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.     

Novel TSAO derivatives modified at positions 3" and 4" of the spiro moiety.

We have explored the introduction of different functional groups at positions 3" and 4" of the spiro moiety of TSAO-T. Alkylation of this spiro moiety afforded mixtures of N and/or C-alkylated derivatives, while acylation occurs, exclusively, on the amino group. Position 3" has been selectively functionalized by halogenation followed by Stille-cross coupling reaction with organostannanes under a variety of experimental conditions.     

Synthesis and characterization of 5-alkoxycarbonyl-4-hydroxymethyl-5-alkyl-pyrroline N-oxide derivatives

The syntheses, analytical properties, and spin trapping behavior of four novel EMPO derivatives, namely 5-ethoxycarbonyl-4-hydroxymethyl-5-methyl-pyrroline N-oxide (EHMPO), 5-ethoxycarbonyl-5-ethyl-4-hydroxymethyl-pyrroline N-oxide (EEHPO), 4-hydroxymethyl-5-methyl-5-propoxycarbonyl-pyrroline N-oxide (HMPPO), and 4-hydroxymethyl-5-methyl-5-iso-propoxycarbonyl-pyrroline N-oxide (HMiPPO), towards different oxygen- and carbon-centered radicals are described.     

Inhibition of anthrax lethal factor: lability of hydroxamate as a chelating group

The metalloprotease activity of lethal factor (LF) from Bacillus anthracis (B. anthracis) is a main source of toxicity in the lethality of anthrax infection. Thus, the understanding of the enzymatic activity and inhibition of B. anthracis LF is of scientific and clinical interests. We have designed, synthesized, and studied a peptide inhibitor of LF, R9LF-1, with the structure NH₂–(d-Arg)₉–Val–Leu–Arg–CO–NHOH in which the C-terminal hydroxamic acid is commonly used in the inhibitors of metalloproteases to chelate the active-site zinc. This inhibitor was shown to be very stable in solution and effectively inhibited LF in kinetic assays. However, its protection on murine macrophages against lethal toxin’s lysis activity was relatively weak in longer assays. We further observed that the hydroxamic acid group in R9LF-1 was hydrolyzed by LF, and the hydrolytic product of this inhibitor is considerably weaker in inhibition of potency. To resist this unique hydrolytic activity of LF, we further designed a new inhibitor R9LF-2 which contained the same structure as R9LF-1 except replacing the hydroxamic acid group with N,O-dimethyl hydroxamic acid (DMHA), –N(CH₃)–O–CH₃. R9LF-2 was not hydrolyzed by LF in long-term incubation. It has a high inhibitory potency vs. LF with an inhibition constant of 6.4 nM had a better protection of macrophages against LF toxicity than R9LF-1. These results suggest that in the development of new LF inhibitors, the stability of the chelating group should be carefully examined and that DMHA is a potentially useful moiety to be used in new LF inhibitors.     

A long-range restriction map of the interleukin-4 and interleukin-5 linkage group on chromosome 5

The genes for two of the hematopoietic growth factors, interleukin-4 and interleukin-5, are located on a small segment of chromosome 5 (q23-31), which is frequently deleted in myeloid disorders. Using pulsed-field gel electrophoresis, we demonstrate physical linkage of these two genes and present a long-range restriction map of the locus. The two genes are closely linked (maximum separation, 310 kb) and appear to be separated by an HTF island. We were unable to physically link these genes to two other closely related hematopoietic growth factor genes, interleukin-3 and granulocyte/macrophage colony-stimulating factor, which also map to this region of the genome. The clustering of these and other growth-related genes suggests that a higher order of genetic organization exists in this region of the chromosome.     

Evolutionary stasis and lability in thermal physiology in a group of tropical lizards

Understanding how quickly physiological traits evolve is a topic of great interest, particularly in the context of how organisms can adapt in response to climate warming. Adjustment to novel thermal habitats may occur either through behavioural adjustments, physiological adaptation or both. Here, we test whether rates of evolution differ among physiological traits in the cybotoids, a clade of tropical Anolis lizards distributed in markedly different thermal environments on the Caribbean island of Hispaniola. We find that cold tolerance evolves considerably faster than heat tolerance, a difference that results because behavioural thermoregulation more effectively shields these organisms from selection on upper than lower temperature tolerances. Specifically, because lizards in very different environments behaviourally thermoregulate during the day to similar body temperatures, divergent selection on body temperature and heat tolerance is precluded, whereas night-time temperatures can only be partially buffered by behaviour, thereby exposing organisms to selection on cold tolerance. We discuss how exposure to selection on physiology influences divergence among tropical organisms and its implications for adaptive evolutionary response to climate warming.     

Unusual bond activation processes in the reaction of group 4 cyclopentadienyl alkyne complexes with azobenzene

The alkyne complexes [Cp′₂M(L)(η²-Me₃SiC₂SiMe₃)] (Cp′ = substituted or unsubstituted cyclopentadienyl; M = Ti, Zr, Hf; L = Py, THF) can serve as metallocene precursors by substitution of the alkyne molecule with other ligands. The reactions of the unsubstituted cyclopentadienyl complexes [Cp₂Zr(THF)(η²-Me₃SiC₂SiMe₃)] (1) and [Cp₂Ti(η²-Me₃SiC₂SiMe₃)] (2) with azobenzene were investigated. In the first case the diazene complex [Cp₂Zr(THF)(N₂Ph₂)] (3) was obtained by alkyne exchange. In the reaction of the titanium complex 2 a NN bond cleavage of azobenzene and a C-H activation of the cyclopentadienyl ligand were observed and the dinuclear imido bridged compound 4 was formed. This mixed valence complex is bridged additionally by a cyclopentadienyl ligand in a η¹:η⁵-coordination mode which is very unusual for titanium complexes. The molecular structures of both compounds were confirmed by X-ray crystallography and compared to former structural data shown in literature.     

Synthesis of 4/5-deoxy-4/5-nucleobase derivatives of 3-O-methyl-D-chiro-inositol as potential antiviral agents

Using D-pinitol (= 3-O-methyl-D-chiro-inositol) as starting material, a concise synthesis of 4/5-deoxy-4/5-nucleobase derivatives 11-19 has been achieved. The key intermediate 9 was obtained in good yield via an epoxidation from mono-methanesulfonate of D-pinitol. The process of opening of the epoxide ring in 9 by nucleobases appeared to be regioselective in presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). All the synthesized carbocyclic nucleosides were assayed against several viruses and tumors such as HIV-1, HSV-1, and HSV-2, and lung and bladder cancer. However, only compounds 14b, 14a, 16a, 16b, and 19 showed mild inhibitory effect against human lung cancer cell lines (PG) with IC50 values ranging from 50 to 100 microM, and the other compounds did not exhibit any significant antiviral activity or cytotoxicity even at concentrations up to 200 microM.     

Unusual sulfonolipids are characteristic of the Cytophaga-Flexibacter group

Capnocytophaga spp. contain a group of unusual sulfonolipids, called capnoids (W. Godchaux III and E. R. Leadbetter, J. Bacteriol. 144:592-602, 1980). One of these lipids, capnine, is 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid; the others are, apparently, N-acylated versions of capnine. The lipids were found, in amounts ranging from 2.5 to 16 mumol of capnoid sulfur per g of cells (wet weight), in two Cytophaga spp. and also in several closely related organisms: several Capnocytophaga spp., Sporocytophaga myxococcoides, two Flexibacter spp., and two Flavobacterium spp. With the exception of the flavobacteria, all of these bacteria have been shown to exhibit gliding motility. The two Flavobacterium spp. belong to a subset of that genus that shares many other characteristics with the cytophagas. Only the Capnocytophaga spp. contained large quantities of capnine as such; in all of the others, most (and possibly all) of the capnoids were present as N-acylcapnines. Capnoid-negative bacteria included some gliding organisms that may not be closely related to the cytophagas: two fruiting myxobacters, a gliding cyanobacterium (Plectonema sp.), Beggiatoa alba, Vitreoscilla stercoraria, Herpetosiphon aurantiacus, and Lysobacter enzymogenes. Nongliding bacteria representing nine genera were also tested, and all of these fell into the capnoid-negative group.     

Study on derivatives of 5-amino-4-acylamino-1H-pyrazole as inhibitors of furin

A series of 5-amino-1H-pyrazoles was synthesized and studied as inhibitors of furin. The most potent compound, 5-amino-4-acetylamino-3-(4-methylphenylamino)1H-pyrazole, was found to retard the activity of furin by mixed-type inhibition with K = 288 microM. These findings permit to plan new ways for chemical modifications of the 5-amino-1H-pyrazole structure and design more potent furin inhibitors of non-peptide nature.     

New lipophilic derivatives of AZT and d4T 5'-phosphonates

5'-Aminocarbonylphosphonyl and aminocarbonylmethylphosphonyl diesters of AZT and d4T were synthesized as potential anti-HIV agents.     

Calix[4]arene supported group 5 imido complexes

The reaction of imidoyl chlorides [V(NR)Cl₃] (R = Ph 1, Tol 2, tBu 3) and calix[4]arene methyl ether H₃Mecalix unexpectedly leads to the formation of the structurally characterized vanadium (IV) complex [VCl(Mecalix)] (4). Calix[4]arene methyl ether stabilized imido complexes of the type [V(NR)(Mecalix)] (R = Ph 7, Tol 8, tBu 9) were afforded from the reaction of [V(NR)Cl₃] (R = Ph 1, Tol 2, tBu 3) and the tris(lithium) or tris(sodium) salt of the calix[4]arene ether. The lithium salt [{Li₃(Mecalix)}₂] (5) is a dimer in the solid state, in which two monomeric trianions are bridged by lithium cations. Imido complexes [M(NR)(Mecalix)] (M = Nb: R = tBu, 12, R = Tol 13, R = Mes 14, R = Dipp 15; M = Ta: R = tBu 16, R = Tol 17) (Tol = 4-C₆H₄Me, Mes = 2,6-C₆H₃Me₂; Dipp = 2,6-C₆H₃iPr₂) have been prepared from structurally characterized [NbCl₂(Mecalix)] (10) and previously known [TaCl₂(Mecalix)] (11) via reaction with two equivalents of the appropriately metallated (Li, K) primary amine. The molecular structures of 13 and 15 confirm the mononuclear nature of these complexes.     

Synthesis and evaluation of 4,5-dihydro-5-methylisoxazolin-5-carboxamide derivatives as VLA-4 antagonists

A novel set of compounds containing a 4,5-dihydro-5-methylisoxazoline have been successfully designed as VLA-4 receptor antagonists. Compound (14p) had a high receptor binding affinity of 4 nM and also found to be metabolically stable in vitro.     

Conformational lability of poly(dG-m5dC):poly(dG-m5dC).

The remarkable conformational lability of poly(dG-m5dC):poly(dG-m5dC) is demonstrated by the observation of an acid-mediated conformational hysteresis. An acid-mediated Z conformation that exists in solutions containing low sodium concentrations that would normally favor the B conformation is described in this report. This Z conformation is reached by an acid-base titration of a B-poly(dG-m5dC):poly(dG-m5dC) solution which is not far from the B-Z transition midpoint. The resulting Z conformation is thermally very stable, with direct melting into single strands at approximately 100 degrees C. In contrast, the B form DNA, initially in solutions of the same ionic strength but without exposure to acidic pH, exhibits a biphasic melting profile, with conversion into the Z form (with high cooperativity) prior to an eventual denaturation into single strands at around 100 degrees C. Cooling experiments reveal that such biphasic transitions are quite reversible. The transition midpoint for the thermally poised B to Z transformation depends strongly on the NaCl concentration and varies with sample batch. The acid-mediated Z form binds ethidium more weakly than its B counterpart, and the ethidium induced Z to B conversion occurs in a step-wise (non-allosteric) fashion without the requirement of a threshold concentration. The acid-mediated as well as the thermally poised Z conformations are reversed by the addition of EDTA, suggesting the involvement of trace amounts of multivalent metal ions.     

Synthesis of 5-enamine-4-thiazolidinone derivatives with trypanocidal and anticancer activity

A series of novel 2-(5-aminomethylene-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropionic acid ethyl esters has been synthesized. Target compounds were evaluated for their trypanocidal activity towards Trypanosoma brucei brucei and Trypanosoma brucei gambiense. Several hit-compounds (8, 10, 12) inhibited growth of the parasites at sub-micromolar concentrations (IC₅₀ 0.027–1.936 µM) and showed significant selectivity indices (SI = 108–1396.2) being non-toxic towards the human primary fibroblasts. The screening of anticancer activity in vitro within NCI DTP protocol allowed to identify active 2-(5-{[5-(2,4-dichlorobenzyl)-thiazol-2-ylamino]-methylene}-4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropionic acid ethyl ester 14 that demonstrated inhibition against all 59 human tumor cell lines with the average GI₅₀ value of 2.57 μM. It was established that the activity type (antitrypanosomal or anticancer) as well as its level depends on the character of enamine fragment in the C5 position of thiazolidinone core.     

Genome organization of the plasmid of IncQ/P4 group and its vector derivatives

The genome organization and functioning of IncQ/P4 plasmids are reviewed. Based on these plasmids, cloning vectors have been constructed for broad host range of gram-negative bacteria. Together with one- and two-replicon vectors for cloning via insertion inactivation of markers, specialized plasmid vectors are described: cosmids, promoter-probe vectors, vectors for direct selection of recombinant molecules. Examples of using broad host range vectors for gene cloning and expression in non-enteric gram-negative bacteria are presented.