D-pantolactone

D-Pantolactone and L-pantolactone are used as chiral intermediates in chemical synthesis, whereas pantoic acid is used as a vitamin B2 complex. All can be obtained from racemic mixtures by consecutive enzymatic hydrolysis and extraction. Subsequently, the desired hydrolysed enantiomer is lactonized, extracted and crystallized (Figure 4.6). The nondesired enantiomer is reracemized and recycled into the plug-flow reactor [33,34]. Herewith, a conversion of 90-95% is reached, meaning that the resolution of racemic mixtures is an alternative to a possible chiral synthesis. The applied y-lactonase from Fusarium oxysporum in the form of resting whole cells immobilized in calcium alginate beads retains more than 90% of its initial activity even after 180 days of continuous use. The biotransformation yielding D-pantolactone in a fixed-bed reactor skips several steps here that are necessary in the chemical resolution. Hence, the illustrated process carried out by Fuji Chemical Industries Co., Ltd is an elegant way for resolution of racemic mixtures. 

Pantothenic acid is produced commerdally by synthesis involving the condensation of d-pantolactone with salt of -alanine. Some of the dietary supplement forms include caldum pantothenate, dexpanthenol, and panthenol. 

The enzyme catalyzing the reduction of ketopantolactone to D-pantolactone was isolated in a crystalline form from the cells of Candida parapsilosis and characterized in some detail [106] (see Tables 4 and 5). It is a novel NADPH-dependent carbonyl reductase with a molecular mass of about 40,000. In addition to the reduction of ketopantolactone, the enzyme catalyzes those of a variety of cyclic diketones, including derivatives of ketopantolactone, isatin, camphorquinone and so on, to give the corresponding (R)-alcohols [106, 107], The enzyme was termed conjugated polyketone reductase , since the enzyme catalyzes only the reduction of conjugated polyketones as follows. 

Reduction of ketopantolactone to D-pantolactone (0 in Fig. 8). This reaction is catalyzed by conjugated polyketone reductase. About 50 or 90 g/1 of D-panto-lactone (98 or 94% e.e., respectively) was produced with a molar yield of nearly 100% on incubation with washed cells of R. minuta or C. parapsilosis, respectively [115, 116]. 

Reduction of ethyl 2 -ketopantothenate to ethyl 2 -d-pantothenate ((g) in Fig. 8). The rate of condensation of ketopantolactone or D-pantolactone with ethyl (3-alanine, yielding ethyl 2 -ketopantothenate (0 in Fig. 8) or ethyl D-panto-thenate, respectively, is quite fast compared to the condensation of ketopantolactone or D-pantolactone with (3-alanine, and the reaction with ethyl (3-alanine proceeds more stoichiometrically [117]. Since the enzymatic hydrolysis of ethyl D-pantothenate has been established [118], if the stereoselective reduction of ethyl 2 -ketopantothenate to ethyl D-pantothenate is possible, both the troublesome resolution and the incomplete condensation might be avoided at the same time. Carbonyl reductase of C. macedoniensis is used for this purpose. Washed cells of the yeast converted ethyl 2 -ketopantothenate (80 g/1) almost specifically to ethyl D-pantothenate (> 98% e.e.), with a molar yield of 97.2% [103]. In a similar manner, 2 -ketopantothenonitrile (50 g/1) was converted to D-pan-tothenonitrile (93.6% e.e.), with a molar yield of 95.6%, on incubation with Sporidiobolus salmonicolor cells as a catalyst [104],... 

Brevibacterium protophormiae produces a similar kind of lactonase, which hydrolyzes various kinds of aromatic lactones as well as the Fusaruim lactonase, but aldonate lactones are not hydrolyzed. The enzyme hydrolyzes only l-pantolactone D-pantolactone is not a substrate. The relative molecular mass of the native enzyme is 62 kDa, and the subunit molecular mass is 26.5 kDa [137],... 

The principle of the optical resolution of racemic pantolactone is shown in Fig. 13. If racemic pantolactone is used as a substrate for the hydrolysis reaction by the stereospecific lactonase, only the d- or L-pantolactone might be converted to d- or L-pantoic acid and the l- or D-enantiomer might remain intact, respectively. Consequently, the racemic mixture could be resolved into D-pan-toic acid and L-pantolactone, or D-pantolactone and L-pantoic acid. In the case of L-pantolactone-specific lactonase, the optical purity of the remaining d-pantolactone might be low, except when the hydrolysis of L-pantolactone is complete. On the other hand, using the D-pantolactone-specific lactonase, d-pantoic acid with high optical purity could be constantly obtained independently of the hydrolysis yield. Therefore, the enzymatic resolution of racemic pantolactone with D-pantolactone-specific lactonase was investigated [138 140]. 

Distribution of D-pantolactone-specific lactonase is narrow only filamentous fungi of three genera, i.e., Fusarium, Gibberella and Cylindrocarpon, show high hydrolytic activity. Washed cells of these fungi, as well as the purified lactonase, catalyze well this stereospecific hydrolysis. For example, D-pantolactone in a racemic mixture of pantolactone (700 mg/ml) was almost... 

D-(-)-Pantolactone was obtained from the complex in the following manner. The 65.4 grams of complex obtained above were treated with 65 ml of chloroform and 5.35 grams of sodium hydroxide contained in 35 ml of water for one hour at room temperature. The aqueous layer was extracted 6 times with 20 ml portions of chloroform in order to remove the brucine. The sodium pantoate contained in the aqueous layer was relactonized by treatment with 11 ml of concentrated hydrochloric acid. Extraction of the crude D-(-)-pantolactone yielded 15.29 grams. This material was then recrystallized from 7 ml of methyl isobutyl ketone and 7 ml hexane thereby yielding 9.77 grams of D-(-)-pantolactone (37% of theory). The aD25 was -44.8°. 

The 2,4-dihydroxy-3,3-dimethyl-butironitrile was treated hydrochloric acid and D,L-3-hydroxy-4,4-dimethyl-dihydro-furan-2-one (D,L-pantolacton) was obtained. The racemic mixture of D- and L-pantolactons was a division of D-and L- isomers by the adding of a-phenylethylamine. So D-pantolacton was isolated. 

Biotechnological Production of D-Pantothenic Acid and its Precursor D-Pantolactone... 

D-Pantolactone (Figure 6.3.1) is an important intermediate in the production of d-pantothenic acid, also called vitamin B5. Deficiency of pantothenic acid can result in symptoms such as pathological changes of the skin and mucosa, disorders in the gastrointestinal tract and nervous system, organ changes, and hormonal disorders. Pantothenic acid is used mainly in feed for chicken and pigs and also as a vitamin supply in human nutrition. Its commercial form, the calcium salt, is produced worldwide on a multi-thousand ton scale. 

D-Pantothenic acid is also traditionally produced by chemical processes which involve efficient but troublesome and costly crystallization of diastereomeric salts of pantoate and chiral amines. After lactonization of the isolated D-pantoate, d-pantolactone is reacted with / -alanine to give D-pantothenate. Because the monovalent salts of pantothenic acid are highly hygroscopic, conversion into the calcium salt is essential for convenient formulation. The third class of synthetic processes for optically active compounds makes use of biotechnology. For natural com-... 

In a second approach hydrocyanic acid was added to hydroxypivaldehyde by use of (R)-selective hydroxynitrile lyase from almonds (PaHNL) [11]. (R)-Cyanohydrin was obtained in 84% yield and 89% ee, and was directly cydized to give crude D-pantolactone by acid-catalyzed hydrolysis. Unfortunately, in contrast with O-protected hydroxy and halogenated pivalaldehydes, the technically available starting compound hydroxypivaldehyde requires use of purified enzyme (and high enzyme loading). 

Finally, biocatalytic resolution was developed for more efficient production of D-pantolactone. Whereas the resolution of O-acyl pantolactone with lipases or esterases [12] did not lead to an industrially attractive process, the hydrolysis of rac-pantolactone by pantolactone hydrolases enabled development of a technically feasible and economic process. 

For a kinetic resolution process use of both d- or an L-specific pantolactone hydrolase is possible in principle (Figure 6.3.3). If the unwanted L-form is hydrolyzed it might take longer for the remaining D-pantolactone to reach a sufficient enantiomeric excess the process is, however, much more robust, e.g. towards competing spontaneous chemical hydrolysis. 

The enzyme has a monomer weight of 30 kDa and a Km and Vmax for L-pan-tolactone of 7 mM and 30 U mg-1, respectively. X-ray fluorescence spectroscopy of crystals, and renaturation of urea/EDTA-denatured Lph in the presence of Zn2+, Mn2+, Co2+, or Ni2+ indicated Lph to be a Zn2+-hydrolase. Kinetic resolution of rac-pantolactone proceeds similarly to the fungal process mentioned above except that L-pantolactone is hydrolyzed and D-pantolactone is left behind. Repeated batches with isolated Lph and enzyme recovery by membrane filtration give d-pantolactone with 50% yield and 90-95% ee over 6 days. 

When the kinetics of the hydrolysis of rac-pantolactone by Lph were investigated a decrease in the reaction velocity was observed this was found to be because of competitive inhibition by D-pantolactone (Eq. 1) [19] and slight product inhibition of Lph. Under the same conditions of pH (7.5) and temperature (30 °C), l-pantolactone was completely converted to L-pantoic acid. This is certainly a disadvantage of Lph-catalyzed kinetic resolution, because space-time yields come to levels as low as 6 g L 1 h 1. 

Several biotechnological synthetic methods for D-pantothenic add and its precursor D-pantolactone have been developed over the past 15 years. Although all have reached preparative scale and might result in cost-effective production processes, they differ considerably in their process characteristics - for example educts and space-time yields, especially when a fermentation and biotransformation are compared. Compared with the chemical process, the biotechnological processes reduce waste production and provide the possibility of a more environmentally friendly yet still competitive means of production of D-pantothenic acid. 

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