Novozyme CALB

This was overcome by acetylation of the same triol intermediate, using Novozym 435 (rmmobihzed CALB) in vinyl acetate and acetonitrile, to afford the monoacetate in 95 % yield and 97 % diastereoselectivity (Scheme 1.46). The monoacetate was then readily converted to the desired c -THF derivative by alcohol activation and cyclization as described above. 

The resolution of a racemic substrate can be achieved with a range of hydrolases including lipases and esterases. Among them, two commercially available Upases, Candida antarctica lipase B (CALB trade name, Novozym-435) and Pseudomonas cepacia lipase (PCL trade name. Lipase PS-C), are particularly useful because they have broad substrate specificity and high enantioselectivity. They display satisfactory activity and good stability in organic media. In particular, CALB is highly thermostable so that it can be used at elevated temperature up to 100 °C. 

Since then, the process has been extended to a wide variety of lactones of different size and to several lipases, as recently reviewed [93-96]. Interestingly, large-membered lactones, which are very difficult to polymerize by usual anionic and coordination polymerizations due to the low ring strain, are successfully polymerized by enzymes. Among the different lipases available, that fi om Candida antarctica (lipase CA, CALB or Novozym 435) is the most widely used due to its high activity. An alcohol can purposely be added to the reaction medium to initiate the polymerization instead of water. The polymerization can be carried out in bulk, in organic solvents, in water, and in ionic liquids. Interestingly, Kobayashi and coworkers reported in 2001 the ROP of lactones by lipase CA in supercritical CO2... 

Nevertheless, the limitations of Novozym 435 have also been clearly identified. Water is the preferred nucleophile, making 100% end-functionalization very challenging, while the preference of CALB for transoid ester bonds limits the potential to reach low polydispersities in a ROP. Multidisciplinary projects focusing both on the enzymology side of the biocatalyst (e.g., by improving the lipase by mutations) and on the polymers required for specific applications can in the near future lead... 

Chemoenzymatic polymerizations have the potential to further increase macro-molecular complexity by overcoming these limitations. Their combination with other polymerization techniques can give access to such structures. Depending on the mutual compatibility, multistep reactions as well as cascade reactions have been reported for the synthesis of polymer architectures and will be reviewed in the first part of this article. A unique feature of enzymes is their selectivity, such as regio-, chemo-, and in particular enantioselectivity. This offers oppormnities to synthesize novel chiral polymers and polymer architectures when combined with chemical catalysis. This will be discussed in the second part of this article. Generally, we will focus on the developments of the last 5-8 years. Unless otherwise noted, the term enzyme or lipase in this chapter refers to Candida antarctica Lipase B (CALB) or Novozym 435 (CALB immobilized on macroporous resin). 

The first enzymatic polymerizations of substituted lactones were performed by Kobayashi and coworkers using Pseudomonas fluorescens lipase or CALB as the biocatalyst [90-92]. A clear enantiopreference was observed for different lactone monomers, resulting in the formation of optically active polymers. More recently, a systematic study was performed by Al-Azemi et al. [93] and Peelers et al. [83] on the ROP of 4-alkyl-substituted CLs using Novozym 435. Peelers et al. studied the selectivity and the rates as a function of the substituent size with the aim of elucidating the mechanism and the rate-determining step in these polymerizations. Enantio-enriched polymers were obtained, but the selectivity decreased drastically with the increase in substituent size [83]. Remarkably for 4-propyl-e-caprolactone, the selectivity was for the (R)-enantiomer in a polymerization, whereas it was S)-selective in the hydrolysis reaction. Comparison of the selectivity in the hydrolysis reaction (Fig. 10b) with that of the polymerization reaction (Scheme 8a) revealed that the more bulky the alkyl substituent, the more important the deacylation step becomes as the rate-determining step. 

Arylalkanols have been one of the most studied groups of compounds, partly because of the different spatial requirements of the substituents, which facilitate the resolution, and such compounds have served well in model studies. Thus, some 1-arylethanols can be very efficiently resolved in hexane using CALB-catalyzed (Novozym 435) acylation with vinyl acetate to give the esters 26-31 in very high ees and with excellent enantioselechvity (E > 200) (Scheme 4.17, top half)... 

Many 1-heteroaryl-1-alkanols are also easily resolved by hydrolase-catalyzed reactions in organic solvents. Thus, some racemic l-(2-pyridyl)-l-alkanols and isoquinonyl-l-ethanols are easily resolved by CALB (Novozym 435) and vinyl acetate in diisopropyl ether to give the acetates 38—44 (Scheme 4.18) [75]. 

Some l-(2-thienyl)-l-alkanols of type 45 can be efficiently resolved by acylation with vinyl butanoate catalyzed by CALB (Novozym 435) in various solvents (Scheme 4.19) [76]. 

With the exception of 2-butanol (E = 9), simple 2-alkanols can be efficiently resolved by using CALB-catalyzed (Novozym 435) acylation in hexane using S-ethyl thiooctanoate or vinyl butyrate as acyl donor to produce the esters 51-59... 

The resolution of anti-3-methyl-l,4-pentanediol (60) proceeds via rapid Novozym 435 (CALB) catalyzed monoacylation with vinyl acetate in TBME under dry conditions followed by a slower enantioselective acylation of the secondary alcohol moiety (Scheme 4.23) [82]. Similarly, diasteromerically pure threo- and erythro-3-methylalkan-2-ols of type 61 and 62, respectively, have been successfully acylated with 2/(-preference ( E > 100 to >1000) using vinyl acetate, propionate or butyrate... 

Successful resolutions of hydrogenated naphthalenes by acylation in organic solvent have been described. Thus, CALB (Novozym 435) catalyzes the acylation with vinyl acetate at 40 °C in hexane of three cis-fused octahydronaphthalenols to furnish after 48 h the acetylated products 72-74 and the remaining alcohol in high ees and with excellent E (Scheme 4.27, products 72-74 shown) [88]. Similarly, a key intermediate in the synthesis of natural products of the marasmane and lactarane... 

Desymmetrizahon by PSL-C-catalyzed octanolysis of the dibenzoate 153 provides the enanhopure monobenzoate R-154 [145]. Although amides are normally not easily hydrolyzed or alcoholyzed by lipases, P-lactams do react, probably because of the strained nature of the four-membered P-lactam ring. Thus CALB-catalyzed (Novozym 435) ring opening of roc-155 by alcoholysis with racemic 2-octanol in DIPE at 60 °C furnished enanhopure recovered P-lactam R-155, whereas the alcoholysis product was obtained only in a low yield, albeit in a high [147]. The recovery of the product 156 was substantially increased by exchanging the nucleophile 2-octanol for an equimolar amount of water in DIPE, as shown in (Scheme 4.43) [148]. 

Acylations of carbohydrate derivatives such as alkyl glucosides and galactosides have also been successfully performed in ionic liquids [63]. Similarly, the flavonoid glycosides naringin and rutin were acylated with vinyl butyrate in ionic liquid media in the presence of a number of lipases, e.g., CaLB (Novozym 435), immobilized TIL, and RmL [119]. The products are of interest for application as strong antioxidants in hydrophobic media. 

Enzymatic kinetic resolution is a key step in the synthesis of the platelet aggregation inhibitor Lotrafiban (Figure 10.11). A disclosed process involves CaLB in tert-butyl alcohol/water (88 12) at 50 °C the substrate concentration was only r> g I 1 owing to its low solubility in this medium [122]. By exploiting the higher solubility in 88% [BMIm][PF6] and the better thermal stability of the biocatalyst in this medium, a higher rate was observed, the reaction was performed at 40 54 1. 1 at 75 °C, and the biocatalyst (Novozym 435) could be recycled 10 times. 

The same concept is applicable to allylic alcohols, ketones, or ketoximes. Enol acetates or ketones were successfully converted in multi-step reactions to chiral acetates in high yields and optical yields through catalysis by Candida antarctica lipase B (CALB, Novozyme 435) and a ruthenium complex. 2,6-Dimethylheptan-4-ol served as a hydrogen donor and 4-chlorophenyl acetate as an acyl donor for the conversion of the ketones (Jung, 2000a). 

The application of enzymes as catalysts in organic chemistry is closely linked to their immobilization. Indeed, many enzymes are only available in an immobilized form. The immobilized enzymes can be used as received, greatly easing their application. Numerous of these readily available immobilized enzymes are now the working horses of biocatalysis. This has even led to the incorrect use of the abbreviation of an enzyme name for a specific enzyme preparation, that is CALB for the immobilized form of Candida antarctica lipase B on cross-linked polymethacrylate (also known as Novozym 435). Vice versa the commercial name of an enzyme preparation-Amano PS-has taken the place of the enzyme (Burkhdderia cepacia lipase on dextrin or diatomaceous earth). Surprisingly, often no attention is paid to the fact that the enzyme is immobilized [1]. 

Table 2.2 Recycling of Novozym 435 (CALB immobilized on a hydrophobic carrier) in the kinetic resolution of 1c in toluene at 60°C.

The same CALB preparation was appUed in many dynamic kinetic resolutions combining two types of catalysts with each other. In the presence of homogeneous transition metal catalysts that catalyze the racemization and heterogeneous acids or bases or immobilized transition metals Novozym 435 was not deactivated [1, 26-28]. This is all the more remarkable since the reactions catalyzed by these catalysts include redox reactions at elevated temperatures (>60°C). When Novozym 435 was applied for the enantioselective synthesis of cyanohydrin acetates (10) from aliphatic aldehydes (7), good results were achieved (Scheme 2.2) for this dynamic kinetic resolution (DKR) [29]. Here NaCN is used as the base for the dynamic racemic formation and degradation of the cyanohydrins (6 and 8). 

Figure 2.3 Enantioselective synthesis of la via DKR catalyzed by CALB on Celite R-633 ( ) and Novozym 435 ( ).

In a comparative study CALB was immobUized on epoxy-activated Sepabeads and amino Sepabeads with long and short spacers (glutaraldehyde was used as the coupling reagent) (see Figure 2.8). Lyophilized CALB and Novozym 435 were also included in the test The specific activity (U/g dry immobihzed CALB) of Novozym 435 was much lower than for CALB immobihzed on the different Sepabeads. In a thermal stabihty screening, Novozym 435 and CALB immobihzed on amino Sepabeads with short spacers displayed equal stabihty, while ah the other CALB preparations were less stable [32]. 

The high molar conversion obtainable in esterifications catalyzed by dry mycelia encouraged us to investigate the partition of water in these heterogeneous systems further. The objechve was to verify whether mycelia were able to affect the thermodynamic equilibrium of the reactions by modifying the parhtioning of the water formed during the esterificahon. The synthesis of two esters by lyophilized mycelia of R. oryzae CBS 112.07 was studied and the results were compared with those obtained with a commercial immobilized CALB (Novozym 435). 

Since this effect was observed with the immobilized preparation of CALB (Novozym 435) it was interesting to perform the same reactions with pure protein preparations of CALB to see if the immobilization of the enzyme influenced the changing f -value. The formulated CALB Novozym 525 F is a water solution of CALB containing 1-10% pure protein. After freeze drying at -80 °C this dry protein... 

Lipase B from Candida antarctica (CALB) has been shown to be an excellent enantioselective biocatalyst for the stereo-selective acylation of racemic alcohols [14, 15]. The most often used commercial preparation of CALB is Novozym 435, where the enzyme is immobilized on a macroporous acrylic resin and the matrix presents about 90% of the total mass. 

In this research the kinetic resolution of 1-phenylethanol catalyzed by commercially available immobilized lipase from CALB was assayed in non-aqueous conditions in SC-CO2 and IL/SC-CO2 systems with the aim of studying the enan-tioselectivity of Novozym 435. The influence of different reaction parameters, such as pressure, the acyl donor/alcohol molar ratio and different ILs, on the enantio-merically pure compound (R)-l-phenylethyl acetate formation via kinetic resolution of 1-phenylethanol was investigated. 

Therefore, the pressure effect on the enantioselectivity of commercially available immobilized CALB (Novozym 435) for the kinetic resolution of 1-phenylethanol was studied in non-aqueous SC-CO2. 

Immobilized lipase B from Candida antarctica (CALB) (Novozym 435) was obtained from Novo Nordisk Novozymes. All chemicals and ionic liquids used were of the highest available purity. 

The single most used lipase for biocatalysis is probably the Candida antarctica B-lipase (CALB) [42]. It is commercialized by Novozymes in liquid formulation as well as in immobilized form under the trade name Novozym 435 (previously SP 435). CALB has high activity on a wide range of substrates (it has some problems with very bulky substrates), often with outstanding selectivities. Formulated as Novozym 435 it is stable up to approx. 90 °C in solvents such as toluene (or solvent-free reaction mixtures). The A-lipase (CALA), currently only commercially available in liquid form, has attractive properties too, including even better thermostability and higher activity on sterically hindered substrates [43]. 

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