Oxidoreductases

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Microorganisms/enzymes have the ability to effect chemical oxidation on a wide variety of substrates, many of which occur with chemo-, regio- or

Figure 4.7 Baeyer-Villiger oxidation.

Figure 4.7 Baeyer-Villiger oxidation.

stereoselectivity, unattainable by conventional chemical methods. In fact, hydroxylations of the alicyclic, aliphatic, and aromatic systems are the single most important reactions effectively catalyzed by the enzyme/microorganisms. Regio- and stereoselective hydroxylations in steroids by the biocata-lysts led to the unprecedented application of microorganisms as reagents in organic synthesis. Similar encouraging results were observed in the reactions incorporating a hydroxyl group on an unactivated carbon in sesquiterpenes, which are of value to the perfumery and pharmaceutical industry. Though these reactions are not high yielding, they provide valuable intermediates in the synthesis of complex organic compounds. These biooxidation compounds are also valuable in assessing the quantitative structure activity relations of the pharmacophore. A number of reports on regiospecific hydroxylation of monoterpene have appeared in the literature. Biocatalytic oxygenation reactions are becoming increasingly important since conventional methodology is either not feasible or makes use of hypervalent metal oxides, which are ecologically undesirable when used on a large scale. As the use of isolated oxygenases will be always hampered due to their requirement of NAD(P)H recycling, many useful oxygenation reactions such as mono- and di-hydrox-ylations, epoxidation, sulphoxidation, and Baeyer-Villiger reactions will continue to be performed using whole cell systems. The study of the stereo-and regioselectivity of these reactions is very valuable to the understanding of the mechanism of the enzymatic transformations, also providing viable alternatives to the general reagents.

4.3.1 Oxidoreductases: Salient Features:

1. This class of enzymes is the largest one, which catalyze oxidation or reduction of the substrate. The general form of the equation being

2. The oxidation-reduction is brought about by the transfer of two reducing equivalents (electrons followed by proton uptake or hydrogen molecule uptake) from one substrate to another.

3. These operate via redox cofactors (NADH, NADPH, FAD, etc.)

4. The transfer of these two reducing equivalents (two electrons) may be stepwise or in a single step. When flavin (FAD) groups are present as the prosthetic groups (flavoproteins, cytochrome oxidases, ferrodoxins, etc.), one equivalent oxidoreductions are effected.

5. These enzymes form the backbone for most of the catabolic processes in the microorganism and the animal systems.

6. These transformations are commonly observed in the catabolism of the endogenous and exogenous compounds (xenobiotics and drugs)

in the blood stream by the enzymes of the liver microsomes. In fact, oxidative transformations are by far the most important in drug metabolism. For example, tetrahydro cannabinol (THC) is oxidized to THC-7-oic acid by the cytochrome P-450 enzymes in the liver microsomes (Fig. 4.8).

7. Since, these reactions involve the transfer of electrons followed by proton uptake, the overall reaction will vary with pH.

8. This class of enzymes includes a. Dehydrogenases: Those that remove a molecule of hydrogen from the substrate. Since these reactions are reversible, these enzymes can be also made to add a molecule of hydrogen to the substrate. These are also referred to as oxidases.

b. Oxidases: Those that remove a molecule of hydrogen from the substrate, the acceptor being oxygen (O2).

c. Peroxidases: Those that use hydrogen peroxide (H2O2) as the oxidant.

d. Oxygenases: Those that incorporate molecular oxygen into the substrate. These are two types, viz., monooxygenases and dioxygenases.

e. Hydroxylases: Those that incorporate oxygen atom, sometimes a hydroxyl group.

9. Since these require cofactors for their activity, when isolated enzymatic transformation is planned, cofactor recycling or supply be-

rif. cm,on

HvdiüW-TIIC
THC-7-nie-a cid

Figure 4.8 Metabolism of tetrahydro cannabinol.

comes another important component of experimental design. To overcome this additional cumbersome procedure, it is usually common that whole cell systems (yeasts, bacteria, and fungi) are used for these reactions, albeit, a small trade-off with the enantiomeric purity in chiral reduction. All the same, it should be borne in mind that an astonishing variety of optically pure compounds are synthesized by this whole cell biocatalytic approach.

4.3.2 Oxygenases-Aromatic Hydroxylation

The two main enzyme systems capable of oxygen insertion into organic molecules are the mono oxygenases (EC.1.13.12) and dioxygenases (EC.1.13.11). These are versatile, not highly specific enzymes (because a lot of isozymes are known). These catalyze the oxidation/ hydroxylation of a variety of substrates.

The ubiquitous monooxygenases are the cytochrome-P-450 (cyto-chrome-b) haemoproteins dependent on NADH or NADPH, in the Eukaryotic organisms. The catalytic cycle is as shown in Scheme 4.1. The crucial step is the transfer of oxygen from the highly electron deficient [FeO]4+ complex to the substrate to yield the corresponding hydroxylated substrate. Instead of molecular oxygen, other oxygen atom donors such as alkyl hydroperoxides, peracids, iodosobenzene, amine oxides, hydrogen peroxide, sodium periodate, or sodium perchlorate can also bring about the hydroxylation. Note:

Scheme 4.1 Catalytic cycle of monooxygenases.
Figure 4.9 Catalytic cycle of FAD.

the site of the reaction on the substrate is the high electron density moiety.

The flavin-dependent monooxygenases use flavin as the cofactor. The catalytic cycle is as shown in the Fig 4.9. The crucial step is the transfer of oxygen to the substrate by the nucleophillic attack of the peroxide anion intermediate of the oxidized enzyme. Hence, the site of reaction on the substrate is the electron deficient center, such as the carbonyl carbon, or an electronegative heteroatom.

These monooxygenases (CytP-450) are known to react with a variety of organic substrates. Some of the widely known being (1) arenes to arenols, (2) alkene to epoxide, (3) alkane C-H to alcohols (hydrox-ylation of saturated carbon), (4) ethers to alcohols, (5) N-alkyl moieties to amines, (6) N-oxide formation of secondary amines, (7) thio ethers to thiols, and (8) S and N oxides from thio ethers and secondary amines, respectively. A graphical account of some of the common transformations is as given in Fig 4.10.

The aromatic hydroxylation by the monooxygenases (CytP-450) is well studied. It has been shown that almost all aromatic hydroxyla-tion reactions proceed through an epoxide intermediate, an '' arene oxide.'' This rearranges in two ways as shown in Fig 4.11.

The arene oxide intermediate formed spontaneously rearranges to a carbo-cation intermediate. The carbocation formed is the most resonance stabilized

Figure 4.10 Reactions catalyzed my monooxygenases.

carbocation. (See Box 4.1, for the mesomeric stabilization of the carbocation.) This then undergoes an NIH shift to give the arenols. Note that the hydroxylation has occurred para to the ring substituent. Alternatively, the arene oxide also opens up in the presence of water to give trans-dihydrodiols, which may also further get oxidized in the presence of cofactors (NADP + ) to yield a catechol derivative (Fig 4.12).

carbocation intermediate

Figure 4.11 Mechanism of aromatic hydroxylation by monooxygenases.

carbocation intermediate

Figure 4.11 Mechanism of aromatic hydroxylation by monooxygenases.

Box 4.1 Mesomeric stabilization of the carbocation.

Phenols and substituted phenols are hydroxylated to give catechol derivatives by polyphenol oxidase enzymes (copper containing monophenol monooxygenases, EC.1.10.3.1).

In the prokaryotic organisms (bacteria), the hydroxylation is brought about by the cycloaddition of molecular oxygen with a double bond to yield a dioxetan. This is then reduced to cis-dihydro diol. Rearomatization by dihydro diol dehydrogenase yields catechol derivative (Fig 4.13).

It is reported that toluene and its derivatives were first oxidized to benzoic acid, which then yielded the catechol via the cis-1,2-diol intermediate (Fig 4.14).

Studies on bacterial metabolism of aromatic compounds indicated benzylic hydroxylation. Dioxygenases are also known to induce a radical formation. Boyd et al. (6). have observed the transformation of 1,2-dihydronapthalene to (1R,2S)-cis-1,2-dihydro napthalene-1,2-diol via the triol intermediate (believed to have arisen by the benzylic hydroxylation), using the growing cell cultures of Pseudomonas

Iruns-dihydrodkils catechol derivative

Figure 4.12 Catechol formation from dihydrodiols.

Iruns-dihydrodkils catechol derivative

Figure 4.12 Catechol formation from dihydrodiols.

Figure 4.13 Mechanism of aromatic hydroxylations by dioxygenases.
Figure 4.14 Biotransformation of toluene and its derivatives.
Figure 4.15 Transformation of 1,2-dihydro naphthalene by P. putida.

putida UV4 (Fig 4.15). A schematic presentation of the mechanism of dioxyegenase action is given in Scheme 4.2.

4.3.3 Summary of the Oxygenation of Aromatic Rings by Mono- and Dioxygenases

1. Electron-rich unsaturation (unsaturation with electron pumping substituents, i.e., OH, OR, NH2, NHR, alkyl) is susceptible to oxidation either by a mono- or dioxygenase more readily than the electron-deficient unsaturation (unsaturation with electron-withdrawing substituents, i.e., NO2, NHCOR, halogens, SO2R groups)

Scheme 4.2 Catalytic cycle of dioxygenases.

7-cthoxy tonm.inn

Figure 4.16 Epoxidation of unsaturation having higher electron density.

7-cthoxy tonm.inn

Figure 4.16 Epoxidation of unsaturation having higher electron density.

because it is from the highly electron deficient [FeO]4+ complex that the transfer of oxygen to the substrate occurs. Therefore, the substrate should be able to readily donate the electrons. This can be appreciated better when we consider the conversion of precocene by Streptomyces griseus to three hydroxylated metabolites, all of which are believed to arise from the epoxide intermediate. However, the same organism could only bring about o-dealkylation of 7-ethoxy coumarin. Note that the C3, C4 unsaturation is being withdrawn due to the lactone carbonyl group in 7-ethoxy coumarin while it is more or less unconjugated in precocene hence having high electron density (Fig 4.16).

2. Aromatic hydroxylation reactions by NADH-dependent monoox-ygenases or dioxygenases appear to proceed more readily in activated (electron rich) rings, whereas deactivated aromatic rings are generally slow or resistant to hydroxylation. For example, when a substituted chlorobenzene (deactivated ring) was incubated with

Figure 4.17 Benzylic hydroxylation.

Asperigillus selerotiorum, only the benzyl alcohol derivative was obtained (Fig 4.17).

4.3.4 Dioxygenases

1. Unlike the NADH-dependent monooxygenases, dioxygenases selectivity for either electron-rich or electron-deficient unsaturation is not as much.

2. Benzylic hydroxylation is also observed.

4.3.5 Epoxidation

Stereoselective epoxidations even on an unactivated alkene are routinely achieved by biocatalytic means.

The enantiospecific epoxidation of di-substituted (terminal) and tri-substituted alkenes have been reported.

Different organisms have varying regioslectivity of alkene epoxidation. Cyclic and internal olefins, aromatic compounds, and alkene units, which are conjugated to an aromatic system, are not epoxidized by hydroxylase of Psuedomonas oleovorans, while the monooxygenases of Cunnighamella blaksleena or Curvularia lunata were able to epo-xidize endocyclic alkenes.

When more than one alkene group is present in the substrate molecule, the rate of epoxidation of the alkene with greater electron density is faster. For instance, 3-oxo-4,8-diene steroid derivatives on incubation with Cunnighamella blakslena gave only the 8,11-epoxide (Fig 4.18). The electron density of the 4,10-alkene being conjugated to the 3-keto group is less as compared to the 8,11-alkene, which is isolated and hence electron dense.

Though epoxidation is highly stereoface selective, when whole cell systems are used, the further transformations of the epoxide to a diol are a competing side reaction.

Figure 4.18 Selective epoxidation of unsaturation with higher electron density.

Epoxides formed during the transformation are normally toxic to the cell. Hence, cosolvent (organic solvents) procedures, wherein the epoxide formed is selectively partitioned into the organic layer, are adopted. This helps in limiting the concentration of the toxic metabolite in the reaction medium (aqueous), thus increasing the yields in the whole cell epoxidations.

(R) epoxides are normally formed. The formation of (R) epoxides is accounted for by preliminary hydrophobic bonding of the alkene to the active site in a such a way as to ensure that the epoxide closure must be from the Si face of C-2.

Chiral epoxides of predictable absolute configurations can also be obtained from halohydrins produced by chloroperoxidase-catalyzed addition of hypohalous acids to double bonds. The enzyme utilizes iodide, bromide, and chloride (Fig 4.19).

4.3.6 Hydroxylation at Allylic and Benzylic Centers

Biocatalytic procedures for the stereo- and regiospecific hydroxylation at activated or unactivated carbon centers are irreplaceable by chemical means, be it in the hydroxylation of steroids or the hydroxylation of benzyllic/allylic centers. In the case of hydroxylation of allylic centers, epoxidation and the corresponding diol formation are significant side reactions.

It is believed that at some stage the hydrocarbon residue is converted into a short-lived (<109 s) free radical.

Since, benzyllic and allylic radicals are resonance stabilized (hence more easily formed), hydroxylations at the benzyllic and allylic carbon atoms is very common. In fact, it is one of the major ways of drug

Figure 4.19 Chloroperoxidase catalyzed epoxidation.

detoxification pathways in the liver microsomes by the mixed function monooxygenases present there (Fig 4.20).

The stability of the radical formed (though for a short period) is of paramount importance in deciding the direction of the reaction.

Geraniol and nerol did not undergo any notable bioconversions when incubated with the cultures of Asperigillus niger, while the corresponding acetates led to exclusive hydroxylation at allylic position (C-8). This may be partly because the hydroxyl is not lipophillic and the monooxygenases being membrane bound demand a certain hy-drophilic/lipophillic balance for the approach and concominant transformation (Fig 4.21).

As discussed earlier both the steric and the electronic factors are to be weighed to predict the outcome of a reaction. Therefore, just the presence of an allylic CH does not vouchsafe allylic hydroxylation. Grindelic acid on incubation with cultures of Asperigillus niger and

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Ltehrisoquin ( ;i n l i-hypertensive )

-tixyge nil se s rV" N

M In

o o monu-oxvucnasca

OH

Hcxobarbiltil (scda live-hypnotic)

A nigcr

OOOH

Figure 4.20 Benzylic and allylic hydroxylations.

Figure 4.21 Enhancement in transformation by lipophillic derivatization.

Sordaria bombioidee, did not yield any allylic hydroxylation, but instead gave the 3-hydroxy grindelic acid (Fig 4.22).

4.3.7 Hydroxylation at Unactivated C—H

In the field of steroid hydroxylations virtually any carbon center in the steroid nucleus can be hydroxylated stereospecifically using a range of microorganisms. To a very large extent, fungi are of greatest use in the hydroxylations of steroids, and five genera in particular have been found to be

Figure 4.22 Transformation of Grindelic acid.

Table 4.2 Hydroxylation of Steroids

Microorganism

Product

Regioselectivity

Asperigillus ochraceus Asperigillus ochraceus spores Asperigillus phoenicis

11a-OH 11a-OH 11a-OH 11a-OH 11 b-OH

Rhizopus nigricans Curvularia lunata Calonectria decora Rhizopus arrhizus

6b-OH

7b/7b-OH

12b, 15 a- dihydroxylation

Cunninghanella elegans

Stereoselectivity

Calonectria decora Curvularia lunata Rhizopus nigricans

Equatorial —OH

Equatorial — OH

Axial and equatorial —OH

Cunninghanella elegans extremely useful, viz., Rhizopus, Calonectria, Asperigillus, Curvularia, and Cunninghamella.

A number of microorganisms show a tendency to hydroxylate in certain positions of the steroid, irrespective of substituent patterns. The regioselectivity and the stereoselectivity of some of the well-known transformations are as given in the Table 4.2.

Functional groups, especially polar groups help in increasing the yield of the hydroxylated products. Probably, these groups enhance the water solubility of the steroid substrates, thus increasing the catalytic efficiency.

The unique selectivity of the organisms can be used effectively to bring about multiple transformations on the substrate. For example, the fermentation of the steroid (shown in Fig 4.23) with Pellicularia

Figure 4.23 Multiple transformations.

Figure 4.23 Multiple transformations.

filamentosa and Bacillus lentus, brought about 11-b-hydroxylation along with a A'-dehydrogenation.

Most of the mixed function oxygenase-catalyzed transformations have a radical intermediate.

The stability of carbon radicals is tertiary > secondary > primary. But, this is slightly modified in the case of bicyclic and fused alicyclic systems: since tertiary carbon radical now is more strained, the stability of carbon radicals in such systems is secondary >> primary > tertiary. We observe the similar trend in steroidal hydroxylations, indicating that a radical intermediate may be formed.

In cases where a tertiary radical can be formed, it is preferred. 1,4-Cineole gives 8-hydroxy cineole on incubation with Streptomyces grieseus as the major product along with 2-exo- and 2-endo-cineoles as minor products (Fig 4.24).

Dioxygenases add molecular oxygen to the substrate. There is probably no formation of a radical intermediate. Hence, streroidal and un-activated carbon hydroxylations by prokaryotic organisms (having dioxygenases) are rare.

Microbial oxidation of alkanes can take place at terminal carbon, in which case an alcohol is the product, or at subterminal position to give either a secondary alcohol or a ketone. Most of these undergo further oxidation and get degraded (metabolized). In order to isolate the primary oxidative products, mutant strains of the microorganisms have to be used.

Monooxygenases are known to bring about hydroxylation at CH position with inversion of stereochemistry.

b-Hydroxylation of short-chain aliphatic carboxylic acids can be accomplished by a number of microorganisms such as Endomyces reesii, Trchosporum fermentans, Torulopsis candida, Micrococus fla-vus, Candida rugosa, baker's yeast (Saccharomyces cerevisiae), Rho-dococus spp., and Pseudomonas putida.

Figure 4.24 Transformation of Cineole.

cine ok:

Figure 4.24 Transformation of Cineole.

The enantiotopic discrimination of hydrogens during oxidation of un-activated C-H bonds by microorganisms is synthetically very useful. For example, (R)-3-hydroxy butanoic acid obtained on incubation of the cultures of Candida rugosa with n-butanoic acid is a versatile homochiral synthon (Fig 4.25).

4.3.8 Reductions of Carbonyl Groups

Enzymes can operate stereospecifically on one of the two enantiotopic or diastereotopic faces of planar groups, such as C=C, C=N, or C=O. With a few exceptions, such as conjugated carbonyl functional groups, stereospecific reduction of an aldehyde or ketone in virtually any molecule can be effected either enzymatically or microbially. The asymmetric reduction of carbonyl compounds by microorganisms, a method outside the traditional arena of chemical synthesis, is now well recognized as an invaluable tool for the preparation of chiral alcohols. Because a single microorganism contains a number of oxidoreductases, it can mediate the reduction of a variety of artificial ketones to produce chiral alcohols of remarkable optical purity. Most of the oxidoreductases use NADH or NADPH as cofactor: hence, use of isolated pure enzyme system necessitates the regeneration of this cofactor, while the use of whole cell systems are devoid of this component. Since the whole cell systems have more than one oxidoreductase with varying enantiospeci-ficities, racemic mixtures are obtained. Be that as it may, various methods such as the following may be adopted to circumvent the problem of the multiple transformations and still use whole cell transformations:

Extensive screening to identify the micoorganism with a single, most active enzyme

Extractive biocatalysis (use of hydrophobic polymer resin for product adsorption)

Recombinant DNA techniques to create a set of strains in which the enzyme of choice is overexpressed or the other enzymes are suppressed (absent)

Expressing individual reductases in a heterologous host, such as Escherichia coli

The Prelog school (7) has investigated the purified NADPH specific dihy-droxyacetone reductase of Mucor javanicus, extensively. This enzyme is

Figure 4.25 Synthesis of (R)-3-hydroxy butanoic acid.

COOH

COOH

Figure 4.25 Synthesis of (R)-3-hydroxy butanoic acid.

Figure 4.26 Prelog rule [R=CH2COOC2H5, (CH2)2CH=CH2, aryl, pyridyl; R'=CH3, ch2cooh, cf3, CH3].

Figure 4.26 Prelog rule [R=CH2COOC2H5, (CH2)2CH=CH2, aryl, pyridyl; R'=CH3, ch2cooh, cf3, CH3].

known to transfer hydrogen exclusively to the Si face of the carbonyl group of most substrates giving (R) alcohols. From the product analysis of the reduction of 2-alkanones, it was concluded that the reaction rate and specificity increased with the increase in alkyl chain length. Also, it was observed that a sufficiently large hydrophobic group is needed for higher stereoselectivity, that is, for getting higher ee values. The stereochemical course of reduction of acyclic ketones by baker's yeast (Saccharomyces cerevisiae), on the other hand, proceeds via hydrogen transfer to the Re face of the prochiral ketone, giving an (S) alcohol.

The stereospecificities of yeast alcohol dehydrogenases-catalyzed reductions of ketones can be predicted by the Prelog rule. This was initially postulated for reductions of decalones by Curvularia lunata. This states that when groups R and R' (the two substituents on either side of carbonyl group) are sterically larger (L) and smaller (S), respectively, as given in Fig 4.26, the hydride equivalent is delivered to the Re face of the carbonyl group as defined by the Cahn-Ingold-Prelog (CIP) priority sequence of oxygen > L > S. This rule also applies to other oxidoreductases, such as horse liver alcohol dehydrogenase.

As indicated earlier, other alcohol dehydrogenases having opposite stereospecificities also are common. Hence, either enantiomer of an alcohol

Figure 4.27 YAD having opposite stereospecificities.
Figure 4.28 Reductions of b-keto acids.

can be produced at will by selecting enzymes with opposite enantiotopic face specificity for the same carbonyl substrate. Yeast contains two fatty acid synthetases with such properties. For example, keto esters are reduced to the R alcohols by the D enzyme and to the S alcohols by the L enzyme (Fig 4.27). The chiralities of the hydroxy products can be predicted for both the enzymes using a rule based on steric size distinctions between R and R'.

The stereochemical course of the reductions of b-keto carboxylic acid derivatives by yeast is influenced by substituents at both the ends of the molecule. While the enantioselective reductions are dependent on the affinity of the competing enzymes of opposite chirality, the enzymes affording (S) alcohols appear to prefer large hydrophobic substituents at the carboxy end, whereas (D) enzymes appear to prefer those at the hydrocarbon end (Fig 4.28).

Substituted cyclic ketones have also been reduced stereospecifically. Brooks et al. (8), in their study designed to establish a relationship between enantioselectivity and size differences of the alkyl substituents at position 2 of cyclic ,3-diketones, found that the enantioselectivity of the reaction depends

Figure 4.29 Reduction of cyclic ketones.

not only on the alkyl substituents but also on the ring size. In cyclopentane series, the (2S,3S) isomers were the major species, whereas the (2R,3S) isomers predominated in the cyclohexane series (Fig 4.29).

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