Plant derived oils bear intrinsic double-bond functionality that can be utilized directly for the thiol–ene reaction. Although terminal unsaturations are far more reactive than internal ones, studies on the reversible addition of thiyl radicals to 1,2-disubstituted alkenes show that this is an important reaction. To investigate the thiol–ene coupling reaction involving these enes, stoichiometric mixtures of a trifunctional propionate thiol with monounsaturated fatty acid methyl esters (methyl oleate or methyl elaidate) supplemented with 2.0 wt.% Irgacure 184 were subjected to 365-nm UV-irradiation and the chemical changes monitored. Continuous (RT– FTIR) and discontinuous (NMR and FT–Raman) techniques were used to follow the progress of the reaction and reveal details of the products formed. Experimental results supported by numerical kinetic simulations of the system confirm the reaction mechanism showing a very fast cis/trans-isomerization of the alkene monomers (<1.0 min) when compared to the total disappearance of double-bonds, indicating that the rate-limiting step controlling the overall reaction is the hydrogen transfer from the thiol involved in the formation of final product. The loss of total unsaturations equals thiol consumption throughout the entire reaction; although product formation is strongly favoured directly from the trans-ene. This indicates that initial cis/trans-isomer structures affect the kinetics. High thiol–ene conversions could be easily obtained at reasonable rates without major influence of side-reactions demonstrating the suitability of this reaction for network forming purposes from 1,2-disubstituted alkenes. To further illustrate the validity of this concept in the formation of cross-linked thiol–ene films a series of globalide/caprolactone based copolyesters differing in degree of unsaturations along the backbone were photopolymerized in the melt with the same trithiol giving amorphous elastomeric materials with different thermal and viscoelastic properties. High thiol–ene conversions (>80%) were easily attained for all cases at reasonable reaction rates, while maintaining the cure behaviour and independent of functionality. Parallel chain-growth ene homopolymerization was considered negligible when compared with the main coupling route. However, the comonomer feed ratio had impact on the thermoset properties with high ene-density copolymers giving networks with higher glass transition temperature values (Tg) and a narrower distribution of cross-links than films with lower ene composition. The thiol–ene systems evaluated in this study serve as model example for the sustainable use of naturally-occurring 1,2-disubstituted alkenes at making semi-synthetic polymeric materials in high conversions with a range of properties in an environment-friendly way. / Vegetabiliska oljor som innehåller dubbelbindningar kan användas direkt för thiolene reaktioner. Trots att terminala dubbelbindningar är mycket mer reaktiva än interna visar dessa studier att den reversibla additionen av thiyl radikaler till 1,2-disubstituerade alkener är en viktig reaktion. För att undersöka tiol–ene reaktionerna, som ivolverar dessa alkener förbereddes stökiometriska blandningar av en trifunktionell propionat tiol och enkelomättade fettsyrametylestrar (metyloleat eller metyl elaidat) samt 2.0 vikt.% Irgacure 184. Dessa blandningar utsattes för 365-nm UV strålning och de kemiska förändringarna studerades. De kemiska förändringarna analyserades med olika kemiska analysmetoder; realtid RT–FTIR, NMR och FT–Raman. Dessa användes för att analysera de kemiska reaktionerna i realtid och följa bildandet av produkterna. Reaktionsmekanismen bekräftades med hjälp av experimentella data och beräkningar av numeriska och kinetiska simuleringar för systemet. Resultaten visar en mycket snabb cis/trans-isomerisering av alkenmonomeren (<1.0 min) jämfört med den totala förbrukningen av dubbelbindningarna, vilket indikerar att det hastighetsbegränsande steget kontrolleras av väteförflyttningen från tiolen till slutprodukten. Förbrukningen av den totala omättade kolkedjan är lika med tiolförbrukningen under hela reaktionen, även om bildandet av produkten gynnas från trans-enen. Detta indikerar att den första cis/trans-isomerstrukturen påverkar kinetiken. Höga tiol-ene utbyten kan enkelt erhållas relativt snabbt utan inverkan av sidoreaktioner. Detta innebär att denna reaktion kan användas som nätverksbildande reaktion för flerfunktionella 1,2-disubstituted alkenmonomerer. Vidare användes fotopolymerisation i smälta på en serie globalid/kaprolaktonbaserade sampolyestrar med varierad grad av omättnad med samma tritiol vilket resulterade i bildandet av amorfa elastomeriska material med olika termiska och viskoelastiska egenskaper. Hög omsättning (>80%) uppnåddes relativt enkelt för samtliga blandningar oberoende av den initiala funktionaliteten. Homopolymerisation av alkenen var försumbar i jämförelse med den tiol–en-reaktionen. Mängden alkengrupper har inverkan på härdplastsegenskaperna där en hög andel alken ger en nätstruktur med högre glastransitionstemperatur (Tg). Tiol–ene reaktionen utvärderades i modellsystem baserade på naturlig förekommande 1,2-disubstituterade alkener för att demonstrera konceptet med tiol-förnätade halvsyntetiska material. / QC 20110915

Hereditary equine regional dermal asthenia (HERDA) is a recessive connective tissue disorder of Quarter Horse lineages. This study correlates previously identified decreases in skin tensile strength in HERDA with abnormal dermal collagen cross linking patterns that are also identified in urine from HERDA horses. Dermal collagen from HERDA horses has significantly less pyridinoline and significantly more deoxypyridinoline than control or carriers. Concentrations of hydroxylysine, the rate limiting substrate for these crosslinks were significantly lower in HERDA versus control and carriers. These characteristics of HERDA skin parallel humans with a similar syndrome of skin fragility, Ehlers Danlos Syndrome TypeVIA. This is the first biochemical evidence explaining the clinical skin fragility that characterizes HERDA and suggests that altered collagen lysine metabolism may be physiologically relevant to the clinical manifestation of HERDA. Evaluations of mature scars indicate that lesion and nonlesioned skin should not be viewed as biologically equivalent in HERDA investigations.

Die Pektat-Lyasen gehören zu einer Proteinfamilie, die meistens von pflanzenpathogenen Mikroorganismen sekretiert werden. Die Enzyme katalysieren den Abbau von Polygalakturonsäure, einem Hauptbestandteil in <br /> pflanzlichen Mittellamellen und Primärzellwänden. Der Abbau der alpha-1,4-verbrückten Galakturonsäurereste erfogt durch eine beta-Eliminierungsreaktion, dabei entsteht ein Produkt mit einer ungesättigten C4-C5 Bindung am nicht reduzierenden Ende, das durch spektroskopische Messungen beobachtet werden kann. Für die enzymatische Reaktion der Pektat-Lyasen ist Calcium nötig und das pH-Optimum der Reaktion liegt bei pH 8.5. Alle bis jetzt bekannten Strukturen der Pektat- und Pektin-Lyasen haben das gleiche Strukturmotiv - eine rechtsgängige parallele beta-Helix. Die Struktur der Pektat-Lyase aus Bacillus subtilis (BsPel) ist im Komplex mit Calcium gelöst worden. BsPel ist ein monomeres Protein mit einer ungefähren Molekularmasse von 43 kDa, das keine Disulfidbrücken enthält. Dies erlaubte sowohl eine effiziente rekombinante Expression des Wildtypproteins, als auch von destabilisierten Mutanten im Cytoplasma von E. coli. Parallele beta-Helices sind relativ große, jedoch verhältnismäßig einfach aufgebaute Proteine. Um detailliertere Informationen über die kritischen Schritte bei der in vitro-Faltung von parallelen beta-Helices zu erhalten, sollte in der vorliegenden Arbeit versucht werden, den Faltungsmechanismus dieses Proteins näher zu charakterisieren. Dabei sollte vor allem die Frage geklärt werden, welche Wechselwirkungen für die Stabilität dieses Proteins einerseits und für die Stabilität von essentiellen Faltungsintermediaten andererseits besonders wichtig sind.<BR><br>Rückfaltung von BsPel, ausgehend vom guanidiniumchlorid-denaturierten Zustand, war bei kleinen Proteinkonzentrationen und niedrigen Temperaturen vollständig möglich. GdmCl-induzierte Faltungsübergänge waren aber nicht reversibel und zeigten eine apparente Hysterese. Kinetische Messungen des Fluoreszenz- und CD-Signals im fernen UV ergaben eine extreme Denaturierungsmittelabhängigkeit der Rückfaltungsrate im Bereich des Übergangmittelpunktes. Der extreme Abfall der Rückfaltungsraten mit steigender Denaturierungsmittelkonzentration kann als kooperative <br /> Entfaltung eines essentiellen Faltungsintermediats verstanden werden. Dieses Faltungsintermediat ist temperaturlabil und kann durch den Zusatz Glycerin im Renaturierungspuffer stabilisiert werden, wobei sich die Hysterese verringert, jedoch nicht vollständig aufgehoben wird. Durch reverse Doppelsprungexperimente konnten zwei transiente Faltungsintermediate nachgewiesen werden, die auf zwei parallelen Faltungswegen liegen und beide zum nativen Zustand weiterreagieren können. Fluoreszenzemissionsspektren der beiden Intermediate zeigten, daß beide schon nativähnliche Struktur aufweisen. Kinetische Daten von Prolin-Doppelsprungexperimenten zeigten, daß Prolinisomerisierung den geschwindigkeitsbestimmenden Schritt in der Reaktivierung des denaturierten Enzyms darstellt. Desweiteren konnte durch Prolin-Doppelsprungexperimenten an Mutanten mit Substitutionen im Prolinrest 281 gezeigt werden, daß die langsame Renaturierung von BsPel nicht durch die Isomerisierung der einzigen cis-Peptidbindung an Prolin 281 verursacht wird, sondern durch die Isomerisierung mehrerer trans-Proline. Die beiden beobachteten transienten Faltungsintermediate sind somit wahrscheinlich zwei Populationen von Faltungsintermediaten mit nicht-nativen X-Pro-Peptidbindungen, wobei sich die Populationen durch mindestens eine nicht-native X-Pro-Peptidbindung unterscheiden.<BR><br>Der Austausch des Prolinrestes 281 gegen verschiedene Aminosäuren (Ala, Ile, Leu, Phe, Gly) führte zu einer starken Destabilisierung des nativen Proteins und daneben auch zu einer Reduktion in der Aktivität, da die Mutationsstelle in der Nähe der putativen Substratbindetasche liegt. Die Rückfaltungskinetiken der Prolinmutanten war bei 10&#176;C annähernd gleich zum Wildtyp und die geschwindigkeitsbestimmenden Schritte der Faltung waren durch die Mutation nicht verändert. Die durch die Mutation verursachte drastische Destabilisierung des nativen Zustands führte zu einem reversiblen Entfaltungsgleichgewicht bei pH 7 und 10&#176;C. GdmCl-induzierte Faltungsübergänge der Mutante P281A zeigten bei Messungen der Tryptophanfluoreszenzemission und der Aktivität einen kooperativen Phasenübergang mit einem Übergangsmittelpunkt bei 1.1 M GdmCl. Durch die Übereinstimmung der Faltungsübergänge bei beiden Messparametern konnten die Faltungsübergänge nach dem Zwei-Zustandsmodell ausgewertet werden. Dabei wurde eine freie Sabilisierungsenthalpie der Faltung für die Mutante von <nobr>-&nbsp;64.2&nbsp;&#177;&nbsp;0.4&nbsp;kJ/mol</nobr> und eine Kooperativität des Übergangs <br /> von <nobr>-&nbsp;58.2&nbsp;&#177;&nbsp;0.3&nbsp;kJ/(mol&#183;M)</nobr> bestimmt.<BR> <br /> <br>BsPel enthält, wie die meisten monomeren rechtsgängigen parallelen beta-Helix-Proteine, einen internen Stapel wasserstoffverbrückter Asparagin-Seitenketten. Die Mehrheit der erzeugten Mutanten mit Substitutionen im Zentrum der Asn-Leiter (N271X) waren als enzymatisch aktives Protein zugänglich. Die Auswirkung der Mutation auf die Stabilität und Rückfaltung wurde an den Proteinen BsPel-N271T und BsPel-N271A näher analysiert. Dabei führte die Unterbrechung des Asparaginstapels im Inneren der beta-Helix zu keiner drastischen Destabilisierung des nativen Proteins. Allerdings führten diese Mutationen zu einem temperatur-sensitiven Faltungsphänotyp und die Hysterese im Denaturierungsübergang wurde verstärkt. Offenbar wird durch die Unterbrechung des Asparaginstapel ein essentielles, thermolabiles Faltungsintermediat destabilisiert. Der Asparaginstapel wird somit bei der Faltung sehr früh ausgebildet und ist wahrscheinlich schon im Übergangszustand vorhanden. / Pectate lyases belong to a family of proteins secreted by plant pathogenic microbes. The enzymes cleave alpha-1,4 linked galacturonic acid by a beta-elimination that results in an unsaturated product, which can be quantified spectrophotometrically. Calcium is essential for the activity and the pH-optimum is near 8.5. All known structures of pectate and pectin lyases have the same structural motif - a right handed parallel beta-helix. The structure of pectate lyase from Bacillus subtilis (BsPel) has been solved in complex with calcium. It is a monomeric protein, with a molecular mass of about 43 kDa and without disulfide bonds. This allows its high-yield recombinant expression in the cytoplasm of Escherichia coli. Parallel beta-helices are relative large proteins, however with a simple folding topology. The objective of this work was to characterize the folding mechanism of BsPel. In particular we investigated the role of the interactions of certain residues in the parallel beta-helix for the stability of the native protein and the stability of essential folding intermediates.<br /> <br /> Refolding of BsPel was possible at low protein concentrations and low temperature. However, denaturation of BsPel was not freely reversible. De- and renaturation curves showed a large apparent hysteresis. Furthermore, the folding rate constant deduced from fluorescence and circulardichroism measurements showed a very strong dependence on denaturant concentrations near the midpoint of the renaturation transition. This can be explained with a cooperative unfolding of an essential folding intermediate. Upon stabilisation of the temperature-sensitive intermediate by addition of glycerol in the renaturation buffer, the hysteresis is reduced, but does not disappear. Reverse double mixing kinetic experiments have shown that two transient folding intermediates are on the folding pathway. These intermediates are on parallel pathways and both can fold to the native state. Fluorescence emission spectra have shown the native-like structure of both intermediates. Furthermore, data from proline double mixing kinetic experiments revealed that isomerization of peptidyl-prolyl bonds was responsible for the slow kinetics in the reactivation of the enzyme. However, the isomerization of the single cis-peptidyl-prolyl bond at Pro281 was not responsible for the slowest folding phase observed, but rather the isomerization of other trans-peptidyl-prolyl bonds. Thus, both transient folding intermediates observed probably represent two populations of folding intermediates with non-native X-Pro-peptide bonds. The difference of the two populations is at least one non-native X-Pro-peptide bond.<br /> <br /> Mutations of the proline 281 against various residues (Ala, Ile, Leu, Phe, Gly) resulted in a strong destabilization of the native protein. Also, the activity of the mutant proteins was strong reduced due to the position of the mutation site near the putative active center of the protein. At 10&#176;C the kinetic folding behavior of the proline mutants was not significant changed. However, the strong destabilization of the native state in the proline mutants resulted in a reversible folding equilibrium at pH 7 and 10&#176;C. The unfolding of the P281A mutant was reversible as determined by fluorescence emission and enzyme activity measurements. The coincidence of these detected transitions is consistent with a two-state equilibrium transition. At pH 7 and 10&#176;C the delta G&#176;(H₂O) for folding of P281A was <nobr>-&nbsp;64.2&nbsp;&#177;&nbsp;0.4&nbsp;kJ/mol,</nobr> with a midpoint of the transition at 1.1 M GdmCl and a cooperativity of <nobr>-&nbsp;58.2&nbsp;&#177;&nbsp;0.3&nbsp;kJ/(mol&#183;M).</nobr><br /> <br /> BsPel has an asparagine ladder in turn 2 of the parallel beta-helix with extensive network of side-chain hydrogen bonds between the Asn residues. Such an Asn-ladder is a conserved feature of many monomeric beta-helices crystallized so far. The middle Asn residue (271) was selected and exchanged for various residues. Most of the mutants were expressed at 25&#176;C as soluble and active proteins but with a significant reduction in yield. Mutants N271T and N271A were selected to study the stability and refolding of these proteins in comparison with the wild-type protein. The substitution in the Asn-ladder did not drastically destabilize the native protein, but caused a temperature-sensitive-folding (tsf) phenotype with an increased hysteresis in the de- and renaturation transition curves. In addition, the disruption of the Asn-ladder resulted in destabilization of an essential, thermosensitive folding intermediate. Thus, the Asn-ladder is formed very early during the folding, probably well before the transition state of folding.

Ο όρος μη γραμμική οπτική (nonlinear optics) αναφέρεται στον κλάδο της φυσικής που μελετά τις μεταβολές που επέρχονται στις οπτικές ιδιότητες της ύλης, όταν αυτή αλληλεπιδρά με ισχυρά ηλεκτρομαγνητικά πεδία. Οι δέσμες λέιζερ (laser) αποτελούν το κατ’ εξοχήν ηλεκτρομαγνητικό πεδίο που χρησιμοποιείται για να προκαλέσει τη μη γραμμική οπτική απόκριση της ύλης, λόγω της ισχυρής ακτινοβολίας. Με την ανακάλυψη του λέιζερ τη δεκαετία του 1960, παράλληλα αναπτύχθηκαν πολλές τεχνικές μέσω των οποίων καθίσταται εφικτός ο προσδιορισμός της μη γραμμικής οπτικής απόκρισης των υλικών. Κίνητρο για αυτή την εκτεταμένη έρευνα αποτελεί το γεγονός ότι υλικά με σημαντική μη γραμμική απόκριση βρίσκουν πολλές εφαρμογές στη φωτονική και την οπτοηλεκτρονική. Ειδικότερα, τέτοια υλικά μπορούν να χρησιμοποιηθούν ως οπτικοί περιοριστές (optical limiters) για την προστασία από υψηλές δέσμες λέιζερ, ως οπτικοί διακόπτες (optical switches), καθώς και ως οπτικές λογικές πύλες (optical logic gates) κ.α. Τα τελευταία χρόνια υπάρχει ιδιαίτερο ερευνητικό ενδιαφέρον στο πεδίο των μοριακών διακοπτών (molecular switches). Ο όρος μοριακός διακόπτης αναφέρεται στην δυνατότητα ύπαρξης ενός μορίου μεταξύ δύο διαφορετικών καταστάσεων, ικανότητα που θα αποτελέσει την αφετηρία για μια λειτουργία “on/off”, σε μοριακό επίπεδο. Υποψήφια μόρια που μπορούν να υποστηρίξουν μια διακοπτική λειτουργία είναι εκείνα που παρουσιάζουν κάτω από ορισμένες συνθήκες αλλαγή σε μια εγγενή ιδιότητά τους, όπως ο φθορισμός, η αγωγιμότητα, η μαγνήτιση, ο στερεο-ισομερισμός, η μη γραμμικότητα κ.α. Σκοπός της παρούσας ειδικής ερευνητικής εργασίας ήταν ο προσδιορισμός της τρίτης τάξης μη γραμμικής οπτικής απόκρισης μοριακών συστημάτων που περιέχουν αζοβενζόλιο, υπό μορφή διαλυμάτων. Τα μόρια αυτά είναι ενδιαφέροντα γιατί αφ’ ενός εμφανίζουν trans/cis ισομερισμό, αφ’ ετέρου παρουσιάζουν μια αλλαγή στη μη γραμμικότητά τους εξαιτίας του φωτο-ισομερισμού του διπλού δεσμού –N=N-. Η δομή της εργασίας έχει ως εξής : Στο πρώτο κεφάλαιο θα γίνει μια περιγραφή της μη γραμμικής αλληλεπίδρασης ύλης-πεδίου, η εξαγωγή της μη γραμμικής κυματικής εξίσωσης, καθώς επίσης και μερικών φυσικών διαδικασιών που περιγράφονται μέσω αυτής. Στο δεύτερο κεφάλαιο θα περιγραφεί η πειραματική τεχνική που ακολουθήθηκε, θα αναφερθούν οι οπτικές παράμετροι που σχετίζονται με τη τρίτης τάξης μη γραμμικότητα και τέλος θα περιγραφεί η διαδικασία ανάλυσης των πειραματικών αποτελεσμάτων. Στο τρίτο κεφάλαιο θα γίνει μια σύντομη περιγραφή της πειραματικής διάταξης καθώς και των κυριότερων οργάνων που την αποτελούν. Στο τέταρτο κεφάλαιο θα παρουσιασθούν τα πειραματικά αποτελέσματα που προέκυψαν από την εργασία. Αρχικά παρουσιάζεται μια σύντομη περιγραφή των μοριακών συστημάτων που μελετήθηκαν. Ακολουθεί η μη γραμμική οπτική απόκριση των συστημάτων για διέγερση με παλμούς χρονικής διάρκειας 35 ps, στα 532 και 1064 nm,και ακολουθεί ο προσδιορισμός της μη μεταβατικής μη γραμμικής απόκρισης για διέγερση με παλμούς διάρκειας 4 ns. Αναφέρονται τα συμπεράσματα από τη μελέτη των συστημάτων και ακολουθεί μια πρόταση για μελλοντική μελέτη. / Extensive research in the field of molecular switches promises a wide variety of switching mechanisms characterized by an eclectic range of intrinsic properties regarding their luminescence, conductivity, magnetic and optical outputs. Because of their controlled alternating properties and the ease of their preparation and modification, molecular switches hold the promise of becoming pivotal components of organic–integrated photonic devices. Among the many available systems, azobenzene dyes represent a particularly promising class of organic switchable materials. The advantage of azobenzene chromophores as molecular switches is based not only on the large geometrical change accompanying the cis–trans isomerization, but also on their photo–stability, and the ease of their preparation and derivatization. In addition, azobenzene chromophores, exhibiting in general large nonlinear optical response, are characterized by an important change in their third-order nonlinear optical response due to the photo-isomerization of the –N=N- double bond. The purpose of this work is hence to describe how the NLO properties of the azobenzene are influenced when surrounded by an always more electron-donating and conjugated environment. In order to do so, we are studying three azobenzene-centered molecules. In two of them, the switch is bonded to two electron-rich groups such as alkylated anilines via either one or two ethynil spacers. In the latter, a Zn-porphyrin was instead linked to the aza-core. The insertion of a porphyrin core was driven by its high polarizability and optical oscillator strength which gives the material remarkable NLO behavior, making it potentially useful for ultra-fast switching technologies. In that view, the nonlinear optical response of these novel azobenzene based molecules dissolved in dichloromethane are studied by means of Z-scan technique using 35 ps laser pulses at 532 and 1064 nm. From the measurements, the nonlinear absorption and refraction and the corresponding third-order susceptibility χ(3) and second hyperpolarizability are determined.

This thesis entitled “Design and Access to Disallowed and Unusual Conformers of Peptides in Crystals and in Solution: Structural Consequences of the Imidate and Thioimidate Isosteres for the Peptide bond” is divided into eight chapters. Imidate Modification The range of disallowed dihedral angles for residues in peptides is governed by their local steric and electrostatic clashes. Rare tolerances of violations in these angles are attributed to distortions in both local and global bond characteristics of the peptides. Discerning the origins of such disallowed angles and the consequent distortions in body of the peptides is essential, for a complete understanding of the protein fold, to improve the crystal database for validation of rare but acceptable residue conformations and for validation and improvement of theoretical models that evaluate the interactions that define the Ramachandran space. Unlike for the ordered secondary structures such as β-sheets α-helices and β-turns currently there are no models for residues constrained in disallowed folds. We reasoned that residues may be stabilized in disallowed folds in peptides if a neighbouring group and The range of disallowed dihedral angles ( , ψ) for residues in peptides is governed by their hence its local unfavorable clashes can be selectively modified to a motif that favors such space Steric clashes of the type H•••Xi±n involving the backbone amide hydrogen (H) contribute to ~60% of disallowed ,ψspace. Conversion of an amide to an imidate (A→I) will remove the corresponding H and hence the steric clashes related to it in peptides. Importantly, this will introduce an H-bond acceptor N (of imidate) in place of an H-bond donor NH (of amide), which will allow formation of unusual H-bonding interactions between the imidate N and the neighbouring Hs and hence constrain residues in otherwise inaccessible dihedral angles. The conversion of A→I is challenging owing to difficulties in selective synthesis, stability and purification of the imidate motif. We address all these concerns by the selective conversion of a backbone amide in peptides to the relatively stable cyclic 5,6-dihydro-4H-1,3-oxazine imidate isostere, by an intra¬molecular nucleophilic cyclo-O-alkylation reaction. Chapter 1:SectionB: Autocyclo-O-Alkylation of N-(3-Bromopropyl)amides into 2-Alkyl-5,6-Dihydro-4H-1,3-Oxazinehydrobromides We are describing the reactivity of N-(3-bromopropyl)amides that are precursors for 2-peptide-5,6-dihydro-4H-1,3-oxazine. The starting materials, 3-bromopropylamides, were synthesized in good yields by coupling the corresponding carboxylic acids and anhydrides with 3-bromopropylaminehydrobromide using standard mixed anhydride peptide coupling protocol. N-(3-bromopropyl)-acylamides are unstable during the isolation. Time-dependent 1H NMR of all the acetamides revealed that they underwent clean auto-cyclization to form the corresponding 2-alkyl-5,6-dihydro-4H-1,3-oxazine hydrobromides following first order rate. The salts were easily isolated in high purity by trituration of the mixtures with ether. The t1/2 of autocycliation of decreased upon increase in electron density on the R-carbon. Notably, the tert-butyl substituent cyclized significantly faster than acetamide which have enolizable hydrogens at the R-carbon. Thus, the cyclization rate is affected predominantly by the inductive effect of the R-carbon substituents. The formamide remained stable and unchanged due to the poor electron-donating ability of hydrogen. Chapter 1: Section C: Intramolecular Hydrogen Bond Assistance Improves Autocyclization in N-(3-Bromopropyl)amides The autocyclisation do not go to 100% completion. This is because the released hydrobromic acid quenches the nucleophilicity of amide carbonyl oxygen. In order to scavenge hydro bromic acid, we used 1 equivalent of DIEA base is acting only acid scavenger which conformed by unaffecting the reaction rate upon increasing equivalents of DIEA. We found that autocyclisation of N-(3-bromopropyl)amides rates in peptides involved in intramolecular backbone H-bonding interactions improve the autocyclization rates significantly than unstructured (random coil) peptides. Even with in the ordered structures the rate depends on the proximity of H-bonding distances as well as the H-bond acceptor strength. Half-life of autocyclisation in various peptide secondary structures are determined from time variant 1H NMR studies performed at 60 mM peptide concentration in CDCl3 at 32 oC. Chapter 2: Section A: Synthesis and Isolation of 5,6 Dihydro-4H-1,3-Oxazine Containing Peptidomimetics We have introduced 5,6-Dihydro-4H-1,3-oxazine as the imidate isostere at C-terminus of a number of peptides through NaH (base) mediated intramolecular cyclo-O-alkylation of N-(3-bromopropyl)amides. The amide to imidate (A→I) modification reaction is faster (1-5.5 h), Exhibiting no electronic and structural effects under these conditions. The side product NaBr can be easily separated by filtration through celite. No side products were observed and there is no need of further purification to get pure 1,3-oxazines in quantitative yields in all the peptidomimetics. Using this synthetic protocol we have synthesised a variety of 1,3-oxazine containing peptide analogues including aliphatic, branched aliphatic, polar side chains and larger peptides. We show that the retention of configuration at Cαof peptides during the base mediated cyclo-O-alkylation reaction. that the C5i.structures are more populated at Aib due to operation of The Thorpe-Ingold effect. The strength of hydrogen bonding interaction in C5i structure is similar to those of the highly buried backbone hydrogen bonding interaction found in the middle of a model 310-helical peptide as indicated by DMSO titration experiments. Chapter 3: Section A: Consequences of "Disallowed" Conformations on Constrained β-Turn Peptides Here we are describe the consequence of disallowed conformations the on a C-terminus of a type-II β-turn. We choose stereochemically constrained Type-II β-turn Pro-Aib dipeptide analogue which is the ideal model to mirror the structural effects of introducing the A→I modification at the C-terminus. The imidate containing peptidomimetic crystallised in dichloromethane and hexane mixture. Analysis of crystal structure revealed that Aib NH is involved in 3-centered H-bonding interactions with the N of oxazine and N of proline. This constrains Aib in a conformation that is natively disallowed to it. The (, ) angles of Aib residue fall in the (180,0) region which is strictly disallowed for natural peptides due to steric clashes involving the back bone amide N-H. More importantly there are two C•••O interactions which are accomidated in the crystal structures. Both oxygen‟s were place in staggered orientation of the Pro oxygen (OPro) between the two β-CH3 groups of Aib, which is again strictly disallowed in natural peptides due to strong C•••Oi-1 hard sphere clashes. However no vdW space violations are observed between these atoms. Chapter 3: Section B: Conformational Effects of “Disallowed Aib on a 310-Helical peptide In order to investigate the origins and consequences of “disallowed” conformations on a folded helical peptide body, the conformationally stable peptide sequence Boc-Leu1-Aib2-Ala3-Leu4-Aib5-Ala6-Phe7-Aib8-OMe (310-helix-OMe)was chosen which is known to adopt 310-helix in crystal structure. Analyses of the ROESY spectra, DMSO titration experiments, and CD spectra of 310-helix-OMe and its Oxa analogue reveal that their solution conformations are identical to those of the crystal structure of 310-helix-OMeSix sequential i+3→i intramolecular backbone H-bonds stabilize the 310-helical peptide fold in both peptides in solvents of varying polarity. The N-terminal and central segments of the helical molecules are quite structurally rigid and are not deformed. The presence of the disallowed Aib*8 residue in Oxa analogue has a clear conformational effect mainly on the residue Phe7. It looks like the Phe7 amide H is involved in shielding, the Aib*8 amide H through a bifurcated hydrogen bonding interactions with the nitrogen of oxazine and carbonyl oxygen of Ala6 residue. Maximum structural distortion effect on the registers closest to the putative imidate bond. Our results show that “disallowed folds need not denature order in the peptide fold”. Chapter 4: Synthetic Methods for Introducing the A → I Modification anywhere along the Peptide Chain Here we describe the incorporation of imidate isostere in the middle of any peptide sequence. In Oxa selectivity is towards 5-exo-cyclo-O-alkylation in 1 : 4. In Thi it is towards 6-exo-cyclo-S-alkylation in 3 : 1 ratio. This is because of better nucleophile of sulphur (S). We saw that Thi is stable to peptide coupling, N-and C-terminus protection, deprotection conditions and can be easily incorporated in middle of peptide. Chapter 5: Section A: Cis-trans Isomerism in the X-Pro Peptide Bond In tertiary amides like X-Pro peptides having high propensity to access cis conformations due to similar environment in both cis and trans around the Cof X. X-Pro peptide bonds, constrained in s-cis conformations are prevalently found in the turn regions of peptides with the residue „X‟ in the i+1position and Pro at the i+2position of the β¬turn. These types of turns are termed as the type VI β-turns. For better understanding of the molecular recognition at specific cis X-Pro peptide bonds in biological events, the structure and dynamics of various constrained cis X-Pro peptide bond analogues with varying steric and electronic perturbations have been studied. Many models have been developed for stabilizing cis conformer by perturbation of molecular recognition surface of proline by employing steric and electronic interaction. In biological functions proline molecular recognition surface and cis X-Pro peptide bond more important. There is need of novel method for stabilizing X-Pro peptide bond in cis conformer without modifying the pyrolidine ring in proline. Chapter 5: Section B: Biasing the cis/trans Equilibrium in X Pro Peptides using Reverse ni → ni-1 * Interactions Here we present our findings that peptidomimetics containing the 5,6-dihydro-4H-1,3¬oxazine (Oxa) and 5,6-dihydro-4H-1,3-thiazine (Thi) functional groups at the C-terminus of Pro selectively and remotely stabilize the s-cis rotamers of the preceding pyrrolyl (Xaa-Pro) 3° amide bonds, while conserving these recognition elements. The cis/trans equilibrium of Xaa-Pro peptide bonds is shifted significantly in favor of the satirically disfavored cis isomers in these peptidomimetics (upto ~90%). We also provide evidence for the influence of an unusual n→ πi-1 * interaction in the cis, and the n)(n repulsion in the trans, conformers of these molecules to beat the origin of such the origin of such cis stabilization. Chapter 6: Steric Interactions in the cis Piv-Pro Peptide Bond The inaccessibility of cis Piv-Pro rotamer in any peptide is believed to be because the steric clashes between substituents on CX and CPro are unavoidable in this conformer. Here we access the cisPiv-Pro conformer in crystal structure of Piv-Pro-Aib-OMe and that it is sufficiently flexible to undergo bond distortions and avoid all steric clashes between substituents on CPiv and CPro . It is however the unavoidable distortions in the dihedral angle of the Prothe cisPiv-Proconformer. The cisPiv -Pro conformer is indeed accessible, if such distortions are accommodated in the peptide. Chapter 7: Steric and Electronic Interactions in the cis Isomer of Piv-Pro Peptide Bond in Solution We have studied the electronic and steric interactions and the conformational equilibrium in two sets of homologous peptides, X-Pro-Aib-OMe (which contain Aib) and X-Pro-NH-Me, where X is acetyl, propionyl, isobutyryl and pivaloyl, in solvents of varying polarities consisting of carbontetrachloride, chloroform or dimethylsulfoxide, by means of their 1H and 13C-NMR, and FT-IR spectra. Formation of n * interactions between the carbonyls that flank the Aib residue, influences the alleviation of steric interactions that are believed to preclude access to the cis conformer of the Piv-Pro peptide bond. The cis Piv-Pro conformer is observable in the Aib containing peptides, at ambient conditions by FT-IR and at temperatures as low as 273 K by NMR. We estimate that the steric interactions contribute < 0.5 kcal/mol to the conformational free energy of X-Pro peptide bond isomerism, irrespective of the steric bulk on the acyl (X) group. The relative strengths of intramolecular hydrogen bonding interactions involving the X-Pro peptide motif in different conformers of these peptides influence their relative conformational stabilities. Chapter 8: Remote Effect of Oxa and Thi Functional Groups on cis-trans Isomerism at X-Pro Peptide Bonds The C5a interaction at Pro residue occurs in the transition states for the intramolecular acid catalysis of cis → trans isomerization in peptidyl prolyl isomerases (PPIs) and enables the decrease in transition energy barrier for the isomerization process. We show that the NPro….HAib interactions in C5a structures can be remotely effected in order to control in equilibrium constant values of the cis/trans isomerism (Kc/t) in X-Pro¬Aib-Oxa and Thi containing peptides. By this method we observed improvement in Kc/t values from 0.18 in esters to 0.56 in Thi and 0.66 in Oxa containing peptides. Analyses of the ROESY spectra, DMSO titration experiments, variable temperature experiments and FT-IR spectra of R-CO-Pro-Aib-Oxa (R = Me, Et, iPr) and its Thi analogues reveals that both interactions (C5a and C5i) are persistent in cis and trans conformers of this peptidomimetics. (for structural formula pl. see the abstract.)

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Chimie dynamique: reconnaissance de nucléobases par des récepteurs synthétiques et isomérie cis-trans d'hydrazones acylées.Ce travail traite du développement des systèmes moléculaires qui peuvent s'adapter à l'addition de substances qui agissent comme un gabarit. Cette approche permet d'isoler une espèce majeure à partir d'un mélange de composés par le biais de la chimie combinatoire dynamique (CCD). La première partie de ma thèse de doctorat inclus l'utilisation d'un ADN simple brin (ADNsb) comme un gabarit pour le transfert d'information par auto-assemblage de récepteurs sans avoir besoin d'enzyme. De nouveaux récepteurs de l'adénine et de la guanine (pinces A et G) solubles dans l'eau ont été conçues dans ce but. Une approche utilisant la résonance magnétique nucléaire (RMN) a été utilisée pour déterminer l'affinité de liaison comme preuve d'une reconnaissance spécifique et efficace. Une évaluation dans l'eau par dichroïsme circulaire (CD) et mesure de la température de fusion par UV (Tm) a été réalisée. Cela a permis de tester respectivement la capacité d'auto-assemblage entre les pinces et un modèle ADNsb, et la force du processus de coopérativité. La deuxième partie de ce travail est axée sur le tri spontanné de motifs pyridine acylhydrazone et sur les configurations intéressantes qu'ils adoptent. Nous avons étudié la synthèse d'une série de motifs pyridine acylhydrazone: dimère, trimères et pentamères. Des études RMN ont permis d'évaluer les changements dans l'équilibre configurationnel cis / trans de ces systèmes dynamiques. Les études ont montré que l'équilibre attendu est biaise la cis acylhydrazone pyridine isomère a été observée par diffraction des rayons X. / Dynamic chemistry: nucleobase recognition by synthetic receptors and cis-trans acylhydrazone isomerism. This work deals with the development of molecular systems which can adapt upon the addition of substances that act as templates. This approach enables one major species to be identified from a mixture of compounds through the use of dynamic combinatorial chemistry (DCC). The first part of my PhD included the use of a single stranded DNA (ssDNA) as a template for information transfer via the self-assembly of receptors without the need for enzymes. New water soluble adenine and guanine receptors (A and G clamps) were designed and synthesised for this purpose. Nuclear magnetic resonance (NMR) titration studies were carried out to calculate the binding affinity and as a proof of specific and efficient recognition. An assessment in water via circular dichroism (CD) and UV temperature melting (Tm) studies was carried out. This tested the ability for self-assembly between the clamps and a ssDNA template and the strength of the cooperative process respectively. The second part of my PhD focused on the self-sorting of acylhydrazone pyridine motifs and the interesting configurations they adopt. The feasibility to synthesise these acylhydrazone pyridine motifs (dimer, trimers and pentamers) was investigated. X-ray and NMR studies showed that the equilibrium was found to be biased in an unusual way, and the cis acylhydrazone pyridine isomer was observed.