Racemization procedures

CPDMO is a new bioreagent for the synthesis of optically pure lactones with excellent enantioselectivity. CPDMO is not only effective in desymmetrization of meso and prochiral compounds (Procedure 2, Section 11.8.2), but excellent in carrying out the kinetic resolution of racemates (Procedure 3, Section 11.8.3). Additional examples of optically pure lactones that can be obtained are summarized in Table 11.4. In the fermenter work (Procedure 4, Section 11.8.4), (R)-2-methyl cyclohexanone was not converted, but evaporated under aeration condition (1 wm). This led to the expected product (5)-7-methyl oxepanone at the end of the experiment. The optically pure lactone could be recovered without sdica-gel chromatography separation. However, the production yield may be improved by using a better condenser. 

Resolving Agent. Scalemic CSA has been used to resolve amines by forming diastereomeric salts which can be separated by fractional crystallization (eq 11). In this instance, after obtaining the desired crystalline diastereomeric salt, the undesired diastereomer was completely transformed into the desired one by a resolution-racemization procedure (eq 12). Additionally, racemic ketones can be resolved by forming enantiomeric iminium salts (eq 13). Two different procedures have been devised depending on the ease of enamine formation. 

Subtilisin Carlsberg, a laundry enzyme, is extraordinarily cheap and stable even at extreme concentrations of substrate and salt at elevated temperature. A major disadvantage of this chemoenzymatic route was the fact that no racemization procedure for the unwanted (R)-enantiomer [(R)-3] could be established [40]. 

A big factor for the success of the process was the close similarity of the substrate to the natural substrates of Subtilisin Carlsberg, an enzyme with extraordinary properties. Since cheap commercial sources for this enzyme were available on the market, the enzyme could be discarded. A major disadvantage of the present chemoenzymatic route was the fact that no satisfactory racemization procedure for the unwanted enantiomer (R)-2 could be established due to elimination and/or hydrolysis side reactions. 

An alternative enzyme/transition metal combination employs transfer hydrogenation catalysts that are capable of racemizing secondary alcohols. The racemization procedure temporarily converts the alcohol into an achiral ketone, which is reduced back to the racemic alcohol. Coupling this racemization procedure to an enzyme-catalyzed acylation reaction affords a dynamic resolution process (Fig. 9-12). Several enzyme/transition metal combinations have been shown to be effective for these reactions, although ruthenium complexes 1-3 appear to be especially effective for the in situ racemization of the alcohol. The product esters are not prone to racemization under the reaction conditions. Early results employing transfer hydrogenation catalysts to effect the racemization of alcohols required the use of added ketone 21, 22. However, it was subsequently shown that added ketone was not required when appropriate transition metal complexes were used as catalysts. Furthermore, the use of 4-chlorophenyl acetate as the acyl donor afforded improved results. 

Backvall and co-workers have reported successful results for a wide range of substrates, some of which are identified in Table 9-1. The procedure works well for secondary alcohols containing aryl and alkyl groups [231, diols1241 and a-hydroxy esters[25]. Although catalyst 1 requires no additional base, Kim, Park and co-workers used triethylamine to facilitate racemization using catalyst 2, Table 9-2126]. In their case, small quantities of oxygen were added to initiate the racemization procedure. In the case of allylic alcohols, careful choice of racemisation catalyst is required in order to minimize the amount of conversion of the substrate into saturated or... 

The enzymatic variant of this concept was reported for the deracemization of d,l-methionine or D,L-leucine (Fig. 16.7-8) 17. Soda and coworkers developed a chemo-enzymatic racemization procedure utilizing boron hydrides for non-enantioselective reduction of the undesired d-AAO product (Fig. 16.7-9)[21, 22). Using the same procedure, the authors achieved conversion of D-proline into L-proline 21. Furthermore, D,L-lactate and 2-hydroxy butyric acid were deracemized by utilizing l-lactate oxidase123. ... 

With new coupling methods and/or reaction techniques, the extent of racemization has usually been determined u g one or more standardized evaluation methods. One of the earliest was due to Anderson where the condensation of Z-Gly-Phe with Gly-OEt was employed and specific rotation of the product measured. Also, racemate may be isolated by fractional crystallization. By this means down to 1-2% racemization may be detected. Next in 1963 appeared the Weygand (Z-Leu-Phe - - Val-OBu , partial hydrolysis and glc determination of D-Phe-Val, sensitive to 0.1-1% racemization) and Young (Bz-Leu - -Gly-OEt, specific rotation of crude material and isolation of racemate, can detect 1-2% racemization) procedures. In 1969 Izumiya s amino acid analyzer method was reported and details were available last year. With this tech-... 

Other Methods. Various procedures for determining tartrates were tested by Fdbregues and Mestre (1948). They preferred a conductometric method for tartrates in lees, based on diluting with ammonium hydroxide to the point of minimum conductivity, adding a known amount of tartaric acid, and repeating the dilution. For wines they favored the racemate procedure. 

The major disadvantage of solid-phase peptide synthesis is the fact that ail the by-products attached to the resin can only be removed at the final stages of synthesis. Another problem is the relatively low local concentration of peptide which can be obtained on the polymer, and this limits the turnover of all other educts. Preparation of large quantities (> 1 g) is therefore difficult. Thirdly, the racemization-safe methods for acid activation, e.g. with azides, are too mild (= slow) for solid-phase synthesis. For these reasons the convenient Menifield procedures are quite generally used for syntheses of small peptides, whereas for larger polypeptides many research groups adhere to classic solution methods and purification after each condensation step (F.M. Finn, 1976). 

One approach called enzymatic resolution, involves treating a racemic mixture with an enzyme that catalyzes the reaction of only one of the enantiomers Some of the most commonly used ones are lipases and esterases enzymes that catalyze the hydrol ysis of esters In a typical procedure one enantiomer of the acetate ester of a racemic alcohol undergoes hydrolysis and the other is left unchanged when hydrolyzed m the presence of an esterase from hog liver... 

Both of the alkaloids anhalamine (62) from l ophophora williamsii and lophocerine (63) from l ophocereus schotti were isolated (after the properties of purified mescaline had been noted) in the search for materials of similar behavior. Interestingly, lophocerine, isolated as its methyl ether, after dia2omethane treatment of the alkaU-soluble fraction of total plant extract, is racemic. It is not known if the alkaloid in the plant is also racemic or if the isolation procedure causes racemization. 

Among chiral dialkylboranes, diisopinocampheylborane (8) is the most important and best-studied asymmetric hydroborating agent. It is obtained in both enantiomeric forms from naturally occurring a-pinene. Several procedures for its synthesis have been developed (151—153). The most convenient one, providing product of essentially 100% ee, involves the hydroboration of a-pinene with borane—dimethyl sulfide in tetrahydrofuran (154). Other chiral dialkylboranes derived from terpenes, eg, 2- and 3-carene (155), limonene (156), and longifolene (157,158), can also be prepared by controlled hydroboration. A more tedious approach to chiral dialkylboranes is based on the resolution of racemates. /n j -2,5-Dimethylborolane, which shows excellent enantioselectivity in the hydroboration of all principal classes of prochiral alkenes except 1,1-disubstituted terminal double bonds, has been... 

Crystallization Method. Such methods as mechanical separation, preferential crystallisation, and substitution crystallisation procedures are included in this category. The preferential crystallisation method is the most popular. The general procedure is to inoculate a saturated solution of the racemic mixture with a seed of the desired enantiomer. Resolutions by this method have been reported for histidine (43), glutamic acid (44), DOPA (45), threonine (46), A/-acetyl phenylalanine (47), and others. In the case of glutamic acid, the method had been used for industrial manufacture (48). 

This procedure is restricted mainly to aminodicarboxyhc acids or diaminocarboxyhc acids. In the case of neutral amino acids, the amino group or carboxyl group must be protected, eg, by A/-acylation, esterification, or amidation. This protection of the racemic amino acid and deprotection of the separated enantiomers add stages to the overall process. Furthermore, this procedure requires a stoichiometric quantity of the resolving agent, which is then difficult to recover efficiendy. Practical examples of resolution by this method have been pubUshed (50,51). 

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

With this epoxidation procedure it is possible to convert the achiral starting material—i.e. the allylic alcohol—with the aim of a chiral reagent, into a chiral, non-racemic product in many cases an enantiomerically highly-enriched product is obtained. The desired enantiomer of the product epoxy alcohol can be obtained by using either the (-1-)- or (-)- enantiomer of diethyl tartrate as chiral auxiliary ... 

The importance of chemical syntheses of a-amino acids on industrial scale is limited by the fact that the standard procedure always yields the racemic mixture (except for the achiral glycine H2N-CH2-COOH and the corresponding amino acid from symmetrical ketones R-CO-R). A subsequent separation of the enantiomers then is a major cost factor. Various methods for the asymmetric synthesis of a-amino acids on laboratory scale have been developed, and among these are asymmetric Strecker syntheses as well. ... 

Figure 10.1 Analysis of racemic 2,5-dimethyl-4-hydroxy-3[2H]-furanone (1) obtained from a strawbeny tea, flavoured with the synthetic racemate of 1 (natural component), using an MDGC procedure (a) dichloromethane extract of the flavoured strawbeny tea, analysed on a Carbowax 20M pre-column (60 m, 0.32 mm i.d., 0.25 p.m film thickness earner gas H2, 1.95 bar 170 °C isothermal) (b) chirospecific analysis of (1) from the sti awbeny tea exti act, ti ansfened foi stereoanalysis by using a pemiethylated /3-cyclodextrin column (47 m X 0.23 mm i.d. canier gas H2, 1.70 bar 110 °C isothemial). Reprinted from Journal of High Resolution Chromatography, 13, A. Mosandl et al., Stereoisomeric flavor compounds. XLIV enantioselective analysis of some important flavor molecules , pp. 660-662, 1990, with permission from Wiley-VCH.

When chiral, drugs and other molecules obtained from natural sources or by semisynthesis usually contain one of the possible enantiomeric forms. However, those obtained by total synthesis often consist of mixtures of both enantiomers. In order to develop commercially the isolated enantiomers, two alternative approaches can be considered (i) enantioselective synthesis of the desired enantiomer or (ii) separation of both isomers from a racemic mixture. The separation can be performed on the target molecule or on one of its chemical precursors obtained from conventional synthetic procedures. Both strategies have their advantages and drawbacks. 

Tlie polymers were prepared by the standard procedure using MAA as functional monomer (see Fig. 6-2) as described elsewhere Mobile phase acetonitrile/acetic acid 90/10 (v/v). Sample 0.2. imol racemate g" Mobile phase acetonitrile/water/acetic acid 96.3/1.2/2.5 (v/v). 

The analytical capability of these matrices has been demonstrated for chiral amines [12, 13]. The procedure is illustrated in Fig. 8-4 for the separation of NapEtNH " CIO . Concentrated methanol/dichloromethane solutions of the racemic mixture were placed on a column containing the chiral macrocycle host. The enantiomers of the ammonium salts were resolved chromatographically with mixtures of methanol and dichloromethane as the mobile phase. The amounts of R and S salts in each fraction were determined by polarimetry. Because the chiral supported macrocycle interacts more strongly with S salts, the R salt passes through the column first and the S salt last, as seen in Fig. 8-4. 

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. 

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