Jack G. Woolley

Biosynthesis of podophyllotoxin in Linum album cell cultures

Abstract. Cell cultures of Linum album Kotschy ex Boiss. (Linaceae) showing high accumulation of the lignan podophyllotoxin (PTOX) were established. Enzymological studies revealed highest activities of phenylalanine ammonia-lyase, cinnamyl alcohol dehydrogenase, 4-hydroxycinnamate:CoA ligase and cinnamoyl-CoA:NADP oxidoreductase immediately prior to PTOX accumulation. To investigate PTOX biosynthesis, feeding experiments were performed with [2-13C]3′,4′-dimethoxycinnamic acid, [2-13C]3′,4′-methylenedioxycinnamic acid (MDCA), [2-13C]3′,4′,5′-trimethoxycinnamic acid, [2-13C]sinapic acid, [2-13C]- and [2,3-13C2]ferulic acid. Analysis of the metabolites by HPLC coupled to tandem mass spectrometry revealed incorporation of label from ferulic acid into PTOX and deoxypodophyllotoxin (DOP). In addition, MDCA was also unambiguously incorporated intact into PTOX. These observations suggest that in L. album both ferulic acid and methylenedioxy-substituted cinnamic acid can be incorporated into lignans. Furthermore, it appears that, in this species, the hydroxylation of DOP is a rate-limiting point in the pathway leading to PTOX.

Enhancement of artemisinin concentration and yield in response to optimization of nitrogen and potassium supply to Artemisia annua

BACKGROUND AND AIMS The resurgence of malaria, particularly in the developing world, is considerable and exacerbated by the development of single-gene multi-drug resistances to chemicals such as chloroquinone. Drug therapies, as recommended by the World Health Organization, now include the use of antimalarial compounds derived from Artemisia annua--in particular, the use of artemisinin-based ingredients. Despite our limited knowledge of its mode of action or biosynthesis there is a need to secure a supply and enhance yields of artemisinin. The present study aims to determine how plant biomass can be enhanced while maximizing artemisinin concentration by understanding the plants nutritional requirements for nitrogen and potassium. METHODS Experiments were carried out, the first with differing concentrations of nitrogen, at 6, 31, 56, 106, 206 or 306 mg L(-1) being applied, while the other differing in potassium concentration (51, 153 or 301 mg L(-1)). Nutrients were supplied in irrigation water to plants in pots and after a growth period biomass production and leaf artemisinin concentration were measured. These data were used to determine optimal nutrient requirements for artemisinin yield. KEY RESULTS Nitrogen nutrition enhanced plant nitrogen concentration and biomass production successively up to 106 mg N L(-1) for biomass and 206 mg N L(-1) for leaf nitrogen; further increases in nitrogen had no influence. Artemisinin concentration in dried leaf material, measured by HPLC mass spectroscopy, was maximal at a nitrogen application of 106 mg L(-1), but declined at higher concentrations. Increasing potassium application from 51 to 153 mg L(-1) increased total plant biomass, but not at higher applications. Potassium application enhanced leaf potassium concentration, but there was no effect on leaf artemisinin concentration or leaf artemisinin yield. CONCLUSIONS Artemisinin concentration declined beyond an optimal point with increasing plant nitrogen concentration. Maximization of artemisinin yield (amount per plant) requires optimization of plant biomass via control of nitrogen nutrition.

Plant cell factories as a source for anti-cancer lignans

The review places podophyllotoxin, a powerful anti-cancer material used in clinical treatment of small cell cancers, in focus. The economical synthesis of podophyllotoxin is not feasible and demand for this material outstrips supply. At present, Podophyllum hexandrum (Indian May apple) is the commercial source but it grows in an inhospitable region (the Himalayas) where it is collected from wild stands. Furthermore, the plant is now an endangered species. Alternative sources of podophyllotoxin are considered, e.g., the supply of podophyllotoxin and related lignans by establishing plant cell cultures that can be grown in fermentation vessels. Increase of product yields, by variation of medium and culture conditions or by varying the channelling of precursors into side-branches of the biosynthetic pathway by molecular approaches, are discussed.

Phygrine, an alkaloid from Physalis species

Abstract A new alkaloid, phygrine, isolated from the roots and aerial parts of Physalis alkekengi has been shown to be bis-hygrine or 1-[1′-methylpyrrolidine-2′-yl]-3-[1″-methyl-2″-(2″-oxopropyl)pyrrolidine-5″-yl]propan-2-one. The alkaloid is also present in P. angulata, P. philadelphica, P. ixocarpa, P. edulis, P. peruviana, P. minima, P. pubescens, P. viscosa and P. pruinosa.

Phenyllactic acid but not tropic acid is an intermediate in the biosynthesis of tropane alkaloids in Datura and Brugmansia transformed root cultures

Abstract(S)-(-)-Tropic acid is the acidic moiety of the tropane ester alkaloids, hyoscyamine and scopolamine (hyoscine). When tropic acid is fed to transformed root cultures of Datura stramonium L. or a Brugmansia (Datura) Candida x B. aurea hybrid, the formation of these alkaloids is inhibited. Phenyllactic acid, from which the tropoyl moiety is derived, is considerably less inhibitory. Label from (RS)-phenyl[1,3-13C2]lactic acid is incorporated at high levels into apoatropine, littorine, aposcopolamine, hyoscyamine, 7β-hydroxyapoatropine, scopolamine and 7β-hydroxyhyoscyamine when fed to these cultures. The presence of an excess concentration of unlabelled tropic acid has little influence on the specific incorporation into these products. It is concluded that free tropic acid is not an intermediate in hyoscyamine biosynthesis but rather that the rearrangement of phenyllactic acid occurs subsequent to its esterification.

The rearrangement of phenyllactate in the biosynthesis of tropic acid

Abstract Phenyl[1,3-13C2]lactic acid was synthesized and fed to Datura stramonium via the wick method and via the roots. Hyoscine (scopolamine) and hyoscyamine were isolated, and examination of their 13C NMR spectra showed spin-spin coupling from the newly formed contiguous centres, indicating that tropic acid is formed by an intramolecular rearrangement of phenyllactate.

The obligatory role of phenyllactate in the biosynthesis of tropic acid

Abstract Phenyl[1-14C] lactic acid and phenyl [2-3H] lactic acid were synthesized by conventional routes and fed in admixture (3H:14C ratio 10:1) to Datura stramonium via the roots. Hyoscine (scopolamine) and hyoscyamine were isolated and found to have essentially the same 3H:14C ratios as the precursor (8.5 and 10.15, respectively). Hydrolysis of hyoscyamine showed that all the activity was present in the tropic acid moiety. The tropic acid was converted to atropic acid and then cleaved by means of osmium tetroxide and sodium metaperiodate to yield formaldehyde containing all the tritium and phenylglyoxylic acid containing all the 14C, a result which shows that the tritium at C-2 of the phenyllactate precursor labelled tropic acid in the hydroxymethyl group. These findings show that phenyllactate is an obligatory intermediate in the biosynthesis of tropic acid and that the rearrangement of the side-chain takes place at the phenyllactate and not the phenylpyruvate level.

The biosynthesis of hyoscyamine: the process by which littorine rearranges to hyoscyamine

The incorporation of isotope from specifically-labelled 3-phenyllactic acid 4 or littorine 7 into 3α-phenylacetoxytropane 10, 3α-phenylacetoxy-6β,7β-epoxytropane and 3α-(2′-hydroxyacetoxy)-tropane 9 has been demonstrated. Transformed root cultures of Datura stramonium or Brugmansia (Datura) Candida x B. aurea incorporated fed (RS)-3-phenyl[1,3-13C2]lactic acid 4 into 3α-phenylacetoxytropane 10 and 3α-phenylacetoxy-6β,7β-epoxytropane wild the efficient retention of both 13C nuclei. In contrast, no label was incorporated into these two compounds from (RS)-3-pheny[2-13C2-2H]lactate 4. From this evidence it can be deduced that 3-phenyllactic acid 4 is not incorporated into 3α-phenylacetoxytropane 10via free phenylacetic acid 6, a route which would result in the loss of the C-1 of 3-phenyllactic acid 4. Furthermore, (RS)-(3′-phenyl[1′,3′-13C2]lactoyl)[methyl-2H3]tropine (littorine 7) was incorporated into 3α-phenylacetoxytropane 10, at up to 4% specific incorporation, with the retention of all the 13C and 2H nuclei. Label was also incorporated into 3α-(2′-hydroxyacetoxy)tropane 9 from (RS)-3-phenyl[1,3-13C2]lactic acid 4 and (RS)-(3′-phenyl[1′,3′-l3C2]lactoyl)[methyl-2H3]tropine 7. We propose, on the basis of these observations, a putative process for the rearrangement of littorine 7 to hyoscyamine 8 and suggest that both 3α-phenylacetoxytropane 10 and 3α-(2′-hydroxyacetoxy)tropane 9 arise as by-products of the rearrangement process.

Screening a diverse collection of Artemisia annua germplasm accessions for the antimalarial compound, artemisinin

The anti-malarial drug artemisinin is commercially extracted from the medicinal plant Artemisia annua (L.). Here we report the screening of seventy A. annua individuals sourced from around the world, identifying individuals containing > 2% artemisinin, concentrations approximately twice as high as have been previously reported. These extremely high yielding individuals have been maintained as propagational clones, and represent promising parental lines for future A. annua breeding programmes.

The biosynthesis of tropic acid. Part 6. Enantioselective, intact incorporation of (R)-(+)-3-phenyllactic acid into the tropic acid ester alkaloids of Datura

(R)-(+)-11 and (S)-(–)3-Phenyl[1,3-13C2; 1-14C] lactic acid 13 were fed separately to Datura stramonium(Solanaceae)via the wick method. In a second feeding experiment (R)-(+)- and (S)-(–)-3-phenyl [1,3-13C2; 2-14C] lactic acid were administered via the roots. In both cases hyoscine (scopolamine) and hyoscyamine 1 and 2, respectively were isolated separately from the roots and aerial parts. The (R)-enantiomer 11 was more efficiently incorporated into the (S)-(–)-tropic acid 8 moiety of both bases. Examination of the 13C NMR spectra of the bases and of tropic acid, obtained by hydrolysis of hyoscyamine, showed 13C–13C spin–spin coupling indicating that the tropoyl ester (–)-hyoscyamine 9 is formed by direct rearrangement of the (R)-(+)-3-phenyllactic acid 11 ester of tropine (littorine).