Synthesis and properties of mRNA cap analogs containing imidodiphosphate moiety--fairly mimicking natural cap structure, yet resistant to enzymatic hydrolysis

We describe synthesis and properties of eight dinucleotide mRNA 5' cap analogs containing imidodiphosphate moiety within 5',5'-tri- or tetraphosphate bridge (NH-analogs). The compounds were obtained by coupling an appropriate nucleoside 5'-imidodiphosphate with nucleotide P-imidazolide mediated by divalent metal chloride in anhydrous DMF. To evaluate the novel compounds as tools for studying cap-dependent processes, we determined their binding affinities for eukaryotic translation initiation factor 4E, susceptibilities to decapping pyrophosphatase DcpS and, for non-hydrolysable analogs, binding affinities to this enzyme. The results indicate that the O to NH substitution in selected positions of oligophosphate bridge ensures resistance to enzymatic decapping and suggest that interactions of NH-analogs with cap binding proteins fairly mimic interactions of unmodified parent compounds. Finally, we identified NH-analogs as potent inhibitors of cap-dependent translation in cell free system, and evaluated their utility as reagents for obtaining 5' capped mRNAs in vitro to be rather moderate.     

Synthesis and biochemical properties of novel mRNA 5' cap analogs resistant to enzymatic hydrolysis

A series of new dinucleotide cap analogs with methylene groups replacing oxygens within the pyrophosphate moieties have been synthesized. All the compounds were resistant to the human scavenger decapping hydrolase, DcpS. Binding constants of the modified caps to eIF4E are comparable to those obtained for m7GpppG. This suggests these methylene modifications in the pyrophosphate chain do not significantly affect cap-binding at least for eIF4E. These cap analogs are also good inhibitors of in vitro translation. mRNAs capped with novel analogs were translated similarly to the mRNA capped with the parent m7GpppG.     

Interactions of 2'-fluoro-substituted dUMP analogues with thymidylate synthase

A series of 2'-fluoro-substituted dUMP/FdUMP analogues were synthesized, their interaction with human recombinant thymidylate synthase investigated, and structural (1)H and (19)F NMR study of the corresponding nucleosides performed. While 2'-F-dUMP (fluorine in the "down" configuration), in striking contrast to 2'-F-ara-UMP (fluorine in the "up" configuration) and 2',2'-diF-dUMP, showed substrate activity, 2'-F-ara-UMP and 2',2'-diF-dUMP were classic inhibitors, and 2',5-diF-ara-UMP behaved as a strong slow-binding inhibitor, suggesting the 2'-F substituent in the "up" position to interfere with the active center cysteine thiol addition to the pyrimidine C(6) and the pyrimidine C(5)-F to prevent this interference. In support, the direct through space heteronuclear coupling J(HF) was observed for the fluorine "up" derivatives, 2'-F-ara-U and 2',5-diF-ara-U, causing the splitting of the H(6) resonance lines. The absence of such splitting in 2',2'-diF-dUrd, indicating an unusual orientation of the base in relation to the furanose, was associated with an exceptionally weak interaction with the enzyme.     

Synthesis and anti-platelet evaluation of 2-benzoylaminobenzoate analogs

Fifty-two 2-benzoylaminobenzoate analogs were synthesized and subjected to anti-platelet aggregation assay using arachidonic acid (AA), collagen (Col), thrombin (Thr), and U46619 as inducers. The results revealed that most of 2-benzoylaminobenzoic acid derivatives showed a selectively inhibitory effect on AA-induced platelet aggregation. As a result of the 2-benzoylaminobenzoic acid derivatives (18, 44, and 46), there were no inhibitory effects on platelet aggregation induced by U46619, but these elicited an inhibitory effect on thromboxane B(2) formation at 1.0microM. These 2-benzoylaminobenzoate analogs were therefore proposed as cyclooxygenase inhibitors.     

Synthesis and properties of mRNA cap analogs containing phosphorothioate moiety in 5',5'-triphosphate chain

Nucleosides and oligonucleotides with an oxygen replaced by sulfur atom are an interesting class of compounds because of their improved stability toward enzymatic cleavage by nucleases. We have synthesized several dinucleotide mRNA cap analogs containing a phosphorothioate moiety in the alpha, beta, or gamma position of 5',5'-triphosphate chain [m7Gp(s)ppG, m7Gpp(s)pG, and m7Gppp(s)G]. These are the first examples of the biologically important 5'mRNA cap analogs containing a phosphorothioate moiety, and these compounds may be useful in a variety of biochemical and biotechnological applications. Incorporation of a sulfur atom in the alpha or gamma position within the dinucleotide cap analog was achieved using PSCl3 in a nucleoside phosphorylation reaction followed by coupling the phosphorothioate of nucleoside with a second nucleotide. Synthesis of cap analogs with the phosphorothioate moiety in beta position was performed using an organic phosphorothioate salt in a coupling reaction with an activated nucleotide. The structures of newly synthesized compounds was confirmed using MS and 1H and 31P NMR spectroscopy. We present here the results of preliminary studies on their interaction with translation initiation factor eIF4E and enzymatic hydrolysis with human and nematode DcpS scavengers.     

Synthesis and antimitotic activity of novel 2-methoxyestradiol analogs

The syntheses and antimitotic activity of several novel 2-methoxyestradiol analogs are described. Structural modifications investigated include introduction of additional unsaturation in rings B and D; inversion at C-13; and substitution at the C-2, C-15, C-16, and C-7 alpha positions. Of 15 analogs synthesized, 2 have demonstrated superior biological activities compared to 2-methoxyestradiol.     

Differential inhibition of mRNA degradation pathways by novel cap analogs

mRNA degradation predominantly proceeds through two alternative routes: the 5'-->3' pathway, which requires deadenylation followed by decapping and 5'-->3' hydrolysis; and the 3'-->5' pathway, which involves deadenylation followed by 3'-->5' hydrolysis and finally decapping. The mechanisms and relative contributions of each pathway are not fully understood. We investigated the effects of different cap structure (Gp(3)G, m(7)Gp(3)G, or m(2)(7,3'-O) Gp(3)G) and 3' termini (A(31),A(60), or G(16)) on both translation and mRNA degradation in mammalian cells. The results indicated that cap structures that bind eIF4E with higher affinity stabilize mRNA to degradation in vivo. mRNA stability depends on the ability of the 5' terminus to bind eIF4E, not merely the presence of a blocking group at the 5'-end. Introducing a stem-loop in the 5'-UTR that dramatically reduces translation, but keeping the cap structure the same, does not alter the rate of mRNA degradation. To test the relative contributions of the 5'-->3' versus 3'-->5' pathways, we designed and synthesized two new cap analogs, in which a methylene group was substituted between the alpha- and beta-phosphate moieties, m(2)(7,3'-O)Gpp(CH2)pG and m(2)(7,3'-O)Gp(CH2)ppG, that are predicted to be resistant to cleavage by Dcp1/Dcp2 and DcpS, respectively. These cap analogs were recognized by eIF4E and conferred cap-dependent translation to mRNA both in vitro and in vivo. Oligonucleotides capped with m(2)(7,3'-O)Gpp(CH2)pG were resistant to hydrolysis by recombinant human Dcp2 in vitro. mRNAs capped with m(2)(7,3'-O)Gpp(CH2)pG, but not m(2)(7,3'-O)Gp(CH2)ppG, were more stable in vivo, indicating that the 5'-->3' pathway makes a major contribution to overall degradation. Luciferase mRNA containing a 5'-terminal m(2)(7,3'-O)Gpp(CH2)pG and 3'-terminal poly(G) had the greatest stability of all mRNAs tested.     

Synthesis of GnRH analogs and their application in targeted gene delivery systems

A set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized using solid phase peptide synthesis and chemical ligation technique. Selective chemical ligation was achieved as a result of hydrazone formation in the course of interaction between NLS hydrazide and GnRH analog modified by pyruvic acid. The efficiency of synthesized peptide carriers was demonstrated in experiments with human cancer cells transfected by reporter luciferase and beta-galactosidase genes or suicide HSV-1 thymidine kinase gene. It was shown that selectivity of action on cancer cells can be achieved as a result of peptide/DNA complex penetration through the cell membrane by GnRH receptor-mediated endocytosis pathway.     

Synthesis and biological activity of N-methylated analogs of endomorphin-2

In this paper, we describe the synthesis of a series of endomorphin-2 analogs containing N-methylated amino acids, consecutively in each position. The μ-opioid receptor binding affinities of the new analogs were determined in the displacement experiments. Their in vivo antinociceptive activity was assessed in the hot-plate test in mice after central (icv) and peripheral (ip) administration. [Sar²]endomorphin-2, which had the highest μ-receptor affinity, also showed the strongest analgesic effect when administered centrally and was the only analog that retained activity after peripheral injection.     

Synthesis and antiviral activity of 2-substituted analogs of triciribine

Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and antineoplastic activity at low or submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore the effect of substitutions at the 2-position of triciribine. 2-Methyl- (2-Me-TCN), 2-ethyl- (2-Et-TCN), 2-phenyl- (2-Ph-TCN), 2-chloro- (2-Cl-TCN), and 2-aminotriciribine (2-NH2-TCN) were designed and synthesized to determine the effects of substitutions at the 2-position which change the steric, electronic, and hydrophobic properties of TCN, while maintaining the integrity of the tricyclic ring system. These compounds were evaluated for activity against human immunodeficiency virus (HIV-1), herpes simplex virus type 1 (HSV-1), and human cytomegalovirus (HCMV) and were found to be either less active than TCN and TCN-P or inactive at the highest concentrations tested, 100 microM. We conclude that substitutions at the 2-position of triciribine adversely affect the antiviral activity most likely because these analogs are not phosphorylated to active metabolites.     

Synthesis and antitumor activities of 3-modified 2-methoxyestradiol analogs

The syntheses of 2-methoxyestradiol analogs with modifications at the 3-position are described. The analogs were assessed for their antiproliferative, antiangiogenic, and estrogenic activities. Several lead substituents were identified with similar or improved antitumor activities and reduced metabolic liability compared to 2-methoxyestradiol.     

Synthesis of pyridazine and thiazole analogs as SGLT2 inhibitors

With anticipation of the improvement in biological aspects in our SGLT2 program, novel pyridazinyl and thiazolyl analogs were designed and efficiently synthesized. The installation of the pyridazine ring at the anomeric carbon of d-glucopyranose was carried out in a stereoselective fashion. On the other hand, a series of thiazolyl analogs was also synthesized through a coupling reaction between perbenzyl gluconolactone 9 and 2-lithiothiazole. Biological activities of the compounds thus prepared were evaluated by the in vitro SGLT2 inhibition assay. Considering assay results, the novel benzylpyridazinyl and benzylthiazolyl analogs, disclosed in this article, could be a quick reference to prospective SGLT2 inhibitors useful for pharmacotherapy.     

Synthesis of carbocyclic 2-substituted adenine nucleoside and related analogs

2-Iodonoraristeromycin, 2-iodoaristeromycin and related analogs were synthesized to investigate their inhibitory activities against human and Plasmodium falciparum S-adenosyl-L-homocysteine hydrolases.     

Synthesis of N2, N2, 7-trimethylguanosine cap derivatives.

Several derivatives of N2,N2-7-trimethylguanosine (m3(2,2,7G)-cap, which was found at the 5' ends of small nuclear RNAs, were synthesized by use of S-phenyl N2,N2,7-trimethylguanosine 5'-phosphorothioate (PhSpm3(2,2,7)G) as a key intermediate. This compound was activated by iodine in the presence of phosphoric acid and diphosphoric acid to give N2,N2,7-trimethylguanosine-5'-diphosphate (ppm3(2,2,7)G) and 5'-triphosphate (ppm3(2,2,7)G), respectively. Similar reactions of PhSpm3(2,2,7)G with ADP and GDP gave capped dinucleoside triphosphates, m3(2,2,7)G5'pppA and m3(2,2,7)G5'pppG, respectively.