Formation of insulin fragments by insulin‐degrading enzyme: the role of zinc(II) and cystine bridges

Insulin is the hormone mainly involved in widespread diseases such as diabetes mellitus. It is widely recognized that metal ions such as zinc(II) as well as insulin degradation and insulin fragments are inexplicably linked to the hormone action. Insulin‐degrading enzyme (IDE) has been identified as the main factor of insulin degradation, but it is still unknown the exact way and location at which IDE action toward insulin occurs and how metal ions can modulate this interaction. Interestingly, some insulin fragments have different biological activity from the intact hormone, and it is not clear how they can be generated from insulin. In this work, the role of zinc(II) and cystine bridges in the degradation of insulin by IDE are investigated by high‐performance liquid chromatography‐mass spectrometry (HPLC‐MS), and the experimental conditions at which peculiar insulin fragments having biological activity are formed by the action of IDE are found and discussed. Docking simulations of IDE/insulin A and B chains are in good accordance with the insulin fragments detected by HPLC‐MS. Copyright © 2013 John Wiley & Sons, Ltd.     

QCM 表面自组装胱氨酸1，3－桥连杯[4]芳烃膜用于识别气相丁胺

Two cystine-bearing 1,3-bridged calix[4]arenes were used as the coatings of the quartz crystal microbalance (QCM) with gold electrodes. The two calix[4]arene derivatives were self-assembled onto the gold electrode surface by the covalent attachment between the di-sulfur and gold. The compound of cystine-bearing bi-phenylalanine 1,3-bridged calix[4]arene (CPC) with longer alkyl chain had better self-assembled capacity onto the fresh surfaces of gold electrode than that of cystine-bearing 1,3-bridged calix[4]arene (CC) with comparably shorter alkyl chain.The modified QCM sensors were used to recognize the butylamine isomers in gas. The results showed that the QCM coated with both compounds had preferential affinity to n-butylamine, then i-butylamine, t-butylamine in the range of low concentrations, indicating that in the recognition process, the steric hindrance effect played an important role when forming complex with guest molecules. When the concentrations of the analytes were increased, the polarity and the magnetism of the butylamine became determinative factors. The reversibility was improved greatly and the equilibrium time was much shorter on the self-assembled film than on the film obtained by dropping coating.     

Melt polycondensation approach for reduction degradable helical polyester based on l‐cystine

Melt polycondensation approach is developed for new classes of reduction responsive disulfide containing functional polyesters based on l ‐cystine amino acid resources under solvent free process. l ‐Cystine was converted into multi‐functional ester‐urethane monomer and subjected to thermoselective transesterification at 120 °C with commercial diols in the presence of Ti(OBu)₄ to produce polyesters with urethane side chains. The polymers were produced in moderate to high molecular weights and the polymers were found to be thermally stable up to 250 °C. The β‐sheet hydrogen bonding interaction among the side chain urethane unit facilitated the self‐assembly of the polyester into amyloid‐like fibrils. The deprotection of urethane unit into amine functionality modified the polymers into water soluble cationic polyester spherical nanoparticles. The reduction degradation of disulfide bond was studied using DTT as a reducing agent and the high molecular weight polymers chains were found be chopped into low molecular weight oligomers. The cytotoxicity of cationic disulfide nanoparticle was studied in MCF‐7 cells and they were found to be biocompatible and non‐toxic to cells upto 50 μg/mL. The custom designed reduction degradable and highly biocompatible disulfide polyesters from l ‐cystine are useful for futuristic biomedical applications. © 2016 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 2864–2875     

Hydrolytic degradation of poly(rac‐lactide) and poly[(rac‐lactide)‐co‐glycolide] at the air–water interface

The understanding of the simultaneous transport and chain‐scission phenomena involved in the hydrolysis of bulk‐degrading polymers requires the experimental separation of chain cleavage and water diffusion. The hydrolytic chain cleavage of poly(rac‐lactide) rac‐(PLA) and poly[(rac‐lactide)‐co‐glycolide] (PLGA) is analysed on the basis of monolayer degradation experiments combined with an improved data reduction procedure. Different, partly contradictory models of the hydrolytic degradation and erosion mechanism of PLA and PLGA, namely random chain scission and chain‐end scission, are discussed in the literature. The instantaneous linear area reduction observed for the polymer Langmuir films indicates a chain‐end scission mechanism. As monolayers of end‐capped and non‐end‐capped polymers degrade with exactly the same rate, the observed differences in the degradation kinetics of bulk samples do clearly result from differences in the water penetration into these polymers. A pronounced ‘auto‐inhibition’ effect is observed for the polymers degraded at initially high pH of the aqueous subphase in the absence of buffers. Copyright © 2007 John Wiley & Sons, Ltd.     

2‐Nitroveratryl as a Photocleavable Thiol‐Protecting Group for Directed Disulfide Bond Formation in the Chemical Synthesis of Insulin

Chemical synthesis of peptides can allow the option of sequential formation of multiple cysteines through exploitation of judiciously chosen regioselective thiol‐protecting groups. We report the use of 2‐nitroveratryl (oNv) as a new orthogonal group that can be cleaved by photolysis under ambient conditions. In combination with complementary S‐pyridinesulfenyl activation, disulfide bonds are formed rapidly in situ. The preparation of Fmoc‐Cys(oNv)‐OH is described together with its use for the solid‐phase synthesis of complex cystine‐rich peptides, such as insulin.     

Multiscale approach to investigate the radiochemical degradation of epoxy resins under high‐energy electron‐beam irradiation

A multiscale investigation of the degradation mechanism of two epoxy systems exposed to electron‐beam irradiation under a helium atmosphere was carried out with a variety of analytical methods, including high‐resolution solution‐ and solid‐state NMR spectroscopy, NMR relaxometry, infrared spectroscopy, sterical exclusion chromatography, and differential scanning calorimetry. As a first step, we studied a linear phenoxy polymer, poly(2‐hydroxyether of bisphenol A), which provided a basis for the investigation of the degradation of a more complex, insoluble epoxy–amine network, diglycidyl ether of bisphenol A/triethylene tetramine. Among different structural modifications, the main degradation process was shown to produce in both cases a chain scission. For the phenoxy resin, the hydroxypropylidene moiety was identified as the fragile site leading to the formation of two phenolic chain ends and acetone and isopropyl alcohol as low‐molecular‐weight products. All methods, ranging from molecular to supramolecular scales, were shown to correlate both qualitatively and quantitatively. Experimental results obtained with diglycidyl ether of bisphenol A/triethylene tetramine evidenced a different degradation scheme occurring at the ethylene amine part and producing a dangling vinyl amine as the major degradation product. A selective increase in the molecular mobility at this site was confirmed by a two‐dimensional, local‐field wide‐line separation experiment. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 865–887, 2006     

Band gap tuning of narrow‐polydispersity two‐dimensional conductive polymers with electroactive side‐chains

A series of two‐dimensional donor–acceptor–donor (D1–A(D2)) type of conducting polymers (CPs) all with electroactive bulky side chain structure has been designed, synthesized, and investigated by introducing the donor–acceptor (D1–A) thiophene–quinoxaline moiety in the main chain alongside and additional donor and hole transporting units in the side chain. All the UV‐vis spectra of the 2D polymers, PTPQT, PFPQT, and PCPQT, each with triphenylamine, fluorene, and carbazole units as the D2 side chain, possess strong intramolecular charge transfer absorption, thus resulting in better light harvesting. Their optical and electronic properties were thoroughly explored experimentally and computationally. The effect of molecular weight of the narrow polydispersity polymers on their optoelectronic property was studied in detail. In summary, the 2‐D CPs show potential for use as an active material in optoelectronic devices. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 1217–1227     

Incorporation of ketone groups into poly(4‐hydroxystyrene)s main chain by radical copolymerization of 2,2‐diphenyl‐4‐methylene‐1,3‐dioxorane with O‐protected hydroxystyrenes and their photodegradable behavior

Here we report the incorporation of ketone groups into poly(4‐hydroxystyrene)s main chain by radical copolymerization of O‐protected hydroxystyrenes such as 4‐ethoxyethoxystyrene and 4‐acetoxystyrene with 2,2‐diphenyl‐4‐methylene‐1,3‐dioxorane (DPMD) followed by removal of the protective groups and the photodegradable behavior of obtained copolymers. The copolymerization of O‐protected hydroxystyrenes with DPMD gave the corresponding copolymers bearing DMPD‐derived ketone groups in the main chain, of which content could be controlled by changing monomer feed ratio. The ethoxyethyl and acetyl groups of the obtained copolymers were completely removed under acidic and basic conditions, respectively, to afford poly(4‐hydroxystyrene)s having ketone moieties in the main chain. The molecular weights of these copolymers decreased under photoirradiation due to the Norrish‐type reactions of the ketone groups distributed in the main chain. These results demonstrate that poly(4‐hydroxystyrene)s having ketone groups in the main chain possess good photo‐scissibility. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Identification and characterization of forced degradation products and stability‐indicating assay for notoginsenosidefc by using UHPLC‐Q‐TOF‐MS and UHPLC‐MS/MS: Insights into stability profile and degradation pathways

Notoginsenoside Fc, a protopanaxadiol‐type saponin, shows multi‐pharmacological activities. Chemical stability evaluation plays a crucial role in drug development. In this study, the forced degradation behavior of Notoginsenoside Fc was investigated under hydrolytic and oxidative conditions. A specific ultra high performance liquid chromatography with quadrupole time‐of‐flight mass spectrometry was developed for the separation, identification, and characterization of the degradation products of Notoginsenoside Fc. Fifty potential degradation products were formed via deglycosylation, dehydration, hydration, isomerization, side‐chain cleaving, oxidation, and superoxidation. Notoginsenoside Fc was subjected to different pH solutions, temperatures, and time periods to assess its stability. A sensitive ultra high performance liquid chromatography‐tandem mass spectrometry was developed for the quantification of Notoginsenoside Fc, notoginsenoside ST‐4, notoginsenoside Ft1, and relative quantification of notoginsenoside Ft2, 20(R)‐notoginsenoside Ft2, notoginsenoside SFt3, and notoginsenoside SFt4. The assay was linear over the concentration range (R² > 0.997) with the lowest limit of quantification of 0.02 μg/mL for Notoginsenoside Fc, Notoginsenoside ST‐4, and Notoginsenoside Ft1. The intra‐day precision, inter‐day precision, and accuracy of the three analytes were within accepted levels. The degradation kinetics of Notoginsenoside Fc in pH 1 and 3 solutions fits to first‐ and second‐order kinetics, respectively. The degradation of Notoginsenoside Fc is pH‐, temperature‐, and time‐dependent.     

GC separation of amino acid enantiomers via derivatization with heptafluorobutyl chloroformate and Chirasil‐L‐Val column

Heptafluorobutyl chloroformate (HFBCF), a recently introduced derivatization reagent, was examined in enantioseparation of amino acids (AAs) by GC. Twenty proteinogenic AAs, plus ornithine, cystine and 4‐fluorophenylalanine (internal standard) were treated with the reagent and separation properties of the derivatives were assessed on a Chirasil‐Val capillary column. Nineteen AA enantiomers were efficiently separated in 43 min except proline, arginine and cystine. The HFBCF derivatives of the studied DL ‐AAs show improved separation over other chloroformate‐based derivatives hitherto reported. A combination of the improved and faster separation with a simple derivatization protocol, involving an immediate one‐step reaction–extraction in two‐phase aqueous‐organic medium, and low elution temperatures extend application of HFBCF to chiral AA analysis.     

TOF‐SIMS study of cystine and cholesterol stones

Two different human stones, cystine and cholesterol from the kidney and gall bladder, were examined by time‐of‐flight secondary ion mass spectrometry using Ga⁺ primary ions as bombarding particles. The mass spectra of kidney stone were compared with those measured for the standard compounds, cystine and cysteine. Similar spectra were obtained for the stone and cystine. The most important identification was based on the existence of the protonated molecules [M + H]⁺ and deprotonated molecules [M‐H]^‐. The presence of cystine salt was also revealed in the stone through the sodiated cystine [M + Na]⁺ and the associated fragments, which might be due to the patient treatment history. In the gallstone, the deprotonated molecules [M‐H]⁺ of cholesterol along with relatively intense characteristic fragments [M‐OH]⁺ were detected. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and characterization of naphthyl‐substituted poly(p‐phenylenevinylene)s with few structural defects for polymer light‐emitting diodes

A series of naphthyl‐substituted poly(p‐phenylenevinylene)s (2N‐PPV, 4N‐PPV, and NAP‐PPV) has been synthesized and characterized by Fourier transform IR, ¹H NMR, and elemental analysis. The polymers possess excellent solubility, high molecular weights, good thermal stability, and high photoluminescence efficiencies. Thermogravimetric analysis reveals the onset of degradation to be 347, 301, and 306 °C for 2N‐PPV, 4N‐PPV, and NAP‐PPV, respectively. The differential scanning calorimetry investigation gives the respective glass‐transition temperature values of 118, 135, and 141 °C. The UV and photoluminescence spectra measurements reveal that the polymers exhibit similar optical properties, indicating that side‐chain substitution has little effect on the optical properties of this series of polymers. Proton NMR measurement of the signal due to tolane–bisbenzyl defects at around 2.7 ppm indicates that all the polymers have negligible amounts of tolane–bisbenzyl defects along the polymer main chain as a result of the steric bulk imposed by the naphthalene side chain. The highest occupied and lowest unoccupied molecular orbital energy levels of the polymers are investigated through cyclic voltammetry. Polymer light‐emitting diodes utilizing the polymers as the emissive layer with a configuration of indium tin oxide/poly(3,4‐ethylenedioxythiophene)/polymer/Ba/Al are fabricated and evaluated. The diodes emit blue‐green to yellow‐green light with maximum peaks at 518, 542, and 486 nm for 2N‐PPV, 4N‐PPV, and NAP‐PPV, respectively. The respective turn‐on electric fields of the diodes are 0.84, 0.69, and 0.83 MV/cm and the respective maximum external quantum efficiencies are 0.08, 0.54, and 0.02%. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 1647–1657, 2004     

Preparation of Selenoinsulin as a Long‐Lasting Insulin Analogue

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin‐degrading enzyme (IDE) without impairing the hormonal function. The [C7U^A,C7U^B] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A‐ and B‐chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se‐Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se‐Ins with IDE was significantly decelerated (τ _1/2≈8 h vs. ≈1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.     

Synthesis,characterization, and degradation of poly(anhydride‐co‐amide)s and their blends with polylactide

In attempt to improve the properties of polyanhydrides based on aliphatic anhydrides, we synthesized novel polyanhydrides containing amide groups in the main chains. In this work, N,N′‐bis(L ‐alanine)‐sebacoylamide (BSAM) was prepared from natural amino acid and sebacic acid (SA) and characterized by IR and ¹H NMR. In addition, polymers of PBSAM, P[1,6‐bis(P‐carboxyphenoxy) hexane (CPH)‐BSAM], and P(CPH‐SA), blends of P(CPH‐SA)/polylactide (PLA), P(CPH‐BSAM)/PLA were also prepared and characterized by IR, gel permeation chromatography, and differential scanning calorimetry. The hydrolytic degradation of polyanhydrides and their blends with PLA (number‐average molecular weight = 2.90 × 10⁵) was evaluated in 0.1 M phosphate buffer pH 7.4 at 37 °C. The results indicate that the existence of amide, aromatic, and ester bonds in the main chain of polymers slows down the degradation rate, and the tendency becomes clearer with the increasing amount of them, and the copolymers and their blends with PLA possess excellent physical and mechanical properties. These can make them more widely used in drug delivery and nerve regeneration. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4311–4317, 2004     

New 2‐methylisothiazolones

New N,N'‐bis(alkoxycarbonyl)‐L‐cystine bis(methylamides) 4a, 4b and N,N'‐bis(benzyloxycarbonyl)‐L‐cystine bis(methylamide) 4c have been synthesized by mixed anhydride method from the essential amino acid L‐cystine 1 in good yield. These cystine bis(methylamides) 4a,b,c have been cyclized with sulfuryl chloride. New 2‐methyl‐4‐amino‐3‐isothiazolone and 5‐chloro‐2‐methyl‐4‐amino‐3‐isothiazolone hydrobromide salts 7, 8 have been obtained by deacylation of 2‐methyl‐4‐(benzyloxycarbonyl)amino‐3‐isothiazolone 5c and 5‐chloro‐2‐methyl‐4‐(benzyloxycarbonyl)amino‐3‐isothiazolone 6c with hydrogen bromide in acetic acid. The microbicidal effect of the new 2‐methy]‐3‐isothiazolones 5a,b,c; 6a,b,c; 7 and 8 compounds obtained by the above method has been investigated.     

Acyl‐Chain Elongation Drives Ketosynthase Substrate Selectivity in trans‐Acyltransferase Polyketide Synthases

Type I modular polyketide synthases (PKSs), which are responsible for the biosynthesis of many biologically active agents, possess a ketosynthase (KS) domain within each module to catalyze chain elongation. Acylation of the KS active site Cys residue is followed by transfer to malonyl‐ACP to yield an extended β‐ketoacyl chain (ACP=acyl carrier protein). To date, the precise contribution of KS selectivity in controlling product fidelity has been unclear. Six KS domains from trans‐acyltransferase (trans‐AT) PKSs were subjected to a mass spectrometry based elongation assay, and higher substrate selectivity was identified for the elongating step than in preceding acylation. A close correspondence between the observed KS selectivity and that predicted by phylogenetic analysis was seen. These findings provide insights into the mechanism of KS selectivity in this important group of PKSs, can serve as guidance for engineering, and show that targeted mutagenesis can be used to expand the repertoire of acceptable substrates.     

Synthesis,characterization, and biodegradability of novel regular‐network polyester‐amines based on 1,1,1‐triethanolamine

Regular‐network polyester‐amines were prepared from 1,1,1‐triethanolamine (Y_N) and various dicarboxylic acids [HOOC? (CH₂)_n?2? COOH, n = 6–14]. A prepolymer prepared by melt polycondensation was cast from dimethylformamide solution and postpolymerized at 220 °C in a nitrogen flow for various periods of time to form a network. The resultant films were transparent, flexible, and insoluble in organic solvents. The network polyester‐amines obtained were characterized by infrared absorption spectra, wide‐angle X‐ray diffraction analysis, density, DSC, and thermomechanical analysis. The biodegradation experiments for the network polyester‐amine films were carried out in enzymatic solution with Rhizopus delemar or Pseudomonas cepacia lipase and in an activated sludge. The degree and rate of biodegradation were estimated by the weight loss of the films. After incubation in Rhizopus delemar lipase solution for 24 h, weight loss was hardly observed for Y_N6–7, whereas it increased greatly for Y_N8–13 (13–51 g/m²), and then it decreased rapidly for Y_N14. The methylene‐chain dependence of degradation was essentially the same as in the case of network polyesters from glycerol and various aliphatic dicarboxylic acids reported previously. Psedomonas cepacia lipases also degraded Y_Nn films, but the rate of degradation was much slower than Rhizopus delemar lipase. In the exposure to activated sludge for 30 days, the network polyester films with medium methylene‐chain lengths (Y_N7–11) showed the lager weight loss, as in the case of the enzymatic degradation, whereas the rate of biodegradation was much slower than that of the enzymatic degradation with Rhizopus delemar lipase. The effect of the protonation of the film with hydrochloric acid on the enzymatic degradation was also examined. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 2896–2903, 2001     

RAFT/MADIX copolymerization of vinyl acetate and 5,6‐benzo‐2‐methylene‐1,3‐dioxepane

The synthesis of well‐defined degradable poly(vinyl acetate) analogues is achieved by RAFT copolymerization of 5,6‐benzo‐2‐methylene‐1,3‐dioxepane (BMDO) and vinyl acetate (VAc) using methyl (ethoxycarbonothioyl)sulfanyl acetate (MEA) as controlling agent. Several monomer mixtures with low BMDO contents (<30 mol %) are employed to prepare different copolymers. In all the cases, the evolution of molar masses and the dispersity values (<1.26) confirm the controlled feature of the polymerization. The livingness of the obtained chains is demonstrated by successful chain extension experiments with VAc, although the presence of dead chains is also shown. The introduction of ester groups into the main chain of these P(VAc‐co‐BMDO) copolymers allows their degradation when treated with a mixture of KOH/MeOH in reflux during 2.5 h. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 104–111     

Enzyme Degradable Polymersomes from Chitosan‐g‐[poly‐l‐lysine‐block‐ε‐caprolactone] Copolymer

The scope of this study includes the synthesis of chitosan‐g‐[peptide‐poly‐ε‐caprolactone] and its self‐assembly into polymeric vesicles employing the solvent shift method. In this way, well‐defined core–shell structures suitable for encapsulation of drugs are generated. The hydrophobic polycaprolactone side‐chain and the hydrophilic chitosan backbone are linked via an enzyme‐cleavable peptide. The synthetic route involves the functionalization of chitosan with maleimide groups and the preparation of polycaprolactone with alkyne end‐groups. A peptide functionalized with a thiol group on one side and an azide group on the other side is prepared. Thiol‐ene click‐chemistry and azide–alkyne Huisgen cycloaddition are then used to link the chitosan and poly‐ε‐caprolactone chains, respectively, with this peptide. For a preliminary study, poly‐l ‐lysin is a readily available and cleavable peptide that is introduced to investigate the feasibility of the system. The size and shape of the polymersomes are studied by dynamic light scattering and cryo‐scanning electron microscopy. Furthermore, degradability is studied by incubating the polymersomes with two enzymes, trypsin and chitosanase. A dispersion of polymersomes is used to coat titanium plates and to further test the stability against enzymatic degradation.     

Loss of PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein

SDS‐PAGE represents a quick and simple method for qualitative and quantitative analysis of protein and protein‐containing conjugates, mostly pegylated proteins. PEG‐maleimide (MAL) is frequently used to site‐specifically pegylate therapeutic proteins via free cysteine residue by forming a thiosuccinimide structure for pursuing homogeneous products. The C–S linkage between protein and PEG‐MAL is generally thought to be relatively stable. However, loss of intact PEG chain in routine SDS‐PAGE analysis of PEG‐maleimide modified protein was observed. It is a thiol‐independent thioether cleavage and the shedding of PEG chain exclusively happens to PEG‐MAL modified conjugates although PEG‐vinylsulfone conjugates to thiol‐containing proteins also through a C–S linkage. Cleavage kinetics of PEG40k‐MAL modified ciliary neurotrophic factor showed this kind of degradation could immediately happen even in 1 min incubation at high temperature and could be detected at physiological temperature and pH, although the rate was relatively slow. This may provide another degradation route for maleimide‐thiol conjugate irrespective of reactive thiol, although the specific mechanism is still not very clear for us. It would also offer a basis for accurate characterization of PEG‐MAL modified protein/peptide by SDS‐PAGE analysis.