Hydrolysis of the Amide Bond

wpe2.jpg (13761 bytes)

The peptide bond is extremely unreactive towards hydrolysis: its half life in solution lies in the order of centuries

 

Hydrolysis of the amide bond is a major target of research in catalytic antibodies. This approach could be of use in the design of catalysts for sequence specific peptide hydrolysis; however, amide hydrolysing antibodies are still rare in spite of many efforts.

In amide hydrolysis breakdown of the tetrahedral intermediate is usually the rate determining step and protonation of the leaving amine is required. Conventional phosphonamide transition state analogue gave in the past only scarce results.

 

Thus the design of new haptens capable of producing antibodies with amidase activity is still a challenge.

We have found that the hydrolysis of  heterocyclic amides of indole is accelerated by monoclonal antibodies raised against sulphonamide haptens. a

Indolo.gif (1030 bytes)

 

meccanismo.gif (1082 bytes)

It has been shown that the base catalyzed hydrolysis of such heterocyclic amides proceeds via rate determinig decomposition of the tetrahedral intermediate, from which the leaving group departs as an anion. b The transition state is reagent-like and thus similar to the transition state itself. c

aptene1.gif (933 bytes)

The carboxymethyl oxime of N-Tosyl 3-formyl indole has been used as transition state analog.

 

A KLH conjugate of the sulphonamide was uesd to immunize Balb/c mice and antibody 312d6 was thus obtained. The antibody catalyzes the hydrolysis of the p-tolyl amides of indole, 3-formyl indole and L-triptophane.

Substrate k0 s-1 kcat s-1 kcat/k0 KM mol l-1
Iindolo.gif (509 bytes) 1.01x10-8 7.5x10-6 750 3.57x10-5
3formil.gif (546 bytes) 5.24x10-5 2.1x10-2 400 1.5x10-4
trp.gif (614 bytes) 8.35x10-7 5.15x10-4 680 1.49x10-3

Click here to see the Lineweaver-Burk plots

 

The antibody is extremely selective for p-tolyl amides, and acts with a dual mechanism. A specific and relatively efficient mechanism occurs only for the original p-methyl substitued substrates. This process is competitively inhibited by the sulphonamide hapten. When methyl is replaced by any other substituent, a second, non specific and less efficient mechanism takes place.

 

hammett.gif (3506 bytes)  

A Hammet plot shows clearly that only the p-methyl substituted substrate is hydrolyzed with a specific mechanism.

This evidence is mirrored by the binding properties of the antibody: only the p-methyl substituted sulphonamide is recongized with high affinity.

 

a. Benedetti F., Berti F., Colombatti A., Ebert C., Linda P., Tonizzo F., ChemComm 1996, 1417.

b. Cipiciani A., Linda P., Savelli G., J. Heterocycl. Chem. 1979, 16, 673. Menger F.M., Donohue J.A., J. Am. Chem. Soc. 1973, 95, 432.

c. Brown R.S., Bennet A.J., Slebocka-Tilk h., Jodhan A., J. Am. Chem. Soc. 1992, 114, 3092.

        

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