Z.-D. Shi et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2321–2324
2323
Globin (FgG) in PBS (pH 7.2) using 1-(3-dimethylami-
nopropyl)-3-ethyl carboimide hydrochloride (EDCI) as
condensation reagent. The immunized rabbits were bled
after 1-month post-immunization and the sera were
precipitated using saturated ammonium sulfate and
separated by ion exchange (DE-52) to remove undesired
serum proteins.8
same conditions using non-specific rabbit immuno-
globulin G, which showed no influence on the reaction
rate.
In summary, herein we report the first antibody-cata-
lyzed aza Diels–Alder reaction through an extension of
10
ourpervious study.
Further kinetic analysis and
exploration of enantioselectivity under catalysis of
monoclonal antibodies are under way.
By this procedure four polyclonal antibodies were
acquired. Kinetic experiments demonstrated that one
polyclonal antibody, Aza-BSA-3, could catalyze the
desired aza Diels–Alder reaction. The rate of the reac-
tion was measured by monitoring the disappearance of
the diene at 242 nm using reversed-phase high perfor-
mance liquid chromatography (HPLC). The catalyzed
reaction was performed under conditions of: diene 1
(370 mM); dienophile 2 (4000 mM); polyclonal antibody
(7.4 mM), 37 ꢀC at pH 7.0 in a PBS (10 mM) buffer.
Initial rates of catalyzed reaction in the presence of
antibodies were measured within 5% completion of the
diene and corrected for background reaction in the
absence of antibody. The data so obtained were
employed to construct a Lineweaver–Burk plot, from
which the kinetic parameters were derived (Fig. 1).
Acknowledgements
We are grateful to the Chinese Academy of Sciences
(KJ951-A1-504), Chinese Natural Science Foundation
(29672048) and State Committee of Science and Tech-
nology for generous financial support.
References and Notes
1. Trost, B. M.; Fleming, I.; Paquette, L. A. Comprehensive
Organic Synthesis, Vol. 5; Pergamon: Oxford, 1991.
2. (a) Oikawa, H.; Yagi, K.; Wantabe, K.; Honma, M.; Ichi-
hara, A. Chem. Commun. 1997, 97. (b) Oikawa, H.;
Katayama, K.; Suzuki, Y.; Ichihara, A. J. Chem. Soc. Chem.
Commun. 1995, 1321.
3. (a) Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M. M.
J. Am. Chem. Soc. 1989, 111, 9261. (b) Braisted, A. C.;
Schlutz, P. G. J. Am. Chem. Soc. 1990, 112, 7430. (c) Gou-
verneur, V. E.; Houk, K. N.; Pascual-Teresa, B.; Beno, B.;
Janda, K. D.; Lerner, R. A. Science 1993, 262, 204.
4. (a) Meekel, A. P.; Resmini, M.; Pandit, U. K. J. Chem. Soc.
Chem. Commun. 1995, 571. (b) Meekel, A. P.; Resmini, M.;
Pandit, U. K. Pure Appl. Chem. 1996, 68, 2025. (c) Hu, Y. J.;
Ji, Y. Y.; Wu, Y. L.; Yang, B. H.; Yeh, M. Bioorg. Med.
Chem. Lett. 1997, 7, 1601.
Because dienophile 2 was in excess, the results were
observed to follow Michaelis–Menten kinetics of
pseudo-first-order reactions, where KM/Vmax and 1/
Vmax are respectively determined as the slope of the line
and the intercept of the vertical axis in Figure 1. The
values of kinetic parameters for the diene 1 were:
KM=833 mM, Vmax=1.82 mM/min, kcat=0.34 minÀ1
.
According to our experiments, the polyclonal antibody
accepted as substrates the diene 1 and the dienophile 2
and produced the expected adduct 4b. The ratio of the
exo adduct 4b to endo adduct 4a was 13:1 underthe
catalysis of Aza-BSA-3, while the ratio of exo adduct to
endo adduct was 1:4 underthe catalysis of mixed protic
acid (1 equiv CF3CO2H and 1 equiv CH3SO3H).9
However, neither exo nor endo adduct could be detected
if the reaction was run without any catalysts. Addition
of an equimolaramount of inhibitor 10 to antibody-
catalyzed reactions resulted in complete inhibition, with
the reaction rate dropping to the background value.
This indicated that catalysis took place utilizing anti-
body binding sites. Controls were performed under the
5. Sellen, M.; Bavckall, J. E.; Helquist, P. J. Org. Chem. 1991,
´
56, 835.
6. Kelly, T. R.; Schmidt, T. E.; Haggerty, J. G. Synthesis
1972, 544.
7. All new compounds gave satisfactory spectral and micro-
analytical data. Selected data forcompound 9: [a]2D0=À95.2
(c 1.2, MeOH); IR (film, cmÀ1): 3031, 2976, 2875, 1747, 1497,
1
1451, 1375; H NMR (300 MHz, CDCl3) d 7.42–7.19 (m, 5H,
–Ph), 5.97 (m, 1H), 5.87 (d, J=8.0 Hz, 1H), 4.24 (q, J=7.2
Hz, 2H), 4.15 (q, J=7.1 Hz, 1H), 3.47 (t, J=7.4 Hz, 2H), 3.46
(m, 1H), 3.31 (s, 3H), 2.73 (m, 1H), 2.34 (m, 1H), 2.05 (m, 1H),
1.90 (m, 1H), 1.61 (m, 1H), 1.32 (d, J=7.2 Hz, 3H), 1.21 (t,
J=7.3 Hz, 3H), 1.03 (m, 1H), 0.92 (m, 1H); MS (EI) m/z 343
(M)+, 105 (100); HRMS calcd forC 21H29O3N (M)+:
343.2161; found: 343.2148. Compound 11: 1H NMR
(600 MHz, CDCl3) d 7.99 (t, J=6.0 Hz, 1H), 7.26–7.18 (m, 5H,
–Ph), 5.83 (m, 1H), 5.65 (d, J=7.8 Hz, 1H), 4.32 (q, J=7.2
Hz, 1H), 3.71 (s, 3H), 3.60–3.53 (m, 2H), 3.44–3.41 (m, 2H),
3.39 (s, 3H), 3.35 (m, 1H), 2.86 (m, 1H), 2.42 (t, J=7.2 Hz,
2H), 2.32 (m, 1H), 2.21 (m, 1H), 1.92 (t, J=7.2 Hz, 2H), 1.70
(m, 1H), 1.51 (m, 1H), 1.30 (d, J=6.6 Hz, 3H), 1.21 (dt, J=3.0
Hz, J=7.3 Hz, 1H), 0.99 (m, 1H); 13C NMR (120 MHz,
CDCl3) 175.0, 173.5, 142.2, 136.7, 130.6, 129.0, 127.8, 127.0,
69.1, 61.4, 58.8, 56.7, 56.5, 51.7, 38.4, 36.0, 33.4, 32.2, 31.6,
25.1, 20.8, 20.3; MS (EI) m/z 414 (M)+, 105 (100). Anal. calcd
forC 24H34O4N2: C, 69.57, H, 8.21, N, 6.76; found: C, 69.40,
H, 8.10, N, 7.02. Compound 5: [a]2D0=À120.1 (c 1.0, MeOH);
1H NMR (600 MHz, CDCl3) d 8.09 (t, J=6.0 Hz, 1H), 7.26–
7.17 (m, 5H, –Ph), 5.78 (m, 1H), 5.66 (d, J=8.4 Hz, 1H), 4.25
(q, J=7.2 Hz, 1H), 3.55 (m, 1H), 3.50 (m, 1H), 3.31 (s, 3H),
Figure 1. Lineweaver–Burk plot for the reaction of diene 1 with dieno-
phile 2 catalyzed by Aza-BSA-3.