5 research outputs found

    Characterization of Self-Assembling Quinoline- Based Foldamers by Fluorescence Anisotropy

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    Foldamers represent a family of synthetic macromolecules which, like their biological counterparts, are able to adopt a well-defined conformation in solution. Oligoquinoline-carboxamides (Qn) are a group of foldamers that adopt a helical conformation in solution. A series of Qn foldamers were prepared by chromatography-free large-scale synthesis and segment-doubling strategy. The C-terminal ester group of the Qn foldamers could be hydrolyzed to yield acid-functionalized foldamers (QnA) which could self-assemble into larger ((QnA)2-Na) complexes by metal coordination with a sodium cation. Moreover, the addition of a bis-acid functionalized tetramer (AQ2PQ2A) to a solution of (QnA)2-Na complexes resulted in insertion oligomeric products. To characterize these complexes in solution, both Qn and QnA were end-labeled with an oligo(phenylene vinylene) dye (OPV) at their N-terminus via a rigid amide bond to yield the OPV-Qn and OPV-QnA fluorescent equivalents. OPV was used to conduct time-resolved fluorescence anisotropy (TRFA) measurements on the OPV-Qn and OPV-QnA foldamers, the (OPV-QnA)2-Na complexes, and the OPV-Qn-Na-(AQ2PQ2A)n oligomers. Analysis of the TRFA of the OPV-Qn foldamers yielded the rotational time () of the fluorescent species, which was found to reflect the hydrodynamic volume (Vh) of the foldamers. The straight line obtained by plotting as a function of the number of (quinoline) units (NUs) demonstrated that the foldamers behaved in solution as rigid cylinders for all lengths examined. The linearity of the -vs-NU plot was employed as a calibration curve against which the rotational time of the QnA-complexes could be compared. Within experimental error, the rotational time of a Qn+m complex was found to equal the sum of the rotational times obtained for Qn and Qm. This result suggests that the complexation of two acid-functionalized oligoquinoline foldamers in solution generated a fully stacked foldamer with a NU equal to the sum of the NUs of its constituting elements. Hetero-complexes between OPV-Q8A and Q16A were also produced by adding a 10-fold excess of Q16A to an OPV-Q8A solution. Complexation was demonstrated by the value of the mixture, that equaled that of an OPV-Q24 foldamer. Dilution experiments on a solution of OPV-Q8A-Na-Q16A complexes led to the dissociation of the complexes into their OPV-Q8A and Q16A constituting elements, as evidenced by the progressive decrease in from the value obtained for OPV-Q24 to that of OPV-Q8 upon decreasing foldamer concentration. Similarly, the addition of increasing amounts of AQ2PQ2A to a solution of OPV-Q8A in chloroform resulted in an increase in demonstrating the formation of complexes between OPV-Q8A and AQ2PQ2A until reached a plateau for large OPV-Q8A/AQ2PQ2A molar ratios. In the plateau region, the rotational time of the oligomeric complexes generated from OPV-Q8A and AQ2PQ2A stabilized by isobutyl or hexyl side chains was equal to that of an OPV-QÂŹn foldamer with n equal to 24 or 30, respectively. The apparent absence of further polymerization, evidenced by the constant value reached for high OPV-Q8A/AQ2PQ2A molar ratios, was attributed to aggregation of longer complexes and their precipitation. This study represents the first example in the scientific literature where TRFA was applied to characterize the NU of helical self-assembling foldamers in solution

    Pseudopeptides and Peptidomimetics Modulating the Proteolytic Activity of Kallikrein-related Peptidase 3

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    The highly prostate specific serine protease kallikrein-related peptidase 3 (KLK3, also known as prostate specific antigen, PSA) is widely used as a biomarker for prostate cancer and it has also been postulated that it may play a part in tumour growth. Especially interestesting is the antiangiogenic effect exerted by proteolytically active KLK3 in cell line models. In order to stimulate the proteolytic activity of KLK3, a series of peptides have been developed by phage display methodology. Even though the peptides are quite potent KLK3 stimulators, they are not directly suitable for in vivo studies or use as drugs. Even though there are many natural and unnatural biologically active peptides, they suffer from rapid clearance via the liver and kidneys and proteolytic degradation of the compounds both in the gastrointestinal tract and other parts of the body. This gives peptides a poor oral bioavailability meaning that they are usually administered as intravenous or intramuscular injections. Several different strategies have been developed in order to access compounds with improved bioavailability including modifications of the peptide structure, development of pseudopeptides and development of small molecular weight peptidomimetics. This thesis concentrates on the further development of the two most potent peptides known to stimulate KLK3, i.e. B-2 and C-4. The main part of the work was concentrated on the replacement of disulphide bridges in the peptides in order to both gain more information on which residues are necessary for obtaining the biological activity and at the same time also gain information on how changes to the geometry of the disulphide bridge affects the activity. A series of different disulphide bridge mimicking building blocks were designed and synthesised with the intention of using them in a protocol for solid-phase synthesis of KLK3 stimulating peptides. Unfortunately, the use of these building blocks in the synthesis of pseudopeptides based on C-4 turned out to be an unsurmountable challenge and the synthesis had to be completed using a different strategy in which the key step was the use of ring-closing metathesis (RCM) for the cyclisation of the partly completed pseudopeptide. Pleasingly, the synthesis of pseudopeptide analogues of the B-2 peptide using the building blocks was more successful. In total three pseudopeptide analogues of C-4 and four of B-2 were synthesised and shown to retain the biological activity of the parent peptides. Based on the information from the synthesised pseudopeptides and a molecular modelling study, a 4-quinolone based peptidomimetic was designed to mimic the C-4 peptide and a synthetic protocol was devised to access this compound. Even though the synthesis of the desired target compound has so far not been successful, the synthetic protocol that was designed has given access to a number of 1,2,8-trisubstituted 4-quinolone derivatives.Prostatacancer Ă€r i nulĂ€get den vanligaste formen av cancer hos mĂ€n i vĂ€stvĂ€rlden, sĂ„ mycket som var sjĂ€tte man har uppskattats insjukna i den under sin livstid. Även om dödligheten berĂ€knas till endast nĂ„gra procent innebĂ€r den stora utbredningen att prostatacancer orsakar det nĂ€st högsta antalet dödsfall i cancer hos mĂ€n efter lungcancer. Pga detta Ă€r det viktigt att hitta nya metoder bĂ„de för att bota prostatacancer och för att sakta ner sjukdomsförloppet. Kallikrein 3 (human kallikrein-related peptidase 3, KLK3; Ă€ven kĂ€nd som prostataspecifik antigen, PSA) Ă€r ett prostataspecifikt enzym som klyver proteiner och peptider (en proteas). KLK3 utsöndras i sĂ€desvĂ€tskan och dess biologiska uppgift Ă€r att klyva de gelbildande proteiner som utsöndras vid ejakulation. Enzymatiskt aktivt KLK3 har visat sig inhibera tillvĂ€xten av blodkĂ€rl bĂ„de in vitro och in vivo vilket har antagits bidra till en lĂ„ngsammare tumörtillvĂ€xt. En elakartad tumör i prostatan sĂ€nker utsöndringen av KLK3 i epitelvĂ€vnaden jĂ€mfört med en frisk prostata. KLK3 inverkar pĂ„ tumörtillvĂ€xten i prostatacancer genom att pĂ„verka de proteolytiska kaskaderna som bryter ned den extracellulĂ€ra matrisen och dĂ€rmed inhibera tumörtillvĂ€xten. Substanser som gör det möjligt att modifiera den proteolytiska aktiviteten hos KLK3 Ă€r mycket intressanta för utvecklingen av nya lĂ€kemedel mot prostatacancer. En serie peptider som stimulerar den proteolytiska aktiviteten hos KLK3 har tidigare utvecklats vid Helsingfors universitet. Även om dessa peptider uppvisar en stark stimulerande effekt pĂ„ KLK3 sĂ„ sönderfaller de snabbt i plasma, de har ocksĂ„ en förhĂ„llandevis stor molekylmassa för att vara peptider vilket starkt begrĂ€nsar deras anvĂ€ndbarhet in vivo. Studier har pĂ„visat vilka delar av dessa peptider som Ă€r viktiga för den biologiska aktiviteten, utgĂ„ende frĂ„n denna information har vi syntetiserat serier med modifierade peptider för att vidare söka utreda vilka modifikationer som Ă€r möjliga att göra utan att den biologiska aktiviteten sjunker avsevĂ€rt. Förhoppningen Ă€r att dessa modifikationer ocksĂ„ förbĂ€ttrar peptidernas stabilitet in vitro och in vivo. De tvĂ„ mest aktiva av de kĂ€nda peptiderna valdes ut och de naturliga disulfidbryggorna i dessa ersattes med en kolvĂ€tekedja samtidigt som andra, mindre förĂ€ndringar, gjordes i övriga delar av peptiden. Vissa av peptiderna i dessa serier visade sig ha en klar biologisk aktivitet som dock var nĂ„got lĂ€gre Ă€n den hos ursprungspeptiderna. En viktig slutsats av studien var att inga omfattande förĂ€ndringar i peptidens struktur Ă€r möjliga. Det slutliga mĂ„let med projektet var att utveckla smĂ„molekyler med en biologisk effekt som motsvarar den hos de ursprungliga peptiderna. Information frĂ„n studierna av peptiderna och de modifierade peptiderna i kombination med datorbaserad molekylmodellering gjorde att vi valde att anvĂ€nda ett 1,2,8-trisubstituerat 4-kinolonskelett som grund för dessa peptidmimetika och ett syntesprotokoll för sĂ„dana substanser utvecklades. Detta syntesprotokoll visade sig vara generellt tillĂ€mpbart och anvĂ€ndes till att syntetisera en serie 1,2,8-trisubstituerade 4-kinoloner med variation i struktur och egenskaper. De syntetiserade substanserna studeras för tillfĂ€llet för sin KLK3-stimulerande effekt

    Structural characterizations of oligopyridyl foldamers, α-helix mimetics

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    International audienceProtein-protein interactions are central to many biological processes, from intracellular communication to cytoskeleton assembly, and therefore represent an important class of targets for new therapeutics. The most common secondary structure in natural proteins is an α-helix. Small molecules seem to be attractive candidates for stabilizing or disrupting protein-protein interactions based on α-helices. In our study, we assessed the ability of oligopyridyl scaffolds to mimic the α-helical twist. The theoretical as well as experimental studies (X-ray diffraction and NMR) on conformations of bipyridines in the function of substituent and pyridine nitrogen positions were carried out. Furthermore, the experimental techniques showed that the conformations observed in bipyridines are maintained within a longer oligopyridyl scaffold (quaterpyridines). The alignment of the synthesized quaterpyridine with two methyl substituents showed that it is an α-helix foldamer; their methyl groups overlap very well with side chain positions, i and i + 3, of an ideal α-helix

    Structural Characterizations of Oligopyridyl Foldamers, α-Helix Mimetics

    No full text
    Protein–protein interactions are central to many biological processes, from intracellular communication to cytoskeleton assembly, and therefore represent an important class of targets for new therapeutics. The most common secondary structure in natural proteins is an α-helix. Small molecules seem to be attractive candidates for stabilizing or disrupting protein–protein interactions based on α-helices. In our study, we assessed the ability of oligopyridyl scaffolds to mimic the α-helical twist. The theoretical as well as experimental studies (X-ray diffraction and NMR) on conformations of bipyridines in the function of substituent and pyridine nitrogen positions were carried out. Furthermore, the experimental techniques showed that the conformations observed in bipyridines are maintained within a longer oligopyridyl scaffold (quaterpyridines). The alignment of the synthesized quaterpyridine with two methyl substituents showed that it is an α-helix foldamer; their methyl groups overlap very well with side chain positions, <i>i</i> and <i>i</i> + 3, of an ideal α-helix
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