New fluorescent probes for carbonic anhydrases

Article abstract of DOI:10.1039/b701421j

We report the synthesis and fluorescence properties of naphthalenesulfonamide derivatives as active site probes for carbonic anhydrases. The Royal Society of Chemistry.

Full text of DOI:10.1039/b701421j

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New fluorescent probes for carbonic anhydrases{
Jayati Banerjee,a Manas K. Haldar,a Sumathra Manokaran,b Sanku Mallik*a and D. K. Srivastava*b
Received (in Austin, TX, USA) 30th January 2007, Accepted 1st April 2007
First published as an Advance Article on the web 20th April 2007
DOI: 10.1039/b701421j
fluorescent probes has been their high quantum yields in the free
forms, and such quantum yields are not significantly altered upon
binding of the probes to the CA sites.9 This feature, aside from
exhibiting lower sensitivity, also provides a high background
fluorescence while performing both equilibrium ligand displace-
ment as well as transient kinetic experiments. In view of the above
limitations, we proceeded to synthesize a variety of naphthalene-
sulfonamide derivatives as fluorescent probes for carbonic
anhydrases and determined their fluorescence properties in the
absence and presence of recombinant human carbonic anhydrase
isozymes I and II.
We report the synthesis and fluorescence properties of
naphthalenesulfonamide derivatives as active site probes for
carbonic anhydrases.
Due to the marked sensitivity of fluorescence spectroscopy,
fluorescent probes have found a wide range of applications as
diagnostics and imaging tools in biomedical sciences. Since the
number of protein targets of human diseases have increased
tremendously in the recent genomics and proteomics era,1 there is
an increasing need for the development of newer fluorescent
probes. Fluorogenic probes are frequently used for designing
enzyme substrates, as well as for the detection of target proteins
and enzymes under both in vivo and in vitro conditions.2
In the effort of synthesizing naphthalenesulfonamide derivatives,
we decided to first prepare aminonaphthalenesulfonamide and
then derivatize the amino group with a variety of substituted
benzaldehyde moieties. The synthesis of 4-aminonaphthalene-1-
sulfonamide was reported in the literature.10 However, in our
hands, this procedure gave very low yields for the intermediates
and the final product, and we were unsuccessful in scaling up the
reactions. Consequently, we developed an efficient synthesis of this
compound (4) starting from inexpensive, commercially-available
4-aminonaphthalene-1-sulfonic acid (1) (Scheme 1, synthetic
details are provided in the ESI{).
Firstly, the amino group of 1 was protected with the Boc-group
employing the standard protocol. This protection also improved
the solubility of compound 2 in organic solvents. The sulfonic acid
was converted to the tert-butyl protected sulfonamide by the
reaction of in situ generated sulfonyl chloride with tert-butylamine.
In the final step, both the Boc and tert-butyl protecting groups
were removed using trifluoroacetic acid to produce 4 in 57%
overall yield. We have performed these reactions on a multigram
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitously-
distributed zinc-containing metalloenzymes.3 These enzymes
catalyze the reversible hydration of CO2 to form HCO32, which
is involved in a variety of biosynthetic reactions, such as
gluconeogenesis, the synthesis of certain amino acids, lipogenesis,
ureagenesis and pyrimidine nucleotide biosynthesis.4 Besides, these
enzymes are involved in pH homeostasis, ion transport, water and
electrolyte balance, bone resorption, calcification and tumorogeni-
city.3,4 Amongst these CA isozymes, CA II has been studied most
extensively.5 Systemic and topical inhibitors of CA II are employed
to lower the elevated intraocular pressure in glaucoma.5 In recent
years, inhibitors for the tumorogenic, hypoxia-induced CA XII
have been synthesized and screened for potential anticancer
activity.5,6
In pursuit of developing inhibitors against carbonic anhydrases,
we resorted to determining binding affinities of inhibitors, both by
performing steady-state kinetic experiments (Ki) as well as by the
ligand displacement method (Kd).7 While attempting to measure
the binding affinities of carbonic anhydrase inhibitors, we noted
that dansylamide has been widely utilized as a fluorescent probe
for carbonic anhydrases.8 Aside from dansylamide, there are a few
reports of other fluorescent probes for carbonic anhydrases in the
literature.9 However, those probes have not been extensively
utilized due to the high costs of their starting materials or
challenging synthetic protocols.9 Another major drawback of all
aDepartment of Pharmaceutical Sciences, North Dakota State
University, Fargo, North Dakota 58105, USA.
E-mail: sanku.mallik@ndsu.edu; Fax: +1 701-231-8666;
Tel: +1 701-231-7888
bDepartment of Chemistry, Biochemistry and Molecular Biology, North
Dakota State University, Fargo, North Dakota 58105, USA.
E-mail: dk.srivastava@ndsu.edu; Fax: +1 701-231-8831;
Tel: +1 701-231-7831
{ Electronic supplementary information (ESI) available: Syntheses, probe
integrity, kinetics and fluorescence data, spectrophotometric and spectro-
fluorometric studies, determinations of dissociation constants and
quantum yields and NMR data. See DOI: 10.1039/b701421j
Scheme 1 Synthesis of the fluorogenic probes.
Chem. Commun., 2007, 2723–2725 | 2723
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scale and the final product is easily purified by recrystallization.
Table 1 Structures of the substituted benzaldehydes used, yields and
dissociation constants of the probes with recombinant human CA I
and CA II
The amine 4 was then reacted with a variety of substituted
benzaldehydes to produce the fluorogenic probes 5a–p (Scheme 1).
The structures of the aldehydes used in the imine formation, as
well as the yields of the probes, are shown in Table 1.
Yield
(%)
Kd(CA I)/
nM
Kd(CA II)/
nM
Product
Aldehyde
Due to the presence of aryl sulfonamide groups, several of these
probes were found to serve as potent inhibitors for both CA I and
CA II (Table 1, for details, see ESI{). All of our synthesized probes
showed strong absorption bands in the UV-Vis region with
absorption maxima in the 330–370 nm range (Table 2). We
observed that under our experimental conditions, the probes are
not in equilibrium with the precursor aldehydes, both in the
absence and presence of CA (see ESI{). When the free forms of
these probes (5 mM in 25 mM HEPES buffer, pH = 7.0, containing
10% DMSO) were excited at the wavelengths of their absorption
maxima, most of the probes (except for compound 5a) showed
very weak fluorescence emission intensity. However, the fluores-
cence emission intensities of these probes were markedly enhanced
in the presence of recombinant human CA I or CA II (Fig. 1A).
We determined the quantum yields of free fluorophores as well as
those bound to the active sites of CA I and CA II (Table 2, details
are provided in ESI{).
As shown in Table 2, several of our synthesized probes showed
significant enhancements in quantum yields when bound to the
enzymes. Many of these quantum yield enhancements (e.g., for 5c,
5f, 5i, 5j, 5k, 5o and 5p) upon binding to the enzymes were
significantly higher than that observed with the parent compound,
dansylamide.8 In fact, the emission intensity of compound 5h was
so weak in solution that we could not determine the quantum
yield. In contrast, the compound turned highly fluorescent in the
presence of recombinant human CA I or CA II. Two of our
synthesized probes showed higher enhancements in quantum yield
upon binding with CA I as compared to that of CA II (e.g., 5b and
5n), while the others showed either a reverse trend (e.g., 5c, 5d, 5i,
5l and 5o) or nearly equal enhancements in the quantum yields
with both of these isozymes (e.g., 5e, 5f, 5g, 5j, 5k and 5p).
A comparison of the fluorescence profiles of free versus enzyme
bound probes reveals that the emission intensities of the free
probes are quenched in aqueous solution. However, this is not a
5a
60
47
47
30
54
39
62
.5000
1460
40
.5000
3300
97
5b
5c
5d
5e
5f
30
30
20
300
3000
47
1000
25
5g
5h
5i
56
76
59
75
107
30
720
60
5j
150
70
130
100
5k
5l
28
45
26
34
80
5m
960
5n
5o
5p
61
48
60
121
235
130
100
130
1500
Table 2 Photophysical properties of the synthesized fluorophores: maximum absorption wavelength (labs, nm), emission maxima (lem, nm) when
excited at labs in 25 mM HEPES buffer containing 10% DMSO (pH = 7.0, 5 mM probe solution) in the presence of recombinant human CA I
(10 mM) and CA II (10 mM), quantum yields (W) in the presence of CA I and CA II and the enhancements in quantum yields in the presence of CA I
and CA II
Probes
labs
lem(free)
lem(CA I )
lem(CA II )
W(CA I)
W(CA II)
W(CA I)/W(free)
W(CA II)/W(free)
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
342
342
333
330
358
336
342
330
330
330
330
336
330
330
330
330
522
516
522
519
528
528
520
—
526
525
528
526
—
516
468
467
470
471
470
473
470
470
470
470
475
470
468
470
475
520
467
465
468
468
466
465
464
464
465
469
463
462
466
465
465
0.022
0.026
0.044
0.014
0.015
0.139
0.020
0.012
0.036
0.073
0.073
0.043
0.018
0.073
0.012
0.116
0.011
0.013
0.062
0.024
0.011
0.144
0.017
0.017
0.045
0.064
0.077
0.063
0.015
0.041
0.015
0.107
1.4
22.4
47.2
30.7
12.1
40.9
28.4
—
39.5
77.7
53.1
20.4
—
1.0
13.3
66.9
55.2
7.9
42.3
23.9
—
49.2
70.0
55.8
30.2
—
17.6
60.6
41.9
525
—
518
31.1
47.7
45.6
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Sulfonamides are known13 to be weak binders of Zn2+ ions in
solution and we did not observe any changes in the fluorescence
emission spectra of the probes in the presence of up to 50 mM of
ZnSO4 in aqueous solution (see ESI{).
In conclusion, we have synthesized several new naphthalene-
sulfonamide derivatives as fluorescent probes for CAs. Due to
their low quantum yields in their free forms in aqueous buffer and
significantly enhanced quantum yields when bound to the
enzymes, they will find applications in a variety of thermodynamic
and kinetic experiments for the binding of enzyme inhibitors to
various isoforms of carbonic anhydrases.
Fig. 1 (A) The emission spectrum of 5f (5 mM) and in the presence of
recombinant CA I and CA II (10 mM, lex = 336 nm) in 25 mM HEPES
buffer (pH = 7.0) containing 10% DMSO. (B) The emission intensities of
5f (5 mM, lex = 336 nm) in 25 mM aqueous HEPES buffer (pH = 7.0)
containing varying amounts of added dioxane are shown. The volume
percentages of added dioxane are indicated on the plot.
This research was supported by the NIH grants 1R01
CA113746 to DKS and SM, and 1R01 GM 63204 to SM.
Notes and references
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Table 3 The absorption maxima of Reichardt’s dye in aqueous buffer
with various amounts of dioxane, the ET(30) values and the
corresponding quantum yields of 5f
Dioxane (%)
labs
ET(30)
W
0
9
16
23
28
33
452
462
470
480
495
505
63.2
61.9
60.8
59.5
57.7
56.6
0.007
0.012
0.019
0.028
0.036
0.043
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self-quenching process, since increasing the concentration of the
probes in buffer solution resulted in regular increases in the
emission intensity (data not shown). Since the active site pockets of
carbonic anhydrases are predominantly hydrophobic,11 the ques-
tion arose whether the enhancements in the fluorescence quantum
yields of these probes upon binding to CA I and CA II were due to
changes in polarity of the solvent media. To probe this, we
determined the fluorescence emission spectra and quantum yields
of 5f in 25 mM HEPES buffer (pH = 7.0) as a function of
increasing concentration of added dioxane (Fig. 1B, Table 3). Note
that as the hydrophobicity of the solvent medium increases (as
determined by the ET(30) values12), the fluorescence emission
intensity as well as the quantum yield keep on increasing. Clearly,
the hydrophobicity of the active site pockets of CA I and CA II is
responsible, at least in part, for the enhanced quantum yields of the
fluorescent probes in Table 1. However, we could not ascertain the
contribution of active site resident Zn2+ ion on the enhancement of
the emission intensity due to precipitation of Zn2+-free (apo) CA II
in the presence of the parent compound dansylamide.
12 M. T. Morgan, M. A. Carnahan, C. E. Immoos, A. A. Ribeiro,
S. Finkelstein, S. J. Lee and M. W. Grinstaff, J. Am. Chem. Soc., 2003,
125, 15485.
13 S. Chaves, S. M. Marques, A. Cachudo, M. A. Esteves and
M. A. Santos, Eur. J. Inorg. Chem., 2006, 3853.
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