Frédéric Fotiadu

CO2 Binding by Dynamic Combinatorial Chemistry: An Environmental Selection

We now report that a dynamic combinatorial selection approach can quantitatively provide, from trivial building blocks, an architecturally complex organic material, in which carbon dioxide is reversibly but covalently incorporated as a guest with a mass content of 20%. Solid-state analyses combined with covalent disconnection and quantization of the liberated components allowed identification of a three-component monomeric unit repeated within a range of assembled oligomeric adducts whose repartition and binding capacity can be finely tuned through the starting stoichiometries. The self-assembly of these architectures occurs through the simultaneous creation of more than 25 covalent bonds per molecular entity. It appears that the thermodynamic selection is directed by the packing efficiency of these adducts, explaining the spectacular building block discrimination between homologues differing by one carbon unit. This selectivity, combined with the reversible nature of the system, provided pure molecular building blocks after a simple chemical disconnection, promoting CO(2) as a green auxiliary to purify polyaldehyde or polyamine from mixtures of homologous structures. Moreover, the gas template could be expelled as a pure compound under thermodynamic control. This cooperative desorption process yielded back the initial libraries of high molecular diversity with a promising reduction of the energetic costs of capture and recycling.

Enantioselective reduction of ketones by borane catalysed by oxazaphospholidine oxides

Abstract New enantioselective catalysts for the asymmetric reduction of ketones by borane are described. Easily prepared by oxidation of chiral oxazaphospholidines, these compounds increase sensitively the reduction rate of numerous ketones and induce ees up to 94% in the case of the 2-chloroacetophenone.

On-line near infrared spectroscopy as a Process Analytical Technology (PAT) tool to control an industrial seeded API crystallization

The final step of an active pharmaceutical ingredient (API) manufacturing synthesis process consists of a crystallization during which the API and residual solvent contents have to be quantified precisely in order to reach a predefined seeding point. A feasibility study was conducted to demonstrate the suitability of on-line NIR spectroscopy to control this step in line with new version of the European Medicines Agency (EMA) guideline [1]. A quantitative method was developed at laboratory scale using statistical design of experiments (DOE) and multivariate data analysis such as principal component analysis (PCA) and partial least squares (PLS) regression. NIR models were built to quantify the API in the range of 9-12% (w/w) and to quantify the residual methanol in the range of 0-3% (w/w). To improve the predictive ability of the models, the development procedure encompassed: outliers elimination, optimum model rank definition, spectral range and spectral pre-treatment selection. Conventional criteria such as, number of PLS factors, R(2), root mean square errors of calibration, cross-validation and prediction (RMSEC, RMSECV, RMSEP) enabled the selection of three model candidates. These models were tested in the industrial pilot plant during three technical campaigns. Results of the most suitable models were evaluated against to the chromatographic reference methods. Maximum relative bias of 2.88% was obtained about API target content. Absolute bias of 0.01 and 0.02% (w/w) respectively were achieved at methanol content levels of 0.10 and 0.13% (w/w). The repeatability was assessed as sufficient for the on-line monitoring of the 2 analytes. The present feasibility study confirmed the possibility to use on-line NIR spectroscopy as a PAT tool to monitor in real-time both the API and the residual methanol contents, in order to control the seeding of an API crystallization at industrial scale. Furthermore, the successful scale-up of the method proved its capability to be implemented in the manufacturing plant with the launch of the new API process.

MmPPOX Inhibits Mycobacterium tuberculosis Lipolytic Enzymes Belonging to the Hormone-Sensitive Lipase Family and Alters Mycobacterial Growth

Lipid metabolism plays an important role during the lifetime of Mycobacterium tuberculosis, the causative agent of tuberculosis. Although M. tuberculosis possesses numerous lipolytic enzymes, very few have been characterized yet at a biochemical/pharmacological level. This study was devoted to the M. tuberculosis lipolytic enzymes belonging to the Hormone-Sensitive Lipase (HSL) family, which encompasses twelve serine hydrolases closely related to the human HSL. Among them, nine were expressed, purified and biochemically characterized using a broad range of substrates. In vitro enzymatic inhibition studies using the recombinant HSL proteins, combined with mass spectrometry analyses, revealed the potent inhibitory activity of an oxadiazolone compound, named MmPPOX. In addition, we provide evidence that MmPPOX alters mycobacterial growth. Overall, these findings suggest that the M. tuberculosis HSL family displays important metabolic functions, thus opening the way to further investigations linking the involvement of these enzymes in mycobacterial growth.

Analysis of the discriminative inhibition of mammalian digestive lipases by 3-phenyl substituted 1,3,4-oxadiazol-2(3H)-ones.

We report here the reactivity and selectivity of three 5-Methoxy-N-3-Phenyl substituted-1,3,4-Oxadiazol-2(3H)-ones (MPOX, as well as meta and para-PhenoxyPhenyl derivatives, i.e.MmPPOX and MpPPOX) with respect to the inhibition of mammalian digestive lipases: dog gastric lipase (DGL), human (HPL) and porcine (PPL) pancreatic lipases, human (HPLRP2) and guinea pig (GPLRP2) pancreatic lipase-related proteins 2, human pancreatic carboxyl ester hydrolase (hCEH), and porcine pancreatic extracts (PPE). All three oxadiazolones displayed similar inhibitory activities on DGL, PLRP2s and hCEH than the FDA-approved anti-obesity drug Orlistat towards the same enzymes. These compounds appeared however to be discriminative of HPL (poorly inhibited) and PPL (fully inhibited). The inhibitory activities obtained experimentally in vitro were further rationalized using in silico molecular docking. In the case of DGL, we demonstrated that the phenoxy group plays a key role in specific molecular interactions within the lipases active site. The absence of this group in the case of MPOX, as well as its connectivity to the neighbouring aromatic ring in the case of MmPPOX and MpPPOX, strongly impacts the inhibitory efficiency of these oxadiazolones and leads to a significant gain in selectivity towards the lipases tested. The powerful inhibition of PPL, DGL, PLRP2s, hCEH and to a lesser extend HPL, suggests that oxadiazolone derivatives could also provide useful leads for the development of novel and more discriminative inhibitors of digestive lipases. These inhibitors could be used for a better understanding of individual lipase function as well as for drug development aiming at the regulation of the whole gastrointestinal lipolysis process.

Use of new chiral tricoordinated phosphorus borane complexes in enantioselective borane reduction of ketones: Complexes structure and mechanistic studies

New tricoordinated phosphorus borane complexes were synthesized and their use as catalysts in enantioselective borane reduction of prochiral aromatic and aliphatic ketones was investigated. The structure of (2R,5S)-2-o-anisyl-3-oxa-1-aza phosphabicyclo[3.3.0]octane—borane complex 1b and (2R,5S)-2,3-diphenyl-1,3-diazaphosphabicyclo[3.3.0]octane—borane complex 6a was established by X-ray diffraction analysis. A relationship has been established between the structure of the oxazaphospholidine borane complexes and the enantioselectivity obtained in the reduction of acetophenone, both with 2 mol% and one equivalent of the catalyst. Among the different oxazaphospholidine borane complexes tested, only the complexes 1–3, including 3-oxa-l-azaphosphabicyclo[3.3.0]octane and 3-oxa-l-azaphosphabicyclo[4.3.0]nonane moieties, were efficient catalysts. A rational mechanism is proposed according to the experimental results, especially from a deuterium labelling study.

Surface properties of unsaturated non-oxidized and oxidized free fatty acids spread as monomolecular films at an argon/water interface.

The interfacial properties of monomolecular films of stearic acid (SA) oleic acid (OA), linoleic acid (LA), ricinoleic acid (RA), 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (13-HPODE) and 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid (13-HODE) were studied by recording the changes occurring in response to monomolecular film compression in their surface pressure and surface potential at the argon/water interface. The oxidized free fatty acids are more expanded than the parent non-oxidized free fatty acids, reflecting a higher hydrophilic-lipophilic balance. The lift-off values of the molecular area of 13-HODE, 13-HPODE and RA were 68, 74 and 106 A2 molecule(-1), respectively, as compared to 47 and 40 A2 molecule(-1) in the case of LA and OA, respectively. Variations in the molecular orientation of free fatty acids can result in large changes in the dipole moment which are not accompanied by appreciable changes in the surface pressure. In the case of the oxidized free fatty acids, the spontaneous desorption into the aqueous phase was found to increase at increasing surface pressures. The desorption rates of OA and LA increased dramatically in the presence of beta-cyclodextrin (beta-CD); whereas the presence of beta-CD only slightly increased the desorption rates of the oxidized free fatty acids.

A Process Analytical Technology (PAT) approach to control a new API manufacturing process: Development, validation and implementation

Pharmaceutical companies are progressively adopting and introducing Process Analytical Technology (PAT) and Quality-by-Design (QbD) concepts promoted by the regulatory agencies, aiming the building of the quality directly into the product by combining thorough scientific understanding and quality risk management. An analytical method based on near infrared (NIR) spectroscopy was developed as a PAT tool to control on-line an API (active pharmaceutical ingredient) manufacturing crystallization step during which the API and residual solvent contents need to be precisely determined to reach the predefined seeding point. An original methodology based on the QbD principles was designed to conduct the development and validation of the NIR method and to ensure that it is fitted for its intended use. On this basis, Partial least squares (PLS) models were developed and optimized using chemometrics methods. The method was fully validated according to the ICH Q2(R1) guideline and using the accuracy profile approach. The dosing ranges were evaluated to 9.0-12.0% w/w for the API and 0.18-1.50% w/w for the residual methanol. As by nature the variability of the sampling method and the reference method are included in the variability obtained for the NIR method during the validation phase, a real-time process monitoring exercise was performed to prove its fit for purpose. The implementation of this in-process control (IPC) method on the industrial plant from the launch of the new API synthesis process will enable automatic control of the final crystallization step in order to ensure a predefined quality level of the API. In addition, several valuable benefits are expected including reduction of the process time, suppression of a rather difficult sampling and tedious off-line analyses.

In vitro stereoselective hydrolysis of diacylglycerols by hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids in adipocytes and shows a substrate preference for the diacylglycerols (DAGs) originating from triacylglycerols. To determine whether HSL shows any stereopreference during the hydrolysis of diacylglycerols, racemic 1,2(2,3)-sn-diolein was used as a substrate and the enantiomeric excess (ee%) of residual 1,2-sn-diolein over 2,3-sn-diolein was measured as a function of DAG hydrolysis. Enantiomeric DAGs were separated by performing chiral-stationary-phase HPLC after direct derivatization from lipolysis product extracts. The fact that the ee% of 1,2-sn-diolein over 2,3-sn-diolein increased with the level of hydrolysis indicated that HSL has a preference for 2,3-sn-diolein as a substrate and therefore a stereopreference for the sn-3 position of dioleoylglycerol. The ee% of 1,2-sn-diolein reached a maximum value of 36% at 42% hydrolysis. Among the various mammalian lipases tested so far, HSL is the only lipolytic carboxylester hydrolase found to have a pronounced stereospecificity for the sn-3 position of dioleoylglycerol.

An ultraviolet spectrophotometric assay for the screening of sn-2-specific lipases using 1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol as substrate

In the present study, we propose a continuous assay for the screening of sn-2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn-2 position and the oleic acid (OA) at the sn-1 and sn-3 positions [1,3-O-dioleoyl-2-O-α-eleostearoyl-sn-glycerol (sn-OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn-1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn-2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn-OEO and the tung oil TAGs, but only CALA hydrolyzed the sn-2 position. The ratio of initial velocities on sn-OEO and tung oil TAGs was used to estimate the sn-2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn-2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn-2 lipases.