Glycoamino Acid Analogues of the Thomsen-Friedenreich Tumor-Associated Carbohydrate Antigen: Synthesis and Evaluation of Novel Antiproliferative Factor Glycopeptides.

Glycoamino acid analogues of the Thomsen-Friedenreich antigen disaccharide, where the 4' and 4″ hydroxyl groups were substituted with fluorine or hydrogen, were synthesized and incorporated into the asialylated antiproliferative factor (as-APF), a biologically active form of APF, a glycopeptide found in the urine of patients with interstitial cystitis. Various strategies were employed to incorporate the fluorine atom at the 4-positions of either the galactose or N-acetylgalactosamine unit of the disaccharide antigen, based on stereochemistry and reactivity. These glycopeptides were evaluated in antiproliferative assays on both primary normal bladder epithelial cells and T24 bladder carcinoma cells. Unlike many previously published substitutions to APF, mono-4'-fluorination of the GalNAc residue did not affect the activity, whereas fluoro-derivatives of the galactose 4″-position or both 4' and 4″ hydroxyls showed a reduced potency relative to the monosubstituted GalNAc derivative. A fourth compound where the 4″ position of galactose was deoxygenated showed a lower potency than the parent and monosubstituted compounds. These results suggest that specific substitutions in the sugar moieties in the APF can be tolerated, and the glycomimetic design of APF analogues can include fluorine in the GalNAc sugar of the disaccharide.

Recognition of the Thomsen-Friedenreich pancarcinoma carbohydrate antigen by a lamprey variable lymphocyte receptor.

Variable lymphocyte receptors (VLRs) are leucine-rich repeat proteins that mediate adaptive immunity in jawless vertebrates. VLRs were recently shown to recognize glycans, such as the tumor-associated Thomsen-Friedenreich antigen (TFα; Galß1-3GalNAcα), with a selectivity rivaling or exceeding that of lectins and antibodies. To understand the basis for TFα recognition by one such VLR (VLRB.aGPA.23), we measured thermodynamic parameters for the binding interaction and determined the structure of the VLRB.aGPA.23-TFα complex to 2.2 Å resolution. In the structure, four tryptophan residues form a tight hydrophobic cage encasing the TFα disaccharide that completely excludes buried water molecules. This cage together with hydrogen bonding of sugar hydroxyls to polar side chains explains the exquisite selectivity of VLRB.aGPA.23. The topology of the glycan-binding site of VLRB.aGPA.23 differs markedly from those of lectins or antibodies, which typically consist of long, convex grooves for accommodating the oligosaccharide. Instead, the TFα disaccharide is sandwiched between a variable loop and the concave surface of the VLR formed by the ß-strands of the leucine-rich repeat modules. Longer oligosaccharides are predicted to extend perpendicularly across the ß-strands, requiring them to bend to match the concavity of the VLR solenoid.

A direct, efficient method for the preparation of siRNAs containing ribo-like North bicyclo[3.1.0]hexane pseudosugars.

An efficient method for the preparation of siRNAs modified with ribo-like North bicyclo[3.1.0]hexane pseudosugars is described. The combined use of 2'-O-(2-cyanoethoxymethyl) (CEM) and 2'-O-TBDMS protection was successfully employed for RNA synthesis with the added advantage that both groups were efficiently removed in a single step. The resulting North ribo-methanocarba-modified siRNAs are compatible with the intracellular RNAi machinery and can mediate specific degradation of target mRNA.

4'-C-methyl-2'-deoxyadenosine and 4'-C-ethyl-2'-deoxyadenosine inhibit HIV-1 replication.

It is important to develop new anti-HIV drugs that are effective against the existing drug-resistant mutants. Because the excision mechanism is an important pathway for resistance to nucleoside analogs, we are preparing analogs that retain a 3'-OH and can be extended after they are incorporated by the viral reverse transcriptase. We show that 4'-C-alkyl-deoxyadenosine (4'-C-alkyl-dA) compounds can be phosphorylated in cultured cells and can inhibit the replication of HIV-1 vectors: 4'-C-methyl- and 4'-C-ethyl-dA show both efficacy and selectivity against HIV-1. The compounds are also effective against viruses that replicate using reverse transcriptases (RTs) that carry nucleoside reverse transcriptase inhibitor resistance mutations, with the exception of the M184V mutant. Analysis of viral DNA synthesis in infected cells showed that viral DNA synthesis is blocked by the incorporation of either 4'-C-methyl- or 4'-C-ethyl-2'-deoxyadenosine. In vitro experiments with purified HIV-1 RT showed that 4'-C-methyl-2'-dATP can compete with dATP and that incorporation of the analog causes pausing in DNA synthesis. The 4'-C-ethyl compound also competes with dATP and shows a differential ability to block DNA synthesis on RNA and DNA templates. Experiments that measure the ability of the compounds to block DNA synthesis in infected cells suggest that this differential block to DNA synthesis also occurs in infected cells.

Using conformationally locked nucleosides to calibrate the anomeric effect: Implications for glycosyl bond stability.

Steric and electronic parameters such as the anomeric effect (AE) and gauche effect play significant roles in steering the North ⇆ South equilibrium of nucleosides in solution. Two isomeric oxa-bicyclo[3.1.0]hexane nucleosides that are conformationally locked in either the North or the South conformation of the pseudorotational cycle were designed to study the consequences of having the AE operational or not, independent of other parameters. The rigidity of the system allowed the orientation of the orbitals involved to be set in "fixed" relationships, either antiperiplanar where the AE is permanently "on", or gauche where the AE is impaired. The consequences of these two alternatives were subject to high-level calculations and measured experimentally by x-ray crystallography, hydrolytic stability of the glycosyl bond, and pKa values.

Synthesis and hybridization properties of modified oligodeoxynucleotides carrying non-natural bases.

The impact of the presence of nonnatural bases on the properties of oligodeoxynucleotides has been studied. First, oligodeoxynucleotides carrying 2'-deoxyzebularine were prepared, and the stability of duplexes carrying this analogue was determined by DNA melting experiments. Melting temperatures and thermodynamic data indicated the preference of 2'-deoxyzebularine for 2'-deoxyguanosine, which behaves as a 2'-deoxycytidine analogue, forming a less stable base pair due to the absence of the amino group at position 4. Moreover, the duplex-hairpin equilibrium of a self-complementary oligodeoxynucleotide carrying several natural and nonnatural bases including 2'-deoxyzebularine as a central mispair, was studied. Depending on the base present in the middle of the sequence, it is possible to affect the stability of the bimolecular duplex modulating the duplex-hairpin equilibrium. Magnesium ions were shown to stabilize preferentially the bimolecular duplex form. The results indicate the importance of the modifications and the role of cations in shifting the structural equilibrium.

Contrasting behavior of conformationally locked carbocyclic nucleosides of adenosine and cytidine as substrates for deaminases.

In addition to the already known differences between adenosine deaminase (ADA) and cytidine deaminase (CDA) in terms of their tertiary structure, the sphere of Zn(+2) coordination, and their reverse stereochemical preference, we present evidence that the enzymes also differ significantly in terms of the North/South conformational preferences for their substrates and the extent to which the lack of the O(4') oxygen affects the kinetics of the enzymatic deamination of carbocyclic substrates. The carbocyclic nucleoside substrates used in this study have either a flexible cyclopentane ring or a rigid bicyclo[3.1.0]hexane scaffold.

Activation of p16 gene silenced by DNA methylation in cancer cells by phosphoramidate derivatives of 2'-deoxyzebularine.

We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.

What are the consequences of freezing the anomeric effect in nucleosides?

The consequences of freezing the orientation of the oxygen's lone pair orbitals--which determines whether the anomeric effect is operative or not--were studied theoretically and experimentally in two oxobicyclo-[3.1.0]hexane nucleosides (1 and 2). The results showed significant differences in the properties of these molecules, which correlated with the magnitude of the n2 --> sigma * delocalization.

Comprehensive structural studies of 2',3'-difluorinated nucleosides: comparison of theory, solution, and solid state.

The conformations of three 2',3'-difluoro uridine nucleosides were studied by X-ray crystallography, NMR spectroscopy, and ab initio calculations in an attempt to define the roles that the two vicinal fluorine atoms play in the puckering preferences of the furanose ring. Two of the compounds examined contained fluorine atoms in either the arabino or xylo dispositions at C2' and C3' of a 2',3'-dideoxyuridine system. The third compound also incorporated fluorine atoms in the xylo configuration on the furanose ring but was substituted with a 6-azauracil base in place of uracil. A battery of NMR experiments in D 2O solution was used to identify conformational preferences primarily from coupling constant and NOE data. Both (1)H and (19)F NMR data were used to ascertain the preferred sugar pucker of the furanose ring through the use of the program PSEUROT. Compound-dependent parameters used in the PSEUROT calculations were newly derived from complete sets of conformations calculated from high-level ab initio methods. The solution and theoretical data were compared to the conformations of each molecule in the solid state. It was shown that both gauche and antiperiplanar effects may be operative to maintain a pseudodiaxial arrangement of the C2' and C3' vicinal fluorine atoms. These data, along with previously reported data by us and others concerning monofluorinated nucleoside conformations, were used to propose a model of how fluorine influences different aspects of nucleoside conformations.

2'-Deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine: a novel inhibitor of DNA methyltransferase that requires activation by human carboxylesterase 1.

2'-Deoxy-N4-[2-(4-nitrophenyl)ethoxycarbonyl]-5-azacytidine (NPEOC-DAC), decitabine with a modification of the N4 position of the azacitidine ring can be used to inhibit DNA methyltransferase. This modification protects the azacitidine ring and can be cleaved by carboxylesterase to release decitabine. NPEOC-DAC was 23-fold less potent at low doses (<10microM) than decitabine at inhibiting DNA methylation, and was also associated with a 3-day delay in its effect. However, at doses > or = 10microM NPEOC-DAC was more effective at inhibiting DNA methylation. Theses differences between decitabine and NPEOC-DAC are dependent on the cleavage of the carboxylester bond, and could be potentially exploited pharmacologically.

The nucleoside analogs 4'C-methyl thymidine and 4'C-ethyl thymidine block DNA synthesis by wild-type HIV-1 RT and excision proficient NRTI resistant RT variants.

HIV-1 can become resistant to nucleoside analogs by developing an enhanced ability to excise the analogs after they have been incorporated. Excision requires that the analog be located at the 3' terminus of the primer. We have describe nucleoside analogs that do not block DNA synthesis at the point of incorporation, but only after additional dNTPs have been added to the DNA. These nucleoside analogs are called "delayed chain terminators" and are relatively effective inhibitors of drug-resistant HIV-1 reverse transcriptases (RTs) that are excision proficient. However, the first delayed chain terminator that we characterized was poorly phosphorylated in cultured cells. We have examined other nucleoside analogs to determine whether these compounds also act as delayed chain terminators, but were more efficiently converted to the triphosphate form by cellular kinases. These analogs contain substitutions on the deoxyribose sugar ring at the 4' carbon (4'C-methyl dT and 4'C-ethyl dT). Unlike true delayed chain terminators, which terminate DNA synthesis in a spatial sense (DNA synthesis is halted only after additional dNTPs have been incorporated after the analog), 4'C-methyl dTTP causes a pause in DNA synthesis at the point of incorporation. However, HIV-1 RT can eventually extend the primer blocked by the 4' C-Me dTMP analog. 4'C-methyl dTTP blocks DNA synthesis in a temporal sense, rather than in a spatial sense. A primer blocked by 4'C-ethyl dTMP is not extended by HIV-1 RT, and this compound acts like a conventional chain terminator, despite the presence of a 3'-OH group. These compounds effectively block the replication of an HIV-1-based vector that replicates using wild-type HIV-1 RT, but only in the presence of herpes simplex virus thymidine kinase (HSV TK). These compounds are effective against many NRTI drug-resistant RT variants; however, the M184V mutant is relatively resistant.

The triphosphate of beta-D-4'-C-ethynyl-2',3'-dideoxycytidine is the preferred enantiomer substrate for HIV reverse transcriptase.

The enantioselective synthesis of the beta-d (1) enantiomer of 4'-C-ethynyl-2',3'-dideoxycytidine confirms an earlier stereochemical assignment that was strictly based on the ability of HIV reverse transcriptase and its M184V mutant to discriminate between the d- and l-configuration of nucleoside 5'-triphosphates.

A 4'-C-ethynyl-2',3'-dideoxynucleoside analogue highlights the role of the 3'-OH in anti-HIV active 4'-C-ethynyl-2'-deoxy nucleosides.

4'-C-ethynyl-2'-deoxynucleosides belong to a novel class of nucleoside analogues endowed with potent activity against a wide spectrum of HIV viruses, including a variety of resistant clones. Although favorable selectivity indices were reported for several of these analogues, some concern still exists regarding the 3'-OH group and its role in cellular toxicity. To address this problem, we removed the 3'-OH group from 4'-C-ethynyl-2'-deoxycytidine (1a). This compound was chosen because of its combined high potency and low selectivity index. The removal of the 3'-OH was not straightforward; it required a different synthetic approach from the one used to synthesize the parent compound. Starting with glycidyl-4-methoxyphenyl ether, the target 4'-C-ethynyl-2',3'-dideoxycytidine analogue (rac-1h) was obtained after 13 steps. In a cellular assay, rac-1h was completely inactive (0.001-10 microM) against HIV(LAI), demonstrating the critical importance of the 3'-OH for antiviral activity. To determine whether the role of the 3'-OH was essential for the phosphorylation of the compound by cellular kinases or for inhibition of DNA polymerization, we synthesized and tested the 5'-triphosphate (rac-1h-TP) for its ability to inhibit HIV reverse transcriptase (RT). rac-1h-TP was slightly more potent than AZT-5'-triphosphate against wild-type HIV RT, suggesting that the role of the 3'-OH is crucial only for the activation of the drug by cellular kinases. The lipase-catalyzed resolution of rac-1h into ent-1h (beta-D-dideoxyribo) and ent-14 (beta-L-dideoxyribo) and the synthesis of the corresponding 5'-triphosphates established the stereochemical assignment based on HIV RT's preference for the beta-D-enantiomer, which was confirmed by assaying against the M184V variant, an RT mutant with a marked preference for incorporating nucleosides in the D-configuration.

Conformationally constrained analogues of diacylglycerol. 24. Asymmetric synthesis of a chiral (R)-DAG-lactone template as a versatile precursor for highly functionalized DAG-lactones.

[structure: see text] Commercially available 2-methylenepropane-1,3-diol was converted to chiral epoxide (R)-2 via Sharpless asymmetric epoxidation in >96% ee. Regiospecific epoxide ring opening and reduction of the intermediate alkyne set the stage for a one-pot lactonization to give (R)-6, a convenient precursor for all functionalized chiral DAG-lactones used as potent PK-C ligands. The synthesis of the most potent DAG-lactones known to date, (Z)-10 and (E)-10, served to confirm PK-C's exclusive preference for the (R)-stereochemistry in this class of compounds.