Stanislaw F. Wnuk

Immune stimulating properties of a novel polysaccharide from the medicinal plant Tinospora cordifolia

Abstract An α-d-glucan (RR1) composed of (1→4) linked back bone and (1→6) linked branches with a molecular mass of >550 kDa and exhibiting unique immune stimulating properties is isolated and characterized from the medicinal plant Tinospora cordifolia. This novel polysaccharide is noncytotoxic and nonproliferating to normal lymphocytes as well as tumor cell lines at 0–1000 μg/ml. It activated different subsets of the lymphocytes such as natural killer (NK) cells (331%), T cells (102%), and B cells (39%) at 100 μg/ml concentration. The significant activation of NK cells is associated with the dose-dependent killing of tumor cells by activated normal lymphocytes in a functional assay. Immune activation by RR1 in normal lymphocytes elicited the synthesis of interleukin (IL)-1β (1080 pg/ml), IL-6 (21,833 pg/ml), IL-12 p70 (50.19 pg/ml), IL-12 p40 (918.23 pg/ml), IL-18 (27.47 pg/ml), IFN- γ (90.16 pg/ml), tumor necrosis factor (TNF)-α (2225 pg/ml) and monocyte chemoattractant protein (MCP)-1 (2307 pg/ml) at 100 μg/ml concentration, while it did not induce the production of IL-2, IL-4, IL-10, interferon (IFN)-α and TNF-β. The cytokine profile clearly demonstrates the Th1 pathway of T helper cell differentiation essential for cell mediated immunity, with a self-regulatory mechanism for the control of its overproduction. RR1 also activated the complements in the alternate pathway, demonstrated by a stepwise increase in C3a des Arg components. Incidentally, RR1 stimulation did not produce any oxidative stress or inducible nitric oxide synthase (iNOS) in the lymphocytes or any significant increase in nitric oxide production. The water solubility, high molecular mass, activation of lymphocytes especially NK cells, complement activation, Th1 pathway-associated cytokine profile, together with a low level of nitric oxide synthesis and absence of oxidative stress confer important immunoprotective potential to this novel α-d-glucan.

Design and synthesis of S-adenosylhomocysteine hydrolase inhibitors as broad-spectrum antiviral agents

Publisher Summary S-Adenosyl-L-methionine (AdoMet) serves as a methyl donor for a variety of cellular enzymatic transmethylation reactions. In AdoMet-dependent transmethylation reactions, the methyl group from AdoMet is transferred to various acceptor molecules, such as proteins, nucleic acids, phospholipids, and small molecules by specific methyltransferases. S-Adenosyl-L-homocysteine (AdoHcy) is a product of all AdoMet-dependent transmethylation reactions. Mammalian AdoHcy hydrolase, which is a homotetramer, contains tightly bound NAD + . One of the physiological roles of AdoHcy hydrolase is to regulate AdoMet-dependent biological methylation reactions. AdoHcy is a potent competitive inhibitor of all AdoMet-dependent methyltransferase This chapter focuses on the rationale for selecting AdoHcy hydrolase as a target for the design of antiviral agents, the way selective inhibitors with improved antiviral effectiveness are designed, the way these inhibitors inactivate the enzyme, and the way this enzyme regulates crucial biological processes. The chapter describes recent relevant nucleoside chemistry that has arisen from laboratories involved in the design and synthesis of AdoHcy hydrolase inhibitors.

Probing the Catalytic Mechanism of S-Ribosylhomocysteinase (LuxS) with Catalytic Intermediates and Substrate Analogues

S-Ribosylhomocysteinase (LuxS) cleaves the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). The catalytic mechanism of LuxS comprises three distinct reaction steps. The first step involves carbonyl migration from the C1 carbon of ribose to C2 and the formation of a 2-ketone intermediate. The second step shifts the C=O group from the C2 to C3 position to produce a 3-ketone intermediate. In the final step, the 3-ketone intermediate undergoes a beta-elimination reaction resulting in the cleavage of the thioether bond. In this work, the 3-ketone intermediate was chemically synthesized and shown to be chemically and kinetically competent in the LuxS catalytic pathway. Substrate analogues halogenated at the C3 position of ribose were synthesized and reacted as time-dependent inhibitors of LuxS. The time dependence was caused by enzyme-catalyzed elimination of halide ions. Examination of the kinetics of halide release and decay of the 3-ketone intermediate catalyzed by wild-type and mutant LuxS enzymes revealed that Cys-84 is the general base responsible for proton abstraction in the three reaction steps, whereas Glu-57 likely facilitates substrate binding and proton transfer during catalysis.

Nucleic Acid Related Compounds. LXXXI. Efficient General Synthesis of Purine (Amino, Azido, and Triflate)-Sugar Nucleosides

Abstract Treatment of 3′,5′-O-(tetraisopropyldisiloxanyl)adenosine and its arabino epimer with trifluoromethanesulfonyl chloride/DMAP gave the 2′-triflates in high yields. Displacements (LiN3/DMF) and deprotection gave 2′-azido-2′-deoxyadenosine and its arabino epimer which were reduced with Bu3SnH/AIBN/DMAC/benzene (or Staudinger reduction) to give 2′-amino-2′-deoxyadenosine and its epimer. Oxidation of 2′,5′-bis-O-(tert-butyldimethylsilyl)adenosine, stereoselective reduction, triflation, azide displacement, deprotection, and reduction gave 3′-amino-3′-deoxyadenosine.

Inhibition of S-ribosylhomocysteinase (LuxS) by substrate analogues modified at the ribosyl C-3 position

S-ribosylhomocysteinase (LuxS) catalyzes the cleavage of the thioether bond of S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), which is the precursor of type 2 autoinducer for bacterial cell-cell communication. In this work, we have synthesized several SRH analogues modified at the ribose C3 position as potential inhibitors of LuxS. While removal or methylation of the C3-OH resulted in simple competitive inhibitors of moderate potency, inversion of the C3 stereochemistry or substitution of fluorine for C3-OH resulted in slow-binding inhibitors of improved potency. The most potent inhibitor showed a K(I)(*) value of 0.43 microM.

Inhibition of LuxS by S-Ribosylhomocysteine Analogues Containing a [4-Aza]Ribose Ring

LuxS (S-ribosylhomocysteinase) catalyzes the cleavage of the thioether linkage of S-ribosylhomocysteine (SRH) to produce homocysteine and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor to a small signaling molecule that mediates interspecies bacterial communication called autoinducer 2 (AI-2). Inhibitors of LuxS should interfere with bacterial interspecies communication and potentially provide a novel class of antibacterial agents. In this work, SRH analogues containing substitution of a nitrogen atom for the endocyclic oxygen as well as various deoxyriboses were synthesized and evaluated for LuxS inhibition. Two of the [4-aza]SRH analogues showed modest competitive inhibition (K(I) ∼40 μM), while most of the others were inactive. One compound that contains a hemiaminal moiety exhibited time-dependent inhibition, consistent with enzyme-catalyzed ring opening and conversion into a more potent species (K(I)(∗)=3.5 μM). The structure-activity relationship of the designed inhibitors highlights the importance of both the homocysteine and ribose moieties for high-affinity binding to LuxS active site.

Palladium-catalyzed direct arylation of 5-halouracils and 5-halouracil nucleosides with arenes and heteroarenes promoted by TBAF.

The 1-N-benzyl-5-iodo(or bromo)uracil undergoes Pd-catalyzed [Pd2(dba)3] direct arylation with benzene and other simple arenes in the presence of TBAF in DMF without the necessity of adding any ligands or additives to give 5-arylated uracil analogues. The TBAF-promoted coupling also occurs efficiently with electron rich heteroarenes at 100 °C (1 h) even with only small excess of heteroarenes. The protocol avoids usage of the arylboronic acid or stannane precursors for the synthesis of 5-(2-furyl, or 2-thienyl, or 2-pyrrolyl)uracil nucleosides, which are used as important RNA and DNA fluorescent probes. The fact that 1-N-benzyl-3-N-methyl-5-iodouracil did not undergo the TBAF-promoted couplings with arenes or heteroarenes suggests that the C4-alkoxide (enol form of uracil) facilitates coupling by participation in the intramolecular processes of hydrogen abstraction from arenes. TBAF-promoted arylation was extended into the other enolizable heterocyclic systems such as 3-bromo-2-pyridone. The π-excessive heteroarenes also coupled with 5-halouracils in the presence of Pd(OAc)2/Cs2CO3/PivOH combination in DMF (100 °C, 2 h) to yield 5-arylated uracils.

Nucleic Acid Related Compounds. 94. Remarkably High Stereoselective Reductions of 2′- and 3′-Ketonucleoside Derivatives To Give Arabino, Ribo, and Xylofuranosyl Nucleosides with Hydrogen Isotopes at C2′ and C3′

Abstract Oxidation of 2′,5′- and 3′,5′- O -( tert -butyldimethylsilyl)-protected ribonucleosides gave the corresponding 3′-keto and 2′-keto derivatives, whose complete oxidation was assayed by total release of the heterocyclic base upon treatment with tetrabutylammonium fluoride/THF. Treatment of the protected ketones with sodium triacetoxyborohydride (generated in situ from sodium borohydride and acetic acid) in acetic acid resulted in hydride delivery at the α face with high stereoselectivity. The xylo/ribo (∼49:1) and arabino/ribo (∼49:1) diastereomers, respectively, were obtained in good to high overall yields upon deprotection. Selective removal of the TBDMS group from O5′ (trifluoroacetic acid/water, 9:1, 0 °C) and treatment of these 5′-hydroxy-(3′- and 2′)-ketones with sodium triacetoxyborohydride effected remarkably selective delivery of hydride at the β face. Deprotection gave the ribo/xylo (∼99:1) and ribo/arabino (∼99:1) nucleosides in high yields. Comparable results were obtained with sodium borodeuteride in acetic acid to give the four 2′[ 2 H] and 3′[ 2 H] arabino, ribo, and xylo isotopomers with > 95% incorporation of deuterium. Development of efficient procedures and comparison with previous methods are discussed.

S-Ribosylhomocysteine analogues with the carbon-5 and sulfur atoms replaced by a vinyl or (fluoro)vinyl unit

Treatment of the protected ribose or xylose 5-aldehyde with sulfonyl-stabilized fluorophosphonate gave (fluoro)vinyl sulfones. Stannyldesulfonylation followed by iododestannylation afforded 5,6-dideoxy-6-fluoro-6-iodo-d-ribo or xylo-hex-5-enofuranoses. Coupling of the hexenofuranoses with alkylzinc bromides gave 10-carbon ribosyl- and xylosylhomocysteine analogues incorporating a fluoroalkene. The fluoroalkenyl and alkenyl analogues were evaluated for inhibition of Bacillus subtilis S-ribosylhomocysteinase (LuxS). One of the compounds, 3,5,6-trideoxy-6-fluoro-d-erythro-hex-5-enofuranose, acted as a competitive inhibitor of moderate potency (K(I)=96microM).

Synthesis and cytotoxicity of 9-(2-deoxy-2-alkyldithio-β-D-arabinofuranosyl)purine nucleosides which are stable precursors to potential mechanistic probes of ribonucleotide reductases

A series of 2[prime or minute]-thionucleosides, as potential inhibitors of ribonucleotide reductases, has been synthesized. Treatment of the 3[prime or minute],5[prime or minute]-O-TPDS-2[prime or minute]-O-(trifluoromethanesulfonyl)adenosine with potassium thioacetate gave the arabino epimer of 2[prime or minute]-S-acetyl-2[prime or minute]-thioadenosine which was deacetylated to give 9-(3,5-O-TPDS-2-thio-[small beta]-d-arabinofuranosyl)adenine in high yield. Treatment of the latter with diethyl azodicarboxylate-C(3)H(7)SH-THF gave 2[prime or minute]-propyl disulfide which was desilylated to give 9-(2-deoxy-2-propyldithio-[small beta]-d-arabinofuranosyl)adenine. Subsequent tosylation (O5[prime or minute]) and displacement of the tosylate with pyrophosphate afforded the 5[prime or minute]-O-diphosphate in a stable form as propyl mixed-disulfide, which upon treatment with dithiothreitol releases 9-(2-thio-[small beta]-d-arabinofuranosyl)adenine 5[prime or minute]-diphosphate. The arabino 2[prime or minute]-mercapto group might interact with the crucial thiyl radical at cysteine 439 leading to the inhibition of ribonucleotide reductases via formation of a Cys439-2[prime or minute]-mercapto disulfide bridge. The 2,6-diamino-, 2-amino-6-chloro- and 2-amino-6-methoxypurine ribosides were also converted to the corresponding 2[prime or minute]-deoxy-2[prime or minute]-propyldithio-[small beta]-d-arabinofuranosyl nucleosides, which might serve as convenient precursors to the arabino epimer of 2[prime or minute]-thioguanosine. Analogously, 2[prime or minute]-deoxy-2[prime or minute]-propyldithioadenosine was prepared from 9-([small beta]-d-arabinofuranosyl)adenine. The nucleoside disulfides show modest cytotoxicity in a panel of human tumor cell lines.