Synthesis and cholera toxin binding properties of a lactose-2-aminothiazoline conjugate.

Article abstract of DOI:10.1021/ol025909w

[structure: see text] During the search for improved monovalent ligands for cholera toxin (CT), a new lactose-2-aminothiazoline conjugate was discovered. In a fluorescence binding assay the compound was found to be one of the strongest relatively simple C

Full text of DOI:10.1021/ol025909w

ORGANIC
LETTERS
2002
Vol. 4, No. 10
1807-1808
Synthesis and Cholera Toxin Binding
Properties of a
Lactose-2-aminothiazoline Conjugate
Ioannis Vrasidas, Johan Kemmink, Rob M. J. Liskamp, and Roland J. Pieters*
Department of Medicinal Chemistry, Utrecht Institute of Pharmaceutical Sciences,
Utrecht UniVersity, P.O. Box 80082, 3508 TB Utrecht, The Netherlands
r.j.pieters@pharm.uu.nl
Received March 21, 2002
ABSTRACT
During the search for improved monovalent ligands for cholera toxin (CT), a new lactose-2-aminothiazoline conjugate was discovered. In a
fluorescence binding assay the compound was found to be one of the strongest relatively simple CT ligands to date with a Kd of 23 µM.
Interference with protein-carbohydrate interactions has great
medicinal potential in a variety of areas,1 including the fight
against bacterial infections2 and toxins.3 We are involved in
a program to design interfering compounds in both monova-
lent and multivalent form. Cholera toxin (CT), the disease-
causing agent produced by the pathogen Vibrio cholerae, is
one of our target proteins. Inhibiting CT binding to its natural
ligand GM1 in the intestinal tract should prevent the onset
of the cholera disease.3 As part of these studies we recently
reported a relatively potent monovalent inhibitor for cholera
toxin.4 For this compound, lactose derivative 1, we deter-
mined a Kd of 248 µM using a direct fluorescence binding
assay. Both the thiourea moiety and the aryl group seemed
to contribute to the enhanced binding as compared to that
of unsubstituted lactose (Kd of 18 mM). To make further
improvements we decided to synthesize other lactose deriva-
tives, such as 2, which contains a more rigid spacer between
the sugar and aryl group yet includes a methylene unit
permitting conformational adjustments.
under modified Sonogashira reaction conditions.5 Compound
4 was obtained in 62% yield after column chromatography
(hexane/EtOAc, 80:20, v/v). The 13C NMR spectrum of 4
showed the characteristic signals at δ 86 and 82 ppm for
the carbons of the triple bond. The amine resulting from Boc
removal of 5 using a CH2Cl2/TFA (5:2) mixture was treated
with acetylated lactose â-isothiocyanate and iPr2NEt in CH2-
Cl2. After the mixture stirred overnight, two new products
were observed by TLC.6 On attempted isolation of both of
them by preparative TLC only a single compound was
obtained. Apparently the higher running compound was
converted to the lower running one on the silica gel plate.
This process could also be induced by adding acetic acid to
a dichloromethane solution of the mixture. Complete conver-
sion to the lower running spot was observed within 3 h. The
The projected synthesis of 2 (Scheme 1) started by
treatment of iodide 3 with Boc-protected propargylamine
(1) Williams, S. J.; Davies, G. J. Trends Biotechnol. 2001, 19, 356.
(2) Zopf, D.; Roth, S. Lancet 1996, 347, 1017.
(3) Fan, E.; Merritt, E. A.; Verlinde, C. L. M. J.; Hol, W. G. J. Curr.
Opin. Struct. Biol. 2000, 10, 680.
(4) Vrasidas, I.; de Mol, N. J.; Liskamp, R. M. J.; Pieters, R. J. Eur. J.
Org. Chem. 2001, 4685.
10.1021/ol025909w CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/18/2002

The affinity of 6 for the cholera toxin B subunit (CTB)
was determined with a direct fluorescence binding assay.4
CTB contains one tryptophan residue (Trp88), the fluores-
cence of which is affected by the binding of carbohydrate
ligands. Upon addition of 6, the decrease of the CTB
fluorescence was practically complete at saturation. This
phenomenon was also observed for 14 and is in contrast to
lactose, which induces only a minor fluorescence quenching.
For 6 the fluorescence at 350 nm fitted well to a simple one-
binding-site model and the Kd value obtained was 23 µM.
This indicates that 6 binds 1 order of magnitude better than
1 and 780-fold better than lactose.4 In comparison to other
monovalent CT ligands 6 performs remarkably well. Excep-
tional remains the close GM1 mimic of Bernardi et al.10 with
a Kd value similar to GM1 itself (50 nM11), but their
simplified analogue has a Kd of 190 µM.12 For the closely
related heat-labile enterotoxin of E. coli, Hol et al. have found
a lead compound from screening a small library, m-nitro-
phenyl-R-D-galactopyranoside, that has a Kd value determined
by ITC of 175 µM, while their best designed derivative binds
with a Kd of 12 µM.13
In summary, the synthesis and characterization of a lactose-
2-aminothiazoline conjugate 6 was described. This is a stable
compound with no tendency to convert to the corresponding
2-aminothiazole under ambient conditions. The class of
2-aminothiazolines is of interest for a variety of biological
activities,14 and the synthetic method described here will be
further explored in this context. Compound 6 proved to be
a high-affinity monovalent CTB ligand of relatively simple
structure and may contribute to the development of an
effective cholera prophylactic. Computational studies to
decipher the origin of its affinity along with work toward
further affinity enhancement for effective competition with
GM1 through multivalency are currently in progress.
Scheme 1a
a (a) HCtCCH2NHBoc, Pd0, CuI, NEt3, CH3CN, 14 h (62%);
(b) TFA, CH2Cl2 (quant); (c) Lac(OAc)7NCS, CH2Cl2, iPr2NEt,
14 h; (d) AcOH, CH2Cl2 (84% for 2 steps); (e) dioxane/MeOH/4
N NaOH, 15:4:2, (45%).
13C NMR spectrum of this compound showed neither a peak
at 184 ppm characteristic for the carbon of a thiourea moiety
nor the expected triple bond signals. The observed spectral
features were consistent with structure 5 (or its tautomer with
the other nitrogen protonated). The structure assignment is
partly based on the reported7,8 reactions of propargylamines
with isothiocyanates and the related reactions of propargylic
isothiocyanates with amines and was confirmed by several
2D-NMR techniques (see Supporting Information for detailed
structural characterization).
The carbohydrate moiety of 5 was deprotected with base
to yield 6. This compound is insoluble in water, but the
compound obtained after treatment with dilute HCl or formic
acid and lyophylization had good water solubility. Presum-
ably both nitrogens were protonated at this stage, since it is
expected that the pKa should exceed 6.9 A similar NMR
analysis as was used for 5 confirmed the structure of 6. A
stability test for 6 of 1 week in aqueous solution showed
the compound to be stable.
Acknowledgment. The Royal Netherlands Academy of
Arts and Sciences (KNAW) is gratefully acknowledged for
support.
Supporting Information Available: Experimental pro-
cedures for the synthesis of 5 and 6 and their structure
assignment including HMBC, HSQC, ROESY, and MS
spectra. This material is available free of charge via the
Internet at http://pubs.acs.org.
OL025909W
(5) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975,
50, 4470.
(6) On TLC two spots with Rf values of 0.36 and 0.27 were observed
(CH2Cl2/MeOH, 95:5 v/v) in a 10:1 ratio. The major product was identified
as 2 by NMR spectra of the mixture.
(10) Bernardi, A.; Checchia, A.; Brocca, P.; Sonnino, S.; Zucotto, F. J.
Am. Chem. Soc. 1999, 121, 2032.
(11) Mertz, J. A.; McCann, J. A.; Picking, W. D. Biochem. Biophys.
Res. Commun. 1996, 226, 140.
(7) Eloy, F.; Deryckere, A. Chim. Ther. 1973, 8, 437.
(8) Ferrand, G.; Maffrand, J. P.; Eloy, F.; Ferrand, J. C. Eur. J. Med.
Chem. 1975, 10, 549.
(9) Hirashima, A.; Tomita, J.; Pan, C.; Taniguchi, E.; Eto, M. Bioorg.
Med. Chem. Lett. 1997, 5, 22121.
(12) Bernardi, A.; Carrettoni, L.; Grosso Ciponte A.; Monti, D.; Sonnino,
S. Bioorg. Med. Chem. Lett. 2000, 10, 2197.
(13) Pickens, J. C.; Merritt, E. A.; Ahn, M.; Verlinde, C. L. M. J.; Hol,
W. G. J.; Fan, E. Chem. Biol. 2002, 9, 215.
(14) Kim, T. H.; Cha, M.-H. Tetrahedron Lett. 1999, 40, 3125.
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