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.     

Biochemical and Molecular Genetic Correlation in Adenylosuccinate Lyase Deficiency

An homology model of human adenylosuccinate lyase structure shows that P100A substitution distorts the amino acid chain of domain I in the proximity of His‐86, which behaves as general acid in the catalysis, and may expose Cys‐98 and Cys‐99 to oxidising agents. This model is in line with the observation that the defective protein is strongly inhibited by 4‐hydroxy‐2‐nonenal, an hydroxyalkenal that is known to form thio‐ether linkage with proteins.     

Synthesis of Oligoribonucleotides Containing 4‐Thiouridine Using the Convertible Nucleoside Approach and the 1‐(2‐Fluorophenyl)‐4‐Methoxypiperidin‐4‐yl Group

Oligoribonucleotides containing 4‐thiouridine were prepared using the Fpmp group for protection of the 2′‐OH. Two uridine derivatives with the 1,2,4‐triazolyl and the 2‐nitrophenyl groups at position 4 were used to obtain 4‐thiouridine by postsynthetic substitution with sodium hydrogen sulfide. Both uridine derivatives allow the preparation of the desired oligonucleotides in good yields.     

Crystal Structure and Conformation of 5‐Fluorouridine: Conformational Preferences for 5‐Fluorinated Pyranosides

Crystals of 5‐fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)Å, α = γ = 90°, β = 96.70° (1), space group P2₁, Z = 2, ρ_obs = 1.56 gm/c.c and ρ_calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I ≥ 3σ). The nucleoside has the anti conformation [χ = 53.1(4)°] with the furanose ring in the favorite C2′–endo conformation. The conformation across the sugar exocyclic bond is g⁺, with values of 49.1(4) and ? 69.3(4)° for Φ_θc and Φ_∞ respectively. The pseudorotational amplitude τ_m is 34.5 (2) with a phase angle of 171.6(4)°. The crystal structure is stabilized by a network of N–H…O and O–H…O involving the N3 of the uracil base and the sugar O3′ and O2′ as donors and the O2 and O4 of the uracil base and O3′ oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5‐fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [χ varies from ? 20 to + 60°] 2) an inverse correlation between the glycosyl bond distance and the χ angle 3) a wide variation of conformations of the sugar ranging froni C2′–endo through C3′–endo to C4′–exo 4) the preferred g⁺ across the exocyclic C4′–C5′ bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.     

Thiosugar Nucleosides. Synthesis and Biological Activity of 1,3,4‐Thiadiazole,Thiazoline and Thiourea Derivatives of 5‐Thio‐d‐Glucose

New acylated 5‐thio‐β‐d‐glucopyranosylimino‐disusbstituted 1,3,4‐thiadiazols 8, and 11 were prepared, via spontaneous rearrangements, by cycloaddition of the glycosyl isothiocyanate 2 with the reactive intermediates 1‐aza‐2‐azoniaallene hexachloroantimonates 4 and 6, respectively. Reaction of 2 with aminoacetone or chloroethylamine afforded the acylated 5‐thio‐β‐d‐glucopyranosyl‐4‐imidazoline‐2‐thione nucleoside 16 and glucopyranosylamino‐2‐thiazoline derivative 18, respectively. Deblocking of 8, 11, 17 and 19 furnished the free nucleoside analogues 9, 12, 18 and 20, respectively. Analogously, treatment of 2 with chloroethylamine in the 1:2 ratio afforded the thioureylendisaccharide 21. No in vitro antiviral activity against HIV‐1, HIV‐2, human cytomegallovirus (HMCV), has been found for the new synthesized compounds.