Adsorption effects of poly(hydroxybutyric acid) depolymerase on chain-folding surface of polyester single crystals revealed by mutant enzyme and frictional force microscopy

Adsorption effects of poly(hydroxybutyric acid) (PHB) depolymerase from Ralstonia pickettii T1 on various polymer single crystals were studied using a catalytically inactive mutant of PHB depolymerase by means of transmission electron microscopy (TEM), atomic force microscopy (AFM), and frictional force microscopy (FFM). Six types of polymer single crystals, poly[(R)-3-hydroxybutyric acid] (P(3HB)), poly[(R)-3-hydroxybutyric acid-co-6 mol% (R)-3-hydroxyvaleric acid] (P(3HB-co-6 mol% 3HV)), poly[(R)-3-hydroxybutyric acid-co-8 mol% (R)-3-hydroxyhexanoic acid] (P(3HB-co-8 mol% 3HH)), poly(l-lactic acid) (PLLA), poly(d-lactic acid) (PDLA), and polyethylene (PE), were prepared to examine the influence of an ester bond and stereoregularity of a polymer on the enzymatic adsorption. The numbers of PHB depolymerase enzymes adsorbed on P(3HB) and P(3HB-co-6 mol% 3HV) single crystals were determined as 171 and 183 enzymes/μm² by AFM, respectively. AFM observation revealed that the concentration of PHB depolymerase enzymes adsorbed onto PLLA and PDLA single crystals is much higher compared to those on a P(3HB) single crystal, whereas the concentration of enzyme adsorbed onto PE and P(3HB-co-8 mol% 3HH) single crystals is much less. In addition, the single crystals of each polymer were characterized by TEM and FFM before and after enzymatic treatment by mutant for 1 h at 37 °C. The surface properties of P(3HB), P(3HB-co-6 mol% 3HV), and P(3HB-co-8 mol% 3HH) single crystals were changed by the enzymatic adsorption, whereas the internal structures were not affected. On the basis of these results, the properties of the binding domain of PHB depolymerase to polymer chain-folding surfaces have been discussed.     

Effects of residual metal compounds and chain-end structure on thermal degradation of poly(3-hydroxybutyric acid)

Thermal degradation behaviours of poly(3-hydroxybutyric acid) (P(3HB); bacterial poly[(R)-3-hydroxybutyric acid] and synthetic poly[(R,S)-3-hydroxybutyric acid] samples, were examined under both isothermal and non-isothermal conditions. The inverse of number-average degree of polymerisation for all P(3HB) samples decreased linearly with degradation time during the initial stage of isothermal degradation at a temperature ranging from 170-190 °C. In addition, crotonyl unit was detected in the residual polymer samples as main ω-chain-end. These results indicate that the dominant thermal degradation reaction for P(3HB) is a random chain scission via cis-elimination reaction of P(3HB) molecules. It was found that the presence of either Ca or Mg ions enhances the depolymerisation of P(3HB) molecules, while that Zn ions hardly catalyse the reaction. As a result, a shift of thermogravimetric curves toward the lower temperature regions was observed for the P(3HB) samples containing high amounts of Ca and Mg compounds.     

Mechanical properties and enzymatic degradation of poly[(R)-3-hydroxybutyrate] fibers stretched after isothermal crystallization near Tg

Poly[(R)-3-hydroxybutyrate] (P(3HB)) fibers with high tensile strength were prepared by stretching the fibers after isothermal crystallization near the glass transition temperature. Two samples with different molecular weights (M_w = 0.7 × 10⁶ and 4.3 × 10⁶) were used to investigate the effect on tensile strength. Increasing the time for isothermal crystallization of P(3HB) fibers resulted in a decrease in the maximum draw ratio. But, the tensile strength of P(3HB) fibers increased remarkably when the isothermal crystallization time was prolonged to more than 24 h. The tensile strength of low-molecular-weight drawn fibers was higher than that of high-molecular-weight fibers. Therefore, it can be concluded that this procedure does not increase the tensile strength of the high-molecular-weight drawn fibers. This is because, in this drawing method, small crystal nuclei grow initially during the isothermal crystallization process. Then, the molecular chains between the small crystal nuclei that acted as the entanglement points are oriented by stretching. In the case of the high-molecular-weight fibers, because the molecular length between the entanglement points of the small crystal nuclei is too long, the molecular chains are not sufficiently oriented by the stretching process. However, in the case of the low-molecular-weight fibers, the molecular length is suitable for generating the extended chains. Based on the result of X-ray analysis of P(3HB) fibers stretched after isothermal crystallization, fibers have the oriented α-form crystal with 2₁ helix conformation and β-form with planar zigzag conformation. The enzymatic degradation of the stretched P(3HB) fibers was performed by using an extracellular PHB depolymerase purified from Ralstonia pickettii T1. The enzymatic erosion rate of β-form was faster than that of α-form in the P(3HB) fibers stretched after isothermal crystallization.     

Mechanical properties, structure analysis and enzymatic degradation of uniaxially cold-drawn films of poly[(R)-3-hydroxybutyrate-co-4-hydroxybutyrate]

Poly[(R)-3-hydroxybutyrate-co-4-hydroxybutyrate] (P(3HB-co-4HB)) films were prepared by uniaxial cold-drawing from an amorphous preform at a temperature below, but close to the glass transition temperature. Molecular and highly-ordered structures and physical properties of cold-drawn films were investigated by tensile testing, wide-angle X-ray diffraction and small-angle X-ray scattering. Enzymatic degradation of P(3HB-co-4HB) films was performed using an extracellular polyhydroxybutyrate depolymerase purified from Ralstonia pickettii T1. Tensile strength, elongation to break and Young’s modulus of P(3HB-co-4HB) with cold-drawn ratio 1200% reached 290 MPa, 58% and 2.8 GPa, respectively. X-ray fibre diagrams of cold-drawn P(3HB-co-4HB) films showed a strong reflection on the equatorial line, indicating a planar zigzag conformation (β-form) together with 2₁ helix conformation (α-form). The β-form seems to contribute to the high tensile strength, and a new mechanism of generation of the β-form is proposed. The enzymatic degradation rate increased with increasing draw ratio, and increased greatly with increasing 4HB content.     

Degradation behaviors of polyester monolayers at the air/water interface: Alkaline and enzymatic degradations

The degradation kinetics of Langmuir monolayer films of a series of biodegradable polyesters has been studied to investigate the effect of degradation medium, alkalinity and enzymes. The degradation behavior of polyester monolayers strongly depended on both degradation medium and surface pressure. As the surface pressure was increased, the degradation rates of poly(l-lactide) (PLLA) and poly[(R)-3-hydroxybutyrate] (P(3HB)) increased in both degradation media. When monolayers were exposed to an alkaline subphase, the degradation of PLLA monolayers occurred at relatively low surface pressures; the PLLA monolayers were hydrolyzed at pH 10.5 regardless of surface pressure, while the alkaline degradation of P(3HB) monolayer occurred over a constant surface pressure of 7 mN/m at pH 11.8. These results have been explained by the difference in hydrophilic/hydrophobic balance of the polymers; PLLA is more hydrophilic than P(3HB). In contrast, the enzymatic degradations of both polymer monolayers occurred at higher constant surface pressures than those of the alkaline treatment; 7 mN/m for PLLA and 10 mN/m for P(3HB). This behavior was attributed to the enzymes being much larger than the alkaline ions: the enzymes need a larger contact area with the submerged monolayers to be activated.     

Improved enantioselective synthesis of natural striatenic acid and its methyl ester

This letter describes the improved and efficient enantioselective synthesis of natural striatenic acid, isolated from Cheilolejeunea serpentina, and its methyl ester starting from a readily available enantiopure building block.     

Pheromone synthesis. Part 244: Synthesis of the racemate and enantiomers of (11Z,19Z)-CH503 (3-acetoxy-11,19-octacosadien-1-ol), a new sex pheromone of male Drosophila melanogaster to show its (S)-isomer and racemate as bioactive

The enantiomers of (11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol were synthesized from the enantiomers of 3,4-epoxy-1-butanol PMB ether. Its racemate was also synthesized. Its (S)-isomer and racemate were shown to possess the same pheromone activity as CH503, a long-lived inhibitor of male courtship in Drosophila melanogaster, although the racemate was less active.     

Isolation, structural elucidation and synthesis of a novel antioxidative pseudo-di-peptide, Hanasanagin, and its biogenetic precursor from the Isaria japonica mushroom

The entomogenous ‘Hanasanagitake’ mushroom, Isaria japonica, is used as a folk medicine and as a traditional health food choice in Japan. A search for naturally occurring antioxidative compounds from the mushroom led to the isolation of a novel pseudo-di-peptide, named Hanasanagin, and a possible biogenetic precursor. The structures of the pseudo-di-peptides were determined as (R)-3,4-diguanidinobutanoyl-(S)-DOPA and (R)-3,4-diguanidinobutanoyl-(S)-tyrosine by spectral analysis, chemical synthesis and enzymatic conversion.     

Synthesis of all the stereoisomers of 6-methyl-2-octadecanone, 6,14-dimethyl-2-octadecanone, and 14-methyl-2-octadecanone, the components of the female-produced sex pheromone of a moth, Lyclene dharma dharma

All of the stereoisomers of the components of the female-produced sex pheromone of a moth, Lyclene dharma dharma, were synthesized. They are (R)- and (S)-6-methyl-2-octadecanone, (6R,14R)-, (6R,14S)-, (6S,14R)-, and (6S,14S)-6,14-dimethyl-2-octadecanone, and (R)- and (S)-14-methyl-2-octadecanone. Enantiomers of citronellal and methyl (S)-3-hydroxy-2-methylpropanoate were the starting materials, and olefin cross metathesis was employed as the key reaction.     

Molecular organogels of the sodium salt of (R)-12-hydroxystearic acid and their templated syntheses of inorganic oxides

(R)-12-Hydroxystearic acid (HSA), a natural product from castor oil, is a well-known low-molecular mass organogelator (LMOG). Here, we demonstrate that the sodium salt of HSA, HSA-S, is an extremely versatile and efficient LMOG. Furthermore, its self-assembled fibrillar networks (SAFINs) in gels with ethanol, benzene, tetrahydrofuran, and dimethyl sulfoxide, as well as the gel of HSA with benzene, are shown to act as templates during the sol-gel polymerization of tetraethyl orthosilicate (TEOS) in the absence or presence of an external catalyst. The templated, fiber-like objects obtained after calcinations have been characterized. The shape of the templated silica is strongly influenced by the catalyst applied. In addition, it has been possible to effect the formation of assemblies of nanoscale objects of Fe₂O₃ and CuO by polymerization of appropriate precursors in HSA-S based gels and in suspensions, respectively, followed by drying and calcination. The procedures employed are efficient and inexpensive protocols to make porous nanomaterials using organogels. Typically, templated syntheses of such materials in organogels have employed less accessible and more structurally complex LMOGs than HSA-S or HSA. Electrostatic interactions via Na⁺ bridges or H-bonding between silicate intermediates and gelator strands are proposed to be a primary driving force for templating.     

Stereoselective total synthesis of the (Z)-isomer of a novel phytotoxic nonenolide from Phomopsis sp. HCCB03520 and its C-6 epimer

The (Z)-isomer of a phytotoxic nonenolide, (6S,7R,9R)-6,7-dihydroxy-9-propylnon-4-eno-9-lactone isolated from Phomopsis sp. HCCB03520 and its C-6 epimer have been synthesized through a common route starting from butyraldehyde. The synthesis involves enantioselective Maruoka allylation, Sharpless asymmetric epoxidation and intramolecular ring closing metathesis as the important steps.     

Synthesis and characterization of the first diorganotin(IV) complexes containing mixed arylazobenzoic acids and having skew trapezoidal bipyramidal geometry

Three diorganotin(IV) complexes of the type, [R₂Sn(L^aH)(L^bH)] (R = ⁿBu or Me and, L^aH and L^bH are two different 5-[(E)-2-(aryl)-1-diazenyl]-2-hydroxybenzoate residues; a: aryl = 4′-Cl-(held constant) and b: aryl = 4′-Me or 4′-Br) have been prepared either by reacting ⁿBu₂SnO, L^aHH′ and L^bHH′ (1:1:1) in anhydrous toluene or by reacting Me₂SnCl₂, L^aHNa and L^bHNa (1:1:1) in anhydrous methanol. The products were characterized by microanalysis, IR, NMR (¹H, ¹³C, ¹¹⁹Sn) and ^119mSn Mössbauer spectroscopy. A full characterization of the structures of the complexes [ⁿBu₂Sn(L^aH)(L^bH)] (1 and 2) and [Me₂Sn(L^aH)(L^bH)] (3) in the solid state were accomplished by single crystal X-ray crystallography. These complexes were found to adopt the usual dicarboxylato structural type with a skew-trapezoidal bipyramidal arrangement around the tin atom.     

Synthesis of a cyclic dinuclear organotin carboxylate via simultaneous debenzylation and decarbonylation reactions: X-ray crystal structure of [(PhCH2)2{O2CC6H4{N(H)N(C6H3-4(O)-5-O)}-o}Sn]2

The complex, [(PhCH₂)₂{O₂CC₆H₄{N(H)N(C₆H₃-4(O)-5-O)}-o}Sn]₂ (1), is obtained as the exclusive reaction product from the reaction of sodium 2-[(E)-2-(3-formyl-4-hydroxyphenyl)-1-diazenyl]benzoate and (PhCH₂)₃SnCl. The reaction possibly proceeds via Dakin type rearrangements where arylazosalicylaldehyde is oxidized to arylazocatechol, followed by facile Sn-C bond cleavage. Complete assignments were achieved by ¹H, ¹³C, 2D ¹H-¹¹⁹Sn HMQC (¹¹⁹Sn chemical shift), 1D gs ¹H-¹⁵N HMQC (¹J(¹⁵N, ¹H) coupling constant) NMR and ESI-MS. The crystal structure of compound 1 as determined by X-ray diffraction analyses shows a cyclic centrosymmetric dinuclear moiety linked into extended chains by pairs of long Sn?O contacts of approximately 3.2 Å. Two polymorphs were identified and their structures differ primarily in the packing arrangement afforded by the benzyl groups. In one polymorph, when viewed along the Sn?Sn vector, the benzyl groups at each Sn-atom are oriented to form an S-shape, while they form a U-shape in the second polymorph.     

Dibutyltin(IV) complexes of the 5-[(E)-2-(Aryl)-1-diazenyl]-2-hydroxybenzoic acid ligand: an investigation of structures by X-ray diffraction, solution and solid state tin NMR, electrospray ionisation MS and assessment of in vitro cytotoxicity

A series of dibutylbis{5-[(E)-2-(aryl)-1-diazenyl]-2-hydroxybenzoato}tin(IV) complexes, Bu₂Sn(LH)₂, have been prepared and characterized by ¹H, ¹³C, ¹¹⁹Sn NMR and ESI mass spectrometry in solution. The structures of the complexes Bu₂Sn(L¹H)₂ (1), Bu₂Sn(L³H)₂ (3), Bu₂Sn(L⁴H)₂ (4), and Bu₂Sn(L⁶H)₂ (6) (L = 5-[(E)-2-(aryl)-1-diazenyl]-2-hydroxybenzoate: aryl = phenyl (L¹H), 3-methylphenyl (L³H), 4-methylphenyl (L⁴H) and 4-bromophenyl (L⁶H)) were determined by X-ray crystallography and ¹¹⁷Sn CP-MAS NMR spectroscopy in the solid state. In general, the complexes were found to adopt a skew-trapezoidal bipyramidal arrangement around the tin atom. In addition, there are weak bridging intermolecular Sn?O contacts in complexes 1 and 3, but not in 4 and 6, where one of the hydroxy oxygen atoms from a neighboring molecule coordinates weakly with the Sn atom, thereby completing a seventh coordination site in the extended Sn coordination sphere. The Sn?O distance is 3.080(2) and 3.439(2) Å in 1 and 3, respectively, which are significantly shorter than the sum of the van der Waals radii of the Sn and O atoms (∼3.8 Å). In 1, this Sn?O interaction links the molecules into polymeric chains. In 3, these interactions link pairs of molecules into head-to-head dimeric units. The in vitro cytotoxicity of compound 2 indicates better results than cisplatin and etoposide against seven well characterized human tumor cell lines.     

Chemical transformations of reaction products of 2-methyl-3-(4-tolyl)-4(3<Emphasis Type="Italic">H</Emphasis>)-quinazolone with benzil and its 4,4′-derivatives

The reaction of 2-methyl-3-(4-tolyl)-4(3H)-quinazolone with benzil produces 2-[(Z)-3-oxo-2,3-diphenylprop-1-enyl]-3-(4-tolyl)-4(3H)-quinazolone, which is readily transformed into 2-(3,3-diphenylsuccinimido)-N-(4-tolyl)benzamide on dissolution in organic water-containing solvents. The rearrangement mechanism was suggested and investigated by the quantum chemical PM3 method. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 1, pp. 146–151, January, 2007.     

Reaction of guaiazulene with o-formylbenzoic acid in diethyl ether (or methanol) in the presence of hexafluorophosphoric acid: comparative studies on H and C NMR spectral properties of 3-guaiazulenylmethylium- and 3-guaiazulenium-ion structures

Reaction of guaiazulene (1) with o-formylbenzoic acid (2) in diethyl ether in the presence of hexafluorophosphoric acid at 25 °C for 90 min gives the corresponding monocarbenium-ion compound, [2-(carboxy)phenyl](3-guaiazulenyl)methylium hexafluorophosphate (3), quantitatively, which upon treatment with aq NaHCO₃ leads to 3-(3-guaiazulenyl)-2-benzofuran-1(3H)-one (5) in 96% isolated yield. Similarly, reaction of 1 with 2 in methanol under the same conditions as the above reaction affords two kinds of inseparable monocarbenium-ion compounds, 3 and (3-guaiazulenyl)[2-(methoxycarbonyl)phenyl]methylium hexafluorophosphate (4) with an equilibrium between them, which upon reaction with a solution of NaBH₄ in ethanol at 25 °C for 30 min leads to 5 in 46% isolated yield and (3-guaiazulenyl)[2-(methoxycarbonyl)phenyl]methane (6) in 37% isolated yield. Along with the ¹H and ¹³C NMR spectral properties of a solution of 5 in trifluoroacetic acid-d₁ at 25 °C, whose molecular structure is converted to a ca. 1:1 equilibrium mixture of 7 possessing a partial structure of the 3-guaiazulenylmethylium-ion and 8 possessing a partial structure of the 3-guaiazulenium-ion, comparative studies on the ¹H and ¹³C NMR spectral properties of 7 and 8 with those of the monocarbenium-ion compound, (3-guaiazulenyl)[4-(methoxycarbonyl)phenyl]methylium hexafluorophosphate (A), 5, and 6 are reported. From these NMR studies, it can be inferred that the positive charge of the 3-guaiazulenylmethylium-ion part of 7 apparently is transferred to the seven-membered ring, generating a resonance form of the 3-guaiazulenylium-ion structure η′, and the same result can be inferred for the previously documented monocarbenium-ion compounds A-I. Moreover, referring to a comparative study on the C-C bond lengths of A observed by the X-ray crystallographic analysis with those of the optimized (3-guaiazulenyl)[4-(methoxycarbonyl)phenyl]methylium-ion structure for A calculated by a WinMOPAC (Ver. 3.0) program using PM3, AM1, or MNDOD as a semiempirical Hamiltonian, the optimized [2-(carboxy)phenyl](3-guaiazulenyl)methylium-ion structure for 3 calculated using PM3 is described.     

Microwave-assisted cleavage of Weinreb amide for carboxylate protection in the synthesis of a (R)-3-hydroxyalkanoic acid

A versatile route for the modular synthesis of (R)-3-hydroxyalkanoic acids, constituents of the naturally biodegradable poly(3-hydroxyalkanoate) polymers, and its application to the synthesis of (R)-3-hydroxydecanoic acid is described. Key steps include a microwave-assisted catalytic transfer hydrogenation and a facile microwave-assisted hydrolysis of an N-methoxy-N-methyl (Weinreb) amide, which enhances the practicality of this protecting group for carboxylic acids.     

Changes in the mechanical properties of compression moulded samples of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) degraded by Streptomyces omiyaensis SSM 5670

Streptomyces omiyaensis SSM 5670 was characterized by its ability to use compression moulded samples of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as its sole carbon source. Biodegradation of PHBV in liquid mineral salts medium was investigated using scanning electron microscopy, gravimetric measurements, capillary viscometry, tensile testing and wide angle X-ray spectroscopy. The biodegradation of PHBV proceeds via surface erosion mechanism, resulting in the formation of pits by microbial attack. PHBV specimens lost about 45% of their original weight after 45 days of exposure. During the degradation process the elastic modulus reduces less than 10%. The formation of pores and microcracks initiated at the degraded pits determines the reduction of the elongation and stress at break. However, the true stress at break is practically independent of the degradation time. No significant changes of PHBV molecular weight or crystallinity were observed during biodegradation. The polymer chain cleavage occurred only at the specimen surface and does not discriminate between crystalline and amorphous states.     

Di-n-octyltin(IV) complexes with 5-[(E)-2-(aryl)-1-diazenyl]-2-hydroxybenzoic acid: Syntheses and assessment of solid state structures by Sn Mössbauer and X-ray diffraction and further insight into the solution structures using electrospray ionization MS, Sn NMR and variable temperature NMR spectroscopy

Reactions of 5-[(E)-2-(aryl)-1-diazenyl]-2-hydroxybenzoic acids (LHH′, where the aryl group is an R-substituted phenyl ring such that for L¹HH′: X = H; L²HH′: X=2′-OCH₃; L³HH′: X = 3′-CH₃; L⁴HH′: X = 4′-CH₃; L⁵HH′:X = 4′-Cl) with ⁿOct₂SnO in 2:1 and 1:1 molar ratios have been investigated. Two types of complexes, ⁿOct₂Sn(LH)₂ and {[ⁿOct₂Sn(LH)]₂O}₂, were isolated and they have been characterized by ¹H, ¹³C, ¹¹⁹Sn NMR, ESI-MS, IR and ^119mSn Mössbauer spectroscopic techniques in combination with elemental analyses. The crystal structures of ⁿOct₂Sn(L¹H)₂ (1), {[ⁿOct₂Sn(L²H)]₂O}₂ (3) and {[ⁿOct₂Sn(L³H)]₂O}₂(4) were determined. The mononuclear complex 1 was found to adopt a skew-trapezoidal bipyramidal arrangement around the tin atom while 3 and 4 are centrosymmetric tetranuclear bis(dicarboxylatotetrabutyldistannoxane) complexes containing a planar Sn₄O₂ core in which two μ₃-oxo O-atoms connect an Sn₂O₂ ring to two exocyclic Sn-atoms. The solution structures were confirmed by ¹¹⁹Sn NMR spectroscopy by observing one tin resonance in compound 1 and two tin resonances in {[ⁿOct₂Sn(L⁵H)]₂O}₂ (5). {[ⁿOct₂Sn(L²H)]₂O}₂ (3) and {[ⁿOct₂Sn(L³H)]₂O}₂ (4) undergo very complex exchange processes in deuteriochloroform solution, which has been confirmed by variable temperature ¹H NMR spectroscopy. The cleavage of the most labile bond in the molecule was studied by ESI mass spectrometry.     

Ru-catalyzed enantioselective preparation of methyl (R)-o-chloromandelate and its application in the synthesis of (S)-Clopidogrel

The preparation of methyl (R)-o-chloromandelate via Ru-catalyzed asymmetric hydrogenation and transfer hydrogenation was investigated. With Ru-(R,R)-2,4,6-triisopropyl C₆H₂SO₂-DPEN as the catalyst and HCOOH-Et₃N azeotrope as the hydrogen donor, up to 92% ee was obtained in an optional condition. The synthesis of (S)-Clopidogrel was also studied.