Effect of Glycosylation on the Catalytic and Conformational Stability of Homologous α-Amylases

summary. A thermostable -amylase from B. licheniformis (BLA) and a mesophilic amylase from B. amyloliquefaciens (BAA) were covalently coupled to oxidized synthetic sucrose polymers (OSP400 and OSP70) and polyglutaraldehyde (PGA) by reductive alkylation to study the effect of neoglycosylation on the activity, kinetic and thermodynamic stability. The catalytic efficiency of the modified enzymes was comparable to that of the native enzyme. Covalent coupling decreased the rate of inactivation at all the temperatures studied, both in the presence and absence of added Ca²⁺. The stability of the native enzyme was found to increase upon modification as observed from the increase in t_1/2 in the absence of Ca²⁺ ions by about 1.5–13.7 times (at 85°C) in the case of BLA and 5.7–8.4 times (at 50°C) for BAA. The highest stability was observed for OSP400 modified enzyme with C_m and T_m values of 0.63 M and 7.92°C for BLA and 0.85 M and 5.3°C for BAA, respectively. The order of stability was OSP400 > OSP70 > PGA > Native for both BLA and BAA. The stability of the modified amylases obtained from the present study were superior compared to most of the single and double mutants obtained by site-directed mutagenesis that were constructed so as to enhance the intrinsic stability of these enzymes.This article is dedicated to Dr. P.V. Sundaram.     

Molecular interactions of zinc(II) cyclams with polycarboxylato ligands

Four new zinc(II) cyclams of the composition {Zn(L)(tp²⁻) · H₂O}_n (1), {Zn(L)(H₂bta²⁻) · 2H₂O}_n (2), [Zn₂(L)₂(ox²⁻)] 2ClO₄ · 2DMF (3), and Zn(L)(H₂btc⁻)₂ · 2DMF (4), where L = cyclam, tp²⁻ = 1,4-benzenedicarboxylate ion, H₂bta²⁻ = 1,2,4,5-benzenetetracarboxylate ion, ox²⁻ = oxalate ion, DMF = N,N-dimethylformamide, and H₂btc⁻ = 1,3,5-benzenetricarboxylate ion, have been synthesized and structurally characterized by a combination of analytical, spectroscopic and crystallographic methods. The carboxylato ligands in the complexes 1-4 show strong coordination tendencies toward zinc(II) cyclams with hydrogen bonding interactions between the pre-organized N-H groups of the macrocycle and oxygen atoms of the carboxylato ligands. The macrocycles in 1, 2, and 4 adopt trans-III configurations with the appropriate R,R,S,S arrangement of the four chiral nitrogen centers, respectively. However, the complex 3 shows an unusual cis V conformation with the R,R,R,R nitrogen configuration. The finding of strong interactions between the carboxylato ligands and the zinc(II) ions may provide additional knowledge for the improved design of receptor-targeted zinc(II) cyclams in anti-HIV agents.     

Entropic interaction chromatography: separating proteins on the basis of size using end-grafted polymer brushes

Partitioning of a macromolecule into the interfacial volume occupied by a grafted polymer brush decreases the configurational entropy (DeltaSbrush(c)) of the terminally attached linear polymer chains due to a loss of free volume. Self-consistent field theory (SCF) calculations are used to show that DeltaSbrush(c) is a strong function of both the size (MWp) of the partitioning macromolecule and the depth of penetration into the brush volume. We further demonstrate that the strong dependence of DeltaSbrush(c) on MWp provides a novel and powerful platform, which we call entropic interaction chromatography (EIC), for efficiently separating mixtures of proteins on the basis of size. Two EIC columns, differing primarily in polymer grafting density, were prepared by growing a brush of poly(methoxyethyl acrylamide) chains on the surface of a wide-pore (1,000-A pores, 64-microm diameter rigid beads) resin (Toyopearl AF-650M) bearing surface aldehyde groups. Semipreparative 0.1-L columns packed with either EIC resin provide reduced-plate heights of 2 or less for efficient separation of globular protein mixtures over at least three molecular-weight decades. Protein partitioning within these wide-pore EIC columns is shown to be effectively modeled as a thermodynamically controlled process, allowing partition coefficients (K(P)) and elution chromatograms to be accurately predicted using a column model that combines SCF calculation of K(P) values with an equilibrium-dispersion type model of solute transport through the column. This model is used to explore the dependence of column separation efficiency on brush properties, predicting that optimal separation of proteins over a broad MWp range is achieved at low to moderate grafting densities and intermediate chain lengths.     

Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

Synthesis and structural characterization of cadmium(II) complexes with 2-(2-pyridyl)imino-N-(2-thiazolin-2-yl)thiazolidine (PyTT)

We have carried out the synthesis of the cadmium coordination compounds [Cd(NO₃)₂(PyTT)(H₂O)] (1) and [CdCl₂{(μ-Cl)₂CdCl(μ-Cl)(μ-PyTT)Cd}₂]_n (2), together with their structural determination by means of X-ray diffraction. The compounds were also characterized through elemental analysis and infrared spectroscopy. The first complex presents a distorted pentagonal bipyramidal geometry with the axial positions occupied by one oxygen atom from a water molecule and a second one from a nitrate ion which acts as a monodentate ligand, whereas the equatorial plane contains three nitrogen atoms from the organic moiety and two oxygen atoms coming from the other nitrate group, which is bidentate. The structure of the second complex consists of parallel sheets linked by van der Waals forces, each one made up of structural units [CdCl₂{(μ-Cl)₂CdCl(μ-Cl)(μ-PyTT)Cd}₂], which possesses two PyTT ligands, 10 bridging chloro ligands and 5 cadmium(II) centres belonging to three environment types: octahedral CdN₂Cl₄, octahedral CdCl₆, on which a centre of symmetry is located, and tetrahedral CdNCl₃, present in a 2:1:2 ratio.     

Syntheses, structures and properties of three cluster-based coordination polymers: influence of the metal ions on the ligand coordination mode and crystal chirality

Three cluster-based coordination polymers, namely [Zn₃(bpy)₃(hip)₂] · 5H₂O (1), [Co₃(bpy)₃(hip)₂] · 5H₂O (2) and [Cd₃(bpy)₃(hip)₂] (3) (bpy=2,2^′-bipyridine, hip=4-hydroxyl-isophthalate) were synthesized and structurally characterized. X-ray single-crystal structural analyses revealed that both 1 and 2 crystallize in the chiral space group P2₁, while 3 crystallizes in the centric space group Pccn. Compounds 1 and 2 are isomorphous and both have (4,4) topological layered structures constructed from trinuclear metal clusters. Compound 3 also shows layered structure of (4,4) topology constructed from trinulear Cd(II) cores. The layers are stacked in a staggered ?ABAB? fashion in 1 and 2 but in an overlapped ?AAA? fashion in 3. There are two types of coordination modes of hip ligand in 1 and 2 but only one in 3. The structural difference between 1 (or 2) and 3 may be attributed to the difference of metal ion nature such as the ionic radius and coordination preference, resulting in the different orientation fashions of the auxiliary bpy ligands, stacking fashions of the layers, as well as chirality of the crystals. The chiral structures of 1 and 2 were also confirmed by measurements of powder second-harmonic-generation (SHG) measurements, which show that 1 and 2 have SHG intensity of 0.50 and 0.02 relative to that of urea, respectively.     

Gold Nanoparticles from Induced Au3+→Au0 Reaction in Polyvinyl Alcohol Molecules in Presence of Sucrose in Hot Water

In hot water (50–60°C), polyvinyl alcohol (PVA) molecules have coordination reaction with Au³⁺ cations, forming an Au³⁺-PVA polymer complex. In the proposed model reaction in small templates, the complex converts to Au⁰ capping in PVA molecules. Adding sucrose (5–10 times the PVA in mass) in a typical batch promotes Au³⁺→Au⁰ reaction, showing absorption coefficient α in Au⁰ surface plasmon band to be enhanced as much as 28 times the value in reaction with PVA. The band shifts at 547 nm from 566 nm (α  =  21.4 cm⁻¹ mol⁻¹) in the PVA sample. Drying Au⁰-PVA/sucrose (2–5 wt% Au⁰) colloid at 60–70°C and then heating at 450°C in air burns off the organic part, leaving behind a light ash colored powder with Au⁰ nanoprisms or nanofibrils (∼30 nm average width). X-ray diffractogram has six reflections, (111), (200), (220), (311), (222), and (400), of Fm3m fcc Au⁰ of lattice parameter a  =  0.4080 nm. The powder has photoluminescence in transversal and longitudinal Au⁰ plasmon bands of 535 and 585 nm, respectively.     

Sonochemical syntheses of strawberry-like nano-structure mixed-ligand lead(II) coordination polymer: Thermal, structural and X-ray powder diffraction studies

Nano-structure of a new Pb(II) two-dimensional coordination polymer, {[Pb(2-pyc)(N₃)(H₂O)]_n (1), 2-Hpyc = 2-pyridinecarboxylic acid} was synthesized by a sonochemical method. The new nano-structure was characterized by scanning electron microscopy, X-ray powder diffraction, IR spectroscopy and elemental analyses. Compound 1 was structurally characterized by single crystal X-ray diffraction and consists of two-dimensional polymeric units. The thermal stability of compound 1 was studied by thermal gravimetric and differential thermal analyses and compared. PbO nano-powder was obtained by calcination of the nano-structure of compound 1 at 400 °C. This study demonstrates the coordination polymers may be suitable precursors for the preparation of nano-scale materials and dependent on their packing they may produce different and interesting morphologies.     

Topological diversity in copper aromatic meta-dicarboxylate coordination polymers with bis(pyridylformyl)piperazine coligands

Hydrothermal synthesis has afforded divalent copper coordination polymers containing bis(4-pyridylformyl)piperazine (4-bpfp) tethers and aromatic meta-dicarboxylate ligands. {[Cu(ip)(4-bpfp)]·2H₂O}_n (1, ip = isophthalate) possesses a (4, 4) rectangular grid structure with an unusual ABCD stacking pattern along a 4₁ screw axis. Sterically bulky substituents in the 5-position of the isophthalate ligands reduced the coordination polymer dimensionality, with [Cu₂(tBuip)₂(4-bpfp)(H₂O)₂]_n (2, tBuip = 5-tert-butylisophthalate) and {[Cu(MeOip)(HMeOip)₂(4-bpfp)]·3H₂O}_n (3, MeOip = 5-methoxyisophthalate) displaying 1D polymeric ladder and chain motifs, respectively. Compound 3 possesses a rare twofold interpenetrated binodal supramolecular hms net with (6³)(6⁹8) topology. Longer meta-disposed acetate pendant arms induced a doubly interpenetrated 3D primitive cubic topology in {[Cu₂(1,3-phda)₂(H₂O)₂(4-bpfp)]}_n (4, 1,3-phda = 1,3-phenylenediacetate), which possesses antiferromagnetically coupled {Cu₂O₂} kernels (J = −6.14(8) cm⁻¹).     

Synthesis, structure and luminescence study of two cadmium(II) coordination polymers with hetero donor ligands: Expansion of network dimensionality from 2D to 3D through hydrogen bonding

Two coordination polymers of cadmium with formula [Cd(pyp)₂(H₂O)₂]_n (1) and {[Cd₂(pyzca)₃(atr)(H₂O)]·H₂O}_n (2) [pypH = 3-pyridinepropionic acid, pyzcaH = 2-pyrazinecarboxylic acid and atrH = 5-aminotetrazole] have been synthesized and structurally characterized by X-ray single crystal diffraction analysis. Both complexes display 2D structures that extend into a 3D network by means of hydrogen bonding. The crystal packing of both complexes is reinforced by π-π interactions between adjacent aromatic rings. The fluorescence study indicates intraligand π-π* charge transfer, which is the reason for emission in both the complexes.