<p>This thesis describes the design and synthesis of molecules with potential roles in biomedicine, with an emphasis on molecular recognition in complex biological environments. The first chapter describes the synthesis and evaluation of compounds for use in nuclide therapy. Carboranes are frequently used in the development of drugs for Boron Neutron Capture Therapy. New routes for monohydroxylation at the B and C atoms of <i>p</i>-carborane have been developed. The Suzuki-Miyaura reaction has been applied to the cross-coupling of <i>bis</i>(neopentyl glycolato)diboron or <i>bis</i>(pinacolato)diboron and 2-I-<i>p</i>-carborane. The synthesized derivatives are important intermediates in the synthesis of a number of potentially biologically active carborane-containing molecules.</p><p>The DNA intercalator doxorubicin has been functionalized to enable ¹²⁵I labelling. The aim of combining the DNA intercalator with ¹²⁵I was to achieve high delivery of cytotoxic radiation to the nucleus. The DNA-binding ability and cellular uptake of the synthesized compounds have been evaluated. One of the compounds bound strongly to DNA and had similar cellular uptake as daunorubicin, which makes the compound very interesting for further biological evaluation.</p><p>The second chapter describes the use of polypeptide conjugates to broaden our knowledge of molecular recognition. The polypeptides consist of 42 amino acids each and are designed to fold into helix-loop-helix motifs that dimerize due to their amphiphilic character. The polypeptides are combined with a variety of small organic molecules. The incorporation of small aromatic molecules to influence the structure and dynamics of a polypeptide has been investigated. By attaching a dansyl group to the side chain of a lysine residue, the dynamics of the protein’s hydrophobic core where affected to such a degree that a native-like fold was formed. The polypeptide conjugates have also been used to study the binding and recognition of native proteins. High-affinity binders for chitinases and acetylcholine esterase have been developed and evaluated.</p>

This thesis describes the design and synthesis of molecules with potential roles in biomedicine, with an emphasis on molecular recognition in complex biological environments. The first chapter describes the synthesis and evaluation of compounds for use in nuclide therapy. Carboranes are frequently used in the development of drugs for Boron Neutron Capture Therapy. New routes for monohydroxylation at the B and C atoms of p-carborane have been developed. The Suzuki-Miyaura reaction has been applied to the cross-coupling of bis(neopentyl glycolato)diboron or bis(pinacolato)diboron and 2-I-p-carborane. The synthesized derivatives are important intermediates in the synthesis of a number of potentially biologically active carborane-containing molecules. The DNA intercalator doxorubicin has been functionalized to enable 125I labelling. The aim of combining the DNA intercalator with 125I was to achieve high delivery of cytotoxic radiation to the nucleus. The DNA-binding ability and cellular uptake of the synthesized compounds have been evaluated. One of the compounds bound strongly to DNA and had similar cellular uptake as daunorubicin, which makes the compound very interesting for further biological evaluation. The second chapter describes the use of polypeptide conjugates to broaden our knowledge of molecular recognition. The polypeptides consist of 42 amino acids each and are designed to fold into helix-loop-helix motifs that dimerize due to their amphiphilic character. The polypeptides are combined with a variety of small organic molecules. The incorporation of small aromatic molecules to influence the structure and dynamics of a polypeptide has been investigated. By attaching a dansyl group to the side chain of a lysine residue, the dynamics of the protein’s hydrophobic core where affected to such a degree that a native-like fold was formed. The polypeptide conjugates have also been used to study the binding and recognition of native proteins. High-affinity binders for chitinases and acetylcholine esterase have been developed and evaluated.

A novel concept for protein recognition has been developed. The recognition unit is a hybrid molecule obtained by conjugation of a small organic molecule to a synthetic polypeptide selected from a 16-membered set of 42 amino acid residue sequences. The sequences are unordered and have no prior relation to the target proteins. The concept is based on the hypothesis that a small set of sequences capable of hydrophobic interactions, hydrogen bonding and electrostatic interactions can yield a binder for any selected protein, provided that the small molecule shows medium affinity or better and is reasonably selective. The concept has been illustrated by the design, synthesis and evaluation of binders for three different proteins, the C-reactive protein, CRP, human Carbonic anhydrase II, HCAII, and Acetylcholine esterase, AChE. Highly efficient binders for CRP have been developed by conjugation of a derivative of the natural ligand, phosphocholine, to the side chain of one of the amino acids in each polypeptide. The binders in the set show a wide range of affinities for CRP and the tightest binder, 4-C10L17-PC6, binds almost irreversibly. Selected binders have been evaluated in human serum, where they capture CRP with high selectivity.High-affinity binders have been developed for HCAII, and the selectivity evaluated by extraction of the protein from blood. The binder 4-C37L34-B, a polypeptide conjugated to a spacered benzenesulphonamide residue, was able to extract Carbonic anhydrases specifically and to discriminate between the two isoforms of human Carbonic anhydrase. The conjugation of an acridine derivative to a polypeptide via a 14 atom spacer has been shown to yield a binder with high affinity and selectivity for AChE. The selectivity was demonstrated by extraction of AChE from Cerebrospinal fluid. This thesis focuses on the development of a fast and reliable procedure for the construction, selection and evaluation of protein binders, with the ambition to develop a technology that is applicable to the development of binders for all proteins.

<p>U okviru ove doktorske disertacije ispitan je hemijski sastav i biolo&scaron;ke aktivnosti metanolnih i vodenih ekstrakata odabranih samoniklih vrsta tribusa Urticeae, rod&nbsp; Urtica:&nbsp; U. <em>dioica</em>&nbsp; subsp.&nbsp; <em>dioica</em>&nbsp; var.&nbsp;<em> pubescens</em>, U.<em>&nbsp; dioica&nbsp;</em> subsp.&nbsp; <em>dioica&nbsp; </em>var.&nbsp; <em>dioica</em>&nbsp; i&nbsp; U. <em>kioviensis</em>&nbsp; i tribusa Parietarieae, rod&nbsp; Parietaria:<em>&nbsp; P. officinalis</em>,&nbsp; <em>P.</em><br /><em>lusitanica</em> L. subsp<em>. lusitanica</em>,<em> P. judaica</em> L. subsp. <em>judaica</em> i<em> P. serbica</em>. Cilj rada bio je da se odredi sadrţaj biolo&scaron;ki aktivnih jedinjenja u ovim, do sada veoma malo ispitanim vrstama famijije Urticaceae, i utvrdi njihov potencijal primene kao pomoćnih lekovitih sredstava i dodataka ishrani.<br />Hemijski sastav ekstrakata ispitivanih vrsta određen je primenom: tečnohromatografskih tehnika (LC-DAD-MS i LC-MS-MS) za kvalitativnu analizu metanolnih ekstrakata, dok je za kvantitativnu analizu odabranih fenolnih jedinjenja primenjena LC-MS-MS tehnika. Spektrofotometrijskim metodama je određen sadržaj<br />ukupnih fenolnih komponenti i flavonoida. Ispitivanja biolo&scaron;kih aktivnosti ekstrakata obuhvatila su: određivanje antioksidantne i antiinflamatorne aktivnosti kao i sposobnost ekstrakata da inhibiraju acetilholinesterazu.&nbsp; Određen je uticaj odabranih metanolnih ekstrakata na imuni odgovor i proliferaciju intestinalnih ćelijskih linija pacova (IEC18) i ĉoveka (Caco2).<br />Dobijeni rezultati ukazuju da odabrane vrste tribusa Urticeae i Parietarieae, odnosno rodova&nbsp; Urtica&nbsp; i Parietaria&nbsp; predstavljaju bogate izvore biolo&scaron;ki aktivnih jedinjenja koja ispoljavaju raznovrsne biolo&scaron;ke aktivnosti. Sa hemotaksonomskog aspekta izdvajaju se sledeća jedinjenja kao potencijalni taksonomski markeri: vi&scaron;i sadržaj 5-O-kafeoilhinske kiseline u ekstraktima herbi vrsta roda&nbsp; Urtica, i visok sadrţaj<br />epikatehina u ekstraktima korena vrsta roda&nbsp; Parietaria. Ekstrakt herbe vrste&nbsp; U. kioviensis&nbsp; se od ostalih izdvaja po tome &scaron;to ne sadrži rutin a sadrži&nbsp; C-glikozide, u najvećoj meri viteksin. Od svih ispitivanih ekstrakata, ekstrakti korena&nbsp; Parietaria&nbsp; vrsta su ispoljili najbolji antioksidantni potencijal u većini izvr&scaron;enih testova. Najsnažniji antiinflamatorni potencijal je ispoljio ekstrakt korena vrste&nbsp; P. officinalis&nbsp; a prate ga ekstrakti korena vrsta roda&nbsp; Urtica.&nbsp; Veoma dobar antiinflamatorni potencijal su ispoljili infuzi herbi vrste U.<em> dioica</em>&nbsp; (čajevi od koprive). Svi ispitani metanolni ekstrakti su ispoljili odličnu inhibiciju enzima acetilholinesteraze a kao najbolji se izdvajaju ekstrakti korena&nbsp; Parietaria&nbsp; vrsta i vrste&nbsp; U.<em> kioviensis</em>. Povećanu sekreciju citokina rat MCP1 i GRO&alpha;&nbsp; izazivaju ekstrakti korena vrsta&nbsp; P. officinalis&nbsp; i&nbsp; P. judaica&nbsp; u<br />bazalnim uslovima i uslovima LPS-stimulisane inlamacije, dok ekstrakti vrste&nbsp; U. dioica&nbsp; povećavaju bazalnu a smanjuju LPS-stimulisanu sekreciju. Stimulaciju sekrecije ova dva citokina, ispitivani ekstrakti vr&scaron;e interakcijom sa adapternim proteinom MyD88 (ali ne intereaguju sa TLR4 receptorom) i NF -&kappa;B signalnim<br />putem. Ekstrakt korena vrste&nbsp; P. <em>officinalis</em>&nbsp; povećava LPS-om indukovanu ekspresiju enzima COX-2 u IEC18 ćelijama, dok je ekstrakt korena vrste&nbsp; U.<em> dioica</em>&nbsp; smanjuje. Efekat epitelizacije ili zarastanja rane na monosloju IEC18 ćelija ispoljavaju ekstrakti herbe i korena vrste&nbsp; P. <em>officinalis</em>. Ispitivani ekstrakti ne menjaju značajno seksreciju citokina hMCP1 i IL-8 u Caco2 ćelijama niti ispoljavaju značajan uticaj na &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; njihovu proliferaciju.</p> / <p>Within this doctoral thesis the chemical composition and biological activity of methanol and aqueous extracts of the selected plant species belonging to the Urticeae and Parietarieae tribe, more specifically to the&nbsp; Urtica&nbsp; and&nbsp; Parietaria&nbsp; genuses was evaluated (Urtica:&nbsp; U.<em> dioica</em>&nbsp; subsp.<em> dioica</em>&nbsp; var.&nbsp; <em>pubescens</em>,&nbsp; U.&nbsp; <em>dioica</em>&nbsp; subsp.&nbsp;<em> dioica</em>&nbsp; var.&nbsp; <em>dioica</em>&nbsp; and&nbsp; U.<em> kioviensis</em>;&nbsp; Parietaria:&nbsp; P<em>. officinalis</em>,&nbsp; P.<em> lusitanica</em>&nbsp; subsp.&nbsp; <em>lusitanica,</em>&nbsp; P. <em>judaica&nbsp;</em> subsp.<em>&nbsp; judaica&nbsp;</em> and<em>&nbsp; P. serbica</em>). The principal aim was to determine the content of biologically active&nbsp; compounds in this, poorly<br />examined species of the Urticaceae family, and determine their potential as additional remedy and dietary supplements.<br />Qualitative analysis of methanol extracts was performed by LC-DAD-MS i LC-MS/MS analysis, and LC-MS/MS for quantitative analysis of selected phenolic compounds. Total phenolics and flavonoids were determined spectrophotometrically. In order to assess the biological potential, the antioxidant and anti-inflammatory activities of the extracts were studied as well as&nbsp; their ability to inhibit acetylcholinesterase. The immuno-modulatory effects of the selected methanol extract on the immune response and proliferation of intestinal epithelial cells (IEC18 and Caco2)<br />was determined.<br />The obtained results suggest that the examined species of the Urticeae and Parietarieae tribe (genuses&nbsp; Urtica&nbsp; and&nbsp; Parietaria) are abundant with the biologically active compounds that express a broad spectrum of biological activities. As a potential chemotaxonomic markers stand out the following&nbsp; compounds: 5-O-caffeoilquinic acid (highly abundant in the herb extracts of the&nbsp; Urtica&nbsp; spp.) and epicatechin (highly abundant in the root extracts of the&nbsp; Parietaria&nbsp; spp.).&nbsp; U.<br />kioviensis&nbsp; herb extracts differs from the rest by high content of vitexin and total lack of rutin. The best antioxidant potential have exhibited the root extracts of the&nbsp; Parietaria&nbsp; species. The strongest anti-inflammatory potential had the root extract of the&nbsp; P. officinalis, followed by root extracts of the&nbsp; Urtica&nbsp; spp. Excellent anti-inflammatory activity have exhibited the aqueous&nbsp; extracts of&nbsp; U.<em> dioica</em>&nbsp; herbs&nbsp; &ndash;&nbsp; stinging nettle teas. All tested methanol extracts have inhibited enzyme acetylcholinesterase, the best inhibitors being root extracts of&nbsp; U.<em> kioviensis&nbsp;</em> and Parietaria&nbsp; species. Root extracts of&nbsp; P<em>. officinalis</em>&nbsp; and&nbsp; P.<em> judaica</em>&nbsp; have increased the basal and LPS-stimulated secretion of rat MCP1 and GRO&alpha;, while&nbsp; U. <em>dioica&nbsp;</em> extracts increased the basal but decreased the LPS-stimulated secretion. The examined extracts interact with the MyD88 (but not the TLR4) and NF-&kappa;B signaling pathway. The root extract of&nbsp; P<em>. officinalis&nbsp; </em>increase LPS-stimulated expression of COX-2 in IEC18 cells, while the root extract of&nbsp; U<em>. dioica</em>&nbsp; decreases it.<br />The herb and root extract of P. <em>officinalis</em>&nbsp; exhibit the wound healing effect. Investigated extracts do not significantly alter the secretion of hMCP1 and IL-8 in Caco2 cells and exhibit no significant effect to their proliferation.</p> / null