Robert B. Grossman

Role of the LolP cytochrome P450 monooxygenase in loline alkaloid biosynthesis.

The insecticidal loline alkaloids, produced by Neotyphodium uncinatum and related endophytes, are exo-1-aminopyrrolizidines with an ether bridge between C-2 and C-7. Loline alkaloids vary in methyl, acetyl, and formyl substituents on the 1-amine, which affect their biological activity. Enzymes for key loline biosynthesis steps are probably encoded by genes in the LOL cluster, which is duplicated in N. uncinatum, except for a large deletion in lolP2. The role of lolP1 was investigated by its replacement with a hygromycin B phosphotransferase gene. Compared to wild type N. uncinatum and an ectopic transformant, DeltalolP1 cultures had greatly elevated levels of N-methylloline (NML) and lacked N-formylloline (NFL). Complementation of DeltalolP1 with lolP1 under control of the Emericella nidulans trpC promoter restored NFL production. These results and the inferred sequence of LolP1 indicate that it is a cytochrome P450, catalyzing oxygenation of an N-methyl group in NML to the N-formyl group in NFL.

Biosynthetic Precursors of Fungal Pyrrolizidines, the Loline Alkaloids

Loline alkaloids are saturated pyrrolizidines with a substituted 1‐amino group and an oxygen bridge between C2 and C7, and are insecticidal metabolites of plant‐symbiotic fungi (endophytes). Cultures of the endophyte, Neotyphodium uncinatum, incorporated labeled L‐proline and L‐homoserine into the 1‐aminopyrrolizidine, N‐formylloline. The A‐ring carbons C1–C3 and the N1 were derived from L‐homoserine; the B‐ring carbons C5–C8 and the ring nitrogen were derived from L‐proline. Incorporation of both deuterium atoms from L‐[4,4‐2H2]homoserine and feeding tests with labeled L‐methionine indicated that L‐homoserine incorporation was not achieved via aspartyl semialdehyde or S‐adenosylmethionine, but probably involved a highly novel NC bond‐forming γ‐substitution reaction.

Behavioural and neurophysiological responses of Spodoptera littoralis to azadirachtin and a range of synthetic analogues

The antifeedant activity of azadirachtin and 56 azadirachtin analogues, including 22, 23‐dihydroazadirachtin, against larvae of Spodoptera littoralis was investigated using behavioural and electrophysiological bioassays. None of the analogues was as active as azadirachtin, although many showed significant antifeedant activity at high concentrations. The majority of the analogues stimulated a dose‐dependent response from a neurone in the medial styloconic maxillary sensilla which correlated with the behavioural activity. Methylation of the hydroxy substitutions on the azadirachtin molecule usually resulted in a decrease in antifeedant activity, as did the addition of bulky groups to the dihydrofuran ring.

On the Sequence of Bond Formation in Loline Alkaloid Biosynthesis

Loline alkaloids are saturated pyrrolizidines with an oxygen bridge between carbon atoms C‐2 and C‐7 and an amino group on C‐1. They are bioprotective alkaloids produced by Epichloë and Neotyphodium species, mutualistic fungal endophytes that are symbiotic with cool‐season grasses. The sequence of bond formation in loline alkaloid biosynthesis was determined by synthesizing deuterated forms of potential intermediates and feeding them to cultures of the endophyte Neotyphodium uncinatum. These cultures incorporated deuterium from labeled N‐(3‐amino‐3‐carboxypropyl)proline and exo‐1‐aminopyrrolizidine into N‐formylloline. The first result suggests that N‐(3‐amino‐3‐carboxypropyl)proline is the first committed intermediate in loline biosynthesis, and the second result demonstrates that the pyrrolizidine rings form before the ether bridge. The incorporation of these two compounds into lolines and the lack of incorporation of several related compounds clarify the order of bond formation in loline alkaloid biosynthesis.

On the structures of plukenetiones B, D, and E and their relationships to other polycyclic polyprenylated acylphloroglucinols

Abstract Three polycyclic polyprenylated benzoylphloroglucinol natural products, plukenetiones B, D, and E, are shown to be diastereomeric to the related compounds, nemorosone II and sampsonione G.

Ether bridge formation in loline alkaloid biosynthesis

Lolines are potent insecticidal agents produced by endophytic fungi of cool-season grasses. These alkaloids are composed of a pyrrolizidine ring system and an uncommon ether bridge linking carbons 2 and 7. Previous results indicated that 1-aminopyrrolizidine was a pathway intermediate. We used RNA interference to knock down expression of lolO, resulting in the accumulation of an alkaloid identified as exo-1-acetamidopyrrolizidine based on high-resolution MS and NMR. Genomes of endophytes differing in alkaloid profiles were sequenced, revealing that those with mutated lolO accumulated exo-1-acetamidopyrrolizidine but no lolines. Heterologous expression of wild-type lolO complemented a lolO mutant, resulting in the production of N-acetylnorloline. These results indicated that the non-heme iron oxygenase, LolO, is required for ether bridge formation, probably through oxidation of exo-1-acetamidopyrrolizidine.

Enzymes from Fungal and Plant Origin Required for Chemical Diversification of Insecticidal Loline Alkaloids in Grass- Epichloë Symbiota

The lolines are a class of bioprotective alkaloids that are produced by Epichloë species, fungal endophytes of grasses. These alkaloids are saturated 1-aminopyrrolizidines with a C2 to C7 ether bridge, and are structurally differentiated by the various modifications of the 1-amino group: -NH2 (norloline), -NHCH3 (loline), -N(CH3)2 (N-methylloline), -N(CH3)Ac (N-acetylloline), -NHAc (N-acetylnorloline), and -N(CH3)CHO (N-formylloline). Other than the LolP cytochrome P450, which is required for conversion of N-methylloline to N-formylloline, the enzymatic steps for loline diversification have not yet been established. Through isotopic labeling, we determined that N-acetylnorloline is the first fully cyclized loline alkaloid, implying that deacetylation, methylation, and acetylation steps are all involved in loline alkaloid diversification. Two genes of the loline alkaloid biosynthesis (LOL) gene cluster, lolN and lolM, were predicted to encode an N-acetamidase (deacetylase) and a methyltransferase, respectively. A knockout strain lacking both lolN and lolM stopped the biosynthesis at N-acetylnorloline, and complementation with the two wild-type genes restored production of N-formylloline and N-acetylloline. These results indicated that lolN and lolM are required in the steps from N-acetylnorloline to other lolines. The function of LolM as an N-methyltransferase was confirmed by its heterologous expression in yeast resulting in conversion of norloline to loline, and of loline to N-methylloline. One of the more abundant lolines, N-acetylloline, was observed in some but not all plants with symbiotic Epichloë siegelii, and when provided with exogenous loline, asymbiotic meadow fescue (Lolium pratense) plants produced N-acetylloline, suggesting that a plant acetyltransferase catalyzes N-acetylloline formation. We conclude that although most loline alkaloid biosynthesis reactions are catalyzed by fungal enzymes, both fungal and plant enzymes are responsible for the chemical diversification steps in symbio.

Insights into the substrate specificity of plant peptide deformylase, an essential enzyme with potential for the development of novel biotechnology applications in agriculture.

The crystal structure of AtPDF1B [Arabidopsis thaliana PDF (peptide deformylase) 1B; EC 3.5.1.88], a plant specific deformylase, has been determined at a resolution of 2.4 A (1 A=0.1 nm). The overall fold of AtPDF1B is similar to other peptide deformylases that have been reported. Evidence from the crystal structure and gel filtration chromatography indicates that AtPDF1B exists as a symmetric dimer. PDF1B is essential in plants and has a preferred substrate specificity towards the PS II (photosystem II) D1 polypeptide. Comparative analysis of AtPDF1B, AtPDF1A, and the type 1B deformylase from Escherichia coli, identifies a number of differences in substrate binding subsites that might account for variations in sequence preference. A model of the N-terminal five amino acids from the D1 polypeptide bound in the active site of AtPDF1B suggests an influence of Tyr(178) as a structural determinant for polypeptide substrate specificity through hydrogen bonding with Thr(2) in the D1 sequence. Kinetic analyses using a polypeptide mimic of the D1 N-terminus was performed on AtPDF1B mutated at Tyr(178) to alanine, phenylalanine or arginine (equivalent residue in AtPDF1A). The results suggest that, whereas Tyr(178) can influence catalytic activity, other residues contribute to the overall preference for the D1 polypeptide.

Pauson-Khand approach to chiral, diastereomerically pure group 4 ansa-metallocene complexes

Abstract The C 2 -symmetric bis(1,6-enynes) threo -1,10-diphenyl-5,6-divinyl-1,9-decadiyne and threo -9,10-divinyl-5,13-octadecadiyne undergo the intramolecular Pauson-Khand reaction regio- and stereoselectively to give C 2 -symmetric bis(enones) with two bicyclo[3.3.0]octyl moieties joined at the C6 position. Experiments aimed at converting the bis(enones) into chiral, diastereopure ansa -bridged group 4 metallocene complexes are described.

Chemistry of insect antifeedants from Azadirachta indica (Part 17): Synthesis of model compounds of azadirachtin. Unusual effect of remote substituents on the course of the oxidative ring contraction reaction.

Abstract The synthesis of model compounds of azadirachtin containing the tetrahydrofurancarboxylate hemiketal functional group was achieved. The course of the oxidative ring contraction reaction that was used to form this functional group was observed to be unusually sensitive to the nature of substituents remote from the reactive centre.