1,3-Diketone-Modified Nucleotides and DNA for Cross-Linking with Arginine-Containing Peptides and Proteins.

Linear or branched 1,3-diketone-linked thymidine 5'-O-mono- and triphosphate were synthesized through CuAAC click reaction of diketone-alkynes with 5-azidomethyl-dUMP or -dUTP. The triphosphates were good substrates for KOD XL DNA polymerase in primer extension synthesis of modified DNA. The nucleotide bearing linear 3,5-dioxohexyl group (HDO) efficiently reacted with arginine-containing peptides to form stable pyrimidine-linked conjugates, whereas the branched 2-acetyl-3-oxo-butyl (PDO) group was not reactive. Reaction with Lys or a terminal amino group formed enamine adducts that were prone to hydrolysis. This reactive HDO modification in DNA was used for bioconjugations and cross-linking with Arg-containing peptides or proteins (e.g. histones).

Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues.

The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.

Borononucleotides as substrates/binders for human NMP kinases: enzymatic and spectroscopic evaluation.

Borononucleotides are a family of natural nucleotide monophosphate analogues with a 5'-boronic acid function. As B-O-P linkages are known to be unstable in solution, we evaluated the ability of borononucleotides to be recognized by nucleoside monophosphate kinases and eventually foil the phosphorylation process. In this context, and with the idea of probing the influence of their size, shape, and flexibility, a library of borononucleotides were synthetized starting from the borononucleotide analogue of thymidine, which was shown to behave as a slow substrate of human TMP kinase. This study thus constitutes a good starting point for the development of new monophosphate mimics as potential substrates or ligands for NMP kinases.

Doubly loaded cycloSaligenyl-pronucleotides - 5,5'-Bis-(cycloSaligenyl-2',3'-dideoxy-2',3'-didehydrothymidine monophosphates).

Recently, we reported on 3,3'-bis-(cycloSaligenyl-2',3'-dideoxy-2',3'-didehydrothymidine monophosphates) (3,3'-bis-(cycloSal-d4TMPs) 4) as the first pronucleotides with a mask-to-drug ratio of 1:2 that is still a novelty in the field of pronucleotides. Here, we report on a new set of compounds of these unique type of cycloSaligenyl prodrugs 5 that bear a biaryl axis at the 5-position of the cycloSal residue. All compounds 5 showed pronounced in vitro activity against HIV-1 and HIV-2 in wild-type CEM cell cultures and better retained their antiviral activities in thymidine kinase-deficient CEM cells than the compound 4 series. Moreover, compound 5b is the first bis-(cycloSal-d4TMP) that even showed complete retention of antiviral activity in TK-deficient CEM cells. The complex hydrolysis behavior of 5 was investigated, and the proposed hydrolysis mechanism was proven by means of (31)P NMR spectroscopy and HPLC analysis.

Synthesis and properties of (alpha-P-borano)-nucleoside 5'-triphosphate analogues as potential antiviral agents.

The alpha-P-borano modification, where one of the alpha-phosphate oxygens is replaced by borane, of chain terminating nucleoside triphosphates are currently being tested in cell culture and are showing promise as effective viral polymerase inhibitors. The goal of this project is to combine the alpha-P-borano and Nanogel drug delivery technology to increase the antiviral potency of chain terminating sugar and base modified purine nucleosides versus the Hepatitis C Viral RNA dependent RNA polymerase (HCV RdRp). Here we show the synthesis of Cordycepin and 2'-O-methyl alpha-P-borano triphosphate via a one-pot phosphorochloridite synthesis under mild conditions. These analogues will be used for future structure-activity relationship (SAR) studies.

Bis-cycloSal-d4T-monophosphates: drugs that deliver two molecules of bioactive nucleotides.

Bis-cycloSal-d4T-monophosphates have been synthesized as potentially anti-HIV active "dimeric" prodrugs of 2',3'-dideoxy-2',3'-didehydrothymidine monophosphate (d4TMP). These pronucleotides display a mask-drug ratio of 1:2, a novelty in the field of pronucleotides. Both bis-cycloSal-d4TMP 6 and bis-5-methyl-cycloSal-d4TMP 7 showed increased hydrolytic stability as compared to their "monomeric" counterparts and a completely selective hydrolytic release of d4TMP. The hydrolysis pathway was investigated via 31P NMR spectroscopy. Moreover, due to the steric bulkiness, compound 6 already displayed strongly reduced inhibitor potency toward human butyrylcholinesterase (BChE), while compound 7 turned out to be devoid of any inhibitory activity against BChE. Partial separation of the diastereomeric mixture of 6 revealed strong dependence of the pronucleotides' properties on the stereochemistry at the phosphorus centers. Both 6 and 7 showed good activity against HIV-1 and HIV-2 in wild-type CEM cells in vitro. These compounds were significantly more potent than the parent nucleoside d4T 1 in HIV-2-infected TK-deficient CEM cells, indicating an efficient TK-bypass.

AknK is an L-2-deoxyfucosyltransferase in the biosynthesis of the anthracycline aclacinomycin A.

The antitumor drug aclacinomycin A is a representative member of the anthracycline subgroup that contains a C(7)-O-trisaccharide chain composed of L-2-deoxysugars. The sugar portion of the molecule, which greatly affects its biological activity, is assembled by dedicated glycosyltransferases; however, these enzymes have not been well-studied. Here we report the heterologous expression and purification of one of these enzymes, AknK, as well as the preparation of dTDP-L-2-deoxysugar donors, dTDP-L-2-deoxyfucose and dTDP-L-daunosamine, and the monoglycosyl aglycone, rhodosaminyl aklavinone. Our experiments show that AknK catalyzes the addition of the second sugar to the chain, using dTDP-L-2-deoxyfucose and rhodosaminyl aklavinone, to create the L-2-deoxyfucosyl-L-rhodosaminyl aklavinone. AknK also accepts an alternate dTDP-L-sugar, dTDP-L-daunosamine, and other monoglycosylated anthracyclines, including daunomycin, adriamycin, and idarubicin, to build alternate disaccharides on variant anthracycline backbones. Remarkably, AknK also catalyzes a tandem addition of a second L-2-deoxyfucosyl moiety, albeit with reduced activity, to the natural disaccharide chain to produce L-deoxyfucosyl-L-deoxyfucosyl-L-rhodosaminyl aklavinone, a variant of the natural aclacinomycin A. These results demonstrate that AknK may be a useful enzyme for the chemoenzymatic synthesis of anthracycline variants.

Synthesis and properties of novel triphosphate analogues: ribonucleoside and deoxyribonucleoside (alpha-P-borano, alpha-P-thio)triphosphates.

The first ribo- and deoxyribo-nucleoside (alpha-P-borano, alpha-P-thio)triphosphates have been synthesized. The chemical and biochemical properties of adenosine (alpha-P-borano, alpha-P-thio)triphosphate and thymidine (alpha-P-borano, alpha-P-thio) triphosphate have been investigated.

Synthesis and targeted delivery of an azidothymidine homodinucleotide conferring protection to macrophages against retroviral infection.

The infectivity and replication of human (HIV-1), feline (FIV), and murine (LP-BM5) immunodeficiency viruses are all inhibited by several nucleoside analogues after intracellular conversion to their triphosphorylated derivatives. At the cellular level, the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and the cytotoxic side effects of nucleoside analogue triphosphates. To overcome these limitations a new nucleoside analogue homodinucleotide, di(thymidine-3'-azido-2',3'-dideoxy-D-riboside)-5'-5'-p1-p2-pyrophosphat e (AZTp2AZT), was designed and synthesized. AZTp2AZT was a poor in vitro inhibitor of HIV reverse transcriptase, although it showed antiviral and cytotoxic activities comparable to those of the parent AZT when added to cultures of a HTLV-1 transformed cell line. AZTp2AZT encapsulated into erythrocytes was remarkably stable. Induction of erythrocyte-membrane protein clusterization and subsequent phagocytosis of AZTp2AZT-loaded cells allowed the targeted delivery of this impermeant drug to macrophages where its metabolic activation occurs. The addition of AZTp2AZT-loaded erythrocytes to human, feline, and murine macrophages afforded almost complete in vitro protection of these cells from infection by HIVBa-L, FIV, and LP-BM5, respectively. Therefore, AZTp2AZT, unlike the membrane-diffusing azidothymidine, acts as a very efficient antiretroviral prodrug following selective targeting to macrophages by means of loaded erythrocytes.

Synthesis and characterization of oligomeric nido-carboranyl phosphate diester conjugates to antibody and antibody fragments for potential use in boron neutron capture therapy of solid tumors.

Antibodies conjugated to oligomeric carboranyl compounds have a high potential as target species for boron neutron capture therapy (BNCT) of solid tumors. As a first step toward developing conjugates with BNCT capabilities, an oligomeric nido-carboranyl phosphate diester (Kane, R. R., Dreschel, K., and Hawthorne, M.F. (1993) J. Am. Chem. Soc. 115, 8853-8854), CB10 (10 nido-carboranes containing 90 boron atoms) with a pseudo-5'-terminal amino group, was conjugated to the anticarcinoembryonic antigen antibody T84.66 and its F(ab') fragment. The homobifunctional linker disuccinimidyl suberate (DSS) was coupled to CB10 via its 5'-terminal amino group followed by removal of excess linker with organic solvent extraction and conjugation with intact antibody. Similarly, the heterobifunctional linker, m-maleimidobenzoyl-N-hydroxysuccinimide (MBS), was coupled to CB10 and conjugated to the hinge region sulfhydryl of the F(ab') fragment of T84.66. The extent of reaction was monitored by the mobility shift of CB10-antibody conjugate on native polyacrylamide gels and the increased susceptibility of the CB10-antibody conjugate to staining with silver nitrate. CB10 was also labeled with radioiodine (131I) in a solid phase reaction with iodogen and used in double-label studies with 125I-labeled antibody. Although free CB10 bound very tightly to gel filtration media such as Sephadex G-25, the CB10-antibody conjugate passed through freely. After separation of CB10-antibody conjugate from free CB10 on Sephadex G-25, molar incorporations of CB10 were calculated. At a molar ratio of 10:1 (CB10:T84.66), greater than 90% of T84.66 and 30% of its F(ab)' fragment were conjugated to CB10.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycerol.

Phospholipid conjugates of antiretroviral nucleoside analogs have been proposed to have several advantageous features when compared to the parent drugs (Hostetler, K.Y. et al. (1990) J. Biol. Chem. 265, 6112-6117). Here we report on the synthesis of one such type of lipid conjugates, i.e., nucleosides diphosphate diacylglycerols. The syntheses of 3'-azido-3'-deoxythymidine diphosphate diacylglycerol, 3'-deoxythymidine diphosphate diacylglycerol and 2',3'-dideoxycytidine diphosphate diacylglycerol (with different acyl chains) were performed starting from phosphatidic acid and the antiviral nucleoside. A high-performance liquid chromatography procedure for a single step purification of the compounds is presented. The compounds were characterized biochemically, using rat liver enzymes and chemically by phosphorus, fatty acid, ultraviolet, IR and 1H-NMR analyses. Preliminary data on the behaviour in aqueous solution of some of the compounds are presented.

[Formation of phosphonoester bonds, catalyzed by DNA polymerases]. / Obrazovanie fosfonoéfirnykh sviazei, kataliziruemoe DNK-polimerazami.

3'-Fluoro-2',3'-dideoxythymidine 5'-(alpha-methylphosphonyl)-beta, gamma-diphosphate (I) and 2'-deoxythymidine 5'-(alpha-methylphosphonyl)-beta,gamma-diphosphate (II) were synthesised. Reverse transcriptases of HIV and avian myeloblastosis virus, rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase and E. coli DNA polymerase I KF incorporated both compounds into the growing DNA chain, KF being the least effective. Compound I revealed termination substrate properties, but II was repeatedly incorporated into the DNA chain, for example, by HIV reverse transcriptase - up to 8 residues. Human placenta DNA polymerases alpha and epsilon incorporated neither I nor II into the DNA chain, although DNA synthesis, catalyzed by all the investigated enzymes, was inhibited in the presence of I or II and compound II was a more effective inhibitor then I. The DNA fragments containing alpha-phosphonomethyl groups were hydrolyzed by 3'----5' exonuclease of DNA polymerase I and not hydrolyzed by ExoIII from E. coli.

Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate.

The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.

Synthesis and anti-HIV evaluation of some phosphoramidate derivatives of AZT: studies on the effect of chain elongation on biological activity.

A series of phosphoramidate derivatives of the anti-HIV drug AZT has been prepared as membrane soluble pro-drugs of the bio-active nucleotide forms and evaluated in vitro against HIV-1. Terminal substituted alkyl amines have a pronounced anti-HIV effect: this effect declines upon increasing the length of the methylene spacer. The results are consistent with a mechanism of action involving intracellular cleavage of the phosphoramidate bond, and release of the nucleotide, or a derivative thereof. Full spectroscopic data are included on the products and their phosphorochloridate precursors.

Synthesis and biological evaluation of dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine and 2', 3'-dideoxycytidine.

The 5'----5' dinucleoside methylphosphonates of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxycytidine (DDC) were prepared and evaluated for their inhibitory properties against different viruses, including human immunodeficiency virus (HIV). The synthesis of the compounds was achieved by reaction of AZT or N4-(4-monomethoxytrityl)-2',3'-dideoxycytidine with in situ prepared methylphosphonic bis (triazolide), followed in the latter case by an acidic treatment. The two title compounds showed in vitro anti-HIV activity, that was 200- to 450-fold less pronounced that that shown by the corresponding monomeric nucleosides AZT and DDC. The decreased antiviral activity may be ascribed to nuclease resistance of the methylphosphonate linkage.

Comparison of polymerase insertion and extension kinetics of a series of O2-alkyldeoxythymidine triphosphates and O4-methyldeoxythymidine triphosphate.

The effect of alkyl group size on ability to act as deoxythymidine triphosphate (dTTP) has been studied for the carcinogen products O2-methyl-, O2-ethyl-, and O2-isopropyl-dTTP by using three types of nucleic acids as template and DNA polymerase I (Pol I) or Klenow fragment as the polymerizing enzymes. Apparent Km and relative Vmax values were determined in primer extension on M13 DNA at a single defined site, in poly[d(A-T)], and in nicked DNA. These data are the basis for calculation of the relative rate of insertion opposite A, relative to dTTP. The insertion rate for any O2-alkyl-dTTP is much higher than for a mismatch between unmodified dNTPs. Unexpectedly, O2-isopropyl-dTTP is more efficiently utilized than O2-methyl-dTTP or O2-ethyl-dTTP on each of the templates. O2-isopropyl-dTTP also substitutes for dTTP over extended times of DNA synthesis at a rate only slightly lower than that of dTTP. Parallel experiments using O4-methyl-dTTP under the same conditions show that it is incorporated opposite A more frequently than is O2-methyl-dTTP. Therefore, both the ring position and the size of the alkyl group influence polymerase recognition. Once formed, all O2-alkyl-T.A termini permit elongation, as does O4-methyl-T.A. In contrast to the relative difficulty of incorporating the O-alkyl-dTTPs, formation of the following normal base pair (C.G) occurs rapidly when dGTP is present. This indicates that a single O-alkyl-T.A pair does not confer significant structural distortion recognized by Pol I.

Inhibition of thymidine kinase by P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate.

Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.

Design of species- or isozyme-specific enzyme inhibitors. 1. Effect of thymidine substituents on affinity for the thymidine site of hamster cytoplasmic thymidine kinase.

5-(Ethylamino)- and 5-acetamido-2'-deoxyuridine 5'-triphosphates were synthesized; the extent and concentration dependence of their inhibitory action on the title enzyme resembled that of the feedback inhibitor TTP. This and other findings provide a tentative indication that bulk tolerance near C-5 of the thymine ring may be more extensive at the TTP site than at the thymidine site. Enzyme-inhibitor dissociation constants (Ki values) were determined for thymidine derivatives monosubstituted at various positions. Competitive inhibition with respect to thymidine (indicative of substituent tolerance in the enzyme-thymidine complex) was produced by 3-amylthymidine (Ki = 65 muM), trans-5-bromo-6-ethoxy-5,6-dihydrothymidine diastereoisomers (Ki = 180 and 310 muM), 5'-C-(acetamidomethyl)- and 5-C-(propionamidomethyl)thymidine epimers (Ki range 65--1100 muM), 3'-acetamido- and 3'-(ethylthio)-3'-deoxythymidines (Ki = 2.5 mM and 12 muM, respectively), and certain 5'-(alkylamino)- and 5'-(alkylthio)-5'-deoxythymidines (Ki range 180--1200 muM). Evidence indicates that bulk tolerance at some, if not most, of the above atoms of thymidine is found in the enzyme-thymidine complexes of human and other mammalian thymidine kinases; attachment of suitable substituents to such atoms could, in principle, lead to thymidine site directed isozyme-specific inhibitors of human cytoplasmic thymidine kinase, which is a candidate target in the design of antineoplastic drugs.

5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate. Synthesis, chemical properties, and effect on Escherichia coli thymidine kinase activity.

5-Iodo-5'-amino-2',5'-dideoxyuridine-5'-N'-triphosphate (AIdUTP), a phosphoramidate analog of 5-iodo-2',5'-dideoxyuridine 5'-triphosphate (IdUTP), was synthesized and some of its chemical and biological properties were investigated. Although AIdUTP is stable in alkaline solutions, below pH 8 it undergoes degradation by a novel phosphorylysis reaction which exhibits first order kinetics. Inclusion of magnesium ion in the reaction mixture decreased the rate of degradation. Protonation of a group on AIdUTP which has a pKa of 6.10, presumably the secondary ionized oxygen on the gamma phosphate, precedes phosphorylysis. The only detectable reaction products are the nucleoside, 5-iodo-5'-amino-2',5'-dideoxyuridine (AIdUrd), and trimetaphosphate. A mechanism for the acid catalyzed phosphorylysis of AIdUTP is proposed. AIdUTP, like TTP, converts Escherichia coli thymidine kinase into an inactive dimer with a sedimentation coefficient of 5.78 S. AIdUTP is, however, 60-fold more potent as an allosteric inhibitor than is TTP at pH 7.8. Although the inhibitory effect of TTP is markedly reduced at high pH, the activity of AIdUTP is lowered only slightly. The allosteric effects of AIdUTP also differ from those of IdUTP, which is an inhibitor at low pH but a strong activator above pH 7.4. 5-Iodo-2'-deoxycytidine 5'-triphosphate, a potent enzyme activator, cannot completely reverse the AIdUTP inhibition, even when present at a 150-fold molar excess.