Secondary enzymatics

It is very likely that the desirable cheesemaking properties of starters are due to a balance between certain, perhaps secondary, enzymatic activities, which have not yet been identified. 

These three methods are commonly used the detection of radioactive phosphate containing [24], the colorimetric detection of a complex of phosphomolybdate and malachite green [25], and the colorimetric detection of a phosphorylated product obtained by a secondary enzymatic phosphate transfer using purine ribonucleoside phosphorylase (PNP) [26] (Figure 2). 

Despite great diversity in form and function, the terpenoids are unified in their common biosynthetic origin. The biosynthesis of all terpenoids from simple, primary metabolites can be divided into four overall steps (a) synthesis of the fundamental precursor IPP (b) repetitive additions of IPP to form a series of prenyl diphosphate homologs, which serve as the immediate precursors of the different classes of terpenoids (c) elaboration of these allylic prenyl diphosphates by specific terpenoid synthases to yield terpenoid skeletons and (d) secondary enzymatic modifications to the skeletons (largely redox reactions) to give rise to the functional properties and great chemical diversity of this family of natural products. As bacosides are triterpenoid derivatives, they may probably foUow the common biosynthetic pathway of terpenoid production [40]. 

Undesired polymer instability originating from secondary enzymatic and metabolic pathways, which are either unknown, or might be phenotypic for an illness. 

The coupling of enzymatic and electrochemical reactions has provided efficient tools, not only for analytical but also for synthetic purposes. In the latter field, the possibilities of enzymatic electrocatalysis, for example, the coupling of glucose oxidation (catalyzed either by GOx or GDH) to the electrochemical regeneration of a co-substrate (benzoquinone or NAD+) have been demonstrated [362-364]. An electroenzymatic reactor has also been developed [363-364] to demonstrate the production of biochemicals on a laboratory scale. NAD(P)+ derivatives immobilized by covalent attachment to polymer matrices or protein backbones have been used in enzyme reactors [365, 366]. Another important coenzyme ubiquinone can be regenerated at an electrode [367, 371] and applied to drive secondary enzymatic reactions with the participation of membrane enzymes (e.g. fumarate reductase). 

Cyclodextrins are macrocyclic compounds comprised of D-glucose bonded through 1,4-a-linkages and produced enzymatically from starch. The greek letter which proceeds the name indicates the number of glucose units incorporated in the CD (eg, a = 6, /5 = 7, 7 = 8, etc). Cyclodextrins are toroidal shaped molecules with a relatively hydrophobic internal cavity (Fig. 6). The exterior is relatively hydrophilic because of the presence of the primary and secondary hydroxyls. The primary C-6 hydroxyls are free to rotate and can partially block the CD cavity from one end. The mouth of the opposite end of the CD cavity is encircled by the C-2 and C-3 secondary hydroxyls. The restricted conformational freedom and orientation of these secondary hydroxyls is thought to be responsible for the chiral recognition inherent in these molecules (77). 

After the first hydrolytic step, secondary alcohols seem to continue biodegradation through ketone, hydroxyketone, and diketone. Diketones then produce a fatty acid and a linear aldehyde which is further oxidized to fatty acid. Finally, these two fatty acids continue biodegradation by enzymatic 3 oxidation [410],... 

More recently, Heise and coworkers have shown that DKR can be combined with enzymatic polymerization for the synthesis of chiral polyesters from racemic secondary diols in one pot [34] (Figure 4.12). 

The enzymatic KR between racemic amines and nonactivated esters using a lipase as biocatalyst is shown in Scheme 7.15. In the same manner as in the transesterification of secondary alcohols, this process fits Kazlauskas rule [32], where normally if the large group (L) has larger priority than medium group (M), the (R)-amide is obtained. In general, major size differences between both groups result in better enantios-electivities ( ). 

Enzymatic oxidations have been reported. Bacilus stearothermophilus, for example, oxidizes secondary alcohols to the ketone. 

The DKR of secondary alcohols can be efficiently performed via enzymatic acylation coupled with simultaneous racemization of the substrates. This method was first used by BackvaU for the resolution of 1-phenylethanol and 1-indanol [38]. Racemization of substrate 18 by a mthenium catalyst (Scheme 5.11) was combined with transesterification using various acyl donors and catalyzed by C.antarctica B Hpase. From aU the acyl donors studied, 4-chlorophenyl acetate was found to be the best. The desired product 19 was obtained in 80% yield and over 99% ee. 

The method is not restricted to secondary aryl alcohols and very good results were also obtained for secondary diols [39], a- and S-hydroxyalkylphosphonates [40], 2-hydroxyalkyl sulfones [41], allylic alcohols [42], S-halo alcohols [43], aromatic chlorohydrins [44], functionalized y-hydroxy amides [45], 1,2-diarylethanols [46], and primary amines [47]. Recently, the synthetic potential of this method was expanded by application of an air-stable and recyclable racemization catalyst that is applicable to alcohol DKR at room temperature [48]. The catalyst type is not limited to organometallic ruthenium compounds. Recent report indicates that the in situ racemization of amines with thiyl radicals can also be combined with enzymatic acylation of amines [49]. It is clear that, in the future, other types of catalytic racemization processes will be used together with enzymatic processes. 

Stereoinversion Stereoinversion can be achieved either using a chemoenzymatic approach or a purely biocatalytic method. As an example of the former case, deracemization of secondary alcohols via enzymatic hydrolysis of their acetates may be mentioned. Thus, after the first step, kinetic resolution of a racemate, the enantiomeric alcohol resulting from hydrolysis of the fast reacting enantiomer of the substrate is chemically transformed into an activated ester, for example, by mesylation. The mixture of both esters is then subjected to basic hydrolysis. Each hydrolysis proceeds with different stereochemistry - the acetate is hydrolyzed with retention of configuration due to the attack of the hydroxy anion on the carbonyl carbon, and the mesylate - with inversion as a result of the attack of the hydroxy anion on the stereogenic carbon atom. As a result, a single enantiomer of the secondary alcohol is obtained (Scheme 5.12) [8, 50a]. 

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