonate gave the key glycoside 3 in good yield 79% (Scheme 2).
Compound 3 is a mixture of two atropisomers, due to restricted
rotation around the central bound.
We wanted to study first the hydrolysis of FMG and MFMG by a
b-galactosidase in solution by the formation of the and fluorescent
products (fluorescein and methylated fluorescein) and to evaluate
the kinetics parameters. The galactosidase used is a genetically
engineered biotinylated b-Gal (b-Gal from Thermo Scientific/
Pierce Protein Research Products) as the latter will be used in our
future experiments in order to probe its enzymatic activities at
the single molecule level. This preliminary study would allow us
to check the substrate purity and to estimate the alteration of
the activity of the biotinylated enzyme.
A literature report suggests that for FMG fluorescence intensity
follows a strict linear relation with substrate concentration within
1 l
M.13 We have kept the concentration of the substrate within
this limit. The initial concentration of the substrate was kept low
enough (absorbance at around 0.05) in order to avoid concentra-
tion related problems.
The treatment of 3 with sodium methylate in dry methanol
gave the methyl ester 4 (MFMG), a neutral compound analogous
to Tokyo greens. The b selectivity was controlled by the participat-
ing group and confirmed in 1H NMR (H-1 5.13 ppm J1,2 = 7.8 Hz)
and 13C NMR (C-1 100.3 ppm). Saponification of 4 with an aqueous
solution of sodium hydroxide gave FMG. FMG is in fact a mixture of
two compounds in equilibrium, as seen in NMR. In DMSO, the less
polar cyclized form is favored (Scheme 3).
Time based kinetic studies of enzymatic assays for both sub-
strates are depicted in Figures 3 and 4. We have used a very dilute
solution of FMG and MFMG (a few hundred nM to 1 lM) in order to
reduce the concentration quenching due to high concentration. The
absorption spectra show a 40 nm red shift of the absorption maxi-
mum on going from substrate to the product for both substrates.
For 1 lM substrate concentration, a 15–20 times enhancement of
the fluorescence intensity was observed at the emission maximum.
As can be seen from the above Figure 4, during first 50–100 s the
curve is clearly linear for all four substrate concentrations ([S])
and the initial velocity (v) of the enzymatic reaction was calculated
from this time period. These data were used for the Lineweaver–
Burk plot (LB plot) of 1/v versus 1/[S]. The Michaelis–Menten con-
stant (Km) values were obtained for both FMG and MFMG from
these LB plots. To the best of our knowledge there is no report for
In a similar way, gluco derivatives (7 and 8) were prepared from
1 and acetobromoglucose (5) in comparable yields (see Section 4
and Scheme 4). Compound 8 was previously prepared by enzymatic
OH
OH
HO
HO
O
Km values of MFMG. Km value obtained for MFMG is 11.85
values for FMG using native b-Gal are reported to be 117.6
and 96.6
M.13 In our experiment using biotinylated b-Gal, we have
obtained a Km value for the same substrate of 2.35 M. So, the sub-
lM. Km
O
HO
HO
O
O
lM
l
OH
OH
l
O
O
O
strate concentration is well below the Km value. Lower Km value for
biotinylated b-Gal indicates higher substrate affinity in comparison
to native enzyme. This result, although it is new, however, it is not
unprecedented. A similar result was reported for the same enzyme
(native and biotinylated b-Gal) with a different substrate (o-nitro-
O
COOH
O
HO
phenyl-b-D
-galacto pyranoside).14 Another important observation
Scheme 3. Equilibrium between open and lactonic form of FMG.

P. K. Mandal et al. / Carbohydrate Research 358 (2012) 40–46
43
0,15
(a)
(b)
Substrate
Product
0,12
0,10
0,08
0,06
0,04
0,02
0,00
0 min
1
4
9
0,10
15
21
26
31
35
40
45
50
55 min
0,05
0,00
350
400
450
500
550
600
350
400
450
500
550
600
Wavelength (nm)
Wavelength (nm)
Figure 3. Time dependent change of the absorption spectra for FMG (a) and MFMG (b) upon enzymatic hydrolysis. In case of MFMG only initial and final spectra are shown
for clarity. A clear isosbestic (both in (a) and (b)) at around 460 nm confirms the mechanism.
800x10 3
200x103
(b)
(a)
600
150
400
100
200
50
0
0
0
1000
2000
3000
4000
0
1000
2000
3000
4000
Time (Sec)
Time (Sec)
Figure 4. Time dependent fluorescence (monitored at 511 nm) measurement plot for different concentrations of FMG (a) and MFMG (b) with a fixed b-Gal concentration.
(red: 1 M, blue: 500 nM, Green: 200nM, black: 100 nM) (excitation at 488 nm, emission monitored at 511 nm).
l
Figure 5. Absorption and emission spectra of FMG, MFMG and fluorescein: (a) Normalized absorption spectra. (Concentration ca. 10ꢁ5 Mꢁ1 in 100 mM phosphate buffer, pH
7.4. (b) Normalized emission spectra with an excitation at 450 nm (concentration ca. 10ꢁ6 Mꢁ1 in 100 mM phosphate buffer, pH 7.4).
we would like to mention here is that the replacement of –COOH
group with the –COOMe group does not alter the absorption or
emission maxima significantly but alters the Km values. This per-
haps indicates that the replacement of –COOH with –COOMe causes

44
P. K. Mandal et al. / Carbohydrate Research 358 (2012) 40–46
significant change in the molecular conformation of the fluorogenic
substrate (presence of a negative charge or coexistence of the cyclic
lactone for FMG).
of FMG suggesting a possible conformational change of the fluoro-
genic substrate. Magnitude of the Michaelis–Menten constant (Km)
for biotinylated b-Gal with FMG is lower than that for the native
enzyme. This observation indicates higher substrate affinity of
the biotinylated enzyme in comparison to the native enzyme. Sim-
ilar translational diffusion coefficients have been obtained for both
substrates and both the products. Thus an enhancement or retar-
dation of the enzymatic kinetics due to difference in translational
mobility of substrate and product is not that apparent.
Absorption and emission spectra of the enzyme substrates
(FMG and FMMG) and fluorescein are shown in Figure 5. Although,
there are a red shift in absorption and a blue shift in emission, on
going from substrate to the product, there is significant overlap of
the spectra both in absorption and in fluorescence. Neither selec-
tive excitation of fluorescein nor a selective observation of fluores-
cein is thus possible to increase the sensitivity. Although these two
galactosides (FMG and MFMG) can be useful tools for galactosi-
dases assays at the ensemble level (a 15–20-fold enhancement of
fluorescence intensity), however, for single molecule fluorescence
experiments these probes are not quite suitable, because these
experiments demand a negligible background signal and signifi-
cantly higher signal to noise ratio. The research for new probes
in this direction is currently underway.
4. Experimental
4.1. General procedures
All compounds were homogeneous by TLC analysis and had
spectral properties consistent with their assigned structures. Opti-
cal rotations were measured with a Perkin–Elmer Model 241 digi-
tal polarimeter at 22 3 °C. Compound purity was checked by TLC
on Silica Gel 60 F254 (E. Merck). Column chromatography was per-
formed on Silica Gel 60 (E. Merck) using cyclohexane (Cyhex),
dichloromethane (DCM) and Ethyl acetate. 1H NMR spectra were
recorded with Brüker AM 250, AM 400 instruments
2.3. Measurement of diffusion coefficient
It is well-known that the rate coefficient, k(t), for enzyme–sub-
strate binding is time dependent due to the influence of diffusion.
Two steps of the enzyme-catalyzed reaction that is (i) reversible
formation of the enzyme–substrate complex (ES), and (ii) irrevers-
ible conversion of the substrate in ES to product, are controlled by
the diffusion rates of the substrate and the product. Hence, we
thought it will be worth measuring the diffusion coefficient of both
the products and both the substrates under identical experimental
condition employing fluorescence correlation spectroscopic tech-
nique. The result is shown in Figure 6 below.
As can be seen from Figure 6 and Table 1, the diffusion behavior
is quite similar for both substrates as well as both products. This is
an important observation as for both substrate–product pair the
diffusion coefficients are similar, so enhancement or retardation
of the enzymatic hydrolysis due to difference of diffusion coeffi-
cient of substrate and/or product is not that much apparent.
OH
O
6'
HO
O
1'
1
2
HO
3
13
16
17
18
OH
15
4
14
12
5
O
11
9
19
O
10
HO
7
20
O
8
.
4.2. Synthesis of substrates
4.2.1. Galacto series
4.2.1.1. Fluorescein methyl ester mono (2,3,4,6-tetra-O-acetyl b-
-galactopyranoside) (3). mixture of (450 mg,
1.292 mmol, 1 equiv), Cs2CO3 (4.21 g, 12.92 mmol, 10 equiv) and
2,3,4,6-tetra-O-acetyl- -galactopyranosyl bromide (2) (4.50 g,
3. Conclusion
D
A
1
We have synthesized two fluorogenic probes for the kinetic
studies of b-galactosidases. Both the substrate–product pairs exhi-
bit similar absorption and fluorescence maxima, suggesting that
methyl substitution at the carboxyl group does not alter photo-
chemical behavior. Both the substrates show strict linearity of fluo-
rescence increase with time. Km value for MFMG is higher than that
a
-D
10.72 mmol, 8.3 equiv) in dry DMF (150 mL) was stirred for 5 h
at room temperature. The precipitate was filtered off and the fil-
trate was concentrated under reduced pressure. The residue ob-
tained was diluted in water and extracted three times with DCM.
The organic layers were combined and washed with water and sat-
urated NaCl, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel with Cyhex/AcOEt 50:50 and
DCM/MeOH 97/3 to give 3 as an orange powder (694 mg, 79%,
two atropoisomers). Rf: 0.31 (DCM/MeOH 100:3) a2D5 +19 (c 1,
CHCl3). 1H NMR (400 MHz, CDCl3)
d (ppm): 8.28 (d, 1H,
J = 7.8 Hz), 7.76 (m, 1H), 7.70 (t, 1H, J = 7.8 Hz), 7.33(d, 1H,
J = 5.7 Hz), 7.13 (m, 1H), 6.91 (dd, 1H, J = 0.87 Hz, J = 8.8 Hz), 6.86
(dd, 1H, J = 4.0 Hz, J = 9.7 Hz), 6.81 (m, 1H), 6.56 (dd, 1H,
J = 1.8 Hz, J = 9.7 Hz), 6.44 (t, 1H, J = 2.0 Hz), 5.54 (m, 2H), 5.18
(m, 2H), 4.21 (m, 3H), 3.68 (2s, 3H), 2.1 (m, 12H). 13C NMR
(100 MHz, CDCl3) d (ppm): 186.2, 170.8, 170.5, 170.4, 169.7,
165.9, 160.7, 159.2, 154.0, 149.9, 134.9, 133.3, 131.6, 130.9,
130.8, 130.7, 130.2, 129.4, 118.9, 117.0, 115.2, 114.7, 106.3,
104.2, 103.8, 99.2, 72.0, 71.1, 68.7, 67.2, 61.9, 52.9, 21.2, 21.1,
21.0, 21.0. MS-ESI-HRMS m/z [M+H]+ calculated for C35H33O14
:
677.18648, found: 677.18711
4.2.1.2. Fluorescein methyl ester mono (b-
(4 MGFM). To a solution of 3 (380 mg, 0.562 mmol, 1 equiv) in
anhydrous MeOH (3 mL) was added sodium (13 mg, 0.56 mmol,
D-galactopyranoside)
Figure 6. Diffusion behavior of Fluorescein (green), 1 (MF) (blue), FMG (brown),
and MFMG (red). For clarity a blown up part of the graph has been shown as inset
(details of the experimental condition is in Section 4).

P. K. Mandal et al. / Carbohydrate Research 358 (2012) 40–46
45
atropoisomers). Rf: 0.31 (DCM/MeOH 100:3) a2D5 2 (c 1, CHCl3). 1H
NMR (400 MHz, CDCl3) d (ppm): 8.26 (d,1H, J = 7.6 Hz), 7.77 (m,
1H, H), 7.69 (t, 1H, J = 7.6 Hz), 7.31 (m, 1H), 7.11 (2d, 1H,
J = 2.3 Hz), 6.90 (d,1H, J = 8.9 Hz), 6.85 (2d, 1H, J = 9.7 Hz), 6.78
(m, 1H), 6.54 (dd, 1H, J = 1.9 Hz, J = 9.7 Hz), 6.42 (2d, 1H,
J = 2.7 Hz), 5.26 (m, 4H), 4.25 (m, 2H), 4.00 (m, 1H), 3.67 (2s, 3H).
13C NMR (100 MHz, CDCl3) d (ppm): 186.2, 170.9, 170.5, 170.5,
169.8, 165.8, 160.7, 159.2, 154.0, 150.0, 133.3, 131.6, 130.9,
130.7, 130.6, 130.2, 129.3, 118.9, 117.0, 115.0, 114.7, 106.2,
104.2, 103.9, 98.5, 72.9, 71.3, 68.5, 67.2, 62.3, 52.9, 21.2, 21.1,
Table 1
Diffusion coefficient of both fluorogenic substrates (FMG and MFMG) and their
enzymatic reaction products (fluorescein, methylated fluorescein)
Probe
Diffusion Coefficient
Reference
(ꢀ10ꢁ10 m2 sꢁ1
)
15
Fluorescein (F)
Fluorescein methyl ester
(MF)
FMG
MFMG (4)
4.36
4.05
This work
4.03
3.45
This work
This work
21.0, 21.0. MS-ESI-HRMS m/z [M+H]+ calculated for C35H33O14
:
677.18648, found: 677.18758.
1 equiv). The mixture was stirred for 2 h at room temperature, neu-
tralized with Amberlite IR120-H+ resin, filtered (the resin was
rinsed with MeOH). The solution was concentrated under reduced
pressure and the residue was chromatographed on silica gel with
DCM/MeOH 90:10 to give 4 as a red solid (180 mg, 47%, two atro-
poisomers). Rf: 0.22 (DCM/MeOH 90:10) a2D5 ꢁ82 (c 1, MeOH). 1H
NMR (400 MHz, MeOD) d (ppm) : 8.30 (2 dd, 1H, J = 7.8 Hz,
J = 1.6 Hz), 7.88 (2t, 1H, J = 7.1 Hz), 7.50 (2t, 1H, J = 7.3 Hz), 7.46
(d, 1H, J = 7.5 Hz), 7.40 (t, 1H, J = 2.3 Hz), 7.08 (m, 2H), 7.04 (d,
1H, J = 2.1 Hz), 6.60 (2d, 1H, J = 2.1 Hz, J = 3.1 Hz), 6.50 (2d, 1H,
J = 1.9 Hz), 5.13 (2d, 1H, J = 7.8 Hz, H01), 3.96 (d, 1H, J = 3.3 Hz),
3.84 (m, 3H), 3.67 (2dd, 1H, J = 9.6 Hz, J = 3.3 Hz), 3.64 (2s, 3H).
13C NMR (100 MHz, MeOD) d (ppm): 186.3, 166.0, 163.2, 160.4,
154.8, 154.7, 134.3, 133.1, 131.6, 131.2, 130.8, 130.4, 130.3,
129.7, 128.6, 117.6, 116.1, 115.5, 104.5, 103.6, 101.3, 76.3, 73.7,
70.9, 69.1, 61.4, 51.9. MS-ESI-HRMS m/z [M+H]+ calculated for
4.2.2.2. Fluorescein methyl ester mono (b-
(7). To a solution of 6 (1.202 g, 1.778 mmol, 1 equiv) in anhy-
D-glucopyranoside)
drous MeOH (30 mL) was added sodium (41 mg, 1.778 mmol,
1 equiv). The mixture was stirred for 2 h at room temperature, neu-
tralized with Amberlyst IR120-H+ resin, (the resin was rinsed with
MeOH), and concentrated under reduced pressure and the residue
was chromatographed on silica gel with DCM/MeOH 90:10 to give
7 as a red solid (503 mg, 56%). Rf: 0.19 (DCM/MeOH 90:10) a2D5 ꢁ98
(c 1, MeOH). 1H NMR (400 MHz, MeOD) d (ppm): 8.33 (d, 1H,
J = 7.8 Hz), 7.88 (2t, 1H, J = 6.9 Hz), 7.81 (2t, 1H, J = 7.1 Hz), 7.46
(d, 1H, J = 7.5 Hz), 7.39 (s, 1H), 7.07 (m, 3H), 7.04 (d, 1H,
J = 2.3 Hz), 6.60 (2dd, 1H, J = 2.6 Hz, J = 9.6 Hz), 6.51 (d, 1H,
J = 2.0 Hz), 5.17 (m, 1H), 3.96 (d, 1H, J = 12.1 Hz), 3.74 (m, 1H),
3.64 (2s, 3H), 3.53 (m, 4H). 13C NMR (100 MHz, CDCl3) d (ppm):
187.4, 167.0, 164.2, 161.4, 155.9, 155.7, 135.4, 134.3, 132.6, 132.3,
131.9, 130.8, 129.7, 118.7, 117.3, 116.5, 116.4 105.7, 104.9, 104.7,
101.7, 101.6, 78.4, 77.8, 74.7, 71.2, 62.4, 53.9. MS-ESI-HRMS m/z
[M+H]+ calculated for C27H25O10: 509.14422, found: 509.14481.
C27H25O10: 509.14422, found: 509.14384.
4.2.1.3.
(FMG).
Fluorescein
Compound 4 (62 mg, 0.122 mmol, 1 equiv) was dis-
L, 0.24 mmol, 2 equiv)
was added and the mixture was stirred for 1 h at room tempera-
ture, neutralized with aq HCl 1 M (0.244 mmol, 244 L, 2 equiv),
mono
(b-D-galactopyranoside)
solved in H2O (2 mL). aq NaOH 1 M (240
l
4.2.2.3. Fluorescein mono (b-
Compound 7 (96 mg, 0.1890 mmol, 1 equiv) was dissolved in H2O
(2 mL). NaOH 1 M (378 L, 0.3780 mmol, 2 equiv) wad added and
the mixture was stirred for 1 h at room temperature, neutralized
with HCl 1 M (378 L, 0.3780 mmol, 2 equiv), and concentrated
D-glucopyranoside) (8).
l
l
and concentrated under reduced pressure. The residue was chro-
matographed on silica gel with AcOEt/MeOH/H2O 70:5:3 to give
a yellow powder (31 mg, 53%). Rf: 0.38 (AcOEt/MeOH/H2O 90:5:3)
a2D5 +20 (c 1, MeOH). 1H NMR (400 MHz, DMSO) d (ppm): 8.00
(d,1H, H18, J = 7.2 Hz), 7.79 (t, 1H, H16, J = 7.3 Hz), 7.73 (t, 1H,
H17, J = 6.8 Hz), 7.29 (d, 1H, H15, J = 6.3 Hz), 7.00 (s, 1H, H13),
6.77 (d, 1H, H2, J = 9.0 Hz), 6.68 (m, 2H, H3, H10), 6.58 (s, 2H, H7,
H8), 4.93 (m, 1H, H10), 3.59 (m, 6H, H20,H30,H40,H50, H60), 2.08 (s,
4H, OH saccharides). 13C NMR (100 MHz, DMSO) d (ppm): 168.9
(C20), 159.8 (C9), 159.2 (C1), 152.6 (C14), 152.0 (C11), 135.9
(C16), 130.4 (C17), 129.3 (C7), 129.1 (C3), 126.2 (C19), 124.9
(C18), 124.0 (C15), 113.5 (C2), 113.1 (C8), 112.4 (C4), 109.6 (C6),
103.4 (C13), 102.4 (C10), 100.9 (C10), 82.8 (C5), 75.62 (C50), 73.2
(C30), 70.1 (C40), 68.1 (C20), 60.4 (C60). MS-ESI-HRMS m/z [M+H]+
calculated for C26H23O10: 495.12857, found: 495.12839.
l
under reduced pressure. The residue was chromatographed on sil-
ica gel with AcOEt/MeOH/H2O 90:5:3 to give 8 as a yellow powder
(64 mg, 69%). Rf: 0.38 (AcOEt/MeOH/H2O 70:5:3) a2D5 ꢁ22 (c 1,
CHCl3). 1H NMR (400 MHz, DMSO) d (ppm): 8.02 (d, 1H, H18
,
J = 6.9 Hz), 7.81 (m, 1H, H16), 7.74 (t, 1H, H17, J = 6.8 Hz), 7.30 (d,
1H, H15, J = 6.5 Hz), 7.03 (s, 1H, H13), 6.80 (d, 1H, H2, J = 8.6 Hz),
6.69 (m, 2H, H3, H10), 6.59 (s, 2H, H7, H8), 5.00 (d,1H, H10,
J = 6.2 Hz), 3.71 (m, 1H, H60a), 3.24 (m, 5H, H20, H30H40, H50,
H60b). 13C NMR (100 MHz, DMSO) d (ppm): 168.4 (C20), 160.1
(C9), 159.2 (C1), 152.0 (C14), 151.9 (C11), 151.8 (C12), 135.9
(C16), 130.4 (C17), 129.4 (C7), 129.2 (C3), 126.4 (C19), 125.00
(C18), 124.3 (C15), 113.5 (C2), 113.3 (C8), 112.6 (C4), 109.6 (C6),
103.6 (C13), 102.5 (C10), 100.3 (C10), 83.5 (C5), 77.3 (C50), 76.7
(C30), 73.4 (C20), 69.9 (C40), 60.9 (C60). MS-ESI-HRMS m/z [M+H]+
calculated for C26H23O10: 495.12857, found: 495.12919 (in agree-
ment with lit data)
4.2.2. Gluco series
Similar experiments starting from aceto-bromoglucose.
4.2.2.1. Fluorescein methyl ester mono (2,3,4,6-tetra-O-acetyl b-
4.3. Absorption and fluorescence experiments
D
-glucopyranoside) (6).
0.5742 mmol, 1 equiv), Cs2CO3 (1.87 g, 5.742 mmol, 10 equiv),
and 2,3,4,6-tetra-O-acetyl- -glucopyranosyl bromide (2.00 g,
A
mixture of
1
(200 mg,
Absorption and florescence measurements were performed in
Uvikon-940 spectrophotometer (Zurich, Switzerland) and LPS 220
spectrofluorometer (PTI, NJ) respectively. The temperature was
maintained using circulating baths (Polystat 34-R2 Fischer Bio-
block Scientific, France) and the temperature was directly mea-
sured using a type K Thermocouple connected to ST-610B digital
Pyrometer (Stafford Instruments, UK). For fluorescence based
experiments 488 nm light was used for excitation and the emission
a-D
4.766 mmol, 8.3 equiv) in dry DMF (70 mL) was stirred for 5 h at
room temperature. The inorganic precipitate was filtered off and
the filtrate was concentrated under reduced pressure. The residue
obtained was diluted in water and extracted three times with DCM.
The organic layers were combined and washed with water and sat-
urated aq NaCl, dried over MgSO4, and evaporated. The residue was
chromatographed on silica gel with Cyhex/AcOEt 50:50 and DCM/
MeOH 100/3 to give 6 as an orange powder (281 mg, 72% two
was monitored at 511 nm. For each experiment, 750
lL of different
concentrations of substrate was allowed to be cleaved by b-Gal

46
P. K. Mandal et al. / Carbohydrate Research 358 (2012) 40–46
(concn <<1 U/mL). Concentrations of substrate and enzyme were
kept in the regime where initial kinetic velocity was proportional
to both substrate and enzyme concentration. All experiments were
performed at 25 °C. All enzymatic assays were performed in phos-
phate buffer (100 mM) at pH 7.4 in the presence of 1 mM MgCl2
and 110 mM mercaptoethanol.
Acknowledgement
P.K.M. thanks Thomas Lesaux for his help during absorption and
fluorescence measurements. P.K.M. gratefully acknowledges Marie
Curie International Incoming Fellowship awarded to him.
Supplementary data
4.4. Fluorescence correlation spectroscopy experiments
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.carres.2012.05.017.
Fluorescence correlation spectroscopy (FCS) was used to calcu-
late the translational diffusion coefficient of the fluorogenic sub-
strates as well as products. For excitation of the fluorophore,
488 nm laser (Ar-ion, Spectra Physics) was used. Excitation laser
as well as fluorescence photons were passed through Olympus UP-
lan Apo 60ꢀ 1.20 W infinity corrected objective. To collect the fluo-
rescence photons 488 nm long pass filter (Semrock) was used.
After passing through the longpass filter the fluorescence photons
were coupled to optical fibers (FG200 LCR multimode fiber, Thorl-
abs) and were detected with two avalanche photodiodes (SPCM-
AQR-14, Perkin–Elmer) coupled to an ALV-6000 correlator (ALV
GmbH). The incident powers at the sample were measured with
a NOVA II powermeter (Laser Measurement Instruments). All the
series of experiments reported in the present work were per-
formed in a regime of laser powers (3–5 mW) in which fluorescein
exhibits a linear dependence of the intensity of fluorescence emis-
sion on the illumination power. The focussed beam was assumed
to adopt a 3D-Gaussian excitation profile characterized by Eq. 1.
References
1. (a)
http://www.biotek.com/resources/articles/beta-galactosidase-plate-
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"
#
2ðx2 þ y2Þ 2z2
I ¼ I0 exp
where,
ꢁ
ꢁ
ð1Þ
x2xy
xz2
x
z and xxy are the sizes of the beam waist in the direction of
propagation of the laser beam and in the perpendicular direction
respectively. FCS autocorrelation curve was fitted successfully with
the following expression (Eq. 2) of the freely diffusing species:
14. Witkowski, A.; Kindy, M. S.; Daunert, S.; Bachas, L. G. Anal. Chem. 1995, 67,
1301–1306.
15. Petrášek, Z.; Schwille, P. Biophys. J. 2008, 94, 1437–1448.
ꢀ
ꢁꢁ1
ꢀ
ꢁꢁ1=2
1
N
s
sD
s
Gð
s
Þ ¼
1 þ
1 þ
ð2Þ
x2sD
ꢀ
ꢀ
where N is the average number of fluorescein molecules contained
in the illuminated volume. sD
through the beam waist, and x = xz/xxy.
=
x2xy/8D is the diffusion time