A unique urinary constituent, 6-hydroxy-6-methyl-3-heptanone, is a pheromone that accelerates puberty in female mice.

BACKGROUND: Olfactorily mediated puberty acceleration in female mice (measured by an increase in uterine weight) has been observed since the 1960s without the active chemosignal being structurally identified. There are many controversies in the literature as to whether this male-originated pheromone is a volatile substance. We investigated the chemical nature of the urinary fractions that are responsible for the characteristic uterine weight increases. RESULTS: The active pheromone was identified as 5,5-dimethyl-2-ethyltetrahydrofuran-2-ol and/or its open-chain tautomer (6-hydroxy-6-methyl-3-heptanone). A series of cyclic vinyl ethers were isolated from chromatographically active fractions of the urine. Because these compounds did not accelerate puberty, we postulated that these ethers were degradation products of a lactol (5,5-dimethyl-2-ethyltetrahydrofuran-2-ol). The lactol was then detected directly in the mouse urine extract using a silylation agent. Synthetic 6-hydroxy-6-methyl-3-heptanone had strong biological activity, whereas its close structural analogs did not. CONCLUSIONS: The male house mouse excretes into its urine a large quantity of a volatile substance that has a unique lactol/hydroxyketone structure. This substance is capable of binding to the less volatile urinary constituents, such as proteins or peptides, and is active in puberty-acceleration bioassays. The controversies regarding the volatility of the puberty-accelerating pheromones can now be explained by considering a complex of volatile lactol/hydroxyketone and urinary proteins.     

Synthesis of fluorinated Thomsen-Friedenreich antigens: direct deoxyfluorination of αGalNAc-threonine tert-butyl esters

Selectively 6-fluorinated analogs of the tumor-associated T(N) antigen Fmoc-Thr(α-O-GalNAc)-OtBu can be efficiently prepared using DAST-mediated de(hydr)oxyfluorination reactions of preformed and orthogonally protected glycosyl amino esters without affecting the labile protecting groups and O-glycosidic linkages. The resulting mono- and difluorinated T(N) analogs are interesting building blocks for non-hydrolyzable mucin-type antigen mimetics, as illustrated by the unprecedented synthesis of two different multiply fluorinated Thomsen-Friedenreich derivatives. The reported deoxyfluoro antigen analogs represent important functional probes for carbohydrate-binding proteins and glycosyl-processing enzymes.     

Structure-activity relationships of alpha-human atrial natriuretic peptide (alpha-hANP) analogs: role of charged groups in alpha-hANP.

To determine whether the addition of a methylene unit in the side chain of the Asp or Arg residue in alpha-human atrial natriuretic peptide (alpha-hANP) influences its biological activity, analogs of alpha-hANP, [Glu13]-alpha-hANP (7-28) (1), [Aad13]-alpha-hANP (7-28) (2), and [Harn]-alpha-hANP(7-28) (where n is any possible combination of 11, 14 and 27) (3-9), where the original Asp or Arg residue was replaced by a homo-amino acid, were synthesized by the solid-phase synthesis method. All the analogs were evaluated for their receptor binding, cyclic guanosine monophosphate (cGMP) accumulation activity in rat vascular smooth muscle cells (VSMC), and for vasorelaxant activity employing rat aorta. 1 and 2 were 0.9 and 0.03 times as potent as alpha-hANP (7-28), respectively, in binding. Har-containing analogs (3-9) were as potent as alpha-hANP (7-28) in binding. Among the Har-containing analogs, [Har11,14]-alpha-hANP (7-28) (6) and [Har11,27]-alpha-hANP (7-28) (7) were remarkably vasorelaxant active, being 4.2 and 5.3 times potent than alpha-hANP (7-28), respectively, in spite of relatively lower cGMP accumulation activity in the case of 7. The roles of the chargeable amino acid residues in biological activity are discussed.     

Rapid Synthesis of Glycosyl Bromides by Ultrasound Irradiation

A convenient and environmentally friendly reactor for the synthesis of glycosyl bromides via ultrasound irradiation was designed. Peracetylated glycosyl bromides were synthesized from free saccharides by means of a one-pot method. Benzoylated and 6-subsituted glycosyl bromides were prepared from protected saccharides. The glycosyl bromides were obtained in isolated yields of 83% to 96%.     

Synthesis and glycosylation of a series of 6-mono-, di-, and trifluoro S-phenyl 2,3,4-tri-O-benzyl-thiorhamnopyranosides. Effect of the fluorine substituents on glycosylation stereoselectivity

A series of 6-mono-, di-, and trifluoro analogs of S-phenyl 2,3,4-tri-O-benzyl-D- or L-thiorhamnopyranoside has been synthesized and used as donors in glycosylation reactions, with activation by the 1-benzenesulfinyl piperidine/triflic anhydride system. The stereochemical outcome of the glycosylation reactions was found to depend on the electron-withdrawing capability of the disarming substituent at the 6-position, i.e., on the number of fluorine atoms present. The results are explained with regard to the increased stability of the glycosyl triflates, shown to be intermediates in the reaction by low-temperature 1H NMR experiments, with increased fluorine content.     

In vivo evaluation and in vitro metabolism of leuprolide in mice--mass spectrometry-based biomarker measurement for efficacy and toxicity

The study of pharmacologically active peptides is central for the understanding of cancer and the development of novel therapeutic approaches. In this context, both qualitative and quantitative determination of bioactive peptides in biological fluids/tissues and their effect on endogenous factors (e.g. hormones) are of great importance. A mass spectrometry-based approach was developed and applied towards the measurement of leuprolide, a peptide drug for the treatment of prostate cancer, in mouse plasma. High-pressure liquid chromatography coupled to a hybrid quadrupole linear ion trap (QqLIT) mass spectrometer, a platform that combines the benefits of triple QqLIT instruments, was employed for the study. Using the described methodology, we established that picomolar concentrations of leuprolide could be measured in mouse plasma (limit of quantification of 0.1 ng/ml). In order to optimize pharmacokinetic properties of analogs of leuprolide, a facile in vivo mouse model was developed and leuprolide concentrations were determined in mouse plasma following intraperitoneal administration. In the same animal model, we demonstrated the versatility of the described MS-based approach by the determination of plasma concentrations of testosterone, an established biomarker for the treatment of prostate cancer. Following dosing with leuprolide, circulating testosterone was increased significantly in comparison to vehicle-treated mice. Finally, in vitro metabolism of leuprolide was evaluated by incubation of leuprolide with mouse kidney membranes, followed by identification of major metabolites by MS. Such studies provide the framework for future evaluation of novel leuprolide analogs with potential therapeutic advantages.     

Efficient synthesis of glycosylated phenazine natural products and analogs with DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors

Inspired by the occurrence and function of phenazines in natural products, new glycosylated analogs were designed and synthesized. DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors were used in an efficient and easily-handled glycosylation protocol compatible with combinatorial chemistry. Benzoylated D-glucose, D-galactose and L-quinovose DISAL glycosyl donors were synthesized in high yields and used under mild conditions to glycosylate methyl saphenate and 2-hydroxyphenazine. The glycosides were screened for biological activity and one compound showed inhibitory activity towards topoisomerase II.     

Enhancement of the T140-based pharmacophores leads to the development of more potent and bio-stable CXCR4 antagonists

A CXCR4 antagonistic peptide, T140, and its bio-stable analogs, such as Ac-TE14011, were previously developed. These peptides inhibit the entry of T cell line-tropic strains of HIV-1 (X4-HIV-1) into T cells. Herein, a series of TE14011 analogs having modifications in the N-terminal region were synthesized to develop effective compounds with increased biostability. Among these analogs, 4F-benzoyl-TE14011 (TF14013) showed the strongest anti-HIV activity derived from CXCR4-antagonism, suggesting that a 4-fluorobenzoyl moiety at the N-terminus of T140 analogs constitutes a novel T140-based pharmacophore for CXCR4 antagonists. Structure-activity relationship (SAR) studies on TE14011 analogs with N(alpha)-acylation by several benzoic acid derivatives have disclosed a significant relationship between the anti-HIV activity and the Hammett constant (sigma) of substituted benzoic acids. TF14013 was found to be stable in mouse serum, but not completely stable in rat liver homogenate due to deletion of the C-terminal Arg14-NH2 from the parent peptide. This biodegradation was completely suppressed by N-alkyl-amidation at the C-terminus. Taken together, the enhancement of the T140-based pharmacophores led to development of a novel CXCR4 antagonist, 4F-benzoyl-TE14011-Me (TF14013-Me), which has very high anti-HIV activity and increased biostability.     

Stereoselective tris-glycosylation to introduce beta-(1-->3)-branches into gentiotetraose for the concise synthesis of phytoalexin-elicitor heptaglucoside

Dodecyl thioglycosides (3, 4, 5) were prepared by conventional transformation of d-glucose and used as new glycosyl donors for a short-step synthesis of phytoalexin elicitor heptaglucoside. A gentio-tetraoside derivative (6) having three hydroxyl groups was synthesized by NIS-TfOH promoted glycosylate in more than 90% yield followed by selective removal of temporary protective groups. Undesired formation of alpha-glycosides at the introduction of beta-(1-->3)-branches into gentio-oligosaccharides was found to be suppressed by use of a thiophilic reagent system, BSP (1-benzenesulfinyl piperidine)-Tf2O, giving the heptaglucoside in only four glycosylation steps.     

A Facile Synthesis of N γ-Glycosyl Asparagine Conjugates and Short N-Linked Glycopeptides

Both free and protected glycosyl azides were efficiently coupled to the side chain of aspartate by the Staudinger reaction for the synthesis of N ^γ-glycosyl asparagine conjugates and short N-linked glycopeptides that can be employed to construct complex N-linked glycopeptides. In the process, a facile two-step protocol was developed for free glycosyl azide synthesis, which includes reported direct transformation of free oligosaccharides into glycosyl amines through reaction with ammonium bicarbonate and then stereospecific diazo transfer reaction to convert glycosyl amines to glycosyl azides.     

Synthesis of Disaccharide Nucleosides by the O‐Glycosylation of Natural Nucleosides with Thioglycoside Donors

Disaccharide nucleosides constitute an important group of naturally‐occurring sugar derivatives. In this study, we report on the synthesis of disaccharide nucleosides by the direct O‐glycosylation of nucleoside acceptors, such as adenosine, guanosine, thymidine, and cytidine, with glycosyl donors. Among the glycosyl donors tested, thioglycosides were found to give the corresponding disaccharide nucleosides in moderate to high chemical yields with the above nucleoside acceptors using p‐toluenesulfenyl chloride (TolSCl) and silver triflate (AgOTf) as promoters. The interaction of these promoters with nucleoside acceptors was examined by ¹H NMR spectroscopic experiments.     

Determination of hydroxycamptothecin affinities to albumin and membranes by steady-state fluorescence anisotropy measurements

Camptothecin (CPT) and its hydroxycamptothecin analogs are fluorescent compounds exhibiting strong anticancer properties. They exist in two forms: active lactone and inactive carboxylate. The deactivation proceeds via hydrolysis in neutral and base solutions. A serious limitation to the clinical application of CPT is the strong affinity of its carboxylate form to human serum albumin (HSA) which destabilizes its active lactone form. However, binding to membranes in blood improves the stability of the lactone form of CPT and its analogs. A high-throughput screening assay based on the steady-state fluorescence anisotropy method was used to determine the protein- and membrane-binding properties of 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxycamptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67). The relative affinities of hydroxycamptothecins to HSA and model membranes in the form of DMPC liposomes were determined, and DB-67 exhibited the most desirable properties including the highest affinity to membranes in its lactone form and low affinity to HSA in its carboxylate form.     

A three-dimensional biomimetic microfluidic chip to study the behavior of hepatic stellate cell under the tumor microenvironment

In this paper, we designed a three-dimensional cell co-cultured microfluidic chip, which generated interstitial flow and oxygen gradient to simulate the complex tumor microenvironment. It consisted of five parallel cell culture channels and one hypoxic channel. These channels were constructed for the culture of mouse liver tumor cells (Hepa1-6), mouse liver stellate cells (JS-1), the simulation of extracellular matrix, complex biochemical factors (hypoxia and interstitial flow), and the supply of cellular nutrients. The 3D-interstitial flow-hypoxia model was used to study the behavior of JS-1 cells under the effect of tumor microenvironment (TME). The results showed that by co-cultured with Hepa1-6 cells, hypoxia of Hepa1-6 cells, and adding TGF-β1 by interstitial flow, the migration of JS-1 cells could be promoted. Similarly, activated JS-1 cells could led to the epithelial-mesenchymal transformation in co-cultured Hepa1-6 cells, which secreted more TGF-β1.     

Synthesis and biological evaluation of nucleosides containing 8-amino-imidazo[1,2-α]pyrazine as an isosteric replacement for adenine

A number of novel C-nucleosides related to purine derivatives are described in which the purine moiety has been replaced by the isosteric heterocycle, 8-aminoimidazo[1,2-α]pyrazine. The nucleosides prepared include the ribo, 3′-deoxy, 2′,3′-dideoxy, and 2′,3′-unsaturated derivatives. These C-nucleosides represent derivatives containing acid stable glycosyl bonds and they can be considered as analogs of adenine- or 3-deazaade-nine-containing nucleosides. Preparation of the parent ribonucleoside was accomplished by reaction of the C-l functionalized sugar, (2ξ)-1-amino-3,6-anhydro-l-deoxy-4,5-O-isopropylidene-7-O-trityl-D-allo-heptitol with 2,3-dichloropyrazine, followed by ring closure to the 8-chloroimidazo[1,2-α]pyrazine nucleoside, conversion to the 8-amino derivative and deblocking. A single crystal X-ray structure of the parent 8-amino-3-(β-D-ribofuranosyl)imidazo[1,2-α]pyrazine is described and the conformation compared to that of formycin. The sugar-modified analogs were prepared by subsequent functional group manipulations on the sugar moiety. Biological evaluation against HIV in H9 T-lymphoid cell culture showed the nucleosides to be devoid of significant antiviral activity compared to DDA. The 3-deazaadenosine analog also demonstrated weak suppression of mouse splenic NK activity toward YAC cells (mouse lymphoma cell targets). The imidazo[1,2-α]pyrazine analog of 3-deazaadenosine showed antiinflammatory activity in vivo in the rat pleurisy carrageenan model in the same range with 3-deazaadenosine.     

Synthesis of optically active alpha-methylene beta-lactams through lipase-catalyzed kinetic resolution

A convenient method for the preparation of the hitherto unknown chiral alpha-methylene beta-lactam derivatives 5a,b is reported. The optically active alpha-methylene beta-lactams 5a-c, and their corresponding amino acids 6a-c have been readily made available through lipase-catalyzed kinetic resolution in high enantiomeric purity (up to 99% ee). The N-substituted beta-lactam derivatives 4a, b and 10 are not accepted by the lipases and were prepared in optically active form by chemical transformation.     

Synthetic 6-O-methylglucose-containing polysaccharides (sMGPs): design and synthesis

With the hope of mimicking the chemical and biological properties of natural 6-O-methyl-D-glucose-containing polysaccharides (MGPs), synthetic 6-O-methyl-D-glucose-containing polysaccharides (sMGPs) were designed and synthesized from alpha-, beta-, and gamma-cyclodextrins (CDs). The synthetic route proved to be flexible and general, to furnish a series of sMGPs ranging from 6-mer to 20-mer. A practical and scalable method was discovered selectively to cleave the CD derivatives and furnish the linear precursors to the glycosyl donors and acceptors. The Mukaiyama glycosidation was adopted to couple the glycosyl donors with the glycosyl acceptors. Unlike in the 3-O-methyl-D-mannose-containing polysaccharide (sMMP) series, the amount of the Mukaiyama acid required in the sMGP series increased with an increase of substrate size; that is, for large oligomers, more than one equivalent of the acid was required.     

Additional evidence for the cyclic GMP signaling pathway resulting in the photophobic behavior of Stentor coeruleus.

We report that exo- and endogenous guanosine 3',5'-cyclic monophosphate (cGMP) specifically influenced the photophobic response. In behavioral experiments the slowly hydrolyzable and membrane-permeable analogs of cGMP (8-bromo-cGMP [Br-cGMP] and N6,2'-o-dibutyryl-cGMP) dramatically prolonged the time for ciliary stop response and decreased the duration of ciliary reversal in a dose-dependent manner. When analogs of adenosine 3',5'-cyclic monophosphate (cAMP) (8-bromo-cAMP or N6,2'-o-dibutyryl-cAMP) were used, no essential effects were detected on the kinetics of the photophobic response. Both nonspecific cyclic nucleotide phosphodiesterase (PDE) activity inhibitors (3-isobutyl-1-methylxanthine [IBMX] and 1,3-dimethylxanthine [theophylline]) and the highly specific cGMP-PDE activity inhibitor 1,4-dihydro-5-[2-propoxyphenyl]-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast) mimicked the effects of cGMP analogs. Treatment of cells with an inhibitor of guanylate cyclase activity (6-anilino-5,8-quinolinedione [LY 83583]) exerted an effect opposite to that of cGMP analogs and PDE activity inhibitors. The positive physiological effect of LY 83583 was significantly diminished in ciliates that were treated simultaneously with Br-cGMP. In an assay of cell cyclic nucleotide content, the exposure of dark-adapted Stentor to light evoked a transient decrease in the basal level of intracellular cGMP. Alterations in internal cGMP levels were more distinct when the intensity of applied illumination was increased. In the presence of IBMX or theophylline the basal content of cGMP was markedly enhanced, and the photoinduced changes in cGMP level were less pronounced. In this paper the possible whole molecular mechanism by which the ciliary orientation in Stentor is controlled by light is presented.     

Homonucleoside phosphonic acid-I: Ageneral synthesis of isosteric phosphonate analogs of nucleoside 3'-phosphates

A general synthetic route to 3'-deoxy-3'-dihydroxyphosphinylmethyl ribonucleosides 3 the isosteric phosphonate analogs of nucleoside 3'-phosphates, is described. This involved alkylation of 1,2;5,6-di-O-isopropylidene-α-D-ribo-hexofuranose-3-ulose 7) with tetraethyl methylenediphosphonate 6, followed by stereoselective catalytic reduction and cleavage of C₆ to generate 3-deoxy-3-diethoxyphosphinylmethyl-1,2-O-isopropylidene-α-D 12a. Benzoylation followed by acetolysis then generated the key crystalline intermediate 1,2-di-O-acetyl-5-O-benzoyl-3-deoxy-3-diethoxyphosphinylmethyl-β-D-ribofuranose 13, This compound, or the related glycosyl chloride, was condensed with several purine and pyrimidine bases and all protecting groups were removed by mild alkaline treatment via a series of intramolecular cyclizations and hydrolysis. In this manner the phosphonate analogs of nucleoside 3'-phosphates derived from adenine, 6-dimethylaminopurine, uracil, thymine, and cytosine were prepared.     

Conformational and electronic effects on the regioselectivity of the glycosylation of different anomers of N-dimethylmaleoyl-protected glucosamine acceptors

In a previous paper (Bohn et al., Carbohydr. Res., 2007, 342, 2522) the relative O3/O4 reactivities of both alpha- and beta-methyl glycosides of N-dimethylmaleoyl (DMM) glucosamine acceptors protected at O6 with three different groups were assessed by us, using two glycosyl donors. The alpha-anomers showed preferential or exclusive substitution at O3, whereas the beta-anomers gave preferential or exclusive substitution at O4. A DFT study of analogs of the reported acceptors indicates that whereas the beta-anomers carry the DMM ring parallel to the C2-H2 bond for steric reasons, the alpha-anomers tilt this ring producing a strong hydrogen bond between the H(O)3 and one of the DMM carbonyl groups. In this way, the O3 group becomes more nucleophilic and thus more reactive: both charge and Fukui functions on O3 and O4 in the model compounds support the experimental results. Surprisingly, the previously mentioned hydrogen bond is not the only driving force for the slant of the DMM group: the axial methoxyl group of the alpha-anomers also plays a role. The ease of rotation around the C2-N2 bond for DMM-protected analogs was assessed with different models. MP2 calculations using higher basis sets yield similar relative energy and charge values to those calculated using DFT.