Amidinophenyl esters

Scheme 5.1.2. Structures of substrates and substrate mimetic moieties for Arg- and Glu-specific proteases. 1. arginine 2. 4-amidinophenyl ester 3. 4-guanidinophenyl ester (OGp) 4. aspartic acid 5. glutamic acid 6. carboxymethyl thioester (SCm).

It is well known that the specificity of an enzyme such as thrombin and plasmin is very close to that of trypsin. In this respect, inverse substrates for trypsin also are expected to be susceptible to the catalysis by these enzymes. In the kinetic analysis of trypsin-like enzymes toward p-amidinophenyl esters, it was found that the inverse concept is also applicable to thrombin, plasmin, urokinase, kallikrein, and trypsins from various origins 74 - 75). These enzymes are not distinctively different from bovine-... 

Further search for inverse substrates other than /7-amidinophenyl esters has been carried out and it has been found that esters derived from p-amin omcthylphcnol and /7-guanidinophenol were also eligible as a substrate of trypsin and trypsin-like enzymes 75 86). We have also found that trimethylaminobutanoic acid p-nitrophenyl ester is an inverse substrate for butyrylcholinesterase 87-88(. Application of the inverse concept to thiol enzymes was also successful p-amidinophenyl esters were found to be substrates for clostripain 74), a thiol enzyme with trypsin-like specificity. Although the design of inverse-type substrates seems not always possible for a variety of hydrolytic enzymes, this new concept could provide potential means for certain enzymes to both fundamental study and application. 

Rather interesting conclusions can be drawn from an examination of the serine protease-catalyzed hydrolysis of a series of esters or anilides of basic substituted benzoic acids and amidinophenyl esters of aromatic carboxylic acids [1-10]. They react with the serine proteases like a substrate by acylation of the serine residue of the active center and by simultaneous release of the alcoholic... 

In order to compare the efficiency of acyl-plasmin to that of the free enzyme, we studied the lysis of microthrombi induced in the lungs of experimental animals [31]. To obtain a rapid onset of fibrinolysis, we prepared the acylated human plasmin by incubation with the amidinophenyl ester of p-chlorobenzoic acid. The p-chlorobenzoyl-plasmin obtained is relatively rapidly deacylated in plasma (ti/2 of deacylation = 10 min). A single administration of a relatively small quantity of acyl-plasmin was able to eliminate microthrombi. The same quantity of free plasmin remained ineffective (Fig. 10). 

A series of acylated recombinant tPAs (r-tPA) was prepared by reaction of r-tPA with 4-amidinophenyl esters of substituted benzoic acids. The different acyl derivatives contained 5-10% of free enzyme. During deacylation, which is markedly influenced by pH and temperature, 80-100% of the amidolytic activity of the enzyme was recovered in active form. 

F. Markwardt, G. Wagner, P. Walsmann, H. Horn, and J. Stiirzebecher. Inhibition of trypsin and thrombin by amidinophenyl-esters of aromatic carboxylic acids. Acta Biol. Med. Germ. 28 K 19 (1972). 

Xaa bonds catalyzed by trypsin. The improvements in the syntheses of acyl amino acid p-guanidino as well as p-amidinophenyl esters [18] promote the application of this new irreversible strategy. Furthermore, the potential of substrate mimetics mediated synthesis could be extended to other suitable proteases [19]. 

For preparation of a wide range of acyl derivatives of trypsinlike enzymes, the 4-amidinophenyl benzoates proved appropriate, and using these fools preparation of acyl enzymes is relatively easy. In the first step the enzyme is acylatcd in the presence of excessive ester concentrations. Thereafter deacylation is stopped by lowering pH and temperature. Ultimately, the acyl enzyme is isolated from the... 

Fig. 3. Time course of the trypsin-catalyzed hydrolysis of p-amidinophenyl acetate (46 R = CH3) at pH 8.0, 25 °C. Concentrations of enzyme and ester are 10 pM and 0.7 mM, respectively...

More recently, these esters increased in significance for the preparation of stable acyl enzymes, which regain their activity by deacylation and, accordingly, have prodrug characteristics. Therefore, we have continued our investigations into amidinophenyl benzoates in order to prepare acylated serine proteases of pharmacological interest (Table 1). 

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