An unusual carbon-carbon bond cleavage reaction during phosphinothricin biosynthesis.

Natural products containing phosphorus-carbon bonds have found widespread use in medicine and agriculture. One such compound, phosphinothricin tripeptide, contains the unusual amino acid phosphinothricin attached to two alanine residues. Synthetic phosphinothricin (glufosinate) is a component of two top-selling herbicides (Basta and Liberty), and is widely used with resistant transgenic crops including corn, cotton and canola. Recent genetic and biochemical studies showed that during phosphinothricin tripeptide biosynthesis 2-hydroxyethylphosphonate (HEP) is converted to hydroxymethylphosphonate (HMP). Here we report the in vitro reconstitution of this unprecedented C(sp(3))-C(sp(3)) bond cleavage reaction and X-ray crystal structures of the enzyme. The protein is a mononuclear non-haem iron(ii)-dependent dioxygenase that converts HEP to HMP and formate. In contrast to most other members of this family, the oxidative consumption of HEP does not require additional cofactors or the input of exogenous electrons. The current study expands the scope of reactions catalysed by the 2-His-1-carboxylate mononuclear non-haem iron family of enzymes.

In vitro characterization of a heterologously expressed nonribosomal Peptide synthetase involved in phosphinothricin tripeptide biosynthesis.

The late stages of biosynthesis of phosphinothricin tripeptide (PTT) involve peptide formation and methylation on phosphorus. The exact timing of these transformations is not known. To provide insight into this question, we developed a heterologous expression system for PhsA, one of three NRPS proteins in PTT biosynthesis. The apparent k(cat)/K(m) value for ATP-pyrophosphate exchange activity for d,l-N-acetylphosphinothricin was 3.5 muM(-1) min(-1), whereas the k(cat)/K(m,app) for l-N-acetyldemethylphosphinothricin was 0.5 microM(-1) min(-1), suggesting the former might be the physiological substrate. Each substrate could be loaded onto the phosphopantetheine arm of the thiolation domain as observed by Fourier transform mass spectrometry (FTMS).

cis-Beta-bis(carbonyl) ruthenium-salen complexes: X-ray crystal structures and remarkable catalytic properties toward asymmetric intramolecular alkene cyclopropanation.

cis-Beta-[Ru(II)(salen(A))(CO)(2)] (salen(A) = N,N'-bis(3-R(1)-5-R(2)-salicylidene)-1,2-cyclohexenediamine dianion; R(1) = R(2) = Bu(t), 1a; R(1) = Pr(i), R(2) = H, 1b; R(1) = Bu(t), R(2) = H, 1c) complexes were prepared by treating Ru(3)(CO)(12) with the respective H(2)salen(A) in 1,2,4-trichlorobenzene and structurally characterized by X-ray crystallography. Complexes 1a-c catalyze intramolecular cyclopropanation of trans-allylic diazoacetates N(2)CHCO(2)CH(2)CH=CHR (3, R = Ph, 4-ClC(6)H(4), 4-BrC(6)H(4), 4-MeC(6)H(4), 4-MeOC(6)H(4), 2-MeC(6)H(4), 2-furanyl) under light irradiation to give cyclopropyl lactones 4 in up to 96% yield and up to 98% ee. DFT calculations on intramolecular cyclopropanation of 3a (R = Ph) with model catalyst cis-beta-[Ru(II)(salen(A0))(CO)(2)] (salen(A0) = N,N'-bis(salicylidene)-1,2-cyclohexenediamine dianion) reveal the intermediacy of both cis-beta- and trans-[Ru(salen(A0))(CHCO(2)CH(2)CH=CHPh)(CO)] bearing salen(A0) in a nonplanar and planar coordination mode, respectively, with the cis-beta-carbene species being a major intermediate in the catalytic carbenoid transfer reaction. The intramolecular cyclopropanation from the cis-beta-carbene species is the most favorable pathway and features an early transition state and an asynchronous concerted [2 + 1] addition mechanism. Enantioselectivities in the reactions involving [Ru(salen(A0))(CHCO(2)CH(2)CH=CHPh)(CO)] were predicted to be 77% ee for the trans-carbene species and 96% ee for the cis-beta-carbene species; the former dramatically increases to 98% ee, whereas the latter slightly increases to 99% ee, upon replacing salen(A0) with salen(A1) (R(1) = R(2) = B(t)). The observed variation in enantioselectivity (90-98% ee) for the conversion of 3a to 4a catalyzed by 1a-c could result from an equilibrium between cis-beta (major) and trans (minor) ruthenium-carbene intermediates.

Biosynthesis of 2-hydroxyethylphosphonate, an unexpected intermediate common to multiple phosphonate biosynthetic pathways.

Phosphonic acids encompass a common yet chemically diverse class of natural products that often possess potent biological activities. Here we report that, despite the significant structural differences among many of these compounds, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethyl-phosphonate, which is synthesized from phosphonoacetaldehyde by a distinct family of metal-dependent alcohol dehydrogenases (ADHs). Although the sequence identity of the ADH family members is relatively low (34-37%), in vitro biochemical characterization of the homologs involved in biosynthesis of the antibiotics fosfomycin, phosphinothricin tripeptide, and dehydrophos (formerly A53868) unequivocally confirms their enzymatic activities. These unique ADHs have exquisite substrate specificity, unusual metal requirements, and an unprecedented monomeric quaternary structure. Further, sequence analysis shows that these ADHs form a monophyletic group along with additional family members encoded by putative phosphonate biosynthetic gene clusters. Thus, the reduction of phosphonoacetaldehyde to hydroxyethyl-phosphonate may represent a common step in the biosynthesis of many phosphonate natural products, a finding that lends insight into the evolution of phosphonate biosynthetic pathways and the chemical structures of new C-P containing secondary metabolites.

Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide.

Phosphinothricin tripeptide (PTT, phosphinothricylalanylalanine) is a natural-product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 21705 (ref. 1) and Streptomyces viridochromogenes DSM 40736 (ref. 2). PTT has attracted widespread interest because of its commercial applications and unique phosphinic acid functional group. Despite intensive study since its discovery in 1972 (see ref. 3 for a comprehensive review), a number of steps early in the PTT biosynthetic pathway remain uncharacterized. Here we report a series of interdisciplinary experiments involving the construction of defined S. viridochromogenes mutants, chemical characterization of accumulated intermediates, and in vitro assay of selected enzymes to examine these critical steps in PTT biosynthesis. Our results indicate that early PTT biosynthesis involves a series of catalytic steps that to our knowledge has not been described so far, including a highly unusual reaction for carbon bond cleavage. In sum, we define a pathway for early PTT biosynthesis that is more complex than previously appreciated.

New insight into the mechanism of methyl transfer during the biosynthesis of fosfomycin.

Hydroxyethylphosphonate is a required intermediate in fosfomycin biosynthesis.

Efficient synthesis of suitably protected beta-difluoroalanine and gamma-difluorothreonine from L-ascorbic acid.

[reaction: see text] Fluorinated amino acids are useful building blocks for the preparation of biologically active peptides and peptidomimetics with increased metabolic stability. We report here the synthesis of two fluorinated amino acids, beta-difluoroalanine and gamma-difluorothreonine, as analogues of Ser and Thr, respectively. These compounds were suitably protected for Fmoc-based solid-phase peptide synthesis. Once incorporated into peptides, they may serve as alternative substrates or inhibitors of lantibiotic synthetases that posttranslationally dehydrate Ser and Thr residues to dehydroalanine and dehydrobutyrine, respectively.

Asymmetric synthesis of multifunctionalized pyrrolines by a ruthenium porphyrin-catalyzed three-component coupling reaction.

[reaction: see text] Chiral multifunctionalized pyrrolines have been synthesized by a ruthenium porphyrin catalyzed three-component coupling reaction. In a one-pot reaction, ruthenium porphyrins catalyzed in situ generation of chiral azomethine ylides from chiral diazo esters and imines. Asymmetric 1,3-dipolar cycloaddition reactions of the chiral azomethine ylides with dipolarophiles afforded the corresponding pyrrolines in good yields and high diastereoselectivity (up to 92% de).

Highly selective intra- and intermolecular coupling reactions of diazo compounds to form cis-alkenes using a ruthenium porphyrin catalyst.

[Ru(2,6-Cl(2)TPP)(CO)] catalyzed intramolecular coupling reactions of bisdiazoacetates and intermolecular coupling reactions of monodiazoacetates to afford the coupling products in up to 76% and 93% yields, respectively. Only the cis isomers were obtained from the reactions. Employing such a ruthenium-catalyzed coupling reaction of a diazo compound as a key step allowed the synthesis of Patulolide B in 67% yield with a ratio of >40:1 against its trans isomer.

Stereoselective synthesis of functionalized pyrrolidines by ruthenium porphyrin-catalyzed decomposition of alpha-diazo esters and cascade azomethine ylide formation/1,3-dipolar cycloaddition reactions.

[reaction: see text] Ruthenium porphyrins catalyze three-component coupling reaction of alpha-diazo esters with a series of N-benzylidene imines and alkenes to form functionalized pyrrolidines in excellent diastereoselectivities. The reaction proceeds via a reactive ruthenium-carbene intermediate and its subsequent reaction with imine to generate azomethine ylide, which reacts with alkenes via 1,3-diploar cycloaddition.