The preparation of polyamide-oligonucleotide probes containing multiple non-radioactive labels.

Oligonucleotide probes containing multiple non-radioactive labels have been prepared by utilising and extending the methods used to prepare polyamide-oligonucleotide conjugates. The probes were prepared by incorporating suitable amino acid residues, such as lysines, in the polyamide, which were then used as sites for the attachment of the non-radioactive labels. The procedures developed give control over the distance of the label from the oligonucleotide, and also the inter-label distance. The labels can be conveniently introduced while the substrate is still on the solid support. Even though fluorescent oligonucleotide probes prepared in this way carrying multiple carboxyfluorescein labels gave low levels of fluorescence due to quenching, the probes containing ten biotin labels gave a detection sensitivity of approximately 5 attomole (3 million molecules).     

Novel solid-phase synthesis of thiol-terminated-poly(alpha-amino acid)-drug conjugate.

A new method using a controlled pore glass solid support for the preparation of a thiol-terminated-polymerdrug, notably poly-L-glutamate-daunomycin having a terminal thiol group, is described. The method consists of first polymerizing an ester-protected glutamic acid onto an amino-disulfide functionalized controlled pore glass support. The ester protecting group is then removed, freeing the gamma-carboxyl groups of the grafted polymer which then allows it to react with daunomycin. Finally, the disulfide bond linking the conjugated polymer-drug to the solid support is broken by thiolysis, thus releasing the desired product. The final product consists of only polymer-drug conjugates with terminal thiol groups (global yield 26%). This novel method is much simpler and more elegant than more conventional preparation methods requiring solution phase techniques.     

Synthesis and antitumor activities of novel 5-deazaflavin-sialic acid conjugate molecules

6-Nitro-5-deazaflavin derivatives bearing O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha- and beta-D-galacto-non-2-ulopyranosylonate)alkyl group (sialosylalkyl group) at N(3) or N(10) and 8-amino-5-deazaflavin substituted with the sialosylalkyl group at the amino group were synthesized and their physicochemical properties as well as antitumor effects on KB and L1210 cells have been investigated. The configurations of the glycosides were determined by 1H NMR and rate of hydrolysis of the glycosidic bond. It has been found that these conjugate molecules show significant antitumor activities. Combination of an 8-amino-5-deazaflavin with the sialosylalkyl group have been found to give rise to significant increase in antitumor activities of the compound. Antitumor effects of 6-nitro-5-deazaflavin-sialic acid conjugate molecules were similar or rather weak in comparison with those of the 6-nitro-5-deazaflavin derivatives without sialosylalkyl group.     

Facile synthesis of benzyl beta-D-galactofuranoside. A convenient intermediate for the synthesis of D-galactofuranose-containing molecules

Benzyl beta-D-galactofuranoside was efficiently obtained from 1,2,3,5,6-penta-O-benzoyl-alpha,beta-D-galactofuranose, via benzyl 2,3,5,6-tetra-O-benzoyl-beta-D-galactofuranoside. Conditions for the O-debenzylation were investigated in order to evaluate the synthetic application of the benzyl group as an anomeric protector of a galactofuranose moiety in synthetic strategies involving galactofuranose.     

Role of small molecules in regulation of D-serine deaminase synthesis.

Cyclic AMP is required for optimal synthesis of D-serine deaminase synthesis from dsdO+ templates and for optimal hyperinducible synthesis from low constitutive dsdO templates both in vitro and in vivo. Neither D-serine, cyclic AMP, nor dsdC activator has an effect on expression of a high constitutive dsdO template. The synthesis of the dsdC activator itself in vitro is independent of cyclic AMP. Guanosine tetraphosphate does not have a significant effect on in vitro D-serine deaminase synthesis from dsdO+ or dsdO templates. A previously described class of dsdO mutants showing partial catabolite sensitivity of constitutive D-serine deaminase synthesis proved to be low dsdO types. They all contain a low constitutive dsdC mutation; the two effects are additive with regard to level of constitutivity, but only that portion of synthesis attributable to the dsdC mutation is cyclic AMP dependent.     

Isolation, determination of structure and synthesis of the acid-labile conjugate of aldosterone.

1. After administration of 600mg of 3H-labelled aldosterone to human volunteers, 57 mg of homogeneous acid-labile conjugate was isolated from the urine and identified as aldosterone 18 beta-D-glucosiduronic acid. 2. Esterification and acetylation of the conjugate gave a tetra-acetate methyl ester, which, by measurement of the optical rotation and nuclear-magnetic-resonance spectrum, was shown to be a beta-glucosiduronate. This tetra-acetate methyl ester was synthesized in approx. 10% yield by the Koenigs-Knorr procedure. 3. Removal of the acetyl and methyl ester groups from the tetra-acetate methyl ester with alkali was accompanied by almost complete isomerization at C-17 to give 17-isoaldosterone 18 beta-D-glucosiduronic acid. 4. To prevent inversion at C-17 during removal of the acetate and ester groups of beta-glucosiduronate (a) the 3,20-disemicarbazone was prepared, (b) the acetate and ester groups were removed from the disemicarbazone by treatment with alkali, and (c) the semicarbazone groups were removed from the product at pH 2.0, and aldosterone 18 beta-D-glucosiduronic acid was obtained in 47% overall yield. 5. In the presence of components used to synthesize beta-glucosiduronate by the Koenigs-Knorr reaction this substance is converted slowly into the alpha-glucosiduronate; this conversion is responsible, in part, for the low yield of beta-glucosiduronate. 6. Two additional conjugates were obtained in the Koenigs-Knorr reaction; a provisional structure was assigned to one substrate. The other substance is a C-18 alpha-glucosiduronate. Removal of the acetyl and ester groups from C-18 alpha-glucosiduronate gave the alpha-glucosiduronic acid in 84% yield and the 17-isoaldosterone alpha-glucosiduronic acid in 12% yield. 7. The rate at which several types of beta-glucuronidase hydrolyse the foregoing steroidal alpha- and beta-glucosiduronic acids is given.     

Fluorescent-fipronil: Design and synthesis of a stable conjugate

Fipronil is a phenyl pyrazole molecule widely used across the world as both insecticide and veterinary drug. The main goal of this work was to synthesize a fluorescently labeled fipronil derivative for cellular imaging without affecting its intrinsic properties. We selected fluorescein as fluorescent probe and we investigated different strategies for stable chemical ligation between both entities, such as thiourea and direct peptide bond. While thiourea bond displayed low stability, direct peptide bond was difficult to achieve due to problems of steric hindrance. The best result was obtained by conjugation using click chemistry, which allowed to obtain fipronil stably labeled with the fluorescent probe.     

Studies on immunoassays of peptide factors. IV. New synthesis of a gastrin/peroxidase conjugate

We have shown that structurally well-defined homogeneous maleoyl-peptides are synthetically accessible. These anchor-modified peptide derivatives allow their selective covalent linkage to thiol-containing proteins via the maleimide-thiol procedure. Correspondingly mercaptosuccinylated horseradish peroxidase was reacted with N alpha-maleoyl-beta-alanyl-human-little gastrin-I-[2-17] to produce the gastrin/peroxidase conjugate in good yields at 1:1 stoichiometry. The conjugate exhibited full enzymatic activity and identical binding affinity to antigastrin antisera as the parent gastrin. This approach proved to be well suited for the preparation of enzyme labeled peptide factors as tracers for immunoassays.     

Primer-dependent synthesis of covalently linked dimeric RNA molecules by poliovirus replicase.

Poliovirus-specific RNA-dependent RNA polymerase (replicase, 3Dpol) was purified from HeLa cells infected with poliovirus. The purified enzyme preparation contained two proteins of apparent molecular weights 63,000 and 35,000. The 63,000-Mr polypeptide was virus-specific RNA-dependent RNA polymerase, and the 35,000-Mr polypeptide was of host origin. Both polypeptides copurified through five column chromatographic steps. The purified enzyme preparation catalyzed synthesis of covalently linked dimeric RNA products from a poliovirion RNA template. This reaction was absolutely dependent on added oligo(U) primer, and the dimeric product appeared to be made of both plus- and minus-strand RNA molecules. Experiments with 5' [32P]oligo(U) primer and all four unlabeled nucleotides suggest that the viral replicase elongates the primer, copying the poliovirion RNA template (plus strand), and the newly synthesized minus strand snaps back on itself to generate a template-primer structure which is elongated by the replicase to form covalently linked dimeric RNA molecules. Kinetic studies showed that a partially purified preparation of poliovirus replicase contains a nuclease which can cleave the covalently linked dimeric RNA molecules, generating template-length RNA products.     

Design and synthesis of a novel synthetic NAPAP-penta-saccharide conjugate displaying a dual antithrombotic action.

The synthesis of a novel antithrombotic consisting of a heparin pentasaccharide conjugated to the active site inhibitor N-(2-naphtalenesulfonyl)-glycyl-(D)-4-aminophenyl-alanyl-piperidin e (NAPAP) (i.e. compound I) is reported. This conjugate shows a unique pharmacological profile both in vitro and in vivo having direct anti-thrombin and ATIII-mediated anti-Xa activity. Furthermore, conjugate I has a prolonged in vivo half-life compared to NAPAP (1.5 h vs 9 min.).     

Profiling solid-phase synthesis products by free-solution conjugate capillary electrophoresis

Solid-phase synthesis of oligomers, both natural and nonnatural, has proved to be invaluable for the development of many areas of biotechnology. A critical step in the solid-phase synthesis of any oligomer is determining the number and concentration of different constituents present in the product mixture resulting from the synthesis, both before and after purification. Most typically, this analysis is performed by reversed-phase high performance liquid chromatography (RP-HPLC), with the separated components detected by UV absorbance. Recently, we described a novel technique, free-solution conjugate electrophoresis (FSCE), for the high-resolution separation and sensitive laser-induced fluorescence (LIF) detection of uncharged, synthetic polymers, PEG in particular. In this report, we apply this bioconjugate capillary electrophoresis technique to analyze products of the solid-phase synthesis of oligomeric polyamides, namely poly(N-substituted glycines), or polypeptoids. When compared to more traditional RP-HPLC analysis, FSCE analysis of oligomeric peptoids results in separation resolutions that are approximately five times higher and separation efficiencies that are increased by 150%. Moreover, when FSCE with LIF detection is applied to the analysis of oligomeric polyamides after HPLC purification, impurities that are not detectable in RP-HPLC analysis are readily separated and detected. With the advent of capillary array electrophoresis (CAE), which allows for automated, parallel analysis of many different samples, we believe that FSCE will be especially applicable to the analysis of combinatorial synthesis products, by allowing researchers to evaluate many different samples in a single, highly parallel, fully automated analysis. This is in contrast to RP-HPLC analysis, in which samples must be analyzed in series.     

Aldolase-catalysed stereoselective synthesis of fluorinated small molecules

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Total stepwise solid-phase peptide-oligonucleotide conjugate synthesis on macroporous polystyrene

An efficient total stepwise solid-phase synthesis of oligonucleotide-peptide conjugates on a macroporous polystyrene is described. Extending our homoserine linker approach, we prepared a range of fluorescein-labelled conjugates containing one of two different peptides together with oligonucleotides containing 2'-deoxynucleoside or 2'-O-methylribonucleoside phosphodiesters, or gapmers containing 2'-deoxyphosphorothioate sequences flanked by 2'-O-methyl wings.     

Hormonotoxins. I. Strategy for synthesis of ovine luteinizing hormone--gelonin conjugate bearing the toxin in the beta-subunit

The amino groups in the beta-subunit of ovine luteinizing hormone (oLH) were modified by thiolation using N-succinimidyl-3-(2-pyridyldithio) propionate so that it may be coupled in a disulfide linkage to similarly modified ribosome inactivating protein, gelonin. The modified beta-subunit was able to hybridize with free LH alpha-subunit and the complex retained full biological activity. However, when gelonin was coupled to the beta-subunit, the resulting conformational changes masked or eliminated the sites necessary for intersubunit recognition of the free alpha-subunit. This has important implications for the design in the synthesis of gonadotropin-toxin/drug conjugates.     

The metabolism of benzyl isothiocyanate and its cysteine conjugate.

1. The corresponding cysteine conjugate was formed when the GSH (reduced glutathione) or cysteinylglycine conjugates of benzyl isothiocyanate were incubated with rat liver or kidney homogenates. When the cysteine conjugate of benzyl isothiocyanate was similarly incubated in the presence of acetyl-CoA, the corresponding N-acetylcysteine conjugate (mercapturic acid) was formed. 2. The non-enzymic reaction of GSH with benzyl isothiocyanate was rapid and was catalysed by rat liver cytosol. 3. The mercapturic acid was excreted in the urine of rats dosed with benzyl isothiocyanate or its GSH, cysteinyl-glycine or cysteine conjugate, and was isolated as the dicyclohexylamine salt. 4. An oral dose of the cysteine conjugate of [14C]benzyl isothiocyanate was rapidly absorbed and excreted by rats and dogs. After 3 days, rats had excreted a mean of 92.4 and 5.6% of the dose in the urine and faeces respectively, and dogs had excreted a mean of 86.3 and 13.2% respectively. 5. After an oral dose of the cystein conjugate of [C]benzyl isothiocyanate, the major 14C-labelled metabolite in rat urine was the corresponding mercapturic acid (62% of the dose), whereas in dog urine it was hippuric acid (40% of the dose). 5. Mercapturic acid biosynthesis may be an important route of metabolism of certain isothiocyanates in some mammalian species.