Assays enantioselective

The purpose of this study is only intended to illustrate and evaluate the decision tree approach for CSP prediction using as attributes the 166 molecular keys publicly available in ISIS. This assay was carried out a CHIRBASE file of 3000 molecular structures corresponding to a list of samples resolved with an a value superior to 1.8. For each solute, we have picked in CHIRBASE the traded CSP providing the highest enantioselectivity. This procedure leads to a total selection of 18 CSPs commercially available under the following names Chiralpak AD [28], Chiral-AGP [40], Chiralpak AS [28], Resolvosil BSA-7 [41], Chiral-CBH [40], CTA-I (microcrystalline cellulose triacetate) [42], Chirobiotic T [43], Crownpak CR(-i-) [28], Cyclobond I [43], DNB-Leucine covalent [29], DNB-Phenylglycine covalent [29], Chiralcel OB [28], Chiralcel OD [28], Chiralcel OJ [28], Chiralpak OT(-i-) [28], Ultron-ES-OVM [44], Whelk-0 1 [29], (/ ,/ )-(3-Gem 1 [29]. 

Determination of the drug substance is expected to be enantioselective, and this may be achieved by including a chiral assay in the specification or an achiral assay together with appropriate methods of controlling the enantiomeric impurity. For a drug product where racemization does not occur during manufacture or storage, an achiral assay may suffice. If racemization does happen, then a chiral assay should be used or an achiral method combined with a validated procedure to control the presence of the other enantiomer. 

It is well known that acrylates undergo transition metal catalyzed reductive aldol reaction, the silanes R3SiH first reacting in a 1,4 manner and the enolsilanes then participating in the actual aldol addition.57,58 A catalytic diastereoselective version was discovered by arrayed catalyst evaluation in which 192 independent catalytic systems were screened on 96-well microtiter plates.59 Conventional GC was used as the assay. A Rh-DuPhos catalyst turned out to be highly diastereoselective, but enantioselectivity was poor.59... 

The best combination turned out to be L5 /A9 (ee = 90% at room temperature and 99% at —78 °C with benzaldehydes and ee = 92-99% with other aldehydes).87 Further improvements were reported later.89 Although only a few dozen reactions were monitored by a JASCO-CD-995 instrument, the CD-based assay is amenable to high-throughput screening of enantioselective catalysts. The chemistry itself lends itself ideally to combinatorial asymmetric metal catalysis, since the principle of asymmetric activation is turning out to be very powerful.89... 

A new hydroxynitrile lyase (HNL) was isolated from the seed of Japanese apricot Prunus mume). It accepts benzaldehyde and a large number of unnatural substrates for the addition of HCN to produce the corresponding (7 )-cyanohydrins in excellent optical and chemical yields. A new high-performance liquid chromatography (HPLC)-based enantioselective assay technique was developed for the enzyme, which promotes the addition of KCN to benzaldehyde in a buffered solution (pH 4.0). Asymmetric synthesis of (7 )-cyanohydrins by a new HNL is described (Figure 8.4). ... 

In another study, an enantioselective assay for the analysis of the enantiomer composition of monomethyl 4-(2, 3 -dichlorophenyl)-2,6-dimethyl-l,4-dihydropyridine-3,5-dicarboxylic acid on the tBuCQN-CSP was proposed (Rs = 3.4) [49], This chiral carboxylic acid is the synthesis intermediate and the primary metabolite... 

Badaloni, E. et al., Enantioselective liquid chromatographic-electrospray mass spectrometric assay of fS-adrenergic blockers application to a pharmacokinetic smdy of sotalol in human plasma, J. Chromatogr. B, 796, 45, 2003. 

High- and Medium-Throughput Screening Systems for Assaying the Enantioselectivity of Enzymatic Reactions... 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

Another colorimetric assay for testing the enantioselectivity of lipases or esterases in ester hydrolysis reactions is based on a different principle (75). To simulate the state of competitive conditions of an enzymatic process, the so-called Quick-ii-Test... 

Fig. 7. Enzyme-coupled assay in which the hydrolase-catalyzed reaction releases acetic acid. The latter is converted by acetyl-CoA synthetase (ACS) into acetyl-CoA in the presence of (ATP) and coenzyme A (CoA). Citrate synthase (CS) catalyzes the reaction between acetyl-CoA and oxaloacetate to give citrate. The oxaloacetate required for this reaction is formed from L-malate and NAD in the presence of L-malate dehydrogenase (l-MDH). Initial rates of acetic acid formation can thus be determined by the increase in adsorption at 340 nm due to the increase in NADH concentration. Use of optically pure (Ry- or (5)-acetates allows the determination of the apparent enantioselectivity i app i81)-...

Fluorescence-based assays either in the measurement of enzyme activity or in the quantification of enantioselectivity all have a high degree of sensitivity, which allows the use of very dilute substrate concentrations and extremely small amounts of enzymes. Basically, there are two different approaches. One involves the use of a substrate of interest to which a fluorescent-active (or potentially active) moiety is covalently attached. The second approach makes use of a fluorescence-based sensor, which gives rise to a signal as a consequence of the enzyme-catalyzed reaction of a substrate of interest. 

One of the first fluorescence-based ee assays uses umbelliferone (14) as the built-in fluorophore and works for several different types of enzymatic reactions 70,86). In an initial investigation, the system was used to monitor the hydrolytic kinetic resolution of chiral acetates (e.g., rac-11) (Fig. 8). It is based on a sequence of two coupled enzymatic steps that converts a pair of enantiomeric alcohols formed by the asymmetric hydrolysis under study (e.g., R - and (5)-12) into a fluorescent product (e.g., 14). In the first step, (R)- and (5)-ll are subjected separately to hydrolysis in reactions catalyzed by a mutant enzyme (lipase or esterase). The goal of the assay is to measure the enantioselectivity of this kinetic resolution. The relative amount of R)- and ( S)-12 produced after a given reaction time is a measure of the enantioselectivity and can be ascertained rapidly, but not directly. 

Fig. 8. Scheme for fluorescence-based assay used in the determination of enantioselectivity of ester hydrolysis (80). 

A number of other ee assays relying on the use of fluorescence have been described, although their general utility in the high-throughput evaluation of enantioselective enzymes remains to be demonstrated in most cases (66-70). A high-throughput ee... 

Isotope labeling forms the basis of a different MS-based ee-assay (92,93). It involves a fundamentally new way to measure enantiopurity. The method has been parallelized for high throughput and used a number of times in the directed evolution of enantioselective enzymes (46,64,94-96). In this MS-based approach. 

It is emphasized that in the case of kinetic resolution, the MS measurements must be performed in the appropriate time window (near 50% conversion). If this is difficult to achieve due to different amounts or activities of the mutants being screened in the wells of microtiter plates, the system needs to be adapted in terms of time resolution. This means that samples for MS evaluation need to be taken as a function of time. Finally, it is useful to delineate the possibility of multi-substrate ee screening using the MS-based assay, which allows for enzyme fingerprinting with respect to the enantioselectivity of several substrates simultaneously. 

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