5‐Stabilized Phosphatidylinositol 3,4,5‐Trisphosphate Analogues Bind Grp1 PH,Inhibit Phosphoinositide Phosphatases,and Block Neutrophil Migration

Metabolically stabilized analogues of PtdIns(3,4,5)P₃ have shown long‐lived agonist activity for cellular events and selective inhibition of lipid phosphatase activity. We describe an efficient asymmetric synthesis of two 5‐phosphatase‐resistant analogues of PtdIns(3,4,5)P₃, the 5‐methylene phosphonate (MP) and 5‐phosphorothioate (PT). Furthermore, we illustrate the biochemical and biological activities of five stabilized PtdIns(3,4,5)P₃ analogues in four contexts. First, the relative binding affinities of the 3‐MP, 3‐PT, 5‐MP, 5‐PT, and 3,4,5‐PT₃ analogues to the Grp1 PH domain are shown, as determined by NMR spectroscopy. Second, the enzymology of the five analogues is explored, showing the relative efficiency of inhibition of SHIP1, SHIP2, and phosphatase and tensin homologue deleted on chromosome 10 (PTEN), as well as the greatly reduced ability of these phosphatases to process these analogues as substrates as compared to PtdIns(3,4,5)P₃. Third, exogenously delivered analogues severely impair complement factor C5a‐mediated polarization and migration of murine neutrophils. Finally, the new analogues show long‐lived agonist activity in mimicking insulin action in sodium transport in A6 cells.     

Identification of a Small‐Molecule Inhibitor of HIV‐1 Assembly that Targets the Phosphatidylinositol (4,5)‐bisphosphate Binding Site of the HIV‐1 Matrix Protein

The development of drug resistance remains a critical problem for current HIV‐1 antiviral therapies, creating a need for new inhibitors of HIV‐1 replication. We previously reported on a novel anti‐HIV‐1 compound, N²‐(phenoxyacetyl)‐N‐[4‐(1‐piperidinylcarbonyl)benzyl]glycinamide ( 14 ), that binds to the highly conserved phosphatidylinositol (4,5)‐bisphosphate (PI(4,5)P₂) binding pocket of the HIV‐1 matrix (MA) protein. In this study, we re‐evaluate the hits from the virtual screen used to identify compound 14 and test them directly in an HIV‐1 replication assay using primary human peripheral blood mononuclear cells. This study resulted in the identification of three new compounds with antiviral activity; 2‐(4‐{[3‐(4‐fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]methyl})‐1‐piperazinyl)‐N‐(4‐methylphenyl)acetamide ( 7 ), 3‐(2‐ethoxyphenyl)‐5‐[[4‐(4‐nitrophenyl)piperazin‐1‐yl]methyl]‐1,2,4‐oxadiazole ( 17 ), and N‐[4‐ethoxy‐3‐(1‐piperidinylsulfonyl)phenyl]‐2‐(imidazo[2,1‐b][1,3]thiazol‐6‐yl)acetamide ( 18 ), with compound 7 being the most potent of these hits. Mechanistic studies on 7 demonstrated that it directly interacts with and functions through HIV‐1 MA. In accordance with our drug target, compound 7 competes with PI(4,5)P₂ for MA binding and, as a result, diminishes the production of new virus. Mutation of residues within the PI(4,5)P₂ binding site of MA decreased the antiviral effect of compound 7 . Additionally, compound 7 displays a broadly neutralizing anti‐HIV activity, with IC₅₀ values of 7.5–15.6 μM for the group M isolates tested. Taken together, these results point towards a novel chemical probe that can be used to more closely study the biological role of MA and could, through further optimization, lead to a new class of anti‐HIV‐1 therapeutics.     

A Tailor‐Made “Tag–Receptor” Affinity Pair for the Purification of Fusion Proteins

A novel affinity “tag–receptor” pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet‐21c expression vector and expressed in E. coli host as soluble protein. A solid‐phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96‐well format for binding to the RKRKRK‐tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK‐tagged GFP with A7C1 emerged as a promising solution (K_a of 2.45×10⁵ M ^?1). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.     

Synthesis and Biological Evaluation of Imidazo[2,1‐b][1,3,4]thiadiazole‐Linked Oxindoles as Potent Tubulin Polymerization Inhibitors

A series of imidazo[2,1‐b][1,3,4]thiadiazole‐linked oxindoles composed of an A, B, C and D ring system were synthesized and investigated for anti‐proliferative activity in various human cancer cell lines; test compounds were variously substituted at rings C and D. Among them, compounds 7 ((E)‐5‐fluoro‐3‐((6‐p‐tolyl‐2‐(3,4,5‐trimethoxyphenyl)‐imidazo[2,1‐b][1,3,4]thiadiazol‐5‐yl)methylene)indolin‐2‐one), 11 ((E)‐3‐((6‐p‐tolyl‐2‐(3,4,5‐trimethoxyphenyl)imidazo[2,1‐b][1,3,4]thiadiazol‐5‐yl)methylene)indolin‐2‐one), and 15 ((E)‐6‐chloro‐3‐((6‐phenyl‐2‐(3,4,5‐trimethoxyphenyl)imidazo[2,1‐b][1,3,4]thiadiazol‐5‐yl)methylene)indolin‐2‐one) exhibited potent anti‐proliferative activity. Treatment with these three compounds resulted in accumulation of cells in G₂/M phase, inhibition of tubulin assembly, and increased cyclin‐B1 protein levels. Compound 7 displayed potent cytotoxicity, with an IC₅₀ range of 1.1–1.6 μM , and inhibited tubulin polymerization with an IC₅₀ value (0.15 μM ) lower than that of combretastatin A‐4 (1.16 μM ). Docking studies reveal that compounds 7 and 11 bind with αAsn101, βThr179, and βCys241 in the colchicine binding site of tubulin.     

Structural and Preliminary Explosive Property Characterizations of New 3,4,5‐Triamino‐1,2,4‐triazolium (Guanazinium) Salts

Two new highly stable energetic salts were synthesized in reasonable yield by using the high nitrogen‐content heterocycle 3,4,5‐triamino‐1,2,4‐triazole and resulting in its picrate and azotetrazolate salts. 3,4,5‐Triamino‐1,2,4‐triazolium picrate (1) and bis(3,4,5‐triamino‐1,2,4‐triazolium) 5,5′‐azotetrazolate (2) were characterized analytically and spectroscopically. X‐ray diffraction studies revealed that protonation takes place on the nitrogen N1 (crystallographically labelled as N2). The sensitivity of the compounds to shock and friction was also determined by standard BAM tests revealing a low sensitivity for both. B3LYP/6–31G(d, p) density functional (DFT) calculations were carried out to determine the enthalpy of combustion (Δ_cH (1) =−3737.8 kJ mol⁻¹, Δ_cH (2) =−4577.8 kJ mol⁻¹) and the standard enthalpy of formation (Δ_fH° (1) =−498.3 kJ mol⁻¹, (Δ_fH° (2) =+524.2 kJ mol⁻¹). The detonation pressures (P (1) =189×10⁸ Pa, P (2) =199×10⁸ Pa) and detonation velocities (D (1) =7015 m s⁻¹, D (2) =7683 m s⁻¹) were calculated using the program EXPLO5.     

Water‐dispersible porous polyisoprene‐block‐poly(acrylic acid) microspheres

Two polyisoprene‐block‐poly(tert‐butyl acrylate) (PI‐b‐PtBA) samples and a poly(tert‐butyl acrylate) (PtBA) homopolymer (hPtBA) were prepared by anionic polymerization and characterized by light scattering, size exclusion chromatography, and NMR. The tert‐butyl groups were removed from one of the diblocks to yield amphiphilic polyisoprene‐block‐poly(acrylic acid) (PI‐b‐PAA). PI‐b‐PAA was then used as the surfactant to disperse dichloromethane containing PI‐b‐PtBA and hPtBA at different weight ratios as oil droplets in water. Solid microspheres containing segregated polyisoprene (PI) and PtBA/hPtBA domains were obtained after dichloromethane evaporation. Permanent microspheres were obtained after PI domain crosslinking with sulfur monochloride. Porous microspheres were produced after the hydrolysis of PtBA and the extraction of the homopoly(acrylic acid) chains. The shape and connectivity of the poly(acrylic acid)‐lined pores were tuned by changes in the PtBA/hPtBA content in the precursor microspheres. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 2785–2793, 2003     

Synthesis,Anti‐tubulin and Antiproliferative SAR of Steroidomimetic Dihydroisoquinolinones

A SAR translation strategy adopted for the discovery of tetrahydroisoquinolinone (THIQ)‐based steroidomimetic microtubule disruptors has been extended to dihydroisoquinolinone (DHIQ)‐based compounds. A steroid A,B‐ring‐mimicking DHIQ core was connected to methoxyaryl D‐ring mimics through methylene, carbonyl, and sulfonyl linkers, and the resulting compounds were evaluated against two cancer cell lines. The carbonyl‐linked DHIQs in particular exhibit significant in vitro antiproliferative activities (e.g., 6‐hydroxy‐7‐methoxy‐2‐(3,4,5‐trimethoxybenzoyl)‐3,4‐dihydroisoquinolin‐1(2H)‐one ( 16 g ): GI₅₀ 51 nM in DU‐145 cells). The broad anticancer activity of DHIQ 16 g was confirmed in the NCI 60‐cell line assay giving a mean activity of 33 nM . Furthermore, 6‐hydroxy‐2‐(3,5‐dimethoxybenzoyl)‐7‐methoxy‐3,4‐dihydroisoquinolin‐1(2H)‐one ( 16 f ) and 16 g and their sulfamate derivatives 17 f and 17 g (2‐(3,5‐dimethoxybenzoyl)‐7‐methoxy‐6‐sulfamoyloxy‐3,4‐dihydroisoquinolin‐1(2H)‐one and 7‐methoxy‐2‐(3,4,5‐trimethoxybenzoyl)‐6‐sulfamoyloxy‐3,4‐dihydroisoquinolin‐1(2H)‐one, respectively) show excellent activity against the polymerization of tubulin, close to that of the clinical combretastatin A‐4, and bind competitively at the colchicine binding site of tubulin. Compounds 16 f and 17 f were also shown to demonstrate in vitro anti‐angiogenic activity. Additionally, X‐ray and computational analyses of 17 f reveal that electrostatic repulsion between the two adjacent carbonyl groups, through conformational biasing, dictates the adoption of a “steroid‐like” conformation that may partially explain the excellent in vitro activities.     

Synthesis and properties of side‐chain liquid‐crystalline polysiloxanes containing hemiphasmidic mesogens

The synthesis of hemiphasmidic monomers 4‐[(3,4,5‐triethoxy)benzoyloxy]‐4′‐[(p‐allyloxy)benzoyloxy]biphenyl (M₁), 4‐[(3,5‐diethoxy)benzoyloxy]‐4′‐[(p‐allyloxy)‐benzoyloxy]biphenyl (M₂), and of the corresponding side‐chain liquid‐crystalline polysiloxanes (P₁, P₂) was carried out. For comparison, rodlike monomer 4‐[(p‐ethoxy)‐benzoyloxy]‐4′‐[(p‐allyloxy)benzoyloxy]biphenyl (M₃) and its polysiloxanes (P₃) were also prepared. The chemical structures of the monomers and polymers obtained were confirmed by FTIR and ¹H‐NMR spectra. Their mesomorphic properties and phase behavior were investigated by differential scanning calorimetry, polarizing optical microscopy, and X‐ray diffraction measurements. The relationship between structures and properties was discussed. It was observed that M₁ and M₃ were enantiotropic nematic phase, M₂ was monotropic mesophase, and their poly(methylsiloxanes) (P₁–P₃) possessed a broad range enantiotropic nematic phases and high thermal stability. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 95: 946–952, 2005     

A DFT Study on the Structures and Energies of Isomers of 4‐Amino‐1,3‐dinitro‐1,2,4‐triazol‐5‐one‐2‐oxide: New High Energy Density Compounds

Isomers of 4‐amino‐1,3‐dinitrotriazol‐5‐one‐2‐oxide (ADNTONO) are of interest in the contest of insensitive explosives and were found to have true local energy minima at the DFT‐B3LYP/aug‐cc‐pVDZ level. The optimized structures, vibrational frequencies and thermodynamic values for triazol‐5‐one N‐oxides were obtained in their ground state. Kamlet‐Jacob equations were used to evaluate the performance properties. The detonation properties of ADNTONO (D=10.15 to 10.46 km s⁻¹, P=50.86 to 54.25 GPa) are higher compared with those of 1,1‐diamino‐2,2‐dinitroethylene (D=8.87 km s⁻¹, P=32.75 GPa), 5‐nitro‐1,2,4‐triazol‐3‐one (D=8.56 km s⁻¹, P=31.12 GPa), 1,2,4,5‐tetrazine‐3,6‐diamine‐1,4‐dioxide (D=8.78 km s⁻¹, P=31.0 GPa), 1‐amino‐3,4,5‐trinitropyrazole (D=9.31 km s⁻¹, P=40.13 GPa), 4,4′‐dinitro‐3,3′‐bifurazan (D=8.80 km s⁻¹, P=35.60 GPa) and 3,4‐bis(3‐nitrofurazan‐4‐yl)furoxan (D=9.25 km s⁻¹, P=39.54 GPa). The  NH₂ group(s) appears to be particularly promising area for investigation since it may lead to two desirable consequences of higher stability (insensitivity), higher density, and thus detonation velocity and pressure.     

Characterization of the Stereochemical Structures of 2H‐Thiazolo[3,2‐a]pyrimidine Compounds and Their Binding Affinities for Anti‐apoptotic Bcl‐2 Family Proteins

In a previous study we reported a class of compounds with a 2H‐thiazolo[3,2‐a]pyrimidine core structure as general inhibitors of anti‐apoptotic Bcl‐2 family proteins. However, the absolute stereochemical configuration of one carbon atom on the core structure remained unsolved, and its potential impact on the binding affinities of compounds in this class was unknown. In this study, we obtained pure R and S enantiomers of four selected compounds by HPLC separation and chiral synthesis. The absolute configurations of these enantiomers were determined by comparing their circular dichroism spectra to that of an appropriate reference compound. In addition, a crystal structure of one selected compound revealed the exocyclic double bond in these compounds to be in the Z configuration. The binding affinities of all four pairs of enantiomers to Bcl‐x_L, Bcl‐2, and Mcl‐1 proteins were measured in a fluorescence‐polarization‐based binding assay, yielding inhibition constants (K_i values) ranging from 0.24 to 2.20 μM . Interestingly, our results indicate that most R and S enantiomers exhibit similar binding affinities for the three tested proteins. A binding mode for this compound class was derived by molecular docking and molecular dynamics simulations to provide a reasonable interpretation of this observation.     

IsoCombretaQuinazolines: Potent Cytotoxic Agents with Antitubulin Activity

A series of novel isocombretaquinazolines (isoCoQ) 4 were quickly prepared by coupling N‐toluenesulfonylhydrazones with 4‐chloroquinazolines under palladium catalysis. These compounds, which can be regarded as isocombretastatin A‐4 (isoCA‐4) analogues that lack the 3,4,5‐trimethoxyphenyl ring, displayed nanomolar‐level cytotoxicity against various human cancer cell lines and were observed to effectively inhibit tubulin polymerization. The isoCoQ compounds 2‐methoxy‐5‐(1‐(2‐methylquinazolin‐4‐yl)vinyl)phenol ( 4 b ), 4‐[1‐(3‐fluoro‐4‐methoxyphenyl)vinyl]‐2‐methylquinazoline ( 4 c ), and 2‐methoxy‐5‐(1‐(2‐methylquinazolin‐4‐yl)vinyl)aniline ( 4 d ), which respectively bear the greatest resemblance to isoCA‐4, isoFCA‐4, and isoNH₂CA‐4, are able to arrest HCT116 cancer cells in the G₂/M cell‐cycle phase at very low concentrations. Preliminary in vitro antivascular assay results show that 4 d is able to disrupt a network of capillary‐like structures formed by human umbilical vein endothelial cells on Matrigel. All these results clearly demonstrate that replacement of the 3,4,5‐trimethoxyphenyl ring of isoCA‐4 with a quinazoline nucleus is a feasible approach toward new and highly promising derivatives with the potential for further development as antitubulin agents.     

Semi‐synthesis and Structure–Activity Relationship of Neuritogenic Oleanene Derivatives

(3S,4R)‐23,28‐Dihydroxyolean‐12‐en‐3‐yl (2E)‐3‐(3,4‐dihydroxyphenyl)acrylate ( 1 a ), which possesses significant neuritogenic activity, was isolated from the traditional Chinese medicine (TCM) plant, Desmodium sambuense. To confirm the structure and to assess biological activity, we semi‐synthesized 1 a from commercially available oleanolic acid. A series of novel 1 a derivatives was then designed and synthesized for a structure–activity relationship (SAR) study. All synthetic derivatives were characterized by analysis of spectral data, and their neuritogenic activities were evaluated in assays with PC12 cells. The SAR results indicate that the number and position of the hydroxy groups on the phenyl ring and the triterpene moiety, as well as the length of the (saturated or unsaturated) alkyl chain that links the phenyl ring with the triterpene critically influence neuritogenic activity. Among all the tested compounds, 1 e [(3S,4R)‐23,28‐dihydroxyolean‐12‐en‐3‐yl (2E)‐3‐(3,4,5‐trihydroxyphenyl)acrylate] was found to be the most potent, inducing significant neurite outgrowth at 1 μm .     

Highly Diverse Protein Library Based on the Ubiquitous (β/α)8 Enzyme Fold Yields Well‐Structured Proteins through in Vitro Folding Selection

Proper protein folding is a prerequisite for protein stability and enzymatic activity. Although directed evolution can be a powerful tool to investigate enzymatic function and to isolate novel activities, well‐designed libraries of folded proteins are essential. In vitro selection methods are particularly capable of searching for enzymatic activities in libraries of trillions of protein variants, yet high‐quality libraries of well‐folded enzymes with such high diversity are lacking. We describe the construction and detailed characterization of a folding‐enriched protein library based on the ubiquitous (β/α)₈ barrel fold, which is found in five of the six enzyme classes. We introduced seven randomized loops on the catalytic face of the monomeric, thermostable (β/α)₈ barrel of glycerophosphodiester phosphodiesterase (GDPD) from Thermotoga maritima. We employed in vitro folding selection based on protease digestion to enrich intermediate libraries containing three to four randomized loops for folded variants, and then combined them to assemble the final library (10¹⁴ DNA sequences). The resulting library was analyzed by using the in vitro protease assay and an in vivo GFP‐folding assay; it contains ～10¹² soluble monomeric protein variants. We isolated six library members and demonstrated that these proteins are soluble, monomeric and show (β/α)₈‐barrel fold‐like secondary and tertiary structure. The quality of the folding‐enriched library improved up to 50‐fold compared to a control library that was assembled without the folding selection. To the best of our knowledge, this work is the first example of combining the ultra‐high throughput mRNA display method with selection for folding. The resulting (β/α)₈ barrel libraries provide a valuable starting point to study the unique catalytic capabilities of the (β/α)₈ fold, and to isolate novel enzymes.     

Synthesis and Characterization of 3,4,5‐Triamino‐1,2,4‐Triazolium 5‐Nitrotetrazolate

3,4,5‐Triamino‐1,2,4‐triazolium 5‐nitrotetrazolate ( 2 ) was synthesized in high yield from 3,4,5‐triamino‐1,2,4‐triazole (guanazine) ( 1 ) and ammonium 5‐nitrotetrazolate. The new compound 2 was characterized by vibrational (IR and Raman) and multinuclear NMR spectroscopy (¹H, ¹³C, ¹⁵N), elemental analysis and single crystal X‐ray diffraction (triclinic, P(‐1), a=0.7194(5), b=0.8215(5), c=0.8668(5) nm, α=75.307(5), β=70.054(5), γ=68.104(5)°, V=0.4421(5) nm³, Z=2, ϱ=1.722 g cm⁻¹, R₁=0.0519 [F>4σ(F)], wR₂(all data)=0.1154). The ¹⁵N NMR spectrum and X‐ray crystal structure (triclinic, P‐1, a=0.5578(5), b=0.6166(5), c=0.7395(5) nm, α=114.485(5)°, β=90.810(5)°, γ=97.846(5)°, V=0.2286(3) nm³, Z=2, ϱ=1.658 g cm⁻¹, R₁=0.0460 [F>4σ(F)], wR₂(all data)=0.1153) of 1 were also determined.     

γ‐FOX‐7: Structure of a High Energy Density Material Immediately Prior to Decomposition

1,1‐Diamino‐2,2‐dinitroethene, C₂H₄N₄O₄ (FOX‐7), is a novel high energy density material with low friction and impact sensitivity and a high activation barrier to detonation. In this study, the previously unknown crystal structure of the γ‐polymorph of trimorphic FOX‐7 is reported. γ‐FOX‐7 is stable from ∼435 K until the compound decomposes just above 504 K. A single crystal of α‐FOX‐7 (P2₁/n, Z=4, a=694.67(7) pm, b=668.87(9) pm, c=1135.1(1) pm, β=90.14(1)°, T=373 K) was first transformed into a single crystal of β‐FOX‐7 (P2₁2₁2₁, Z=4, a=698.6(1) pm, b=668.6(2) pm, c=1168.7(3) pm, T=423 K) and then into a single crystal of γ‐FOX‐7 at 450 K. The γ‐FOX‐7 crystal was then subsequently quenched to 200 K. The structure of γ‐FOX‐7 (P2₁/n, Z=8, a=1335.4(3) pm, b=689.5(1) pm, c=1205.0(2) pm, β=111.102(8)°, T=200 K) consists of four planar layers, each containing two crystallographically independent FOX‐7 molecules found in the asymmetric unit.     

A Fragment‐Based Approach to Probing Adenosine Recognition Sites by Using Dynamic Combinatorial Chemistry

A new strategy that combines the concepts of fragment‐based drug design and dynamic combinatorial chemistry (DCC) for targeting adenosine recognition sites on enzymes is reported. We demonstrate the use of 5′‐deoxy‐5′‐thioadenosine as a noncovalent anchor fragment in dynamic combinatorial libraries templated by Mycobacterium tuberculosis pantothenate synthetase. A benzyl disulfide derivative was identified upon library analysis by HPLC. Structural and binding studies of protein–ligand complexes by X‐ray crystallography and isothermal titration calorimetry informed the subsequent optimisation of the DCC hit into a disulfide containing the novel meta‐nitrobenzyl fragment that targets the pantoate binding site of pantothenate synthetase. Given the prevalence of adenosine‐recognition motifs in enzymes, our results provide a proof‐of‐concept for using this strategy to probe adjacent pockets for a range of adenosine binding enzymes, including other related adenylate‐forming ligases, kinases, and ATPases, as well as NAD(P)(H), CoA and FAD(H₂) binding proteins.     

Discovery and evolution of double P‐E loops in a tetragonal Fe‐doped KTa0.57Nb0.43O3 single crystal

Double polarization‐electric field (P‐E) loops were observed in Fe‐doped KTa_0.57Nb_0.43O₃ (Fe‐KTN) crystals because of the restraining effect of the defect dipoles on domain reorientation. In Fe‐KTN crystals, the positively charged O^2? vacancies and negatively charged dopant Fe³⁺ ions form defect dipoles, providing a restoring force for domain reorientation. Moreover, built‐in macro‐polarization was observed depending on the orientation of the defect dipole polarization. The response of the defect dipoles to an external electric field and the evolution of the double P‐E loops were investigated. The restraining effect of the defect dipoles on domain reorientation was found to affect normal piezoelectric activities. The domains could maintain the state close to the polarization direction, resulting in an extremely high d₃₃ value (287 pC/N).     

Dynamic mechanical properties of polyimide/poly(silsesquioxane)‐like hybrid films

Polyimide/poly(silsesquioxane)‐like (PI/PSSQ‐like) films have three‐dimensional structure with linear PI blocks and a crosslinked PSSQ‐like structure. They have higher thermal stability and char yields than pure PI from 4,4′‐diaminodiphenyl ether and 3, 3′‐oxydiphthalic anhydride (ODPA). In a series of X‐PIS [PI modified with p‐aminophenyltrimethoxysilane (APTS), where X is the molecular weight of each PI block] hybrid films, decreasing the PI block chain length enhances the storage modulus, tensile modulus, and glass‐transition temperature (T_g) but reduces the α‐relaxation damping peak intensity, density, and elongation. The change in the former three properties may be caused by an increase in the crosslinking density and rigidity of the network structure. The changes in the next two properties are caused by an increase in the free volume or the PI interblock separation and decreases in the interblock PI chain interaction. The change in the last property (i.e., a decrease in elongation) is related to the increase in the rigidity of the network structure. The activation energy of the α transition depends on the chain length of the PI block. A maximum value is reached at the chain length of 10,000 because of two different factors: crosslinking density and free volume. In a series of X‐PIS‐y‐PTS [X‐PIS modified with phenyltrimethoxysilane (PTS), where y is the weight‐ratio percentage of PTS to APTS–polyamic acid] films with a constant PI block length, the storage modulus, tensile modulus, T_g, density, and α‐relaxation damping peak intensity decrease with the PTS content. This could be because of the increase of the PSSQ‐like domain size with the PTS content, which leads to the introduction of more free volume or interblock separation and to a decrease in the interblock chain interaction force. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 2500–2516, 2001     

The AT2 Domain of KirCI Loads Malonyl Extender Units to the ACPs of the Kirromycin PKS

The antibiotic kirromycin is assembled by a hybrid modular polyketide synthases (PKSs)/nonribosomal peptide synthetases (NRPSs). Five of six PKSs of this complex assembly line do not have acyltransferase (AT) and have to recruit this activity from discrete AT enzymes. Here, we show that KirCI is a discrete AT which is involved in kirromycin production and displays a rarely found three‐domain architecture (AT₁‐AT₂‐ER). We demonstrate that the second AT domain, KirCI‐AT₂, but not KirCI‐AT₁, is the malonyl‐CoA‐specific AT which utilizes this precursor for loading the acyl carrier proteins (ACPs) of the trans‐AT PKS in vitro. In the kirromycin biosynthetic pathway, ACP5 is exclusively loaded with ethylmalonate by the enzyme KirCII and is not recognized as a substrate by KirCI. Interestingly, the excised KirCI‐AT₂ can also transfer malonate to ACP5 and thus has a relaxed ACP‐specificity compared to the entire KirCI protein. The ability of KirCI‐AT₂ to load different ACPs provides opportunities for AT engineering as a potential strategy for polyketide diversification.