Chemosensors for the marine toxin saxitoxin

Article abstract of DOI:10.1021/ja027507p

Eleven anthracylmethyl crown ethers have been synthesized and evaluated as fluorescence sensors for the marine toxin saxitoxin. Fluorescence enhancement data are consistent with a 1:1 binding complex for all crowns. The binding constants are in the range

Full text of DOI:10.1021/ja027507p

Published on Web 10/17/2002
Chemosensors for the Marine Toxin Saxitoxin
Robert E. Gawley,*,‡ Sandra Pinet,‡ Claudia M. Cardona,‡ Probal K. Datta,‡
Tong Ren,‡ Wayne C. Guida,§ Jason Nydick,§ and Roger M. Leblanc*,‡
Contribution from the Department of Chemistry, and NIEHS Marine and Freshwater Biomedical
Sciences Center, UniVersity of Miami, P.O. Box 249118, Coral Gables, Florida 33124-0431, and
Department of Chemistry, Eckerd College, 4200 54th AVenue South Street,
Petersburg, Florida 33711
Received June 28, 2002
Abstract: Eleven anthracylmethyl crown ethers have been synthesized and evaluated as fluorescence
sensors for the marine toxin saxitoxin. Fluorescence enhancement data are consistent with a 1:1 binding
complex for all crowns. The binding constants are in the range of 104 M-1 in ammonium phosphate buffer
(pH 7.1) in 80% ethanol solvent. Selectivity for sensing saxitoxin versus several organic analytes has been
demonstrated for the first time. Possible modes of binding are presented, and relevance to saxitoxin
monitoring programs are discussed.
which is mouse bioassay.8 Unfortunately, not all countries can
afford such monitoring facilities, and as recently as 1987, a
Introduction
Harmful algal blooms (red tides) produce a wide variety of
secondary metabolites,1 but saxitoxin is virtually alone among
them in being capable of causing human mortality by consump-
tion of tainted shellfish.2 The most severe symptom of saxitoxin
consumption, also known as paralytic shellfish poisoning,3 is
respiratory paralysis.4,5 Saxitoxin and its congeners are known
as paralytic shellfish poisons (PSPs).6 The tragedy of human
mortality events due to PSP consumption is that recovery is
virtually guaranteed if the disease is properly diagnosed and
treated by mechanical ventilation for 24 h.7 Currently, govern-
ments of many countries monitor shellfish beds for the presence
of saxitoxin by using one of several tests, the most reliable of
harmful algal bloom (HAB) on the Pacific coast of Guatemala
resulted in numerous deaths, including 50% of the children who
were reported ill.9 Despite massive monitoring efforts by the
respective governments, disease outbreaks in the United States
and Portugal have been documented as recently as the past
decade.5 In the spring of 2002, the lay press reported a number
of saxitoxin poisonings from puffer fish caught near Cape
Canaveral, Florida. This is the first time this toxin has been
reported in the Atlantic, south of New England!
* To whom correspondence should be addressed. E-mail: rgawley@
miami.edu,rml@miami.edu(afterJanuary1,2003,e-mail: rgawley@uark.edu).
‡ University of Miami.
§ Eckerd College.
(1) (a) Fenical, W.; Baden, D.; Burg, M.; de Goyet, C. d. V.; Grimes, D. J.;
Katz, M.; Marcus, N.; Pomponi, S.; Rhines, P.; Tester, P.; Vena, J. From
Monsoons to Microbes: Understanding the Ocean’s Role in Human Health;
National Academy Press: Washington, DC, 1999. (b) Botana, L. M., Ed.
Seafood and Freshwater Toxins. Pharmacology, Physiology, and Detection;
Marcel Dekker: New York, 2000. (c) Yasumoto, T.; Murata, M. Chem.
ReV. 1993, 93, 1897-1909. (d) Shimizu, Y. Chem. ReV. 1993, 93, 1685-
1698.
(2) (a) Meyer, K. F. New Engl. J. Med. 1953, 249, 843-852. (b) Ayres, P. A.;
Cullum, M. Paralytic Shellfish Poisoning, Technical Report No. 4; Ministry
of Agriculture, Fisheries and Food, 1978.
(3) Sommer, H.; Meyer, K. F. Arch. Pathol. 1937, 560-598.
(4) Gessner, B. D.; Middaugh, J. P. Am. J. Epidemiol. 1995, 141, 766-770.
(5) (a) Gessner, B. D.; Bell, P.; Doucette, G. J.; Moczydlowski, E.; Poli, M.
A.; Dolah, F. V.; Hall, S. Toxicon 1997, 35, 711-722. (b) de Carvalho,
M.; Jacinto, J.; Ramos, N.; de Oliveira, V.; Melo, T. P. E.; de Sa, J. J.
Neurol. 1998, 245, 551-554.
(6) (a) Shimizu, Y. In Handbook of Natural Toxins; Tu, A. T., Ed.; Marcel
Dekker: New York, 1988; Vol. 3, pp 63-85. (b) Hall, S.; Strichartz, G.;
Moczydlowski, E.; Ravindran, A.; Reichardt, P. B. In Marine Toxins:
Origin, Structure, and Molecular Pharmacology; Hall, S., Strichartz, G.,
Eds.; American Chemical Society: Washington, DC, 1990; Vol. 418, pp
29-65. (c) Kao, C. Y. In Algal Toxins in Seafood and Drinking Water;
Falconer, I. R., Ed.; Academic Press: London, 1993; pp 75-104.
(7) Baden, D. G.; Fleming, L. E.; Bean, J. A. In Handbook of Clinical
Neurology: Intoxications of the NerVous System, Part III; de Wolff, F.
A., Ed.; Elsevier: Amsterdam, 1995; Vol. 21, pp 141-175.
Saxitoxin detection programs may use a number of tech-
niques,10 but mouse bioassay is by far the most common, and
is the current benchmark technique.8 New approaches include
insect bioassay,11 tissue biosensors,12 molecular pharmacology,13
(8) Fernandez, M.; Cembella, A. D. In Manual on Harmful Marine Microalgae,
IOC Manuals and Guides, No. 33; Hallegraeff, G. M., Anderson, D. M.,
Cembella, A. D., Eds.; UNESCO: Paris, 1995; pp 213-228.
(9) Rodrigue, D. C.; Etzel, R. A.; Hall, S.; de Porras, E.; Velasquez, O. H.;
Tauxe, R. V.; Kilbourne, E. M.; Blake, P. A. Am. J. Trop. Med. Hyg. 1990,
42, 267-271.
(10) (a) Sullivan, J. J.; Wekell, M. M.; Hall, S. In Hanbook of Natural Toxins;
Tu, A. T., Ed.; Marcel Dekker: New York, 1988; Vol. 3, pp 87-106. (b)
Vanegmond, H. P.; Vandentop, H. J.; Paulsch, W. E.; Goenaga, X.; Vieytes,
M. R. Food Addit. Contam. 1994, 11, 39-56. (c) Quilliam, M. A. J. AOAC
Int. 1997, 80, 131-136.
(11) McElhiney, J.; Lawton, L. A.; Edwards, C.; Gallacher, S. Toxicon 1998,
36, 417-420.
9
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J. AM. CHEM. SOC. 2002, 124, 13448-13453
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Chemosensors for Saxitoxin
A R T I C L E S
Figure 1. Crown sensors used in this study.
neurophysiology,14 whole-cell bioassay,15 HPLC with postcol-
umn oxidation of the C4-C12 bond and aromatization,16 and
HPLC-linked mass spectroscopy.17 For both economic and
ethical reasons, an alternative to mouse bioassay is desired.18
Herein, we report our efforts to date in the development of a
chemosensor for saxitoxin using fluorescence sensing,19 with
the ultimate aim of incorporating the sensor into an optical
fiber.20
Synthesis. The crown ether sensors used in this study are
shown in Figure 1. Crown 1 was reported in 198521 and was
the better of two STX sensors evaluated in preliminary studies.19
(12) (a) Cheun, B. S.; Watanabe, E. Agro-Food-Ind. Hi-Tech 1998, 9, 36-39.
(b) Cheun, B. S.; Takagi, S.; Hayashi, T.; Nagashima, Y.; Watanabe, E. J.
Nat. Toxins 1998, 7, 109-120.
(16) (a) Oshima, Y. In Manual on Harmful Marine Microalgae, IOC Manuals
and Guides, No. 33; Hallegraeff, G. M., Anderson, D. M., Cembella, A.
D., Eds.; UNESCO: Paris, 1995; pp 81-94. (b) Oshima, Y.; Sugino, K.;
Yasumoto, T. In Mycotoxins and Phycotoxins ‘88: A Collection of InVited
Papers at the SeVenth International IUPAC Symposium on Mycotoxins and
Phycotoxins; Tokyo, Japan, 16-19 August, 1988; Elsevier: Amsterdam,
1989; pp 319-326. (c) Sullivan, J. J. In Marine Toxins. Origin, Structure,
and Molecular Pharmacology; Hall, S., Strichartz, G., Eds.; American
Chemical Society: Washington, DC, 1990; Vol. 418, pp 66-77. (d)
Oshima, Y. J. AOAC Int. 1995, 78, 528-532. (e) Lawrence, J. F.; Wong,
B. J. Chromatogr. A 1996, 755, 227-233.
(13) (a) Vieytes, M. R.; Cabado, A. G.; Alfonso, A.; Louzao, M. C.; Botana,
A. M.; Botana, L. M. Anal. Biochem. 1993, 211, 87-93. (b) Dolah, F. M.
V.; Finley, E. L.; Haynes, B. L.; Doucette, G. J.; Moeller, P. D.; Ramsdell,
J. S. Nat. Toxins 1994, 2, 189-196. (c) Dolah, F. M. V. In Workshop
Conference on Seafood Intoxications: Pan American Implications of
Natural Toxins in Seafood; Baden, D. G., Ed.; University of Miami:
Rosenstiel School of Marine and Atmospheric Science, 1995; pp 50-56.
(d) Llewellyn, L. E.; Doyle, J.; Negri, A. P. Anal. Biochem. 1998, 261,
51-56. (e) Doucette, G. J.; Logan, M. M.; Ramsdell, J. S.; Van Dolah, F.
M. Toxicon 1997, 35, 625-636. (f) Negri, A.; Llewellyn, L. Toxicon 1998,
36, 283-298.
(17) Mirocha, C. J.; Cheong, W.; Mirza, U.; Kim, Y. B. Rapid Commun. Mass
Spectrom. 1992, 6, 128-134.
(18) Gallacher, S.; Mackintosh, F.; Shanks, A. M.; O’Neill, S.; Riddoch, I.;
Howard, F. G. J. Shellfish Res. 1998, 17, 1647-1651.
(14) Kerr, D. S.; Briggs, D. M.; Saba, H. I. Toxicon 1999, 37, 1803-1825.
(15) Jellett, J. F.; Doucette, L. I.; Belland, E. R. J. Shellfish Res. 1998, 17, 1653-
1655.
9
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A R T I C L E S
Scheme 1 a
Gawley et al.
to 1.0 × 10-4 M, at concentrations of 1.0 × 10-7, 2.5 × 10-7
,
5.0 × 10-7, and 7.5 × 10-7 M, and so forth, over this
concentration range. The concentration of crown ether was held
constant at 1.0 × 10-6 M and the buffer at 6 × 10-3 M, with
irradiation at λ ) 366 nm.
The raw data and the normalized binding isotherm for 1 are
shown in Figure 2a, along with the emission spectra corre-
sponding to the first and last datapoints (Figure 2b), at [STX]
) 0 and 1.0 × 10-4 M. It was not possible to determine the
stoichiometry of binding using either a continuous variation or
mole ratio plot,25 for the following reasons: (i) at high
concentrations, the fluorescence intensity was not linear; (ii) at
lower concentrations, F/F0 was too small. Therefore, as a
working hypothesis, we assume that the binding is 1:1.
The binding constant was determined from the experimental
data using a nonlinear least-squares fit to eq 1,26 where F and
F0 are the observed fluorescence intensities in the presence and
absence of STX, respectively, integrated from 380 to 600 nm;
kcrown and k11 are constants related to fluorescence intensities
of the crown and the presumed 1:1 crown‚STX complex,
respectively; K11 is the binding constant for the 1:1 complex;
and [STX] is the equilibrium concentration of unbound sax-
itoxin.
a Reagents and conditions: (a) HOAc, DCC, CH2Cl2, RT; (b) Et3N,
BrCH2CO2t-Bu, toluene, reflux; (c) CH2dCHCO2t-Bu, i-PrOH, 85°; (d)
CH2dC(CO2t-Bu)2, i-PrOH, rt; (e) Cbz-Asp-ROMe, EDCI, CH2Cl2, rt; (f)
Cbz-Glu-ROMe, EDCI, CH2Cl2, RT; (g) 96% HCO2H, 75°; (h) K2CO3,
MeOH/H2O, rt.
Crowns 2-11 were prepared to compare structural effects on
sensitivity and, in some cases, as derivatives suitable for
incorporation into peptidic libraries. Crown 1 was prepared by
the method of de Silva;21 the others were prepared by routine
transformations as outlined in Scheme 1.
k11
1 +
K11[STX]
(
)
kcrown
F
F0
)
(1)
1 + K11[STX]
Fluorescence Titrations. Our initial work19 began with crown
ether 1.21 This sensor responds to alkali cations in (strictly
anhydrous!) methanol solution. Aminomethylanthracenes fluo-
resce poorly as free bases because the nitrogen lone pair
quenches the excited state by photoinduced electron transfer
(PET).22 The sensing mechanism invoked for sensors such as 1
is protonation, hydrogen bonding, or coordination to the
nitrogen, thereby inhibiting electron-transfer such that the
anthracene fluorophore emits normally. Because the C-8 guani-
dinium in STX has a pKa of 8.24,23 and because we wanted to
eliminate the possibility of simple proton-transfer enhancing
fluorescence, we did our binding studies in ethanol/water solvent
mixtures, buffered to pH 7.1 with 6 × 10-3 M ammonium
phosphate. Control experiments showed that this buffer had no
effect on the fluorescence intensity of 1 in 80% ethanol. In water,
this type of crown sensor is insensitive to metal ions such as
A good fit was obtained (R ) 0.991), and revealed a binding
constant of (1.38 ( 0.46) × 104 M-1. Figure 2a also shows
data obtained from similar experiments with arginine, adenine,
guanidinium hydrochloride, and o-bromophenol as controls.
None produced any fluorescence enhancement in crown 1.
Arginine and guanidinium hydrochloride were tested because
guanidinium ions are known to bind to crowns, and STX has
two guanidinium moieties. Adenine was chosen because it is a
biomolecule and has a purine ring system, as does STX.
o-Bromophenol was chosen because it has a pKa of 8.45,27
similar to that of saxitoxin. Clearly, none shows any evidence
of binding, indicating that crown 1 binds saxitoxin selectively
over all of these analytes and that the observed fluorescence
response is not due to simple proton transfer to the benzylic
nitrogen of the crown.
With these preliminary results in hand, we examined a series
of 10 more diazacrowns, 2-11 for binding. There were two
objectives in evaluating these compounds: probing the effect
of structural variation on binding and designing derivatives that
are suitable for incorporation into combinatorial libraries. The
results of the binding studies are listed in Table 1, along with
the data from 1 for comparison. Least-squares fit of all the
titrations resulted in excellent correlations (r g 0.96). Individual
binding isotherms and emission spectra are reported in the
Supporting Information.
Na+, K+, and Ca2+ 21,24
.
To determine the binding constant, fluorescence spectra were
recorded at concentrations of [STX] ranging from 1.0 × 10-7
(19) A preliminary report of some of this work has appeared: Gawley, R. E.;
Zhang, Q.; Higgs, P. I.; Wang, S.; Leblanc, R. M. Tetrahedron Lett. 1999,
40, 5461-5465. Corrigendum 6135.
(20) (a) Wang, S.; Zhang, Q.; Datta, P. K.; Gawley, R. E.; Leblanc, R. M.
Langmuir 2000, 16, 4607-4612. (b) Kele, P.; Orbulescu, J.; Calhoun, T.
L.; Gawley, R. E.; Leblanc, R. M. Langmuir 2002, 18, 8523-8526.
(21) (a) Bissell, R. A.; Calle, E.; de Silva, A. P.; de Silva, S. A.; Gunaratne, H.
Q. N.; Habib-Jiwan, J.-L.; Peiris, S. L. A.; Rupasinghe, R. A. D. D.;
Samarasinghe, T. K. S. D.; Sandanayake, K. R. A. S.; Soumillion, J.-P. J.
Chem. Soc., Perkin Trans. 2 1992, 1559-1564. (b) de Silva, A. P.; de
Silva, S. A. J. Chem. Soc., Chem. Commun. 1986, 1709-1710.
(22) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.;
McCoy, C. P.; Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515-
1566.
(25) (a) Connors, K. A. In Binding Constants: The Measurement of Molecular
Complex Stability; Wiley-Interscience: New York, 1987; pp 24-28. (b)
Bruneau, E.; Lavabre, D.; Levy, G.; Micheau, J. C. J. Chem. Educ. 1992,
69, 833-838. (c) Beltran-Porter, A.; Beltran-Porter, D.; Cervilla, A.;
Ramirez, J. A. Talanta 1983, 30, 124-126.
(26) Connors, K. A. In Binding Constants: The Measurement of Molecular
Complex Stability; Wiley-Interscience: New York, 1987; pp 339-343.
(27) Hand, C. W.; Blewitt, H. L. In Acid-Base Chemistry; Macmillan: New
York, 1986; pp 246-254.
(23) (a) Bordner, J.; Thiessen, W. E.; Bates, H. A.; Rapoport, H. J. Am. Chem.
Soc. 1975, 97, 6008-6012. (b) Wong, J. L.; Oesterlin, R.; Rapoport, H. J.
Am. Chem. Soc. 1971, 93, 7344-7345.
(24) Kele, P.; Orbulescu, J.; Calhoun, T. L.; Gawley, R. E.; Leblanc, R. M.
Tetrahedron Lett. 2002, 43, 4413-4416.
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A R T I C L E S
Figure 2. (a) Binding isotherm for crown 1 with STX, and comparison data for adenine, arginine, guanidinium hydrochloride and o-bromophenol. Crown
concentration is 1.0 × 10-6 M, and the guest concentration is varied from 1.0 × 10-7 to 1.0 × 10-4 M. The solvent is 80:20 ethanol/water for STX,
guanidinium and o-bromophenol and 98:2 ethanol/water for adenine and arginine. Both solvent mixtures were buffered to pH 7.1 with ammonium
phosphate: 6 × 10-3 M in 80/20 and 2 × 10-4 M in 98:2 EtOH/H2O. (b) Emission spectra for crown 1 (1.0 × 10-6 M) in the absence and presence of 1.0
× 10-4 M STX, corresponding to the first and last datapoints for the binding isotherm in Figure 2a.
Table 1. Binding Constants Determined by Fluorescence Titration
and Assuming a 1:1 STX‚Crown Complex (Buffered 80:20
Ethanol/Water)
1
1
cmpd
K11
×
104 (M-
)
r
cmpd
K11
×
104 (M-
)
r
1
2
3
4
5
6
1.38 ( 0.46
3.58 ( 1.11
.990
.982
.974
.986
.996
.991
7
8
9
10
11
1.51 ( 0.87
.50 ( 0.21
.974
.995
.993
.972
.988
3.26 ( 1.32a
2.25 ( 0.73a
0.88 ( 0.24
1.63 ( 0.50
1.80 ( 0.52a
1.63 ( 0.97
1.13 ( 0.56
a Average of two runs.
Discussion
Comparison of monoazacrown 1 with the diazacrown ana-
logue 2 shows a slight increase in binding, and acylation of the
basic nitrogen, as in 3, gives no further change. The three tert-
butyl esters, 4-6, have similar binding constants, as does N-
and O-protected aspartate derivative 7. Curiously, the homologue
8, a similarly protected glutamate derivative, has a somewhat
lower binding constant. Also interesting is the fact that
incorporation of a carboxylic acid moiety, which ought to be
ionized at pH 7.1, as in 9-11, has no measurable increase on
binding. The excellent fits of the experimental isotherms to the
theoretical 1:1 binding equation strongly suggests that a 1:1
complex is indeed formed in all cases.
It is of interest to determine the nature of the binding. The
mechanism by which these types of sensors are believed to work
is through photoinduced electron transfer, PET.28 In PET, the
fluorophore fails to fluoresce because the excited state is
quenched by electron transfer, unless the relative energies of
the fluorophore are perturbed by a binding event. For example,
in aminomethylanthracenes, PET can be inhibited by protonation
or hydrogen bonding.22 Figure 3 shows the X-ray crystal
Figure 3. ORTEP drawing of 2‚NaCl at the 20% probability level. Two
waters of hydration and the chloride anion are deleted for clarity.
structure of crown 2, with sodium complexed to the crown
heteroatoms in this way. In the solid state, the sodium is in
close contact with all the heteroatoms in the crown except the
benzylic nitrogen.
Our initial thinking was that saxitoxin, molecular formula
C10H19N7O4, would be an excellent candidate for fluorescence
sensing by quenching of PET, because of the large number (11)
of potential hydrogen-bond donors. Moreover, STX is a bis-
guanidinium dication, and it has long been known that guani-
dinium ions bind to crown ethers.29 This hypothesis is supported
by the fact that we observe a fluorescence response.
(28) (a) Bissell, R. A.; de Silva, A. P.; Gunaratne, H. Q. N.; Lynch, P. L. M.;
Maguire, G. E. M.; McCoy, C. P.; Sandanayake, K. R. A. S. Top. Curr.
Chem. 1993, 168, 223-264. (b) Valeur, B.; Bourson, J.; Pouget, J. In
Fluorescent Chemosensors for Ion and Molecule Recognition; Czarnik, A.
W., Ed.; American Chemical Society: Washington DC, 1993; Vol. 538,
pp 25-44.
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Gawley et al.
Figure 4. Global minima for STX docked into crowns 1 and 2, with distances (in Å) between STX hydrogen bond donors and crown heteroatoms indicated.
(a) Crown 1‚STX; (b) Crown 2‚STX.
Several crystal structures of 18-crown-6 and its aza analogues
with urea, uronium ions, amides, and amidines show hydrogen
bonding with crown heteroatoms,30 but several of the analytes
tested (Figure 1) against crown 1 have hydrogen bond donors
but provide no detectable fluorescence enhancement. Attempts
to grow crystals of a crown‚STX complex have, thus far, failed.
Although NMR titrations can be used to evaluate structural
features in a complex, such studies are not possible at the
concentrations we are studying (10-6 M). As an alternative, we
have used computational techniques to probe possible modes
of binding.
Monte Carlo docking searches using the explicit hydrogen
atom AMBER* force field were performed using the “low-mode
docking” search procedure, which performs atomic movement
on the crown‚STX complex in a manner such that atoms are
moved along a trajectory that is consistent with the low-
frequency vibrational modes of the complex.31 Further, STX
was subjected to explicit translation and rotation in the crown
ether binding site, and explicit torsional variation was performed
for the STX and crown ether side chains that can undergo free
rotation. The Supporting Information contains details of these
calculations and the software employed. The global minima for
the docking of STX with crowns 1 and 2, each of which were
found multiple times in the search, are shown in Figure 4.
The lack of apparent participation by the benzylic nitrogen
in the binding of sodium in the solid state (Figure 3) and of
STX in these computational models (Figure 4) is interesting. If
the structures in Figures 3 and 4 bear any resemblance to the
structure of the crown complexes in solution, it would seem
that coordination or hydrogen bonding to the benzylic nitrogen
is too simple an explanation to account for the PET. Molecular
orbital calculations and time-resolved fluorescence measure-
ments are in progress to clarify this point.
The parameters utilized by AMBER* for this study contained
no low-quality torsional parameters, which could have adversely
affected the accuracy of computed conformational energies. The
point charges used for the calculations were derived from
charges fitted to the electrostatic potential derived from the
6-31G** wave function. Moreover, since AMBER itself was
parametrized using electrostatic potential fitted charges, we
believe that the energies calculated are reliable within the
molecular mechanics paradigm. Nonetheless, we performed a
second Monte Carlo search using the OPLS all-atom force field
(OPLS-AA) for 1‚STX. The lowest-energy structures (i.e., the
global minimum and all structures within 1 kcal/mol of it)
possessed hydrogen bonds between the C-8 guanidinium of STX
and the crown ether oxygens, while some of the higher-energy
structures possessed hydrogen bonds between the C-2 guani-
dinium of STX and the crown oxygens.
Relevance to Saxitoxin Monitoring Programs. The bench-
mark method for detecting saxitoxin and its congeners in
shellfish is mouse bioassay. The current legal limit for STX in
shellfish is 80 µg/100 g of shellfish, and the mouse bioassay
can reliably detect down to 40 µg of STX/100 g of shellfish. In
the AOAC technique, extraction of shellfish that contains 40
µg of STX/100 g of shellfish produces an aqueous solution that
is ∼10-6 M in STX, and this is what is injected into the mouse.
As it stands now, our crown sensors show excellent fluorescence
enhancement at STX concentrations of 10-4 M. Many show
significant (10-20%) enhancement at concentrations of 5 ×
10-6 M, which is very encouraging for development of better
sensors. A more sensitiVe STX sensor, having a larger binding
constant, would have a steep binding isotherm in the low-
concentration range, and this is what we will be seeking as this
work proceeds. Nevertheless, we are already very close to the
limit of detection by the mouse bioassay!
Selectivity of binding of STX to all our crowns has already
been demonstrated relative to ammonium ion, and crown 1 is
selective for STX relative to arginine, adenine, guanidinium ion,
and o-bromophenol. Very recently, we have found that coumaryl
crown ethers have binding constants to STX in water that are
an order of magnitude higher than those in aqueous ethanol,
and which are selective for STX in the presence of sodium and
(29) Kyba, K. P.; Helgeson, R. C.; Madan, K.; Gokel, G. W.; Tarnowski, T. L.;
Moore, S. S.; Cram, D. J. J. Am. Chem. Soc. 1977, 99, 2564-2571.
(30) (a) Watson, W. H.; Galloy, J.; Grossie, D. A.; Vo¨gtle, F.; Mu¨ller, W. M.
J. Org. Chem. 1984, 49, 347-353. (b) Harkema, S.; van Hummel, G. J.;
Daasvatn, K.; Reinhoudt, D. N. J. Chem. Soc., Chem. Commun. 1981, 368-
369. (c) Uiterwijk, J. W. H. M.; Harkema, S.; Reinhoudt, D. N.; Daasvatn,
K.; den Hertog, H. J., Jr.; Geevers, J. Angew. Chem., Int. Ed. Engl. 1982,
21, 450-451.
(31) Kolossvary, I.; Guida, W. C. J. Comput. Chem. 1999, 20, 1671-1684.
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A R T I C L E S
potassium ions in aqueous solution.24 Thus, it appears that crown
ether sensors such as those reported herein will prove useful in
testing for STX in environmental samples. Such experiments
are being planned, and will be reported in due course.
Syracuse) for much practical advice on handling saxitoxin, and
to Dr. Sherwood Hall (FDA) for generous gifts of saxitoxin.
We are grateful to Professor Franc¸isco Raymo for the use of
his fluorometer and for many helpful discussions. T.R. is grateful
to the University of Miami for purchase of the CCD diffrac-
tometer used in this work.
Acknowledgment. This work was supported by the United
States Department of Agriculture (Grants No. 9802839 and No.
0191270), the Environmental Protection Agency (Grant No.
R826655), and the National Institute of Environmental Health
Sciences, NIH, through Grants ES05705 to the University of
Miami Marine and Freshwater Biomedical Sciences Center,
ES07320, and ES05931 (NRSA traineeship and individual
fellowship to C.M.C.). Its contents are solely the responsibility
of the authors and do not necessarily represent the official views
of the funding agencies. We are grateful to Dr. Sherwood Hall
(FDA, Office of Seafood) and Professor Greg Boyer (SUNY-
Supporting Information Available: Experimental details for
the preparation of crowns 2-11, proton and carbon NMR
spectra, mass spectra, crystal data for crowns 2 and 9, plots of
the binding isotherms for crowns 2-11, details of the molecular
modeling calculations (PDF) and an X-ray crystallographic file
(CIF). This material is available free of charge via the Internet
at http://www.pubs.acs.org.
JA027507P
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J. AM. CHEM. SOC. VOL. 124, NO. 45, 2002 13453