Nucleosides with self-complementary hydrogen-bonding motifs: synthesis and base-pairing studies of two nucleosides containing the imidazo[4,5-d]pyridazine ring system.

Synthesis and base-pairing studies of two 2'-deoxyribonucleosides, containing a common heterocyclic base, 7(4)-amino-5(6)H-imidazo[4,5-d]pyridazin-4(7)one (1 and 2), have been reported. The synthesis was accomplished by base-promoted deoxyribosylation of ethyl 5(4)-cyanoimidazole-4(5)-carboxylate (6), followed by ring-closure with hydrazine hydrate. The 1H NMR-based base-pair studies were conducted using DMF-d7 as a solvent by measuring changes in chemical shifts of the amino, hydrazide, imidazole H-2, and the sugar H-1' protons of the nucleosides with variations in concentrations and temperatures. Large downfield chemical shifts were observed for the NH, NH2, and to a lesser extent for the H-1' protons when the temperature was lowered from 25 to 0 degrees C, and then further down to -50 degrees C in 10 degree intervals. The observed experimental data are consistent with the results of molecular modeling studies. Nucleoside 2 exhibited low level antiviral activity against HIV-1 in CEM-SS cells with an IC50 of 89.2 microM. No cellular toxicity was observed at the highest concentration of the compound tested.

Broad spectrum antimicrobial activity of leukocyte extracts from the American alligator (Alligator mississippiensis).

Leukocytes were isolated from whole blood of wild alligators by differential sedimentation. The leukocytes were disrupted in 5% AcOH and the crude extracts processed by ultrafiltration. The extracts were subjected to solvent exchange (0.1% AcOH) and the fraction that contained macromolecules between 1 and 10 kDa were subjected to further analyses. The acid extracts of the alligator leukocytes exhibited substantial antimycotic activities against six of eight species of Candida yeast tested. In addition, the alligator leukocyte extracts were effective as antimicrobial agents against 10 of 12 bacterial species, and displayed moderate activity against two enveloped viruses (human immunodeficiency virus-1 and herpes simplex virus-1(HF)). Kinetic analyses revealed that the antimycotic effects of the leukocyte extract occurred rapidly, with 64% fungal growth inhibition within 3 min of exposure. The molecule(s) responsible for the antimicrobial activities were sensitive to proteases, heat-stable, acid soluble, and in the 1-10 kDa range. These data suggest that alligator leukocytes express cationic peptides that are responsible for their antimicrobial properties.

Antiviral activity of serum from the American alligator (Alligator mississippiensis).

Serum from wild alligators was collected and tested for antibiotic activity against three enveloped viruses using cell-based assays. Alligator serum demonstrated antiviral activities against human immunodeficiency virus type 1 (HIV-1; IC50=0.9%), West Nile virus (WNV; IC50=4.3%), and Herpes simplex virus type 1 (HSV-1; IC50=3.4%). The inhibitory concentration (IC50) is defined as the concentration of serum that inhibits 50% of viral activity. The antiviral effects of the alligator serum were difficult to evaluate at high concentrations due to the inherent toxicity to the mammalian cells used to assay viral activities. The TC50 (serum concentration that reduces cell viability to 50%) values for the serum in the HIV-1, WNV, and HSV-1 assays were 32.8, 36.3 and 39.1%, respectively. Heat-treated serum (56 degrees C, 30 min) displayed IC50 values of >50, 9.8 and 14.9% for HIV-1, WNV and HSV-1 viruses, respectively. In addition, the TC50 values using heat-treated serum were substantially elevated for all three assays, relative to untreated serum (47.3 to >50%). Alligator serum complement activity has been shown to be heat labile under these conditions. HIV-1 antiviral action was heat-sensitive, and thus possibly due to the action of serum complement, while the anti-WNV and anti-HSV-1 activities were not heat labile and thus probably not complement mediated.