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The performance of a HSA-based HPLC column has been improved by modulating the binding properties of the anchored protein using lithocholic acid as a selective modifier in the mobile phase. In particular, retention decreased for both drugs binding to sites I or II, while the enantioselectivity was maintained for most analytes. The advantages of using lithocholic acid in the mobile phase were impressive in the case of profens, for which a considerable reduction in analysis time and a significant increase in enantioselectivity were obtained. The results were rationalized by considering lithocholic acid as a direct competitor for site II binding drugs and indirect competitor for site I binding drugs.

Pig liver esterase (PLE) afforded smooth chemical resolution of racemic N -substituted 4-(benzoyloxy)-3-carbomethoxypiperidines. The enzyme showed good chemo- and enantioselective properties, thus allowing discrimination between the carbomethoxy and benzoate ester groups, the latter being more easily hydrolyzed. The proposed methodology also represents a practical means for the procurement of N -substituted 4-hydroxy-3-carbomethoxypiperidines in enantiomerically pure form.

Herpes simplex virus 1 (HSV-1) entry into cells and cell-cell fusion mediated by HSV-1 glycoproteins require four glycoproteins, gD, gB, gH, gL. Of these, gH is the only one that so far exhibits structural-functional features typical of viral fusion glycoproteins, i.e., a candidate fusion peptide and, downstream of it, a heptad repeat (HR) segment able to form a coiled coil, named HR-1. Here, we show that gH carries a functional HR-2 capable of physical interaction with HR-1. Specifically, mutational analysis of gH aimed at increasing or decreasing the ability of HR-2 to form a coiled coil resulted in an increase or decrease of fusion activity, respectively. HSV infection was modified accordingly. A mimetic peptide with the HR-2 sequence inhibited HSV-1 infection in a specific and dose-dependent manner. Circular dichroism spectroscopy showed that both HR-2 and HR-1 mimetic peptides adopt mainly random conformation in aqueous solution, while a decrease in peptide environmental polarity determines a conformational change, with a significant increase of the α-helical conformation content, in particular, for the HR-1 peptide. Furthermore, HR-1 and HR-2 mimetic peptides formed a stable complex, as revealed in nondenaturing electrophoresis and by circular dichroism. The mixture of HR-1 and HR-2 peptides reversed the inhibition of HSV infection exerted by the single peptides. Complex formation between HR-1 and HR-2 was independent of the presence of adjacent gH sequences and of additional glycoproteins involved in entry and fusion. Altogether, HR-2 adds to the features typical of class 1 fusion glycoproteins exhibited by HSV-1 gH.

A human serum albumin (HSA)-based HPLC column has been modified in situ by disulfiram, an alcohol-deterrent drug reported to bind cys34, the only free cysteine in HSA, under physiological conditions.

The circular dichroism spectrum of (+)-5,6,7,8-tetrahydro-8,9,9-trimethyl-5,8-methanoquinoline, a molecule containing a pyridine chromophore perturbed by an alkyl moiety has been carried out down to 170 nm through the region of the allowed π→π* transitions.

C.d. and u.v. absorption bands of (R)-3,3-dimethyl-2-methylaminobutane in the 250–180 nm spectral region are reported and assigned to Rydberg-type transitions on the basis of c.d. data at low temperature.

The c.d. spectra of three chiral open-chain saturated ethers are described in the spectral region 140–200 nm.

Circular dichroism spectra recorded far into the vacuum u.v. region for solutions of a simple ether demonstrate that the positions of the bands depend on the extent of hydrogen bonding with the solvent.

This dissertation is divided into three parts. The first part aimed to investigate the cannabidiol anxiolytic effect in treatment-naïve individuals with social anxiety disorder through simulation of public speaking. Twenty-four never-treated social anxiety disorder subjects were allocated to receive 0 or 600 mg cannabidiol (CBD; n=12) in a double-blind randomized design. The same number of controls performed the simulation of a public speaking test without receiving any medication. Pretreatment with CBD significantly reduced anxiety, cognitive impairment, and discomfort in speech performance and significantly decreased alertness in their anticipatory speech. The placebo group displayed higher anxiety, cognitive impairment, discomfort, and alertness when compared with controls as assessed with the Visual Analogue Mood Scale (VAMS). The SSPS-N scores showed significant increases during testing of the placebo group that was almost abolished in the cannabidiol group. No significant differences were observed between the cannabidiol and control groups in SSPS-N scores or in cognitive impairment, discomfort, and alertness factors of the VAMS. The second part evaluated healthy subjects\' x y during a public speaking test following a high rimonabant oral dose, to understand better the possible pharmacological approaches for anxiety disorder treatment. Twenty four participants were randomly allocated to receive 0 or 90 mg rimonabant (n=12) in a double-blind design. No significant adverse effects were reported in either group. Participants who received rimonabant showed increased anxiety levels compared to placebo during anticipatory speech and performance measurements. Rimonabant treatment did not affect sedation, cognitive impairment, discomfort, blood pressure, heart rate, self-statements during public speaking, or bodily symptoms scales. Increased anxiety may reflect lower endocannabinoid activity in CB1 receptors and CB1 p \' possible role in modulation of anxiety and anxiety disorders. The third part aimed to monitor cannabinoid blood concentrations during sustained abstinence from chronic daily cannabis smoking. Thirty male chronic daily cannabis smokers resided on a secure clinical research unit for up to 33 days, with blood collected once daily. ?9-tetrahydrocannabinol (THC), 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) whole blood concentrations were quantified by two-dimensional gas chromatography-mass spectrometry. Twenty-seven of 30 participants were THC-positive on admission, with a median (range) concentration 1.4 ng/mL (0.3-6.3). THC decreased gradually with only 1 of 11 participants negative at 26 days; 2 of 5 participants remained THC-positive (0.3 ng/mL) for 30 days. 5.0% f p p h TH >=1 0 g/ L f 12 y M 11-OH-THC w 1 1 g/ L w h >=1 0 g/ L 24h THCCOOH detection rates were 96.7 on admission, decreasing slowly to 95.7 and 85.7% on days 8 and 22, respectively; four of 5 participants remained THCCOOH positive (0.6-2.7 ng/mL) after 30 days and one remained positive on discharge at 33 days. THC was quantified in some participants for 30 days, albeit in low concentrations, due to the large cannabinoid body burden from extended exposure Esta tese é dividida em três partes. A primeira parte consiste em investigar o efeito ansiolítico do canabidiol na ansiedade social através do teste de simulação de falar em público. Vinte e quatro sujeitos com ansiedade social, nunca tratados, receberam placebo ou canabidiol (CBD) 600 mg (n=12) em um estudo randomizado e duplo-cego. O mesmo número de indivíduos saudáveis realizaram o teste de simulação de falar em público sem receber medicação. A administração do CBD reduziu significativamente a ansiedade, sedação física e outros sentimentos e atitudes durante a fase de estresse, e diminui o nível de alerta na fase pré-estresse. O grupo placebo apresentou níveis elevado de ansiedade, sedação física, outros sentimentos e atitudes, e alerta comparado com o grupo controle. A pontuação do SSPS-N evidenciou aumento significativo durante o teste no grupo placebo, enquanto que o CBD reduziu estes níveis. Não houve diferenças significativas entre os grupos CBD e controle na SSPS-N e nos fatores sedação física, outros sentimentos e atitudes e alerta, da Visual Analogue Mood Scale (VAMS). A segunda parte do estudo avaliou a ansiedade em indivíduos saudáveis que receberam alta dose oral de rimonabanto e submetidos ao teste de simulação de falar em público, para melhor entendimento do possível mecanismo farmacológico para tratamento de transtornos de ansiedade. Vinte e quatro sujeitos saudáveis receberam placebo ou rimonabanto 90 mg (n=12) em um randomizado e duplo-cego. Não foi observado efeitos adversos significativo em ambos grupos. O grupo rimonabanto apresentou maiores níveis de ansiedade na fase pré-estresse e durante o estresse. Não houve diferença significativa quanto aos demais fatores avaliados entre os grupos. O aumento na ansiedade após administração do rimonabanto pode-se ao fato de haver diminuição no sistema endocanabinóide nos receptores CB1 e a possível modulação na ansiedade clínica e patológica. A terceira parte objetivou quantificar canabinóides no sangue total em usuários crônicos de cannabis durante abstinência supervisionada. Trinta usuários crônicos de cannabis, do sexo masculino, permaneceram no centro de pesquisa por até 33 dias, com coleta de sangue uma vez ao dia. ?9-tetrahidrocanabinol (THC), 11-hidróxi-THC (11-OH-THC) e 11-nor-9-carbóxi-THC (THCCOOH) foram quantificados no sangue por meio da cromatografia gasosa-espectrometria de massa bidimensional. Vinte e sete de 30 usuários foram positivos para THC no ingresso do estudo, com concentração mediana (variação) de 1.4 ng/mL (0.3-6.3). Níveis de THC diminuíram gradativamente com somente 1 de 11 participantes negativo no dia 26; 2 de 5 indivíduos permaneceram positivos para THC (0.3 g/ L p 30 5 0% j TH >=1 0 g/ L p 12 ç mediana de 11-OH-TH f 1 1 g/ L g >=1 0 g/ L pó 24h. A taxa de detecção de THCCOOH foi 96.7% no ingresso, diminuindo gradativamente para 95.7 e 85.7% nos dias 8 e 22, respectivamente; 4 de 5 sujeitos permaneceram positivo para THCCOOH (0.6-2.7 ng/mL) após 30 dias e um permaneceu positivo no 33º dia. Foi detectado THC em alguns indivíduos por 30 dias, porém em baixas concentrações, devido a extensa eliminação do canabinóide em decorrência da exposição crônica

Fenoterol is a selective β 2 -adrenergic receptor (AR) agonist used in the treatment of asthma, which is under scrutiny as possible drug against congestive heart failure. Fenoterol is currently used as a racemic mixture of the (R,R′)- and (S,S′)-enantiomers, whose stereochemistry was previously determined by electronic circular dichroism (ECD) analysis through the application of a semi-empirical sector rule. [1] Recently, a series of fenoterol derivatives has been synthesized and tested: [2-3] their absolute configuration was determined by chemical correlation with synthetic precursors, but no further stereochemical characterization has been carried out. In addition, stereoselectivity in the binding to β 2 - AR has been demonstrated, with (R,R′)- and (R,S′)-fenoterol displaying higher affinity compared to (S,R′)- and (S,S′)-fenoterol, and a tridimensional quantitative structure-activity relationship model (3D- QSAR) based on comparative molecular field analysis (CoMFA) has been developed to rationalize the binding of fenoterol derivatives to β 2 -AR. [2-3] The relationship between chiroptical properties and absolute stereochemistry of fenoterol and its derivatives has been investigated [4] by experimental ECD analysis and quantum chemical (QC) calculations using time-dependent density functional theory (TD-DFT). [5-6] The stereoisomers of three compounds have been considered: fenoterol (1), (4′′-methoxy-1′′-naphthyl)fenoterol (2), and (4′′-methoxy- 1′-desmethyl)fenoterol (3). Due to the high conformational flexibility of the investigated structures and the consequent large pool of equilibrium conformers, DFT geometry optimizations were carried out using the resolution of identity (RI) approximation and the B97D functional with empirical dispersion corrections; [7] TD-DFT calculations were then performed using the PBE0 functional. The accuracy of this protocol in reproducing the experimental ECD spectra of fenoterol derivatives will be evaluated. [1] F. Beigi, C. Bertucci, W. Zhu, K. Chakir, I.W. Wainer, R.P. Xiao, D.R. Abernethy, Chirality, 2006, 18, 822-827. [2] K. Jóźwiak, K. Chakir, M.J. Tanga, I. Berzetei-Gurske, L. Jimenez, J.A. Kozocas, A. Woo, W. Zhu, R.P. Xiao, D.R. Abernethy, I.W. Wainer, J. Med. Chem., 2007, 50, 2903-2915. [3] K. Jóźwiak, A.Y.H. Woo, M.J. Tanga, L. Toll, L. Jimenez, J.A. Kozocas, A. Plazinska, R.P. Xiao, I.W. Wainer, Bioorg. Med. Chem., 2010, 18, 728-736. [4] ISCRA project IsC08_SCCFPM (ID: HP10CU0YHL) granted by CINECA, Bologna, Italy. [5] J. Autschbach, Chirality, 2009, 21, E116-E152. [6] L. Goerigk, H. Kruse, S. Grimme, in: N. Berova, P.L. Polavarapu, K. Nakanishi, R.W. Woody (Eds.), Comprehensive chrioptical spectroscopy, vol. 1, 2011, Hoboken: Wiley & Sons, pp. 643-673. [7] S. Grimme, WIREs Comput. Mol. Sci., 2011, 1, 211-228.

At the molecular level, the activity of drugs relies on a sequence of biorecognition phenomena, such as the binding to biological macromolecules, which define their pharmacological, toxicological and pharmacokinetic profiles. Physiologically-relevant interactions are usually very stereospecific: the occurrence of high binding affinities for specific stereochemical configurations or conformational arrangements of a drug is a very important research topic in the field of medicinal chemistry. When a drug is bound with high affinity to its target or to a carrier protein, e.g. a serum albumin, the conformational restraint due to the interaction may result in the insurgence of induced circular dichroism (ICD), which can be detected experimentally by spectroscopic analysis: nice examples are given by the ICD spectra of diazepam and ketoprofen complexed with human serum albumin (HSA). [1] Quantum mechanical (QM) calculations based on time-dependent density functional theory (TD-DFT) can be used to investigate the theoretical ICD spectra of all the possible conformational arrangements of the ligand, and may result very helpful in the identification of possible binding modes. [2] The present communication will report the application of TD-DFT calculations to the investigation of the binding modes of ketoprofen and benzodiazepines to serum albumins: in particular, the stereospecific binding of the M conformation of benzodiazepines to HSA is confirmed, and the peculiar species-dependent ICD spectra observed for the binding of ketoprofen to different serum albumins can be explained by the selection of different mutual arrangements of the phenyl moieties in the binding pockets. [1] M. Pistolozzi, C. Bertucci, Chirality 20, 552 (2008). [2] S. Ionescu, I. Matei, C. Tablet, M. Hillebrand, Phys. Chem. Chem. Phys. 15, 11604 (2013).

This study shows that the accuracy of ab initio MD simulations in the description of short-range solute-solvent interactions, although very demanding in terms of computational effort, may eventually be necessary to characterize subtle solvent-related perturbations to the calculated chiroptical properties of chiral molecules: in fact, TD-DFT calculations without explicit treatment of solvation would have led to a wrong assessment of absolute configuration for austdiol.

Time-dependent density functional theory (TD-DFT) has become the standard method for chiroptical properties calculations on small-to-medium sized molecules, due to the reasonable balance between chemical accuracy and computational efficiency [1]. However, the intrinsic shortcomings of TD-DFT methods and the high demands in terms of computational resources are still limiting a broader applicability to pharmaceutically relevant systems [2]. The description of solvation is arguably one of the most challenging tasks in computational chiroptical spectroscopy [3]; solvation may exert its influence on chiroptical properties at several levels, such as electric perturbations to the electronic transitions and conformational perturbations due to weak intermolecular interactions. TD-DFT calculations are then very likely to fail in predicting the correct properties if solvation is not accurately described. The fungal metabolite austdiol is the building block of a series of newly discovered natural products from Mycoleptodiscus indicus, named mycoleptones A–C; the stereochemistry of mycoleptone A was fully characterised by a combination of ECD and NMR spectroscopies, TD-DFT calculations and X-ray crystallography [4]. Austdiol, however, shows the importance of a proper description of solvation effects for a correct stereochemical characterisation: when solvation is only described with a continuum solvation model, the wrong absolute configuration (AC) is predicted from the calculated [α]D values. When solvation is treated explicitly through ab initio molecular dynamics (AIMD) simulations of the solvation dynamics of austdiol in methanol, the description of the chiral environment around the carbonyl chromophores is dramatically improved, and TD-DFT calculations are able to reproduce the experimental chiroptical properties with high accuracy. The improvement is mainly due to a small conformational change involving the hydroxyl group in α-position to the ketone moiety of austdiol, confirming the extreme sensitivity of chiroptical properties to small perturbations to the molecular geometry. The constant improvement of theoretical models and methods, both in terms of reliability and computational requirements, is a very promising path towards chemical accuracy, which will also benefit stereochemical and pharmaceutical analysis in the investigation of non-trivial effects on chiroptical properties in solvated systems and in the characterisation of the relationship between the AC and the biological activity of complex chiral drugs. [1] Autschbach, J. Computing Chiroptical Properties with First-Principles Theoretical Methods: Background and illustrative examples. Chirality 2009, 21, E116-E152. [2] Goerigk, L.; Kruse, H.; Grimme, S. Theoretical Electronic Circular Dichroism Spectroscopy of Large Organic and Supramolecular Systems. In Comprehensive chiroptical spectroscopy, vol. 1; Berova, N., Polavarapu, P. L., Nakanishi, K., Woody, R. W., Eds.; Hoboken: Wiley & Sons, 2012; pp 643-673. [3] Mennucci, B.; Cappelli, C.; Cammi, R.; Tomasi, J. Modeling Solvent Effects on Chiroptical Properties. Chirality 2011, 23, 717-729. [4] Andrioli, W.J.; Conti, R.; Araújo, M.J.; et al.. Mycoleptones A–C and Polyketides from the Endophyte Mycoleptodiscus indicus. J. Nat. Prod. 2014, 77, 70-78.

β-lactam rings play a key role in medicinal chemistry: β-lactam derivatives are widely used in therapy as antibiotics, cholesterol absorption and prostate-specific antigen inhibitors. Stereochemical characterisation is crucial for the application of β-lactam rings as chiral intermediates for the asymmetric synthesis of a variety of compounds of medicinal interest, such as proteinogenic and non-proteinogenic amino acids and taxanes. [1] The absolute stereochemistry of a series of synthetic (3R)-3-hydroxy-4-aryl-β-lactams was investigated by means of chiroptical spectroscopies, i.e. polarimetry and electronic circular dichroism (ECD), and quantum mechanical (QM) calculations using time- dependent density functional theory (TD-DFT). The absolute configuration at C3 was assigned by chemical correlation with synthetic precursors; the overall stereochemistry was assessed by application of the β-lactam sector rule, which relates the sign of the specific rotatory power ([α] D ) and the lowest-energy ECD band with the absolute configuration at C4. [1] The assessed absolute configurations were then confirmed by TD-DFT calculations. The β-lactam sector rule allowed a correct stereochemical characterisation of the investigated β-lactams, with the exception of a thienyl-substituted derivative (1). Conformationally-averaged TD-DFT calculations yielded accurate predictions of ECD spectra in 2-propanol and [α] D values in chloroform, which allowed to assign the correct absolute configuration to compound 1. [α] D values in acetone were successfully predicted by TD-DFT calculations with explicit treatment of solvent molecules at a QM level. [2] A detailed analysis of the geometric features of the β-lactam ring for all the equilibrium conformers, as obtained by DFT geometry optimisation, helped to identify regular patterns for the arrangement of atoms around the carbonyl chromophore, which is responsible for the chiroptical properties of β-lactam derivatives. This study showed the importance of conformational flexibility for the definition of chiroptical properties, and highlighted strengths and weaknesses of the different methods for the stereochemical characterisation of chiral molecules in solution. References [1] Barbaro, G., Battaglia, A., Guerrini, A., Bertucci, C., Geremia, S., Tetrahedron: Asymmetry 9 (1998), 3401-3409. [2] Tomasi, J., Mennucci, B., Cammi, R., Chem. Rev. 105 (2005), 2999-3093.

Difenoconazole (DFZ) is a chiral antimycotic agent belonging to the class of triazoles, which is extensively used as a pesticide in agriculture and is also employed as a drug in the treatment of fungal skin infections. The interest in the stereochemical characterization of pesticides is growing steadily in the field of environmental chemistry, similarly to what happened in medicinal chemistry. The stereoisomers of chiral pesticides may show stereoselective activities and toxicities, which can play a fundamental role in the development of enhanced formulations with increased potency and decreased environmental impact. The fungicidal bioactivity of DFZ shows this behaviour: the stereoisomer with the highest activity towards pathogens is also the least toxic towards non-target organisms and the least persisting in the environment, while the least active stereoisomer is also the most toxic and polluting. [1] The replacement of the commercial mix of stereoisomers with a stereoisomerically enriched formulation of the most active DFZ stereoisomer is therefore a promising strategy to reduce the environmental pollution arising from the use of this pesticide. The stereochemical characterization of the 4 DFZ stereoisomers (2 chiral centres) was carried out by a multidisciplinary approach which combines experimental spectroscopic techniques, such as 1D- and 2D- NMR and electronic circular dichroism (ECD), and ab initio calculations of the theoretical ECD spectra by time-dependent density functional theory (TD-DFT). [2] The relative configuration of the 2 enantiomeric pairs was determined by NMR, and the ECD spectra of all stereoisomers were recorded. Calculations were then carried out on one enantiomer for each of the 2 enantiomeric pairs: (2S,4S)- and (2S,4R)- DFZ. A total number of 37 input structures were considered in the calculations; a B97D/TZ2P level was used for DFT geometry optimizations, while a PBE0/TZ2P was used for TD-DFT calculations. The combination of this computational protocol with experimental spectroscopies allowed to assign the absolute configuration to each of the 4 DFZ stereoisomers, therefore assessing the stereochemistry of the most active stereoisomer as (2R,4S) and of the most toxic stereoisomer as (2S,4S). [1] [1] F. Dong, J. Li, B. Chankvetadze, Y. Cheng, J. Xu, X. Liu, Y. Li, X. Chen, C. Bertucci, D. Tedesco, R. Zanasi, Y. Zheng, Environ. Sci. Technol., 2013, 47, 3386-3394. [2] ISCRA project IsC08_SCCFPM (ID: HP10CU0YHL) granted by CINECA, Bologna, Italy.