Ultrasonic degradation of poly(γ-benzyl-l-glutamate), an archetypal highly extended polymer

Ultrasonic degradation provides a convenient means of studying the degradation of macromolecules in transient elongational flows. Of particular interest, because of their being a limiting architectural case, is the degradation of highly extended polymers. A classic example of a polymer with this type of architecture is poly(γ-benzyl-l-glutamate) or PBLG. Here, we monitor results of the ultrasonic degradation of PBLG using size-exclusion chromatography with a multiplicity of physical detectors. Under the given experimental conditions, the limiting molar mass (M_lim) of PBLG was found to be approximately 114,000 g/mol. The fractal dimension of PBLG, which exemplifies the highly extended nature of the macromolecule, was measured by both light scattering and viscometric means. Results from both methods demonstrated a virtual invariance in chain conformation as a function of degradation. Additional support for these observations was provided by the dimensionless ratio of the viscometric and root-mean-square radii.     

Surface modification of microporous polypropylene membranes by the grafting of poly(γ-stearyl-l-glutamate)

A polypeptide, poly(γ-stearyl-l-glutamate) (PSLG), was grafted on the surface of hydrophobic polypropylene hollow fiber membranes through the ring opening polymerization of N-carboxyanhydride (NCA) of γ-stearyl-l-glutamate initiated by amino groups which was generated by ammonia plasma. X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FT-IR), together with water contact angle and bovium serum albumin adsorption measurements were used to characterize the modified membrane surface. The XPS and FT-IR spectra demonstrated that polypeptide was actually grafted on the membrane surface despite of the low degree of graft polymerization due to the hydroxyl groups on the membrane surface. To subject the ammonia plasma-treated membrane with γ-(aminopropyl)triethanoxysilane (γ-APS) which can react with hydroxyl groups and leave amino groups, the degree of graft polymerization could be improved. The bovium serum albumin adsorption measurement was conducted to further examine the surface properties of modified and original membranes. Potential applications of the PSLG grafted membranes are expected for enantiomer separation and/or enzyme immobilization.     

New organotin(IV) complexes with l-Arginine, Nα-t-Boc-l-Arginine and l-Alanyl-l-Arginine: Synthesis, structural investigations and cytotoxic activity

Novel diorganotin(IV) derivatives of l-Arginine (HArg), N_α-(tert-Butoxycarbonyl)-l-Arginine (Boc-Arg-OH) and l-Ala-l-Arg (H₂Ala-Arg), H₂NC(NH)NH(CH₂)₃CH(NHR′)CO₂H, where R′ = H in HArg, R′ = C(O)OC(CH₃)₃ in Boc-Arg-OH, R′ = H₂NCH(CH₃)CO in H₂Ala-Arg and triorganotin(IV) derivatives of Boc-Arg-OH have been synthesized and structurally characterized. The complexes were investigated by FT-IR and ¹¹⁹Sn Mössbauer in the solid state and by ¹H, ¹³C, ¹¹⁹Sn and ¹H-¹H COSY NMR spectroscopy, in solution. The spectroscopic characterization leading to the proposed molecular structures was accomplished on the basis of these experiments. l-Arginine appears to behave as a chelating ligand through carboxylate and -NH₂ groups in Me₂Sn(Arg)₂, while in N_α-t-Boc-l-Arginine complex, the N_α-protected amino group being exempted from coordination, only the carboxylate groups are effectors of bonding to the organometallic moieties. FT-IR spectra give a clear indication that guanidino groups in all the complexes are not involved in coordination, since ν(CN-H) frequency of the terminal guanidino group is fairly constant and unshifted relative to the free ligand. The biological activity of organotin(IV)-complexes was also investigated by use of human HT29 colorectal carcinoma cells. The cytotoxic activity of the compounds was determined by the MTT quantitative colorimetric assay, capable of detecting viable cells in comparison with that exerted by cisplatin. A marked cytotoxic activity for nearly all complexes, is evident being higher than that exerted by cisplatin, while no significant improvement of activity was observed for Me₂Sn(Arg)₂ and Me₂Sn(Ala-Arg), which was confirmed by IC₅₀ values. Then, we assessed whether the cytotoxicity induced by organotin(IV) complexes was associated with the induction of apoptosis. Light microscopy analysis, performed to study the morphological changes induced in HT29 cells, confirmed the results obtained with MTT test. No significant morphological alterations were observed in HT29 cells after treatment with Me₂Sn(Ala-Arg) and Me₂Sn(l-Arg)₂. Cells treated with ⁿBu₂Sn(Boc-Arg)₂, ⁿBu₂Sn(Ala-Arg), ⁿBu₃Sn(Boc-Arg) and Me₃Sn(Boc-Arg), appeared rounded, isolated and detached from culture substrate, indicating the commitment to apoptotic cell death.     

Direct access to new β-d-galactofuranoconjugates: application to the synthesis of galactofuranosyl-l-cysteine and l-serine

Galactofuranose post-translational modifications, although quite rare, were detected in some biomolecules produced by parasites. While hexopyranosides were already linked to various peptides and proteins, few hexofuranosides have been artificially conjugated to amino acids. We thus report herein a robust glycosylation methodology to obtain S-alkyl, O-serine and S-cysteine-β-d-galactofuranosides starting from readily available galactofuranose donors. O-Acetyl, thioimidoyl and acetimidoyl donors were compared in terms of yields and selectivity when reacted with mercaptans, l-cysteine and l-serine. Acetimidates turned out to be the best notably for amino acids glycosylation.     

Blends as a strategy towards tailored hydrolytic degradation of poly(?-caprolactone-co-d,l-lactide)-poly(ethylene glycol)-poly(?-caprolactone-co-d,l-lactide) co-polymers

Binary blends were prepared from poly(?-caprolactone) (PCL), and P(CL-co-d,l-lactic acid)-P(ethylene glycol)-P(CL-co-d,l-lactic acid) co-polymers, where the d,l-LA content in the side chains varied from 0 to 70 mol%. Blend discs were fabricated by melt-molding, and the effect of blend composition on hydrolytic degradation was studied. Variations in medium pH were monitored, and morphological changes were observed using scanning electron microscopy. Blending of these co-polymers was found to constitute a simple means by which intermediate rates of water absorption and mass loss were obtained, compared to those observed in pure co-polymer preparations. In one of the blends, prepared from the two components containing 70 or 0 mol% d,l-LA in the side chains and thereby exhibiting large differences in degradation rate, hydrolysis resulted in the formation of a porous material over time. Furthermore, all blend samples maintained their initial shape throughout the study. Such materials may be interesting for further investigations for applications in cellular therapy and controlled release.     

A practical synthesis of fully protected l-γ-carboxyglutamic acid

We have developed a new synthetic route for the preparation of Fmoc protected l-γ-carboxyglutamic acid in 60% overall yield (>99% ee) via a six-step synthesis from d-Garner’s aldehyde. An aldol condensation and the selective cleavage of the acetonide protective group are key steps.     

Structure-based study of antiamyloidogenic cyclic d,l-α-peptides

Protein misfolding and aggregation are the hallmarks of many devastating diseases. We have previously shown that cyclic d,l-α-peptide CP-2 reacts and stabilizes less toxic forms of amyloid β (Aβ), and protects the cells from Aβ-induced toxicity. Here, we performed extensive structure-based studies on CP-2 to elucidate the contribution of each residue to the total antiamyloidogenic activity and determine the interactions that are involved between CP-2 and Aβ. We showed that the hydrophobicity of CP-2 analogs correlates with their antiamyloidogenic potency, however, aromatic interactions are even more important for this activity. The antiamyloidogenic activity of CP-2 analogs also correlates with their ability to self-assemble, as shown by the critical micelle concentration measurements. The cell survival studies performed on rat pheochromocytoma PC-12 cells suggest that incorporation of an additional aromatic residue to the CP-2's sequence increases its protective effect against Aβ42-induced toxicity.     

l-Galactose as a natural product: isolation from a marine octocoral of the first α-l-galactosyl saponin

Saponine 1 (6′-O-acetyl-3β-pregna-5,20-dienyl-α-l-galactopyranoside), that contains a l-galactose moiety linked to the aglycone through an infrequent α-glycosidic bond, has been isolated from the marine octocoral Muricea c.f. purpurea. This constitutes the first report on the occurrence of l-Gal as a nonpolymeric natural product. A CD procedure for the absolute stereochemical assignment of saponins, based on the CD analysis of its perbenzoylated derivative, is proposed.     

Increasing the inhibitory potency of l-arabino-imidazolo-[1,2]-piperidinose towards β-d-glucosidase and β-d-galactosidase

The synthesis of some potent inhibitors of two retaining β-glycosidases was achieved by introducing aglycon-mimics into the imidazole moiety of l-arabino azasugar 1. The strongest inhibition was observed with the phenyl-ethyl substituent at C(2) of 1 against β-d-galactosidase and β-d-glucosidase, whereas the hydroxymethyl group at C(2) increased only slightly the inhibitory properties.     

Synthesis of quercetin 3-O-β-d-apiofuranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→6)]-β-d-glucopyranoside

A concise method to construct a unique 2,6-branched trisaccharide was established by regioselective glycosylation of three free hydroxyl groups on a 3-O-protected glucose moiety, and successfully used in the synthesis of quercetin 3-O-β-d-apiofuranosyl-(1→2)-[α-l-rhamnopyranosyl-(1→6)]-β-d-glucopyranoside, a flavonol O-glycoside isolated from glandless cotton seeds which showed notable antidepressant activities.     

Synthesis and RNA-selective hybridization of α-l-ribo- and β-d-lyxo-configured oligonucleotides

Three α-l-ribofuranosyl analogues of RNA nucleotides (α-l-RNA analogues) have been synthesized and incorporated into oligonucleotides using the phosphoramide approach on an automated DNA synthesizer. The 4′-C-hydroxymethyl-α-l-ribofuranosyl thymine monomer was furthermore synthesized. Relative to the unmodified duplexes, incorporation of a single α-l-RNA monomer into a DNA strand leads to reduced thermal stability of duplexes with DNA complements but unchanged thermal stability of duplexes with RNA complements, whereas incorporation of more than one α-l-RNA monomer lead to moderately decreased thermal stability also of duplexes with RNA complements. Efficient hybridization with an RNA complement and no melting transition with a DNA complement were observed with stereoregular chimeric oligonucleotides composed of a mixture of α-l-RNA and affinity enhancing α-l-LNA monomers (α-l-ribo-configured locked nucleic acid). Furthermore, duplexes formed between oligodeoxynucleotides containing an α-l-RNA monomer and complementary RNA were good substrates for Escherichia coli RNase H. RNA-selective hybridization was also achieved by the incorporation of 1-(4-C-hydroxymethyl-β-d-lyxofuranosyl)thymine monomers into a DNA strand, whereas stable duplexes were formed with both complementary DNA and RNA when these monomers were incorporated into an RNA strand.     

Sequential fluorometric quantification of γ-aminobutyrate and l-glutamate using a single line flow-injection system with immobilized-enzyme reactors

A single line flow-injection system with immobilized-enzyme reactors is proposed for the sequential quantification of γ-aminobutyrate (GABA) and l-glutamate. A co-immobilized l-glutamate oxidase and catalase reactor and an immobilized GABase reactor were introduced into the flow line in series. Sample and reagent were injected into the flow line using an open sandwich method. GABA was selectively detected by GABase when α-ketoglutarate at a high concentration and NADP⁺ were injected as the reagents with a sample. When GABA at a high concentration and NADP⁺ were injected as the reagents with a sample, l-glutamate only was determined by the series of enzymatic reactions. NADPH produced by the immobilized-enzyme reactors was monitored fluorometrically at 455 nm (excitation at 340 nm). Linear relationships between the responses and concentrations of GABA or l-glutamate were observed in the ranges of 5.0 × 10⁻⁶-5.0 × 10⁻⁴ M and 1.0 × 10⁻⁵-5.0 × 10⁻⁴ M, respectively. The relative standard deviations for ten successive injections were less than 2% at the 0.5 mM level. This analytical method was applied to the sequential quantification of GABA and l-glutamate that were produced and consumed, respectively, by lactic acid bacteria, and the results showed good agreement with those obtained using liquid chromatography.     

Hierarchical self-assembly in diblock copolypeptides of poly(γ-benzyl-l-glutamate) with poly poly(l-leucine) and poly(O-benzyl-l-tyrosine)

Block copolypeptides with their inherent nanometer length scale of phase separation, provide means of manipulating the type (α-helices, β-strands) and persistence of peptide secondary structures. Two such examples are employed based on the α-helical poly(γ-benzyl-l-glutamate) (PBLG) polypeptide as one block and poly(l-leucine) (α-helical) or poly(O-benzyl-l-tyrosine) (POBT) (β-strands) as the second block. Although both secondary structures are present in the copolypeptides the effect of nano-scale confinement is to induce folding in the POBT β-sheets and to maintain the defected α-helices of PBLG and PLEU with a limited lateral coherence.     

A novel amperometric biosensor based on a co-crosslinked l-lysine-α-oxidase/overoxidized polypyrrole bilayer for the highly selective determination of l-lysine

An amperometric biosensor for the determination of l-lysine based on l-lysine-α-oxidase immobilized by co-crosslinking on a platinum electrode previously modified by an overoxidized polypyrrole film is described. The optimization of experimental parameters, such as pH and flow rate, permitted to minimize significantly substrate interferences even using a low specific, commercial enzyme. The relevant biases introduced in the measurement of lysine were just about 1% for l-arginine, l-histidine and l-ornithine, roughly 4% for l-phenylalanine and l-tyrosine. The developed approach allowed linear lysine responses from 0.02 mM up to 2 mM with a sensitivity of 41 nA/(mM × mm²) and a detection limit of 4 μM (S/N = 3). No appreciable loss in lysine sensitivity was observed up to about 40 days. Allowing polypyrrole layer to remove interference from electroactive compounds, the present method revealed suitable to detect l-lysine in a pharmaceutical and cheese sample, showing a good agreement with the expected values.     

Enzymatic synthesis of ω-carboxy-β-hydroxy-(l)-α-amino acids

Commercially available ω-carboxy-aldehydes and glycine have been subjected to the catalytic action of an l-threonine aldolase from Escherichia coli to give the corresponding β-hydroxy-α-(l)-amino acids as a mixture of erythro/threo epimers.Specifically, the reaction with glyoxylic acid (2) gave the epimeric β-hydroxy-(l)-aspartates (t,e)-9 that could be isolated by ion-exchange chromatography in 67% yield. Following esterification and N-Boc protection, the two epimers could be isolated as pure compounds.Similarly, the aldolase-catalyzed addition of glycine to succinic semialdehyde (4) gave the expected mixture of β-hydroxy-l-α-aminoadipic acids (t)-12 and (e)-12 in 34% yield.     

A convenient new route to protected and free 2,6-anhydro-d-glycero-d-gulo-heptoses (1-formyl-β-d-glucopyranosides)

A new and efficient process has been developed for the synthesis of 1-formyl-β-d-glucopyranosides from d-glucose.     

Solvent-induced morphological diversification in poly(l-lactide-b-?-caprolactone) block copolymer thin films

Biodegradable block copolymer of poly(l-lactide-b-?-caprolactone) (P(LA-b-CL)) was dissolved in various solvents with different solubility as well as volatility, and spin-cast on a highly oriented pyrolytic graphite (HOPG) to prepare thin films. The surface morphologies were observed by using atomic force microscopy (AFM) in a dynamic force (tapping) mode. Particle like morphology was found in the thin films prepared form the dichloromethane and acetone. Higher volatility of dichloromethane and acetone resulted in the reflection of the particle like objects in the solution to HOPG substrate. In contrast, the P(LA-b-CL)s in toluene and 1,4-dioxane exhibited different morphologies compared to those in dichloromethane and acetone. Lower volatility of toluene and 1,4-dioxane assisted the epitaxial crystallization of PCL component along the HOPG lattice, that was revealed by enzymatic degradation of PLLA component by proteinase K. Thus, adjusting the solubility and solvent volatility for the film formation provided morphological divergence of the P(LA-b-CL) block copolymer, and this technique would be applicable for the surface patterning of biodegradable polymers.     

Facile one-step synthesis of N-α-Boc-1-alkyl-l-histidines

N-α-Boc-l-Histidine upon direct τ(N-1) ring alkylation with various alkyl halides in the presence of sodium hydride in DMF or CH₃CN easily afforded N-α-Boc-1-alkyl-l-histidines 2a-f. The reaction works equally well in either DMF or CH₃CN as solvent, however, CH₃CN is preferred due to ease of reaction work-up.     

Effect of partial crosslinking on morphology and properties of the poly(β-hydroxybutyrate)/poly(d,l-lactic acid) blends

Poly(β-hydroxybutyrate) (PHB) is a bio-based and biodegradable aliphatic polyester, however its application is limited by some disadvantages such as high price, brittleness, poor processability and low melt-strength due to serious thermal degradation. Partial crosslinking initiated by dicumyl peroxide (DCP) was applied in this work to improve the performance of poly(β-hydroxybutyrate)/poly(d,l-lactic acid) (PHB/PDLLA) blends. The partial crosslinking of the blends and its effect on the properties, morphology, rheology and thermal behavior of the blends were investigated. The tensile strength and impact toughness of the PHB were increased by incorporation of the PDLLA, which were improved further after the partial crosslinking because of an increased compatibility between the PHB and the PDLLA phases. The rheological study revealed that the storage modulus (G′) and complex viscosity (η*) of the blends were increased after addition of the DCP. On the other hand, the crystallization of PHB in the blends was restricted to a certain extent by the formation of partially crosslinked network while its crystal form was not modified.     

Synthesis of chiral thiourea–phosphine organocatalysts derived from l-proline

A novel class of thiourea–phosphine was prepared from l-proline as a chiral renewable resource. The original structure of the chiral framework offers an interesting potential to the construction of bifunctional organocatalysts for asymmetric transformations.