Synthesis, cytostatic, and antiviral activity of novel 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, 6-[2-(alkylsulfanyl)ethyl]-, and 6-[2-(dialkylamino)vinyl]purine nucleosides

An efficient and facile synthesis of a large series of diverse 6-[2-(dialkylamino)vinyl]-, 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, and 6-[2-(alkylsulfanyl)ethyl]purine nucleosides (35 examples of both ribo- and 2'-deoxyribonucleosides) was developed. The key transformations involved conjugate nucleophilic additions of amines, alcoholates, or thiolates to Tol-protected 6-alkylylpurine or 6-vinylpurine nucleosides. 6-[(2-Dialkylamino)vinyl]- and some 6-[(2-dialkylamino)ethyl]purine ribonucleosides exerted significant cytostatic effects and some anti-HCV activity with low selectivity.     

Synthesis, cytostatic and anti-HCV activity of 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides

An efficient and facile synthesis of a large series of diverse 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides (55 examples of both ribo- and 2'-deoxyribonucleosides), aimed at identifying novel homologues of natural nucleosides, was developed. The key transformation involved nucleophilic substitutions of Tol-protected 6-(mesyloxymethyl)purine nucleosides by primary or secondary amines, alcoholates or thiolates. While the 2'-deoxyribonucleosides were inactive, the ribonucleosides exerted considerable cytostatic effects and some anti-HCV activity with low selectivity.     

Stereo- and biochemical profiles of the 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerenes

The 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerene were studied by means of circular dichroism (CD), deuterium labeling, 1H-NMR, molecular-dynamics (MD) calculations, and a lectin-binding assay. The CD spectra of the O-acetylated derivatives allowed clear discrimination of the isomers, while the 1H-NMR spectra, with assistance from deuterium labeling and MD calculations, served to disclose the unique conformation and molecular geometry of each acetylated isomer in chloroform solution. The deprotected 5-6- and 6-6-isomers, which gave colloidal suspensions in aqueous mixtures, displayed marked activity in blocking lectin-induced hemagglutination by concanavalin A.     

Differential inflammatory activation of IL-6 (-/-) astrocytes

IL-6 is a major immunomodulatory cytokine with neuroprotective activity. The absence of interleukin-6 (IL-6) results in increased vulnerability of dopaminergic neurons to the neurotoxicant, MPTP, and a compromised reactive microgliosis. To determine how astrogliosis may contribute to nigrostriatal degeneration in IL-6 (-/-) mice, the inflammatory profiles of astrocytes of IL-6 genotype were compared. Fourteen cytokines and four chemokines were simultaneously assayed in the supernatants of LPS-stimulated primary astrocyte cultures. In a time course of 6, 18 and 48 h and LPS stimulations of 0, 0.1, 1, 10 and 100 ng/ml, IL-6 (-/-) astrocytes secreted significantly greater amounts of the pro-inflammatory cytokines IL-1alpha, IL-1beta and TNFalpha than did IL-6 (+/+) cells. Elevated levels of IL-10 and IL-12p40 were only detected at 48 h post-stimulation with greater IL-10 in IL-6 (-/-) supernatants and greater IL-12p40 in IL-6 (+/+) supernatants. IL-6 (+/+) astrocytes produced more G-CSF and GM-CSF when compared with IL-6 (-/-) astrocytes. Chemokine levels were greater in supernatants of IL-6 (+/+) astrocytes than IL-6 (-/-) cells prior to 48 h post-stimulation. At that time, higher levels of MIP-1alpha were maintained in IL-6 (+/+) supernatant, while similar levels of MCP-1 in supernatants of both IL-6 (+/+) and IL-6 (-/-) cells were measured. Additionally, LPS (100 ng/ml) resulted in greater levels of KC and Rantes in IL-6 (-/-) astrocyte supernatants compared with IL-6 (+/+) supernatants at that time. These results suggest that the autocrine modulatory activities of IL-6 affect multiple cytokine secretory pathways, which could participate in neurodegenerative processes.     

Preparation of 5- and 6-(aminomethyl)fluorescein.

5(6)-Carboxyfluorescein is protected as the diacetate then reduced to 5(6)-(hydroxymethyl)fluorescein diacetate. The separated isomers are subjected to a Mitsunobu reaction with dibenzyl imidodicarbonate, yielding diprotected 5- and 6-(aminomethyl)fluorescein diacetate. Methanolysis of the acetates followed by deprotection with HBr/acetic acid gives 5- and 6-(aminomethyl)fluorescein hydrobromide.     

Isomerisation and conformation studies of (+)- and (-)6-hydroxy-5, 6-dihydrouridine.

(1) "Uridine hydrates" i.e. (+)- and (-)6-hydroxy-5, 6-dihydrouridine were formed under gamma irradiation in a deaerated aqueous solution of uridine. (2) The structures of two diastereoisomers were determined by spectroscopic measurements (infrared, ultraviolet and NMR) and verified by stereospecific synthesis; uridine hydrates were prepared by mild reduction of trans(+)- and (-)iodohydrins with acetic acid and zinc power. (3) The carbon 6 epimerisation of uridine hydrates 6R or 6S was performed in triated water (pH 5.5, 30 degrees C) and at the same time tritium incorporation on carbon 5 was noted. The mechanism of these reactions could be explained by the opening of the N1-C6 bond of the pyrimidine ring, followed by ketoenolisation reaction of carbons 4 and 5. (4) The 250 MHz NMR analysis has allowed us to determine the nucleoside conformations. Nucleosides had mainly the S(C2' endo) conformation. A slight preference of gauche-gauche (gg) rotamer of the exocyclic hydroxymethyl group was noted and the aglycone was in the anti conformation.     

Complex formation of double-stranded DNA (6-4) photoproducts and anti-(6-4) photoproduct antibody Fabs.

DNA (6-4) photoproducts are major constituents of ultraviolet-damaged DNAs. We prepared double-stranded (ds) (6-4) DNA photoproducts and analyzed formation of their complexes with anti-(6-4) photoproduct antibody Fabs. Elution profiles of the mixtures of ds-(6-4) DNAs and Fabs from anion-exchange and gel-filtration columns indicate that Fab 64M-2 deprives 14mer ds-(6-4) DNA of single-stranded (ss) (6-4) DNA and shows no interaction with 18 mer ds-(6-4) DNA (A18). Fab 64M-5 with an approximately 100-fold higher affinity than Fab 64M-2 forms a complex with the ds-(6-4) DNA (A18), but partly dissociates another 18 mer ds-(6-4) DNA (A18-3), with a lowered G-C content, into ss-DNAs. From these results, antibody 64M-5 possibly accommodates the T(6-4)T photolesion moiety of the ds-(6-4) DNA (A18) by flipping out the moiety from its neighboring segments.     

Radiolabeled guanine derivatives for the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase: 6-(4-[(18)F]Fluoro-benzyloxy)-9H-purin-2-ylamine and 6-(3-[(131)I]Iodo-benzyloxy)-9H-purin-2-ylamine

Two radiolabeled analogues of 6-benzyloxy-9H-purin-2-ylamine (O(6)-benzylguanine; BG) potentially useful in the in vivo mapping of O(6)-alkylguanine-DNA alkyltransferase (AGT) were synthesized. Fluorine-18 labeling of the known 6-(4-fluoro-benzyloxy)-9H-purin-2-ylamine (FBG; 6) was accomplished by the condensation of 4-[(18)F]fluorobenzyl alcohol with 2-aminopurin-6-yltrimethylammonium chloride (4) or 2-amino-6-chloropurine in average decay-corrected radiochemical yields of 40 and 25%, respectively. Unlabeled 6-(3-iodo-benzyloxy)-9H-purin-2-ylamine (IBG; 7) was prepared from 4 and 3-iodobenzyl alcohol. Radioiodination of 9, prepared from 7 in two steps, and subsequent deprotection gave [(131)I]7 in about 70% overall radiochemical yield. The IC(50) values for the inactivation of AGT from CHO cells transfected with pCMV-AGT were 15 nM for IBG and 50 nM for FBG. The binding of [(18)F]6 and [(131)I]7 to purified AGT was specific and saturable with both exhibiting similar IC(50) values (5-6 microM).     

Miscoding properties of 6alpha- and 6beta-diastereoisomers of the N(2)-(estradiol-6-yl)-2'-deoxyguanosine DNA adduct by Y-family human DNA polymerases

Treatment with estrogen increases the risk of breast, ovary, and endometrial cancers in women. DNA damage induced by estrogen is thought to be involved in estrogen carcinogenesis. In fact, Y-family human DNA polymerases (pol) eta and kappa, which are highly expressed in the reproductive organs, miscode model estrogen-derived DNA adducts during DNA synthesis. Since the estrogen-DNA adducts are a mixture of 6alpha- and 6beta-diastereoisomers of dG-N(2)-6-estrogen or dA-N(6)-6-estrogen, the stereochemistry of each isomeric adduct on translesion synthesis catalyzed by DNA pols has not been investigated. We have recently established a phosphoramidite chemical procedure to insert 6alpha- or 6beta-isomeric N(2)-(estradiol-6-yl)-2'-deoxyguanosine (dG-N(2)-6-E(2)) into oligodeoxynucleotides. Using such site-specific modified oligomer as a template, the specificity and frequency of miscoding by dG-N(2)-6alpha-E(2) or dG-N(2)-6beta-E(2) were explored using pol eta and a truncated form of pol kappa (pol kappaDeltaC). Translesion synthesis catalyzed by pol eta bypassed both the 6alpha- and 6beta-isomers of dG-N(2)-6-E(2), with a weak blockage at the adduct site, while translesion synthesis catalyzed by pol kappaDeltaC readily bypassed both isomeric adducts. Quantitative analysis of base substitutions and deletions occurring at the adduct site showed that pol kappaDeltaC was more efficient than pol eta by incorporating dCMP opposite both 6alpha- and 6beta-isomeric dG-N(2)-6-E(2) adducts. The miscoding events occurred more frequently with pol eta, but not with pol kappaDeltaC. Pol eta promoted incorporation of dAMP and dTMP at both the 6alpha- and 6beta-isomeric adducts, generating G --> T transversions and G --> A transitions. One- and two-base deletions were also formed. The 6alpha-isomeric adduct promoted slightly lower frequency of dCMP incorporation and higher frequency of dTMP incorporation and one-base deletions, compared with the 6beta-isomeric adduct. These observations were supported by steady-state kinetic studies. Taken together, the miscoding property of the 6alpha-isomeric dG-N(2)-6-E(2) is likely to be similar to that of the 6beta-isomeric adduct.     

S6K1(-/-)/S6K2(-/-) mice exhibit perinatal lethality and rapamycin-sensitive 5'-terminal oligopyrimidine mRNA translation and reveal a mitogen-activated protein kinase-dependent S6 kinase pathway

Activation of 40S ribosomal protein S6 kinases (S6Ks) is mediated by anabolic signals triggered by hormones, growth factors, and nutrients. Stimulation by any of these agents is inhibited by the bacterial macrolide rapamycin, which binds to and inactivates the mammalian target of rapamycin, an S6K kinase. In mammals, two genes encoding homologous S6Ks, S6K1 and S6K2, have been identified. Here we show that mice deficient for S6K1 or S6K2 are born at the expected Mendelian ratio. Compared to wild-type mice, S6K1(-/-) mice are significantly smaller, whereas S6K2(-/-) mice tend to be slightly larger. However, mice lacking both genes showed a sharp reduction in viability due to perinatal lethality. Analysis of S6 phosphorylation in the cytoplasm and nucleoli of cells derived from the distinct S6K genotypes suggests that both kinases are required for full S6 phosphorylation but that S6K2 may be more prevalent in contributing to this response. Despite the impairment of S6 phosphorylation in cells from S6K1(-/-)/S6K2(-/-) mice, cell cycle progression and the translation of 5'-terminal oligopyrimidine mRNAs were still modulated by mitogens in a rapamycin-dependent manner. Thus, the absence of S6K1 and S6K2 profoundly impairs animal viability but does not seem to affect the proliferative responses of these cell types. Unexpectedly, in S6K1(-/-)/S6K2(-/-) cells, S6 phosphorylation persisted at serines 235 and 236, the first two sites phosphorylated in response to mitogens. In these cells, as well as in rapamycin-treated wild-type, S6K1(-/-), and S6K2(-/-) cells, this step was catalyzed by a mitogen-activated protein kinase (MAPK)-dependent kinase, most likely p90rsk. These data reveal a redundancy between the S6K and the MAPK pathways in mediating early S6 phosphorylation in response to mitogens.     

Transgenic mice with -6A haplotype of the human angiotensinogen gene have increased blood pressure compared with -6G haplotype

Hypertension is a serious risk factor for cardiovascular disease, and the angiotensinogen (AGT) gene locus is associated with human essential hypertension. The human AGT (hAGT) gene has an A/G polymorphism at -6, and the -6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 kb of the promoter with -6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared with -6G. We have found that the hAGT gene has three additional SNPs (A/G at -1670, C/G at -1562, and T/G at -1561). Variants -1670A, -1562C, and -1561T almost always occur with -6A, and variants -1670G, -1562G, and -1561G almost always occur with -6G. Therefore, the hAGT gene may be subdivided into either -6A or -6G haplotypes. We show that these polymorphisms affect the binding of HNF-1α and glucocorticoid receptor to the promoter, and a reporter construct containing a 1.8-kb hAGT gene promoter with -6A haplotype has 4-fold increased glucocorticoid-induced promoter activity as compared with -6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either the -6A or -6G haplotype of the hAGT gene and the human renin gene. Our ChIP assay shows that HNF-1α and glucocorticoid receptor have stronger affinity for the chromatin obtained from the liver of transgenic mice containing -6A haplotype. Our studies also show that transgenic mice containing -6A haplotype have increased plasma AGT level and increased blood pressure as compared with -6G haplotype. Our studies explain the molecular mechanism involved in association of the -6A allele of the hAGT gene with hypertension.     

Regression of a mammary adenocarcinoma in STAT6-/- mice is dependent on the presence of STAT6-reactive T cells

Polarization of the immune response toward a type 1 cytokine profile has been posited to be associated with a therapeutic antitumor immune response. STAT6-/- mice are unable to generate a type 2 immune response, and instead mount an enhanced type 1 response. STAT6-/- mice are significantly more resistant to 4T1, a mammary adenocarcinoma cell line, resisting a 10-fold higher tumor dose compared with wild-type (wt) BALB/c mice. An analysis of the T cells from tumor-bearing STAT6-/- mice revealed that they contained a population primed by a peptide (STAT6(531-539)) of the STAT6 protein expressed in 4T1. The adoptive transfer of T cells from STAT6(531-539)-vaccinated STAT6-/- mice significantly reduced the number of 4T1 pulmonary metastases in recipient mice. Additionally, the role of these STAT6(531-539)-reactive T cells against s.c. 4T1 tumor challenge was determined by tumor-challenging wt BALB/c mice reconstituted with STAT6-/- bone marrow, thereby assessing whether a polarized type 1 immune response in the absence of STAT6-reactive T cells was sufficient to reject a 4T1 tumor challenge. T cells from the STAT6-/- bone marrow chimeras failed to recognize the STAT6(531-539), and these mice proved to be as susceptible as wt BALB/c mice to 4T1 challenge. This demonstrated that the absence of STAT6(531-539)-reactive T cells correlated with the inability to reject 4T1 challenge. Additionally, these data emphasize that the enhanced ability to mount a type 1-polarized immune response is inconsequential if a sufficient antitumor immune response is not primed by the tumor.     

Increased vascular smooth muscle contractility in TRPC6-/- mice

Among the TRPC subfamily of TRP (classical transient receptor potential) channels, TRPC3, -6, and -7 are gated by signal transduction pathways that activate C-type phospholipases as well as by direct exposure to diacylglycerols. Since TRPC6 is highly expressed in pulmonary and vascular smooth muscle cells, it represents a likely molecular candidate for receptor-operated cation entry. To define the physiological role of TRPC6, we have developed a TRPC6-deficient mouse model. These mice showed an elevated blood pressure and enhanced agonist-induced contractility of isolated aortic rings as well as cerebral arteries. Smooth muscle cells of TRPC6-deficient mice have higher basal cation entry, increased TRPC-carried cation currents, and more depolarized membrane potentials. This higher basal cation entry, however, was completely abolished by the expression of a TRPC3-specific small interference RNA in primary TRPC6(-)(/)(-) smooth muscle cells. Along these lines, the expression of TRPC3 in wild-type cells resulted in increased basal activity, while TRPC6 expression in TRPC6(-/-) smooth muscle cells reduced basal cation influx. These findings imply that constitutively active TRPC3-type channels, which are up-regulated in TRPC6-deficient smooth muscle cells, are not able to functionally replace TRPC6. Thus, TRPC6 has distinct nonredundant roles in the control of vascular smooth muscle tone.     

Coordination features of difunctionalized beta-cyclodextrins with carnosine: ESI-MS and spectroscopic investigations on 6A,6D-di-(beta-alanyl-L-histidine)-6A,6D-dideoxy-beta-cyclodextrin and 6A,6C-di-(beta-alanyl-L-histidine)-6A,6C-dideoxy-beta-cyclodextrin and their copper(II) complexes

The synthesis and characterization of two beta-cyclodextrins (beta-CD) functionalized with two units of carnosine (beta-alanyl-L-histidine) through the amino group, 6A,6C-(beta-alanyl-L-histidine)-6A,6C-dideoxy-beta-cyclodextrin (ACCDAH) and 6A,6D-(beta-alanyl-L-histidine)-6A,6D-dideoxy-beta-cyclodextrin (ADCDAH), are reported. NMR and C.D. data of the ligands indicate a different interaction of dipeptide chains with upper rim and cavity of beta-CD. Analogously, spectroscopic and electrospray ionization mass spectrometry data show different copper(II) complex species formed by the two regioisomers. The ability of carnosine-cyclodextrin derivatives to bind copper ions in a head-to-tail fashion induces the formation of oligomeric species (up to hexamers) in the case of ACCDAH, where the two carnosine moieties are adjacent, while in the ADCDAH case the mutual interaction between the peptidic chains of two ADCDAH molecules allows the almost exclusive formation of a copper-assisted self-assembled dimeric species.     

Synthesis and biological evaluation of 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles as transforming growth factor-β type 1 receptor kinase inhibitors

A series of 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles has been synthesized and evaluated for their ALK5 inhibitory activity. The 1-(6-methylpyridin-2-yl)-1,2,3-triazoles were assembled by Cu(I)-catalyzed azide–alkyne 1,3-dipolar cycloaddition. Following this, quinoxaline was introduced through Pd-catalyzed direct arylation. The synthesized 1-(6-methylpyridin-2-yl)-5-(quinoxalin-6-yl)-1,2,3-triazoles revealed significant selectivity differences with respect to p38α MAP kinase. In particular, 12k showed 80.8% ALK5 inhibitory activity at a concentration of 10 μM and IC₅₀ value of 4.69 μM, but did not show p38α MAP kinase inhibitory activity (?1.94% inhibition at a concentration of 10 μM).     

Conformational preferences of modified nucleic acid bases N6-methyl-N6-(N-threonylcarbonyl) adenine and 2-methylthio-N6-(N-threonylcarbonyl) adenine by the quantum chemical PCILO calculations

Conformational preferences of the hypermodified nucleic acid bases N6-methyl-N6-(N-threonylcarbonyl) Adenine, m6tc6 Ade, and 2-methylthio-N6-(N-threonylcarbonyl) Adenine, mS2 tc6 Ade, have been studied theoretically using the quantum chemical PCILO (Perturbative Configuration Interaction using Localized Orbitals) method. The multidimensional conformational space has been searched using selected grid points formed by combining the various torsion angles which take the favoured values obtained from energy variation with respect to each torsion angle individually. In m6 tc6 Ade and mS 2tc6 Ade alike the threonylcarbonyl substituent preferably orients away (distal) from the imidazole moiety of the adenine ring. And as in the simpler N6-(N-threonylcarbonyl) Adenine, tc6 Ade, the atoms in the ureido group as well as the amino acid carbon atoms C(12) and C(13) remain coplanar with the purine base. As in tc6 Ade, this conformation is stabilized by the intramolecular hydrogen bond between N(11)H of the amino acid and N(1) of the adenine base. The N6-methyl protons, in m6 tc6 Ade, take trans-staggered orientation with respect to the C(6)-N(6) bond. The preferred orientation of the 2-methylthio group is cis to the C(2)-N(3) bond in mS 2tc6 Ade. This is in marked contrast to the modified nucleic acid base 2-methylthio-N6-(delta 2-isopentenyl) Adenine, mS 2i6 Ade, where the 2-methylthio group orients trans to the C(2)-N(3) bond, causing a change in the preferred orientation of the isopentenyl component on methylthiolation. The present results thus indicate that unlike in the isopentenyl adenine the role of further chemical substitutions in threonylcarbonyl adenine may be indirect and less pronounced.     

Genetic properties of transposons Tn6-1, Tn6-2 and Tn19-1

The level and range transposition of the transposons Tn6-1, Tn6-2, Tn19-1, and their ability to influence plasmid transfer has been studied. The widest range of transposition was shown for transposon Tn6-2. Insertions of each of the studied transposons into different conjugative plasmids genomes resulted in change of frequencies of plasmids transfer and change of plasmids mobilization activity.     

Mechanistic studies on type I and type II dehydroquinase with (6R)- and (6S)-6-fluoro-3-dehydroquinic acids

(6R)- and (6S)-6-Fluoro-3-dehydroquinic acids are shown to be substrates for type I and type II dehydroquinases. Their differential reactivity provides insight into details of the reaction mechanism and enables a novel enzyme-substrate imine to be trapped on the type I enzyme.     

An Improved Synthesis of N6-(6-Aminohexyl)FAD

Abstract

We report an improved synthesis of N ⁶-(6-aminohexyl)FAD (1) using an efficient one-pot conversion of inosine to the N-trifluoroacetyl protected N ⁶-(6-aminohexyl)adenosine 3. The 5′-O-phosphorylated AMP derivative 4, activated as the imidazolide, was coupled with commercial sodium riboflavin phosphate by using 18-crown-6 in DMF.     

Somatic hypermutation and class switch recombination in Msh6(-/-)Ung(-/-) double-knockout mice

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, Msh2, or Msh6 affect SHM and CSR. To determine whether Msh2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6(-/-)Ung(-/-) mice. SHM and CSR were affected in the same degree and fashion as in Msh2(-/-)Ung(-/-) mice; mutations were mostly C,G transitions and CSR was greatly reduced, making Msh2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6(-/-)Ung(-/-) mice the 5' end and the 3' region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are "footprints" of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.