Biomedical subjects
Publications and source records attributed to Stanislaw F Wnuk.
A new reaction of the efficient difluorocarbene-generating reagent trimethylsilyl fluorosulfonyldifluoroacetate (TFDA) is reported in which molecules containing an N-alkylimidazole or benzimidazole structure undergo an unexpected one-pot conversion to N-difluoromethylthioureas. [reaction: see text].
Cross-metathesis of suitably protected 5'-deoxy-5'-methyleneadenosines with racemic and chiral N-Boc-protected six-carbon amino acids bearing a terminal double bond in the presence of the Hoveyda-Grubbs catalyst gave adenosylhomocysteine analogues with the C5'-C6' double bond. Bromination with pyridinium tribromide and dehydrobromination with DBU followed by standard deprotections yielded the 5'-(bromo)vinyl analogue. [structure: see text]
Ribonucleotide reductases (RNRs) supply the 2'-deoxyribonucleotide building blocks for DNA synthesis in mammalian cells and for herpes viruses. The viral-encoded RNRs have unique protein sequences that differ from mammalian enzyme primary structures. Selective inhibition of a viral RNR might provide an approach to new anti-herpes agents with minimal effects on the mammalian host RNRs. This review summarizes efforts to develop anti-herpes agents that selectively target viral-encoded RNRs.
Treatment of the 6-N-cyclopropyl-2',3'-di-O-isopropylideneadenosine 5'-aldehyde with sulfone-stabilized phosphonate or fluorophosphonate reagents followed by stannyldesulfonylations and subsequent iodo- or protiodestannylation gave 6-N-cyclopropyl-5'-deoxy-5'-(iodomethylene)adenosine 8b or its 5'-fluoromethylene analogue 11. Treatment of the 5'-aldehyde with hydroxylamine or dibromomethylene- or cyanomethylene-stabilized Wittig reagents and deprotections gave the oxime 4b, 5'-cyanomethylene 5b, and 5'-dibromomethylene 13b analogues. Dehydrobromination of 13b gave acetylenic compound 14b. From the tested 6-N-cyclopropyladenosine analogues modified at the 5' carbon, the 5'-iodomethylene 8b had the most potent activity against Trypanosoma brucei in vitro with an IC50 of 12 microg/mL. The IC50 value was 19 microg/mL for both the 5'-fluoromethylene 11 and the 5'-cyanomethylene 5b compounds. The (E)-5'-deoxy-5'-(iodomethylene)adenosine 2a, a known inhibitor of AdoHcy hydrolase not modified with a cyclopropyl ring at 6-amino group, also inhibited T. brucei with an IC50 of 9 microg/mL. In contrast to some other adenosine analogues modified at C5', the 6-N-cyclopropyladenosine analogues described here do not exhibit an inhibitory effect on AdoHcy hydrolase and displayed only marginal antiviral activity.
[reaction: see text] The 1-fluoro-1-haloalkenes undergo Pd-catalyzed Negishi cross-couplings with primary alkylzinc bromides to give multisubstituted fluoroalkenes. The alkylation was trans-selective giving pure Z-fluoroalkenes in most cases. The highest yields were obtained with Pd2(dba)3 and PdCl2(dppb) catalysts but the best stereochemical outcome was obtained with less reactive Pd(PPh3)4. The tertiary alkylzincs also produced desired fluoroalkenes in high yields. Coupling of beta,beta-dichlorostyrene with organozinc reagent resulted in the formation of monocoupled product.
Moffatt oxidation of 2',3'-O-isopropylidene-L-adenosine and treatment of the resulting crude 5'-aldehyde with hydroxylamine followed by deprotection gave L-adenosine 5'-carboxaldehyde oximes, whose enantiomers are known to be potent inhibitors of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. The L-adenosine and its 5'-aldehyde oxime derivatives were found to be inactive as inhibitors of AdoHcy hydrolase. Docking calculations showed that binding of L-adenosine to AdoHcy hydrolase is weaker (higher energy) and less specific (larger number of clusters) compared to D-Ado.
The oxidative treatment of vinyltris(trimethylsilyl)germanes with hydrogen peroxide (NaOH/H(2)O/THF) or tert-butyl peroxide (KH/THF) generates reactive germanol or germanoxane species that undergo Pd-catalyzed cross-couplings with aryl and alkenyl halides and aryl triflates in the presence of Pd(PPh(3))(4). Vinylgermanes having either a conjugated or isolated double bond serve as versatile transmetalation reagents. The E-germanes undergo coupling with retention of stereochemistry under aqueous and anhydrous conditions, while coupling of Z-germanes occurs with less stereoselectivity to produce a mixture of E/Z products.
An alpha-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 microg/ml. It activated different subsets of the lymphocytes such as natural killer (NK) cells (331%), T cells (102%), and B cells (39%) at 100 microg/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)-1beta (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- gamma (90.16 pg/ml), tumor necrosis factor (TNF)-alpha (2225 pg/ml) and monocyte chemoattractant protein (MCP)-1 (2307 pg/ml) at 100 microg/ml concentration, while it did not induce the production of IL-2, IL-4, IL-10, interferon (IFN)-alpha and TNF-beta. 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 alpha-D-glucan.
Sonogashira coupling of (E)-6'-iodohomovinyl nucleosides 1 with (trimethylsilyl)acetylene gave the conjugated 8'-(trimethylsilyl)enyne derivatives of the adenosine 2a and uridine 2b with expected E-stereochemistry. Desilylation of 2a,b with tetrabutylammonium fluoride followed by treatment with N-iodosuccinimide/AgNO(3) afforded 8'-iodoenynes 4a,b. Analogous coupling of (Z)-6'-iodohomovinyl nucleosides 7a,b produced (Z)-8'-(trimethylsilyl)enynes 8a,b, which were deprotected with aqueous trifluoroacetic acid to give the Z-enynes 9a,b. Stereoselective coupling of the adenosine 4'-acetylenic 11 and ethyl (Z)-3-bromoacrylate followed by deprotection gave the conjugated enyne system attached in the reverse orientation at C4' 13. Because of their diverse stereochemical attributes, deprotected enyne analogues 5a, 6a, 9a, and 13 derived from adenosine require a different vicinity for binding with S-adenosyl-l-homocysteine (AdoHcy) hydrolase and/or addition of enzyme-bound water across the conjugated enyne system. Enyne 5a and 8'-iodoenyne 6a produced time-dependent and concentration-dependent inhibition of AdoHcy hydrolase (K(i), 0.55 and 118.5 microM, respectively). No reduction in NAD(+) content of the enzyme and no iodide ion released were observed upon incubation of 6a with the enzyme, while incubation of 5a produced 30% reduction in the NAD(+) content of the enzyme. No specific antiviral activity was noted for 5a,b, 6a,b, 9a,b, and 13 against any of the viruses tested; the E-iodoenynes 6a and 6b inhibited HIV-1 virus (IC(50), 1.1 and 1.8 microM; selectivity index, 7 and 3, respectively). The E-enyne 5a showed activity against cytomegalovirus at a concentration (EC(50), 30 microM) that was 3- to 10-fold lower than the cytotoxic concentration.
[reaction: see text] Radical-mediated silyl- and germyldesulfonylations of various vinyl and (alpha-fluoro)vinyl sulfones with tris(trimethylsilyl)silane and germanium hydrides provide access to vinyl and (alpha-fluoro)vinyl silanes and germanes. Upon oxidative treatment with hydrogen peroxide in basic aqueous solution, the vinyl tris(trimethylsilyl)silanes and -germanes undergo Pd-catalyzed cross-couplings with aryl halides.
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.
Vinylogously extended deoxyeritadenine derivatives were synthesized as acyclic/ carbocyclic analogues of the 6'-halo(homovinyl)adenosines, which are known to be potent inhibitors of S-adenosyl-L-homocysteine hydrolase. Swern oxidation of 9-[3-(t-butyldimethylsilyloxy)-4-hydroxybutyl]adenine (4) followed by Wittig olefination and desilylation gave access to ethyl 6-(adenin-9-yl)-4-hydroxy-2(E)-hexenoate (7) and 5-(adenin-9-yl)-1,1-dibromo-1-penten-3-ol (9). No inhibition of AdoHcy Hydrolase was observed with 7 and 9.
Stereoselective pyramidalization of free radicals by a vicinal fluorine substituent, the beta-fluorine effect, was invoked to rationalize a 77:23 anti/syn ratio of 2-deuterio-1-fluorocyclopentanes obtained by radical reduction of trans-2-fluoro-1-bromocyclopentane with tributyltin deuteride (Dolbier, W. R., Jr.; Bartberger, M. D. J. Org. Chem. 1995, 60, 4984-4985). We have evaluated analogous reductions of the four possible stereoisomers of some adenine 2'(3')-fluoro-3'(2')-O-phenoxythiocarbonyl nucleoside derivatives. In all cases, the steric effect of adenine on the beta face directs deuterium transfer from the stannane to C2'(C3') on the alpha face of the furanose ring. However, the beta-fluorine effect enhances ratios of deuterium transfer anti to the vicinal fluorine substituent.
Moffatt oxidation of 2',3'-O-isopropylideneuridine (1a) and treatment of the crude 5'-aldehyde with formylmethylene-stabilized Wittig reagent gave the vinylogously extended 7'-aldehyde2a. Condensation of 2a with ethoxycarbonyl- or dibromomethylene phosphorane reagents gave the conjugated dienes 6a and 4a, respectively. Deacetonization gave diene ester 7a [5'(E),7'(E); with s-trans conformation] and dibromodiene 5a [5'(E)], respectively. Analogously, 2',3'-O-isopropylideneadenosine (1b) was Wittig-extended into the conjugated dibromodiene 5b [5'(E)] and dienoic ester 7b [5'(E),7'(E)]. Furthermore, palladium-catalyzed coupling of the vinyl 6'(E)-stannanes 14 with (E) and (Z) ethyl 3-iodoacrylate gave stereodefined access to dienoic esters 7 (E,E) and 16 (E,Z). Incubation of AdoHcy hydrolase with 100 microM of 5b resulted in partial inhibition of the enzyme without any apparent change in the enzyme's nicotinamide adenine dinucleoside (NAD(+)) content. In contrast, 7b and 16b produced time- and concentration-dependent inactivation of S-adenosyl-L-homocysteine (AdoHcy) hydrolase producing significant decreases in the enzyme's NAD(+) content. However, 7b and 16b upon incubation with AdoHcy hydrolase were not metabolized suggesting that these compounds are type I mechanism-based inhibitors. No specific antiviral activity was noted for 5a,b, 7a,b, and 16a,b against any of the viruses tested; dibromodiene 5b proved cytotoxic at a concentration > or =6.7 microM and cytostatic at > or =11 microM, while dienoic esters 16a,b showed activity against both varicella-zoster virus (at 10 microM, 16a) and cytomegalovirus (at 10 microM, 16a; 18 microM, 16b).
Treatment of the alpha carbanions generated from several alpha-(pyrimidin-2-ylsulfonyl)alkylphosphonates with Selectfluor gave high yields of the alpha-fluoro-alpha-(pyrimidin-2-ylsulfonyl)alkylphoshonates, which were desulfonylated [Bu(3)SnH/2,2'-azobisisobutyronitrile (AIBN)/benzene or toluene/Delta] to give alpha-fluoroalkylphosphonates. "Catalytic" tin hydride, generated from tributyltin chloride and excess polymethylhydrosiloxane in the presence of potassium fluoride, also effected removal of the pi-deficient alpha-(pyrimidin-2-ylsulfonyl) group from the phosphonate esters. Substitution of Bu(3)SnD for Bu(3)SnH gave access to alpha-deuterium-labeled phosphonates. Prolonged treatment of alpha-(pyridin-2-ylsulfonyl)alkylphosphonate with excess Bu(3)SnH/AIBN or catalytic tin hydride also effected desulfonylation but in moderate yields. This represents a mild new methodology for removal of the synthetically useful pi-deficient heterocyclic sulfone moiety and an alternative route for the preparation of alpha-fluorinated phosphonates. Desulfonylation is suggested to proceed via attack of tin radical at an oxygen (or sulfur) atom of the sulfonyl group to give a stabilized alpha-phosphonyl radical intermediate. The latter was found to undergo 5-exo-trig ring closure to give the corresponding 2-methylcyclopentylphosphonates. Treatment of diethyl 1-bromohex-6-enylphosphonate with Bu(3)SnH/AIBN produced an analogous mixture of ring-closure products. Treatment of [(2-bromo-5- methoxyphenyl)(fluoro)(pyrimidin-2-ylsulfonyl)]methylphosphonate with Bu(3)SnH resulted in an intramolecular radical [1,5]-ipso substitution reaction and migration of the pyrimidinyl ring to give fluoro[5-methoxy-2-(pyrimidin-2-yl)phenyl]methylphosphonate.
Thermolysis of a 2'-[(16)O]-O-benzoyl-[(17)O]-5'-O-(tert-butyldimethylsilyl)-O(2),3'-cyclouridine derivative gave the more stable 3'-[(17)O]-O-benzoyl-[(16)O]- 5'-O-(tert-butyldimethylsilyl)-O(2),2'-cyclouridine isomer, which was converted into 3'-[(17)O]-2'-azido-2'-deoxyuridine by deprotection and nucleophilic ring opening at C2' with lithium azide. The 5'-diphosphate was prepared by nucleophilic displacement of the 5'-O-tosyl group with tris(tetrabutylammonium) hydrogen pyrophosphate. Model reactions gave (16)O and (18)O isotopomers, and base-promoted hydrolysis of an O(2),2'-cyclonucleoside gave stereodefined access to 3'-[(18)O]-1-(beta-D-arabinofuranosyl)uracil. Inactivation of ribonucleoside diphosphate reductase with 2'-azido-2'-deoxynucleotides results in appearance of EPR signals for a nitrogen-centered radical derived from azide, and 3'-[(17)O]-2'-azido-2'-deoxyuridine 5'-diphosphate provides an isotopomer to perturb EPR spectra in a predictable manner.
In vitro selection is a powerful approach for generating novel aptamers and catalysts. Currently, several methods are being developed to extend this technique to proteins. In principle, selection methods could be applied to any library whose members can be replicated. Here, we describe a bifunctional tRNA that fuses translation products to their mRNAs. The utility of peptide-tRNA-mRNA fusions for in vitro selection was illustrated by the selective enrichment of tagged peptides-together with their mRNAs-by affinity chromatography. Our system can generate libraries larger than 10(11). Because library members can be copied and amplified, they provide a means for applying in vitro selection procedures to peptides and proteins. Furthermore, because the system is amenable to translation with misacylated tRNAs, a wide range of unusual monomers could be used to make libraries of nonstandard polymers for selection experiments.
Conjugated diene 5-7 and enyne 8 analogs derived from adenosine and uridine were synthesized employing Pd-catalyzed cross-coupling reactions.