An Efficient Synthesis of Optically Active [4-13C] Labelled Quorum Sensing Signal Autoinducer-2.

A new synthetic route for the quorum sensing signal Autoinducer-2 (AI-2) is described and used for the preparation of [4-13C]-AI-2 starting from [1-13C]-bromoacetic acid. The key step in this process was the enantioselective reduction of an intermediate ketone. This synthesis provides, selectively, both enantiomers of the labelled or unlabelled parent compound, (R) or (S)-4,5-dihydroxypentane-2,3-dione (DPD) and was used for an improved synthesis of [1-13C]-AI-2.

The effect of new compounds in stabilizing downstream monoclonal antibody (mAb) process intermediates.

Determining the stability of downstream process (DSP) intermediates is an extremely important parameter used to maintain product quality attributes within their acceptance ranges. The IgG4 monoclonal antibody studied (mAb1) showed aggregation under acidic conditions, inhibiting the use of low pH treatment to inactivate endogenous retroviruses, and poor virus filtration performance. Both manufacturing steps are included in mAb DSP for viral clearance. The impact of several new compounds on the aggregation and stabilization of mAb1 in process intermediate pools encountered during these critical DSP steps was investigated. Results showed that, in the presence of a protein stabilizer at pH 3.2, 27% less aggregation was observed compared to controls, during the low pH treatment for viral inactivation. The impact of a novel protein stabilizer on virus filter throughput during mAb1 filtration was compared to L-arginine using an innovative high-throughput automation technique. Compared to control experiments without additives, conditions were found where a 70% increase in filter volumetric throughput was achieved in the presence of the novel stabilizer, and a 56% decrease in volumetric throughput observed with L-arginine. These findings present the possibility of using these novel compounds to stabilize proteins during DSP and permitting the use of platform DSP elements such as low pH treatment and high-throughput virus filtration to challenging and unstable proteins.

Synthesis and biological activity of a potent optically pure autoinducer-2 quorum sensing agonist.

Quorum sensing (QS) regulates population-dependent bacterial behaviours, such as toxin production, biofilm formation and virulence. Autoinducer-2 (AI-2) is to date the only signalling molecule known to foster inter-species bacterial communication across distantly related bacterial species. In this work, the synthesis of pure enantiomers of C4-propoxy-HPD and C4-ethoxy-HPD, known AI-2 analogues, has been developed. The optimised synthesis is efficient, reproducible and short. The (4S) enantiomer of C4-propoxy-HPD was the most active compound being approximately twice as efficient as (4S)-DPD and ten-times more potent than the (4R) enantiomer. Additionally, the specificity of this analogue to bacteria with LuxP receptors makes it a good candidate for clinical applications, because it is not susceptible to scavenging by LsrB-containing bacteria that degrade the natural AI-2. All in all, this study provides a new brief and effective synthesis of isomerically pure analogues for QS modulation that include the most active AI-2 agonist described so far.

Syntheses of the plant auxin conjugate 2-O-(indole-3-acetyl)-myo-inositol IAInos.

The plant hormone conjugate 2-O-(indole-3-acetyl)-myo-inositol (IAInos) has been selectively prepared for the first time by two routes from myo-inositol. One of the syntheses depended upon the construction of the 3-indoleacetyl group by a Fischer indole synthesis on an unreactive axial hydroxyl group, while the other via a direct acylation of the equatorially orientated hydroxy group created by conformational constraint of the cyclohexane ring. The latter synthesis produced IAInos in 5 steps and 29% overall yield.

Peptidomimetic ß-Secretase Inhibitors Comprising a Sequence of Amyloid-ß Peptide for Alzheimer's Disease.

Alzheimer's disease is a grave social problem in an aging population. A major problem is the passage of drugs through the blood-brain barrier. This work tests the hypothesis that the conjugation of peptidomimetic ß-secretase inhibitors with a fragment of amyloid-ß peptide facilitates entrance into the central nervous system. HVR-3 (compound 4), one of the conjugation products, was found to be as potent as OM00-3, a known peptidomimetic inhibitor, 4-fold more selective toward ß-secretase 1 in relation to ß-secretase 2 and 3-fold more resistant to in vitro metabolization in human serum. Its intravenous administration to mice and Wistar rats generated an active metabolite recovered from the rodent's brains.

The Aza-Wharton reaction: syntheses of cyclic allylic amines and vicinal hydroxyamines from the respective acylaziridines.

The Wharton reaction, initially described for acyl epoxides, has been studied using the structurally similar aziridines. By this reaction, a range of cyclic allylic amines and vicinal amino alcohols have been prepared stereoselectively and, in some cases, enantiomerically pure.

Use of aziridines for the stereocontrolled synthesis of (-)-LL-C10037α, (+)-MT35214, and (+)-4-epi-MT35214.

Strategies for the synthesis of the title compounds have been developed using a diastereoselective aziridination reaction of 4-O-substituted cyclohexenones. Aziridination using a chiral amine permitted resolution of a 4-hydroxycyclohexane derivative, and this resulted in the synthesis of both enantiomers of the title compound. Alternatively, the chiral 4-hydroxycyclohexenone starting material was derived from quinic acid. In both cases stereoselective epoxidation and opening of the aziridine ring with hydrazoic acid afforded the 2-azidocyclohexenone, which was transformed to the 2-acetamido group present in the natural product.

Metal-free direct amination/aromatization of 2-cyclohexenones to iodo-N-arylanilines and N-arylanilines promoted by iodine.

An iodine mediated aromatization leading to a one-pot synthesis of iodo-N-arylanilines and N-arylanilines is reported. This highly regioselective aliphatic-aromatic transformation can be performed with various combinations of 2-cyclohexenones and anilines. The presence of a directing group is crucial for achieving high yields.

Stereochemical diversity of AI-2 analogs modulates quorum sensing in Vibrio harveyi and Escherichia coli.

Bacteria coordinate population-dependent behaviors such as virulence by intra- and inter-species communication (quorum sensing). Autoinducer-2 (AI-2) regulates inter-species quorum sensing. AI-2 derives from the spontaneous cyclisation of linear (S)-4,5-dihydroxypentanedione (DPD) into two isomeric forms in dynamic equilibrium. Different species of bacteria have different classes of AI-2 receptors (LsrB and LuxP) which bind to different cyclic forms. In the present work, DPD analogs with a new stereocenter at C-5 (4,5-dihydroxyhexanediones (DHDs)) have been synthesized and their biological activity tested in two bacteria. (4S,5R)-DHD is a synergistic agonist in Escherichia coli (which contains the LsrB receptor), while it is an agonist in Vibrio harveyi (LuxP), displaying the strongest agonistic activity reported so far (EC(50)=0.65µM) in this organism. Thus, modification at C-5 opens the way to novel methods to manipulate quorum sensing as a method for controlling bacteria.

An efficient synthesis of the precursor of AI-2, the signalling molecule for inter-species quorum sensing.

Autoinducer-2 (AI-2) is a signalling molecule for bacterial inter-species communication. A synthesis of (S)-4,5-dihydroxypentane-2,3-dione (DPD), the precursor of AI-2, is described starting from methyl glycolate. The key step was an asymmetric reduction of a ketone with (S)-Alpine borane. This new method was highly reproducible affording DPD for biological tests without contaminants. The biological activity was tested with the previously available assays and compared with a new method using an Escherichia coli reporter strain thus avoiding the use of the pathogenic Salmonella reporter.

Synthesis of potassium (2R)-2-O-alpha-d-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute.

Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-thio-d-glucopyranoside, as a mixture of anomers, was employed for the stereoselective synthesis of the potassium salt of (2R)-2-O-alpha-d-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-2,3-dihydroxypropanoic acid (alpha-d-glucosyl-(1-->6)-alpha-d-glucosyl-(1-->2)-d-glyceric acid, GGG), a recently isolated compatible solute. The alpha-anomer was by far the major product of both glycosylation reactions using NIS/TfOH as activator.

Stereoselective alkylation of tartrate derivatives. A concise route to (+)-O-methylpiscidic acid and natural analogues.

The lithium enolate of (2S,3S,5S,6S)-dimethoxy-2,3-dimethyl-1,4-dioxane-5,6-dithiocarboxylate undergoes stereoselective mono- and/or dialkylations to afford two new stereogenic centers. The alkylation products obtained possessed a cis stereochemistry, which was confirmed by the synthesis of natural 4'-O-methylpiscidic acid dimethyl ester .

Highly stereoselective aldol reaction for the synthesis of gamma-lactones starting from tartaric acid.

A simple stereoselective process for the synthesis of highly substituted gamma-lactones was developed based on aldol reactions between the enolate of dioxanes derived from tartaric acid and aldehydes. A range of aromatic and aliphatic aldehydes were reacted, in most cases achieving good yields and stereoselectivity. The limitations of this reaction were identified and a transition state is proposed.

Improved anomeric selectivity for the aroylation of sugars.

By manipulating the solvent and using bulky TMEDA as a base, good yields and improved anomeric selectivities were obtained for the aroylation of D-glucose over similar esterifications using pyridine. The reaction has been extended to mannose and the beta-anomer of pergalloylated mannose was predominantly obtained in one step by direct aroylation of the parent sugar.

Aziridines as a protecting and directing group. Stereoselective synthesis of (+)-bromoxone.

[reaction: see text] The directing ability of an aziridine group for the epoxidation of adjacent double bonds is demonstrated. The aziridine group is also used to effectively protect a double bond in a cycloenone system for a short synthesis of the title compound.

Aldol reactions of dioxanes derived from tartaric acid. A total synthesis of (+)-nephrosteranic acid.

[reaction: see text]. A general enantioselective synthesis of the paraconic acids was developed. The key step was a highly stereoselective aldol reaction between a dioxane dithioester derived from l-tartaric acid and a suitable aldehyde.

Photochemistry of flavothione and hydroxyflavothiones: mechanisms and kinetics.

In this work we present a detailed study of the mechanism of photochemistry and thermal reactions, as well as of the kinetics of flavothione (FLT) in ethanol. Furthermore, we analyzed how the hydroxysubstitution pattern of FLT influenced both the kinetics and the mechanism relative to the parent FLT. We show that the primary photochemical reaction of FLT in the absence of oxygen is hydrogen (H)-atom abstraction from the solvent by way of the excited triplet state of FLT. Several products result from thermal reactions of the resulting semireduced FLTH* radical, including more than one dimer. A full mechanism is proposed, and the relevant rate constants are evaluated. On the other hand, in the presence of oxygen and a low concentration of FLT, we found that the principal photoproduct is the parent flavone (FL). The reaction leading to photoxidation is not via 1O2 attacking a thione, but instead, it is via a reaction of the FLTH* radical with ground state oxygen. The kinetic data also demonstrate that the relative values of concentrations of reactants and the rate constants of the reactions can control the dominance of one mechanism over others. We also have examined the photochemical mechanisms and kinetics for several hydroxyflavothiones (n-OHFLT) and compared them with FLT itself. We have found that the photochemical mechanism radically changes depending on the positions of substitution. These differences are directly related to the ordering of the excited states of the n-OHFLT. Specifically, FLT with lowest 3n,pi* states (FLT, 6-hydroxyflavothione, 7-hydroxyflavothione and 7,8-dihydroxyflavothione) efficiently abstract H atoms to give the semireduced radical of the thione. The radical can (1) dimerize to form two different dimers; (2) react with oxygen to produce the parent FL; and (3) recombine with the solvent radical to yield the original FLT. In contrast, FLT with lowest 3pi,pi* states (3-hydroxyflavothione, 3,6-dihydroxyflavothione and 3,7-dihydroxyflavothione) behave as photosensitizers of oxygen to form singlet oxygen, which then reacts with the ground state of the substituted FLT. Finally, when T2(pi,pi*) is above S1(n,ppi*), as for 5-hydroxyflavothione and 5,7-dihydroxyflavothione, both the S1(n,pi*) --> T1(n,pi*) intersystem crossing and photodegradation are inefficient.

Stereoselective radical reactions of some tartaric and glyceric acid derivatives.

[reaction: see text] Free radicals generated by the decarboxylation of dimethoxydioxanecarboxylic acids derived from L-(+)-tartaric acid and L-glyceric acid added to some maleimides and acrylates with high stereoselectivity. This method provides easy access to some chiral building blocks.