10752 J. Am. Chem. Soc., Vol. 122, No. 44, 2000
Shulman et al.
chloride (0.037 mL, 0.55 mmol). The mixture was warmed slowly to
room temperature over 3.5 h and then worked up with aqueous NH4Cl
and ether followed by column chromatography (silica gel, ethyl
acetate-hexane, from 3:7 to 1:1) to give a mixture of racemic isomers,
9a and 9b (11%), in the form of a yellowish oil. 1H NMR (300 MHz,
partial): 5.91-5.85 (br d, 1H), 3.45 (m, 4H), 1.4 (s, 9H). MS (FAB):
356 (MNa+).
acetylcyclohexanone (833 µM) within a pH range between 4 and 10.
The initial rate of formation of the typical UV absorption at 340 nm
was measured.
Antibody-Catalyzed Reactions. All antibody-catalyzed reactions
were carried out in PBS (50 mM phosphate, 100 mM NaCl, pH 7.4)
containing 10% organic cosolvent. In the aldol reactions, the organic
cosolvent was CH3CN (5%) and acetone (5%), while in the retroaldol
reactions only CH3CN (10%) was used. The progress of the reactions
was monitored by HPLC using an RP Supelcosil LC18 column, with
the initial rates being calculated by regressional analysis. Antibody
active sites’ concentrations ranged between 4 and 12 µM for 38C2,
and between 30 and 120 µM for 24H6. The rate of the uncatalyzed
reactions was subtracted when significant.
Preparation of Aldol Substrates. General Procedure. The ap-
propriate ketone (1 mmol) was added to a freshly prepared, cold (-78
°C) solution of LDA (1.05 mmol) in dry THF (2 mL), and the mixture
was stirred at the same temperature for 30 min. A solution of the
appropriate aldehyde (1 mmol in 2 mL of THF) was added dropwise,
and the progress of the reaction was monitored by TLC. Upon
completion, the reaction was quenched with saturated aqueous NH4Cl.
The mixture was allowed to warm to room temperature and then
extracted with ethyl acetate and dried over MgSO4. The solvent was
removed under reduced pressure, and the residue was purified by flash
chromatography.
Haptens 2a and 2b. The above-described isomeric mixture of 9a
and 9b (1.5 g, 4.5 mmol) was dissolved in dichloromethane (20 mL)
under argon. Excess trifluoroacetic acid was added, and the reaction
was stirred at 40 °C for 24 h. The solvent was removed under reduced
1
pressure to produce the crude unprotected amine (0.685 g). H NMR
(300 MHz, partial): 5.8-6.0 (m, 1H), 3.5-2.9 (m, 5H), 2.05 (s, 0.7H)
2.04 (s, 1H). MS (FAB): 234 (MH+), 256 (MNa+).
The crude amine (0.685 g) was dissolved in pyridine (15 mL),
glutaric anhydride (0.67 g, 6.7 mmol) was added, and the mixture was
stirred at room temperature for 1 h and then worked up with
dichloromethane and water followed by purification using preperative
TLC (silica gel, hexanes-ethyl acetate, 7:3) to give a mixture of racemic
1
isomers, 2a and 2b (50%), in the form of a yellow oil. H NMR (300
MHz): 5.89 (m, 1H), 3.6-1.9 (m, 23H). MS (FAB): 348 (MH+), 370
(MNa+).
Protein Conjugates of Haptens 2a and 2b. The hapten mixture
was conjugated to the carrier proteins, KLH and BSA, by activation
of its carboxylic groups with EDC and sulfo-NHS.33 Two stock
solutions were prepared as follows: (A) EDC (29.5 mg, 0.154 mmol)
in water (0.051 mL), and (B) SulfoNHS (31.7 mg, 0.146 mmol) in
DMF-water (1:1, 0.097 mL). The above-described isomeric mixture
of 2a and 2b (4 mg, 0.0115 mmol) was dissolved in DMF (0.195 mL).
Solution A (0.005 mL) and solution B (0.013 mL) were added, and
the mixture was stirred at room temperature overnight. This mixture
(0.095 mL) was added to a cold (4 °C) protein solution that was
prepared from KLH (5 mg) and PBS (pH 7.4, 0.9 mL), and the mixture
was gently shaken at 4 °C overnight. The same procedure was repeated
with BSA.
Immunization and Production of Antibodies. 129GIX+ mice were
immunized with the KLH conjugate in complete Freund’s adjuvant.
Mice with a high serum titer were used to generate hybridomas by
fusion of their spleen cells with myeloma cells.34 Twenty-four hybri-
doma cells producing anti-2 antibodies were selected on the basis of
ELISA tests for further studies.35 Antibodies from each cell line were
produced in larger amounts from ascites fluid and then purified by
ammonium sulfate precipitation and protein-G affinity chromatography.
Antibody Screening with 1,3-Diketones. Each of the 24 anti-2
antibodies [7 µM in phosphate-buffered saline (PBS, 50 mM phosphate,
100 mM NaCl, pH 7.4) containing 10% acetonitrile] was mixed with
acetylcyclohexanone (513 µM). The mixture was incubated at 25 °C
for 24 h, and the formation of a vinylogous amide was monitored by
observing the growth of the characteristic absorption at 340 nm using
a UV spectrometer. Antibody 24H6 was selected for further studies
because it exhibited the most significant absorption at 340 nm.
Irreversible Inhibition of 24H6 by Reductive Amination. Anti-
body 24H6 (12 µM in PBS, pH 7.4, containing 3% v/v acetone) was
incubated at 25 °C for 48 h. An aqueous solution of Na(CN)BH3 (38
µL, 0.83 M) was then added, and the mixture was kept at room
temperature for 2 h before undergoing extensive dialysis (the 100-µL
antibody solution was dialyzed repeatedly 10 times against 250 mL
each of the same buffer solution). The modified antibody was assayed
as described above, and the typical absorption at 340 nm was not
observed. Similar results were obtained with cyclohexanone instead
of acetone. In a control experiment we used acetonitrile instead of
acetone and found no change in the 340-nm absorption.
Physical Data of Representative Aldol Products. 6-Phenyl-4-
hydroxyhex-5-en-2-one (II-1c).36 1H NMR: 7.3 (m, 5H), 6.6 (d, J )
16 Hz, 1H), 6.2 (dd, J ) 16, 6 Hz, 1H), 4.7 (m, 1H), 3.1 (d, J ) 3.3
Hz, 1H), 2.75 (d, J ) 6 Hz, 2H), 2.2 (s, 3H). MS (EI): 190 (M+).
1
6-(4′-Chlorophenyl)-4-hydroxyhex-5-en-2-one (II-1d). H NMR:
7.25 (br s, 4H), 6.5 (d, J ) 15.6 Hz, 1H), 6.1 (dd, J ) 15.6, 6.1 Hz,
1H), 4.7 (m, 1H), 2.7 (m, 2H), 2.2 (s, 3H). MS (EI): 258 (M+).
6-(4′-Methoxyphenyl)-4-hydroxyhex-5-en-2-one (II-1b). 1H NMR:
7.3 (d, J ) 8.6 Hz, 2H), 6.8 (d, J ) 8.6 Hz, 2H), 6.5 (d, J ) 15.9 Hz,
1H), 6.1 (dd, J ) 15.9, 6.5 Hz, 1H), 4.7 (m, 1H), 3.78 (s, 3H), 2.97 (d,
J ) 3.7 Hz, 1H, OH), 2.74 (d, J ) 6 Hz, 2H), 2.2 (s, 3H). MS (EI):
220 (M+).
6-(4′-Trifluoromethylphenyl)-4-hydroxyhex-5-en-2-one (II-1e). 1H
NMR: 7.5 (m, 4H), 6.7 (d, J ) 16.0 Hz, 1H), 6.3 (dd, J ) 16.0, 5.7
Hz, 1H), 4.75 (m, 1H), 3.18 (d, J ) 3.0 Hz, 1H), 2.7 (m, 2H), 2.2 (s,
3H). MS (CI): 241 (MH+-H2O).
6-(4′-N-Dimethylaminophenyl)-4-hydroxyhex-5-en-2-one (II-1a).3b
1H NMR: 7.24 (d, J ) 8.6 Hz, 2H), 6.6 (d, J ) 8.6 Hz, 2H), 6.5 (d,
J ) 15.9 Hz, 1H), 6.0 (dd, J ) 15.9, 6.6 Hz, 1H), 4.7 (m, 1H), 2.9 (s,
6H), 2.7 (d, J ) 6.0 Hz, 2H), 2.2 (s, 3H). MS (EI): 233 (M+).
6-(4′-Nitrophenyl)-4-hydroxyhex-5-en-2-one (II-1f).3a 1H NMR:
8.05 (d, J ) 8.6 Hz, 2H), 7.3 (d, J ) 8.6 Hz, 2H), 6.6 (d, J ) 15.9 Hz,
1H), 6.3 (dd, J ) 15.9, 5.2 Hz, 1H), 4.7 (m, 1H), 3.25 (d, J ) 3.7 Hz,
1H), 2.65 (m, 2H), 2.1 (s, 3H). MS (EI): 235 (M+).
6-(2′-Nitrophenyl)-4-hydroxyhex-5-en-2-one (II-2). 1H NMR: 7.95
(d, J ) 8.3 Hz, 1H), 7.5 (m, 3H), 7.1 (d, J ) 15.2 Hz, 1H), 6.2 (dd, J
) 15.2, 6.9 Hz, 1H), 4.8 (m, 1H), 3.3 (br, 1H), 2.8 (d, J ) 6.9 Hz,
2H), 2.2 (s, 3H). MS (-DCI): 235 (M-). MS (+DCI): 218 (MH+
H2O).
-
4-(4′-Nitrophenyl)-4-hydroxybutan-2-one (II-5a).37 1H NMR: 8.13
(d, J ) 8.5 Hz, 2H), 7.5 (d, J ) 8.5 Hz, 2H), 5.2 (t, J ) 7.2 Hz, 1H),
3.6 (br s, 1H), 2.8 (d, J ) 6.25 Hz, 2H), 2.2 (s, 3H). MS (EI): 209
(M+). MS (CI): 210 (MH+).
4-(2′-Nitrophenyl)-4-hydroxybutan-2-one (II-3).37b 1H NMR: 7.93
(d, J ) 8.3 Hz, 1H), 7.88 (d, J ) 8.0 Hz, 1H), 7.65 (t, J ) 8.0 Hz,
1H), 7.4 (t, J ) 8.3 Hz, 1H), 5.68 (br d, J ) 9.3 Hz, 1H), 3.7 (d, J )
3.0 Hz, 1H), 3.1 (dd, J ) 17.9, 2.2 Hz, 1H), 2.7 (dd, J ) 17.9, 9.3 Hz,
1H), 2.2 (s, 3H). MS (CI): 210 (MH+).
Evaluation of the pKa of the Active-Site Lysine. Antibody 24H6
(10 µM in 50 mM phosphate-citrate buffer) was incubated with
4-(3′-Nitrophenyl)-4-hydroxybutan-2-one (II-4).37b,38 1H NMR:
8.18 (s, 1H), 8.1 (d, J ) 8.0 Hz, 1H), 7.6 (d, J ) 8.0 Hz 1H), 7.43 (t,
(33) Staros, J. V., Wright, R. W., Swingle, D. M. Anal. Biochem. 1986,
156, 220.
(34) Goding, J. W. Monoclonal Antibodies: Principles and Practice, 2nd
ed.; Academic Press: New York, 1986.
(35) Clark, B.; Eengvali, E. In ELISA: Theoretical and Practical Aspects
in Enzyme-Immunuassay; Mggio, E. T., Ed.; CRC Press: Boca Raton, FL,
1980; Chapter 8.
(36) Keck, G. E.; Wager, C. A.; Sell, T.; Wager, T. T. J. Org. Chem.
1999, 64, 2172.
(37) (a) Sugasawa, T.; Toyoda, T.; Sasakura, K. Synth. Commun. 1979,
9, 583. (b) Grayson, D. H.; Tuite, M. R. J. J. Chem. Soc., Perkin Trans. 1
1986, 12, 2137,.
(38) Zhang, Y.; Xu, W. Synth. Commun. 1989, 19, 1291.