A catalytic antibody programmed for torsional activation of amide bond hydrolysis

Article abstract of DOI:10.1002/chem.200204620

Amidase antibody 312d6, obtained against the sulfonamide hapten 4a that mimics the transition state for hydrolysis of a distorted amide, accelerates the hydrolysis of the corresponding amides 1a-3a by a factor of 10³ at pH 8. The mechanisms of

Full text of DOI:10.1002/chem.200204620

FULL PAPER
A Catalytic Antibody Programmed for Torsional Activation of
Amide Bond Hydrolysis
Ranjana Aggarwal,[a] Fabio Benedetti,*[a] Federico Berti,*[a] Sabrina Buchini,[a]
Alfonso Colombatti,[b] Francesca Dinon,[a] Vinicio Galasso,[a] and Stefano Norbedo[a]
Abstract: Amidase antibody 312d6, ob-
tained against the sulfonamide hapten
4a that mimics the transition state for
hydrolysis of a distorted amide, acceler-
ates the hydrolysis of the corresponding
amides 1a 3a by a factor of 103 at pH 8.
The mechanisms of both the uncatalyzed
and antibody-catalyzed reactions were
studied. Between pH 8 and 12 the un-
catalyzed hydrolysis of N-toluoylindoles
1a and 3a shows a simple first-order
dependence on [OHÀ], while hydrolysis
of 3a is zeroth-order in [OHÀ] below
pH 8. The pH profile for hydrolysis of
the corresponding tryptophan amide 2a
is more complex due to the dissociation
of the zwitterion into an anion with
pKa 9.74; hydrolysis of the zwitterionic
and the anionic form of 2a both show
simple first-order dependence on
[OHÀ]. Absence of 18O exchange be-
tween H218O/18OHÀ and the substrate, a
normal SKIE for both 1a (kH/kD ˆ 1.12)
and 3a (kH/kD ˆ 1.24) and the value of
the Hammett constant 1 for hydrolysis
of p-substituted amides 3a e are con-
sistent with an ester-like mechanism in
which formation of the tetrahedral in-
termediate is rate-determining and the
amine departs as anion. The 312d6-
catalyzed hydrolysis of 3a was studied
between pH 7.5 and 9, and its independ-
ence of pH in this range indicates that
water is the reacting nucleophile. Hy-
drolysis of 3a is only partially inhibited
by the sulfonamide hapten, and this
indicates that non-specific catalysis by
the protein accompanies the specific
process. Only the nonspecific process is
observed in the hydrolysis of amides 3
with para substituents other than meth-
yl. Binding studies on the corresponding
series of p-substituted sulfonamides
5a e confirm the high specificity of
antibody 312d6 for p-methyl substituted
substrates.
Keywords:
amides
¥
catalytic
antibodies
¥
heterocycles
¥
hydrolysis ¥ sulfonamides
intrinsic stability of the amide bond. For example, the
hydrolysis of unactivated peptides, which is largely dominated
by the reaction with water between pH 5 and 9, is exceedingly
slow in this pH range, with a half-life of centuries at 378C.[5]
Hence, an extremely efficient catalyst is needed for the
reaction to be even detectable.
Introduction
The discovery of antibody catalysis was a major advance in
research on artificial enzymes,[1] and provided access to a new
class of protein catalysts with enzyme-like properties.[2] Over a
hundred catalytic antibodies have been described so far,
covering a wide range of reactions and applications, from the
asymmetric synthesis of carbon carbon bonds on the prep-
arative scale[3] to the in vivo activation of prodrugs.[4] Despite
this outstanding progress, amide hydrolysis still represents an
ambitious target for catalytic antibodies, not only because of
the importance of this reaction in nature, but also due to the
Although spontaneous protease activity by auto-antibodies
has been demonstrated in several diseases, including haemo-
phylia,[6] only a few antibodies designed to catalyze the
hydrolysis of the amide bond have been obtained by the
conventional approach based on transition-state analogues.
These include anti-phosphonamidate antibody 43C9[7] and
polyclonal antiserum PCA 270-29,[8] both of which catalyze
the hydrolysis of activated p-nitroanilides; primary-amide-
hydrolyzing antibodies 13D11[9] and BL25,[10] elicited against a
phosphinate and a boronate hapten, respectively; and an
amidase antibody that requires an external nucleophilic
cofactor.[11] b-Lactamase activity has been described in an
antiserum designed for carbonate hydrolysis,[12] and in anti-
bodies obtained by the anti-idiotype approach[13] or by
reactive immunization.[14] The intense research effort in this
area has also fostered significant progress in the design of
transition-state analogues[10, 15] and in the development of new
immunization and selection strategies.[14, 16]
[a] Prof. F. Benedetti, Dr. F. Berti, Dr. R. Aggarwal, S. Buchini,
F. Dinon, Prof. V. Galasso, Dr. S. Norbedo
Dipartimento di Scienze Chimiche
Universita degli Studi di Trieste
¡
Via Giorgieri 1, 34127 Trieste (Italy)
Fax : (‡39)040-558-2402
E-mail: benedett@univ.trieste.it
berti@dsch.univ.trieste.it
[b] Prof. A. Colombatti
Dipartimento di Scienze e Tecnologie Biomediche
Universita degli Studi di Udine
P.le Kolbe 4, 33100 Udine (Italy)
¡
Fax : ( ‡ 39)0432494301
3132
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/chem.200204620
Chem. Eur. J. 2003, 9, 3132 3142

3132 3142
With respect to ordinary amides, heterocyclic amides such
as N-acyl- and N-benzoylpyrroles, -indoles and -carbazoles
are much more reactive towards hydrolysis due to the
aromatic character of the amide nitrogen atom, which lowers
the basicity of the amine.[17] We thus chose the hydrolysis of N-
toluoylindole (1a) as model system (Scheme 1) on which to
test an approach to hapten design that combines stabilization
of the transition state with destabilization of the substrate.
this reason, phosphinates[9, 15b] and phosphonamidates,[7, 8, 21]
which are ionized at physiological pH, have been preferred as
haptens for the generation of amidase antibodies. However,
we anticipated that, for the highly reactive amides 1 3, a
neutral pathway, corresponding to the uncatalyzed addition of
water, might operate at near-neutral pH (see below). We
reasoned that a sulfonamide would be a better mimic for the
neutral species that are present along this pathway and thus
might favour this mechanism in the resulting antibodies.
The second element of design embedded in the structure of
the hapten 4 is destabilization of the amide substrate. Twisting
the amide group out of planarity reduces amide resonance[22]
and increases the reactivity of the carbonyl group towards
nucleophiles.[23] Torsional activation of amide hydrolysis,
originating from binding the peptide substrate in a distorted,
nonplanar conformation, has been suggested to be important
in enzyme catalysis,[24] and increased reactivity of twisted
amides towards hydrolysis has been demonstrated in model
systems.[23, 25] Based on this concept, it was suggested that
antibodies raised against a hapten that mimics the structure of
a twisted amide should force the substrate to adopt a
distorted, more reactive conformation and thus catalyze the
reaction by substrate destabilization.[26] This approach was
followed by Hansen et al. in the design of conformationally
restricted dipeptide analogues in which the peptide bond was
replaced by a spiro[4.4]nonyl[26] or cyclobutanol[27] analogue.
More recently Gouverneur et al. described the synthesis of a
twisted phosphinate hapten, designed to elicit antibodies for
the hydrolysis of N-benzoylpyrroles.[15b] No antibodies, how-
ever, were described by either group.
R
R
R
k1
k2
+ OH–
k–1
N –
N
N
+
O
HO
O-–
R'
R'
ArCOOH
1 R = H
2 R = CH2CH(NH3)+COO-
3 R = CHO
R
N
S
O
a: R' = CH3
b: R' = H
c: R' = OCH3
d: R' = Cl
e: R' = NO2
O
R'
4 R = H
5 R = CHO
Scheme 1. Hydrolysis of N-benzoylindoles and sulfonamide transition-
state analogues.
In a previous communication[18] we showed that hydrolysis
of amide 1a (Scheme 1) is accelerated by antibody 312D6,
obtained against 4a, a sulfonamide hapten designed to induce
catalysis by transition-state mimicry and torsional activation.
We now report full details of the experiments together with
new results that indicate that N-benzoylindoles 1 3 are
hydrolyzed with an ester-like mechanism. The results of a
study on the effects of substituents R' on the catalyzed and
uncatalyzed reactions and on the binding of sulfonamides 5
are also described and reveal a tight specificity of the antibody
for p-methyl substituted benzamides.
Figure 1 compares the X-ray crystal structure of N-benze-
nesulfonylindole[28] (4b) with the equilibrium structures of the
benzamides 1b and 3b, optimized with the B3LYP func-
tional[29] and the standard 6-31G(d,p) basis set using the
Gaussian98 suite of programs.[30] The relevant structural
parameters are reported in Table 1. In compound 1b the
amide is nearly planar, with the carbonyl group slightly
twisted (128) with respect to the plane of the indole ring, and a
twist angle of 38.28 with respect to the plane of the phenyl
group. The amide 3b adopts a similar conformation with more
Results and Discussion
À
pronounced twists around both the N C(O) (21.18) and
À
Ph C(O) bonds (43.28). As a consequence, in both amides,
Design and synthesis of the transition-state analogue: In the
base-catalyzed hydrolysis of amides, the transition state for
the rate-determining addition of hydroxide ion (step 1 of
Scheme 1) was shown to be similar to the tetrahedral
intermediate.[19] The same holds also for the transition state
for breakdown of the same intermediate (step 2), which can
be the rate-determining step in the hydrolysis of amides of
lower basicity.[17c] Hence, the sulfonamide 4 was selected as
hapten to mimic the tetrahedral
the two rings adopt a conformation intermediate between
coplanar and bisected, defined by torsion angles f and y of
12.3 and 38.08 for 1b and 21.4 and 43.08 for 3b (Figure 1,
Table 1). In the sulfonamide 4b, in contrast, a nearly
orthogonal conformation with f ˆ 79 and y ˆ 778 is imposed
on the rings by the tetrahedral structure of the sulfonyl
group.[31] We thus anticipated that antibodies raised against
intermediate and the related
transition states.[20]
Calculations indicate that
sulfonamides adequately repro-
duce the geometry and confor-
mation of tetrahedral inter-
mediates for the base-catalyzed
hydrolysis of amides, but not
the charge distribution.[15e] For
Figure 1. B3LYP/6-31G(d,p) optimized structures of amides 1b (left) and 3b (middle) and X-ray crystal structure
of sulfonamide 4b (right).[28]
Chem. Eur. J. 2003, 9, 3132 3142
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3133

F. Benedetti, F. Berti et al.
FULL PAPER
Table 1. Selected dihedral angles for amides 1b, 3b and sulfonamide 4b.
Immunization and screening: Three Balb/C mice were immu-
nized with the KLH conjugate 7 by following a standard
protocol.[33] After three months and three immunization
rounds, the mouse with the highest level of circulating anti-
10 antibodies was sacrificed, and monoclonal antibodies were
obtained from its spleen cells. Seventeen binders were
selected with an ELISA screening based on binding to the
BSA conjugate 10. Antibodies were purified by immunoaf-
finity chromatography on protein G and ion-exchange chro-
matography, and the purified immunoglobulins were assayed
for their ability to accelerate the hydrolysis of amide 1a at
pH 7.5 in PBS and at pH 8 in TRIS buffer. Two antibodies
showed a significant activity over background, and the most
active catalyst, namely, antibody 312D6, an IgG2a, was
selected for further characterization and subcloned.
1b[a] (X ˆ C)
À 168.0
3b[a] (X ˆ C)
À 158.9
4b[b] (X ˆ S)
À 165.3
À 36.1
À 39.1
79.0
C2-N1-X-O1
C2-N1-X-O1'
C2'-C1'-X-O1
f: C2-N1-X-C1'
y: N1-X-C1'-C2'
À 141.8
12.3
À 136.8
21.4
38.0
43.0
77.0
[a] From DFTcalculations, this work. [b] From the X-ray crystal struc-
ture.[28]
the sulfonamide 4, on binding the substrates 1 3, would force
them to adopt a twisted conformation (Scheme 2) in which the
highly distorted amide is expected to be more reactive.
R
R
N
antibody
Uncatalyzed hydrolysis of indole amides: Kinetic data for the
hydrolysis of amides 1 and 3 were obtained at 258C, while the
hydrolysis of tryptophan amide 2a was studied at 378C. Faster
reactions were followed spectrophotometrically, while slower
hydrolyses were monitored by HPLC. All the reactions were
run in 10 mm buffer (MES, pH 6 6.5; phosphate, pH 6.5 8;
TRIS, pH 8 10; NaOH, pH > 10) containing 10% dioxan,
and at constant ionic strength (0.1m NaCl). The observed rate
constants for the hydrolysis of amides 1 and 3 were
independent of buffer concentration in the range from 0.005
to 0.05m. For these substrates the uncorrected kobs values were
used. In contrast, hydrolysis of the tryptophan amide 2a
shows a significant dependence on the concentration of both
phosphate buffer (at pH 7.5) and TRIS buffer in the whole pH
range explored. For this substrate the observed rate constants
were extrapolated to zero buffer concentration.
N
φ
bind and twist
O
O
ψ
R'
R'
Scheme 2. Torsional activation by antibody 312d6.
Based on this analysis, the p-toluenesulfonamide conju-
gates 7 and 10 were synthesized (Scheme 3), starting from
3-formylindole. This was converted to the corresponding
OH
CHO
CHO
ArSO2Cl
NaBH4
99%
NaH/THF
81%
N
H
N
N
SO2Ar
5a
SO2Ar
8
The possible general pathways for the base-catalyzed
hydrolysis of an amide are shown in Scheme 4. Path a is
commonly followed in the hydrolysis of unactivated amides
and requires protonation of the amine leaving group by a
donor DH. If the basicity of the amine is decreased, path a is
disfavoured, and the amine departs as anion via path b and/or
path c. In the latter, breakdown of the tetrahedral intermedi-
ate is catalyzed by a second hydroxide ion.[17]
COOH
succinic
87%
74%
O
H2N
anhydride
O
COR
Ar = p-CH3C6H5
COR
N
O
O
N
N
SO2Ar
6 R = OH
7 R = KLH
SO2Ar
9 R = OH
EDC
EDC
10 R = BSA
Due to the low basicity of indole,[34] path a can be excluded
for amides 1 3. A number of studies on the base-catalyzed
hydrolysis of amides of indole, and of the related heterocycles
pyrrole and carbazole,[17] have shown that these substrates
may follow both paths b and c (Scheme 4), the balance
between the two being determined by the structure of the
substrate and by the medium. To determine the mechanism of
the uncatalyzed reactions, we studied the pH dependence of
the hydrolysis of amides 1a, 2a and 3a. The corresponding pH
profiles are compared in Figure 2. Between pH 8 and 11.5, the
hydrolysis of both amides 1a and 3a displays a simple first-
order dependence on base concentration. The experimental
points are well aligned on straight lines with slopes of 0.99 for
1a and 1.03 for 3a, and no curvature or inflections due to
concurrent processes second-order in [OHÀ] can be detec-
Scheme 3. Synthesis of haptens and conjugates.
anion with NaH in THF and sulfonylated to give the p-
toluenesulfonamide (5a) which, by reaction with O-carbox-
ymethylhydroxylamine gave the oxime 6. Conjugate 7 was
obtained by condensation of 6 with keyhole limpet hemocya-
nin (KLH). Borohydride reduction of the aldehyde 5a and
reaction of the resulting alcohol 8 with succinic anhydride
gave the hemisuccinate 9, which was conjugated to bovine
serum albumin (BSA) to give 10. Both conjugations were
carried out by a standard EDC/MES buffer method,[32] and
gave two highly modified conjugates that were purified by gel
filtration chromatography over Sephadex G-25 and dialysis
against water. A spectrophotometric analysis of the conjugate
10 yielded 31 molecules of bound sulfonamide per protein
molecule.
i) ]
ted.[17c,f Path c of Scheme 4 can thus be excluded in favour
of path b. The values of kobs between pH 9 and 11.5 were used
to calculate the second-order rate constants kOH for the
3134
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www.chemeurj.org
Chem. Eur. J. 2003, 9, 3132 3142

Catalytic Antibodies
3132 3142
''
zwitterionic (kO' H† and anionic 2a (k † (Table 2) and a value
OH
of 9.74 for pKa2 of tryptophan, in good agreement with
literature values.[35] Consistent with classical electrostatic
theory, in the base-catalyzed hydrolysis of tryptophan amide
2a, the anion is more than ten times less reactive than the
zwitterion, as a consequence of unfavourable interactions
between the reacting anions.
Table 2. Kinetic parameters for the OHÀ-catalyzed (kOH), uncatalyzed
(kW), and 312d6-catalyzed (kcat, KM) hydrolysis of indole amides 1a 3a.
Amide
kOH
[mÀ1 sÀ1
kW Â 105
kcat  105
KM
[mm]
kcat/k0
kcat/KM
[mÀ1 sÀ1
]
]
[sÀ1
]
[sÀ1
]
1a[a]
0.48
0.80
0.0421
16.5
36
52
36
1490
> 750[e]
> 650[e]
10
0.35
2a[b,c]
2a[b,d]
3a[a]
1.30
1455
160
1120[f]
91
[a] At 298 K. [b] At 310 K. [c] Zwitterion. [d] Anion. [e] Lower limit; for
k0 ˆ kobs at pH 8. [f] k0 ˆ kw.
Figure 2. pH rate profiles for the uncatalyzed hydrolysis of N-benzoy-
!
^
*
lindoles 1a ( ), 2a ( ) and 3a ( ) and for the 312d6-catalyzed hydrolysis
Having demonstrated that the hydrolysis of amides 1
3
*
of 3a ( ).
follows the mechanism shown in Scheme 1, corresponding to
path b of Scheme 4, it remains to be established whether the
rate-limiting step is the formation or the spontaneous break-
down of the tetrahedral intermediate.
hydroxide-catalyzed processes according to the simple Equa-
tion (1).
kobs ˆ kOH[OHÀ]
(1)
R'
N
HO O–
+H
R
RR'NH + R"COO–
RR'N- + R"COO–
R"
a
Values of 0.48 and 16.5mÀ1 sÀ1 were obtained for 1a and 3a,
respectively (Table 2).
In the profile for the amide 3a (Figure 2), a plateau region
can be observed below pH 7.5, corresponding to the sponta-
neous, uncatalyzed hydrolysis of this more reactive substrate.
The plateau value gives directly the rate constant for the
hydrolysis of 3a by water (kW ˆ 1.30  10À5 sÀ1).
D-H
R'
R'
N
k1
k2
–
R"
N
R"
+ OH
b
c
R
R
k–1
HO O–
O
OH–
Scheme 4. Mechanisms for the base-catalyzed hydrolysis of amides.
The pH profile for the hydrolysis of tryptophan amide 2a is
more complex (Figure 2) and can be divided into three
regions: below pH 9 and above pH 10.4, a simple first-order
dependence on the concentration of hydroxide ion is ob-
served, while in the intermediate region, the behaviour is
more complex. The first-order process at higher pH is also
intrinsically slower than that at low pH. The discontinuity in
the profile can be simply explained with the transition of the
amino acid from the zwitterionic form which is present at pH
values below its second pKa, to the anionic form present at
higher pH. The profile can be described by a linear
combination of the equations for the zwitterionic and anionic
forms of the substrate, with the ratio between the two forms as
the combination coefficient. This is represented by Equa-
To distinguish between these two possibilities, additional
information was obtained from 1) 18O-exchange experiments,
2) D2O solvent kinetic isotope effect (SKIE: kH/kD), and
3) substituent effects in the series of 3-formylindole benza-
mides 3a e.
Amides 1a and 3a were incubated in 99% H218O at 258C
and pH 7.5, and the reaction mixture was analyzed by
electrospray mass spectrometry at several time intervals. No
measurable 18O incorporation was detected in the uncon-
verted substrates upon incubation for three half-life times for
the activated amide 3a and one half-life time for the less
reactive 1a. In the mechanism shown in Scheme 1, incorpo-
ration of 18O in the amide should take place if addition of
18OHÀ (k1) is followed by proton equilibration in the
tetrahedral intermediate, which is assumed to be fast,[17b]
and partitioning of the intermediate between k2 and expulsion
of 16OHÀ (kÀ1). The absence of exchange indicates that
breakdown of the tetrahedral intermediate to products (k2) is
faster than kÀ1. Application of the steady-state approximation
to the tetrahedral intermediate of Scheme 1 gives Equa-
tion (3). If k2 ꢁ kÀ1, then Equation (3) simplifies to Equa-
tion (2) in which KW is the dissociation constant of water, kO' H
''
and k are the second-order kinetic constants for the base-
OH
catalyzed hydrolysis of the neutral and anionic forms of 2a,
respectively, and pKa2 refers to the equilibrium constant for
the dissociation of the zwitterion into the anion.
2ÀpH†
10ÀꢀpKa
''
''
logkobs À logKW ˆ logk
‡
(logkO' H À (logk ) ‡ pH (2)
OH
OH
1 ‡ 10ÀꢀpKa
2 ÀpH†
Fitting the experimental data to Equation (2) gave values of
0.80 and 0.0421mÀ1 sÀ1 for the rate constants for hydrolysis of
Chem. Eur. J. 2003, 9, 3132 3142
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3135

F. Benedetti, F. Berti et al.
FULL PAPER
tion (4), that is, formation of the tetrahedral intermediate is
the slow step.
k1k2 ‰OHÀŠ
kobs
ˆ
(3)
(4)
kÀ1 ‡ k2
kobs ˆ k1[OHÀ]
The hydrolysis of amides 1a and 3a was monitored in L2O
(L ˆ H/D), at [OLÀ] ˆ 3.2  10À7 m (pH 7.5) and 258C, where
both reactions exhibit normal SKIEs of kH/kD ˆ 1.12 and 1.24,
respectively. In the proposed mechanism (Scheme 1) there are
no protons in flight, and thus only a secondary SKIE is
expected, due to the reorganization of solvent water mole-
cules around the reacting species. Approximate values of the
SKIE for k1 and k2 can be calculated by applying an isotopic
fractionation analysis[36] to the transition states for the two
steps. Figure 3 depicts the structures of the transition states for
formation (TS1) and breakdown (TS2) of the tetrahedral
Figure 4. Hammett plots for hydrolysis of amides 3a e at pH 8 in TRIS
buffer. a) uncatalyzed; k ˆ kobs. b) 312d6-catalyzed; aspecific catalysis
'
*
*
(
): k ˆ kcat ; specific catalysis ( ): k ˆ kcat/KM
.
HOH
HOH
HOH
Tol
HOH
HOH
Tol
HOH
O
O
Table 3. Kinetic parameters for hydrolysis of amides 3a e in 10%
aqueous dioxan at 298 K.
N(Ind)
N(Ind)
OH
HOH
OH
Amide
pH
Uncatalyzed
312d6-catalyzed
kobs  105
kcat  105
KM
[mm]
kc'at
[mÀ1 sÀ1
HOH
[sÀ1
]
[sÀ1
]
]
[a]
TS1
TS2
3a
7.5
1.53
980
78
150[b]
170
140
250
1.2
920[b]
Figure 3. Transition states for formation (TS1) and breakdown (TS2) of
the tetrahedral intermediate in the hydrolysis of N-toluoylindole.
3a
3a
3a
3b
3c
3d
3e
8.0
8.5
9.0
8.0
8.0
8.0
8.0
2.63
6.18
17.4
7.27
2.26
14.4
123
1990
1330
1520
2.0
4.7
13.2
4.4
1.5
5.3
intermediate with associated water molecules, as proposed by
Brown et al. for the hydrolysis of N-toluoylpyrrole.[17b]
Assuming both transition states to be similar to the tetrahe-
dral intermediate, weighting factors of 0.6 and 0.9 were chosen
to describe two limit structures for TS1, corresponding to 60
and 90% advancement along the reaction coordinate, re-
spectively. Similarly, two limit structures for TS2 with 10 and
40% advancement were represented with weighting factors of
0.1 and 0.4. By applying these weighting factors to Gold and
Grist×s[37] fractionation factors for alkoxide, hydroxide and
their solvating water molecules, an approximate SKIE of
0.91 1.14 is calculated for k1, while the corresponding SKIE
for k2 is 0.61 0.87. Within the experimental error, the SKIEs
measured for the hydrolysis of 1a (kH/kD ˆ 1.12) and 3a (kH/
kD ˆ 1.24) are in good agreement with those predicted for k1,
and support the hypothesis that formation of the tetrahedral
intermediate is the slow step.
75.7
[a] Observed pseudo-first-order rate constant of the uncatalyzed reaction.
[b] FAB fragment.
ester-like mechanism (Scheme 1) in which the rate-determin-
ing step is the addition of OHÀ to form a tetrahedral
intermediate from which the amine departs as anion. This,
however, is consistent with the pKa of the leaving group being
comparable to that of a phenol in amide 3 (3-formylindole has
a pKa of 12.4)[38] or to that of an alcohol in substrates 1 and 2.
The 312d6-catalyzed hydrolysis of indole amides: The 312d6-
catalyzed hydrolyses of N-toluoylindole (1a) and N-toluoyl-
tryptophan (2a) were monitored at pH 8 in TRIS buffer at 25
and 378C, respectively. Both reactions follow saturation
kinetics and are reversibly inhibited by the sulfonamide
hapten 4a, a clear indication that the reaction is taking place
inside the binding site of the antibody. The rate constants kcat
and Michaelis constants KM were obtained by a standard
Lineweaver Burk treatment of the kinetic data and are
reported in Table 2.
The hydrolysis of para-substituted benzamides 3a e was
monitored at pH 8. The observed rate constants kobs are
reported in Table 3, while the Hammett graph obtained by
plotting logkobs against the Hammett constants sp for the
corresponding substituents is shown in Figure 4a. The exper-
imental points are well aligned on a straight line of slope 1 ˆ
1.56 (rˆ 0.99), similar to that obtained by Cipiciani et al. for k1
(formation of the tetrahedral intermediate) in the closely
related hydrolysis of N-benzoylpyrroles (1 ˆ 1.48).[17g]
The behaviour of the more reactive amide 3a is more
complex. The hydrolysis of this substrate is not completely
inhibited by the sulfonamide 4a, and even in the presence of a
20-fold molar excess of the hapten, a residual acceleration is
In conclusion, the pH profiles, absence of 18O exchange,
secondary SKIE, and Hammett values 1 strongly support the
hypothesis that, in base, amides 1 3 are hydrolyzed with an
3136
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3132 3142

Catalytic Antibodies
3132 3142
present (Figure 5). The uninhibited process can be attributed
to nonspecific catalysis by polar groups present outside the
binding site, on the protein surface.[39] Partitioning of the
observed acceleration between the two catalytic effects thus
becomes necessary. The rate of the nonspecific process can be
obtained from kinetic experiments run in the presence of
200 mm of inhibitor 4a, which suppresses the specific catalysis
(Figure 5), and the corresponding observed rate constant kc'at
is reported in Table 3. The kinetic parameterskcat and KM for
Figure 2). Data in Table 3 clearly indicate that the 312d6-
catalyzed hydrolysis of indole amide 3a is pH-independent
between pH 7.5 and 9, and a slope of 0.07 was obtained by
fitting the experimental points to a straight line (Figure 2).
Thus, in the antibody-catalyzed reaction, water is the attack-
ing nucleophile, possibly activated by a general base/acid
present in the binding site, which does not change its
protonation state in this pH interval. By averaging the
experimental values obtained in this pH interval, a value of
1455 Â 10À5 sÀ1 is obtained for kcat, which corresponds to a
rate-acceleration factor of 1120 when the rate constant for the
spontaneous, pH-independent hydrolysis of amide 3a (kw ˆ
1.3 Â 10À5 sÀ1) is taken as the corresponding uncatalyzed rate
constant (Table 2).
For the less reactive amides 1a and 2a, spontaneous
hydrolysis could not be detected; it is possible, however, to
estimate a lower limit for the rate-acceleration factor if the
observed pseudo-first-order rate constants for the hydroxide-
catalyzed reaction at the same pH are used for the uncata-
lyzed process. Values of 750 and 650 are thus obtained as
lower limits for the rate acceleration factor for the hydrolysis
of 1a and 2a, respectively (Table 2).
Concentration-dependent inhibition by the sulfonamide 4a
is observed for the hydrolysis of all substrates, as expected for
a competitive inhibitor. However, due to the relatively high
concentration of antibody required by the kinetic assay
(10 mm), it was not possible to obtain a meaningful value of
the inhibition constant Ki from kinetic experiments. For this
reason the apparent binding constants Kd,app of the antibody
for hapten 4a and its 3-formyl derivative 5a were measured by
a competitive ELISA assay[40] and are 1.0 and 1.3 mm for 4a
and 5a, respectively. Similar values were obtained for the
apparent binding constant of the BSA conjugate 10 by a direct
ELISA assay[41] (0.86 mm) and by a IAsys resonant mirror
experiment[42] (1.4 mm). Considering that analytical methods
based on immobilized substrates may overestimate the
dissociation constant,[43] it seems safe to assume the value of
1 mm as an upper limit for Ki. For an antibody accelerating a
reaction by simple transition-state complementarity, it can
been derived that KM/Ki ˆ kcat/k0.[44] For the 312d6-catalyzed
hydrolysis of simple indole amides 1a and 3a, by introducing
the upper limit of 1 mm for Ki, we obtain for 1a KM/Ki ꢂ 36,
and for 3a KM/Ki ꢂ 160. If we compare these values with the
corresponding kcat/k0 ratios of Table 2, it appears that a
substantial fraction of hapten complementarity is reflected in
catalytic activity, and this corroborates the choice of sulfona-
mides as transition-state analogues for the torsionally acti-
vated hydrolysis of amides.
Figure 5. Variation of absorbance at 320 nm for the hydrolysis of amide 3a
(100 mm) in TRIS buffer at pH 8. Dashed line: uncatalyzed reaction. Solid
line: with 312d6 (10 mm). Dotted line: with 312d6 (10 mm) and sulfonamide
4a (200 mm). Approximately 20% of the catalyzed reaction is shown.
the specific process were then obtained from the inhibited
fraction of the reaction, which follows the Michaelis Menten
rate law (Figure 6). Similar values of kcat and KM were
obtained for catalysis by the whole antibody and the Fab
fragment at pH 7.5 (Table 3). The approach was extended to
study the pH dependence of the 312d6-catalyzed hydrolysis of
3a between pH 7.5 and 9 in TRIS buffer (Table 3 and
The value of the Michaelis constant KM for tryptophan
amide 2a is more than one order of magnitude higher than the
corresponding values observed for amides 1a and 3a (Ta-
ble 2); this may reflect unfavourable electrostatic interactions
between the antibody and the charged groups of the amino
acid, which are not present in the haptens 4 and 5.
Antibody 312d6 is not inhibited by the reaction products, as
demonstrated by its ability to perform repeated catalytic
cycles. In a typical experiment, the antibody was incubated
with a 15-fold excess of amide 3a until hydrolysis of the
substrate was complete; a second batch of the same substrate
Figure 6. Saturation plot for the specific 312d6-catalyzed hydrolysis of
amide 3a in PBS buffer, pH 7.5. The corresponding Lineweaver Burk plot
is shown in the inset.
Chem. Eur. J. 2003, 9, 3132 3142
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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F. Benedetti, F. Berti et al.
FULL PAPER
was then added (again 15-fold excess) and completely hydro-
lyzed at the same initial rate. The absence of product
inhibition indicates that both the p-toluoyl residue and the
3-substituted indole ring are essential for recognition by the
antibody. This is also confirmed by the tight selectivity
displayed by 312d6. The antibody was tested on a number
of amides in which the p-toluic acid residue was replaced
by acetate and cyclohexanecarboxylate, and the indole ring
was replaced by another heterocycle (imidazole, carbazole,
benzimidazole), none of which was recognized as a sub-
strate.
sulfonamide was revealed by an anti-mouse IgG conjugated to
horseradish peroxidase (HRP), which resulted in an absorp-
tion at 450 nm upon incubation with the HRP substrate
tetramethylbenzidine (TMB); competition by free sulfona-
mide results in a decrease in absorption at this wavelength.
Figure 7 shows that competition between free p-methyl
sulfonamide 5a and bound p-methyl sulfonamide 9 is
observed for concentrations of 5a above 10À7 m, while none
of the transition state analogues 5b e competes up to at least
10À4 m, that is, on replacing the p-methyl substituent with
hydrogen, methoxy, chlorine or nitro the affinity for the
antibody decreases by a factor of 1000 or more.
To further study the specificity of the antibody, the
benzamides 3a e were hydrolyzed at pH 8 in the presence
of 312d6. In all cases the reaction was accelerated with respect
to background, but only the p-tolyl substrate 3a exhibited
clean saturation kinetics; in all the other cases a simple first-
order dependence (second-order overall) on the concentra-
tions of antibody and substrate was found. Furthermore, none
of the reactions of substrates 3b e was inhibited by the
sulfonamide hapten 4a, and this indicates that only 3a is
hydrolyzed with a specific mechanism, while the acceleration
observed in substrates 3b e is due to the aspecific effect
already noted for 3a. The (pseudo-)second-order rate con-
stants for the aspecific reactions kc'at (Table 3) were used to
construct the Hammett plot of Figure 4b, in which the second-
order rate constant kcat/KM for the specifically catalyzed
hydrolysis of 3a is also reported. All the points for the
aspecifically catalyzed reaction lie on a straight line of slope
1 ˆ 1.56, identical to that of the uncatalyzed process (1.57).
Again it is clear that only in the p-methyl-substituted
substrate 3a is the aspecific process accompanied by recog-
nition and specific catalysis.
In general, high specificity is common for anti-hapten
monoclonal antibodies and is an advantage for analytical
applications requiring low cross-reactivity. Conversely, the
development of immunoassays with broad specificity may
prove surprisingly difficult,[45] and, for catalytic antibodies,
special approaches in hapten design and immunization
strategy are often required to obtain catalytic species with
broad selectivity.[46] In the case of antibody 312d6, which is by
no means a strong binder, with a Kd,app value for the hapten
approaching the mm range, the presence of the methyl group in
the para position is probably crucial for properly aligning the
substrate. Larger groups prevent a correct fit of the molecule
in the binding pocket, while smaller groups allow additional
freedom for the ligand and hence a loss of binding inter-
actions. In this respect, the behaviour of 312d6 resembles that
of antibody 43C9.[7g] This antibody, designed for the hydrolysis
of p-nitroanilides and esters, failed to display any activity
when the p-nitro group in the substrate was replaced with a
hydrogen atom.
Binding studies confirm that the absence of specific
catalysis in the hydrolysis of amides 3b e is due to the high
specifity of the antibody for p-methyl-substituted substrates.
Figure 7 reports the results of a competitive ELISA experi-
ment carried out on the series of indole sulfonamides 5a e,
with the same substituents as the corresponding amide
substrates 3a e. In this experiment the free p-substituted
sulfonamides 5a e and the immobilized p-methylsulfona-
mide BSA conjugate 10 were allowed to compete for
binding to 312d6. Binding of the antibody to the immobilized
Conclusion
We have demonstrated that cataytic antibodies with amidase
activity can be obtained by immunization with a simple,
neutral sulfonamide hapten mimicking the transition state for
hydrolysis of a twisted amide. The presence of the tetrahedral
sulfonyl group and the nearly orthogonal conformation of the
indole and benzene rings in the hapten 4 combine to provide
in the resulting antibodies transition-state stabilization and
substrate destabilization by binding the amide in a nonplanar
conformation. Combination of these effects results, in anti-
body 312d6, in a rate acceleration factor kcat/k0 ˆ 103 for the
hydrolysis of N-(p-methylbenzoyl)indoles 1a 3a. Twisting
the amide bond out of planarity can in principle result in very
large accelerations.[23a] B3LYP/6-31G(d,p) calculations on
model amides 1b and 3b indicate that conformations in
which the amide bond is twisted by 908 from coplanarity are 8
to 9 kcalmolÀ1 higher in energy with respect to the minimum-
energy conformations of Figure 1. Clearly, only a fraction of
this energy is expressed as acceleration of the hydrolysis by
antibody 312d6. Whether this is due to incomplete twisting of
the amide bond by the antibody in the ground state or by
unfavourable binding of the transition state remains to be
established.
Figure 7. ELISA assay showing binding of 312d6 to para-substituted
sulfonamides 5a e in competition with binding to the immobilized
hapten BSA conjugate 10. Binding to the immobilized hapten was
revealed with a secondary anti-mouse IgG conjugated to HRP.
3138
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Chem. Eur. J. 2003, 9, 3132 3142

Catalytic Antibodies
3132 3142
À1
(ethyl acetate); IR (KBr): nÄ ˆ 1705 (C O), 1672 cm (C O); 1H NMR
(400 MHz, CD3CN): d ˆ 2.47 (s, 3H, CH3), 7.43 (m, 2H, Ind-5,6), 7.70 (d,
2H, Ar), 8.15 (s, 1H, Ind-2), 8.22 (d, 1H, Ind-4), 8.28 (d, 1H, Ind-7), 10.0 (s,
1H, CHO); 13C NMR (CD3CN): d ˆ 20.8, 116.0, 121.5, 121.6, 125.2, 126.2,
126.3, 129.5, 129.9, 130.3, 136.9, 139.6, 140.1, 168.7, 186.6; EIMS (70 eV):
ˆ
ˆ
Experimental Section
General: Optical rotations were measured on
a Perkin Elmer 241
polarimeter fitted with a 10 cm cell. IR spectra were recorded using a
Thermo-Nicolet FT-IR Avatar 320. 1H and 13C NMR spectra were recorded
at 400 and 104 MHz, respectively, on a Jeol EX400 spectrometer, using the
residual solvent peak as an internal reference. Chemical shifts are given in
parts per million. Coupling constants Jare given in Hz. EI mass spectra vere
measured on a VG 70/70 EIMS spectrometer. ES mass spectra were
recorded on a Perkin Elmer API1 spectrometer. Elemental analyses were
recorded on a Carlo Erba EA1110 elemental analyzer. UV/Vis spectra and
spectrophotometric kinetic measurements were performed on a Perkin
Elmer Lambda2 or on a Helios b spectrophotometer. HPLC analyses were
run on a Hewlett Packard 1100 HPLC system. ELISA experiments were
always carried out on Nunc maxisorp immunomodules; secondary anti-
body/HRP conjugates were purchased from Pierce ltd. Plates were washed
on an SLTplate washer, and ELISA measurements were obtained on an
SLTSpectra Vision microplate reader. THF was freshly redistilled from
sodium/benzophenone. Flash column chromatography was performed on
silica gel 60H (230 400 mesh) Merck 9385; thin-layer chromatography was
performed on silica-coated Merck kieselgel 60F254 0.25 mm plates and
visualized by UV irradiation at 254 nm.
‡
‡
‡
m/z (%): 263 (35) [M] , 119 (100) [ArCO] , 91 (23) [C7H7] ; elemental
analysis (%) calcd for C17H13NO2: C 77.55, H 4.97, N 5.32; found: C 77.4, H
4.96, N 5.35.
1-Benzoylindole-3-carbaldehyde (3b): 60% from indole-3-carbaldehyde
and benzoyl chloride; m.p. 848C (diisopropyl ether) (lit.: 83.58C[49]); IR
(KBr): nÄ ˆ 1670 (CO), 1705 cmÀ1 (CO); H NMR (400 MHz, CDCl3): d ˆ
1
7.3 7.8 (m, 7H, Ind-5,6, Ar), 7.9 (s, 1H, Ind-2), 8.2 8.4 (m, 2H, Ind-4,7),
10.0 (s, 1H, CHO); 13C NMR: d ˆ 116.1, 122.0, 125.6, 126.6, 128.7, 129.3,
129.4, 130.1, 133.0, 133.5, 136.8, 137.6, 168.5, 185.7; EIMS (70 eV): m/z (%):
‡
‡
‡
249 (100) [M] , 144 (25) [M À PhCO] , 105 (94) [PhCO] , 77 (53);
elemental analysis (%) calcd for C16H11NO2: C 77.10, H 4.45, N 5.62; found:
C 77.21, H 4.49, N 5.66.
1-(4-Methoxybenzoyl)indole-3-carbaldehyde (3c): 90% from indole-3-
carbaldehyde and 4-methoxybenzoyl chloride; m.p. 95 978C (diisopropyl
ether); IR (KBr): nÄ ˆ 1702, 1685 cmÀ1 (CO); 1H NMR (400 MHz, CDCl3):
d ˆ 3.9 (s, 3H, OCH3), 7.0 7.1 (dd, 2H, Ar), 7.3 7.4 (m, 2H, Ind-5,6), 7.7
7.8 (dd, 2H, Ar), 8.0 (s, 1H, Ind-2), 8.1 8.2 (m, 1H, Ind-4), 8.3 8.4 (m, 1H,
Ind-7), 10.0 (s, 1H, CHO); 13C NMR: d ˆ 56.3, 115.0, 116.5, 122.4, 122.6,
125.5, 125.9, 126.8, 126.9, 132.7, 137.6, 138.4, 164.3, 168.4, 186.3; EIMS
General procedure for synthesis of heterocyclic carboxamides: All the
heterocyclic amides were synthesized according to ref. [47] The acyl
chloride (15 mmol) was slowly added over 20 min with mechanical stirring
to a slurry of the heterocyclic compound (10 mmol) and finely powdered
sodium hydroxide (1 g) in dry dichloromethane (30 mL) containing
Aliquat 337 (40 mg). The mixture was then extracted with 1% aqueous
ammonium chloride and water. The organic fraction was dried over
anhydrous sodium sulfate, and the solvent was removed. The crude product
was then purified by crystallization or by flash chromatography.
‡
‡
(70 eV): m/z (%): 279 (50) [M] , 144 (8) [M À ArCO] , 136 (75)
‡
‡
[ArCOH] , 135 (100) [ArCO] ; elemental analysis (%) calcd for
C17H13NO3: C 73.11, H 4.69, N 5.02; found: C 72.23, H 4.94, N 4.90.
1-(4-Chlorobenzoyl)indole-3-carbaldehyde (3d): 90% from indole-3-car-
baldehyde and 4-chlorobenzoyl chloride; m.p. 121 1238C (petroleum
ether/ethyl acetate); IR (KBr): nÄ ˆ 1709, 1667 cmÀ1 (CO); 1H NMR
(400 MHz, CDCl3): d ˆ 7.4 7.5 (m, 2H, Ind-5,6), 7.5 7.6 (dd, 2H, Ar),
7.7 7.8 (dd, 2H, Ar), 7.9 (s, 1H, Ind-2), 8.2 8.4 (m, 2H, Ind-4,7), 10.0 (s,
1H, CHO); 13C NMR: d ˆ 116.0, 122.1, 122.4, 125.6, 126.6, 127.0, 129.4,
130.8, 131.3, 136.7, 137.0, 139.5, 167.3, 185.5; EIMS (70 eV): m/z (%): 285/
283 (10/43) [M] , 141/139 (30/100) [p-ClPhCO] , 113/111 (7/25) [p-ClPh] ;
elemental analysis (%) calcd for C16H10ClNO2: C 67.74, H 3.55, N 4.94;
found: C 67.83, H 3.59, N 4.87.
1-(4-Methylbenzoyl)indole (1a): 95% from indole and 4-methylbenzoyl
chloride; m.p. 958C (diisopropyl ether) (lit.: 948C[48]) IR (KBr): nÄ ˆ
1660 cmÀ1 (C O); 1H NMR (400 MHz, CDCl3): d ˆ 2.5 (s, 3H, CH3), 6.7
ˆ
‡
‡
‡
(d, 1H, Ind-4),7.31 (m, 1H, Ind-5), 7.33 (d, 2H, Ar),7.35 (d, 1H, Ind-7), 7.38
(m, 1H, Ind-6), 7.60 (d, 1H, Ind-3), 7.66 (d, 2H, Ar), 8.35 (d, 1H, Ind-2);
13C NMR: d ˆ 21.73, 108.36, 116.43, 120.94, 123.89, 124.89, 127.80, 129.33,
129.52, 130.84, 131.77, 136.14, 142.70, 169.05; EIMS (70 eV): m/z (%): 235
1-(4-Nitrobenzoyl)indole-3-carbaldehyde (3e): 65% from indole-3-carbal-
dehyde and 4-nitrobenzoyl chloride; m.p. 185 1888C (lit.: 188 190[50]); IR
(KBr): nÄ ˆ 1705, 1690 cmÀ1 (CO); 1H NMR (400 MHz, [D6]DMSO): d ˆ
7.5 7.6 (m, 2H, Ind-5,6), 8.1 8.5 (m, 7H, Ar‡ Ind), 10.0 (s, 1H, CHO);
13C NMR ([D6]DMSO): d ˆ 116.3, 121.6, 123.9, 124.1, 125.8, 126.7, 130.9,
131.0, 136.6, 138.9, 140.5, 150.2, 166.0, 187.5; EIMS (70 eV): m/z (%): 294
(30) [M] , 150 (100) [p-NO2PhCO] , 144 (7) [M À p-NO2PhCO] , 104 (35)
[C7H4O]; elemental analysis (%) calcd for C16H10N2O4: C 65.31, H 3.43, N
9.52; found: C 65.77, H 3.39, N 9.98.
‡
‡
‡
‡
(20) [M] , 119 (100) [p-MePhCO] , 91 (35) [C7H7] , 65 (15) [C5H5] ;
elemental analysis (%) calcd for C16H13NO: C 81.7, H 5.56, N 5.95; found: C
81.4, H 5.72, N 5.79.
Benzyl (2S)-2-amino-3-[1-(4-methylbenzoyl)indol-3-yl] propanoate: 77%
from Na-benzyloxycarbonyl-l-tryptophan benzyl ester and 4-methylben-
‡
‡
‡
zoyl chloride; m.p. 129 1318C, (chloroform/petroleum ether); [a]D25
ˆ
À1.31 (c ˆ 0.19, DMSO); IR (KBr): nÄ ˆ 3400 (NH), 1730 (CO),
1680 cmÀ1 (CO); H NMR (400 MHz, [D6]DMSO): d ˆ 2.40 (s, 3H, CH3),
1
3.03, 3.18 (dd, 1H, J ˆ 14.3, 9.6 Hz; dd, 1H, J ˆ 14.3, 4.6 Hz; b-CH2), 4.43
(m, 1H, a-CH), 4.97 (s, 2H, CH2Ph), 5.10 (s, 2H, CH2Ph), 7.24 7.45 (m,
16H, Ar and NH), 7.57 (d, 2H, Ar, J ˆ 7.9), 7.65 (d, 1H, J ˆ 7.51 Hz, Ar),
8.28 (d, 1H, J ˆ 8.1 Hz, Ar); 13C NMR: d ˆ 21.6 (CH3), 27.9 (b-CH2), 54.1
(a-CH), 67.0 (CH2Ph), 67.4 (CH2Ph), 115.8, 116.4, 118.8, 123.8, 125.2, 125.9,
127.9, 128.1, 128.2, 128.5, 128.6, 129.1, 129.2, 129.4, 130.2, 130.7, 131.5, 134.7,
General procedure for synthesis of the heterocyclic sulfonamides: All
sulfonamides were synthesized by either the procedure described above for
the carboxamides (method A), or by the following method B: sodium
hydride (35 mmol) was added at room temperature to a solution of indole-
3-carbaldehyde (5 g, 35 mmol) in anhydrous THF (200 mL) under an argon
atmosphere. After 15 min at room temperature, the sulfonyl chloride
(35 mmol) in THF (20 mL) was added. The mixture was heated under
reflux for 1 h, cooled, poured into water (100 mL) and extracted with
dichloromethane (3 Â 100 mL). The organic phases were dried over
anhydrous sodium sulfate and evaporated to yield the crude sulfonamide,
which was purified by chromatography.
ˆ
ˆ
ˆ
136.2, 142.6, 155.6 (C O), 168.3 (C O), 171.3 (C O); ESMS m/z (%): 547
(90) [M‡H] , 564 (48) [M‡NH4] ; elemental analysis (%) calcd for
‡
‡
C34H30N2O5: C 74.71, H 5.53, N 5.12; found: C 73.52, H 5.61, N 4.97.
(2S)-2-Amino-3-[1-(4-methylbenzoyl)indol-3-yl]propanoic acid (2a): 80%
from benzyl (2S)-2-amino-3-[1-(4-methylbenzoyl)indol-3-yl]propanoate by
atmospheric-pressure hydrogenation over 5% Pd/C overnight in methanol;
m.p. 225 2278C (ethanol); [a]D25 ˆ À12.50 (c ˆ 0.032; DMSO); IR (KBr):
nÄ ˆ 3700 2000 (OH), 1700, 1650 cmÀ1 (CO); 1H NMR (400 MHz,
[D6]DMSO): d ˆ 2.35 (s, 3H, CH3), 2.95 and 3.45 (dd, 1H, J ˆ 14.8,
8.6 Hz; dd, 1H, J ˆ 14.8, 4.6 Hz; b-CH2), 4.15 (m, 1H, a-CH), 7.2 7.5 (m,
5H, Ar), 7.71 (d, 2H, J ˆ 7.5 Hz, Ar), 8.28 (d, 1 h, J ˆ 7.6, Ar); 13C NMR:
d ˆ 21.1 (CH3), 26.5 (b-CH2), 53.6 (a-CH), 115.8, 116.7, 119.5, 123.5, 124.6,
126.9, 129.2, 129.4, 130.6, 131.3, 135.9, 142.2, 167.9, 173.5; ESMS m/z (%):
1-[(4-Methylphenyl)sulfonyl]indole-3-carbaldehyde (5a): Method B; 85%
from indole-3-carbaldehyde and toluene-4-sulfonyl-chloride; m.p. 1498C
(ethyl acetate) (lit.: 148 150[51]); IR (KBr): nÄ ˆ 1160, 1360 (SO2),
1660 cmÀ1 (CO); 1H NMR (400 MHz, CDCl3): d ˆ 2.4 (s, 3H, CH3), 7.26
(d, 2H, Ar), 7.36 (m, 1H, Ind-6), 7.39 (m, 1H, Ind-5), 7.83 (d, 2H, Ar), 7.95
(d, 1H, Ind-4), 8.23 (s, 1H,Ind-2), 8.25 (d, 1H, Ind-7), 10.2 (s, 1H, CHO);
13C NMR: d ˆ 21.7, 113.3, 122.4, 122.6, 125.1, 126.3, 127.2, 130.3, 134.4, 135.3,
‡
136.2, 146.2, 185.3; EIMS (70 eV): m/z (%): 299 (38) [M] , 271 (3), 155 (52)
[ArSO2] , 139 (3), 116 (14), 91 (100); elemental analysis (%) calcd for
C16H13NSO3: C 64.2, H 4.38, N 4.68, S 10.7; found: C 64.1, H 4.35, N 4.58, S
10.6.
‡
‡
‡
‡
323 (65) [M‡H] , 361 (63) [M‡K] , 645 (25) [2M‡H] ; elemental analysis
(%) calcd for C19H18N2O3: C 70.79, H 5.63, N 8.69; found: C 70.46, H 5.87, N
8.64.
1-(4-Methylbenzoyl)indole-3-carbaldehyde (3a): 92% from indole 3-car-
baldehyde and 4-methylbenzoyl chloride in dry THF; m.p. 142 1438C
1-[(4-Methoxyphenyl)sulfonyl]indole-3-carbaldehyde (5c): method A;
85%; m.p. 1378C(lit.: 138 140[52]); IR (KBr): nÄ ˆ 1150, 1380 (SO2),
Chem. Eur. J. 2003, 9, 3132 3142
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¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3139

F. Benedetti, F. Berti et al.
FULL PAPER
1680 cmÀ1 (CO); 1H NMR (400 MHz, CDCl3): d ˆ 3.8 (s, 3H, OCH3), 7.0 (d,
2H, Ar), 7.4 (m, 2H, Ind-5,6), 7.90 (d, 2H, Ar), 7.95 (d, 1H, Ind-5), 8.23 (s,
1H, Ind-2), 8.26 (d, 2H Ind-7), 10.2 (s, 1H, CHO); 13C NMR: d ˆ 55.8,
113.2, 114.9, 122.2, 122.5, 125.0, 126.2, 128.5, 129.6, 135.1, 136.2, 164.5,
2H, CH2), 2.7 (t, 2H, CH2), 5.3 (s, 2H, CH2), 6.8 8.3 (m, 9H), 10.8 (s, 1H,
COOH); 13C NMR: d ˆ 21.6, 28.9 (2C), 58.2, 113.7, 117.1, 119.7, 123.5, 125.1,
125.7, 126.9, 129.4, 130.0, 135.2 (2C), 145.1, 172.1, 177.6; elemental analysis
(%) calcd for C20H19NSO6: C 59.8, H 4.77, N 3.49, S 7.99; found: C 59.3, H
4.74, N 3.44, S 7.98.
‡
‡
185.3; EIMS (70 eV): m/z (%): 315 (45) [M] , 171 (100) [ArSO2] , 107 (55)
‡
[C6H4OCH3] ; elemental analysis (%) calcd for C16H13NO4S: C 60.94, H
KLH conjugate 7: Sulfonamide 6 (1 mg) in 500 mL 0.1m MES buffer at
pH 4.5, containing NaCl (0.9m) and DMF (20 vol%), was added to a
solution of KLH (2 mg) in 500 mL of milliQ water. EDC (0.5 mg) was
added, and the mixture stirred for 2 h at room temperature. The white
precipitate was removed by centrifugation, and the solution dialyzed
against PBS at 48C. The conjugate was purified by gel filtration over G-25,
and an apparent value of 210 molecules of bound hapten per molecule of
KLH was obtained by spectrophotometric analysis.
4.16, N 4.44; found: C 62.12, H 4.34, N 4.19.
1-[(4-Chlorophenyl)sulfonyl]indole-3-carbaldehyde (5d): Method A;
78%; m.p. 152 1548C (ethanol); IR (KBr): nÄ ˆ 1130, 1370 (SO2),
1670 cmÀ1 (CO); 1H NMR (400 MHz, CDCl3): d ˆ 7.4 (m, 2H, Ind-5,6),
7.5 (d, 2H, Ar), 7.9 (d, 2H, Ar), 8.0 (d, 1H, Ind-4), 8.2 (s, 1H, Ind-2), 8.3 (d,
1H, Ind-7), 10.1 (s, 1H, CHO); 13C NMR: d ˆ 113.1, 122.7, 125.3, 126.3,
126.5, 128.5, 130.0, 135.1, 135.7, 135.8, 141.6, 185.2; EIMS (70 eV): m/z (%):
‡
‡
321/319 (10/28) [M] , 177/175 (27/100) [p-ClC6H4SO2] , 116 (35), 113/111
BSA conjugate 10: The conjugate was obtained, as described for the
immunogenic conjugate 7, from BSA (2 mg), hemisuccinate 9 (2 mg) and
EDC (2 mg). After dialysis and gel filtration, spectrophotometric analysis
of the conjugate gave an apparent value of 34 molecules of bound hapten
per molecule of BSA.
‡
(16/65) [C6H4Cl] ; elemental analysis (%) calcd for C15H10NSO3Cl: C
56.34, H 3.15, N 4.38; found: C 57.5 H 3.42 N 4.12.
1-[(4-Nitrophenyl)sulfonyl]indole-3-carbaldehyde (5e): 70% from indole-
3-carbaldehyde and 4-nitrobenzenesulfonyl chloride; method A (70%) or
method B (61%). Yellow crystals from butanol; m.p. 1158C; IR (KBr): nÄ ˆ
1100, 1380 (SO2), 1380, 1520, (NO2), 1680 cmÀ1 (CO); 1H NMR (400 MHz,
CDCl3): d ˆ 7.40 (m, 1H, Ind-6), 7.45 (m, 1H, Ind-5), 7.94 (d, 1 h, Ind-4),
8.15 (d, 2H, Ar), 8.20 (s, 1H, Ind-2), 8.26 (d, 1H, Ind-7), 8.35 (d, 2H, Ar),
10.1 (s, 1H, CHO); 13C NMR: d ˆ 34.8, 62.7, 113.0, 123.0, 124.4, 124.9, 125.4,
127.7, 126.9, 128.1, 128.4, 135.5, 185.0; EIMS (70 eV): m/z (%): 330 (90)
Immunization and production of monoclonal antibodies: BALB/c mice
were immunized with conjugate 7. 0.1 mg per mouse of protein was
emulsified with complete Freund adjuvant and injected intraperitoneally.
Four repeated injections every 10 14 d with the same amount of protein
emulsified with incomplete Freund adjuvant were administered. Three
days after the last booster injection, the spleens were removed and the
splenocytes fused with the cell line P3X63Ag8/NS-1.[54] Culture fluids of the
resulting hybridomas were screened for anti-10 activity in ELISA.
Seventeen hybridomas that recognized 10 and reacted with the antigens
in ELISA assay were selected and subcloned twice before using.
‡
‡
[M] , 329 (30), 145 (100) [M À p-NO2C6H4SO2] , 144 (50), 116 (75);
elemental analysis (%) calcd for C15H10N2SO5: C 54.6, H 3.05, N 8.48;
found: C 54.8, H 3.2, N 8.36.
[((1E)-{1-[(4-Methylphenyl)sulfonyl]indol-3-yl}methylene)amino]oxyace-
tic acid 6: 2n sodium hydroxide (1.1 mL) was added to sulfonamide 5a
(100 mg, 0.33 mmol) and O-carboxymethyl hydroxylamine hydrochloride
(100 mg, 0.92 mmol) in ethanol (15 mL). The solution was heated at reflux
for 1 h, and then concentrated to a final volume of 3 mL. Water (4 mL) was
added, and the pH adjusted to 10.5. The solution was extracted with ethyl
acetate (2 Â 5 mL), acidified with hydrochloric acid to pH 2 and, upon
cooling to 48C, the crude product precipitated. The yield was 87% after
crystallization from water/acetone. M.p. 195.5 (decomp); IR (KBr): nÄ ˆ
Screening and competition ELISA: The microtitration plates were coated
with conjugate 10 diluted in 50 mm carbonate buffer, pH 9.6, to a final
concentration of 1 mg per mL. Each microwell was filled with 200 mL of the
solution, and the plates were incubated overnight at 48C. The wells were
washed with 0.05% Tween 20 in PBS buffer, and saturated with 1% gelatin
from cold-water fish skin in PBS at 48C for 90 min. The same solution was
used to prepare all the working solutions of 312d6 (1:80 from ascitic fluids,
1:5 from supernatants) and of the competing haptens. Titrations or
competition experiments were performed in a final volume of 200 mL per
well at 48C for 4 h. After washing, the plates were always incubated with an
anti-mouse IgG HRP conjugate (working dilution 1:10000) for 2.5 h at
room temperature. The plates were then washed again, and 100 mL of a
0.1 mg per mL solution of TMB in 100 mm citrate buffer, pH 4, containing
0.01% hydrogen peroxide, was added to each well. The chromogenic
reaction was stopped after 30 60 min at room temperature by adding
50 mL of 2m sulfuric acid. The optical density at 450 nm was then read.
1160, 1360 (SO2), 1700 (C N), 1730 (CO), 2500 3150 cmÀ1 (OH); 1H NMR
ˆ
(400 MHz, [D6]DMSO): d ˆ 2.35 (s, 3H, CH3), 4.8 (s, 2H, CH2), 7.3 7.7 (m,
4H, Ar‡Ind-5,6), 7.8 8.3 (m, 5H, Ar‡Ind-4,7), 8.4 (s, 1H, Ind-2), 8.65 (s,
1H); 13C NMR (CD3COCD3): d ˆ 21.5, 71.2, 114.2, 116.5, 124.1, 125.0,
126.5, 127.9, 128.1, 130.4, 131.1, 135.5, 136.5, 145.3, 146.8, 171.0; EIMS
‡
‡
(70 eV): m/z (%): 296 (16) [M À C2H2O3] , 155 (45) [ArSO2] , 142 (6), 114
(5), 91 (100); elemental analysis (%) calcd for C18H16N2SO5: C 58.1, H 4.33,
N 7.52, S 8.61; found: C 58.0, H 4.36, N 7.51, S 8.59.
1-[(4-Methylphenyl)sulfonyl]indol-3-ylmethanol (8): Sodium borohydride
(0.54 g, 14 mmol) in water (25 mL) was added dropwise to a solution of
sulfonamide 5a (4 g, 13 mmol) in methanol (50 mL). After 30 min the
solution was neutralized with 10% ammonium chloride and extracted with
chloroform. The organic phase was dried over anhydrous sodium sulfate,
and the solvent evaporated under reduced pressure. The crude oil solidified
on wetting with isopropyl ether. 99% Yield after crystallizazion from
water/acetone. M.p. 1108C (lit.: 1078C[53]); IR (KBr): nÄ ˆ 1175, 1380 (SO2),
3400 cmÀ1 (OH); 1H NMR (400 MHz): d ˆ 1.8 (s, 1H, OH), 2.3 (s, 3H,
CH3), 4.8 (s, 2H, CH2), 7.8 9.2 (m, 9H); 13C NMR: d ˆ 21.5, 57.1, 113.7,
119.9, 122.3, 123.3, 123.8, 125, 126.8, 129.4, 129.9, 135.2, 135.4, 145.0;
elemental analysis (%) calcd for C16H15NSO3: C 63.8, H 5.02, N 4.65, S 10.6;
found: C 63.5, H 4.94, N 4.61, S 10.7.
Purification of monoclonal antibodies: Purified antibody 312d6, as well as
all other antibodies tested in this work, was obtained from the supernatant
of the hybridoma cell culture (RPMI Sigma with 15% FCS) and from
ascitic fluids. Isolation from large volumes of supernatants started with
ammonium sulfate precipitation of the antibody (up to 45% saturation of
ammonium sulfate), and was followed by dialysis against PBS buffer and
purification on Gamma-bind‡ Sepharose (Recombinant Protein G, Phar-
macia). Ascitic fluids were loaded directly on the Gamma-bind‡ column
after filtration and removal of the fatty fraction. The column was washed
with PBS buffer until all the unbound proteins were removed, and then the
antibody fraction was eluted with 0.5m ammonium acetate, pH 3. The pH
was immediately raised to neutral with 1m TRIS pH 9, and then the
antibody fractions were dialyzed against 10 mm TRIS, pH 8.5. The Gamma-
bind purified antibody fraction was then submitted to further purification
on a DEAE-Sepharose Fast Flow ion-exchange column (Pharmacia). The
antibody, dissolved in 10 mm TRIS buffer at pH 8.5, was loaded on the
column, which was washed with the same buffer. The antibody was then
eluted in a gradient of sodium chloride. The antibody thus purified by
affinity and ion exchange was finally dialyzed against the kinetic buffer.
The purity of the antibody was assessed by SDS-PAGE, and was always
>95%. The concentration of antibody was determined either spectropho-
tometrically or by a bicynchoninic acid (BCA) test using a reference mouse
IgG solution for calibration.
4-{1-[(4-Methylphenyl)sulfonyl]-1H-indol-3-yl}methoxy-4-oxobutanoic
acid (9): Sulfonamide 8 (2.5 g, 8.8 mmol) and succinic anhydride (2.62 g,
26 mmol) were heated at reflux in dry pyridine for 4 h. The solvent was
evaporated, and the oily residue dissolved in the minimum amount of ethyl
acetate. The solution was extracted with 0.5m sulfuric acid, and the aqueous
phase discarded. The organic phase was extracted with 0.5% aqueous
sodium hydrogencarbonate saturated with sodium carbonate. The basic
solution was extracted with ethyl acetate, whereby the organic phase was
discarded, then acidified to pH 4 and extracted with ethyl acetate. The
organic phase was dried and evaporated to give a brown oil, which was
purified by chromatography (chloroform/methanol 95:5). 74% Yield after
crystallization from toluene. M.p. 1108C; IR (KBr): nÄ ˆ 1170, 1370 (CO),
Preparation and purification of the Fab fragment of 312d6: 5 mg of
antibody 312d6, purified from the ascitic fluids and dialyzed against 100 mm
phosphate buffer, pH 7.3, containing EDTA 1.25 mm, was incubated with
1
2800 3400 cmÀ1 (OH); H NMR (400 MHz): d ˆ 2.3 (s, 3H, CH3), 2.6 (t,
3140
¹ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2003, 9, 3132 3142

Catalytic Antibodies
3132 3142
50 mg of papain for 6 h at 378C. Papain was inactivated by addition of
iodoacetamide, and the Fab fragment was purified by ion exchange
chromatography over DEAE/Trisacryl‡ M at pH 7.5 (phosphate), and
then by gel filtration over sephacryl S-100 HR.
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Kinetics of uncatalyzed reactions: The hydrolyses were monitored
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(amide 2a). Preliminary scans of the whole UV spectrum showed that all
the reactions were accompanied by a significant spectral change, with at
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320 nm for 1a, 3a, 3c, 3d, 310 nm for 2a and 3b and 340 nm for 3e. All the
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metrically at the same wavelengths used for the uncatalyzed reactions, or
by HPLC for 1a. The observed initial rates for the first 5% of reaction were
obtained from the slopes of plots of the concentration of substrate versus
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background reaction. Hydrolyses of the substrates were always monitored
for the whole reaction to verify that the overall spectral change
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Acknowledgements
This work was supported by the CNR (Progetto Finalizzato Biotecnologie)
and by the European Union (contract ERB FMRX-CT98-0193). S.N.
thanks the Centre of Excellence for Biocrystallography of the University of
Trieste for a postdoctoral grant. R.A. thanks CIB for a grant. We are
grateful to Dr. Fabio Hollan for mass spectra, and to Dr. BenoÓt Gigant,
Laboratoire d×Enzymologie et Biochimie Structurales, CNRS, Gif sur
Yvette, for setting up the conditions of the papain digestion. We are
grateful to Thermo Labsystems and to Dr. Roberta Collino for IASYS
affinity measurements.
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Received: November 29, 2002 [F4620]
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