Isolation and Structure Elucidation of Additional Alkaloids from the Tropical Rainforest Tree Galbulimima baccata.

The structures of five new natural products (GB 27-GB 31, 1-5), isolated as minor components from the bark of Galbulimima baccata, have been determined by 2D NMR spectroscopy in combination with DFT calculations. Among the alkaloids, GB 31 (5) belongs to Class I, GB 27 (1) and 28 (2) belong to Class II, and GB 30 (4) belongs to Class III GB alkaloids. GB 31 is the first non-nitrogen-containing GB "alkaloid", being a biosynthetic oxidation product of himbacine, himandravine, or himbeline. GB 29 (3) has an entirely new natural product scaffold but belongs to Class IV (miscellaneous alkaloids). The isolation of a new Galbulimima scaffold has revealed a new pathway in the biosynthesis of the GB alkaloids. The new molecules isolated have shed further light on the biogenetic relationship among these structurally unique and complex groups of alkaloids. We present, for the first time, a unified biogenesis for the GB alkaloids that were first isolated in the 1950s and now number over 40 examples. This work also brings full circle the story of Galbulimima alkaloids. A life-long project of Wal Taylor involving one of his first students (Lew Mander) and one of his last students (Peter Karuso), a story stretching over six decades, has come to a final conclusion.

Syntheses of Gibberellins A15 and A24, the Key Metabolites in Gibberellin Biosynthesis.

Gibberellins (GAs) are essential phytohormones involved in numerous aspects of plant growth and development. Notably, the biochemistry and genetics of GA biosynthesis, which is associated with their endogenous regulation, have been largely resolved; however, a crucial unsolved question remains: the precise mechanism of the stepwise oxidation and subsequent removal of C-20 from C20 precursors, leading to bioactive C19 gibberellins, is still unresolved. To satisfy numerous requests from biologists, practical preparations of certain GAs that were isolated in miniscule quantities are highly demanded. Herein, we report the first practical syntheses of GA15 and GA24, the key C20 metabolites in gibberellin biosynthesis, from commercially available GA3. The protocols are robust and offer the capacity to produce GA24 and GA15 under gram scales in high overall yields and thus aid in further biological and related studies.

Structures of New Alkaloids from Rain Forest Trees Galbulimima belgraveana and Galbulimima baccata in Papua New Guinea, Indonesia, and Northern Australia.

Following on our 60-year research on the chemical constituents of the rain forest trees Galbulimima belgraveana and Galbulimima baccata, we report the isolation of seven new alkaloids: GB14 (14), GB22 (15), GB25 (16), GB21 (17), GB23 (18), GB24 (19), and GB26 (20). Their structures were elucidated by a combination of spectroscopic analyses and single-crystal X-ray crystallography, as well as structure degradation and interconversion. The newly isolated alkaloids are precursors or derivatives of the known family members from our early studies and could be intermediates in the biosynthesis of the Galbulimima alkaloids. Therefore, the present study has expanded the range of structures in this family of alkaloids and provided some missing links in the biosynthetic sequences.

Functional analysis of Arabidopsis CYP714A1 and CYP714A2 reveals that they are distinct gibberellin modification enzymes.

Endogenous levels of bioactive gibberellins (GAs) are controlled by both biosynthetic and inactivation processes, and some cytochrome P450s are involved in this control mechanism. We have previously reported that CYP714B1 and CYP714B2 encode the enzyme GA 13-oxidase, which is required for GA1 biosynthesis, and that CYP714D1 encodes GA 16α,17-epoxidase, which inactivates the non-13-hydroxy GAs in rice. Arabidopsis has two CYP714 members, CYP714A1 and CYP714A2. To clarify the possible role of these genes in GA metabolism, enzymatic activities of their recombinant proteins were analyzed using a yeast expression system. We found that the recombinant CYP714A1 protein catalyzes the conversion of GA12 to 16-carboxylated GA12 (16-carboxy-16ß,17-dihydro GA12), a previously unidentified GA metabolite. Bioassays of this GA product showed that CYP714A1 is an inactivation enzyme in Arabidopsis. This was confirmed by the extreme GA-deficient dwarf phenotype shown by CYP714A1-overexpressing plants. Intriguingly, the recombinant CYP714A2 protein catalyzed the conversion of ent-kaurenoic acid into steviol (ent-13-hydroxy kaurenoic acid). When GA12 was used as a substrate for CYP714A2, 12α-hydroxy GA12 (GA111) was produced as a major product and 13-hydroxy GA12 (GA53) as a minor product. Transgenic Arabidopsis plants overexpressing the CYP714A2 gene showed semi-dwarfism. GA analysis showed that the levels of non-13-hydroxy GAs, including GA4, were decreased, whereas those of 13-hydroxy GAs, including GA1 (which is less active than GA4), were increased in the transgenic plants. Our results suggest that the CYP714 family proteins contribute to the production of diverse GA compounds through various oxidations of C and D rings in both monocots and eudicots.

Flowering in snow tussock (Chionochloa spp.) is influenced by temperature and hormonal cues.

Snow tussocks (Chionochloa spp.) in New Zealand exhibit extreme mast (episodic) seeding which has important implications for plant ecology and plant-insect interactions. Heavy flowering appears to be triggered by very warm/dry summers in the preceding year. In order to investigate the physiological basis for mast flowering, mature snow tussock plants in the field and younger plants in a glasshouse and shadehouse were subjected to a range of manipulative treatments. Field treatments included combinations of warming, root pruning and applications of two native gibberellins (GAs) GA3, which is known to be highly floral inductive and GA4, which is associated with continued floral apex development in another long-day grass. Warming, GA3 alone and especially warming+GA3, significantly promoted flowering, as did applications of GA4 alone and GA4+CCC (2-chloroethyltrimethylammonium chloride, which is a known synergist of GA3-induced flowering in the annual grass, Lolium temulentum L.). Our results provide support for the concept that mast flowering events in tussock species are causally related to high temperature-induced increases in endogenous gibberellin levels. It is likely that GAs (endogenous or applied) promote the continued development of a previously long-day induced floral apex. In addition to the promotion of flowering, applied GA3 also disturbed the plant's innate resource threshold requirements, as shown by the death, over winter, of many non-flowering tillers. Applied GA4 did not show this effect, likely due to its rapid catabolic metabolism to an inactive form. High temperature-induced flowering mediated by elevated levels of endogenous floral-promotive GAs could have important implications for regulating the evolutionary interaction between these masting plants and their seed predators.

Improving a natural enzyme activity through incorporation of unnatural amino acids.

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

Endogenous diterpenes derived from ent-kaurene, a common gibberellin precursor, regulate protonema differentiation of the moss Physcomitrella patens.

Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurene via ent-kaurenoic acid. GAs are ubiquitously present in seed plants. The GA signal is perceived and transduced by the GID1 GA receptor/DELLA repressor pathway. The lycopod Selaginella moellendorffii biosynthesizes GA and has functional GID1-DELLA signaling components. In contrast, no GAs or functionally orthologous GID1-DELLA components have been found in the moss Physcomitrella patens. However, P. patens produces ent-kaurene, a common precursor for GAs, and possesses a functional ent-kaurene synthase, PpCPS/KS. To assess the biological role of ent-kaurene in P. patens, we generated a PpCPS/KS disruption mutant that does not accumulate ent-kaurene. Phenotypic analysis demonstrates that the mutant has a defect in the protonemal differentiation of the chloronemata to caulonemata. Gas chromatography-mass spectrometry analysis shows that P. patens produces ent-kaurenoic acid, an ent-kaurene metabolite in the GA biosynthesis pathway. The phenotypic defect of the disruptant was recovered by the application of ent-kaurene or ent-kaurenoic acid, suggesting that ent-kaurenoic acid, or a downstream metabolite, is involved in protonemal differentiation. Treatment with uniconazole, an inhibitor of ent-kaurene oxidase in GA biosynthesis, mimics the protonemal phenotypes of the PpCPS/KS mutant, which were also restored by ent-kaurenoic acid treatment. Interestingly, the GA(9) methyl ester, a fern antheridiogen, rescued the protonemal defect of the disruption mutant, while GA(3) and GA(4), both of which are active GAs in angiosperms, did not. Our results suggest that the moss P. patens utilizes a diterpene metabolite from ent-kaurene as an endogenous developmental regulator and provide insights into the evolution of GA functions in land plants.

Synthesis and bioactivity of the gibberellin, 18-hydroxy-GA1 (GA132).

As part of a study to confirm putative structural assignments to new gibberellins and to furnish sufficient quantities for biological investigations, a twenty step synthesis of 18-hydroxy GA1 from gibberellic acid (GA3) is described, allowing the confirmation of structure for a new gibberellin, GA132, that occurs in developing grains of barley (Hordeum vulgare). The early part of the sequence involved cleavage of the C(3)-C(4) bond in the A-ring of a 3-oxo intermediate. The ring was then reformed as part of a "domino" process involving the conjugate addition of alkoxide to an alpha-methylene lactone moiety followed by an intramolecular aldol reaction. The bioactivities of the new GA, and its 18-hydroxy-GA4 relative, have been confirmed in dwarf barley growth and alpha-amylase induction assays.

Characterization of gibberellin receptor mutants of barley (Hordeum vulgare L.).

The sequence of Gid1 (a gene for a gibberellin (GA) receptor from rice) was used to identify a putative orthologue from barley. This was expressed in E. coli, and produced a protein that was able to bind GA in vitro with both structural specificity and saturability. Its potential role in GA responses was investigated using barley mutants with reduced GA sensitivity (gse1 mutants). Sixteen different gse1 mutants each carried a unique nucleotide substitution in this sequence. In all but one case, these changes resulted in single amino acid substitutions, and, for the remaining mutant, a substitution in the 5' untranslated region of the mRNA is proposed to interfere with translation initiation. There was perfect linkage in segregating populations between new mutant alleles and the gse1 phenotype, leading to the conclusion that the putative GID1 GA receptor sequence in barley corresponds to the Gse1 locus. Determination of endogenous GA contents in one of the mutants revealed enhanced accumulation of bioactive GA(1), and a deficit of C(20) GA precursors. All of the gse1 mutants had reduced sensitivity to exogenous GA(3), and to AC94377 (a GA analogue) at concentrations that are normally 'saturating', but, at much higher concentrations, there was often a considerable response. The comparison between barley and rice mutants reveals interesting differences between these two cereal species in GA hormonal physiology.

Selective deactivation of gibberellins below the shoot apex is critical to flowering but not to stem elongation of Lolium.

Gibberellins (GAs) cause dramatic increases in plant height and a genetic block in the synthesis of GA(1) explains the dwarfing of Mendel's pea. For flowering, it is GA(5) which is important in the long-day (LD) responsive grass, Lolium. As we show here, GA(1) and GA(4) are restricted in their effectiveness for flowering because they are deactivated by C-2 hydroxylation below the shoot apex. In contrast, GA(5) is effective because of its structural protection at C-2. Excised vegetative shoot tips rapidly degrade [14C]GA(1), [14C]GA(4), and [14C]GA(20) (>80% in 6 h), but not [14C]GA(5). Coincidentally, genes encoding two 2beta-oxidases and a putative 16-17-epoxidase were most expressed just below the shoot apex (<3 mm). Further down the immature stem (>4 mm), expression of these GA deactivation genes is reduced, so allowing GA(1) and GA(4) to promote sub-apical stem elongation. Subsequently, GA degradation declines in florally induced shoot tips and these GAs can become active for floral development. Structural changes which stabilize GA(4) confirm the link between florigenicity and restricted GA 2beta-hydroxylation (e.g. 2alpha-hydroxylation and C-2 di-methylation). Additionally, a 2-oxidase inhibitor (Trinexapac Ethyl) enhanced the activity of applied GA(4), as did limiting C-16,17 epoxidation in 16,17-dihydro GAs or after C-13 hydroxylation. Overall, deactivation of GA(1) and GA(4) just below the shoot apex effectively restricts their florigenicity in Lolium and, conversely, with GA(5), C-2 and C-13 protection against deactivation allows its high florigenicity. Speculatively, such differences in GA access to the shoot apex of grasses may be important for separating floral induction from inflorescence emergence and thus could influence their survival under conditions of herbivore predation.

Model studies toward the synthesis of the bioactive diterpenoid, harringtonolide.

In model studies towards the synthesis of harringtonolide, the construction of the tropone moiety via arene cyclopropanation was investigated. The installation of the lactone ring was accomplished by way of a Diels-Alder cycloaddition of various indenones and a-pyones. The incorporation of the key bridge methyl group and subsequent control of its stereochemistry is also outlined.

Studies toward the total synthesis of nominine.

The construction of the hetisane group of alkaloids, of which the extensively bridged nominine 17 is the simplest member, poses the ultimate challenge for those interested in the synthesis of the C20 diterpene alkaloids. We describe the synthesis of an advanced intermediate toward this goal. The key steps include reductive acylation, reductive deoxygenation, Birch reduction, and an intramolecular Lewis acid-catalyzed 1,6-addition of a carbamate to a dienone.

A formal total synthesis of the marine diterpenoid diisocyanoadociane.

[Structure: see text] A formal total synthesis of diisocyanoadociane, a marine diterpenoid with potent antimalarial properties, has been completed. The synthesis begins with a phenanthrenoid precursor that is transformed into a pyrene-derived intermediate by means of an intramolecular Michael reaction. Nitrogen functionality is introduced via a double Curtius reaction.

Synthesis and confirmation of structure for the gibberellin GA131 (18-hydroxy-GA4).

A general method for the hydroxylation of the 18-methyl group in gibberellins has been developed, as demonstrated by the successful synthesis of 18-hydroxy GA(4) (GA(131)) by means of a tandem process involving the conjugate addition of alkoxides to the alpha-methylene lactone moiety of a ring A-seco-gibberellin followed by an intramolecular aldol reaction.

ELONGATED UPPERMOST INTERNODE encodes a cytochrome P450 monooxygenase that epoxidizes gibberellins in a novel deactivation reaction in rice.

The recessive tall rice (Oryza sativa) mutant elongated uppermost internode (eui) is morphologically normal until its final internode elongates drastically at the heading stage. The stage-specific developmental effect of the eui mutation has been used in the breeding of hybrid rice to improve the performance of heading in male sterile cultivars. We found that the eui mutant accumulated exceptionally large amounts of biologically active gibberellins (GAs) in the uppermost internode. Map-based cloning revealed that the Eui gene encodes a previously uncharacterized cytochrome P450 monooxygenase, CYP714D1. Using heterologous expression in yeast, we found that EUI catalyzed 16alpha,17-epoxidation of non-13-hydroxylated GAs. Consistent with the tall and dwarfed phenotypes of the eui mutant and Eui-overexpressing transgenic plants, respectively, 16alpha,17-epoxidation reduced the biological activity of GA(4) in rice, demonstrating that EUI functions as a GA-deactivating enzyme. Expression of Eui appeared tightly regulated during plant development, in agreement with the stage-specific eui phenotypes. These results indicate the existence of an unrecognized pathway for GA deactivation by EUI during the growth of wild-type internodes. The identification of Eui as a GA catabolism gene provides additional evidence that the GA metabolism pathway is a useful target for increasing the agronomic value of crops.

Overexpression of a petunia zinc-finger gene alters cytokinin metabolism and plant forms.

Shoot branching and plant height are among the key factors that define the overall architecture of plants. We found that overexpression of a cDNA for a zinc-finger protein of petunia, designated Lateral shoot-Inducing Factor (LIF), in transgenic petunia plants resulted in a dramatic increase in lateral shoots and reduced plant height. LIF overexpression also caused a decrease in the number of cells in the stem, leaf, and flower, accompanied by enlargement of cells. trans-Zeatin was decreased while N6-(Delta2-isopentenyl)adenine was increased in the leaves of LIF-overexpressed petunia. Most of the riboside, ribotide, and glucoside forms were also increased. Expression analysis using a LIF::GUS fusion gene and RT-PCR suggested that LIF is specifically expressed around the bases of axillary buds and weakly in basal part of flowers in wild-type petunia. GFP-LIF-GUS fusion proteins were translocated into the nucleus when transiently expressed in onion epidermal cells. LIF overexpression resulted in enhanced branching also in tobacco and Arabidopsis, indicating the conservation of the response to LIF overexpression among dicotyledonous plants. On the basis of these results we discuss about possible functions of LIF.

Total synthesis of sordaricin.

An enantioconvergent total synthesis of sordaricin (3), the diterpene aglycon of an important class of antifungal compounds, is described. Two approaches were explored, the first of which utilized a possible biogenetic intramolecular [4 + 2] cycloaddition to form the complete carbon skeleton of the target molecule as a single regioisomer 30. A second approach employed a tandem cycloreversion/intramolecular [4 + 2] cycloaddition process to afford not only the desired product 30 but also significant quantities of the undesired regioisomer iso-30. An investigation into the reasons for the difference in regioselectivity between these two reactions revealed the intervention of a cycloreversion/cycloaddition pathway at elevated temperatures leading to the formation of iso-30. Experimental evidence supports the hypothesis that iso-30 is the more thermodynamically stable of the two regioisomers.

Gibberellin structure and function: biological activity and competitive inhibition of gibberellin 2- and 3-oxidases.

Some gibberellin (GA) analogues, especially with C-16,17 modifications of GA(5), can inhibit growth of plants apparently by acting as competitors with the endogenous substrate of GA biosynthetic enzymes. Here, we directly confirm the competitive action of GA derivatives but also show that some analogues may retain significant bioactivity. A recombinant 3-oxidase from pea, which converts GA(20) to bioactive GA(1), was inhibited by GA(5), and 16,17-dihydro-GA(5) derivatives, especially if the C-17 alkyl chain length was increased by up to three carbons or if the C-13 hydroxyl was acetylated. Genetic confirmation that GA(5) analogues target 3-oxidases in vivo was provided by comparing the growth response of a WT (LE) pea with a 3-oxidase mutant (le-1). Two pea 2-oxidases that inactivate bioactive GAs, were inhibited by GA(1) and GA(3) but were generally insensitive to GA(5) analogues. alpha-Amylase production by barley half-seeds in response to GA analogues provided a method to study their action when effects on GA biosynthesis were excluded. This bioactivity assay showed that 16,17-dihydro GA(5) analogues have some inherent activity but mostly less than for GA(5) (5-50-fold), which in turn was 100-fold less active than GA(1) and GA(3). However, although C-17 alkyl derivatives with one or two added carbons showed little bioactivity and were purely 3-oxidase inhibitors, adding a third carbon (the 17-n-propyl-16,17-dihydro GA(5) analogue) restored bioactivity to that of GA(5). Furthermore, this analogue has lost its capacity to inhibit stem elongation of Lolium temulentum (Mander et al., Phytochemistry 49:1509-1515, 1998a), although it strongly inhibits the 3-oxidase. Thus, the effectiveness of a GA derivative as a growth retardant will reflect the balance between its bioactivity and its capacity to inhibit the terminal enzyme of GA biosynthesis. The weaker growth inhibition in dicots including pea (approximately 10%) than in monocots such as L. temulentum (>35%) is suggestive of taxonomic differences in the bioactivity of GAs and/or their effects on GA biosynthesis.

Synthesis of the himandrine skeleton.

The hexacyclic himandrine skeleton 5, which is present in the most complex alkaloids of the tropical rain forest tree Galbulimima belgraveana, has been prepared for the first time. The synthesis begins from the known [3.2.1]benzobicyclooctene intermediate 10. Key steps include a Diels-Alder cycloaddition, Curtius rearrangement, Birch reduction, an intramolecular nucleophilic amination, and a palladium-mediated alkene amination. [reaction: see text]

Bridgehead arylation: a direct route to advanced intermediates for the synthesis of C-20 diterpene alkaloids.

[reaction: see text] Rapid access to the ABCE ring system of the C(20) diterpene alkaloids was achieved by silver(I)-promoted intramolecular Friedel-Crafts arylation of a functional group-specific 5-bromo-3-azabicyclo[3.3.1]nonane derivative.