Antibodies with broad specificity to azaspiracids by use of synthetic haptens

Article abstract of DOI:10.1021/ja066971h

The development of general, sensitive, portable, and quantitative assays for the azaspiracid (AZA) class of marine toxins is urgently needed. Use of a synthetic hapten containing rings F-I of AZA to generate antibodies that cross-react with the AZAs via t

Full text of DOI:10.1021/ja066971h

Published on Web 11/08/2006
Antibodies with Broad Specificity to Azaspiracids by Use of Synthetic
Haptens
Craig J. Forsyth,*,† Jianyan Xu,† Son T. Nguyen,† Ingunn A. Samdal,§ Lyn R. Briggs,
Thomas Rundberget,§ Morten Sandvik,§ and Christopher O. Miles*,§,
Department of Chemistry, UniVersity of Minnesota, National Veterinary Institute, Oslo, Norway, and
AgResearch Ruakura, Hamilton, New Zealand
Received September 29, 2006; E-mail: forsyth@chem.umn.edu; chris.miles@agresearch.co.nz & chris.miles@vetinst.no
The azaspiracids (Figure 1) are natural products that accumulate
in shellfish and have severe acute and suspected chronic toxicologi-
cal effects on humans.1 The seasonal recurrence of azaspiracid
contamination in European cultured shellfish coupled with the
potential for global distribution of toxin-harboring strains of the
dinoflagellate Protoperidinium crassipes2 has spurred worldwide
surveillance programs.3 Existing methods for detecting and quan-
tifying azaspiracids have been restricted to mass spectrometry and
mouse toxicity assays.4 The development of general, sensitive,
portable, and quantitative assays is urgently needed. Attempts to
use azaspiracids isolated from environmental samples as haptens
for antibody development that can be translated to an ELISA format
for the detection of the natural products are underway.5 Meanwhile,
herein we report the successful detection of natural azaspiracids
by antibodies raised against a synthetic hapten representing the
common C28-C40 domain of the azaspiracids.
Initially reported in 1998,6 the azaspiracids were shrouded in
Figure 1. Structures of the azaspiracids with variable functionality R1-
R4 at C3-C24 and the hapten 1.
structural ambiguity. Neither the relative stereochemistry among
the discrete cyclic domains nor the absolute configuration of any
was fully established until recently.7,8 We hypothesized that
antibodies raised against two enantiomeric forms of a synthetic
C28-C40 azaspiracid hapten could correlate the absolute config-
uration of this domain between synthetic and natural products via
enantiospecific recognition. This immuno-stereochemical determi-
nation strategy would utilize the C26-C40 domain that is invariant
among the azaspiracids. An antibody that universally cross-reacts
with the azaspiracid natural products via their common C28-C40
domain would serve important roles in development of matrices
for immunoaffinity purification and sensitive, quantitative detection
via ELISA.9
Scheme 1. Retrosynthesis of 1
In 2001, enantioselective total syntheses of dextrarotatory
enantiomers of N-protected forms of the C26-C40 domain of the
azaspiracids were reported,10a,b whereas a C26-C40 degradation
product of azaspiracid 1 (AZA1) was reported to be levarotatory.11
Consequently, a levarotatory derivative of the C26-C40 domain
was initially targeted for hapten development. The complete
stereochemistry of AZA1 has since been established via correlation
of natural product degradation fragments with synthetic products,
culminating in a total synthesis by Nicolaou and co-workers in
2004.7
Our original synthesis of the C26-C40 domain relied upon a
stepwise Lewis acid-induced spiroaminal formation followed by a
double intramolecular hetero-Michael addition (DIHMA) to as-
semble the bridged F-G rings.10 We have since developed more
efficient entries.12 DIHMA disconnection of ketal 1 (Scheme 1)
yields ynone 2, the spiroaminal of which would arise from a
Staudinger reduction/aza-Wittig13 (S-aW) initiated closure from
azido-hydroxy-ketone 3. A chelation-controlled Mukaiyama aldol
reaction14 of silyl enol ether 4 and aldehyde 5 would install the
requisite stereochemistry and functionality in 3.
The synthesis of 1 began with addition of the alkyl lithium
derived from C27-C31 iodide 6 to the C32-C34 aldehyde 7 to
generate a diastereomeric mixture of C32 alcohols (Scheme 2).
Subsequent oxidation-reduction enhanced the desired (32R)-
configuration, and a silylation-desilylation sequence provided
primary alcohol 9. Oxidation of 9 gave aldehyde 5. Early
incorporation of the C33 p-methoxy benzyl ether emergent in 5
facilitated an R-chelated Mukaiyama aldol reaction14 with methyl
† University of Minnesota.
§ National Veterinary Institute.
AgResearch Ruakura.
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10.1021/ja066971h CCC: $33.50 © 2006 American Chemical Society

C O M M U N I C A T I O N S
Scheme 2. Synthesis and Protein Conjugation of 1a
Figure 2. ELISA inhibition curve for antibodies derived from hapten 1
with natural azaspiracid 1.
predisposed in ynone (32R,34R)-2. Deprotection of the C- and
N-termini provided carboxylic acid 1, which was then conjugated
to cBSA16 and ovalbumin (OVA) to yield conjugates 17 and OVA-
1, respectively.
Sheep were repeatedly immunized with cBSA conjugate 17 and
the antisera harvested.9b ELISA plates were coated with OVA-1
and blocked with polyvinylpyrrolidone. Competitive ELISA analy-
ses were performed by adding appropriate amounts of antiserum
and analyte to the ELISA wells. The bound antiserum was detected
using a commercial anti-sheep-HRP conjugate, and the absorbance
was measured after reaction with a colorimetric substrate.9b ELISA
analyses showed that the antibodies in the antiserum recognized
and bound to OVA-1 in the absence of AZA1 and that this binding
was inhibited by addition of 1. Addition of AZA1 also caused
concentration-dependent inhibition of antibody binding (Figure 2),
indicating that the antibodies produced against 1 recognized AZA1.
A partially purified extract containing a ca. 2:2:2:1 mixture of
AZA1, AZA2, AZA3, and AZA6 was available from Norwegian
mussels, and its AZA content was determined by LC-MS17 against
the AZA1 standard. Addition of this mixture caused a degree of
inhibition of antibody binding consistent with its total AZA content,
rather than just its content of AZA1. This result suggests that the
antibodies also have a similar affinity for AZA2, AZA3, and AZA6
as they do for AZA1 and that such antibodies are suitable for
analysis of AZAs in shellfish samples, although assay optimization
will be required.
The broad recognition of the antibodies was further confirmed
by immunoaffinity chromatography. The IgG fraction from the
antiserum was purified using HiTrap Protein G HP columns
(Amersham Biosciences, A.B.) and coupled to cyanogen bromide-
activated Sepharose 4B (A.B.) according to the manufacturer’s
instructions. The mixture of AZA1, AZA2, AZA3, and AZA6 was
applied together with a standard of yessotoxin (YTX)18 to a column
containing the immobilized anti-AZA IgG. The column was washed
with buffer and eluted with methanol. LC-MS analysis of the
fractions showed the YTX was present in the buffer wash and was
not retained on the immunoaffinity column, but that AZA1, AZA2,
AZA3, and AZA6 were retained on the column with buffer and
eluted by the methanol. This shows that the antibodies produced
against hapten 1 did not bind to the structurally dissimilar polyether
algal toxin YTX, but did bind to a wide array of naturally occurring
AZA analogues. In addition to demonstrating the broad specificity
of the antibodies for AZA congeners, the immunoaffinity columns
have considerable potential for concentrating and purifying shellfish
extracts prior to LC-MS analysis and could be useful for identifying
novel AZA metabolites.
a (a) (i) t-BuLi, pentanes/Et2O, -78 °C then 7, 90%, (ii) SO3·Pyr,
i-Pr2NEt, DMSO, CH2Cl2, 0 °C, 70%, (iii) LiI, Et2O, -40 °C, then LiAlH4,
-100 °C, 100%, (iv) TBSCl, DMAP, CH2Cl2, 96%; (b) HF·Pyr, Pyr, THF,
69%; (c) SO3·Pyr, i-Pr2NEt, DMSO, CH2Cl2, 0 °C, 100%; (d) 4, MgBr2·OEt2,
CH2Cl2, -78 °C to -25 °C, 12 h, 79%; (e) TESOTf, 2,6-lutidine, CH2Cl2,
-10 °C, 92%; (f) DDQ, t-BuOH, H2O, CH2Cl2, 5 min, 93%; (g) Et3P,
PhH, 1.5 h, ∼100%; (h) CbzCl, K2CO3, 4 Å MS, CH2Cl2, 14 h, 73%; (i)
CF3COOAg, NIS, acetone, 10 min, 95%; (j) ethyl 6-oxo-hexanoate, CrCl2,
NiCl2 (0.2%), THF, 12 h, 70%; (k) MnO2, K2CO3, CH2Cl2, 85%; (l) TBAF,
THF, 12 h, 76%; (m) LiOH, t-BuOH, THF, H2O, 12 h, 90%; (n) H2, Pd/
CaCO3 (5%), EtOAc, 2 h, 80%; (o) N-hydroxysuccinimide, DIPCDI, DMF;
(p) cationized bovine serum albumin (cBSA).
ketone derivative 4 to yield C33,34 syn-aldol 10. Thereafter, the
C34 hydroxyl of 10 was silylated in parallel with the C32 group in
anticipation of F-G ring closure. Intervening spiroaminal formation
was preceded by liberation of the C33 hydroxyl group from 11.
The resultant azido-keto-alcohol 3 was poised to cascade into
sprioaminal 12 via a one-pot Staudinger reduction/intramolecular
aza-Wittig reaction13-imine capture sequence. This was ac-
complished by treating 3 with Et3P in benzene under anhydrous
conditions to provide spiroaminal 12 and its C36 epimer in 75%
combined yield and ca. 3:1 ratio, respectively. The thermodynami-
cally favored (36S)-configuration in 12 was established via 1H NMR
spectroscopy.10a Conversion of the amine and alkyne of 12 into
carbamate and iodo alkyne functionalities, respectively, provided
iodide 14. This was coupled with ethyl 6-oxo-hexaoate under NHK
conditions,15 and the resultant propargyl alcohol was oxidized to
ynone 2. Fluoride-initiated bis-conjugate addition of the liberated
C32 and C34 oxygens upon the C28 Michael acceptor gave ketal
15. The resultant (28S)-configuration of ketal 15 is geometrically
The absolute configurational assignment of C28-C40 of the
azaspiracids made initially via chiroptical correlation of synthetic
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C O M M U N I C A T I O N S
products10 with a C26-C40 AZA1 degradation product11 is
corroborated by the recognition of natural AZA1 by the levarotatory
hapten-derived antibody [(-)-AZAAb]. Further corroboration
derives from fragment19 and total synthesis.7 The complete array
of naturally occurring azaspiracids (Figure 1) has not yet been
assayed against (-)-AZAAb. It is anticipated, however, that minor
structural variations within the C1-C26 region will not substantially
affect molecular recognition between the natural products and
antibodies raised against the common F-I ring terminal domain.
Preliminary studies with the azaspiracids mixture are consistent with
this proposal. Optimization of the C26-linker structure in subsequent
haptens is expected to enhance derived antibody sensitivity toward
the natural toxins.
The results from this and an earlier study9a highlight the
advantages of applying synthetic organic chemistry to produce
carefully designed haptens containing conserved substructures of
complex toxin molecules. Such haptens can be used as immunogens
to generate antibodies that bind a whole family of toxin molecules.
Additional advantages of this approach are that it is possible to
generate antibodies to natural toxins whose availability is severely
limited, as in the present study, and this approach may also be useful
where the whole toxin molecule is unstable in vitro or in vivo.
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Acknowledgment. The project described was supported by
grant number ES10615 (C.J.F.) from the National Institute of
Environmental Health Sciences (NIEHS), NIH, by Norwegian
Research Council grant 139593/140, by the BIOTOX project (partly
funded by the European Commission, through 6th Framework
Programme contract no. 514074, topic Food Quality and Safety),
and by New Zealand Foundation for Research Science and
Technology contract C10X0406 (International Investment Op-
portunities Fund). Sheep for antibody development in Norway were
provided and cared for by The Animal Production Experimental
Centre, The Norwegian University of Life Sciences, Ås. All animal
manipulations in New Zealand were performed under the authority
of the AgResearch Ruakura Animal Ethics Committee in accordance
with the Animal Welfare Act (1999: Part 6). Animal experiments
in Norway were approved by the Norwegian Animal Research
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Drs. J. Hao and J. Aguade for seminal contributions, Drs. M. A.
Quilliam and P. Hess for standards of AZA1, and L. Nguyen and
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Supporting Information Available: Preparation procedures and
characterization data for compounds 1-16.
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