1,4-Reductive addition of glutathione to quinone epoxides. Mechanistic studies with h.p.l.c. with electrochemical detection under anaerobic and aerobic conditions. Evaluation of chemical reactivity in terms of autoxidation reactions

The nucleophilic addition of GSH to quinonoid compounds, characterized as a 1,4-reductive addition of the Michael type, was studied with p-benzoquinone- and 1,4-naphthoquinone epoxides with different degree of methyl substitution. Identification and evaluation of molecular products from the above reaction were assessed by h.p.l.c. with either reductive or oxidative electrochemical detection, based on the redox properties retained in the molecular products formed. It was found that the degree of methyl substitution of the quinone epoxide, from either the 1,4-naphthoquinone- or p-benzoquinone epoxide series, determined their rate of reaction with GSH. The reductive addition implied the rearrangement of the quinone structure with opening of the epoxide ring yielding as the primary product a hydroxy-glutathionyl substituted adduct of either p-benzohydroquinone or 1,4-naphthohydroquinone. The primary product undergoes elimination reactions and redox transitions which bring about a number of secondary molecular products. The distribution pattern of the latter depends on the degree of methyl substitution of the quinone epoxide studied and on the concentration of O2 in the solution. The occurrence of the hydroxy-substituent in position alpha, adjacent to the carbonyl group, enhances the autoxidation properties of the compound resulting in an augmented O2 consumption and H2O2 production. Therefore, it could be expected that the chemical reactivity of the products originating from the thiol-mediated nucleophilic addition to quinone epoxides would be of toxicological interest.     

The reactivity of affinity labels: A kinetic study of the reaction of alkyl halides with thiolate anions—a model reaction for protein alkylation

Factors have been investigated which govern the electrophilic reactivity of alkyl halides with thiolate anions in aqueous solution. In the series of alkyl halides studied, some are potential metal-directed affinity labels, while others are frequently used in protein modification. Previous data on the kinetics of this type of alkylation are compared with the present results. The influence of electronic, polar, and steric factors on alkyl halide reactivity is seen. The following order of reactivity for alkyl halides bearing different α substituents was observed: RCH₂CH(X)COOCH₃ > RCH₂CH(X)CONH₂ > RCH₂CH(X)COOH > RCH₂CH₂X > RCH₂CH(X)CH₂OH. The metal-directed affinity labels are imidazole derivatives, some of which have substituents in their imidazole ring. The effect of the imidazole ring and of ring substitution on reactivity is seen. The nucleophilic reactivity of thiols is highly pH dependent since the thiolate anion (RS^?) is the reactive species, but only minor differences emerged between different free thiolates.     

Emollient, humectant, and fluorescent alpha,beta-unsaturated thiol esters for long-acting skin applications

We describe compounds in which an emollient or a humectant bears an α,β-unsaturated thiol ester capable of reacting with nucleophilic amino acids in stratum corneum proteins. These compounds should serve as long-lasting moisturizers for skin. The emollient derivatized was octadecyl propanoate, and the humectant was poly(ethylene glycol). These hydrophobic and hydrophilic compounds, as well as a fluorescent, dansyl-containing thiol ester, were found to react within minutes with the thiol N-acetylcysteamine upon addition of a catalytic amount of an organic base in chloroform. The structures of the products resulting from conjugate addition to the unsaturated thiol esters were determined by NMR spectroscopy. In the case of the α,β,γ,δ-unsaturated (sorboyl) thiol ester, both the 1,4-addition product and the β,γ-unsaturated-1,6-addition product formed, followed by diadduct. An in vivo test of the fluorescent α,β-unsaturated thiol ester showed that this compound persisted on skin for 3 weeks vs. 6 days for the non-bonding control compound.     

Incorporation of UDP-[C]Glucose into Xyloglucan by Pea Membranes

The water-insoluble 1,4-β-linked products formed from UDP-[¹⁴C]glucose by pea membranes were dissolved in hot dimethyl-sulfoxide/paraformaldehyde and fractionated on columns of controlled pore glass beads calibrated with dextran standards. The products eluted with a peak size close to 70 kilodaltons in dextran equivalents. Similar elution profiles were obtained for products formed in brief or extended incubations and at high or low substrate concentrations. Methylation analysis indicated that only a few [¹⁴C]glucose units had been added to an endogenous acceptor to form this product. In the presence of UDP-xylose at concentrations equal to or less than UDP-[¹⁴C]glucose, incorporation from the latter was enhanced and the products elongated with time to a size range where the major components eluted between dextran 264 and 500 kilodaltons. Treatment with endo-1,4-β-glucanase resulted in a mixture of oligosaccharides, including the xyloglucan subunit Glc₄Xyl₃, which were hydrolyzed further by mixed glycosidases to labeled glucose and isoprimeverose (xylosyl-1,6-α-d-glucose). In pulse-chase experiments, the low molecular weight product formed from UDP-[¹⁴C]glucose alone was clearly a precursor for high molecular weight products formed subsequently in the presence of both UDP-glucose and UDP-xylose. It is concluded that the 1,4-β-transglucosylation activity detected in these tests was due to an enzyme that is required for biosynthesis of the backbone of xyloglucan.     

Transformations of Aromatic Compounds by Nitrosomonas europaea

Benzene and a variety of substituted benzenes inhibited ammonia oxidation by intact cells of Nitrosomonas europaea. In most cases, the inhibition was accompanied by transformation of the aromatic compound to a more oxidized product or products. All products detected were aromatic, and substituents were often oxidized but were not separated from the benzene ring. Most transformations were enhanced by (NH₄)₂SO₄ (12.5 mM) and were prevented by C₂H₂, a mechanism-based inactivator of ammonia monooxygenase (AMO). AMO catalyzed alkyl substituent hydroxylations, styrene epoxidation, ethylbenzene desaturation to styrene, and aniline oxidation to nitrobenzene (and unidentified products). Alkyl substituents were preferred oxidation sites, but the ring was also oxidized to produce phenolic compounds from benzene, ethylbenzene, halobenzenes, phenol, and nitrobenzene. No carboxylic acids were identified. Ethylbenzene was oxidized via styrene to two products common also to oxidation of styrene; production of styrene is suggestive of an electron transfer mechanism for AMO. Iodobenzene and 1,2-dichlorobenzene were oxidized slowly to halophenols; 1,4-dichlorobenzene was not transformed. No 2-halophenols were detected as products. Several hydroxymethyl (-CH₂OH)-substituted aromatics and p-cresol were oxidized by C₂H₂-treated cells to the corresponding aldehydes, benzaldehyde was reduced to benzyl alcohol, and o-cresol and 2,5-dimethylphenol were not depleted.     

Preparations and properties of tripodal and linear tetradentate N,S-Donor ligands and their complexes containing the MoO22+core

New linear and tripodal tetradentate ligands, LH₂, are reported and their syntheses are described. The new linear ligands L = HSCH₂CH₂SCH₂CH₂NRCH₂CR₂SH, R = H, CH₃) and the new tripodal ligands N(CH₂CH₂SH)₂CH₂Z, Z = CH₂NH₂, CH₂N(CH₃)₂, CH₂N(C₂H₅)₂, CH₂SCH₃ and CO₂^- were synthesized. The known linear ligands HSCH₂CH₂NCH₃(CH₂)_nNCH₃CH₂CH₂SH (n = 2, 3) and HSCR₂CH₂NHCH₂CH₂NHCH₂CR₂SH (R = H, CH₃) were also utilized. These ligands react with MoO₂(acac)₂ in CH₃OH to yield MoO₂L complexes in high yield. Infra-red and ¹H nmr spectra provide evidence to supplement X-ray crystallographic results reported elsewhere for selected numbers of the series. Octahedral structures with cis MoO₂²⁺ groupings are assigned. Solution ¹H nmr studies are consistent with a trans placement of the two thiolate donors in agreement with the X-ray studies.     

Identical Ring Cleavage Products during Anaerobic Degradation of Naphthalene, 2-Methylnaphthalene, and Tetralin Indicate a New Metabolic Pathway

Anaerobic degradation of naphthalene, 2-methylnaphthalene, and tetralin (1,2,3,4-tetrahydronaphthalene) was investigated with a sulfate-reducing enrichment culture obtained from a contaminated aquifer. Degradation studies with tetralin revealed 5,6,7,8-tetrahydro-2-naphthoic acid as a major metabolite indicating activation by addition of a C₁ unit to tetralin, comparable to the formation of 2-naphthoic acid in anaerobic naphthalene degradation. The activation reaction was specific for the aromatic ring of tetralin; 1,2,3,4-tetrahydro-2-naphthoic acid was not detected. The reduced 2-naphthoic acid derivatives tetrahydro-, octahydro-, and decahydro-2-naphthoic acid were identified consistently in supernatants of cultures grown with either naphthalene, 2-methylnaphthalene, or tetralin. In addition, two common ring cleavage products were identified. Gas chromatography-mass spectrometry (GC-MS) and high-resolution GC-MS analyses revealed a compound with a cyclohexane ring and two carboxylic acid side chains as one of the first ring cleavage products. The elemental composition was C₁₁H₁₆O₄ (C₁₁H₁₆O₄-diacid), indicating that all carbon atoms of the precursor 2-naphthoic acid structure were preserved in this ring cleavage product. According to the mass spectrum, the side chains could be either an acetic acid and a propenic acid, or a carboxy group and a butenic acid side chain. A further ring cleavage product was identified as 2-carboxycyclohexylacetic acid and was assumed to be formed by β-oxidation of one of the side chains of the C₁₁H₁₆O₄-diacid. Stable isotope-labeling growth experiments with either ¹³C-labeled naphthalene, per-deuterated naphthalene-d₈, or a ¹³C-bicarbonate-buffered medium showed that the ring cleavage products derived from the introduced carbon source naphthalene. The series of identified metabolites suggests that anaerobic degradation of naphthalenes proceeds via reduction of the aromatic ring system of 2-naphthoic acid to initiate ring cleavage in analogy to the benzoyl-coenzyme A pathway for monoaromatic hydrocarbons. Our findings provide strong indications that further degradation goes through saturated compounds with a cyclohexane ring structure and not through monoaromatic compounds. A metabolic pathway for anaerobic degradation of bicyclic aromatic hydrocarbons with 2-naphthoic acid as the central intermediate is proposed.     

Organic sulfur compounds resulting from the interaction of iron sulfide,hydrogen sulfide and carbon dioxide in an anaerobic aqueous environment

The reaction of iron sulfide (FeS) with H₂S in water, in presence of CO₂ under anaerobic conditions was found to yield H₂ and a variety of organic sulfur compounds, mainly thiols and small amounts of CS₂ and dimethyldisulfide. The same compounds were produced when H₂S was replaced by HCl, in the H₂S-generating system FeS/HCl/CO₂. The identification of the products was confirmed by GC-MS analyses and the incorporation of H₂ in the organic sulfur compounds was demonstrated by experiments in which all hydrogen compounds were replaced by deuterium compounds. Generation of H₂ and the synthesis of thiols were both dependent upon the relative abundance of FeS and HCl or H₂S, i.e. the FeS/HCl- or FeS/H₂S-proportions. Whether thiols or CS₂ were formed as the main products depended also on the FeS/HCl-ratio: All conditions which create a H₂ deficiency were found to initiate a proportional increase in the amount of CS₂. The quantities of H₂ and thiols generated depended on temperature: the production of H₂ was significantly accelerated from 50°C onward and thiol synthesis above 75°C. The yield of thiols increased with the amount of FeS and HCl (H₂S), given a certain FeS/HCl-ratio and a surplus of CO₂. A deficiency of CO₂ results in lower thiol systhesis. The end product, pyrite (FeS₂), was found to appear as a silvery granular layer floating on the aqueous surface. The identity of the thiols was confirmed by mass spectrometry, and the reduction of CO₂ demonstrated by the determination of deuterium incorporation with DCl and D₂O. The described reactions can principally proceed under the conditions comparable to those obtaining around submarine hydrothermal vents, or the global situation about 4 billion years ago, before the dawn of life, and could replace the need for a reducing atmosphere on the primitive earth.     

Unraveling Curcumin Degradation: AUTOXIDATION PROCEEDS THROUGH SPIROEPOXIDE AND VINYLETHER INTERMEDIATES EN ROUTE TO THE MAIN BICYCLOPENTADIONE*

Curcumin is a dietary anti-inflammatory and chemopreventive agent consisting of two methoxyphenol rings connected by a conjugated heptadienedione chain. Curcumin is unstable at physiological pH and rapidly degrades in an autoxidation reaction to a major bicyclopentadione product in which the 7-carbon chain has undergone oxygenation and double cyclization. Early degradation products (but not the final bicyclopentadione) mediate topoisomerase poisoning and possibly many other activities of curcumin, but it is not known how many and what autoxidation products are formed, nor their mechanism of formation. Here, using [¹⁴C₂]curcumin as a tracer, seven novel autoxidation products, including two reaction intermediates, were isolated and identified using one- and two-dimensional NMR and mass spectrometry. The unusual spiroepoxide and vinylether reaction intermediates are precursors to the final bicyclopentadione product. A mechanism for the autoxidation of curcumin is proposed that accounts for the addition and exchange of oxygen that have been determined using ¹⁸O₂ and H₂¹⁸O. Several of the by-products are formed from an endoperoxide intermediate via reactions that are well precedented in lipid peroxidation. The electrophilic spiroepoxide intermediate formed a stable adduct with N-acetylcysteine, suggesting that oxidative transformation is required for biological effects mediated by covalent adduction to protein thiols. The spontaneous autoxidation distinguishes curcumin among natural polyphenolic compounds of therapeutic interest; the formation of chemically diverse reactive and electrophilic products provides a novel paradigm for understanding the polypharmacological effects of curcumin.     

Formation of dimethylsulfide and methanethiol from methoxylated aromatic compounds and inorganic sulfide by newly isolated anaerobic bacteria

Formation of gas and of methylated sulfur compounds was observed in anaerobic enrichment cultures with methoxylated aromatic compounds as substrates. Via direct dilution of mud samples in defined reduced media supplemented with trimethoxybenzoate or syringate two new strains of anaerobic homoacetogenic bacteria (strain TMBS4 and strain SA2) were obtained in pure culture. Both strains produced dimethylsulfide and methanethiol during growth on methoxylated aromatic compounds. Growth tests and determination of stoichiometries demonstrated that the volatile sulfur compounds were formed from the methyl group at the aromatic ring and the sulfide added as reducing agent to the medium (R = aromatic residue): 2 R - O - CH₃ + H₂ S 2 R - OH + (CH₃)₂SDimethylsulfide was the major organic sulfur compound formed, whereas methanethiol appeared only as intermediate in small quantities. The isolates grew also with trihydroxybenzenes such as gallate, phloroglucinol, or pyrogallol without formation of methylated sulfur compounds. The aromatic compounds were degraded to acetate. The freshwater strain TMBS4 also fermented pyruvate. Other aliphatic or aromatic compounds were not utilized. External electron acceptors (sulfate, nitrate, fumarate) were not reduced. Both strains were mesophilic and formed rod-shaped, non-motile, Gram-negative cells. Spore formation was not observed. Tentatively, both isolates can be affiliated to the genus Pelobacter.Abbreviations TMB 3,4,5-trimethoxybenzoate - MT methanethiol - DMS dimethylsulfide     

Enzymic degradation of allantoate in developing soybeans

A Mn²⁺-dependent enzymic breakdown of allantoate has been detected in crude and partially purified extracts of developing soybeans. The products detected were CO₂, NH₃, glyoxylate, labile glyoxylate derivatives, and low levels of urea. Urea is initially produced at less than 10% the rate of urease-independent CO₂ release indicating that the activity is not allantoate amidinohydrolase (i.e. urea is not directly cleaved off allantoate). The urease-independent CO₂ releasing activity has an apparent K_m of 1.0 millimolar for allantoate. Ethylenediaminetetraacetate, borate, and acetohydroxamate (all at 10 millimolar) inhibit the enzymic production of NH₃, CO₂, and labile glyoxylate derivatives from allantoate. However, the potent urease inhibitor, phenyl phosphordiamidate does not inhibit CO₂ and NH₃ release indicating that the action of acetohydroxamate is not due to its inhibition of urease. That the allantoatedegrading activity was more than 5-fold greater in seed coats than in embryos is consistent with the data of Rainbird et al. (Plant Physiol 1984 74: 329-334) which indicate that available ureides are metabolized before reaching the embryo. 2-Ethanolthio, 2′ureido, acetic acid (NH₂COHNCHCO₂HSCH₂CH₂OH), the first allantoate-derived product detected by HPLC analysis, is an addition produced of mercaptoethanol with an unidentified enzymically produced ureido intermediate that is not derived from ureidoglycolate or oxalurate.     

Synthesis of hydrocarbons under presumed prebiotic conditions using high-frequency discharge

Summary Various hydrocarbons were synthesized by high-frequency discharge in a primordial terrestrial model atmosphere. The products were extracted by benzene or methanol and analyzed by GC-MS. The mean carbon chain length of the hydrocarbons formed by the discharge through pure CH₄ gas was less than 6. Benzene was also obtained. Some isomers were obtained for each of the hydrocarbons containing a given number of carbons. When a small amount of C₂H₂ was added to the CH₄, longer chain compounds were formed, as compared with discharge in CH₄ only. However, when the amount of C₂H₂ was increased, unextractable high molecular weight compounds were produced. The amounts of products decreased as the mixing ratio of CO₂ to CH₄ increased. No hydrocarbons were detected when the ratio of CO₂/CH₄ exceeded 1.     

Characterization of Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) with Municipal Anaerobic Sludge

The biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in liquid cultures with municipal anaerobic sludge showed that at least two degradation routes were involved in the disappearance of the cyclic nitramine. In one route, RDX was reduced to give the familiar nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX). In the second route, two novel metabolites, methylenedinitramine [(O₂NNH)₂CH₂] and bis(hydroxymethyl)nitramine [(HOCH₂)₂NNO₂], formed and were presumed to be ring cleavage products produced by enzymatic hydrolysis of the inner C—N bonds of RDX. None of the above metabolites accumulated in the system, and they disappeared to produce nitrous oxide (N₂O) as a nitrogen-containing end product and formaldehyde (HCHO), methanol (MeOH), and formic acid (HCOOH) that in turn disappeared to produce CH₄ and CO₂ as carbon-containing end products.     

Involvement of Hydroxyl Radical Formation and Dna Strand Breakage in the Cytotoxicity of Anthraquinone Antitumour Agents

Four 9,10-anthraquinones (AQ) mono- or bis-substituted with the -NH(CH₂)₂ NH(CH₂)₂OH group were studied. 1-AQ, 1,5-AQ and 1,8-AQ but not 1,4-AQ (100°M) generated pBR322 plasmid DNA single strand breaks in the presence of purified NADPH dependent cytochrome P450 reductase. 1-AQ, 1,5-AQ and 1,8-AQ (at 100 °M) stimulated hydroxyl radical formation in MCF-7 S9 cell fraction (as measured by dimethyl pyrolline N-oxide spin trapping) and MCF-7 DNA strand breaks as measured by alkaline filter elution. In contrast 1,4-AQ did not stimulate hydroxyl radical formation and produced considerably less strand breaks in MCF-7 cells compared to the other AQ's. It would appear that the position of the -NH(CH₂)₂ NH(CH₂)₂OH groups on the chromophore is an important determinant in the metabolic activation of cytotoxic anthraquinones. This may contribute to the cytotoxicity (ID₅₀ values) of 1-AQ (0.06 °M), 1-8-AQ (0.5 °M) and 1,5-AQ (12.3 °M) but not the 1,4-AQ (1.2 °M).     

The reaction of carbonyl cyanide phenylhydrazones with thiols

Carbonyl cyanide phenylhydrazone and its ring-substituted analogs react with thiols (thioglycolic acid, 2-mercaptoethanol, dithiothreitol) and amino-thiols (cysteine, glutathione) to give corresponding N-(substituted phynyl)-N′-(alkythiodicyano)-methylhydrazine derivatives. These addition products decompose to the original components in alkaline solution. On the other hand, in the presence of an excess of thiols in aqueous buffered systems the addition reactions are practically quantitative with respect to phenylhydrazones, follow pseudo-first-order kinetics and can be investigated spectrophotometrically. These reactions are of the bimolecular Ad_N type where the non-dissociated form of carbonyl cyanide phenylhydrazones function as an electrophilic component, while the RS^? ion plays the role of nucleophilic component in the case of thiols (the attack of the azomethine group).The reactivity of carbonyl cyanide phenylhydrazones with respect to thiols increases in the order carbonyl cyanide phenylhydrazone < carbonyl cyanide m-chlorophenylhydrazone < carbonyl cyanide p-trifluoromethoxyphenylhydrazone which corresponds to the order of decreasing values of the pK_a constants. On the other hand, the reactivity of thiols increases with their basicity.The reactivity of carbonyl cyanide phynylhydrazone with thiols is comparable with the reactivity of phynyl isothiocyanate and N-ethylmaleimide. It was demostrated that carbonyl cyanide phenylhydrazone is an efficient inhibitor of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12).The results obtained are discussed in relation to the biological activity of carbonyl cyanide phenylhydrazones.     

Deodorizing Mechanism of (–)-Epigallocatechin Gallate against Methyl Mercaptan

The deodorizing mechanism of (-)-epigallocatechin gallate (EGCg), the main constituent of a green tea extract, against methyl mercaptan (CH₃SH) was investigated. EGCg showed deodorizing activity against CH₃SH by a chemical reaction between EGCg and CH₃SH. The non-volatile reaction products were identified to be compounds introducing a methylthio and/or a methylsulfinyl group into the B ring of EGCg, and gaseous oxygen was necessary for deodorizing activity. From these results, it was assumed that the deodorizing mechanism of EGCg was due to the addition of a methylthio group to the ortho-quinone generated by atmospheric oxygen. It was also found that secondary compounds produced by the reaction between EGCg and CH₃SH had a stronger deodorizing activity than that of EGCg itself.     

Reactions of methylcobalamin with tin and lead compounds

Reactions of CH₃[Co] with (CH₃)_nM^(4?n)+ (n = 2, 3; M = Sn, Pb) at concentrations high enough to detect (CH₃)₄M in the head space (yields 7.08×10^?5?2.06×10^?5%), indicate that dismutation is the major route of production. Similarly, kinetic reactions at lower concentrations show that no demethylation of CH₃[Co] by (CH₃)₃M⁺ (M = Sn, Pb) occurs after 60 days. From the methylation of SnCl₂ by CH₃[Co] at pD 1.0 and under aerobic conditions, the following hydrolysis species were observed in the 400 MHz ¹H NMR spectrum: CH₃- Sn(OH)Cl₂·2H₂O (63.6%), [CH₃Sn(OH)(H₂O)₄]²⁺ (17.6%) and CH₃Sn(OH)₂Cl·nH₂O (18.8%). No methylation products were observed from similar reactions with Pb(II) salts.     

Theoretical investigation of the ring opening process of verdoheme to biliverdin in the presence of dioxygen

The conversion of ferrous verdoheme to ferric biliverdin in the presence of O₂ was investigated using the B3LYP method. Both 6-31G and 6-31G (d) basis sets were employed for geometry optimization calculation as well as energy stabilization estimation. Three possible pathways for the conversion of iron verdoheme to iron biliverdin were considered. In the first route oxygen and reducing electron were employed. In this path formation of ferrous verdoheme-O₂ complex was followed by the addition of one electron to the ferrous-oxycomplex to produce ferric peroxide intermediate. The ferric peroxide intermediate experienced an intramolecular nucleophilic attack to the most positive position at 5-oxo carbons on the ring to form a closed ring biliverdin. Subsequently the ring opening process took place and the iron (III) biliverdin complex was formed. Closed ring iron biliverdin intermediate and open ring iron biliverdin formed as a product of verdoheme cleavage were respectively 13.20 and 32.70 kcal mol⁻¹ more stable than ferric peroxide intermediate. Barrier energy for conversion of ferric peroxide to closed ring Fe (III) biliverdin and from the latter to Fe (III) biliverdin were respectively 8.67 and 3.35 kcal mol⁻¹. In this path spin ground states are doublet except for iron (III) biliverdin in which spin state is quartet. In the second path a ferrous-O₂ complex was formed and, without going to a one electron reduction process, nucleophilic attack of iron superoxide complex took place followed by the formation of iron (III) biliverdin. This path is thermodynamically and kinetically less favorable than the first one. In addition, iron hydro peroxy complex or direct attack of O₂ to macrocycle to form an isoporphyrin type intermediate have shown energy surfaces less favorable than aforementioned routes.     

Use of Self-Assembled Monolayers of Different Wettabilities To Study Surface Selection and Primary Adhesion Processes of Green Algal (Enteromorpha) Zoospores

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH₃) or hydroxyl (OH) groups or mixtures of CH₃- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and “searching” above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to “sense” that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.     

3-18F-fluoropropane-1-thiol and 18F-PEG4-1-thiol: Versatile prosthetic groups for radiolabeling maleimide functionalized peptides

The efficient radiosynthesis of biomolecules utilizing minute quantities of maleimide substrate is important for availability of novel peptide molecular imaging agents. We evaluated both 3-¹⁸F-fluoropropane-1-thiol and 2-(2-(2-(2-¹⁸F-fluoroethoxy)ethoxy)ethoxy)ethane-1-thiol (¹⁸F-fluoro-PEG₄ thiol) as prosthetic groups for radiolabeling under physiological conditions. The precursor employed a benzoate for protection of the thiol and an arylsulfonate leaving group. The radiofluorination was fully automated on an Eckert & Ziegler synthesis system using standard Kryptofix₂₂₂/K₂CO₃ conditions. In order to minimize the amount of biological molecule required for subsequent conjugation, the intermediates, S-(3-¹⁸F-fluoropropyl) benzothioate and ¹⁸F-fluoro-PEG₄ benzothioate, were purified by HPLC. The intermediates were isolated from the HPLC in yields of 37–47% and 28–35%, respectively, and retrieved from eluate using solid phase extraction. Treatment of the benzothioates with sodium methoxide followed by acetic acid provided the free thiols. The desired maleimide substrate in acetonitrile or phosphate buffer was then added and incubated at room temperature for 15 min. The final radiolabeled bioconjugate was purified on a separate HPLC or NAP-5 column. Maleimides utilized for the coupling reaction included phenyl maleimide, an Evans Blue maleimide derivative, a dimeric RGDfK maleimide (E[c(RGDfK)]₂), two aptamer maleimides, and PSMA maleimide derivative. Isolated radiochemical yields (non-decay corrected) of maleimide addition products based on starting ¹⁸F-fluoride ranged from 6 to 22% in a synthesis time of about 90 min.¹⁸F-thiol prosthetic groups were further tested in vivo by conjugation to E[c(RGDfK)]₂ maleimide in a U87MG xenograft model. PET studies demonstrated similar tumor accumulation of both prosthetic groups. ¹⁸F-fluoro-PEG₄-S-E[c(RGDfK)]₂ displayed a somewhat favorable pharmacokinetics compared to ¹⁸F-fluoropropyl-S-E[c(RGDfK)]₂. Bone uptake was low for both indicating in vivo stability.