Second Generation of cycloSal‐Pronucleotides with Esterase‐Cleavable Sites: The ”Lock‐In”‐Concept

A conceptual extension of the cycloSal‐pronucleotide approach is presented. The characteristic feature of the new cycloSal‐derivatives of the anti‐HIV active nucleoside analogue d4T 1 is the incorporation of an enzymatically cleavable carboxylic ester moiety with the intention to trap the triesters inside cells (”lock‐in”‐concept). CycloSal‐triesters bearing different ester groups in the 3‐or 5‐position of the cycloSal‐moiety are described. Surprisingly, only acetyl‐and levulinyl esters are cleaved readily in CEM cell extracts while alkyl esters were found to be stable. Nevertheless, in in‐vitro anti‐HIV assays most of the compounds achieve the thymidine–kinase bypass, thus proving that they act at least as nucleotide delivery systems.     

Toward the Development of Metal‐Free Synthetic Nucleases: Cleavage of a Model Substrates by 1,4‐Diazabicyclo[2.2.2]Octane Derivatives

Artificial ribonucleases of A_nBCL series were synthesized by solid‐phase method. They consist of a hydrophobic alkyl radical A (n = 3–12 carbon atoms), an “RNA‐binding domain” B (bisquaternary salt of 1,4‐diazabicyclo[2.2.2]octane), a “catalytic domain” C (histidine residue) and a “linker” L that joins the domains B and C. The effect of the alkyl radical on the catalytic properties of the chemical catalyst was studied using three activated phosphate ester substrates: p‐nitrophenyl phosphate, bis‐p‐nitrophenyl phosphate, and thymidine‐3′‐p‐nitrophenyl phosphate.     

Nucleosides. IX. Synthesis of Purine N 3,5′‐Cyclonucleosides and N 3,5′‐Cyclo‐2′,3′‐seconucleosides via Mitsunobu Reaction as TIBO‐like Derivatives

The Mitsunobu reaction was applied to prepare, in one step, purine N ³,5′‐cyclonucleosides 10a–d. A subsequent ring opening in the ribose moiety of the resultant N ³,5′‐nucleosides by sodium periodate led to the corresponding N ³,5′‐cyclo‐2′,3′‐seconucleosides. These products consist of 5‐, 6‐, and 7‐membered tricyclic system which is the basic skeleton of TIBO derivatives, known antiviral agents.     

Synthesis of D‐Altritol Nucleosides with a 3′‐O‐Tert‐Butyldimethylsilyl Protecting Group

Four D‐altritol nucleosides with a 3′‐O‐tert‐butyldimethylsilyl protecting group are synthesized (base moieties are adenine, guanine, thymine and 5‐methylcytosine). The nucleosides are obtained by ring opening reaction of 1,5:2,3‐dianhydro‐4,6‐O‐benzylidene‐D‐allitol. Optimal reaction circumstances (NaH, LiH, DBU, phase transfer, microwave irridation) for the introduction of the heterocycles are base‐specific. For the introduction of the 3′‐O‐silyl protecting group, long reaction times and several equivalents of tert‐butyldimethylsilyl chloride are needed.     

Detection of Prothrombin and Osteopontin in a Renal Stone Found in a Hyperuricemic Patient Using 2D‐PAGE and LC‐MS Analysis

The liquid chromatography‐mass spectrometry (LC‐MS) following on from the two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) technique was applied for the analysis of proteins in a renal stone found in a hyperuricemic patient. This technique was sensitive enough to detect small quantities of proteins even in a renal stone.     

Clinical and Biochemical Manifestations and Molecular Characterization of the Mutation HPRT Jerusalem

A novel point mutation (I137T) was identified in the hypoxanthine‐guanine phosphoribosyltransferase (HPRT) encoding gene, in a patient with partial deficiency of the enzyme. The mutation, ATT to ACT (substitution of isoleucine to threonine), occurred at codon 137, which is within the region encoding the binding site for 5‐phosphoribosyl‐1‐pyrophosphate (PRPP). The mutation caused decreased affinity for PRPP, manifested clinically as a Lesch–Nyhan variant (excessive purine production and delayed acquisition of language skills). The partial HPRT deficiency could be detected only by measuring HPRT activity in intact fibroblasts (uptake of hypoxanthine into nucleotides).     

In Vitro Selection of Single‐Stranded DNA Aptamers that Bind Human Pro‐Urokinase

Single‐chain pro‐urokinase is an inactive proenzyme form of human urokinase (urinary plasminogen activator) with a Mr of 50,000 which is converted to the active two‐chain form by catalytic amounts of plasmin. It is used for thrombolytic therapy of acute myocardial infarction and acute ischemic stroke. We have isolated single‐stranded DNA molecules with significantly increased binding affinity for human pro‐urokinase by SELEX (systematic evolution of ligands by exponential enrichment) procedure from a pool of 10¹⁵ molecules containing 24 randomized positions which are flanked by defined regions. ssDNA from this library was hybridized with helper «fixture», thus allowing the central random chain to fold into complex three‐dimentional shapes. Sequencing data from pro‐urokinase aptamers obtained after 12 selection cycles displayed a highly conserved 12–14 base region.     

Novel Mutations and Hot‐Spots in Patients with Purine Nucleoside Phosphorylase Deficiency

Purine nucleoside phosphorylase (PNP) deficiency results in severe immune dysfunction and early death from infections. Lymphopenia, reduced serum uric acid, and abnormal PNP enzymatic activity assist in the diagnosis of PNP‐deficient patients. Analysis of the gene encoding PNP in these patients reveals several recurring mutations. Identification of these hot‐spots for mutation may allow faster confirmation of the diagnosis in suspected cases.     

Synthesis of Some Novel 6‐Benzyl(or Substituted Benzyl)‐2‐β‐d‐Glucopyranosyl‐1,2,4‐Triazolo[4,3‐b][1,2,4]Triazines as Potential Antimicrobial Chemotherapeutics

Glucosidation of the new 8‐amino‐6‐benzyl(or substituted benzyl)‐2,8‐dihydro‐1,2,4‐triazolo[4,3‐b][1,2,4]triazin‐7(3H)‐ones (3a–d) with 2,3,4,6‐tetra‐O‐acetyl‐α‐d‐glucopyranosyl bromide 4 gave the corresponding N‐glucosides 5a–d. Chemical transformations leading to new functionalities have also been achieved to give compounds 7–12. Antimicrobial activity of compounds 5a–c against Aspergillus fumigatus, Penicillium italicum, Syncephalastrum racemosum, Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Escherichia coli is described.     

The Solution Synthesis of Antisense Oligonucleotide‐Peptide Conjugates Directly Linked via Phosphoramide Bond by Using a Fragment Coupling Approach

To improve antisense oligonucleotide penetration inside cells, conjugates of oligonucleotides and cell‐penetrating peptides, covalently linked through a phosphoramide bond, were prepared by a fragment coupling approach in the liquid phase. Two methods were used for this synthesis, i.e., phosphorylation of a peptide amino group by an oligonucleotide terminal phosphate 1‐hydroxybenzotriazole ester in aqueous media or condensation of phosphate and amino groups in presence of triphenylphosphine, 2,2′‐dithiopyridine and 4‐dimethylaminopyridine in organic media. Several oligonucleotides, including a 18‐mer antisense oligodeoxyribonucleotide complementary to an internal coding region of the reporter gene of the green fluorescent protein (GFP) were prepared. Peptides derived from the third helix of the homeodomain of Antennapedia, the influenza envelope hemagglutinin subunit as well as melittin and polymyxin B were used for the conjugates' synthesis. The peptides with various amino acid composition were chosen to confirm that these coupling methods are of a general use.