N-acetylhomocysteine Thiolactone Research Articles

Ring‐opening (co)polymerization of cardanol glycidyl ether

AbstractThe anionic ring‐opening copolymerization of commercially available bio‐based cardanol glycidyl ether (CGE) was investigated without any prior purification. As a first step, anionic‐ring‐opening homopolymerization was attempted through active chain‐end and monomer‐activated mechanisms. Both strategies were unsuccessful. Conversely, in a second step, the anionic alternating ring‐opening copolymerization (AAROP) of CGE with the renewable N‐acetyl homocysteine thiolactone (NHTL) was successfully carried out in the presence of a strong base. Anisole, a solvent classified as sustainable and rarely used in anionic ring‐opening polymerization, proved to be a suitable for the AAROP. This polymerization is an unusual example of synthesis of linear polyesters with cardanol‐based monomers. The copolymers were carefully characterized by 1H, 13C, COSY, HSQC, 1H‐15N NMR and MALDI‐ToF, demonstrating an alternating structure. Then, CGE was copolymerized with NHTL and another additional epoxide. The cardanol‐derived monomers enable the preparation of functionalizable poly(ester‐alt‐thioether) bearing multiple allyl and alkene groups. The AAROP method in anisole offers new opportunity for green anionic polymerization through the use of sustainable chemicals, witnessed by the valorization of cardanol‐derived compounds and expands the scope of synthesized renewable polyesters.

Thiolactomide: A New Homocysteine Thiolactone Derivative from Streptomyces sp. with Neuroprotective Activity.

A new homocysteine thiolactone derivative, thiolactomide (1), was isolated along with a known compound, N-acetyl homocysteine thiolactone (2), from a culture extract of soil-derived Streptomyces sp. RK88-1441. The structures of these compounds were elucidated by detailed NMR and MS spectroscopic analyses with literature study. In addition, biological evaluation studies revealed that compounds 1 and 2 both exert neuroprotective activity against 6-hydroxydopamine (6-OHDA)-mediated neurotoxicity by blocking the generation of hydrogen peroxide in neuroblastoma SH-SY5Y cells.

Headspace study of chiral interconversion of N-acetyl-homocysteine thiolactones

The rate constants for (L)-N-acetyl homocysteine thiolactone enantiomerization have been obtained from batch-wise studies and by dynamic gas chromatography of racemic mixtures. Results from the batch-wise experiments show that the kinetics of racemization at 150 °C is the same for vials made of glass, silanized glass or Teflon-coated glass so that the vial surface exhibited no effect on the kinetics of racemization. From the temperature dependence of the rate constants the preexponential factor, activation energy, the activation Gibbs energy and activation entropy have been obtained from transition state theory. The catalytic effect of G-DP, G-BP and B-DP GC chiral stationary phases on racemization has been observed and quantified by the values of rate constants; B-DP exhibited the greatest activity. The Eyring activation parameters obtained from batch-wise experiment were compared with theoretical values acquired from quantum chemical modelling. Agreement between the experimental and calculated values of activation Gibbs energy, activation enthalpy and activation entropy is good. The dynamic gas chromatography of racemic mixture on chiral B-DP, G-DP and G-BP capillary columns indicate that the rate constants of forward and reverse reactions are different in chiral environments. The greatest accelerating effect in the process of enantiomerization has been identified for G-BP both in the batch-wise experiments and by the dynamic gas chromatography.

Alternating copolymerization of bio-based N-acetylhomocysteine thiolactone and epoxides

The anionic ring-opening polymerizations (AROP) of bio-based N-acetyl homocysteine thiolactone (NHTL) and different epoxides were carried out using benzyl alcohol and 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (BEMP) as initiating system. This polymerization is a rare example of AROP in the presence of an acidic moiety (acetamido group). Well-defined alternating poly(ester-alt-sulfide)s are obtained with number-average molar masses Mn ranging from 1.7 to 13.0 kg mol−1 and dispersities as low as 1.14. The presence of one acetamido function in the lateral group on each repeating unit of the copolymers derived from NHTL results in very significant increases (up to 94 °C) of the glass transition temperature Tg compared to similar poly(ester-alt-sulfide) derived from petro-based γ-butyrothiolactone (BTL). These functional poly(NHTL-alt-epoxide)s are valuable structures with numerous potential applications due to the presence in each repeating unit of one cleavable ester group and one redox-sensitive thioether group.

Bio-based poly(ester-alt-thioether)s synthesized by organo-catalyzed ring-opening copolymerizations of eugenol-based epoxides and N-acetyl homocysteine thiolactone

A series of fully bio-based poly(ester-alt-thioether)s are efficiently synthesized by organo-catalyzed alternating ring-opening copolymerizations of eugenol-derived monomers, vanillin glycidyl ether and N-acetyl homocysteine thiolactone.

Oxyhalogen-Sulfur Chemistry: Kinetics and Mechanism of Oxidation of N-Acetyl Homocysteine Thiolactone by Acidified Bromate and Aqueous Bromine

N-acetyl homocysteine thiolactone (NAHT), medically known as citiolone, can be used as a mucolytic agent and for the treatment of certain hepatic disorders. We have studied the kinetics and mechanisms of its oxidation by acidic bromate and aqueous bromine. In acidic bromate conditions the reaction is characterized by a very short induction period followed by a sudden and rapid formation of bromine and N-acetyl homocysteine sulfonic acid. The stoichiometry of the bromate-NAHT reaction was deduced to be: BrO3(-) + H2O + CH3CONHCHCH2CH2SCO → CH3CONHCHCH2CH2(SO3H)COOH + Br(-) (S1) while in excess bromate it was deduced to be: 6BrO3(-) + 5CH3CONHCHCH2CH2SCO + 6H(+) → 3Br2 + 5CH3CONHCHCH2CH2(SO3H)COOH + 2H2O (S2). For the reaction of NAHT with bromine, a 3:1 stoichiometric ratio of bromine to NAHT was obtained: 3Br2 + CH3CONHCHCH2CH2SCO + 4H2O → 6Br(-) + CH3CONHCHCH2CH2(SO3H)COOH + 6H(+) (S3). Oxidation occurred only on the sulfur center where it was oxidized to the sulfonic acid. No sulfate formation was observed. The mechanism involved an initial oxidation to a relatively stable sulfoxide without ring-opening. Further oxidation of the sulfoxide involved two pathways: one which involved intermediate formation of an unstable sulfone and the other involves ring-opening coupled with oxidation through to the sulfonic acid. There was oligooscillatory production of aqueous bromine. Bromide produced in S1 reacts with excess bromate to produce aqueous bromine. The special stability associated with the sulfoxide allowed it to coexist with aqueous bromine since its further oxidation to the sulfone was not as facile. The direct reaction of aqueous bromine with NAHT was fast with an estimated lower limit bimolecular rate constant of 2.94 ± 0.03 × 10(2) M(-1) s(-1).

Kinetics and Mechanism of the Oxidation of N-Acetyl Homocysteine Thiolactone with Aqueous Iodine and Iodate

The kinetics of N-acetyl homocysteine thiolactone (NAHT) oxidation by aqueous iodine and iodate were studied by spectrophotometric techniques. The iodate-NAHT reaction is slow and results in the formation of N-acetyl homocysteine thiolactone sulfoxide as the sole product (NAHTSO). The stoichiometry of the reaction was deduced as: IO3(-) + 3NAHT → I(-) + 3NAHTSO (S1). In excess iodate conditions, the iodide produced in S1 is oxidized to give iodine: IO3(-) + 5I(-) + 6H(+) → 3I2 + 3H2O (S2). Thus in excess iodate conditions the overall stoichiometry of the reaction is a linear combination of S1 and S2 that eliminates iodide, 5S1 + S2: 2IO3(-)+ 5NAHT+ 2H(+) → I2 + 5NAHTSO + H2O. There was a 1:1 stoichiometry for the NAHT - I2 reaction: NAHT+ I2 + H2O → NAHTSO +2I(-) + 2H(+) (S3). All reactions, S1, S2 and S3 occur simultaneously and since they are all comparable in rate; complex dynamics were observed. Iodide catalyzes S1 and S2 but inhibits S3. Iodide is a product of both S1 and S3. It has the most profound effect on the overall global dynamics observed. The overall reaction scheme which involved S1, S2 and S3 was modeled by a simple 12-reaction mechanistic scheme which gave a very good fit to experimental data.

An Efficient Thiol‐Ene Chemistry for the Preparation of Amphiphilic PHA‐Based Graft Copolymers

We present a straightforward method to prepare amphiphilic graft copolymers consisting of hydrophobic poly(3-hydroxyalkanoates) (PHAs) backbone and hydrophilic α-amino-ω-methoxy poly(oxyethylene-co-oxypropylene) (Jeffamine®) units. Poly(3-hydroxyoctanoate)-co-(3-hydroxyundecenoate) (PHOU) was first methanolyzed to obtain the desired molar mass. The amino end groups of Jeffamine were converted into thiol by a reaction with N-acetylhomocysteine thiolactone and subsequently photografted. This "one-pot" functionalization prevents from arduous and time-consuming functionalization of the hydrophilic precursor or tedious modifications of PHAs, thus simplifying the process. The amphiphilic nature of modified PHAs leads to water-soluble copolymers exhibiting thermoresponsive behavior.

Disulphide cross-linking of thiolated alpha-chymotrypsin to form macromolecules.

Abstract Thiolation of α-chymotrypsin with N-acetyl homocysteine thiolactone (AHTL) occurs in the presence of Ag+ and imidazole with the introduction of 4–5·5 SH groups per mole. Oxidation of the thiolated enzyme with ferricyanide gave a macromolecule consisting of thiolated enzyme residues cross-linked through intermolecular disulphide bonds. The number of residues linked in this manner varied with each batch of thiolated enzyme but was within the range 16–44 residues. The light scattering data indicated that the macromolecule was rod-shaped.

Reversible gelatin-based hydrogels: Finetuning of material properties

The present work reports on the synthesis and evaluation of a crosslinkable thiolated gelatin derivative. The effect of varying two parameters including the pH of the reaction buffer and the thiolating agent applied (i.e. N-acetylhomocysteine thiolactone versus Traut’s reagent) on the obtained modification degree was studied in a first part. The gelatin derivatives synthesized starting from N-acetylhomocysteine thiolactone and Traut’s reagent were characterized in depth using size exclusion chromatography and UV–VIS spectrophotometry. In a subsequent part of the present work, hydrogel films were prepared starting from the thiolated gelatin derivative developed using N-acetylhomocysteine thiolactone. The contributions of both the chemical and the physical crosslinking of the hydrogels developed were studied in depth using rheology, swelling experiments and texturometry. The results indicate that the physical structuring, inherent to gelatin, contributes to a large extent to the mechanical properties. However, the chemical crosslinking mostly determines the final hydrogel properties and can be controlled to a large extent. The gelatin-based gels are flexible, strong and transparent. A major advantage of disulfide-crosslinked hydrogels is the fact that the crosslinking is reversible. The latter could be interesting in view of future applications as cell carriers for tissue engineering.

Peptomer aluminum oxide nanoparticle conjugates as systemic and mucosal vaccine candidates: synthesis and characterization of a conjugate derived from the C4 domain of HIV-1MN gp120.

Peptomers are polymers composed of peptides that are specifically cross-linked in a head-to-tail fashion. Recently, a peptomer composed of an amphipathic peptide from the C4 domain of HIV-1MN gp120 was shown to display a prominent alpha-helical conformation that, as an immunogen, elicited rabbit antibodies recognizing native and recombinant gp120 [Robey et al. (1995) J. Biol. Chem. 270, 23918-23921]. For the present study, we synthesized a conjugate composed of the C4 peptomer covalently linked to calcinated aluminum oxide nanoparticles. The nanoparticles were first reacted with (3-aminopropy])-triethoxysilane to provide an amine load of 15.9 mmol of R-NH2/g of solid. The amine-modified aluminum oxide nanoparticles then were reacted with N-acetylhomocysteine thiolactone at pH 10 to place a reactive thiol on the nanoparticles. A bromoacetylated C4 peptomer, modified at the epsilon-amines of lysine residues, then was reacted with the thiolated nanoparticles to give the peptomer covalently linked to aluminum oxide via a thioether bond. The peptomer load was determined to be 16 mg of peptomer/g of particles, a 55% theoretical yield. Particle shape and size of the peptomer-conjugated alumina were analyzed by electron microscopy and displayed a mean maximum diameter of 355 nm and a mean minimum diameter of 113 nm, well within the desired size range of 300 nm believed to be optimal for mucosal immunization purposes. Experimentally determined values of mean particle diameters, specific surface area, and specific peptomer load provided the information necessary to calculate the mean antigen load, which was determined to be 53000 +/- 42000 peptomer epitopes per particle. Peptomer-alumina conjugates, such as that described here, could form the basis of a new class of biomaterial that combines a chemically defined organic immunogen with a nontoxic chemically defined inorganic adjuvant.

Facile synthesis of fluorinated methionines

L-Di- and tri-fluoromethylhomocysteines (6) and (7) were efficiently prepared from N-acetylhomocysteine thiolactone (3) and the protected monofluoromethylhomocysteine (2) was synthesized from the protected methionine sulphoxide (1).

Polymeric thiols as enzyme activators of serum creatine phosphokinase.

Several hydrophilic polymeric thiols were prepared from amino-activated polymeric supports by reaction with N-acetylhomocystein ethiolactone. Supports include agaroses, cellulose, Glycophase controlled-pore glass, and Matrex acrylic beads. Thiol content in these polymers was 3-72 mumol SH/g dry polymer. Several were effective solid-phase activators of the sulfhydryl-dependent enzyme creatine phosphokinase at concentrations comparable to that of monomeric thiol required for enzyme activation. The kinetic activation curves for the polymeric and the monomeric (thioglucose) activators were similar, suggesting unhindered interaction of the enzyme with the polymeric activator.

High-performance liquid chromatographic determination of N-acetylcysteine in human serum following acetaminophen overdosage

An analytical method has been developed for the determination of N-acetylcysteine in human serum following acetaminophen overdosage in humans. Serum samples were treated with dithiothreitol and the protein-freed product was derivatized with 2,4-dinitrofluorobenzene. N-Acetylhomocysteine thiolactone was used as an internal standard. Following diethyl ether extraction, the components were separated on a reversed-phase column with retention times of 7.4 and 9.9 min for N-acetylcysteine and internal standard, respectively. Ultraviolet detection at 365 nm was employed and little interference was noted from other serum components. The method has been applied to quantitation of N-acetylcysteine given as treatment for acetaminophen intoxication.

Influence of N-acetylhomocysteine thiolactone on cultured retinal cells in canine neuronal lipofuscinosis. An ultrastructural study.

An SH- group donor drug, N-acetylhomocysteine thiolactone (0.2 mg/ml), was added to retinal cultures from an adult dog affected with ceroid lipofuscinosis (NCL). This substance was applied every 3rd day to the culture medium. After 1, 2 and 3 weeks in culture the cells were fixed for electron microscopy. Ultrastructurally the NCL-specific lipopigments revealed extensive fading out or vacuolation, revealing randomly arranged lamellar or 'fingerprint' membranes as well as structures resembling degenerated mitochondria. Additionally extensive fragmentation of the pigment bodies was present. The significance of these results are discussed.

Sulfhydrylcellulose: a new medium for chromatography of mercurated polynucleotides.

We have synthesized a new medium, sulfhydrylcellulose, for affinity chromatography of mercurated polynucleotides. It is the product of reaction between aminoethylcellulose and N-acetylhomocysteine thiolactone. Sulfhydrylcellulose carries up to 90 mumol of SH groups/g and is inexpensive, easy to prepare, and stable. Because it binds mercurated RNA specifically and reversibly and exhibits no size discrimination, sulfhydrylcellulose should have wide applications.

Immobilised lipoamide dehydrogenase. 3. Preparation and properties of an immobilised polythiolated enzyme.

1 The dimeric flavoprotein pig heart lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) has been polythiolated by reaction with N-acetylhomocysteine thiolactone at pH 10.5 for 24 h. Under these conditions 5–6 mol additional thiol groups are introduced per mol FAD. 2 The free polythiolated enzyme has a 50–60% lower specific activity for lipoamide, a reduced affinity for a specific anti-lipoamide dehydrogenase antibody but an unchanged apparent Km (lipoamide). The free polythiolated enzymes containing 6 mol–SH/mol FAD and 7 mol–SH/mol FAD were 270% and 640% respectively more stable to thermal inactivation at 90°C than the free native enzyme. 3 Immobilisation of the polythiolated enzyme to thiolated 6-aminohexyl-Sepharose reduced the specific activity with lipoamide to less than 10% of that of the free native enzyme, raised the apparent Km (lipoamide) to 8.3 mM and lowered its affinity for the specific antibody. The thermal stability at 90°C was enhanced by up to 25-fold. 4 Immobilisation of the polythiolated enzyme to a short spacer group, L-cysteinyl-Sepharose, reduced the specific activity with lipoamide but enhanced the stability at 90°C and in 70% (v/v) dioxane by 800% and 770% respectively relative to the free native enzyme. These data are discussed in terms of the effects of proximity to the matrix backbone. 5 The marked improvement in stability of the polythiolated enzyme was matched by lipoamide dehydrogenase immobilised directly to CNBr-activated Sepharose at pH 7.5 and pH 9.0. However, in this case the specific activity of the immobilised enzyme was 300–350% less than that of the polythiolated enzyme. These data are discussed in terms of multiple attachment of the enzyme to the matrix and the possibility of disulphide cross-links in the polythiolated enzyme.

Chemical modification of enzymes I. Two-step cross-linking of bovine pancreatic ribonuclease

A new procedure for the statistical cross-linking of enzymes is described. In the first step the enzyme polythiolated with N-acetylhomocysteine thiolactone, whilst in the second step the mercapto functions are intramolecularly cross-linked with different bifunctional reagents or by oxidation. With bovine pancreatic ribonuclease A, eight mercapto groups were introduced. After cross-linking with bis-iodoacetamides of different length, RNase with an optimum relative activity (c-CMP) of 144% was obtained. In addition, the pH optimum was shifted upwards by 0.8 pH units.

Mercaptodextran, a metal-chelating and disulphide-reducing polythiol of high molecular weight

A high molecular weight polythiol, mercaptodextran has been synthesized by thiolating various dextrans with N-acetyl homocysteine thiolactone. The thiol groups are unusually stable towards autoxidation. They are highly reactive and readily reduce disulphide bonds. Because of these properties mercaptodextran may be used to keep autoxidizable thiols in the reduced form. Mercaptodextran has no radioprotective effect, since it does not penetrate the cellular membrane. Mercaptodextran has a very high affinity for heavy metal ions. Competitive binding experiments show that mercapto-dextran has much higher affinity for silver, mercuric, cupric and auric ions than most other thiols (glutathione, penicillamine, N-acetyl-DL-penicillamine, cysteamine, mercapto-propionyl glycine, cysteine and diethyl dithiocarbamate) and other chelating agents (EDTA). Only 2,3-dimercaptopropanol (BAL) shows comparable binding properties. The stability constants for the Hg-BAL and Hg-mercaptodextran complexes are both about 10 20, and about 100-times higher than for the Hg-penicillamine complex. The high metal-binding ability combined with a low toxicity suggest a possible use of mercaptodextran in acute metal-intoxications.

Thiolation of proteins with homocysteine thiolactone: Preparation of immunoglobulin g heavily labelled with methylmercury

1. 1.|Homocysteine thiolactone (HTL) is proposed as a reagent for introducing thiol groups into proteins. HTL is analogous to N-acetylhomocysteine thiolactone (AHTL), except that the unblocked amino group permits a polymeric accumulation of homocysteinyl residues; hence several thiol groups may be introduced for each protein group attacked. 2. 2.|Relevant differences in properties between HTL and AHTL were established in the absence of protein. 3. 3.|Thiolation of immunoglobulin G (IgG) was best performed with imidazole catalysis at pH 9.6–10, in the presence of methylmercuric hydroxide to block and label the thiol groups. At a lower pH, mutual interaction of HTL molecules and precipitation of by-products promoted irreversible denaturation of the protein. A high concentration of all reactants and a large excess of HTL relative to protein favoured a high mercury content in the final product. 4. 4.|A single treatment of IgG with HTL and mercurial could effect binding of mercury equivalent to 11% of the weight of the protein, or more, but further labelling was possible. The methylmercury label was stable to gel filtration and to concentration of the protein by ultrafiltration, and there had been no degradation to inorganic mercury. 5. 5.|The nature of the labelled product is discussed, and parallels and distinctions are drawn between treatment of proteins with HTL and the introduction of polypeptidyl residues with N-carboxyamino acid anhydrides.