Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aim of this work was to study the vegetative propagation of louro-pardo (Cordia trichotoma (Vell.) Arrab. ex Steud.) from root cuttings and minicuttings. Three trials were conducted. The first trial, roots were gotten from adult trees of louro-pardo. Root cuttings with 5.0 cm of length were classified in two diameters: thick (2.0 - 3.0 cm) and thin (< 2.0 cm). The root cuttings were immersed for 10 s. in an indolbutiric acid (IBA) solution of 0, 4000, 8000 or 12000 mg L-1. At 180 days, the percentages of sprouting and rooting, the number and length of shoots and roots were evaluated. Root cuttings from adult trees of louro-pardo showed 1.25% of sprouting, but they did not root. The second trial, root cuttings were gotten from three year-old plants of louro-pardo and root cuttings classified in small (1.0 - 1.5 cm) and large (1.6 - 2.5 cm) and immersed for 10 s. in an IBA solution of 0, 2000, 4000 or 6000 mg L-1. At 30 and 60 days, the percentages of rooted cuttings; sprouted cuttings; and survival of cuttings; the number and length of sprouted and rooted cuttings were evaluated. Root cuttings treated with 6000 mg L-1 of IBA had the best response in terms of sprouting and rooting. Large root cuttings showed the highest percentage of sprouting (26%), the highest number of sprouts (0.30) and the longhest sprouts and roots (2.27 and 5.40 cm) at 60 days of evaluation. The third trial, root cuttings were classified according to the position in the root system as basal, medium and apical. Cutting measuring 1.0; 3.0 or 5.0 cm of length were immersed for 10 s. in an IBA solution of 6000 mg L-1. At 60 and 90 days, the percentages of rooted and sprouted cuttings; and survival; the number and length of sprouted and rooted cuttings were evaluated. Basal and medium cuttings had the best response in terms of sprouting and rooting. Root cuttings measuring 3.0 and 5.0 cm were better than those with 1.0 cm of length. In this study, we also carried out trials with minicuttings of louro-pardo. The first trial, ministumps from plantlets of asexual and seminal origin with six and 12 month-old were fertilized with different nutrient solutions. The production of minicuttings per ministump was evaluated after 30 and 60 days. Minicuttings from these ministumps were treated or not with 1000 mg L-1 of naphthalene acetic acid (NAA). At 30 days, the percentage of sprouting, rooting and survival; the number of sprouts; and the number and length of roots were evaluated. The second trial, minicuttings from ministumps of asexual and seminal origin were treated or not with 2000 mg L-1 of IBA. The same evaluations were done at 30 and 60 days. The concentration of the nutrient solution did not affect the production of minicuttings per ministumps. The concentrations of NAA and IBA did not affect minicutting rooting. Overall, ministumps from asexual origin showed adequate capability for vegetative propagation of louro-pardo. These ministumps have the highest production of minicutting associated with the highest rooting potential without any treatment. / O objetivo deste estudo foi avaliar a propagação vegetativa de louro-pardo (Cordia trichotoma (Vell.) Arrab. ex Steud.) por estaquia radicular e miniestaquia. Foram realizados três experimentos de estaquia radicular. No primeiro experimento, raízes de árvores adultas de louro-pardo foram seccionadas em estacas com 5,0 cm de comprimento e classificadas quanto ao diâmetro em grossas (2,0 - 3,0 cm) e finas (< 2,0 cm). As estacas radiculares foram tratadas em solução de 0, 4000, 8000 ou 12000 mg L-1 de ácido indolbutírico (AIB). Aos 180 dias, avaliaram-se as porcentagens de brotação e enraizamento, o número e comprimento de brotos e raízes. Estacas radiculares adultas de louro-pardo apresentaram 1,25% de brotação e não formaram raízes. No segundo experimento, raízes de plantas de louro-pardo de origem seminal e com três anos de idade foram seccionadas em estacas com 5,0 cm de comprimento, classificadas quanto ao diâmetro em grossas (1,6 - 2,5 cm) e finas (1,0 - 1,5 cm) e tratadas em solução de 0, 2000, 4000 ou 6000 mg L-1 de AIB. Aos 30 e 60 dias, avaliaram-se as porcentagens de brotação e enraizamento, a sobrevivência das estacas, o número e o comprimento de brotos e raízes. Estacas radiculares juvenis, tratadas em solução de 6000 mg L-1 de AIB, apresentaram a maior porcentagem de enraizamento e o maior número de raízes, aos 30 e 60 dias. Estacas radiculares grossas apresentaram a maior porcentagem de brotação (26%), o maior número de brotos (0,30) e o maior comprimento de brotos e raízes (2,27 e 5,40 cm), aos 60 dias de avaliação. No terceiro experimento, raízes de plantas de louro-pardo de origem seminal e com três anos de idade foram classificadas quanto à posição de coleta em basais, medianas e apicais, seccionadas em estacas de 1,0; 3,0 e 5,0 cm de comprimento e tratadas com 6000 mg L-1 de AIB. Aos 60 e 90 dias, avaliaram-se as porcentagens de brotação e enraizamento, a sobrevivência das estacas, o número e comprimento de brotos e raízes, e o comprimento de brotos e raízes por volume de estaca. Estacas radiculares juvenis basais e medianas apresentaram as melhores respostas de enraizamento, aos 60 e 90 dias. Estacas radiculares juvenis com 3,0 e 5,0 cm de comprimento se mostraram superiores às estacas radiculares com 1,0 cm. Neste estudo, também foram conduzidos experimentos de miniestaquia de louro-pardo. No primeiro experimento, minicepas de origem assexuada e de origem seminal foram fertirrigadas com diferentes concentrações de solução nutritiva. A produção de miniestacas por minicepa foi avaliada aos 30 e 60 dias. Para o enraizamento, as miniestacas foram tratadas ou não em solução de 1000 mg L-1 de ácido naftalenoacético (ANA). Aos 30 dias, avaliaram-se as porcentagens de brotação e enraizamento, a sobrevivência das miniestacas, o número e comprimento de brotos e raízes. No segundo experimento, miniestacas com e sem folhas obtidas de minicepas de origem assexuada e seminal foram tratadas ou não com 2000 mg L-1 de AIB. Aos 30 e 60 dias, foram avaliadas as variáveis descritas anteriormente. A concentração da solução nutritiva não influenciou a produção de miniestacas/minicepas. O uso de ANA e AIB não afetou o enraizamento das miniestacas. As minicepas de origem assexuada mostraram-se adequadas à propagação vegetativa de louro-pardo, pois apresentaram maior produtividade em minijardim clonal associada ao maior potencial de enraizamento das miniestacas, sem a necessidade de tratamento com auxina.

This study had as objective to study the in vitro regeneration of cedar (Cedrela fissilis Vell.), a native tree of economic interest, belonging to Meliaceae family. For seeds desinfestation, different treatments with sodium hypochlorite in different concentration and immersion times were tested combined with differents culture mediums. To establish the nodal segments in vitro, benlate at 300 mg.l-1 and streptomicine sulphate at 10 mg.l-1 were added to the culture medium WPM reduced to ¾ of its salts concentration, isolated or not. After that, the hypocotyl segments from the 30 days in vitro cultured plants were used as explant in sprouts multiplication in culture mediums MS and WPM combined or not with the cytokinins BAP and KIN at 5μM.l-1 . It was also tested, in hypocotyl segments, the influence of BAP (0; 1,25; 2,5 and 5,0 μM.l-1) combined with the IBA or NAA (0,55μM.l- 1), in culture medium WPM during two subcultives. 60 days hypocotyl segments from cultive in BAP (0;1,25; 2,5 and 5,0 μM.l-1) were transferred to WPM medium added of only GA3 at 3μM.l-1 remaining during more than 30 days. Seeds desinfestated with sodium hypochlorite at 2% during 10 minutes showed a lower index of fungal contamination (8,7%) and the higher index of germination (80%), the mediums MS and WPM allowed the germination of 72,5%. The nodal segments from 3 years old plants showed an average index of bacterial contamination (13% 21 days after the inocultation) when cultivated in WPM culture medium added of benlate (300mg.l-1) plus streptomicine sulphate (10 mg.l-1) and survival index of 83%. The explants from the hypocotyl region showed average indexes of regeneration of 2,8 when cultivated in WPM medium supplemented with BAP at 5μM.l-1, on the other hand, in MS medium, the average tax of formation was 2,5 sprouts per explant. When tested in WPM culture medium, in different BAP concentrations, only at 30 days of cultive there was an interaction between the cytokinins doses and the presence of auxins IBA and NAA. BAP at 5μM showed explants with higher taxes of multiplication, regardless the auxins addition to the culture medium. In the following subcultives there was not an interaction between the concentrations and the effect of auxin presence, where the medium without auxin, associated to BAP at 5μM, promoted a higher average of sprouts per explant 2,29 (60 days). The average number of knots at 30 days of cultive was of 2,84 when evaluated the medium with ANA plus 2,5μM of BAP. At 60 days, the culture medium without the auxine promoted an average of 4,15 knots per explant, differing statistically from the mediums with auxin. At 90 days, the number of knots were 5,59 when BAP was used at 5μM plus IBA. The average length of the sprouts in the medium with NAA showed to be the more efficient (0,86 cm) at 30 days of cultive. At 60 days, the average length of the sprouts was of 0,95 and, at 90 days the average length of the sprouts was of 1,14. The formation of callus in the base of the explants happened when cytokinins were added to the medium, and the roots formation happened in the absence of these and in the presence of the auxin, generating complete plants. Explants exposed to GA3 at 60 days of cultive showed a partial reversion, showing an increase in formed roots and average length of the sprouts. / O presente trabalho teve como objetivo o estudo da regeneração in vitro de cedro (Cedrela fissilis Vell.), uma árvore nativa de interesse econômico, pertencente à família Meliaceae. Para a desinfestação de sementes, foram testadas diferentes concentrações e tempos de imersão em hipoclorito de sódio e para a germinação, diferentes meios de cultura. Para o estabelecimento in vitro de segmentos nodais, foram adicionados ao meio de cultura WPM, reduzido a ¾ de sua concentração original, benlate (300 mg.l-1) e sulfato de estreptomicina (10 mg.l-1). Após, segmentos do nó cotiledonar advindos de plântulas germinadas in vitro com 30 dias de idade, foram utilizados para a multiplicação de brotações nos meios de cultura MS e WPM, em combinação ou não com 5μM.l-1 de BAP ou KIN . Foi testado também, a influência da citocinina BAP (0; 1,25; 2,5 e 5,0 μM.l-1) combinada ou não com as auxinas AIB ou ANA (0,5μM.l-1) em meio de cultura WPM durante dois subcultivos. Os segmentos do nó cotiledonar com 60 dias de cultivo em meio WPM e BAP (0; 1,25; 2,5 e 5,0 μM.l-1) foram transferidos para meio WPM adicionado apenas de GA3 a 3μM.l-1 permanecendo por mais 30 dias. Os resultados demonstram que as sementes desinfestadas com hipoclorito de sódio a 2% durante 10 minutos apresentaram a menor taxa de contaminação por fungos (8,7%) e a maior percentagem de germinação (80%), os meios MS e WPM proporcionaram percentagem de germinação de 72,5%. Os segmentos nodais de plantas de três anos de idade apresentaram uma taxa média de contaminação por bactérias de 13% após 21 dias de inoculação quando cultivados em meio de cultura WPM adicionado de benlate (300mg.l-1)mais sulfato de estreptomicina (10 mg.l-1)e taxa de sobrevivência de 83%. Os explantes provenientes da região do nó cotiledonar apresentaram regeneração média de brotações de 2,8 quando cultivados em meio WPM suplementado com BAP a 5μM.l-1, enquanto que, no meio MS, a taxa média de brotações foi de 2,5 por explante. Quanto testados em meio de cultura WPM, em diferentes concentrações de BAP, apenas aos 30 dias de cultivo, houve interação entre as doses da citocinina e a presença das auxinas AIB e ANA. O BAP a 5μM apresentou explantes com maiores taxas de multiplicação de brotações independente da adição das auxinas ao meio de cultura. Nos subcultivos subseqüentes, não houve interação entre as concentrações e o efeito da presença de auxina, em que o meio sem a presença desta, adicionado o BAP a 5μM, proporcionou a maior média de brotações por explante 2,29 (60 dias). O número médio de nós aos 30 dias de cultivo foi de 2,84 no meio contendo ANA mais 2,5 μM de BAP. Enquanto que, aos 60 dias, o meio de cultura sem auxina proporcionou uma média de 4,15 nós por explante, diferindo estatisticamente dos meios contendo auxina. Aos 90 dias de cultivo, o número de nós foi de 5,59 quando utilizado BAP a 5μM mais AIB ao meio de cultura. Quanto ao comprimento médio das brotações, o meio contendo ANA mostrou ser o mais eficiente (0,86 cm) aos 30 dias de cultivo. Aos 60 dias, o comprimento médio das brotações foi de 0,95 e, aos 90 dias o comprimento médio de brotações foi de 1,14. A formação de calos na base dos explantes ocorreu quando foi adicionada citocinina ao meio, e a formação de raízes ocorreu na ausência desta e na presença de auxina, gerando plantas completas. Explantes expostos ao GA3 aos 60 dias de cultivo mostraram parcial reversão, apresentando um aumento na percentagem de raízes formadas e comprimento médio de brotações.

Submitted by Eduardo Oliveira (eduardojorge.com@gmail.com) on 2018-06-22T02:31:55Z No. of bitstreams: 1 Cidade “verde” ou cidade “vermelha” AIB e ANL em Petrópolis.pdf: 9180649 bytes, checksum: b72faf8b1eacf5d20e77a632a1f5fdcb (MD5) / Approved for entry into archive by Diego Andrade (diego.andrade@fgv.br) on 2018-07-30T18:47:02Z (GMT) No. of bitstreams: 1 Cidade “verde” ou cidade “vermelha” AIB e ANL em Petrópolis.pdf: 9180649 bytes, checksum: b72faf8b1eacf5d20e77a632a1f5fdcb (MD5) / Made available in DSpace on 2018-08-06T17:03:23Z (GMT). No. of bitstreams: 1 Cidade “verde” ou cidade “vermelha” AIB e ANL em Petrópolis.pdf: 9180649 bytes, checksum: b72faf8b1eacf5d20e77a632a1f5fdcb (MD5) Previous issue date: 2018-06-15 / O enfrentamento entre grupos militantes antagonistas é um fenômeno presente em vários momentos da história política brasileira, especialmente no período republicano. Devido a diferentes contextos, pode-se entender, igualmente, que diferentes motivos podem determinar a origem de cada um destes conflitos. O primeiro destes grandes confrontos se deu entre membros da Ação Integralista Brasileira (AIB) e Aliança Libertadora Nacional (ANL), em 1935. Foram diversos enfrentamentos, envolvendo vários de militantes, registrados em diferentes pontos do país. Considere-se, porém, que embora houvesse causas comuns que motivavam a oposição entre as siglas em todo território nacional, havia também fatores locais, que diferenciavam as características deste enfrentamento de acordo com o cenário em que ele se desenvolvia. Partindo desta hipótese, a presente pesquisa toma como objeto os eventos relacionados ao enfrentamento AIB-ANL na cidade de Petrópolis (RJ). Foi, simultaneamente, um importante pólo para a militância integralista e, igualmente, um importante centro para a atuação da Aliança. Considera-se, aqui, que tanto a mobilização quanto as ações dos militantes foram, de alguma maneira, bastante distintas das formas de enfrentamento observadas em outros pontos do país, especialmente no tocante à greve geral que se seguiu à morte de um militante aliancista. / The confrontation between antagonistic militant groups is a phenomenon present in several moments of Brazilian political history, especially in the republican period. Due to different contexts, it can also be understood that different reasons can determine the origin of each of these conflicts. The first of these greats clashes occurred between members of the Ação Integralista Brasileira (AIB) and the Aliança Nacional Libertadora (ANL) in 1935. There were several clashes, involving many militants, registered in different parts of the country. It consider, however, that although there had common causes that motivated the opposition between the groups throughout the national territory, there were also local factors, which differentiated the characteristics of this confrontation according to the scenario in which it developed. Based on this hypothesis, this research takes as object the events related to the AIB-ANL confrontation in the city of Petrópolis (RJ). It was, simultaneously, an important pole for integralist militancy and also an important center for the ANL's activities. It is considered here that both the mobilization and the actions of the militants were in some way quite different from the forms of confrontation observed in other parts of the country, especially regarding the general strike that followed the death of a ANL militant.

This thesis entitled “Investigating the Intercarbonyl X···C′ (X = O/S/N) Interactions in Short Peptides and Peptidomimetics. Evidence of Charge→π* Interactions. Synthesis and Characterization of Thioimidate Isostere Containing Peptidomimetics” is divided into two chapters. First chapter is further subdivided into four sections where investigation of the nature of intercarbonyl X···C′ (X = O/S/N) interactions in short peptides and peptidomimetics has been described. The second chapter also has been subdivided into three parts where the syntheses and characterization of thioimidate (1,3-thiazine) and imidate (1,3-oxazine) isostere containing peptidomimetics have been discussed. Chapter 1: Section A: Revisiting the earlier models for the intercarbonyl O···C′ interactions The proximity between carbonyls is ubiquitous in crystals. Here we review the key reports that have assigned an n→π* nature to interactions between carbonyl oxygen (O) atoms and adjacent carbonyl carbon (C′) atoms (O···C′). Based on earlier hypotheses (by Burgi-Dunitz) that suggest that “the minimum energy trajectory of a nitrogen nucleophile adding to the C′ of carbonyl is at N···C′ distances of ≤ 3.2 Å and along N···C′=O angles of 109±10o”, the optimum trajectory for addition of an O to an adjacent C′ has also been assigned to be the same (O···C′ distance ≤ 3.2 Å and O···C′=O angle is 109±10o). Additionally, all O and C′ atoms within these boundary conditions in crystal structures were assigned a status of interacting and those outside of the same as non-interacting. Based on quantum mechanical models for electronic orbitals that contain the valence electrons of such proximal O and C′ atoms – derived through NBO (Natural Bond Order) calculations (on crystal structures) – it has been proposed that the filled non-bonding lone pair orbital of the O (donor) overlaps with the empty π* orbital of the carbonyl C′ (acceptor), in these O···C′ interactions. Hence, these have been termed as n→π* interactions. Using DFT (Density Functional Theory) calculations energies for these interactions have been predicted to range from 0.5 to 5.0 kcal mol-1, which are similar to those for other strong non-covalent interactions such as H-bonding, weak cation-π, etc. This n→π* interaction model is assumed to prevail between adjacent carbonyls (Oi-1···C′i) at Xaa-Pro dipeptide motifs and to be exclusively responsible for the changes in equilibrium constant values (Kc/t) for the trans to cis isomerisation reaction at Xaa-Pro peptide bond in chosen analogue molecules. Based on this assumption, these Kc/t values have been used as direct experimental equivalents for the energies of these n→π* interactions. Simultaneous to such review of literature, this chapter highlights several anomalies in this n→π* model for the intercarbonyl O···C′ interactions. We discuss the alternate models that also exist for the O···C′ proximities and show that several features – such as improved pyramidalization at the acceptor carbonyl; decrease in Kc/t values at Xaa-Pro peptide bonds; and small changes in 13C NMR chemical shift values for the acceptor carbonyls; etc. – that accompany the shortening of O···C′ distance, can be explained without invoking the n→π* interaction model. Moreover, we discuss key observations such as the presence of near-symmetric antiparallel short contacts between carbonyl groups (C=O) in crystal structures, which cannot be explained by the quantum mechanical n→π* model for the O···C′ interactions. Chapter 1: Section B: Spectroscopic and kinetic investigations into the nature of X···C′ (X = O/S) interactions in N-acyl homoserine lactones (AHLs) In this section the key interactions involving the adjacent carbonyls in model N-acyl homoserine lactones (AHLs) (which are signalling molecules in quorum sensing) in solution, their electronic nature and their influence on solvolysis of the lactone ring have been investigated. Earlier, in the crystal structures of two sterically encumbered synthetic AHL analogues N-trimethyl acetyl homoserine lactone and N-tribromoacetyl homosrine lactone the presence of an n→π* orbital overlap type interaction between Oacyl and C′lact had been suggested. Based primarily on this, the operation of similar OacylC′lact interaction was proposed in all AHLs in their solution conformations as well. More intriguingly, the interaction was hypothesized to decrease the rates of lactone hydrolysis, rendering AHLs with longer biological half-life. This is contrary to physical organic understanding of nucleophilic catalysis of addition to carbonyls. Here we synthesize a variety of AHLs and analyze their NMR and FT-IR data in solution. The spectral data reveal that the role of the N-acyl group in AHLs is to withdraw eˉs from lactone C=O inductively and to improve electronic shielding at C′lact. Lack of appreciable changes in C=O stretching frequencies of lactone and 13C NMR chemical shift values of C′lact indicate the absence of electronic perturbation of the π* of the lactone. Similar non-variance of spectral bands with improvement in nucleophilicity of the N-acyl group indicates the absence of any evidence for n→π* nature for the O···C′ interactions (between the lone pair of eˉs from Oacyl to π* at C′lact). Further the spectroscopic data indicate that any change in charge at the acyl O is felt by C′lact and this weak interaction releases energy in the order of ≤ 0.05 kcal mol-1. The combined influence of the electron withdrawing N-acyl group and the weak Oacyl···C′lact interaction in AHLs is that, increasing the charge at Oacyl increases the rate of solvolysis of lactone. Analysis of the conformation of the lactone ring in the LuxR receptor-bound and unbound crystal structure forms reveals the flattening of the puckered ring in the LuxR bound state – facilitated by several interactions with the receptor. Conserved interactions between LuxR and AHLs lock the N-acyl carbonyl motif such that they are orthogonal to the lactone carbonyl and intramolecular interaction between Oacyl and C′lact is precluded. We propose the design of flat cyclic analogues of γ-butyrolactone bearing electron withdrawing side chains as potential molecules for taking advantage of bacterial quorum sensing in environmental applications and biotechnology. Chapter 1: Section C: Spectroscopic investigation into the nature of intercarbonyl X•••C′ (X = O/S/N) interactions: Carbamyl-cisPro model systems In this section we investigate the nature of intercarbonyl X···C′ interactions in carbamyl-Pro model systems using spectroscopic methods like FT-IR and 1D NMR. Further we derive the enthalpic and entropic contributions towards the free energy for trans to cis isomerization (Kc/t) at these model carbamyl-Pro systems. Our results reveal that changes in Kc/t values cannot always be used as proof for the presence or absence of electronic interactions, and hence to unambiguously suggest the nature of these interactions. Cis/trans isomerism exists at Xaa-Pro amide and carbamate motifs, and it was proposed that in acyl-Pro systems the O···C′ interactions are responsible for the stability of either cis or trans depending upon their direction of operation (Forward direction: O of Xaa is the donor of electrons to π* at C′ of Pro; Reverse direction: O of Pro is the donor of electrons to π* at C′ of Xaa). Investigation of the carbamyl-Pro systems can shed further light on this hypothesis. Hence we undertook the first spectroscopic and Van’t Hoff analysis of homologous carbamyl-Pro model systems. The Kc/t of the homologous series surprisingly increased with increase in the bulk at R (R varies from Me to tBu). The spectroscopic data revealed the presence of charge→σ* interactions at carbamyl groups. This interaction locks the carbamyl motif in the s-transoid conformation, along the C′-O σ-bond. Such conformational lock is observed to be greater in carbamyl groups where R has at least one Cα-H bond. Interestingly, we observe the absence of X···C′ electronic interactions that may selectively stabilize the cisPro conformer in these molecules. Van’t Hoff analyses on the other hand showed that as the number of Me substituents in R increases (R = Me to iPr), there is a favorable increase in entropy ( So) associated with the transPro to cisPro conformational isomerism. As a result, the population of the cisPro conformer improves significantly as the steric bulk at R increases. We note that the enthalpy of cisPro is however relatively small and remains unfavourable as R-bulk increases (Me to iPr). These data reveal the influence of electrostatic interactions between charged groups, on the change in entropy associated with cis/trans isomerism at carbamayl-Pro motifs. This not only opposes the n→π* model, but also provides an example for the important point that changes in Kc/t can/should not be taken as direct evidences of any single electronic interaction. Importantly, this study provides another example where electronic interactions between charged, polarized carbonyl motif rather than nonbonding lone pair eˉs of carbonyl motifs influence cis/trans isomerism at Xaa-Pro systems. Chapter 1: Section D: Investigation of the stereoelectronic nature of the X···C′ (X = O/S) contacts In this section we provide experimental evidence for the existence of inverse correlation between the charge on the O nucleophile and the O···C′ distances. We show that O and C′ atoms (of adjacent carbonyls), which are separated at distances > 3.20 Å in carefully chosen analogues, come together to σ-bonding distances when the charge on O is increased to -1. Additionally, the influence of backbone steric factors on these charge→π* interactions is investigated. A partial covalent nature was proposed for the O···C′ interactions. Our study showed that the shortest intercarbonyl O···C′ distances between the O of 1°, 2° and 3° amide carbonyls and proximal C′ in molecules found in the Cambridge Structural Database (CSD) (v5.36, November 2014) show an inverse linear correlation with the partial negative charge (δ‾) on the amide carbonyl O rendered by natural amide carbonyl polarization. These data suggest the interaction of charge on the nucleophilic O with π* of the acceptor carbonyl. Further on increasing the charge on nucleophilic carbonyl O to -1 in the model compound, we achieved the formation of σ-bond through non-native (natively disallowed) Oi‾¹→C′i-1 interaction. Here we provide the first experimental evidences that suggest the interaction between charge of O and π* at adjacent C′ (the charge→π* interaction) and the latent covalent nature of the O···C′ interactions. This charge→π* model explains the origins of variations in O···C′ distances (3.20 Å–1.43 Å) in proteins and complexes that occur to suit biological functions; and the mutual interactions between antiparallel carbonyls. Further the effect of 3 key steric factors – namely the allowed τ (N-Cα-C′) angle, entropy and allowed (ϕ,ψ) angles – on the non-native Oi→C′i-1 interactions were investigated in the model compounds. Our kinetic data revealed that, the allowed τ angles have the greatest influence on charge→π* interaction, followed by entropy. Importantly the allowed (ϕ,ψ) torsional angles for residues, that govern protein folding pathways, have little influence on the O···C′ electronic interactions. Chapter 2: Section A: Design and synthesis of novel 1,3-Thiazine containing peptidomimetics This section describes the first synthesis of peptidomimetics containing the 1,3-thiazine isostere (thioimidate isostere for the peptide bond), at the C-terminus and also at the middle of the peptide. The synthesis of the 6-membered heterocycles – 1,3-oxazine (Oxa) – have earlier been reported. Oxa motifs constrain preceding amino acid backbones into natively disallowed conformations. Here we present the first synthesis of peptidomimetics containing the 1,3-thiazine (Thi) (the thioimidate analogue of Oxa) motif, by the treatment of N-(3-hydroxypropyl)thioamides with MsCl/Et3N, which leads to intramolecular S-alkylation / cyclization. When placed at the C-terminus of acyl-Pro motifs the Thi group selectively improves the stability of the rare s-cis conformation of the acyl-Pro peptide bond. Further this method has been used to synthesize peptidomimetics in which an endogenous peptide bond is replaced with the Thi isostere. These Thi analogues are shown to be stable to standard conditions of peptide coupling and N- and C- terminus protection, deprotection and can be extended selectively at their N- or C- termini. Chapter 2: Section B: Epimerization in 1,3-Thiazine containing peptidomimetics The epimerization in 1,3-thiazine containing peptidomimetics and its mechanism has been described in this section. Further the aggregation behaviour of these thiazines, in solution and crystal structures, has been studied. It has been well-documented that epimerization (Racemization) occurs at the chiral centers at the C(2) exo methine of 1,3-thiazolines and 1,3-thiazoles. Similar epimerizations in 1,3-thiazines have however not been explored. Here we report our observation of epimerization in chiral aminoacid (non Pro) containing 1,3-thiazine peptidomimetics. Our studies revealed that, the epimerization happens at C2 positions of chiral (non-Pro) amino acids-derived 1,3-thiazine containing peptiomimetics. And NH of chiral (non-Pro) amino acid fused to Thi ring at C2 position is necessary for the epimerization. Further we investigated the Boc-Xaa*-Thi analogues in solution, which showed two resonances for the carbamate N-H (HN) and the H of Xaa*, irrespective of the side chain in Xaa, in CDCl3 a weakly polar solvent. The integral ratios of the major : minor peak increased with increase in concentration for Boc-Val*-Thi, indicating the formation of H-bonded aggregates. Even in the polar aprotic (DMSO-d6) and polar protic (D2O) solvents the two sets of resonances were observed for Boc-Val*-Thi in 1H NMR. But when the thioimidate N is protonated (N of Thi is no longer a H-bond acceptor), showed only a single set of resonances. Formation of intermolecular H-bonds involving N of Thi in solution is thus evident in the aggregates. This is further suggested by the crystal structures obtained for the peptide mimetics Boc-Val*-Thi, Boc-Leu*-Thi and Boc-Phe*-Thi in which the racemic pair, instead of one enantiomer of it, are present in the unit cell and are locked in a pair of intermolecular 10 membered H-bonding interactions between NThi and HLeu* similar to an antiparallel β-sheet. A mechanism for racemization is proposed, where this strong H-bond assists enamination/racemization process. Chapter 2: Section C: Influence of a disallowed conformation of Aib on the structure of a 310-helical fold In this section, the effect of the presence of a disallowed conformation of Aib at the C-terminus of a 310-helical peptide, on the structure and fold of the rest of the peptide body has been studied in solution. We constrain the C-terminal Aib in the Aib-rich octapeptide (N-tert-butoxycarbonyl-Leu1-Aib2-Ala3-Leu4-Aib5-Ala6-Phe7-Aib8-CO2Me (1), which adopts a complete 310-helical conformation throughout the peptide body in the crystal structure and in solution) in one of its disallowed conformations using a method earlier developed in our group. This involves the synthetic modification of the C-terminal ester (Aib8-CO2Me) in 1 to an Oxa (Aib*8-Oxa) in 2 and the study of its effect on the peptide body. Analyses of the solution FT-IR, CD, ¹H, 2D (TOCSY, HSQC, HMBC and ROESY) and solvent polarity dependent NMR data reveal that 2 adopts a 310-helical conformation similar to that of 1. The C-terminal CO2Me → Oxa (E → O) modified Aib*8-Oxa motif is constrained in a unique conformation where the two Cβ atoms of Aib*8 are staggered with respect to the Aib*8 C=O and are both interacting with the two Hβ of Phe7. Here the Aib* backbone is constrained by a 5-membered ring NOxa∙∙∙HAib* H-bond, in a C5i structure. Solvent polarity dependent ¹H NMR data indicate the formation and persistence of C5i H-bond at the Aib*8-Oxa motif in 2. Analyses of the ROESY, solvent polarity dependent ¹H NMR and CD spectra reveal that four crucial changes in ROESY cross peaks occur at the Phe7-Aib*8 motif of 2, compared to that in 1. From these spectroscopic data it has been confirmed that there is no change in the structure of 2 from Leu1 to Ala6. Whatever the crucial changes happened are at Phe7-Aib*8 motif of 2. Hence our study showed that the significant structural consequences of this disallowed conformation of Aib* are primarily observed to occur in the residue in its immediate vicinity, rather than in the whole peptide body. Presence of a disallowed fold at a residue need not result in disruption of the structure, or the overall fold, in the rest of the peptide body.

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