Synthesis of (+/−)–1′-Aza-carbocyclic-pyrimidine-2′,3′-dideoxynucleoside analogues as potential anti-HIV agents

1′-Aza-carbocyclic-2′, 3′-dideoxyuridine, 3′-deoxythymidine and 2′, 3′-dideoxycytidine were synthesized from 1-aminopyrrolidine intermediate 13 and evaluated as anti-HIV agents in MT-4 cells.     

Synthesis and anti-HIV activity of 3′-deoxy-3′-(N-hydroxyamino) analogues of nucleosides

3′-Deoxy analogues of thymidine and uridine bearing a 3′-N-alkyl-(or N-aralkyl) -N-hydroxyamino group either on the or the β face of the furanose ring have been prepared. One of these (13), exhibited a moderate anti-HIV activity.     

1′,2′-Dideacetylboronolide, an α-pyrone from Iboza riparia

The structural elucidation of 1′,2′-dideacetylboronolide, 5,6-dihydro-6-(3′-acetoxy-1′,2′-dihydroxyheptyl)2-pyrone, a new α-pyrone isolated from the leaves of Iboza riparia has been performed. Additionally, three sterols, sitosterol, stigmasterol and campesterol, have been identified in this species.     

Affinity Modulation of Platelet Integrin αIIbβ3 by β3-Endonexin, a Selective Binding Partner of the β3 Integrin Cytoplasmic Tail

Platelet agonists increase the affinity state of integrin α_IIbβ₃, a prerequisite for fibrinogen binding and platelet aggregation. This process may be triggered by a regulatory molecule(s) that binds to the integrin cytoplasmic tails, causing a structural change in the receptor. β₃-Endonexin is a novel 111–amino acid protein that binds selectively to the β₃ tail. Since β₃-endonexin is present in platelets, we asked whether it can affect α_IIbβ₃ function. When β₃-endonexin was fused to green fluorescent protein (GFP) and transfected into CHO cells, it was found in both the cytoplasm and the nucleus and could be detected on Western blots of cell lysates. PAC1, a fibrinogen-mimetic mAb, was used to monitor α_IIbβ₃ affinity state in transfected cells by flow cytometry. Cells transfected with GFP and α_IIbβ₃ bound little or no PAC1. However, those transfected with GFP/β₃-endonexin and α_IIbβ₃ bound PAC1 specifically in an energy-dependent fashion, and they underwent fibrinogen-dependent aggregation. GFP/β₃-endonexin did not affect levels of surface expression of α_IIbβ₃ nor did it modulate the affinity of an α_IIbβ₃ mutant that is defective in binding to β₃-endonexin. Affinity modulation of α_IIbβ₃ by GFP/β₃-endonexin was inhibited by coexpression of either a monomeric β₃ cytoplasmic tail chimera or an activated form of H-Ras. These results demonstrate that β₃-endonexin can modulate the affinity state of α_IIbβ₃ in a manner that is structurally specific and subject to metabolic regulation. By analogy, the adhesive function of platelets may be regulated by such protein–protein interactions at the level of the cytoplasmic tails of α_IIbβ₃.     

2′-Azido-2′-deoxy- and 2′-amino-2′-deoxy-β-d-arabino-furanosyl purine nucleosides

A general method for the preparation of 2′-azido-2′-deoxy- and 2′-amino-2′-deoxyarabinofuranosyl-adenine and -guanine nucleosides is described. Selective benzoylation of 3-azido-3-deoxy-1,2-O-isopropylidene-α-d-glucofuranose afforded 3-azido-6-O-benzoyl-3-deoxy-1,2-O-isopropylidene-α-d-glucofuranose (1). Acid hydrolysis of 1, followed by oxidation with sodium metaperiodate and hydrolysis by sodium hydrogencarbonate gave 2-azido-2-deoxy-5-O-benzoyl-d-arabinofuranose (3), which was acetylated to give 1,3-di-O-acetyl-2-azido-5-O-benzoyl-2-deoxy-d-arabinofuranose (4). Compound 4 was converted into the 1-chlorides 5 and 6, which were condensed with silylated derivatives of 6-chloropurine and 2-acetamido-hypoxanthine. The condensation reaction gave α and β anomers of both 7- and 9-substituted purine nucleosides. The structures of the nucleosides were determined by n.m.r. and u.v. spectroscopy, and by correlation of the c.d. spectra of the newly prepared nucleosides with those published for known purine nucleosides.     

Transglycosylation Catalyzed by Almond β-glucosidase and Cloned Pichia etchellsii β-glucosidase II using Glycosylasparagine Mimetics as Novel Acceptors

The stability of almond β-glucosidase in five different organic media was evaluated. After 1 hour of incubation at 30°C, the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively. Transglucosylation involving p-nitrophenyl β-D-glucopyranoside as donor and β-1-N-acetamido-D-glucopyranose, which is a glycosylasparagine mimic, as acceptor was explored under different reaction conditions using almond βglucosidase and cloned Pichia etchellsii β-glucosidase II. The yield of disaccharides obtained in both reactions turned out to be 3%. Both enzymes catalyzed the formation of (1→3)- as well as (1→6)- regioisomeric disaccharides, the former being the major product in cloned β-glucosidase II reaction while the latter predominated in the almond enzyme catalyzed reaction. Use of β-1-N-acetamido-D-mannopyranose and β-1-N-acetamido-2-acetamido-2-deoxy-D-glucopyranose as acceptors in almond β-glucosidase catalyzed reactions, however, did not afford any disaccharide products revealing the high acceptor specificity of this enzyme.     

Synthesis and biological activity of 1β-methyl-2-[5′-isoxazoloethenylpyrrolidin-3′-ylthio]carbapenems

A new series of 1β-methylcarbapenems 1a–i bearing isoxazoloethenyl groups on the pyrrolidine ring has been prepared and evaluated for in vitro antibacterial activity and stability to DHP-I. Most compounds showed excellent antibacterial activity and high stability to DHP-I superior to that of meropenem. Of these new carbapenems, 1a,b,h exhibited the best combination of antibacterial activity and DHP-I stability.     

Synthesis,Structure, and Biological Applications of α‐Fluorinated β‐Amino Acids and Derivatives

This review gives a broad overview of the state of play with respect to the synthesis, conformational properties, and biological activity of α‐fluorinated β‐amino acids and derivatives. General methods are described for the preparation of monosubstituted α‐fluoro‐β‐amino acids (Scheme 1). Nucleophilic methods for the introduction of fluorine predominantly involve the reaction of DAST with alcohols derived from α‐amino acids, whereas electrophilic sources of fluorine such as NFSI have been used in conjunction with Arndt? Eistert homologation, conjugate addition or organocatalyzed Mannich reactions. α,α‐Difluoro‐β‐amino acids have also been prepared using DAST; however, this area of synthesis is largely dominated by the use of difluorinated Reformatsky reagents to introduce the difluoro ester functionality (Scheme 9). α‐Fluoro‐β‐amino acids and derivatives analyzed by X‐ray crystal and NMR solution techniques are found to adopt preferred conformations which are thought to result from stereoelectronic effects associated with F located close to amines, amides, and esters (Figs. 2–6). α‐Fluoro amide and β‐fluoro ethylamide/amine effects can influence the secondary structure of α‐fluoro‐β‐amino acid‐containing derivatives including peptides and peptidomimetics (Figs. 7–9). α‐Fluoro‐β‐amino acids are also components of a diverse range of bioactive anticancer (e.g., 5‐fluorouracil), antifungal, and antiinsomnia agents as well as protease inhibitors where such fluorinated analogs have shown increased potency and spectrum of activity.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Determination of 3′-azido-2′,3′-dideoxyuridine in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography

3′-Azido-2′,3′-dideoxyuridine (AZDU, Azddu, CS-87) is a nucleoside analog of 3′-azido-3′-deoxythymidine (zidovudine, AZT) that has been shown to inhibit human immunodeficiency virus (HIV-1). AZDU is a potential candidate for treatment of pregnant mothers to prevent prenatal transmission of HIV/AIDS to their unborn children. A rapid and efficient high-performance liquid chromatography (HPLC) method for the determination of AZDU concentrations in rat maternal plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. Tissue samples were homogenized in distilled water, protein precipitated and extracted using a C-18 solid-phase extraction (SPE) method prior to analysis. Plasma and amniotic fluid samples were protein precipitated with 2 M perchloric acid prior to analysis. Baseline resolution was achieved using a 4.5% acetonitrile in 40 mM sodium acetate (pH 7) buffer mobile phase for amniotic fluid, placenta and fetus samples and with a 5.5% acetonitrile in buffer solution for plasma at flow-rates of 2.0 ml/min. The HPLC system consists of a Hypersil ODS column (150×4.6 mm) with a Nova-Pak C-18 guard column with detection at 263 nm. The method yields retention times of 6.2 and 12.2 min for AZDU and AZT in plasma and 8.3 and 17.6 min for AZDU and AZT in amniotic fluid, fetal and placental tissues. Limits of detection ranged from 0.01 to 0.075 μg/ml. Recoveries ranged from 81 to 96% for AZDU and from 82 to 96% for AZT in the different matrices. Intra-day (n=6) and inter-day (n=9) precision (% RSD) and accuracy (% Error) ranged from 1.48 to 6.25% and from 0.50 to 10.07%, respectively.     

Effects of all-trans-retinoic acid on skeletal pattern, 5′HoxD gene expression,and RAR β2/β4 promoter activity in embryonic mouse limbs

Mouse embryos were exposed to all-trans-retinoic acid on day 11 or day 12 of development and the resulting skeletal pattern alterations compared with early effects on Hoxd-11 and Hoxd-13 expression domains and RAR-β2/β4 promoter activity. The effects on skeletal pattern showed a clear correlation between the timing of retinoic acid exposure and the sequence of mesenchymal condensation. Ectopic RAR-β2/β4 promoter activity was detected within 2 hr of exposure to retinoic acid, and was present throughout the limb bud after 5 hr; it remained high in the apical ectodermal ridge and proximal mesenchyme after 12 hr, by which time the abnormal digital pattern could be seen. HoxD gene expression domains in the distal handplate were narrowed by 5 hr after maternal retinoic acid administration on day 11. Following retinoic acid treatment on both day 11 and day 12, the normal downregulation of Hoxd-11 and Hoxd-13 in the digital mesenchymal condensations was retarded. There was no evidence to suggest that RAR-β2/β4 promoter activity mediates the effects of RA on HoxD gene expression, but ectopic promoter activity is a useful indicator of at least some of the sites in which RA levels are raised. We suggest (1) that the apical ectodermal ridge is the most functionally significant of these sites, (2) that raised retinoic acid levels in the ridge result in altered gene expression and/or altered cell proliferation within this epithelium, (3) that both altered HoxD gene expression domains and altered skeletal pattern formation are secondary to this effect. There was a good correlation between the effects of retinoic acid on Hoxd-11 and Hoxd-13 expression and delay of skeletal differentiation, suggesting that this may be a direct effect. © 1996 Wiley-Liss, Inc.     

Synthesis and conformational study of poly(0,0′-dicarbobenzoxy-L-β-3,4-dihydroxyphenyl-α-alanine)

High-molecular-weight poly(0,0′-dicarbobenzoxy-L -β-3,4-dihydroxyphenyl-α-alanine) was prepared by the N-carboxyanhydride method. From the results obtained by a study of the optical rotation, nuclear magnetic resonance, and solution infrared absorption, the conformation of poly(0,0′-dicarbobenzoxy-L -β-3,4-dihydroxyphenyl-α-alanine) depended greatly on the solvent taking a right-handed helix with [θ]₂₂₅ = ?13,600 ～ ?18,900 in alkyl halides, a left-handed helix with [θ]₂₂₈ = 22,100 ～ 24,800 in cyclic ethers or trimethylphosphate, and a random coil structure in dichloroacetic acid, trifluoroacetic acid, or hexafluoroacetone sesquihydrate. The polypeptide underwent a right-handed helix-coil transition in chloroform/dichloroacetic acid (or trifluoroacetic acid) mixed solvents and a left-handed helix-coil transition in dioxane/dichloroacetic acid (or trifluoroacetic acid) mixed solvents. The results were compared with those of poly(0-carbobenzoxy-L -tyrosine).     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

Mycobacterium tuberculosis class II apurinic/apyrimidinic‐endonuclease/3′‐5′ exonuclease III exhibits DNA regulated modes of interaction with the sliding DNA β‐clamp

The class‐II AP‐endonuclease (XthA) acts on abasic sites of damaged DNA in bacterial base excision repair. We identified that the sliding DNA β‐clamp forms in vivo and in vitro complexes with XthA in Mycobacterium tuberculosis. A novel ₂₃₉QLRFPKK₂₄₅ motif in the DNA‐binding domain of XthA was found to be important for the interactions. Likewise, the peptide binding‐groove (PBG) and the C‐terminal of β‐clamp located on different domains interact with XthA. The β‐clamp‐XthA complex can be disrupted by clamp binding peptides and also by a specific bacterial clamp inhibitor that binds at the PBG. We also identified that β‐clamp stimulates the activities of XthA primarily by increasing its affinity for the substrate and its processivity. Additionally, loading of the β‐clamp onto DNA is required for activity stimulation. A reduction in XthA activity stimulation was observed in the presence of β‐clamp binding peptides supporting that direct interactions between the proteins are necessary to cause stimulation. Finally, we found that in the absence of DNA, the PBG located on the second domain of the β‐clamp is important for interactions with XthA, while the C‐terminal domain predominantly mediates functional interactions in the substrate's presence.     

Synthesis of 11β-H-prostaglandin E2

11β-³H-Prostaglandin E₂ was synthesized by the stereoselective reduction of the PGD₂ derivative using sodium borotritide.     

β-Alanine containing cyclic peptides with turned structure: The “pseudo type II β-turn.” VI

In the present paper we describe the synthesis, purification, single crystal x-ray analysis, and nmr solution characterization, combined with restrained molecular dynamic simulations, of the cyclic hexapeptide cyclo-(L -Pro-L -Phe-β-Ala)₂. The peptide was synthesized by classical solution methods and the cyclization of the free hexapeptide was accomplished in good yields in diluted methylene chloride solution using N,N-dicyclohexyl-carbodiimide. The compound crystallizes in the monoclinic space group P2₁ from methanol-dichloro-methane solution. The two identical halves of the molecule adopt in the solid state two different conformations. One β-Ala-L -Pro peptide bond is trans, while the second is cis. The molecule is present in dimethylsulfoxide d₆ solutions as a mixture of conformational families. One of these corresponds to a C₂ symmetrical molecule with both β-Ala-Pro cis peptide bonds, while the second major conformation is very similar to that observed in the solid state. All Pro-Phe segments, both in the solid state and the symmetrical and unsym-metrical solution conformations, display ?,ψ angles close to that of position i + 1 and i + 2 of type II β-turns. In addition, the segments preceeded by a trans β-Ala-Pro peptide bond are characterized by a typical i ← i + 3 hydrogen bond, which is absent in the conformer containing a cis β-Ala-Pro peptide bond. The latter conformation corresponds to a new structural domain we define as the “pseudo type II β-turn.” © 1994 John Wiley & Sons, Inc.     

Physical properties of type i collagen in solution: Structure of α-Chains and β- and γ-components and two-component mixtures

The structure of thermally denatured Type I collagen has been studied using laser light scattering. The results indicate that the diffusion coefficients of α-chains and β- and γ-components are 1.550 ± 0.08 × 10^?7, 1.000 ± 0.05 × 10^?7, and 0.835 ± 0.04 × 10^?7 cm²/sec, respectively, at temperatures between 20 and 40°C. It is concluded from diffusion data that these species have hydrodynamic radii of about 13.8 nm (α-chain), 21.5 nm (β-component), and 25.7 nm (γ-component), consistent with previous studies of thermal denaturation by light scattering. It is also concluded, based on volume calculations, that a large volume increase occurs when the triple helix unfolds. Homodyne correlation functions for two component mixtures of α-chains and β-and γ-components appeared to decay exponentially. In all but one case discussed the correlation function could be fitted with a single component having a translational diffusion coefficient which was an intensity weighted average of the diffusion coefficient of each component present.     

Specific localization of 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca/Mg)-ATPase, and acetylcholinesterase in human erythyrocyte membrane

A procedure was developed for the detection of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in myelin. This assay was sufficiently sensitive to detect the low levels of 2′,3′-cyclic nucleotide 3′-phosphohydrolase in human erythrocytes. The 2′,3′-cyclic nucleotide 3′-phosphohydrolase of human erythrocytes was determined to be exclusively associated with the inner (cytosolic) side of the membrane. Leaky ghostsand resealed ghosts were assayed for 2′,3′-cyclic nucleotide 3′-phosphohydrolase, (Ca²⁺/Mg²⁺-ATPase, and acetylcholinesterase activity, and the 2′,3′-cyclic nucleotide 3′-phosphohydrolase profile is the same as that of the (Ca²⁺/Mg²⁺)-ATPase, an established inner membrane maker.