Pseudomonas sp. strain 9816-4 grows with naphthalene as the sole source of carbon and energy (9). The initial reaction is catalyzed by a multicomponent enzyme system designated naphthalene dioxygenase (NDO) (11,12,23,24). NDO catalyzes the NAD(P)H-dependent enantiospecific incorporation of dioxygen into naphthalene to form (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (26, 27) ( Fig. 1). An analogous reaction is catalyzed by toluene dioxygenase (TDO) from Pseudomonas putida F1, where enantiomerically pure (ϩ)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) is the first detectable oxidation product (17,31,60). TDO also catalyzes the enantiospecific oxidation of naphthalene to (ϩ)-cis-naphthalene dihydrodiol (18,39).In addition to the enantiospecific oxidation of naphthalene and toluene, NDO and TDO from the above strains oxidize many related aromatic compounds to optically active dihydrodiols (10,18,28,30). Other bacterial dioxygenases show similar properties, and more than 130 chiral arene cis-dihydrodiols have been produced from a small number of strains (7,35,48). The high enantiomeric purity of these compounds has led to their use as chiral synthons in the enantiospecific synthesis of a wide variety of biologically active natural products (7,8,46,57). The present studies focus on another facet of this interesting group of dioxygenases, that is, their ability to catalyze reactions other than the formation of arene cis-dihydrodiols. For example, the TDO expressed by P. putida F39/D oxidizes indan to (1R)-indanol and oxidizes indene to cis-(1S,2R)-indandiol and (1S)-indenol (55). Similar reactions have been reported for TDO from P. putida UV4, although the 1-indenol produced by this strain is the (1R)-enantiomer (3, 5).We now report the identification and absolute stereochemistry of the products formed from indan and indene by NDO from Pseudomonas sp. strain 9816-4 and confirm earlier observations on the desaturation of indan to indene by NDO (22). MATERIALS AND METHODSOrganisms. Pseudomonas sp. strain 9816/11 is a mutant which oxidizes naphthalene stoichiometrically to (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (40). This organism is a derivative of Pseudomonas sp. strain 9816-4 (9, 59), which harbors the genes for naphthalene catabolism on an 83-kb NAH plasmid designated pDTG1 (45). Pseudomonas sp. strain 9816/C84, a cured strain, was used as a control in experiments with strain 9816/11. Escherichia coli strain JM109 (DE3)[pDTG141] contains the structural genes (nahAaAbAcAd) for NDO in plasmid pT7-5 (50). Expression of NDO in this strain is inducible by the addition of isopropylthiogalactopyranoside (IPTG). E. coli JM109(DE3)[pT7-5] was used as a control in experiments with strain JM109(DE3) [pDTG141].Biotransformation experiments. Strain 9816/11 was grown at 30ЊC in mineral salts basal medium (MSB) (49) with 0.2% (wt/vol) pyruvate as a carbon source in the presence of 0.05% (wt/vol) salicylate or anthranilate. These aromatic acids induce the s...

A two-step reaction sequence between artemisinin and methanolic ammonia followed by treatment with Amberlyst 15 yielded 11-azaartemisinin in 65% yield. Substituting a variety of primary alkyl- and heteroaromatic amines for ammonia in the reaction sequence yields N-substituted 11-azaartemisinins in similar or greater yield. When Amberlyst 15 is replaced by a mixture of sulfuric acid/silica gel, both 11-azaartemisinin and the expected metabolite, 10-azadesoxyartemisinin, are formed in 45% and 15% yields, respectively. In vitro and in vivo test data for a number of novel N-substituted 11-azaartemisinins, against drug-resistant strains of Plasmodium falciparum, show they possess antimalarial activities equal to or greater than that of artemisinin. The most active derivative, N-(2'-acetaldehydo)-11-azaartemisinin, 17, was 26 times more active in vitro and 4 times more active in vivo than artemisinin.

Bacterial strains expressing toluene and naphthalene dioxygenase were used to examine the sequence of reactions involved in the oxidation of 1,2-dihydronaphthalene. Toluene dioxygenase of Pseudomonas putida F39/D oxidizes 1,2-dihydronaphthalene to (؉)-cis-(1S,2R)-dihydroxy-1,2,3,4-tetrahydronaphthalene, (؉)-(1R)-hydroxy-1,2-dihydronaphthalene, and (؉)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, naphthalene dioxygenase of Pseudomonas sp. strain NCIB 9816/11 oxidizes 1,2-dihydronaphthalene to the opposite enantiomer, (؊)-cis-(1R,2S)-dihydroxy-1,2,3,4-tetrahydronaphthalene and the identical (؉)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. Recombinant Escherichia coli strains expressing the structural genes for toluene and naphthalene dioxygenases confirmed the involvement of these enzymes in the reactions catalyzed by strains F39/D and NCIB 9816/11. 1-Hydroxy-1,2-dihydronaphthalene was not formed by strains expressing naphthalene dioxygenase. These results coupled with time course studies and deuterium labelling experiments indicate that, in addition to direct dioxygenation of the olefin, both enzymes have the ability to desaturate (dehydrogenate) 1,2-dihydronaphthalene to naphthalene, which serves as a substrate for cis dihydroxylation.Pseudomonas putida F1 and Pseudomonas sp. strain NCIB 9816-4 initiate the oxidation of toluene and naphthalene by the addition of both atoms of molecular oxygen and two hydrogen atoms to the aromatic nucleus to form (ϩ)-cis-(1S,2R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol) (14, 21) and (ϩ)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene (cis-naphthalene dihydrodiol) (19,20), respectively. These reactions are catalyzed by multicomponent enzyme systems designated toluene dioxygenase (TDO) (16, 31) and naphthalene dioxygenase (NDO) (13).Current interest in TDO and NDO stems from the fact that, in addition to the enantiospecific reactions shown above, both enzymes also oxidize a wide range of substrate analogs to optically active products (5,6,10,11,25). Many of these compounds have been used as chiral synthons to synthesize a number of compounds of interest to the pharmaceutical and specialty chemical industries (references 9, 10, and 17 and references cited therein).In addition to forming arene cis-dihydrodiols, the TDO expressed by strain F39/D catalyzes the monohydroxylation of indan to (1R)-indanol (7, 29). The same reaction is catalyzed by TDO expressed by P. putida UV4, a strain studied extensively by Boyd and his colleagues (5, 6). In contrast, NDO from strain NCIB 9816/11 oxidizes indan to (1S)-indanol and also catalyzes the desaturation of indan to indene. The latter compound is then oxidized by the enzyme to (1S)-indenol and cis-(1R,2S)-indandiol (15). The TDOs expressed by F39/D and UV4 do not catalyze the desaturation of indan to indene (6,29). Strain UV4 does, however, oxidize 1,2-dihydronaphthalene (compound I, Fig. 1) to (ϩ)-(1R)-hydroxy-1,2-dihydronaphthalene (compound II), (ϩ)-cis-(1S,2R)-dihydroxy-1,2,3,4-tetrahydronaphthalene (compo...

No abstract

The biotransformation of 1-indanone and 2-indanone to hydroxyindanones was examined with bacterial strains expressing naphthalene dioxygenase (NDO) and toluene dioxygenase (TDO) as well as with purified enzyme components. Pseudomonas sp. strain 9816/11 cells, expressing NDO, oxidized 1-indanone to a mixture of 3-hydroxy-1-indanone (91%) and 2-hydroxy-1-indanone (9%o). The (R)-3-hydroxy-1-indanone was formed in 62% enantiomeric excess (ee) (R:S, 81:19), while the 2-hydroxy-1-indanone was racemic. The same cells also formed 2-hydroxy-1-indanone from 2-indanone. Purified NDO components oxidized 1-indanone and 2-indanone to the same products produced by strain 9816/11. P. puta F39/D cells, expressing TDO, oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 76% ee (R:S, 12:88) but did not oxidize 1-indanone eficiently. Purified TDO components also oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 90%Yo ee (R:S, 5:95) and failed to oxidize 1-indanone. Oxidation of 1-and 2-indanone in the presence of [18O]oxygen indicated that the hydroxyindanones were formed by the incorporation of a single atom of molecular oxygen (monooxygenation) rather than by the dioxygenation of enol tautomers of the ketone substrates. As alternatives to chemical synthesis, these biotransformations represent direct routes to 3-hydroxy-1-indanone and 2-hydroxy-l-indanone as the major products from 1-indanone and 2-indanone, respectively.