1644
Can. J. Chem. Vol. 80, 2002
Scheme 1. (a) n-BuLi, THF, –78°C, HO(CH2)5NHCbz (4), 59%.
(b) H2, 10% Pd/C, EtOH, 85%.
Fig. 1. Comparison of antibody F123 activity with hydroxy ester
substrate 6 (᭺); with ester 7 (᭹). In each case, antibody was
0.46 M; substrates were 100 M; carried out in PBS buffer
(pH 7.4) containing 10% DMSO and 0.5% Triton X-100; total
reaction volume was 225 L.
Results and discussion
The synthesis of hapten 2 (Scheme 1) was achieved by
substitution of the phenyl moiety of the S*R* diastereomer
of phosphonate 3 (4) with the anion of alcohol 4 to give 5 in
a 59% yield.
Hydrogenolysis of 5 in ethanol with hydrogen and 10%
Pd/C afforded amine 2 in 85% yield.
Amine 2 was coupled to carboxyl residues of the carrier
protein keyhole limpet hemocyanin (KLH) using the water
soluble coupling reagent 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDC), emulsified in
Freund’s complete adjuvant and used to immunized Balb/c
mice. After three subsequent booster injections with KLH·2
in Freund’s incomplete adjuvant, spleens of mice were har-
vested and MAbs were produced using standard hybridoma
methodology (5). Three MAbs (F123, F125, and F150) were
selected in an immunoassay based on their selective binding
to 2 coupled to bovine serum albumin (BSA). MAbs were
purified by sheep anti-mouse affinity chromatography and
dialyzed against PBS buffer, pH 7.4. Antibody concentration
was determined by measuring optical density at 280 nm
(where 1.2 absorbance units correspond to 1 mg mL–1 anti-
body in solution (6)) and using 190 kDa for the molecular
weight of IgM monomer.
The three MAbs that showed selective binding to 2 were
tested for catalytic activity against substrate 6 while moni-
toring the release of p-nitrophenolate. Of these, only F123
(an IgM monoclonal antibody as determined by isotyping)
showed significant activity and was selected for further
study. The F123-catalyzed reaction with 6 was shown to
obey Michaelis–Menten kinetics with a Km of 255 M, and a
kcat of 0.01 min–1 (based on p-nitrophenol release and calcu-
lated on an active site basis). The observed rates were cor-
rected for the background hydrolysis in buffer.
ity purified control MAb raised against an unrelated hapten
was run in parallel with F123. The control MAb showed no
activity with substrate 6. It should be noted that while this
evidence is supportive, it is by no means definitive. In addi-
tion, substrate 7, which lacks the secondary hydroxyl neces-
sary for lactonization, was tested as a substrate for F123 and
showed little or no p-nitrophenolate release compared with 6
(Fig. 1). This provided strong evidence that the catalytic
MAb was not simply behaving as an esterase by hydrolyzing
the p-nitrophenyl ester moiety. An alternate reason as to
why 7 is not processed by F123 could simply be lack of rec-
ognition by the antibody. However, these two control exper-
iments together provide substantial evidence that it is indeed
the antibody that is carrying out the catalysis.
The 4-nitrophenyl phosphonate derivative 8 was synthe-
sized by base hydrolysis of phenyl phosphonate 3 using
refluxing 3 N NaOH–THF, producing the corresponding
phosphonic acid derivative in a 73% yield. Conversion of the
phosphonic acid derivative to the acid chloride with oxalyl
chloride, followed by coupling of this phosphoryl chloride
intermediate with p-nitrophenol, gave a 1.2:1 ratio of
diastereomers of 8 in 69% yield.
Owing to a lack of compound 2, compound 8 was chosen
for inhibition studies as we felt it would sufficiently mimic
the putative transition state for the cyclization of substrate 6
to give lactone 1. Inhibition assays with 8 (using a 1:1 mix-
ture of diastereomers) were performed by varying the con-
centration of 8 at fixed levels of substrate 6 and MAb F123
To illustrate that the activity of antibody F123 with 6 is
not due to an esterolytic contaminant of the serum, an affin-
© 2002 NRC Canada