Rate of enzymatic hydrolysis

Another interesting target for this type of inhibitors is the dipeptidyl peptidase IV (DPP IV). This exodipeptidase, which can cleave peptides behind a proline residue is important in type 2 diabetes as it truncates the glucagon-like peptide 1. Taking into account the P2-Pi( Pro)-P,1 cleavage and the requirement for a free terminal amine, the synthesis of a suicide inhibitor was planned. It looked as if the the e-amino group of a P2 lysine residue could be cyclized because of the relative little importance of the nature of the P2 residue on the rate of enzymatic hydrolysis of known synthetic substrates. Therefore, anew series of cyclopeptides 11 was synthesized (Fig. 11.8). 

Rates of Enzymatic Hydrolysis of Hexosides and Pentosides with the Same Ring... 

In neutral or alkaline buffer solution at 37°, the hydrolytic breakdown of A-formylbenzamide (4.166, R=H) produced only benzamide (4.167). In contrast, the higher homologues were hydrolyzed at the two amide bonds, with benzamide (4.167) and benzoic acid (4.168) formed in a 3 2 ratio. Plasma-catalyzed hydrolysis occurred predominantly at the distal amide bond to produce benzamide. Under these conditions, hydrolysis was very rapid for N-formylbenzamide (80% hydrolysis in 15 min, i. e., ca. 500-fold faster than under abiotic conditions). The rate of enzymatic hydrolysis was also markedly influenced by the length of the A-acyl group, and decreased in the order H>Me>Bu>Pr>Et. 

R = Me, Et, and PhCH2, respectively Fig. 8.1). In 80% human plasma at pH 7.4 and 37°, these model prodrugs were hydrolyzed with tm values of 3.5, 16, and 2.6 min, respectively [59]. Such rates of enzymatic hydrolysis are comparable to those of various carbamoylmethyl esters of benzoic acid (Table 8.2). It is important to note that the direct liberation of benzoic acid by Reaction a (Fig. 8.1) was severalfold faster than the competitive Reaction b. Reaction c was very slow in human plasma (tm > 100 h). In HO -catalyzed hydrolysis, the opposite regioselectivity was seen, with the terminal ester bridge being cleaved markedly faster than the central one. No data appears to be available on chemical hydrolysis at neutral pH. 

In a series of ten morphine 3-benzoates, large differences in rates of enzymatic hydrolysis were seen [122], In 80% human plasma at pH 7.4 and 37°, the unsubstituted 3-benzoate had a tm value of 0.6 h, whereas esters of 2,6-disubstituted benzoic acid were much more resistant to enzymatic attack (t1/2 ranging from 60 h for the dimethylbenzoate to 300 h for the dichloro-and dimethoxybenzoates). Although these results point to marked steric hindrance, electronic effects cannot be excluded but escape characterization because of the limited series. Furthermore, and as mentioned repeatedly in this text, the possibility of binding to plasma proteins is a complicating factor that should be kept in mind. 

Higher homologues of glycine have also been evaluated as pro-moieties of active alcohols or phenols. Thus, the highly lipophilic a-tocopherol was de-rivatized with a series of m-aminoalkanecarboxylic acids in the search for a water-soluble, injectable provitamin E [146], Good to high water solubility was indeed achieved. The esters were substrates of liver esterases and showed marked structure-dependent differences in their rate of enzymatic hydrolysis. 

Benzoic acids substituted with a basic side chain also are also of interest as pro-moieties whose physicochemical properties and rates of enzymatic hydrolysis can readily be modulated. A number of drugs have been converted to prodrugs with this type of pro-moiety, e.g., hydrocortisone, prednisolone, acyclovir, chloramphenicol, and paracetamol [148] [149], These prodrugs appear well suited as parenteral formulations, being water-soluble, stable in slightly acidic solution, and readily hydrolyzed enzymatically. As examples, we consider here the hydrolysis in human plasma of a number of (aminomethyl)ben-zoates of metronidazole (8.109-8.115, Sect. 8.5.5.1, Table 8.9) [138], These prodrugs are very rapidly activated, which may be beneficial for parenteral administration. However, this type of pro-moiety may be cleaved too rapidly after oral administration to be of interest for poorly absorbed drugs. 

A device for retention of a dopamine prodrug into the central nervous system has been designed by the synthesis on N-methyl-dihydronicotinoyl amides of dopamine 3,4-0-diesters, such as 23, which are converted into a quaternary pyridinium derivative by oxidation, and thereby retained inside the blood-brain barrier [19]. Only modest dopamine-like activity was observed, and this may due to a slow rate of enzymatic hydrolysis of the amide bond joining the dopamine and the pyridinium moieties. 

The (R)-amine 7 is a liquid, whereas the (S)-amide 6 is a solid. This difference in physical properties in effect enabled easy separation of the amine antipodes by filtration. The desired (S)-amide 6 needs to be deacylated prior to the synthesis of the final compound, xemilofiban 1. The slow rate of deacylation of the (S)-amide by the enzyme PGA can be enhanced significantly by using a high concentration of the enzyme. The temperature can also be raised to increase the rate of enzymatic hydrolysis. The (S)-amide 6 obtained was deacylated to liberate the free (S)-amine 9 using excess PGA (Fig. 5). 

Acetyl DL-methionine which is used as a substrate for amino acylase activity determination was prepared by acetylation of DL-methionine with acetic anhydride in acetic acid [5]. The rate of enzymatic hydrolysis was determined by measuring the liberated amino acid by ninhydrin method [6] where ascorbic acid was used as oxidizing agent instead of sodium cyanide. The activity curve of pure amino acylase enzyme is shown in Fig. 1 as a continuous line. For determining the effect of metal ions on the activity of amino acylase the following procedure was adopted. 

Figure 18 Relation between the rate of enzymatic hydrolysis by PHA depolymerase from Ralstonia pickettii and the fraction of second monomer unit for random copolymers of 3HB with different HA units, (a) P(3HB-co-3HHx) (o) P(3HB-co-3HP) and (a) P(3HB-co-4HB). Reprinted from Abe, H. Doi, Y. int. J. Bioi. Macromoi. 1999, 25,185. Copyright (1999), with permission from Elsevier.

Fan LT, Lee YH, Beardmore DR. (1981). The influence of major structural features of cellulose on rate of enzymatic hydrolysis. Biotechnol Bioeng, 23(2), 419 24. 

Grethlein HE. (1985). The effect of pore size distribution on the rate of enzymatic hydrolysis of ceUulosic suhstrutes. Nat Biotechnol, 3(2), 155-160. 

The rates of enzymatic hydrolysis (proteinase K) for branched PLLA (prepared from pentaerythritol with four branches and from polyglycerin with 22 branches) were found to be dependent on the average molecular weight of the PLLA segment in the branched molecules, not on the overall molecular weight of the samples [140]. 

The rate of enzymatic hydrolysis depends on the structural features of cellulose, as well as on the composition of the cellulolytic complex. Sttuctural features such as crystalhnity and accessible surface area determine the... 

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