Amino acids Kinetic acidity

Sensitivity levels more typical of kinetic studies are of the order of lO molecules cm . A schematic diagram of an apparatus for kinetic LIF measurements is shown in figure C3.I.8. A limitation of this approach is that only relative concentrations are easily measured, in contrast to absorjDtion measurements, which yield absolute concentrations. Another important limitation is that not all molecules have measurable fluorescence, as radiationless transitions can be the dominant decay route for electronic excitation in polyatomic molecules. However, the latter situation can also be an advantage in complex molecules, such as proteins, where a lack of background fluorescence allow s the selective introduction of fluorescent chromophores as probes for kinetic studies. (Tryptophan is the only strongly fluorescent amino acid naturally present in proteins, for instance.)... 

Polyethylene (Section 6 21) A polymer of ethylene Polymer (Section 6 21) Large molecule formed by the repeti tive combination of many smaller molecules (monomers) Polymerase chain reaction (Section 28 16) A laboratory method for making multiple copies of DNA Polymerization (Section 6 21) Process by which a polymer is prepared The principal processes include free radical cationic coordination and condensation polymerization Polypeptide (Section 27 1) A polymer made up of many (more than eight to ten) amino acid residues Polypropylene (Section 6 21) A polymer of propene Polysaccharide (Sections 25 1 and 25 15) A carbohydrate that yields many monosacchande units on hydrolysis Potential energy (Section 2 18) The energy a system has ex elusive of Its kinetic energy... 

Enzymatic hydrolysis of A/-acylamino acids by amino acylase and amino acid esters by Hpase or carboxy esterase (70) is one kind of kinetic resolution. Kinetic resolution is found in chemical synthesis such as by epoxidation of racemic allyl alcohol and asymmetric hydrogenation (71). New routes for amino acid manufacturing are anticipated. 

Swarc,M. The Kinetics and Mechanism of N-carboxy-a-amino-acid Anhydride (NCA) Polymerization to Poly-amino Acids. Vol. 4, pp. 1—65. 

Upon carefully controlled hydrolysis with hydrochloric acid at room temperature, the corresponding serine methyl esters 4 are obtained in reasonable yields. Higher yields of 4 arc obtained by hydrolyzing with dilute trifluoroacetic acid5. In some cases, the diastereomeric ratio of 4 does not exactly correspond to the d.r. of the adduct 3, which is attributed to different kinetics in the hydrolysis of the diastereomers 4. Subsequent treatment of the methyl ester with excess 5 N hydrochloric acid and methyloxirane as an acid scavenger results in the free amino acid 54,7. 

Remes et al. (1976) also investigated the kinetics of the N-azo coupling of nine a-amino acids. They are aware of earlier investigations in which the major products were pentaz-1,4-dienes, but they claim that under their reaction conditions (pH 8.00-10.25, thirty-fold excess of amino acid) only the triazenes are formed. The rates were found to be first-order with respect to diazonium ion which is consistent with their conclusion however, in the opinion of the present author the results suggest a significant (say, 10%) contribution of pentazdiene formation to the total rate process. No significant correlation was found between the rate constants and the acidity constants of the nine amino acids. 

Metal template reactions, 1, 416, 433 equilibrium kinetic, 1, 434 thermodynamic, 1, 434 Metal tolerance amino acid complexes, 2, 964 plants, 2, 963 Metal toxicity... 

Finally, as an old example of kinetic resolution of racemic mixtures, mention must be made on the report of Kise and Tomiuchi on the significant effect of acetonitrile on the enantioselectivity of different proteases toward the kinetic resolution of aromatic amino acid ethyl esters (5-8). For instance, (l)-DOPA (8) was obtained with 99% ee in the presence of 90% v/v acetonitrile [9]. 

Table 1.6 I nfluence of the organic solvent on the enantioselectivity ofthe protease from A oryzae subtilisin in the kinetic resolution of the racemic amino acid (12) (expressed as the ratio of the initial rate of acylation of the pure enatiomers, Vs/vr).

In another study a hyperthermophilic esterase from Aeropyrum pemix K1 (APE1547) was used as a catalyst in the hydrolytic kinetic resolution of rac-3-octanol acetate [53]. Following a single round of epPCR, a mutant displaying a 2.6-fold increase in enantioselectivity was identified having five amino acid substitutions, which were shown to be spatially distal to the catalytic center. 

Hydantoinases belong to the E.C.3.5.2 group of cyclic amidases, which catalyze the hydrolysis of hydantoins [4,54]. As synthetic hydantoins are readily accessible by a variety of chemical syntheses, including Strecker reactions, enantioselective hydantoinase-catalyzed hydrolysis offers an attractive and general route to chiral amino acid derivatives. Moreover, hydantoins are easily racemized chemically or enzymatically by appropriate racemases, so that dynamic kinetic resolution with potential 100% conversion and complete enantioselectivity is theoretically possible. Indeed, a number of such cases using WT hydantoinases have been reported [54]. However, if asymmetric induction is poor or ifinversion ofenantioselectivity is desired, directed evolution can come to the rescue. Such a case has been reported, specifically in the production of i-methionine in a whole-cell system ( . coli) (Figure 2.13) [55]. 

In another study that appeared prior to the advent of CASTing, the traditional combination of epPCR and DNA shuffling was used to enhance the enantioselectivity of the hydrolytic kinetic resolution of p-nitro phenyl glycidyl ether and other epoxides catalyzed by the EH from Agrobacterium radiobacter [59]. Several mutants were obtained with up to 13-fold improved enantioselectivity. The amino acid exchanges took place around the active site. 

The phosphotriesterase from Pseudomonas diminuta was shown to catalyze the enantioselective hydrolysis of several racemic phosphates (21), the Sp isomer reacting faster than the Rp compound [65,66]. Further improvements using directed evolution were achieved by first carrying out a restricted alanine-scan [67] (i.e. at predetermined amino acid positions alanine was introduced). Whenever an effect on activity/ enantioselectivity was observed, the position was defined as a hot spot. Subsequently, randomization at several hot spots was performed, which led to the identification of several highly (S)- or (R)-selective mutants [66]. A similar procedure was applied to the generation of mutant phosphotriesterases as catalysts in the kinetic resolution of racemic phosphonates [68]. 

In nature, aminotransferases participate in a number of metabolic pathways [4[. They catalyze the transfer of an amino group originating from an amino acid donor to a 2-ketoacid acceptor by a simple mechanism. First, an amino group from the donor is transferred to the cofactor pyridoxal phosphate with formation of a 2-keto add and an enzyme-bound pyridoxamine phosphate intermediate. Second, this intermediate transfers the amino group to the 2-keto add acceptor. The readion is reversible, shows ping-pong kinetics, and has been used industrially in the production ofamino acids [69]. It can be driven in one direction by the appropriate choice of conditions (e.g. substrate concentration). Some of the aminotransferases accept simple amines instead of amino acids as amine donors, and highly enantioselective cases have been reported [70]. 

The synthesis of a-amino acids by reaction of aldehydes or ketones with ammonia and hydrogen cyanide followed by hydrolysis of the resulting a-aminonitrile is called the Strecker synthesis. Enzymatic hydrolysis has been applied to the kinetic resolution of intermediate a-aminonitriles [90,91]. The hydrolysis of (rac)-phenylglycine nitrile... 

The main application of the enzymatic hydrolysis of the amide bond is the en-antioselective synthesis of amino acids [4,97]. Acylases (EC 3.5.1.n) catalyze the hydrolysis of the N-acyl groups of a broad range of amino acid derivatives. They accept several acyl groups (acetyl, chloroacetyl, formyl, and carbamoyl) but they require a free a-carboxyl group. In general, acylases are selective for i-amino acids, but d-selective acylase have been reported. The kinetic resolution of amino acids by acylase-catalyzed hydrolysis is a well-established process [4]. The in situ racemization of the substrate in the presence of a racemase converts the process into a DKR. Alternatively, the remaining enantiomer of the N-acyl amino acid can be isolated and racemized via the formation of an oxazolone, as shown in Figure 6.34. 

Figure 6.38 Dynamic kinetic resolution of amino acid amides.

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