Polyhydroxybutyrate synthesis in Camelina: Towards coproduction of renewable feedstocks for bioplastics and fuels.

Plant-based co-production of polyhydroxyalkanoates (PHAs) and seed oil has the potential to create a viable domestic source of feedstocks for renewable fuels and plastics. PHAs, a class of biodegradable polyesters, can replace conventional plastics in many applications while providing full degradation in all biologically active environments. Here we report the production of the PHA poly[(R)-3-hydroxybutyrate] (PHB) in the seed cytosol of the emerging bioenergy crop Camelina sativa engineered with a bacterial PHB biosynthetic pathway. Two approaches were used: cytosolic localization of all three enzymes of the PHB pathway in the seed, or localization of the first two enzymes of the pathway in the cytosol and anchoring of the third enzyme required for polymerization to the cytosolic face of the endoplasmic reticulum (ER). The ER-targeted approach was found to provide more stable polymer production with PHB levels up to 10.2% of the mature seed weight achieved in seeds with good viability. These results mark a significant step forward towards engineering lines for commercial use. Plant-based PHA production would enable a direct link between low-cost large-scale agricultural production of biodegradable polymers and seed oil with the global plastics and renewable fuels markets.

Geometric and electronic structure of a crystallographically characterized thiolate-ligated binuclear peroxo-bridged cobalt(III) complex.

In order to shed light on metal-dependent mechanisms for O-O bond cleavage, and its microscopic reverse, we compare herein the electronic and geometric structures of O2-derived binuclear Co(III)- and Mn(III)-peroxo compounds. Binuclear metal peroxo complexes are proposed to form as intermediates during Mn-promoted photosynthetic H2O oxidation, and a Co-containing artificial leaf inspired by nature's photosynthetic H2O oxidation catalyst. Crystallographic characterization of an extremely activated peroxo is made possible by working with substitution-inert, low-spin Co(III). Density functional theory (DFT) calculations show that the frontier orbitals of the Co(III)-peroxo compound differ noticeably from the analogous Mn(III)-peroxo compound. The highest occupied molecular orbital (HOMO) associated with the Co(III)-peroxo is more localized on the peroxo in an antibonding π*(O-O) orbital, whereas the HOMO of the structurally analogous Mn(III)-peroxo is delocalized over both the metal d-orbitals and peroxo π*(O-O) orbital. With low-spin d6 Co(III), filled t2g orbitals prevent π-back-donation from the doubly occupied antibonding π*(O-O) orbital onto the metal ion. This is not the case with high-spin d4 Mn(III), since these orbitals are half-filled. This weakens the peroxo O-O bond of the former relative to the latter.

The use of an acetoacetyl-CoA synthase in place of a ß-ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells.

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing ß-ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the ß-ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10(6)  Da. These results are a major step forward in engineering a high biomass C4 grass for the commercial production of PHB.

Total synthesis of iejimalide B.

Iejimalide B, a structurally unique 24-membered polyene macrolide having a previously underutilized mode of anticancer activity, was synthesized according to a strategy employing Julia-Kocienski olefinations, a palladium-catalyzed Heck reaction, a palladium-catalyzed Marshall propargylation, a Keck-type esterification, and a palladium-catalyzed macrolide-forming, intramolecular Stille coupling of a highly complex cyclization substrate. The overall synthesis is efficient (19.5% overall yield for 15 linear steps) and allows for more practical scaled-up synthesis than previously reported strategies that differed in the order of assembly of key subunits and in the method of macrocyclization. The present synthesis paves the way for efficient preparation of analogues for drug development efforts.

Iejimalides A and B inhibit lysosomal vacuolar H+-ATPase (V-ATPase) activity and induce S-phase arrest and apoptosis in MCF-7 cells.

Iejimalides are novel macrolides that are cytostatic or cytotoxic against a wide range of cancer cells at low nanomolar concentrations. A recent study by our laboratory characterized the expression of genes and proteins that determine the downstream effects of iejimalide B. However, little is known about the cellular target(s) of iejimalide or downstream signaling that lead to cell-cycle arrest and/or apoptosis. Iejimalides have been shown to inhibit the activity of vacuolar H(+)-ATPase (V-ATPase) in osteoclasts, but how this inhibition may lead to cell-cycle arrest and/or apoptosis in epithelial cells is not known. In this study, MCF-7 breast cancer cells were treated with iejimalide A or B and analyzed for changes in cell-cycle dynamics, apoptosis, lysosomal pH, cytoplasmic pH, mitochondrial membrane potential, and generation of reactive oxygen species. Both iejimalides A and B sequentially neutralize the pH of lysosomes, induce S-phase cell-cycle arrest, and trigger apoptosis in MCF-7 cells. Apoptosis occurs through a mechanism that involves oxidative stress and mitochondrial depolarization but not cytoplasmic acidification. These data confirm that iejimalides inhibit V-ATPase activity in the context of epithelial tumor cells, and that this inhibition may lead to a lysosome-initiated cell death process.

Properties of square-pyramidal alkyl-thiolate Fe(III) complexes, including an analogue of the unmodified form of nitrile hydratase.

The syntheses and structures of three new coordinatively unsaturated, monomeric, square-pyramidal thiolate-ligated Fe(III) complexes are described, [Fe(III)((tame-N(3))S(2)(Me2))](+) (1), [Fe(III)(Et-N(2)S(2)(Me2))(py)](1-) (3), and [Fe(III)((tame-N(2)S)S(2)(Me2))](2-) (15). The anionic bis-carboxamide, tris-thiolate N(2)S(3) coordination sphere of 15 is potentially similar to that of the yet-to-be characterized unmodified form of NHase. Comparison of the magnetic and reactivity properties of these reveals how anionic charge build up (from cationic 1 to anionic 3 and dianionic 15) and spin-state influence apical ligand affinity. For all of the ligand-field combinations examined, an intermediate S = 3/2 spin state was shown to be favored by a strong N(2)S(2) basal plane ligand field, and this was found to reduce the affinity for apical ligands, even when they are built in. This is in contrast to the post-translationally modified NHase active site, which is low spin and displays a higher affinity for apical ligands. Cationic 1 and its reduced Fe(II) precursor are shown to bind NO and CO, respectively, to afford [Fe(III)((tame-N(3))S(2)(Me))(NO)](+) (18, nu(NuO) = 1865 cm(-1)), an analogue of NO-inactivated NHase, and [Fe(II)((tame-N(3))S(2)(Me))(CO)] (16; nu(CO) stretch (1895 cm(-1)). Anions (N(3)(-), CN(-)) are shown to be unreactive toward 1, 3, and 15 and neutral ligands unreactive toward 3 and 15, even when present in 100-fold excess and at low temperatures. The curtailed reactivity of 15, an analogue of the unmodified form of NHase, and its apical-oxygenated S = 3/2 derivative [Fe(III)((tame-N(2)SO(2))S(2)(Me2))](2-) (20) suggests that regioselective post-translational oxygenation of the basal plane NHase cysteinate sulfurs plays an important role in promoting substrate binding. This is supported by previously reported theoretical (DFT) calculations.

Effects of Iejimalide B, a marine macrolide, on growth and apoptosis in prostate cancer cell lines.

Iejimalide B, a marine macrolide, causes growth inhibition in a variety of cancer cell lines at nanomolar concentrations. We have investigated the effects of Iejimalide B on cell cycle kinetics and apoptosis in the p53+/AR+ LNCaP and p53-/AR- PC-3 prostate cancer cell lines. Iejimalide B, has a dose and time dependent effect on cell number (as measured by crystal violet assay) in both cell lines. In LNCaP cells Iejimalide B induces a dose dependent G0/G1 arrest and apoptosis at 48 h (as measured by Apo-BrdU staining). In contrast, Iejimalide B initially induces G0/G1 arrest followed by S phase arrest but does not induce apoptosis in PC-3 cells. qPCR and Western analysis suggests that Iejimalide B modulates the steady state level of many gene products associated with cell cycle (including cyclins D, E, and B and p21(waf1/cip1)) and cell death (including survivin, p21B and BNIP3L) in LNCaP cells. In PC-3 cells Iejimalide B induces the expression of p21(waf1/cip1), down regulates the expression of cyclin A, and does not modulate the expression of the genes associated with cell death. Comparison of the effects of Iejimalide B on the two cell lines suggests that Iejimalide B induces cell cycle arrest by two different mechanisms and that the induction of apoptosis in LNCaP cells is p53-dependent.

Total synthesis of iejimalide B. An application of the Shiina macrolactonization.

The potent anticancer compound iejimalide B (1) was prepared by a total synthesis through a strategy that features Julia olefinations, Wittig olefinations, a Carreira enantioselective alkynylation, a Heck reaction, a Marshall propargylation reaction, a Stille coupling, and a Shiina macrolactonization.

Periodic trends within a series of five-coordinate thiolate-ligated [MII(SMe2N4(tren))]+ (M = Mn, Fe, Co, Ni, Cu, Zn) complexes, including a rare example of a stable CuII-thiolate.

A series of five-coordinate thiolate-ligated complexes [M(II)(tren)N4S(Me2)]+ (M = Mn, Fe, Co, Ni, Cu, Zn; tren = tris(2-aminoethyl)amine) are reported, and their structural, electronic, and magnetic properties are compared. Isolation of dimeric [Ni(II)(SN4(tren)-RS(dang))]2 ("dang"= dangling, uncoordinated thiolate supported by H bonds), using the less bulky [(tren)N4S](1-) ligand, pointed to the need for gem-dimethyls adjacent to the sulfur to sterically prevent dimerization. All of the gem-dimethyl derivatized complexes are monomeric and, with the exception of [Ni(II)(S(Me2)N4(tren)]+, are isostructural and adopt a tetragonally distorted trigonal bipyramidal geometry favored by ligand constraints. The nickel complex uniquely adopts an approximately ideal square pyramidal geometry and resembles the active site of Ni-superoxide dismutase (Ni-SOD). Even in coordinating solvents such as MeCN, only five-coordinate structures are observed. The MII-S thiolate bonds systematically decrease in length across the series (Mn-S > Fe-S > Co-S > Ni-S approximately Cu-S < Zn-S) with exceptions occurring upon the occupation of sigma* orbitals. The copper complex, [Cu(II)(S(Me2)N4(tren)]+, represents a rare example of a stable CuII-thiolate, and models the perturbed "green" copper site of nitrite reductase. In contrast to the intensely colored, low-spin Fe(III)-thiolates, the M(II)-thiolates described herein are colorless to moderately colored and high-spin (in cases where more than one spin-state is possible), reflecting the poorer energy match between the metal d- and sulfur orbitals upon reduction of the metal ion. As the d-orbitals drop in energy proceeding across the across the series M(2+) (M= Mn, Fe, Co, Ni, Cu), the sulfur-to-metal charge-transfer transition moves into the visible region, and the redox potentials cathodically shift. The reduced M(+1) oxidation state is only accessible with copper, and the more oxidized M(+4) oxidation state is only accessible for manganese.

Synthesis of carbamate derivatives of iejimalides. Retention of normal antiproliferative activity and localization of binding in cancer cells.

The syntheses of six iejimalide carbamate derivatives are described. Their biological activity and those of the unmodified iejimalides A and B against breast and prostate cancer cell lines were determined. These results show that the serine hydroxyl group of iejimalides A and B is a permissive site that can be functionalized to form carbamate derivatives without significant loss of normal biological activity. This method of derivatization will be valuable for cellular target identification, mechanism of action studies, and drug development efforts. A fluorescent derivative does not exhibit binding to the cytoskeletal features of cancer cells.

Spectroscopy of non-heme iron thiolate complexes: insight into the electronic structure of the low-spin active site of nitrile hydratase.

Detailed spectroscopic and computational studies of the low-spin iron complexes [Fe(III)(S2(Me2)N3 (Pr,Pr))(N3)] (1) and [Fe(III)(S2(Me2)N3 (Pr,Pr))]1+ (2) were performed to investigate the unique electronic features of these species and their relation to the low-spin ferric active sites of nitrile hydratases. Low-temperature UV/vis/NIR and MCD spectra of 1 and 2 reflect electronic structures that are dominated by antibonding interactions of the Fe 3d manifold and the equatorial thiolate S 3p orbitals. The six-coordinate complex 1 exhibits a low-energy S(pi) --> Fe 3d(xy) (approximately 13,000 cm(-1)) charge-transfer transition that results predominantly from the low energy of the singly occupied Fe 3d(xy) orbital, due to pure pi interactions between this acceptor orbital and both thiolate donor ligands in the equatorial plane. The 3d(pi) --> 3d(sigma) ligand-field transitions in this species occur at higher energies (>15,000 cm(-1)), reflecting its near-octahedral symmetry. The Fe 3d(xz,yz) --> Fe 3d(xy) (d(pi) --> d(pi)) transition occurs in the near-IR and probes the Fe(III)-S pi-donor bond; this transition reveals vibronic structure that reflects the strength of this bond (nu(e) approximately 340 cm(-1)). In contrast, the ligand-field transitions of the five-coordinate complex 2 are generally at low energy, and the S(pi) --> Fe charge-transfer transitions occur at much higher energies relative to those in 1. This reflects changes in thiolate bonding in the equatorial plane involving the Fe 3d(xy) and Fe 3d(x2-y2) orbitals. The spectroscopic data lead to a simple bonding model that focuses on the sigma and pi interactions between the ferric ion and the equatorial thiolate ligands, which depend on the S-Fe-S bond angle in each of the complexes. These electronic descriptions provide insight into the unusual S = 1/2 ground spin state of these complexes: the orientation of the thiolate ligands in these complexes restricts their pi-donor interactions to the equatorial plane and enforces a low-spin state. These anisotropic orbital considerations provide some intriguing insights into the possible electronic interactions at the active site of nitrile hydratases and form the foundation for further studies into these low-spin ferric enzymes.

The first example of a nitrile hydratase model complex that reversibly binds nitriles.

Nitrile hydratase (NHase) is an iron-containing metalloenzyme that converts nitriles to amides. The mechanism by which this biochemical reaction occurs is unknown. One mechanism that has been proposed involves nucleophilic attack of an Fe-bound nitrile by water (or hydroxide). Reported herein is a five-coordinate model compound ([Fe(III)(S(2)(Me2)N(3)(Et,Pr))](+)) containing Fe(III) in an environment resembling that of NHase, which reversibly binds a variety of nitriles, alcohols, amines, and thiocyanate. XAS shows that five-coordinate [Fe(III)(S(2)(Me2)N(3)(Et,Pr))](+) reacts with both methanol and acetonitrile to afford a six-coordinate solvent-bound complex. Competitive binding studies demonstrate that MeCN preferentially binds over ROH, suggesting that nitriles would be capable of displacing the H(2)O coordinated to the iron site of NHase. Thermodynamic parameters were determined for acetonitrile (DeltaH = -6.2(+/-0.2) kcal/mol, DeltaS = -29.4(+/-0.8) eu), benzonitrile (-4.2(+/-0.6) kcal/mol, DeltaS = -18(+/-3) eu), and pyridine (DeltaH = -8(+/-1) kcal/mol, DeltaS = -41(+/-6) eu) binding to [Fe(III)(S(2)(Me2)N(3)(Et,Pr))](+) using variable-temperature electronic absorption spectroscopy. Ligand exchange kinetics were examined for acetonitrile, iso-propylnitrile, benzonitrile, and 4-tert-butylpyridine using (13)C NMR line-broadening analysis, at a variety of temperatures. Activation parameters for ligand exchange were determined to be DeltaH(+ +) = 7.1(+/-0.8) kcal/mol, DeltaS(+ +) = -10(+/-1) eu (acetonitrile), DeltaH(+ +) = 5.4(+/-0.6) kcal/mol, DeltaS(+ +) = -17(+/-2) eu (iso-propionitrile), DeltaH(+ +) = 4.9(+/-0.8) kcal/mol, DeltaS(+ +) = -20(+/-3) eu (benzonitrile), and DeltaH(+ +) = 4.7(+/-1.4) kcal/mol DeltaS(+ +) = -18(+/-2) eu (4-tert-butylpyridine). The thermodynamic parameters for pyridine binding to a related complex, [Fe(III)(S(2)(Me2)N(3)(Pr,Pr))](+) (DeltaH = -5.9(+/-0.8) kcal/mol, DeltaS = -24(+/-3) eu), are also reported, as well as kinetic parameters for 4-tert-butylpyridine exchange (DeltaH(+ +) = 3.1(+/-0.8) kcal/mol, DeltaS(+ +) = -25(+/-3) eu). These data show for the first time that, when it is contained in a ligand environment similar to that of NHase, Fe(III) is capable of forming a stable complex with nitriles. Also, the rates of ligand exchange demonstrate that low-spin Fe(III) in this ligand environment is more labile than expected. Furthermore, comparison of [Fe(III)(S(2)(Me2)N(3)(Et,Pr))](+) and [Fe(III)(S(2)(Me2)N(3)(Pr,Pr))](+) demonstrates how minor distortions induced by ligand constraints can dramatically alter the reactivity of a metal complex.

Enhancing reactivity via structural distortion.

To examine how small structural changes influence the reactivity and magnetic properties of biologically relevant metal complexes, the reactivity and magnetic properties of two structurally related five-coordinate Fe(III) thiolate compounds are compared. (Et,Pr)-ligated [Fe(III)(S(2)(Me2)N(3)(Et,Pr))]PF(6) (3) is synthesized via the abstraction of a sulfur from alkyl persulfide ligated [Fe(III)(S(2)(Me2)N(3)(Et,Pr))-S(pers)]PF(6) (2) using PEt(3). (Et,Pr)-3 is structurally related to (Pr,Pr)-ligated [Fe(III)(S(2)(Me2)N(3)(Pr,Pr))]PF(6) (1), a nitrile hydratase model compound previously reported by our group, except it contains one fewer methylene unit in its ligand backbone. Removal of this methylene distorts the geometry, opens a S-Fe-N angle by approximately 10 degrees, alters the magnetic properties by stabilizing the S = 1/2 state relative to the S = 3/2 state, and increases reactivity. Reactivity differences between 3 and 1 were assessed by comparing the thermodynamics and kinetics of azide binding. Azide binds reversibly to both (Et,Pr)-3 and (Pr,Pr)-1 in MeOH solutions. The ambient temperature K(eq) describing the equilibrium between five-coordinate 1 or 3 and azide-bound 1-N(3) or 3-N(3) in MeOH is approximately 10 times larger for the (Et,Pr) system. In CH(2)Cl(2), azide binds approximately 3 times faster to 3 relative to 1, and in MeOH, azide dissociates 1 order of magnitude slower from 3-N(3) relative to 1-N(3). The increased on rates are most likely a consequence of the decreased structural rearrangement required to convert 3 to an approximately octahedral structure, or they reflect differences in the LUMO (vs SOMO) orbital population (i.e., spin-state differences). Dissociation rates from both 3-N(3) and 1-N(3) are much faster than one would expect for low-spin Fe(III). Most likely this is due to the labilizing effect of the thiolate sulfur that is trans to azide in these structures.