We disclose a study on nucleoside triphosphate (NTP) analogues in which the γ-phosphate is covalently modified by two different biodegradable masking units and d4T as nucleoside analogue that enable the delivery of d4TTP with high selectivity in phosphate buffer (pH 7.3) and by enzyme-triggered reactions in human CD4 + Tlymphocyte CEM cell extracts. This allows the bypass of all steps normally needed in the intracellular phosphorylation. These TriPPPronucleotides comprising an acyloxybenzyl (AB; ester) or an alkoxycarbonyloxybenzyl (ACB; carbonate) in combination with an ACB moiety are described as NTP delivery systems. The introduction of these two different groups led to the selective formation of γ-(ACB)-d4TTPs by chemical hydrolysis and in particular by cell extract enzymes. γ-(AB)-d4TTPs are faster cleaved than γ-(ACB)-d4TTPs. In antiviral assays, the compounds are highly active against HIV-1 and HIV-2 in wild-type CEM/O cells and more importantly in thymidine kinase-deficient CD4 + T-cells (CEM/TK − ).

We elaborate on a novel strategy for enhancing the binding affinity of an active-site directed inhibitor by attaching a tether group, designed to interact with the surface-exposed histidine residue(s) of enzymes. In this approach, we have utilized the recombinant form of human carbonic anhydrase-II (hCA-II) as the enzyme source and benzenesulfonamide and its derivatives as inhibitors. The steady-state kinetic and the ligand binding data revealed that the attachment of iminodiacetate (IDA)-Cu(2+) to benzenesulfonamide (via a triethylene glycol spacer) enhanced its binding affinity for hCA-II by about 40-fold. No energetic contribution of either IDA or triethylene glycol spacer was found (at least in the ground state of the enzyme-inhibitor complex) when Cu(2+) was stripped off from the tether group-conjugated sulfonamide derivative. Arguments are presented that the overall strategy of enhancing the binding affinities of known inhibitors by attaching the IDA-Cu(2+) groups to interact with the surface-exposed histidine residues will find a general application in designing the isozyme-specific inhibitors as potential drugs.

A new method for intramolecular oligosaccharide synthesis that is conceptually related to the general molecular clamp approach is introduced. Exceptional α-selectivity has been achieved in a majority of applications. Unlike other related concepts, this approach is based on the bisphenol A template, which allows one to connect multiple building blocks to perform templated oligosaccharide synthesis with complete stereoselectivity. This principle was demonstrated by the synthesis of an α,α-linked trisaccharide.

The enzyme inhibitors are usually designed by taking into consideration the overall dimensions of the enzyme's active site pockets. This conventional approach often fails to produce desirable affinities of inhibitors for their cognate enzymes. To circumvent such constraints, we contemplated enhancing the binding affinities of inhibitors by attaching tether groups, which would interact with the surface exposed amino acid residues. This strategy has been tested for the inhibition of human carbonic anhydrase II. Benzenesulfonamide serves as a weak inhibitor for the enzyme, but when it is conjugated to iminodiacetate-Cu2+ (which interacts with the surface-exposed His residues) via a spacer group, its binding affinity is enhanced by about 2 orders of magnitude. This "two-prong" approach is expected to serve as a general strategy for converting weak inhibitors of enzymes into tight-binding inhibitors.

Nucleoside reverse transcriptase inhibitors (NRTIs) are widely used as antiviral and anticancer agents, although they require intracellular phosphorylation into their antivirally active form, the triphosphorylated nucleoside analogue metabolites. We report on the synthesis and characterization of a new class of nucleoside triphosphate analogues comprising a C-alkyl-phosphonate moiety replacing the γ-phosphate. These compounds were converted into bioreversibly modified lipophilic prodrugs at the γ-phosphonate by the attachment of an acyloxybenzyl (ester) or an alkoxycarbonyloxybenzyl (carbonate) group. Such compounds formed γ-C-(alkyl)-nucleoside triphosphate analogues with high selectivity because of an enzyme-triggered delivery mechanism. The latter compounds were very stable in CD4+ T-lymphocyte (CEM cell) extracts, and they were substrates for HIV-reverse transcriptase without being substrates for DNA-polymerases α, β, and γ. In antiviral assays, the excellent antiviral activity of the prodrugs that was found in CEM/0 cells was completely kept in CEM/TK– cells. The activity was improved by 3 logs as compared to the parent nucleoside d4T.

Benzenesulfonamide and iminodiacetate (IDA)-conjugated Cu(2+) independently interact at the active site and a peripheral site of carbonic anhydrases, respectively [Banerjee, A. L., Swanson, M., Roy, B. C., Jia, X., Haldar, M. K., Mallik, S., and Srivastava, D. K. (2004) J. Am. Chem. Soc. 126, 10875-10883]. By attaching IDA-bound Cu(2+) to benzenesulfonamide via different chain length spacers, we synthesized two "two-prong" ligands, L1 and L2, in which the distances between Cu(2+) and NH(2) group of sulfonamide were 29 and 22 A, respectively. We compared the binding affinities of L1 and L2, vis-a-vis their parent compound, benzenesulfonamide, for recombinant human carbonic anhydrase I (hCA-I) by performing the fluorescence titration and steady-state kinetic experiments. The experimental data revealed that whereas the binding affinity of L1 for hCA-I was similar to that of benzenesulfonamide, the binding affinity of L2 was approximately 2 orders of magnitude higher, making L2 one of the most potent ligands or inhibitors of hCA-I. Since the enhanced binding or inhibitory potency of L2 is diminished (to the level of benzenesulfonamide) either in the presence of EDTA or upon treatment of the enzyme with diethyl pyrocarbonate, it is proposed that Cu(2+) of L2 interacts with one of the surface-exposed histidine residues of the enzyme. A cumulative account of the experimental data leads to the suggestion that the differential binding of L1 versus L2 to hCA-I is encoded in the chain length of the spacer moiety.

The antiviral efficacy of many nucleoside analogues is strongly dependent on their intracellular activation by host cellular kinases to yield ultimately the bioactive nucleoside analogue triphosphates (NTP). The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. We developed a nucleoside triphosphate (NTP) delivery system (the TriPPPro approach), in which the γ-phosphate is covalently modified by two different biodegradable masking units, one is the acyloxybenzyl (AB) moiety and the other is the alkoxycarbonyloxybenzyl (ACB) group. Such compounds formed NTPs with high selectivity by an enzyme-triggered mechanism in human T-lymphocyte CEM cell extracts loosing first the AB moiety, followed by the ACB group. This enables the bypass of all steps of the intracellular phosphorylation. This approach was applied here to convert some modestly active or even inactive nucleoside analogues into powerful biologically active metabolites. Potent antiviral activity profiles were obtained depending on the lipophilicity of the TriPPPro-NTP prodrugs against HIV-1 and HIV-2 replication in cultures of infected wild-type CD4+ CEM T-cells and more importantly in thymidine kinase-deficient CD4+ T-cells (CEM/TK–). This TriPPPro strategy offers high potential for future antiviral and antitumoral chemotherapies.

Remote 6-O-picolinyl or 6-O-picoloyl substituents often provide high β-selectivity due to H-bond-mediated aglycone delivery (HAD). Herein it has been demonstrated that if the nitrogen atom of the 6-O-picolinyl or picoloyl moiety is temporarily blocked by coordination to a metal center (Pd), it cannot engage in HAD-mediated β-glycosylation. Hence, the stereoselectivity of 6-O-picolinyl/picoloyl-assisted glycosylations can be "switched" to α-selectivity.