Pantolactone resolution

R)-Pantolactone (9) is prepared ia either of two ways by resolution of the (R,5)-pantolactone mixture (18), or by stereoselective reduction of ketopantolactone (19) by chemical or microbial methods (19). 

Despite the progress made in the stereoselective synthesis of (R)-pantothenic acid since the mid-1980s, the commercial chemical synthesis still involves resolution of racemic pantolactone. Recent (ca 1997) synthetic efforts have been directed toward developing a method for enantioselective synthesis of (R)-pantolactone by either chemical or microbial reduction of ketopantolactone. Microbial reduction of ketopantolactone is a promising area for future research. 

Figure 4.6 Classical kinetic resolution with subsequent reracemization of unconverted enantiomer Synthesis of pantoic acid from pantolactone applying a stirred-tank reactor, extraction module and racemization step...

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. 

Optical Resolution of Racemic Pantolactone with Lactonase.75... 

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],... 

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]. 

Fig. 14. Comparison of enzymatic and conventional chemical resolution processes for DL-pantolactone. PL, pantolactone PA, pantoic acid...

The overall process for this enzymatic resolution is compared with the conventional chemical process in Fig. 14. The enzymatic process can skip several tedious steps which are necessary in chemical resolution and this is a considerable practical advantage. There have been several reports on the application of enzymatic asymmetric hydrolysis to the optical resolution of pantolactone [141, 142], In these cases, esterified substrates, such as O-acetyl or O-formyl pantolactone, and lipases were used as the starting materials and catalysts, respectively. Since the lactonase of F. oxysporum hydrolyzes the intramolecular ester bond of pantolactone, it is not necessary to modify the substrate, pantolactone. This is one of the practical advantages of this enzyme. 

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. 

In the nineties a process for the kinetic resolution of pantolactone by enantio-selective hydrolysis was developed by Yamada and coworkers with Fuji Chemical Industries, Japan (now Daiichi Fine Chemical) using the fungus Fusarium oxy-sporum AKU3702 [13]. Like most of the Fusarium strains and strains of the related species Gibberella, Cylindrocarpon, Penicillium, and Aspergillus, Fusarium oxysporum... 

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. 

Fig. 6.3.3. Production of D-pantothenic acid by resolution of D,L-pantolactone by either a d- or an L-specific lactono-hyd rolase.

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. 

Resolution of rac-Pantolactone by Bacterial Hydrolysis of L-Pantolactone The Development of a Novel Biocatalyst 505... 

The atom efficiency of a kinetic resolution is increased if the starting material is not an ester but a lactone. Indeed, kinetic resolutions of lactones are used on an industrial scale. Fuji/Daiichi Chemicals produces D-pantothenic acid on a multi-ton scale based on such a resolution. D-Pantolactone is hydrolysed at pH 7 by a hydrolase from Fusarium oxysporum yielding D-pantoic acid with an ee of 96% while L-pantoic acid was barely detectable. The immobilized Fusarium oxysporum cells were recycled 180 times and retained 60% of their activity, demonstrating the great stability of this catalytic system [47-50]. 

The major industrial route to calcium pantothenate starts from isobutyralde-hyde, which is condensed with formaldehyde. Hydrocyanation and hydrolysis affords the racemic pantolactone (Fig. 8.20). The resolution of pantolactone is carried out by diastereomeric crystallization with a chiral amine, such as (+)-2-aminopinane (BASF), 2-benzylamino-l-phenylethanol (Fuji) or (lR)-3-endo-ami-nonorbomeol (Roche). The undesired enantiomer is racemized and recycled. 

Availability. Although commercially available via the degradation of pantothenic acid, (i )-pantolactone is also conveniently prepared by enantioselective reduction of its corresponding keto lactone employing homogeneous catalysis," " or by microbial methods. The (5)-enantiomer has been prepared by inversion of the natural product in 90% yield and 97% ee via triflate activation, acetate displacement, and Lithium Hydroxide hydrolysis. The enantiomers were also prepared by resolution of the race-mate with (R)- and (5)-phenethylamine. A gas chromatographic method exists for ee determination. ... 

Pantenoic acid is used as a vitamine B2 complex, d- and L-pantolactone are used as chiral intermediates in chemical synthesis. The enantioselective hydrolysis is carried out in the aqueous phase with a substrate concentration of 2.69 M = 350 g L 1 (Fig. 19-17). For the synthesis whole cells are immobilized in calcium alginate beads and used in a fixed bed reactor. The immobilized cells retain more than 90 % of their initial activity after 180 days of continuous use. At the end of the reaction l-pantolactone is extracted and reracemized to d,L-pantolactone, which is recycled to the reactor. The D-pantenoic acid is chemically lactonized to D-pantolactone and extracted. By applying cells from Brevibacterium protophormia the L-lactone is available. The biotransformation eliminates several steps that are necessary in the chemical resolution process (Fig. 19-18). 

Kinetic Resolution of Pantolactones and Derivatives thereof by a Lactonase from Fusarium oxysporum (E. C. 3.1.1.25) 1433... 

The present industrial processes used to produce the crucial intermediate (R)-(-)-pantolactone (22) are based on resolution of racemic material (3 ). A different and very promising approach has been reported by a Japanese group (26). Independently, Roche workers also investigated this approach which involves asymmetric reduction of ketolactone using rhodium catalysts derived from chiral phosphines (27 . In this manner, 22 can be obtained in very high chemical and optical yieldS ... 

About 6,0001 of the animal feed additive calcium-D-pantothenate are produced annually via D-pantolactone (d-112) (Scheme 35, left side).D-Pantolactone itself is an important chiral intermediate for chemical synthesis and a chiral resolution agent for optically pure amines. Optically pure d-112 is for instance produced by Fuji Chemical Industries by using the D-specific 1,4-lactone hydroxy-acylhydrolase from Fusarium oxysporum [100-102], an enzyme that catalyzes the stereospecific hydrolysis of various kinds of lactones. Treatment of mc-112 leads to an exclusive hydrolysis of d-112 the hydroxy acid d-113 can be easily separated from the remaining lactone l-1 12 and is subsequently chemically con-... 

Contrary to the Fuji process, BASF described the characterization and cloning of an L-specific pantolactone hydrolase from Agrobacterium tumefa-ciens [103,104]. This enzyme exclusively opens up the undesired lactone l-1 12, providing a more direct route to d-1 12 (Scheme 35, right side). In addition, this new process is expected to be much more robust toward the competing spontaneous chemical hydrolysis, which could theoretically cause a diminished optical yield in the Fuji process. The enzymatic resolution of d/l-112 in repeated batches with membrane filtration techniques provided d-1 12 in 50% yield and with 90-95% ee. By immobilization onto Eupergit C it was possible to obtain a stable biocatalyst which was easy to use in repeated batch reactions. 

Resolution of pantoic acid (2). This precursor of (R)-pantothenic acid (4) has been resolved with (+ )-l. The salts of the (R)-acid and the (S)-acid with this amine show a marked difference in solubility in water at 60°, which makes separation relatively simple. In fact, (- )-(R)-pantolactone (3) can be obtained in 90% yield with a purity of 94%. ... 

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