Isothiocyanate Group Research Articles

Cyanine dye labeling reagents containing isothiocyanate groups.

New isothiocyanate derivatives of cyanine dyes were synthesized as fluorescent covalent labeling reagents for proteins and other biomolecules. These dyes have maximum absorbance in the red and near infrared regions of the spectrum, have high extinction coefficients and have adequate quantum yields. Incorporating two alkyl sulfonate groups in the dye structures increases their water solubility, which is beneficial for labeling biological molecules in aqueous solution. Reactivities of proteins with these new cyanines are similar to their reactivities with fluorescein isothiocyanate. These new labeling reagents are complementary to the fluorescein and rhodamine reagents, expanding the possibilities of multicolor analyses. Sheep anti-mouse-IgG antibody was labeled with a pentamethine cyanine dye (CY5.8-ITC) and used with a fluoresceinated antibody as a second reagent for detecting human T-cell subsets by flow cytometry.

A quantitative analysis of the retrograde axonal transport of 4 different fluorescent dyes in peripheral sensory and motor neurons and lack of anterograde transport in the corticospinal system

Many fluorescent dye compounds are transported by axons in retrograde and anterograde directions. In the present study the uptake and retrograde axonal transport of 4 chemically related fluorescent dyes was evaluated in the peripheral nervous system of adult mice. Anterograde transport was studied in the corticospinal tract of adult rats. In addition to confirming the previously reported intra-axonal transport of Rhodamine-B-isothiocyanate, we report the transport of Rhodamine-X-isothiocyanate, Sulforhodamine-101-acid chloride and Lissamine rhodamine-B-sulfonyl chloride. By using the fluorescence intensity of the labeled motor and sensory neurons as well as cell counts of fluorescently labeled motor neurons and percent of labeled dorsal root ganglia (DRG) cells, we were able to quantitate the amount of retrograde transport of a given fluorescent compound. The two dyes with isothiocyanate groups available for conjugation were transported in higher amounts compared to the dyes containing sulfonyl chloride groups. No anterograde transport in the corticospinal system was observed. We conclude that the 4 dyes described are useful for retrograde neuroanatomical tracing experiments. We describe methods for quantifying the amount of retrograde transport by peripheral motor and sensory neurons.

Streptomyces albus G produces an antibiotic complex identical to paulomycins A and B.

An antibiotic complex active against multiply resistant strains of staphylococci and other Gram-positive bacteria was isolated from cultures of Streptomyces albus G. Silica gel and Sephadex LH-20 column chromatography gave two congeners with Mr values of 786 and 772, which differed by one -CH2-group. The two homologues contained an isothiocyanate group, and proved to be identical with paulomycins A and B produced by Streptomyces paulus; the FAB mass spectra, in addition, proved the same two congeners to be present in proceomycin obtained from Streptomyces alboniger.

Preparation of Three-Armed Star Polystyrene by Reaction of Prepolymer Having a Terminal Functional Group with Triisocyanate

Polystyrene prepolymers having hydroxyl or amino groups at their ends were prepared by anionic polymerization techniques. The linking reactions of these prepolymers with triisocyanate derived from lysine were carried out under various conditions and their efficiencies of conversion to three-armed star polymers were determined by GPC measurements. The reaction of the prepolymer having an –OH group with triisocyanate under catalysis of tri-n-butyltin oxide as well as the direct reaction of the intermediate polystyryl anion with triisocyanate was found to be most effective for preparation of star polystyrene having three arms. The star polymer thus prepared was successfully isolated by fractionation procedures after treating the reaction mixture with reactive polystyrene gels containing sulfonyl chloride or isothiocyanate groups. The number of arms of the isolated star polymer was confirmed to be virtually three by the ratio of the number-average molecular weight of the star polymer to that of the corresponding prepolymer.

ChemInform Abstract: Monosaccharide Isothiocyanates and Thiocyanates: Synthesis, Chemistry, and Preparative Applications

Publisher Summary This chapter presents the synthesis, chemistry, and preparative applications of monosaccharide isothiocyanates and thiocyanates. The ability of the isothiocyanate group to participate in nucleophilic reaction provides an attractive approach to nucleoside analogs and other nitrogen and sulfur heterocycles. Sugar thiocyanates are generally good precursors for the synthesis of thio and deoxy sugars and they are readily synthesized by a variety of methods from the corresponding halides. The thermodynamically more-stable isothiocyanate may also be formed by a secondary, isomerization reaction. It is found that that the treatment of semicarbazide with a nitroso acid at 0° affords the 1,2,3,4-thiatriazole derivative by addition and cyclization. The same 1,2,3,4-thiatriazole derivative 39 was obtained by the treatment of isothiocyanate 2 with hydrazoic acid. It is observed that isothiocyanate reacts with glycine, as well as with β-alanine, in tetrahydrofuran (THF) solution with the formation of corresponding N -(carboxyalky1)- d -gluconamides by nucleophilic attack of the amino group on the hard site, with simultaneous elimination of a molecule of thiocyanogen. The ability of aryl and alkyl isothiocyanates to react with reducing agents such as triethyl phosphite, triethylphosphine, or triphenyltin hydride to form the corresponding isocyanide is also elaborated in the chapter.

Monosaccharide Isothiocyanates and Thiocyanates: Synthesis, Chemistry, and Preparative Applications

This chapter presents the synthesis, chemistry, and preparative applications of monosaccharide isothiocyanates and thiocyanates. The ability of the isothiocyanate group to participate in nucleophilic reaction provides an attractive approach to nucleoside analogs and other nitrogen and sulfur heterocycles. Sugar thiocyanates are generally good precursors for the synthesis of thio and deoxy sugars and they are readily synthesized by a variety of methods from the corresponding halides. The thermodynamically more-stable isothiocyanate may also be formed by a secondary, isomerization reaction. It is found that that the treatment of semicarbazide with a nitroso acid at 0° affords the 1,2,3,4-thiatriazole derivative by addition and cyclization. The same 1,2,3,4-thiatriazole derivative 39 was obtained by the treatment of isothiocyanate 2 with hydrazoic acid. It is observed that isothiocyanate reacts with glycine, as well as with β-alanine, in tetrahydrofuran (THF) solution with the formation of corresponding N-(carboxyalky1)-d-gluconamides by nucleophilic attack of the amino group on the hard site, with simultaneous elimination of a molecule of thiocyanogen. The ability of aryl and alkyl isothiocyanates to react with reducing agents such as triethyl phosphite, triethylphosphine, or triphenyltin hydride to form the corresponding isocyanide is also elaborated in the chapter.

Kinetic analysis of competitive reactions of isothiocyanates with hydroxyl and alkoxide ions

The reaction of isothiocyanates with hydroxyl and alkoxide ions has been followed spectrophotometrically in alkaline aqueous-alcoholic solutions. Both the nucleophiles are added to the carbon atom of isothiocyanate group in a competition reaction. The effects of concentration of the nucleophiles, temperature, and acid-base properties of the alcoholic group have been followed on the reaction course. The possibility of analogous reactions in real biological systems is discussed.

Plasma polymerization of phenyl isothiocyanate

AbstractThin polymer films were obtained by plasma polymerization of phenyl isothiocyanate. Polymerizations were carried out in rf (13.56 MHz) glow discharge generated in an electrodeless flow system. It was found that this monomer produces uniform films with a wide range of thicknesses, from hundreds of nanometers to tens of micrometers. The deposition rate appeared to be dependent on the substrate distance from the monomer inlet. The chemical structure of plasma polymer was characterized by using elemental analysis, IR spectroscopy, gas chromatography, and mass spectrometry. Elemental analysis showed that the composition of polymer depends on the substrate position in the reactor. It was observed that sulphur content decreased with increasing the substrate distance from the monomer inlet, whereas nitrogen content appeared to increase. The IR data revealed significant decrease in —NCS groups content in the polymer as compared with the monomer spectrum and indicated for the appearance of new absorption bands corresponding to the CN and CH aliphatic, groups. The results account for a strong fragmentation of monomer in plasma involved in decomposition of isothiocyanate group and phenyl ring. The soluble fraction of polymeric material was examined by gas chromatography and then the separated products were analyzed by mass spectrometry. The soluble fraction was found to be composed of numerous low molecular‐weight compounds. Identification of their structure revealed the presence of residual monomer, thiophenol, cyanobenzene, diphenyl, diphenyl sulphide, diphenyl disulphide, phenyl thiocyanate, dicyanobenzene, phenatroline, and some other oligomeric products. Formation of these compounds proves high susceptibility of NCS group in the monomer towards different fragmentation reactions. The surface free energy and electrical conductivity of polymer films were evaluated. The surface free energy value was very close to those estimated for plasma polymers deposited from other benzene derivatives. The low electrical conductivity manifested by the investigated polymeric material indicated for its dielectric character. The photoelectrical measurements revealed some photoconductivity effect in this material.

Synthesis of 14C‐ and 3H‐labelled 4‐(4‐nitrophenyl)aminophenylisothiocyanate (GO 9333 / CGP 4540; amoscanate

AbstractAmoscanate labelled with carbon‐14 on the isothiocyanate carbon atom was prepared in an overall yield of 13% at a specific activity of 4.13 μCi/mg from potassium [14C]thiocyanate. The 4‐nitro[U‐14C]phenyl ring labelled compound was synthesized in 20.4% overall yield from [U‐14C]aniline at a specific activity of 12.2 μCi/mg. The corresponding tritiated compound was prepared from 4‐amino[2‐3H]acetanilide at 112 μCi/mg. Labelling with tritium in the aromatic ring bearing the isothiocyanate group was achieved by catalysed halogen‐tritium replacement. However, for pharmacokinetic and metabolism studies in experimental animals, the 14C‐ and 3H‐labels associated with the phenylisothiocyanate moiety subsequently proved disadvantageous because of the instability of the labels in vivo.

Inhibition of anion transport across the red cell membrane by dinitrophenylation of a specific lysine residue at the H 2DIDS binding site of the band 3 protein

The inhibition of anion transport by dinitrophenylation of the red cell membrane is brought about by the modification of a single lysine residue located on the 17-kDa segment of the band 3 protein. This residue is identical with Lys a, which is also capable of reacting with one of the two isothiocyanate groups of 4,4'-diisothiocyano dihydro-stilbene-2,2'-disulfonate (H 2DIDS). The rate of reaction between Lys a and 1-fluoro-2,4-dinitrobenzene is reduced when a second lysine residue on the 35-kDa segment of the band 3 protein becomes dinitrophenylated. This latter residue is not identical with Lys b which is known to be present on the 35-kDa segment and involved in the cross-linking of this segment with the 17-kDa segment by H 2DIDS.

Cycloadditions of N,N'-dicyclohexylcarbodiimide to 2-phenylethenyl isothiocyanates

2-Phenylethenyl- and 2-(4-nitrophenyl)ethenyl isothiocyanates undergo a 2 + 2 cycloaddition with N,N'-cyclohexylcarbodiimide to form 1,3-thiazetedine derivatives; this reaction takes place at the C=S bond of the isothiocyanate group. The olefinic C=C bond was not involved.

Metabolism of some naturally occurring isothiocyanates in the rat.

The metabolism of methyl, ethyl, butyl and allyl isothiocyanate, which occur as glucosinolates in a number of plants, was studied. Oral administration of the substances to the rat was followed by their renal excretion as mercapturic acids, which were isolated as dicyclohexylamine salts. Chemical structure was determined by synthesis and 1H-n.m.r. spectra. The mercapturic acids were very labile dithiocarbamidic acid esters, formed by the addition of the isothiocyanate group to the SH group of the cysteine component.

SITS: a covalently bound fluorescent retrograde tracer that does not appear to be taken up by fibers-of-passage

Experiments in the rat indicate that the plasma membrane marker SITS is a sensitive retrograde tracer that is not taken up in detectable amounts by preterminal axons. SITS contains a reactive isothiocyanate group that binds covalently to proteins under mild aqueous conditions, and its use is fully compatible with autoradiographic, HRP, and immunohistochemical methods, as well as with certain other fluorescent tracers. In addition, it neither diffuses from retrogradely labeled cells, nor is degraded after prolonged survival times.

An affinity label for alpha 2-adrenergic receptors in rat brain.

Clonidine, a potent and highly selective alpha 2-adrenergic agonist of the central nervous system, was modified. Insertion of the strong alkylating isothiocyanate group (NCS) group, at its aromatic residue, makes clonidine a potential affinity label of the alpha 2-adrenergic receptors. In displacement of [3H]clonidine and p-[3H]aminoclonidine from rat brain membrane preparations, clonidine-NCS demonstrates high affinity for the alpha 2-adrenergic receptors (Kd = 50 mM). The covalent labelling of the central alpha 2-receptors requires higher concentrations of the irreversible ligand (1-70 microM), thus indicating possible non-productive interactions at the environment of the receptor site. Only partial protection of the receptors is observed with a reversible alpha 2-agonist. The new clonidine analog appears to be a general ligand for the alpha 2-adrenergic receptors and might serve as a potential affinity probe for these receptors.

Paulomycins A and B. Isolation and characterization.

Paulomycin A, C34H46N2O17S and paulomycin B, C33H44N2O17S are two antibiotics produced by Streptomyces paulus strain 273 (UC 5142). Both antibiotics, which are mainly active against a variety of Gram-positive bacteria, contain an isothiocyanate group and in this respect they are related to senfolomycins A and B and proceomycin.

Thiol derivatives of cellulose as supports for the immobilization of non-thiol enzymes

Thiosulfate derivatives, which can be reduced with mercaptoacetic acid, are suitable intermediates for the preparation of thiol derivatives of polymers. Thiosulfate derivatives of cellulose were prepared via chlorodeoxy- or via 3-chloro-2-hydroxy-propylcellulose, while mercaptodeoxycellulose prepared via chlorodeoxy derivative had more convenient properties for the immobilization of non-thiol enzymes (acetylcholine esterase, butyrylcholine esterase and trypsin). Before immobilization SH groups were introduced into choline esterases by i) reduction of the cystine residues, ii) reaction with methyl 4-mercaptobutyrimidate, and the isothiocyanate groups were introduced into trypsin on reaction with 3-isothiocyanatopropyl 1-isocyanate. The immobilization of the enzymes treated in this way was carried out under the conditions of the oxidation of thiol groups (i), thiol-disulfide exchange reaction (ii), or an addition nucleophilic reaction of isothiocyanates with thiols. In contrast to the proteolytic activity of the immobilized trypsin the esterolytic activity of immobilized choline esterases attained satisfactory values.

Localization of the two protease binding sites in human alpha 2-macroglobulin.

The distance between the two protease binding sites in human plasma alpha 2-macroglobulin has been estimated using singlet-singlet energy transfer experiments. alpha-Chymotrypsin was labeled covalently with donor (dansyl chloride) or acceptor (fluorescein isothiocyanate) groups, and the efficiency of transfer between these dyes was measured within the alpha 2-macroglobulin . (alpha-chymotrypsin)2 complex. The distance between the surface exterior of the protease molecules was calculated to be 4 to 11 A, depending on the assumption made about the equivalence of the binding sites. A catalytically active dimer of alpha-chymotrypsin was prepared using the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate. In contrast with the alpha-chymotrypsin monomer, it binds to alpha 2-macroglobulin with a 1:1 stoichiometry. However, the 1:1 alpha 2-macroglobulin . dimeric alpha-chymotrypsin complex is still able to bind 1 mol of the alpha-chymotrypsin monomer. Energy transfer experiments performed with this ternary complex showed that the distance between the alpha 2-macroglobulin-bound alpha-chymotrypsin molecules is not higher than 4 A, i.e. the two protease binding sites in alpha 2-macroglobulin should be about 44 A apart (center to center) if the anhydrous radius of alpha-chymotrypsin is 20 A.

Stereochemistry of the acyclic phosphono- and phosphino-thiocyanide isomerisation into corresponding isothiocyanidates : Synthesis of optically active thiocyanidates > P(O)SCN isothiocyanidates > P(O)NCS, isocyanidates > P(O)NCO and related phosphorylated derivatives of carbonic acid

Optically active t-butylphenylphosphinothiocyanidate ButPhP(O)SCN 3 and t-butyl-O-methyl-phosphonothiocyanidate But(MeO)P(O)SCN 7 have been prepared by condensation of the corresponding sulphenyl chlorides > P(O)SCl with trimethylsilylcyanide and isomerised into optically active t-butyl-O-methyl-phos-phonoisothiocyanidates But(MeO)P(O)NCS 8. Chirality at P in and the optical purity of the chiral phosphino (phosphono) thiocyanidates and isothiocyanidates have been determined by chemical correlations. It has been demonstrated that the thiocyanate ion and amine catalysed thiocyanidate-isothiocyanidate isomerisation > P(O)SCN → > P(O)NCS occurs stereospecifically with inversion of configuration at the P center. This result can be rationalized by postulation of a phosphorane intermediate, formed by nucleophilic attack of the SCN- anion on phosphorus, in which thiocyanate and isothiocyanate groups occupy apical positions. In connection with these studies a number of novel optically active phosphorylated derivatives of carbonic acid, > P(O)NHCSNHR, > P(O)NCCl2, > P(O)NCO and > P(O)NHCOOBut, have also been synthesised.

New polymer syntheses, 3. Binding of nucleosides to basic polypeptides via isocyanato‐isothiocyanates

AbstractNew aliphatic α‐isocyanato‐ω‐isothiocyanates of various chain lengths were synthesized from ω‐isothiocyanatocarboxylic acid chlorides and trimethylsilyl azide. The 5′‐OH group of 2′‐O,3′‐O‐isopropylideneuridine and of N6‐benzoyl‐2′‐O,3′‐O‐isopropylideneadenosine was added selectively onto the isocyanate group of aromatic and aliphatic α‐isocyanato‐ω‐isothiocyanates. The isothiocyanate group of the resulting nucleoside derivatives was then reacted with the free amino groups of polylysine, isopolylysine, polyornithine and isopolyornithine. The resulting polypeptides with pending nucleosides were characterized by elemental analyses and 360 MHz 1H NMR spectra. A new “one‐pot‐procedure” was developed to protect the primary amino group (N6) of 2′‐O,3′‐O‐isopropylideneadenosine.

Two rapid fluorescence procedures for the detection of some thio pungent compounds in plant tissues.

Two rapid flourescence procedures are described for detecting sulphydryl, disulphide and isothiocyanate groups of scented and pungent principles present in the vacuolar sap of onion, garlic and cabbage. To localize compounds containing sulphydryl groups, fresh or fixed frozen sections of the plants were treated with mercurochrome. After the fluorochromization, strongly-positive sulphydryl sites emitted an intense orange-red fluorescence, while weakly-positive sites emitted a distinctive red-brown fluorescence. Disulphide groups were detected by first reducing with thioglycolic acid to thiol groups before treating with mercurochrome. To effect isothiocyanate localization, frozen sections were exposed to ammonia: isothiocyanates were converted to thioureas and the engendered amino groups were revealed with fluorescamine.