Academic literature on the topic 'Proteins Enantiomers'

Biological polymers such as nucleic acids and proteins are constructed of only one—the d or l—of the two possible nonsuperimposable mirror images (enantiomers) of selected organic compounds. However, before the advent of life, it is generally assumed that chemical reactions produced 50:50 (racemic) mixtures of enantiomers, as evidenced by common abiotic laboratory syntheses. Carbonaceous meteorites contain clues to prebiotic chemistry because they preserve a record of some of the Solar System’s earliest (∼4.5 Gy) chemical and physical processes. In multiple carbonaceous meteorites, we show that both rare and common sugar monoacids (aldonic acids) contain significant excesses of the d enantiomer, whereas other (comparable) sugar acids and sugar alcohols are racemic. Although the proposed origins of such excesses are still tentative, the findings imply that meteoritic compounds and/or the processes that operated on meteoritic precursors may have played an ancient role in the enantiomer composition of life’s carbohydrate-related biopolymers.

ABSTRACT Sphingomonas paucimobilis B90A contains two variants, LinA1 and LinA2, of a dehydrochlorinase that catalyzes the first and second steps in the metabolism of hexachlorocyclohexanes (R. Kumari, S. Subudhi, M. Suar, G. Dhingra, V. Raina, C. Dogra, S. Lal, J. R. van der Meer, C. Holliger, and R. Lal, Appl. Environ. Microbiol. 68:6021-6028, 2002). On the amino acid level, LinA1 and LinA2 were 88% identical to each other, and LinA2 was 100% identical to LinA of S. paucimobilis UT26. Incubation of chiral α-hexachlorocyclohexane (α-HCH) with Escherichia coli BL21 expressing functional LinA1 and LinA2 S-glutathione transferase fusion proteins showed that LinA1 preferentially converted the (+) enantiomer, whereas LinA2 preferred the (−) enantiomer. Concurrent formation and subsequent dissipation of β-pentachlorocyclohexene enantiomers was also observed in these experiments, indicating that there was enantioselective formation and/or dissipation of these enantiomers. LinA1 preferentially formed (3S,4S,5R,6R)-1,3,4,5,6-pentachlorocyclohexene, and LinA2 preferentially formed (3R,4R,5S,6S)-1,3,4,5,6-pentachlorocyclohexene. Because enantioselectivity was not observed in incubations with whole cells of S. paucimobilis B90A, we concluded that LinA1 and LinA2 are equally active in this organism. The enantioselective transformation of chiral α-HCH by LinA1 and LinA2 provides the first evidence of the molecular basis for the changed enantiomer composition of α-HCH in many natural environments. Enantioselective degradation may be one of the key processes determining enantiomer composition, especially when strains that contain only one of the linA genes, such as S. paucimobilis UT26, prevail.

The paper presents studies on the presence or formation of d - enantiomers of amino acids in animal tissues or organs, in meat products during its production processes. It is shown that the process of epimerization of L - amino acid residues with the formation of D-enantiomers affect the reduction of the properties of food products, including the formation of oncoassociated subsequent effects on the human body.Modern control of the quantitative and qualitative composition of d-enantiomers of amino acids in food products, monitoring for stratification of the increased risk of toxic compounds in food are becoming an urgent medical and social problem. The studies planned in this paper are aimed at developing approaches to the creation of food products that reduce the oncogenic alimentary load on human health by solving the problem of technological modification of production, eliminating or minimizing post - translational modifications in proteins that contribute to the formation of d-enantiomers of amino acids. These studies will create a scientific and technological database associated with the risk assessment of carcinogenesis in protein matrices of animal origin. Based on the presented analysis, the task of developing and testing a method to control the accumulation of D-isomers in the course of various technological processes of meat production is extremely popular.

‘How did life start on Earth?’ and ‘Why were left-handed amino acids selected for the architecture of proteins?’ A new attempt to answer these questions of high public and interdisciplinary scientific interest will be provided by this review. It will describe most recent experimental data on how the basic and molecular building blocks of life, amino acids, formed in a prebiotic setting. Most amino acids are chiral, that is that they cannot be superimposed with their mirror image molecules (enantiomers). In processes triggering the origin of life on Earth, the equal occurrence, i.e. the parity between left-handed amino acids and their right-handed mirror images, was violated. In the case of amino acids, the balance was tipped to the left – as a result of which life's proteins today exclusively implement the left-handed form of amino acids, called l-amino acid enantiomers. Neither plants, nor animals, including humans, make use of d-amino acids for the molecular architecture of their proteins (enzymes). This review addresses the molecular asymmetry of amino acids in living organisms, namely the preference for left-handedness. What was the cause for the violation of molecular parity of amino acids in the emergence of life on Earth? All the fascinating models proposed by physicists, chemists, and biologists will be vividly presented including the scientific conflicts. Special emphasis will be given to amino acid enantiomers that were subjected to chiral photons. The interaction between racemic molecules and chiral photons was shown to produce an enantiomeric enrichment that will be discussed in the context of absolute asymmetric synthesis. The concluding paragraphs will describe the attempt to verify any of those models with the chirality-module of the Rosetta mission. This European space mission contains probe Philae that was launched on board the Rosetta spacecraft with the aim of landing on the icy surface of comet 67P/Churyumov-Gerasimenko and analysing whether chiral organic compounds are present that could have been brought to the Earth by comet impacts.

Chemical communication involves the production and release of specific chemicals (pheromones and other semiochemicals) by the emitter, and the detection and olfactory processing of these signals leading to appropriate behavioral responses in the receiver. In contrast to most of the scarab species investigated to date, the Japanese and Osaka beetles have the ability to detect the allospecific pheromone, which plays a pivotal role in the isolation mechanism between these two species. Each species produces a single enantiomer of japonilure [(Z)-5-(dec1-enyl)oxacyclopentan-2-one], but they have evolved the ability to detect both enantiomers, one as an attractant and the other as a behavioral antagonist (stop signal). There is growing evidence in the literature that the inordinate sensitivity and selectivity of the insect olfactory system is achieved by a combination of various olfactory-specific proteins, namely, odorant-binding proteins (OBPs), odorant receptors (ORs), and odorant-degrading enzymes. The relationship between the pheromone structures and the primary sequences of the proteins suggest that OBPs play a part in the selectivity of the olfactory system in scarab beetles by "filtering" chemical signals during the early olfactory processing (perireceptor events). Nevertheless, it is unlikely that pheromone-binding proteins are "chiral filters" as the Japanese and Osaka beetles each possess only one single binding protein. Upon interaction with negatively charged membranes, OBPs undergo conformational changes that may lead to the release of the ligands.

Nitrogen-inversion rates and diffusion coefficients were measured using 1H NMR for 14 drug-like molecules. The slow nitrogen-inversion rates interconverting the enantiomers of these molecules lay within a postulated intermediate range in terms of their ability to bind to proteins bounded by diffusion constraints, potentially affecting the availability, hence efficacy, of these compounds if they were utilized as drugs. The postulated intermediate range is based on a capture-volume concept, whereby the nitrogen inversion during the time a ligand takes to pass through a volume surrounding the protein binding site, as calculated by the diffusion rate, determines if it will influence ligand binding to the protein. In the systems examined here, the measured nitrogen-inversion rates and the times required to traverse the capture volume differed by a few orders of magnitude. Potentially more consequential are intermediate nitrogen-inversion rates in epimeric cases—since the energies of the interconverting species are unequal, a heavy bias against the eutomer might occur. The implications of an intermediate nitrogen-inversion rate are significant for in silico drug design, drug efficacy, molecular modeling of drug–protein binding, pharmacokinetics, drug enantiomer evaluation, etc. Due consideration of the process should thus be taken into account for drug development directions and in vitro evaluation.

Mexiletine is a relatively new class IB antiarrhythmic agent, similar in structure and pharmacological effects to lidocaine. It is effective mainly against ventricular arrhythmias and can be administered by the oral route, mexiletine is a chiral drug which is used clinically as the racemic mixture. The enantiomers of numerous chiral drugs have been shown to differ in their disposition in the body due to their stereoselective pharmacodynamic and/or pharmacokinetic properties. The relative antiarrhythmic potencies of the individual enantiomers of mexiletine have not been studied, nor have their pharmacokinetics been properly elucidated. Thus, the present study was aimed at developing a highly sensitive and stereoselective assay for mexiletine enantiomers which will be utilized to study their pharmacokinetics and in vitro serum protein binding. A high-performance liquid-chromatographic assay was developed using the Pirkle[sup R] ionic chiral stationary phase. The enantiomers were resolved as their 2-naphthoyl derivatives. The HPLC mobile phase consisted of 5.5% 2-propanol in hexane and was delivered at a flow rate of 1.4 mL/min. Detection of the enantiomeric derivatives was accomplished with a fluorescence detector [230 nm (Ex) and 340 nm (Em)]. Recovery of the enantiomers from plasma after pH adjustment to above 12 was found to be substantially low and stereoselectively in favour of the S(+)-enantiomer when compared with their recovery from water. This was attributed to a greater plasma protein binding of the R(-)-isomer despite the high plasma pH. Recovery was improved (83%), and the natural enantiomeric ratio restored by precipitation of the plasma proteins with barium hydroxide/zinc sulfate. Linear calibration curves (r² >0.999) were obtained in plasma over the concentration range 5 to 750 ng/mL for each enantiomer. Similar correlations (r >0.999) were obtained in saliva from 10 to 1,500 ng/mL and in urine from 0.25 to 7.5 ug/ml and 10 to 500 ng/ml. The inter- and intra-assay coefficients of variation were less than 4% for all the biological fluids. The minimum detectable quantity of each enantiomer in plasma was 5 ng/mL at a signal-to-noise ratio of 5:1, representing 100 pg injected onto the column. Protein binding of the enantiomers was determined with serum from five healthy male subjects. The mean percent free fraction of R(-)-mexiletine, 19.80 ± 2.64% was significantly (P<0.001) less than that of S(+)-mexiletine, 28.32 ± 1.45%. Binding was independent of concentration over the therapeutic range. Five healthy male subjects (same as above) were given 300 mg of (±) -mexi letine hydrochloride (capsules) orally. The plasma concentration-time data were analyzed by AUTOAN and NONLIN computer programs. The enantiomer kinetics were best described by a triexponential function in three of the five subjects and a biexponential function in the remaining two. There was no statistically significant difference in the absorption and distribution rate constants, peak plasma concentrations, time to peak plasma concentrations and plasma AUCs of the enantiomers. Bioavailability of the enantiomers was not determined due to lack of approval from the Health Protection Branch (Canada) to administer intravenous (±) -mexiletine to healthy volunteers. The terminal elimination half-life from plasma data for R (-)-mexiletine, 9.1 ± 2.9 hours, was significantly (P<0.02) less than that for the S( + )-isomer, 11.0 ± 3.8 hours. The cumulative urinary excretion of S(+)-mexiletine was 9.14 ± 3.07%, which was significantly (P<0.01) greater than that of R(-)-mexiletine, 7.40 ± 2.40%. Renal clearance of the enantiomers was consistent with cumulative urinary excretion, 0.72 ± 0.26 mL., min-1.Kg-1 and 0.61 ± 0.20 mL.min⁻¹.Kg⁻¹respectively (P<0.05). The non-renal elimination rate constant (mainly metabolism) was significantly (P<0.001) greater for R(-)-mexiletine, 0.0763 ± 0.0273 h⁻¹, than for the S( + )-iosmer, 0.0634 ± 0.0270 h⁻¹. The saliva AUCS were 18.2. ± 6.3 ug.mL ⁻¹.hr and 14.1 ± 5.0 ug. mL⁻¹.hr for S(+)-mexiletine and the R(-)-isomer respectively (difference significant, P<0.01). The enantiomer concentration ratios (R/S) in the three biological fluids showed evidence of a cross-over [R(-)> S(+) to S(+)>R(-)] between 0-2 hours in urine and saliva and 8-10 hours in plasma. This indicated an apparent discrepancy in the cross-over time in plasma relative to urine and saliva. However, the free enantiomer concentration ratios in plasma were similar to the enantiomer ratios in urine and saliva. Thus the disposition of mexiletine enantiomers in man is stereoselective with serum protein binding and metabolism favouring the R(-)-isomer. Renal elimination and salivary secretion, on the other hand, favour the S(+)-isomer. The similarity between the free enantiomer ratios in plasma, urine and saliva suggests that the stereoselective renal elimination and salivary secretion of the enantiomers are a reflection of their stereoselective protein binding.
Pharmaceutical Sciences, Faculty of
Graduate

Mestrado em Bioquímica - Métodos Biomoleculares
A quiralidade é a uma propriedade importante na indústria farmacêutica, uma vez que um enantiómero de um fármaco pode exercer o efeito terapêutico desejado enquanto o outro pode ser inerte ou mesmo nefasto. Embora vários fármacos sejam comercializados na sua forma racémica, as entidades regulatórias aconselham o desenvolvimento de fármacos enantiomericamente puros e mais seguros. Neste contexto, a indústria farmacêutica procura formas baratas e eficientes de produzir fármacos enantiomericamente puros, sendo este o objetivo da presente tese. A separação enantiomérica do ácido mandélico (AM), aqui utilizado como um fármaco racémico modelo, será tentada recorrendo a sistemas aquosos bifásicos (SABs) constituídos por seletores quirais de origem natural (proteínas e açúcares). Serão usadas duas abordagens: (i) a introdução de proteínas como seletores quirais em diferentes tipos de SABs; e (ii) o uso de (D)-sacarose simultaneamente como seletor quiral e componente de fase em SABs. Na primeira abordagem, foram utilizados diferentes tipos de SABs (polímero+polímero, polímero+sal, sal+líquido iónico (LI), polímero+LI e polímero+açúcar) e duas proteínas (albumina de soro bovino – BSA – e citocromo C – Cit c). A escolha das proteínas assentou em resultados de molecular docking que indicaram interações distintas entre diferentes proteínas e os enantiómeros do AM. Nestas fases, os sistemas constituídos por PPG400+(D)-Sacarose+BSA (excesso enantiómerico de -5.9± 0.5%) e PPG400+dihidrogeno fosfato de colínio+Cit c (excesso enantiomérico de -9.0 ± 1.2%) revelaram-se os mais eficientes. As proteínas e os constituintes de fase dos SABs afetaram a separação enantiomérica de ácido mandélico. Uma vez que a docagem molecular não considera as interações com os componentes de fase, esta abordagem revelou ser incapaz de prever o desempenho das proteínas como seletores quirais em SABs. Com o objetivo de ultrapassar as limitações de seletividade enantiomérica e melhorar a simplicidade operacional da tecnologia proposta, a (D)-sacarose foi usada simultaneamente como formador de fase e seletor quiral em SABs. Depois de uma otimização cuidada, foi possível obter um excesso enantiomérico máximo de -12.3 ± 0.5% com um SAB constituído por polímero e (D)-sacarose.
Chirality is an important property for the pharmaceutical industry, since one enantiomer of a drug can exert a therapeutic action, while the other may be inert or even nefarious. While several drugs are commercialized as racemates, regulatory bodies strongly encourage the development of safer enantiopure drugs. In this context, pharmaceutical industry seeks for cheap and efficient ways of obtaining enantiopure pharmaceuticals and this is the main objective of this thesis. The enantiomeric separation of mandelic acid (MA), here used as a model racemic drug, using aqueous biphasic systems (ABS) composed of natural chiral selectors (proteins and sugars) will be proposed. Two different approaches were used: (i) the introduction of proteins as chiral selectors in several types of ABS; and (ii) ABS formed by D-Sucrose as both phase former and chiral selector. Within the first approach, different types of systems (polymer+polymer, polymer+salt, polymer+sugar, and ionic liquids (ILs)+salt, ILs+polymer) and of proteins (bovine serum albumin –BSA - and cytochrome C – Cyt C) were used. These two proteins were chosen based on molecular docking results that shown distinctive interactions with the two MA enantiomers among eleven screened proteins. PPG400+(D)-sucrose+BSA system (enantiomeric excess of -5.9 ± 0.5%) and PPG+cholinium dihydrogenphosphate+Cyt C (enantiomeric excess of -9.0 ± 1.2% were the most efficient ABS developed up to this stage. Both the protein and ABS phase formers affected the enantioseparation of MA. Since molecular docking does not encompass the interactions with the ABS phase formers, it was limited at predicting the proteins’ performance as chiral selectors in ABS. In order to surpass the limited enantioselectivity displayed and to improve the operational simplicity of the proposed technology, (D)-sucrose was employed as both chiral selector and phase former in ABS. After a proper optimization, it was possible to achieve a maximum enantiomeric excess of -12.3 ± 0.5% with an ABS composed of polymer and (D)-sucrose.

Mexiletine (MexitilR) is an orally effective antiarrhythmic drug used clinically as a racemate of the R(-)- and S(+)-enantiomers. A stereoselective high-performance liquid chromatographic assay was developed for the determination of mexiletine enantiomers in serum, saliva, red blood cells and urine. The mexiletine enantiomers were resolved as their N-anthroyl derivatives on a PirkleR phenylglycine ionic chiral HPLC column. The present study examined the serum free (unbound) and total drug kinetics for the mexiletine enantiomers in twelve healthy volunteers following oral administration of 200 mg of racemic mexiletine hydrochloride. To further characterize serum free mexiletine in the body, the concentrations of mexiletine enantiomers in saliva and in red blood cells were examined. Since mexiletine was largely eliminated by metabolic processes, p-hydroxy-mexiletine and hydroxymethyl-mexiletine metabolites were examined in the urine of four healthy subjects. Following oral drug administration, the disposition of mexiletine enantiomers was described by one or two-compartment open models. The mean peak serum total mexiletine concentration of 217 ± 68 ng/ml for R(-)-mexiletine was found to be significantly greater (p<0.01) than a mean value of 196 ± 56 for S(+)-mexiletine. The mean serum total R(-)-mexiletine concentrations were also found to be significantly greater than those for S(+)-mexiletine during the first six hours. The absorption, rapid and terminal disposition kinetic parameters between the two enantiomers were not significantly different. From urinary data, the mean percentages of mexiletine enantiomers recovered from the urine over 72 hours were found to be 3.5 ± 3.4% and 3.7 ± 3.9% for R(-)- and S(+)-mexiletine, respectively. The mean terminal elimination half-lives were found to be 5.8 ± 1.5 h and 5.6 ± 1.4 h for R(-)- and S(+)-mexiletine, respectively. Both the urinary recoveries and the half-1ives for the enantiomers were not significantly different. Comparative in vitro studies on the serum protein binding of mexiletine enantiomers by ultrafiltration and by equilibrium dialysis indicated a serum pH-dependent stereoselective protein binding of mexiletine enantiomers. A serum pH range from 6.3 to 9.4 was correlated with the serum protein binding of mexiletine enantiomers from ≈30% to ≈80%. Within this pH range, the serum free drug R(-)/S(+) ratio was found to decrease from 1.0 to 0.7. At serum pH 7.4, the serum protein binding of mexiletine enantiomers was similar, and was not dependent on the therapeutic concentration range of 0.25 to 3.0 µg/ml. The in vivo serum protein binding of mexiletine enantiomers was found to be non-stereoselective. The mean serum free fractions of 0.57 ± 0.07 and 0.56 ± 0.06 for R(-)- and S(+)-mexiletine, respectively, were not significantly different. The overall mean serum free R(-)/S(+) mexiletine ratio of 1.09 was also indicative of a non-stereoselective binding of mexiletine enantiomers. Following the collection of unstimulated saliva, the overall mean saliva / serum free mexiletine area under the concentration-time curve ratios of 6.10 ± 2.82 and 7.49 + 3.48 for R(-)- and S(+)-mexiletine, respectively, were found to be significantly different (p<0.01). The overall mean saliva R(-)/S(+) ratio of 0.89 ± 0.02 (mean ± S.E.) over 48 hours suggested that the disposition of mexiletine enantiomers in saliva was stereoselective. In addition, saliva mexiletine concentrations were found to correlate poorly with serum free mexiletine concentrations. In vitro studies on the distribution of mexiletine enantiomers into red blood cells indicated a distribution equilibrium of ≈40 minutes. The mean red blood cell mexiletine area under the concentration-time curve of 2.3 ±1.5 µg/ml/h and 2.8 ± 2.1 µg/ml/h for R(-)- and S(+)-mexiletine, respectively, were not significantly different. The overall mean red blood cell mexiletine R(-)/S(+) ratio of 0.91 ± 0.13 suggested a similar distribution of the enantiomers into the red blood cells. A stereoselective HPLC assay was developed for the simultaneous determination of mexiletine, p-hydroxy-mexiletine, and hydroxymethyl-mexiletine enantiomers in urine. Mexiletine and hydroxymethyl -mexiletine enantiomers were resolved on a PirkleR isoleucine covalent HPLC column as their N-anthroyl derivatives, while p-hydroxy-mexiletine enantiomers were resolved as their 0-ethyl-N-anthroyl derivatives. For mexiletine and p-hydroxy-mexiletine, chromatographic retention was found to favour the R(-)-enantiomer, thus leading to the initial elution of the S(+)-enantiomer. For hydroxymethyl-mexiletine, the order of elution was found to be reversed. The mean cumulative amounts of p-hydroxy-mexiletine enantiomers recovered from the urine over 72 hours were found to be 1.31 ± 0.33 mg and 1.27 + 0.39 mg for the R(-) and S(+)-enantiomers, respectively. The two values were not significantly different. The mean cumulative amounts of R(-)-hydroxymethyl-mexiletine (2.94 ± 1.70 mg) recovered from the urine over 72 hours were found to be significantly (p<0.01) greater than a value of 1.17 ± 0.60 mg for the S(+)-enantiomer, suggesting the presence of a stereoselective metabolic pathway for hydroxymethyl-mexiletine.
Pharmaceutical Sciences, Faculty of
Graduate

Antibiotics have been used widely for the treatment of bacterial infections for over half a century. However, the emergence of resistance to antibiotics has aroused public health concern, leading to the development of antimicrobial peptides (AMPs) as potential alternative therapeutic agents against bacterial infections. AMPs are naturally found in many species and have important roles in our innate immune defense systems. AMPs are usually cationic amphipathic peptides with membrane destabilizing property. They have a relatively broad spectrum of antimicrobial activity and pathogens are less likely to develop resistance against AMPs. The major challenge of using AMPs as therapeutic agents is their toxicity towards mammalian cells. The biological stability of AMPs to protease in human body is another concern. To address the latter problem, instead of the naturally occur L-enantiomers, Denantiomeric AMPs were introduced to enhance their stability. This study aimed to test the hypothesis that the D-enantiomeric AMPs are more resistant than the Lenantiomeric AMPs against proteolytic degradation. Three pairs of synthetic D-/LAMPs (D-LAO160-P13/LAO160-P12; D-LAO160-H/LAO160-H; and D-LAK-120-HP13/LAK-120-HP13) were employed to test for their stability when treated with trypsin, serum and gastric fluid, and the samples were analyzed by high performance liquid chromatography (HPLC). Generally, all the D-enantiomeric AMPs were found to be resistant towards proteolysis. Besides, to compare the cytotoxicity of D-/LAMPs, MTT and LDH assays of the D/L-LAK120-HP13 pair were carried out on two different cell lines, A549 cells (human lung adenocarcinoma epithelial cells) and RAW264.7 cells (mouse macrophage cells). Significant difference in cytotoxicity of D-LAK120-HP13 and LAK120-HP13 on RAW264.7 cells were obtained from MTT assay, but not in LDH assays or on A549 cells. Further analysis has to be done to validate the findings obtained from this research.
published_or_final_version
Pharmacology and Pharmacy
Master
Master of Medical Sciences

Submitted in fulfillment of the requirements for the Doctor of Philosophy degree in Chemistry, Durban University of Technology, Durban, South Africa, 2016.
In drug discovery and development projects, metabolism of new chemical entities (NCEs) is a major contributing factor for the withdrawal of drug candidates, a major concern for other chemical industries where chemical-biological interactions are involved. NCEs interact with a target macro-molecule to stimulate a pharmacological or toxic response, known as pharmacodynamics (PD) effect or through the Adsorption, Distribution, Metabolism, and Excretion (ADME) process, triggered when a bio-macromolecule interacts with a therapeutic drug. Therefore, the drug discovery process is important because 75% of diseases known to human kind are not all cured by therapeutics currently available in the market. This is attributed to the lack of knowledge of the function of targets and their therapeutic use in order to design therapeutics that would trigger their pharmacological responses. Accordingly, the focus of this work is to develop cost saving strategies for medicinal chemists involved with drug discovery projects. Therefore, studying the synergy between in silico and in vitro approaches maybe useful in the discovery of novel therapeutic compounds and their biological activities. In this work, in silico methods such as structure-based and ligand-based approaches were used in the design of the pharmacophore model, database screening and flexible docking methods. Specifically, this work is presented by the following case studies: The first involved molecular docking studies to predict the binding modes of catechin enantiomer to human serum albumin (HSA) interaction; the second involved the use of docking methods to predict the binding affinities and enantioselectivity of the interaction of warfarin enantiomers to HSA. the third case study involved a combined computational strategy in order to generate information on a diverse set of steroidal and non-steroidal CYP17A1 inhibitors obtained from literature with known experimental IC50 values. Finally, the fourth case study involved the prediction of the site of metabolisms (SOMs) of probe substrates to Cytochrome P450 metabolic enzymes CYP 3A4, 2D6, and 2C9 making use of P450 module from Schrödinger suite for ADME/Tox prediction. The results of case study I were promising as they were able to provide clues to the factors that drive the synergy between experimental kinetic parameters and computational thermodynamics parameters to explain the interaction between drug enantiomers and thetarget protein. These parameters were correlated/converted and used to estimate the pseudo enantioselectivity of catechin enantiomer to HSA. This approach of combining docking methodology with docking post-processing methods such as MM-GBSA proved to be vital in estimating the correct pseudo binding affinities of a protein-ligand complexes. The enantioselectivity for enantiomers of catechin to HSA were 1,60 and 1,25 for site I and site II respectively. The results of case study II validates and verifies the preparation of ligands and accounting for tautomers at physiological pH, as well as conformational changes prior to and during docking with a flexible protein. The log KS = 5.43 and log KR = 5.34 for warfarin enantiomer-HSA interaction and the enantioselectivity (ES = KS/KR) of 1.23 were close to the experimental results and hence referred to as experimental-like affinity constants which validated and verified their applicability to predict protein-ligand binding affinities. In case study III, a 3D-QSAR pharmacophore model was developed by using 98 known CYP17A1 inhibitors from the literature with known experimental IC50 values. The starting compounds were diverse which included steroidal and non-steroidal inhibitors. The resulting pharmacophore models were trained with 69 molecules and 19 test set ligands. The best pharmacophore models were selected based on the regression coefficient for a best fit model with R2 (ranging from 0.85-0.99) & Q2 (ranging from 0.80-0.99) for both the training and test sets respectively, using Partial Least Squares (PLS) regression. On the other hand, the best pharmacophore model selected was further used for a database screening of novel inhibitors and the prediction of their CYP17A1 inhibition. The hits obtained from the database searches were further subjected to a virtual screening workflow docked to CYP17A1 enzyme in order to predict the binding mode and their binding affinities. The resulting poses from the virtual screening workflow were subjected to Induced Fit Docking workflow to account for protein flexibility during docking. The resulting docking poses were examined and ranked ordered according to the docking scores (a measure of affinity). Finally, the resulting hits designed from an updated model from case study III were further synthesized in an external organic chemistry laboratory and the synthetic protocols as well as spectroscopic data for structure elucidation forms part of the provisional patent specification. A provisional patent specification has been filed (RSA Pat. Appln. 2015/ 07849). The case studies performed in this thesis have enabled the discovery of non-steroidal CYP17A1 inhibitors.
D

Carvedilol, 1-(9H-carbazol-4-yloxy)-3-[[2-(2-methoxyphenoxy)ethyl]amino]-2- propanol, is a new antihypertensive drug which has recently been introduced on the market in Canada under the trade name, COREG. It contains a chiral centre in its structure and therefore exists as two enantiomers. The drug is marketed as the racemate, however, the two enantiomers possess different pharmacological actions. (-)-(S)-Carvedilol is a much more potent Pi-blocker than (+)-(R)-carvedilol, whereas both enantiomers exhibit the same oci-adrenergic antagonism. This study consisted of the development of sensitive stereoselective assays for carvedilol using high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) and the determination of the stereoselective protein binding of carvedilol. An attempt to derivatize carvedilol enantiomers with fluorogenic reagents was undertaken. The reaction with 2-naphthoyl chloride, 2-anthroyl chloride, and (+)-(S)-naproxen chloride resulted in incomplete derivatization. Mass spectrometric analysis of the reaction products revealed the formation of the mono-derivative of carvedilol with 2-naphthoyl chloride at the secondary amine group on the side chain and also the di-derivative at both the amine group and the hydroxyl group of carvedilol. The reaction with (+)-(S)-1-(1-naphthyl)ethyl isocyanate was also examined and was incomplete and produced multiple derivatives. A direct chiral HPLC method was therefore developed, without the need for derivatization, using (S)-indoline-2-carboxylic acid and (R)-1-(a-naphthyl)ethylamine as the stationary phase. The assay was validated for carvedilol enantiomers in serum. A limit of quantitation (LOQ) of 1 ng/ml for both enantiomers was obtained. A new stereoselective CE method was also developed for the analysis of the enantiomers of carvedilol in serum. Several types and concentrations of cyclodextrins were tested. Near baseline resolution was obtained using 10 mM hydroxypropyl-pcyclodextrin as the chiral selector. The electrophoretic conditions were optimized. The chiral CE method for carvedilol was validated for the drug enantiomers in serum. An LOQ of 50 ng/ml per enantiomer was obtained. The HPLC and the CE assays were compared by analyzing a series of serum samples containing racemic carvedilol in different concentrations using the two methods. The concentrations obtained by the two assays were not found to be significantly different. The stereoselective binding of carvedilol enantiomers to serum proteins was investigated by equilibrium dialysis. Carvedilol is highly bound to serum proteins (> 98%). The free fractions obtained after dialysis of serum containing carvedilol were 0.6% for (+)-(R)-carvedilol and 0.9% for (-)-(S)-carvedilol with an R/S ratio of 0.67. The binding of the two enantiomers was found to be significantly different, with (-)-(S)-carvedilol being less bound. Binding to isolated serum proteins was also determined. Using 4% human serum albumin (HSA) in isotonic phosphate buffer, the unbound fractions obtained were 2.4% and 2.6% for (+)-(R)-and (-)-(S)-carvedilol, respectively, with an R/S ratio of 0.92. The stereoselective binding to HSA was not found to be significantly different. On the other hand, the binding of carvedilol to 100 mg% cti-acid glycoprotein (AAG) in isotonic phosphate buffer was found to be highly stereoselective. The unbound fractions obtained were 1.5% for (+)-(R)-carvedilol and 2.5% for (-)-(S)-carvedilol, with an R/S ratio of 0.60. The results suggest that AAG is the major protein responsible for the stereoselective binding of carvedilol enantiomers in serum. Carvedilol enantiomers were not found to exhibit concentration-dependent binding in human serum over the concentration range of 0.5-4 ug/ml per enantiomer. However, binding to 4% HSA was found to be significantly different above 2 ug/ml for each enantiomer. The free fractions increased substantially above concentrations of 3 ug/ml of each enantiomer when the binding to 100 mg% AAG was tested suggesting that the AAG binding sites are saturable. In order to simulate an increase in the levels of AAG as would occur in myocardial infarction or surgery, the binding of carvedilol enantiomers to 4% HSA combined with 100 mg% AAG or 400 mg% AAG was compared. A decrease in the free drug concentration was observed for both enantiomers when the levels of AAG increased from 100 to 400 mg% and the R/S ratio changed from 0.82 to 0.67. In summary, comparative HPLC and CE assay methods were developed for the stereoselective analysis of carvedilol enantiomers. While the fluorescent detection used for the HPLC method allowed for lower detection limits required for determination of free (unbound) enantiomer concentrations in serum, the CE method, with a higher limit of quantitation, was used for the determination of total enantiomer concentrations (free and bound). However, the actual sensitivity of the CE method, considering the amount of sample injected, was greater by approximately 10 fold than the HPLC method. Using a combination of both methods, the stereoselective protein binding characteristics were established for the enantiomers of carvedilol using human serum and purified protein fractions of human serum.

碩士
國立臺灣大學
食品科技研究所
87
Rehydrated squids, prepared by pickling the dried squid in a Na-hydrogen carbonate solution, is usually served as raw materials for cooking and is one of the major consumption ways of squids in Taiwan. However, alkali treatments of proteins usually cause the crossing-linking reaction, degradation, browning reaction and, especially, racemization of compositional amino acids in proteins of squids those are originally rich in essential amino acids and thus, lead to the decline of protein nutritional values. Enatiomers or epimers of analytes were reported to be completely separated and identified through the modifications of partition coefficient between buffer solution and neutral analytes micelles formed by the application of surfactants in 1984. Therfore, the techniques of micellar electrokinetic capillary electrophoresis(MECC) for separating enatiomers or epimers have been improved and become much mature since then. In the present study, MECC was used to analyze the 9-fluorenylmethyl chloroformate (FMOC) derivatized amino acids by altering the factors such as wavelength, voltage, surfactant level and organic modifier level in the electrolyte solution. The optimal separation conditions was 50 mM SDS-12 mM β-CD-50 mM phosphate buffer (pH 7.5) of electrolyte solution, 15 kV for applied voltage, 25 ℃ for operating temperature, and a silicated capillary with 50μm in diameter and 67cm in length. Enantiomeric separation of enantiomers of methionine, valine, isoleucine, leucine, phenylalanine and tryptophan were complete, however, separations of enantiomers of threonine and lysine were failed in the such model system. Addition of 45 mM β-cyclodextrin and 1 M urea improved the enantiomeric separation of lysine and proline (Rs > 1.0). Using rehydrated squids, prepared under various temperatures (room temperature, 50℃) and alkaline levels (sodium carbonate/sodium hydrogen carbonate buffer, pH 9.0, 10.0 and 11.0) as real system, enantiomeric separations of FMOC derivatized amino acids were conducted to elucidate the effects of processing condition on the formation of amino acid enatiomers. Results showed that the enantiomerations of valine, phenylalanine, methionine, lysine, leucine, isoleucine, glutamic acid, aspartic acid alanine and arginine in the compositional amino acids of rehydrated squids in both control and experimental group were not apparent suggesting the nutritional loss due to enantiomeration of the commercial rehydrated squids, pickled with NaHCO3 (pH 8.9) at ambient temperature ,was negligible.

Understanding the relation between amino acid sequences and protein structures is one of the most important problems in modern molecular biology. However, due to the complexities in the protein structure, this task is really daunting. Hence, understanding the structural features of proteins and the rules of folding is central to the design of novel and more effective biomaterials. With the inception of the de novo design of synthetic mimetics for protein structural elements, the study of designed peptides is a subject of intense current research. The de novo design of polypeptide structures provides insights into the factors that govern the folding of peptides and proteins. The rational design of synthetic peptide models for secondary structural motifs in proteins depends on the ability to control the polypeptide chain stereochemistry. An approach, which seems to be useful, is the introduction of constrained genetically coded amino acids like Proline or the introduction of non-protein constrained amino acids like Aib which are capable of restricting the range of available backbone conformations of the polypeptide chain. The use of such residues would then permit the design of well defined and intended structural motifs like the β-turns which serve as chain reversal areas of the polypeptide chain. Templates incorporating multiple repeats of such conformationally constrained residues would in turn further enhance the choice of conformational parameters for the polypeptide chain towards folding. Crystal structure determination of the oligopeptides by X-ray diffraction gives insight into the specific conformational states, modes of aggregation, hydrogen bond interactions and solvation of peptides. Precise structural analysis and good characterization of geometrical parameters and stereochemical details of these molecules provide valuable inputs for peptide design and are indispensable for exploring strategies to design peptide sequences which serve as synthetic mimics for folding motifs in proteins. Many of the above points have been investigated in this thesis which incorporates study of designed peptides containing heterochiral and homochiral diproline segments followed by protein database analysis. This thesis reports results of x-ray crystallographic studies of twenty two (22) oligopeptides containing heterochiral or homochiral diproline segments. Apart from the crystal data, protein database analysis has also been carried out to investigate what actually is found in nature. Given in brackets are the compound names used in the thesis for the peptides solved. 1) Piv-DPro-LPro-NHMe ( DPPN ) [C16H27N3O3 ] 2) Piv-DPro-LPro-LVal-OMe ( DPPV ) [C21H35N3O5 . 0.09 H2O] 3) Piv-DPro-LPro-LPhe-OMe ( DPPF ) [C25H35N3O5 . H2O] 4) Piv-DPro-LPro-DAla-OMe ( DPPDA ) [C19H31N3O5] 5) Piv-LPro-DPro-LAla-OMe ( PDPA ) [C19H31N3O5] 6) Piv-DPro-LPro-LVal-NHMe ( DPPVN ) [C21H36N4O4 . H2O] 7) Piv-DPro-LPro-LLeu-NHMe ( DPPLN ) [C22H38N4O4 . 0.34H2O] 8) Piv-DPro-LPro-LPhe-NHMe ( DPPFN ) [C25H36N4O4 . H2O] 9) Piv-DPro-LPro-Aib-NHMe ( DPPUN ) [C20H34N4O4] 10) Piv-DPro-LPro-DAla-NHMe ( DPPDAN ) [C19H32N4O4] 11) Piv-DPro-LPro-DVal-NHMe ( DPPDVN ) [C21H36N4O4 .1.43 H2O] 12) Piv-DPro-LPro-DLeu-NHMe ( DPPDLN ) [C22H38N4O4 . H2O] 13) Piv-LPro-DPro-LAla-NHMe ( PDPAN ) [C19H32N4O4] 14) Piv-LPro-DPro-LVal-NHMe ( PDPVN ) [C21H36N4O4] 15) Piv-LPro-DPro-LLeu-NHMe ( PDPLN ) [C22H38N4O4 . H2O] 16) Piv-LPro-DPro-LVal-OMe ( PDPVO ) [C21H35N3O5 . H2O] 17) Racemic mixture of Piv-DPro-LPro-DVal-NHMe + Piv-LPro-DPro-LVal-NHMe ( PPVVN ) [C21H36N4O4 . 0.74H2O] 18) Racemic mixture of Piv-DPro-LPro-DLeu-NHMe + Piv-LPro-DPro-LLeu-NHMe ( PPLLN ) [C22H38N4O4 . H2O] 19) Racemic mixture of Piv-DPro-LPro-DPhe-NHMe + Piv-LPro-DPro-LPhe-NHMe ( PPFFN ) [C25H36N4O4 . 2 H2O] 20) Piv-LPro-LPro-LPhe-OMe ( PPFO ) [C25H35N3O5 . 0.5 H2O] 21) Piv-LPro-LPro-LVal-NHMe ( PPVN ) [C21H36N4O4 . H2O] 22) Piv-LPro-LPro-Aib-NHMe ( PPUN ) [C20H34N4O4. H2O] Results from the X-ray crystallographic analysis of the above peptides provided substantial information regarding role of diproline templates on the folding of the polypeptide chain. The thesis is divided into the following eight chapters : Chapter 1 gives a general introduction to the stereochemistry of polypeptide chains and the secondary structure classification: helices, β-sheets and β-turns. This section also provides a brief overview of the use of non standard and D-amino acids into peptide design. Discussions on DProline, puckering states of the Proline ring, diproline segments and racemic mixtures of peptides are also presented. A brief discussion on X-ray diffraction and solution to the phase problem is also given. Chapter 2 describes the structural characterization in crystals of the five following designed peptides: Piv-DPro-LPro-NHMe (DPPN), Piv-DPro-LPro-Xxx-OMe [Xxx = LVal (DPPV); LPhe (DPPF); DAla (DPPDA)] and Piv-LPro-DPro-LAla-OMe (PDPA) containing the heterochiral diproline segment with an aim towards understanding the directive influence of short range interaction on polypeptide folding. Except PDPA, in all the structures, a type II’ β-turn was observed at the DPro-LPro segment with the formation of a 4→1 intramolecular hydrogen bond between the atoms of the polypeptide backbone. In PDPA, the expected type II β-turn occurred at the LPro-DPro segment. Thus, the DPro-LPro segment preferably adopts a type II’ β-turn conformation when present at the C-terminus which is mimicked by the methyl ester group. The use of pivalyol group at the N-terminus is to ensure the trans geometry of the peptide bond between pivalyol and the first Proline. Crystal parameters DPPN: C16H27N3O3; P21; a = 10.785(1) Å, b = 15.037(1) Å, c = 11.335(1) Å; β = 109.96(1)°; Z = 4; R = 0.0388, wR2 = 0.1047. DPPV: C21H35N3O5 . 0.09 H2O; P212121; a =10.676(1) Å, b = 16.608(1) Å, c = 39.887(1) Å, Z = 12; R = 0.0688, wR2 = 0.1701. DPPF: C25H35N3O5 . H2O; P21; a = 9.538(1) Å, b = 10.367(1) Å, c = 13.102(1) Å; β = 93.04(1) °; Z = 2; R = 0.0504, wR2 = 0.1455. DPPDA: C19H31N3O5; P21; a = 11.269(1) Å, b = 9.945(1) Å, c = 18.550(2) Å; β = 97.46(1)°; Z = 4; R = 0.0563, wR2 = 0.1249. PDPA: C19H31N3O5; P212121; a = 9.043(1) Å, b = 10.183(2) Å, c = 23.371(1) Å; Z = 4; R = 0.0753, wR2 = 0.1603. Chapter 3 describes the crystal structures of the four following designed peptides containing the heterochiral diproline segment followed by a L-residue or an achiral amino acid residue like Aib : Piv-DPro-LPro-Xxx-NHMe [Xxx = LVal (DPPVN); LLeu (DPPLN); LPhe (DPPFN) and Aib (DPPUN)]. In the first three peptides the DPro-LPro segennt adopts a type II’ β-turn conformation with the formation of a type I β-turn at the LPro-Xxx segment. The peptide backbone overall therefore adopts a consecutive β-turn structure. When the L-amino acids at the C-terminus are replaced by the achiral amino acid Aib, the overall folded structure adopted by the peptide backbone still remains unchanged with the formation of a consecutive β-turn. All the structures are stabilized by two intramolecular 4→1 hydrogen bonds between the C=O group and the nitrogen atom of the polypeptide backbone. Crystal parameters DPPVN: C21H36N4O4 . H2O; P21; a = 9.386(1) Å, b = 12.112(1) Å, c = 10.736(1) Å; β = 99.53(1) °; Z = 2; R = 0.0528, wR2 = 0.1337. DPPLN: C22H38N4O4 . 0.34H2O; P21; a =9.231(1) Å, b = 17.558(1) Å, c = 15.563(1) Å; β = 91.94(1) °; Z = 4; R = 0.0555, wR2 = 0.1422. DPPFN: C25H36N4O4 . H2O; P212121; a = 10.473(1) Å, b = 15.980(1) Å, c = 15.994(1) Å; Z = 4; R = 0.0620, wR2 = 0.1826. DPPUN: C20H34N4O4; P212121; a = 10.571(2) Å, b = 11.063(1) Å, c = 18.536(1) Å; Z = 4; R = 0.0578, wR2 = 0.1256. Chapter 4 describes the crystal structures of the seven designed peptides containing heterochiral diproline segment. Three of these contain sequences of the type DPro-LPro-DXxx [DXxx = DAla (DPPDAN); DVal (DPPDVN); DLeu (DPPDLN)] and three contains the enantiomeric peptides of the ones that are mentioned earlier in sequences of the type LPro-DPro-LXxx [LXxx = LAla (PDPAN); LVal (PDPVN); LLeu (PDPLN)]. In order to investigate the effect of the C-terminal protecting group, a final peptide Piv-LPro-DPro-LVal-OMe (PDPVO) was crystallographically characterized. All the peptides containing the DXxx residues adopted different backbone conformations. For DAla, a structure simultaneously having a β-turn and an α-turn was obtained which is the first example in designed peptides of an isolated α-turn. In the case of DVal, an open / extended structure devoid of any intramolecular hydrogen bonding was obtained whereas for DLeu, type II β-turn occurred at the LPro-DLeu segment instead of the expected type II’ turn at the DPro-LPro segment. In the case of enantiomeric peptides, all the three peptides adopted folded structures with exact mirror image conformation being generated for LAla and nearly identical mirror image conformation in the case of LLeu. The enantiomeric peptide of DVal which contained LVal residue following the diproline segment also adopted a folded conformation with the formation of type II β-turn at the LPro-DPro segment as expected. X-ray crystallographic characterization of PDPVO resulted in the peptide adopting an overall extended / open structure. Thus, the chirality of the C-terminal residue seems to have a profound effect on the conformation of the heterochiral diproline segments. The role of the C-terminal protecting group cannot also be undermined. Crystal parameters DPPDAN: C19H32N4O4; P1; a = 5.964(1) Å, b = 9.354(1) Å, c = 9.961(1) Å; α = 75.44(1), β = 78.90(1) °, γ = 77.04(1); Z = 1; R = 0.0728, wR2 = 0.1528. DPPDVN : C21H36N4O4 .1.43 H2O; P212121; a = 8.744(8) Å, b = 11.609(1) Å, c = 23.577(2) Å; Z = 4; R = 0.0625, wR2 = 0.1856. DPPDLN : C22H38N4O4 . H2O; P212121; a = 10.531(3) Å, b = 11.659(3) Å, c = 20.425(6) Å; Z = 4; R = 0.0444, wR2 = 0.1239. PDPAN: C19H32N4O4; P1; a = 5.964(1) Å, b = 9.354(2) Å, c = 9.961(2) Å; α = 75.44(1), β = 78.90(1) °, γ = 77.04(1); Z = 1; R = 0.0745, wR2 = 0.1572. PDPVN : C21H36N4O4; P212121; a = 9.743(1) Å, b = 11.423(1) Å, c = 21.664(3) Å; Z = 4; R = 0.0803, wR2 = 0.1899. PDPLN : C22H38N4O4 . H2O; P212121; a = 10.462(4) Å, b = 11.572(4) Å, c = 20.262(7) Å; Z = 4; R = 0.0968, wR2 = 0.2418. PDPVO : C21H35N3O5 . H2O; P212121; a = 8.784(4) Å, b = 11.587(5) Å, c = 23.328(1) Å; Z = 4; R = 0.0888, wR2 = 0.1465. Chapter 5 describes the crystal structures of the three designed peptides containing racemic mixtures [Racemic mixture of Piv-DPro-LPro-DVal-NHMe + Piv-LPro-DPro-LVal-NHMe (PPVVN); Racemic mixture of Piv-DPro-LPro-DLeu-NHMe + Piv-LPro-DPro-LLeu-NHMe (PPLLN); Racemic mixture of Piv-DPro-LPro-DPhe-NHMe + Piv-LPro-DPro-LPhe-NHMe (PPFFN)] having the heterochiral diproline segment in their sequences and three peptides having a homochiral diproline segment [Piv-LPro-LPro-LPhe-OMe (PPFO); Piv-LPro-LPro-LVal-NHMe (PPVN); Piv-LPro-LPro-Aib-NHMe (PPUN)]. The inability of the pure enantiomers to crystallize in the case of Phe (chapter 4) invoked the use of peptide racemates for obtaining a crystal state conformation for the said compound. In all the cases, the L-enantiomer of Xxx crystallized in the asymmetric unit. A type II β-turn was obtained in the case of PPVVN at the LPro-DPro segment and a type II’ β-turn was obtained for PPLLN at the DPro-LLeu segment. in the case of Phe, an open structure devoid of any intermolecular hydrogen bonding an very similar to DPPDVN (chapter 4) was obtained. In the case of homochiral diproline segment containing peptides, PPFO crystallized with two molecules in the asymmetric unit, both of which adopted a type VIA1 hydrogen bonded β-turn conformation with a cis peptide bond between the diproline segment. In the case of Valine (PPVN) however, a structure devoid of any intramolecular hydrogen bonding was obtained. In the final peptide PPUN, a type II β-turn conformation is observed at the LPro-Aib segment. The analysis revealed that the hydration of the peptide can cause dramatic changes in its backbone conformation. In homochiral LPro-LPro sequences, the tendency to form hydrogen bonded turns competes with the formation of semi-extended polyproline structures. The results also emphasize the subtle role of sequence effects in modulating the conformations of short, constrained peptide segments. The possibility of trapping distinct conformational segments of the diproline segments in crystals by generating racemic centro-symmetric crystals in which packing effects may be appreciably different from those observed in the crystals of individual pure enantiomeric peptides has been clearly exploited in this chapter to obtain a crystal in the case of Phe. These results suggest that the energetic differences between these states is small. Conformational choice can therefore be readily influenced by environmental and sequence effects. Crystal parameters PPVVN: C21H36N4O4 . 0.74H2O; C2/c; a = 36.667(17) Å, b = 10.092(5) Å, c = 13.846(6) Å; β = 107.27(1) °; Z = 8; R = 0.1317, wR2 = 0.3141. PPLLN: C22H38N4O4 . H2O; P21/c; a = 10.555(1) Å, b = 11.687(1) Å, c = 20.108(2) Å; β = 95.47(1) °; Z = 4; R = 0.0761, wR2 = 0.2034. PPFFN: C25H36N4O4 . 2 H2O; P21/c; a = 8.883(5) Å, b = 18.811(10) Å, c = 16.033(9) Å; β = 96.28(1) °; Z = 4; R = 0.1218, wR2 = 0.2848. PPFO : C25H35N3O5 . 0.5 H2O; P212121; a = 10.199(1) Å, b = 20.702(2) Å, c = 23.970(2) Å; Z = 8; R = 0.0716, wR2 = 0.1901. PPVN : C21H36N4O4 . H2O; P212121; a = 9.454(1) Å, b = 11.119(1) Å, c = 23.021(2) Å; Z = 4; R = 0.0551, wR2 = 0.1587. PPUN: C20H34N4O4. H2O; P21; a = 6.276(1) Å, b = 14.011(2) Å, c = 12.888(1) Å; β = 96.80(1) °; Z = 2; R = 0.0475, wR2 = 0.1322. Chapter 6 describes the pyrrolidine ring puckering states of the Proline residue present in diproline segments in the peptides solved in this thesis, the Cambridge structural database (CSD) [only acyclic diproline containing peptides have been taken into account] and in a non-redundant dataset of proteins in the Protein Data Bank (PDB). The five membered pyrrolidine ring of Proline can be best characterized in terms of the following five endocyclic torsion angles χ1, χ2, χ3,χ4 and θ. Using various values of these endocyclic torsion angles the following puckering states were identified : [1] Cγ-exo (A) [2] Cγ-endo (B) [3] Cβ-exo (C) [4] Cβ-endo (D) [5] Twisted Cγ-exo-Cβ-endo (E) [6] Twisted Cγ-endo-Cβ-exo (F) [7] Planar (G) [8] Cα-distorted (H) [9] Twisted Cβ-exo-Cα-endo (I) [10] Cδ-endo (K) [11] N-distorted (L) [12] Twisted Cδ-endo- Cγ-exo (N). In the case of peptides solved in this thesis for heterochiral diproline segments, the Cγ-exo / Cβ-exo (AC) combination turns out to more preferred than the other combinations. The Cγ-endo / Cγ-endo (BB) state is the second most populated state. The overall investigation of Proline rings in peptides show that the states Cγ-exo (A), Cβ-exo (C) and Twisted Cγ-endo-Cβ-exo (F) are the most preferred states of occurrence of the pyrrolidine ring conformation. In the case of proteins, the overall percentage distribution of various combinations indicates that the AA (Cγ-exo / Cγ-exo), AE (Cγ-exo / Twisted Cγ-exo-Cβ-endo) and FF (Twisted Cγ-endo-Cβ-exo / Twisted Cγ-endo-Cβ-exo) categories are the most preferred combinations. For Proline rings in proteins, the states Cγ-exo (A), Twisted Cγ-exo-Cβ-endo (E) and Twisted Cγ-endo-Cβ-exo (F) are the most preferred states of occurrence of the pyrrolidine ring conformation. Chapter 7 describes the analysis of diproline segments in a non-redundant dataset of proteins In this chapter, the possible conformational states for the diproline segment (LPro-LPro) found in proteins taken from a non-redundant dataset has been investigated an identified with an emphasis on the cis and trans states for the peptide bond between the diproline segment. The occurrence of diproline segments in type VIA1 turns (cis Pro-Pro peptide bond) and other regular secondary structures like type III β-turns and α-helices has been studied. This has been followed up by the amino acid distribution flanking the diproline segment and the conformation adopted by Xaa-Pro and Yaa-Pro segments in proteins. It is observed that for cis Pro-pro peptide bond, the conformation adopted by the first Proline lies in PII region whereas the second Proline inevitably adopts a conformation in the Bridge region, leading to the formation of the type VIA1 β-turn structure. But in the trans case, the conformation adopted by the first Proline is overwhelmingly populated in the PII (Polyproline) and right-handed α-helical region. For position i+2, the major conformation adopted by Proline is P II and α with a substantial amount of occurrences in Bridge and the C7 (γ-turn) region. The analysis also reveals that the cis-cis configuration of the peptide bond is very rare when considering the diproline segment. With a cis-trans peptide linkage, PII-PII conformation is the most stable and favoured conformation for the Pro-Pro segment in proteins. With trans peptide bond linkage between the Proline residues, α- α and PII-Bridge conformations are equally likely for the diproline segment. The population in trans-cis and cis-trans states are comparable indicating that the energy differences between these states is small. However, trans-trans is the most populated state with a percentage occurrence of 85.43%. The analysis and comparison of conformational states for the Xaa-Pro-Yaa sequence reveals that the Xaa-Pro peptide bond exists preferably as the trans conformer rather than the cis conformer. The same is valid for Pro-Yaa segment, with the cis conformer being populated to even lesser extent. The data shows that α- α, PII-α, PII-PII and extended-PII are the most populated states for Xaa-Pro and Pro-Yaa segments as compared to PII-PII and PII-α and states observed for the Pro-Pro segment. Chapter 8 describes the analysis of single and multiple β-turns in a non-redundant dataset of proteins. The analysis on β-turns in proteins has shed a new light into the propensity values for amino acid residues at various positions of β-turns which in certain cases have undergone appreciable change in values than previously observed. One of the other notable feature of the analysis is the fact that the data displays a higher occurrence of unprimed β-turns of type I and type II as compared to their primed counterparts of type I’ and type II’ as previously observed. In fact, the results show that type I β-turn is the highest occurring turn both in isolated as well as in consecutive β-turn examples. The analysis of multiple β-turns in proteins has revealed many new categories like the (I,I+1,I+3), (I,I+2,I+3) and combination of turns which can be used for the design of the loops, especially in the case of β-hairpins. Among the multiple turns, double turns occur more frequently than the other consecutive turns like triple and quadruple turns. It is also important to note that the number of examples of a hydrogen bonded turn being followed by a hydrogen bonded turn is very less with type IV turn preceding a primed turn in most of the cases. Thus, the data available from consecutive β-turn analysis and the type-dependent amino acid positional preferences and propensities derived from the present study may be useful for modeling various single and consecutive turns, especially in designing loop regions of β-hairpins.

Circular dichroism (CD) is the ideal technique for studying chiral molecules in solution. It is uniquely sensitive to the asymmetry of the system. These features make it particularly attractive for biological systems. CD is by definition the difference in absorption, A, of left and right circularly polarized light (CPL): . . . CD = Ae − Ar . . . . . . 1 . . . CPL has the electric field vector of the electromagnetic radiation retaining constant magnitude in time but tracing out a helix about the propagation direction. Following the optics convention we take the tip of the electric field vector of right CPL to trace out a right-handed helix in space at any instant of time (1, 2). CD spectra can in principle be measured with any frequency of electromagnetic radiation. In practice, most CD spectroscopy involves the ultraviolet-visible (UV-visible) regions of the spectrum and electronic transitions, though increasing progress is being made with measuring the CD spectra of vibrational transitions using infrared radiation. We shall limit our consideration to electronic CD spectroscopy since the practical considerations for vibrational CD differ from those for electronic CD. For randomly oriented samples, such as solutions, a net CD signal will only be observed for chiral molecules (ones that cannot be superposed on their mirror images (3)). Oriented samples of achiral molecules, such as crystals, will also give a CD spectrum unless the optical axis of the sample aligns with the propagation direction of the radiation. However, such spectra are seldom useful. CD is now a routine tool in many laboratories. The most common applications include proving that a chiral molecule has indeed been synthesized or resolved into pure enantiomers and probing the structure of biological macromolecules, in particular determining the α-helical content of proteins. Figure 3 gives an example of a CD spectrum. The key points to remember are that a CD signal is observed only at wavelengths where the sample absorbs radiation, i.e. under absorption bands, and the signal may be positive or negative depending on the handedness of the molecules in the sample and the transition being studied.

The dihydropyridine (DP) Ca2+ channel antagonist, nifedipine (NF), inhibits platelet aggregation .in vitro and ex vivo by an undefined mechanism. Inhibition of Ca2+ influx via Ca2+ channels is a postulated mechanism, but voltage-dependent Ca2+ channels have not been demonstrated in platelets. We previously observed that NF blocked thromboxane A2 (TXA2)-induced platelet aggregation and secretion. In order to further evaluate the mechanism of DP inhibition of platelet activation, we studied the effects of NF and BAY K 8644, (BAY), a DP with opposite (agonist) effects on muscle cells, on human platelet aggregation and secretion induced by the TXA2 mimic, U46619. We also observed the effects of DP on biochemical consequences of platelet activation: cytoplasmic ionized Ca2+ ([Ca2+]i) by fura-2 fluorescence; phosphorylation of 40,000 Dalton protein (40KP) substrate of protein kinase C by SDS-PAGE and [32p] counting; TXA2 formation by RIA of TXB2. 1μM BAY and 10μM NF inhibited the 2nd wave of platelet aggregation and secretion induced by ADP or epinephrine and blocked aggregation and secretion induced by U46619. A Schild plot gave a slope of -1 indicating competitive inhibition of U46619 by BAY (K1[=0.7μM).BAY and NF also blocked U46619-induced phosphorylation of 40KP, rise in [Ca2+]i and TXB2 formation. The (+)-(R) enantiomer of BAY (BAY+) was responsible for BAY inhibition. BAY, BAY(+), and the R enantiomer of another DP, 202-791, all functioned as competitive antagonists of [3H]-U4661 9 binding (K1[ for BAY=2.8 μM-comparable to known receptor antagonists, 13-azaprostanoic acid and BM 13.177; K1 for BAY(+)=0.69μM). Neither BAY nor NF inhibited[3H]-yohimbine binding to α adrenergic receptors.NF, BAY, BAY(+) and BAY(-) in nM concentrations slightly stimulated platelet aggregation,secretion and biochemical events induced by U46619 similar to their effects on muscle. Therefore, DP's do not inhibit platelet activation by blocking voltage-dependent Ca2+ channels. The mechanism of DP inhibition of TXA2-induced platelet activation is stereoselective, competitive binding to the TXA2/PGH2 receptor. DP's may exert similar effects on TXA2-induced vascular smooth muscle contraction.