Dissertations / Theses on the topic 'Novel Bile Acid Conjugates'

Bioconjugation is the process of linking or connecting a biological molecule with another moiety. This moiety may include another biomolecule: in this case a hybrid is formed, in which the properties of the parent molecules are integrated, yielding a single entity with two different functions. With the aim to discover new nucleoside-based compounds of bio-medical interest, we consider bioconjugation as a powerful approach. In light of the well-known therapeutic potential of nucleosides as antitumorals and antivirals, in the first work reported in this thesis a conjugate between 2'-deoxyadenosine and a molecule called "photocage" was synthesized: the latter is in fact able to release under irradiation nitric oxide, whose activity in various bioregulatory systems has been proven in recent studies. Furthermore, two other bioconjugates were synthesized between the same photocage and the ursodeoxycholic and chenodeoxycholic bile acids, using click chemistry reactions. The novel bioconjugates were subjected to cytotoxicity tests and subsequently to photobiological studies that confirmed the combination of chemo- and phototherapeutic effects. In the second work the amphiphilic nature of bile acids was considered, which makes them suitable carriers for poorly lipophilic molecules, as in our case the nucleosides, increasing their cellular uptake. Therefore, new bioconjugates were made using CuAAC reactions between 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyuridine, adenosine and guanosine alkyne derivatives, and azide derivatives of cheno-, urso-, nor-cheno-, nor-urso- and taurourso-deoxycholic acids. Biological and structure-activity relationship tests on new hybrids have shown that cytoselectivity is mainly driven by the nature of the bile acid and can be fine-tuned by the nature of the nucleoside. The ability of bile acids as carrier molecules has also been exploited in the third work: here the ursodeoxycholic acid and other lipophilic molecules have been conjugated to an antisense oligonucleotide able to associate with the exon of the human dystrophin gene which is more frequently responsible for Duchenne Muscular Dystrophy, in order to improve exon skipping approach. For the synthesis of the oligonucleotide the classic phosphoramidite chemistry in solid phase was used, using the oligosyntesizer "Äkta Oligopilot Plus". By inserting suitable amino linkers to the 3'- and 5'-ends of the oligonucleotide and by the activation of the carboxylic groups present on the lipophilic molecules as N-succinimide esters, it was possible to carry out the bioconjugation by means of amide bonds. Following the tests performed in vitro and in vivo, an increase in the amount of dystrophin in the muscle cells treated with the oligonucleotide conjugated with ursodeoxycholic acid has emerged compared to those treated with the unconjugated oligonucleotide. In the last work of this thesis, bioconjugation was used to synthesize biomimetic compounds of UDP-GlcNAc, a natural substrate of glycosyltransferases, in which the β-phosphate was replaced by an alkyl chain or a triazole ring. These new compounds were then studied as glycosyltransferase inhibitors, as the dysregulation of these enzymes is related to various diseases, including cancer and neurodegenerative syndromes. Uridine monophosphate was conjugated with glucose, N-acetylglucosamine or a pyrrolidine ring substituted in various ways. Enzymatic, spectroscopic and computational studies were in agreement, highlighting how the absence of β-phosphate should be adequately counterbalanced in glycomimetics to have inhibition at micromolar concentrations.
La bioconiugazione è il processo di collegamento di una molecola biologica con un'altra entità chimica. Quest’ultima può includere un'altra biomolecola: in questo caso si forma un ibrido, nel quale le proprietà delle molecole genitrici si integrano, producendo una singola entità con due differenti funzioni. Allo scopo di scoprire nuovi composti di interesse bio-medico a base di nucleosidi, abbiamo considerato la bioconiugazione una valida strategia. Alla luce del ben noto potenziale terapeutico dei nucleosidi come antitumorali e antivirali, nel primo lavoro riportato in questa tesi è stato sintetizzato un coniugato tra 2'-deossiadenosina e una molecola definita “fotogabbia”: quest’ultima è infatti in grado di rilasciare sotto stimolo luminoso ossido nitrico, la cui attività in vari sistemi bioregolatori è stata provata in recenti studi. Sono stati inoltre sintetizzati altri due bioconiugati tra la stessa fotogabbia e gli acidi biliari ursodesossicolico e chenodesossicolico, utilizzando reazioni di click chemistry. I nuovi bioconiugati sono stati sottoposti a test di citotossicità e successivamente a studi fotobiologici che ne hanno confermato la combinazione di effetti chemo- e fototerapeutici. Nel secondo lavoro è stata considerata la natura anfifilica degli acidi biliari, che li rende adeguati carrier per molecole scarsamente lipofiliche, come nel nostro caso i nucleosidi, aumentandone l’uptake cellulare. Sono quindi stati realizzati nuovi bioconiugati utilizzando reazioni CuAAC tra derivati alchinici di 2'-deossiadenosina, 2'-deossiguanosina, 2'-deossiuridina, adenosina e guanosina, e derivati azidici degli acidi cheno-, urso-, nor-cheno-, nor-urso- e taurourso-desossicolico. Test biologici e di relazione struttura-attività sui nuovi ibridi hanno mostrato come la citoselettività sia guidata principalmente dalla natura dell'acido biliare e possa essere affinata dalla natura del nucleoside. L’abilità degli acidi biliari come molecole carrier è stata sfruttata anche nel terzo lavoro: qui l’acido ursodesossicolico e altre molecole lipofiliche sono stati coniugati ad un oligonucleotide antisenso in grado di associarsi all’esone del gene della distrofina umana che è più di frequente responsabile della Distrofia Muscolare di Duchenne, in modo da migliorarne l’exon skipping. Per la sintesi dell'oligonucleotide è stata utilizzata la chimica classica del fosforoammidito in fase solida, impiegando l'oligosintetizzatore "Äkta Oligopilot Plus". Attraverso l’inserimento di adeguati linker amminici alle estremità 3' e 5' dell’oligonucleotide e all’attivazione dei gruppi carbossilici presenti sulle molecole lipofiliche come esteri N-succinimmidici, è stato possibile realizzare la bioconiugazione per mezzo di legami ammidici. In seguito ai test eseguiti in vitro e in vivo, è emerso un effettivo aumento della quantità di distrofina nelle cellule muscolari trattate con l’oligonucleotide coniugato con l’acido ursodesossicolico rispetto a quelle trattate con l’oligonucleotide non coniugato. Nell’ultimo lavoro di questa tesi, la bioconiugazione è stata utilizzata per realizzare composti biomimetici di UDP-GlcNAc, substrato naturale di glicosiltransferasi, nei quali il β-fosfato è stato rimpiazzato da una catena alchilica o da un anello triazolico. Questi nuovi composti sono poi stati studiati come inibitori di glicosiltransferasi, in quanto la disregolazione di questi enzimi è connessa a vari disturbi, tra cui cancro e malattie neurodegenerative. L’uridina monofosfato è stata coniugata con glucosio, N-acetilglucosammina o con un anello pirrolidinico sostituito in vari modi. Test enzimatici, spettroscopici e computazionali sono risultati in accordo, evidenziando come l’assenza del β-fosfato debba essere adeguatamente controbilanciata nei glicomimetici per avere inibizione a concentrazioni micromolari.

Cancer is not only difficult to treat but the patients also suffer from the pain associated with anticancer treatments. Targeted chemotherapeutics can reduce the adverse effects by reducing the dose required for tumor cell kill. Cancers of various origins often have characteristic marker molecules that distinguish them from the normal tissues. Folate receptors are such marker molecules present in ovarian and cervical cancers. The hypothesis for the current study is that amphiphiles constructed out of folic acid, the natural ligand for the folate receptor, can deliver paclitaxel, a chemotherapeutic compound, to folate receptor expressing cancer cells. To test this hypothesis, amphiphilic molecules were synthesized out of folic acid and fatty acids or long chain aliphatic amines. The gamma carboxylic group of folic acid was converted to an N-alkyl substituted amide. The alkyl group had various chain lengths varying from eleven methylene groups to seventeen methylene groups giving rise to a number of amphiphiles. The amphiphiles formed micelles in aqueous solutions. The critical micellization concentrations of the amphiphiles were measured by pyrene fluorescence and were found to be in the range of 10–70μM. HeLa and Caco-2 cells were taken as in vitro tumor models. Folate receptor expression was verified in HeLa and Caco-2 cells by western blot analysis. HeLa showed more than forty fold expression of the receptor when compared to Caco-2 and was chosen as receptor positive cell line while Caco-2 served as a negative control. Uptake of the folate labeled delivery system in the cell lines was tested by a fluorescent probe (aminocoumarin) labeled amphiphile. To test the specificity of the delivery system towards the receptor positive HeLa cells, the receptors were knocked down (70%) by folate receptor specific siRNA. Fluorescent amphiphile uptake in the knockdown cells was comparable to that of the negative control, Caco-2. Finally cytotoxicity studies were performed for paclitaxel formulated with the folate labeled amphiphiles and compared to free drug treatment in HeLa and Caco-2. IC50 values in HeLa for formulations with the folate labeled amphiphiles were ten folds less than those observed for free drug treatment whereas in Caco-2 no significant difference was noted.

Primary bile acids are a group of cholesterol metabolites that are synthesized in the liver and stored in the gallbladder. The main role of bile acids is to aid in the digestion of dietary fats and lipids. More recently, a signaling role for bile acids has been characterized. Secondary bile acids are formed when the bacteria of the colon metabolize primary bile acids. Secondary bile acids that escape enterohepatic re-uptake to the liver can have a deleterious effect in the colon. Deoxycholic acid (DCA) and ursodeoxycholic acid (UDCA) are two secondary bile acids that have been studied for their biological effects in the colon. More recently, these bile acids have been found to induce intracellular signaling in the colon. Although much is known about bile acids and their link to tumorigenesis or chemoprevention, there is very little known about how they induce the intracellular signaling of cells. In short, it is our goal to understand the mechanism of how bile acids like DCA and UDCA induce the activation of signaling pathways. For several years, bile acids have slowly been characterized as hormonal molecules due to their ability to naturally bind to the farnesoid x receptor (FXR) and pregnane x receptor (PXR). Moreover, DCA and UDCA have also been researched for their gender specific role. In animal models, UDCA was found to be a chemopreventive agent in males, but a tumor-promoting agent in females. In our studies, we identified a novel nuclear receptor that bile acids exert their effect on. Estrogen receptor β (ERβ) is highly predominant in the colon and a regulator of colorectal cancer. Using human colorectal adenocarcinoma cell lines, we identified a key serine residue (Serine-87) on ERβ that is highly regulated by the MAPK pathway. We found that DCA and UDCA induce the phosphorylation and subsequent up-regulation of estrogen receptor regulated genes. Moreover, we found that phosphorylation was occurring mainly in the nucleus of our cells, indicative of activation of ERβ. Remarkably, we also identified a tumorigenic role for UDCA. UDCA-treated cells showed increased migration, which was blocked when the MAPK and ERβ pathways were inhibited. Collectively, this data reveals a novel signaling mechanism for bile acids in colorectal cancer and uncovers an adverse role for UDCA.

Polymer conjugation is a strategy able to enhance the tumour selectivity of anticancer drugs and to prolong their half life. Seventeen polymer-drug conjugates containing traditional chemotherapy (e.g. paclitaxel) have entered clinical trials and novel conjugates containing experimental drugs (e.g. the anti-angiogenic agent TNP-470) are emerging. Dopamine (DA) is an endogenous regulator of angiogenesis (the process of new blood vessels formation) and is able to retard the development of angiogenesis-dependent tumours when administered at a low non-toxic dose. However, the clinical application of DA in cancer treatment is hindered by its short half life (2 min) and severe side effects. DA is also a natural substrate of tyrosinase, a physiological enzyme present in the skin and over-expressed in malignant melanoma. Derivatives of DA have been designed as a new class of melanoma-specific anticancer prodrugs but their development is still limited to the preclinical stage. This study focuses on the development of novel polymer conjugates of DA and DA derivatives with the aim of enhancing their therapeutic potential as anti- angiogenic drugs and melanoma-specific agents, respectively. Polyglutamic acid (PGA) was selected as the polymeric carrier and the conjugation of DA was optimised using a carbodiimide and N-hydroxysuccinimide mediated coupling. The synthesis of the PGA-DA conjugate was confirmed by IH-NMR and 13C_NMR spectroscopic analysis. The total DA content was quantified by IH-NMR spectroscopic analysis and found to be in the range 10.8-14.9% w/w. The free DA content was determined by HPLC analysis and always found to be below 1 % of the total DA. PGA-DA as an anti-angiogenic drug. DA exerts its anti-angiogenic effect by binding to its D2 receptor and inhibiting the pro-angiogenic action of the vascular endothelial growth factor (VEGF). Thus, the anti-angiogenic activity of PGA-DA was assessed in two VEGF- dependent systems: an in vitro scratch assay, concerning the migration of human umbilical vein endothelial cells (HUVEC) and an in vivo Miles assay, evaluating vascular permeability. After a short incubation (1 h), both DA and PGA-DA reduced HUVEC migration to a similar extent (-50% VEGF inhibition). In contrast, after a longer incubation time (24 h), DA proved inactive while PGA-DA was even more effective (complete inhibition of VEGF). Similarly, a single injection of PGA-DA reduced the VEGF-induced vascular permeability for longer timeframes (24 h, 60% reduced dye extravasation) than DA (inactive at 24 h). Binding and degradation studies were performed to investigate the mechanism of action of PGA-DA. The inability of PGA-DA to bind to the D2 receptor and its degradation in the presence of cathepsin B suggested that the anti- angiogenic activity of the conjugate was mediated by the release of DA. PGA-(DA derivative) as melanoma-specific conjugates. The ability of DA to act as a substrate of tyrosinase was exploited in the development of an enzymatically activated conjugate. DA was chemically modified to act as a linker between the polymer and the drug. A model drug (p-nitroaniline (pNA)) or a cytotoxic drug (nitrogen-mustard) were coupled to DA. The ability of these DA derivatives to undergo enzymatic activation was assessed by a UV -Vis assay and oximetry. There were two main findings: (i) the linker- mustard was a substrate of tyrosinase; (ii) the linker-pNA was not recognised by tyrosinase as a substrate. Computational studies were used to probe this difference and showed that pNA prevented the activation of the DA derivative by altering its conformation in the active site of tyrosinase. An attempt to conjugate PGA to a DA derivative was also carried out using the method optimised for DA. Further characterisation is required to confirm the identity of the product. To conclude, the relevance of this work is two-fold: (i) polymer conjugation extended the anti-angiogenic activity of DA, making its translation to the clinic a plausible target; (ii) the development of the first melanoma-specific polymer-drug conjugate was undertaken.

Chapter 1: Functional and Responsive Supramolecular Gels In this chapter ‘supramolecular gels’ derived from small organic molecules with molecular mass of typically less than 2000 daltons are discussed. Representative examples of various low molecular weight gelators based on their natural availability and also divergent functionalities are mentioned (Scheme 1). Scheme 1 Advances in the recent years have been very rapid in the field of supramolecular chemistry of gels giving rise to ‘Tunable responsive gels’. Control of the gel property in a reversible fashion has been the highlight of responsive gels. A few of the gels which are responsive towards various stimuli such as pH, photoirradiation, cations, anions, neutral species have been discussed. Advances and scope of supramolecular gels in various applications have also been mentioned in detail with respective examples. Utilities of supramolecular gels in synthesis of nanostructures, in biology and medicine, enzyme recognition, catalysis etc are discussed. (Scheme 2). Chapter 2: Charge transfer triggered organogels of bis(bile acid)anthracene conjugates and 2,4.7-trinitrofluorenone. In this chapter the study involves the synthesis of a special class of anthracene based steroidal derivatives. The appending of two amphiphilic bile acid units imparts a unique hydrophobic/ hydrophilic balance on the chromophore. The 2,3-didecyloxyanthracene (DDOA) was reported to be a gelator of various organic solvents but none of the three bile acid derivatives of anthracene synthesized was a gelator on its own. It was also observed that dialkoxy (propyl, heptyl, decyl) derivatives of anthracene formed strong charge-transfer gels in the presence of 2,4,7-trinitrofluorenone (TNF). The addition of electron deficient TNF to the steroidal derivatives of anthracene resulted in the gelation of some specific organic solvents. The driving force behind the gel formation resulted from the charge-transfer (CT) interaction between the electron rich anthracene and electron deficient fluorenone. Figure 1. Chemical structures of 2,3-bis(bile acid)anthracenes and TNF (centre), a scanning electronic microscopy image of xerogels prepared from bis(deoxycholyl)anthracene and TNF (left) and a photograph of the gel of bis(deoxycholyl)anthracene and TNF in n-octanol. Thermochromic property (during sol to gel phase transition), absorption and variable temperature fluorescence measurements supported CT interaction. Thermal stability studies and dynamic rheology experiments confirmed that CT gels were thermally most stable and mechanically stronger with equi-molar amounts of the two components. Stiffness values obtained from rheological experiments also suggested that the gels were viscoelastic solids. Chapter 3(A): Tb(III) sensitization in an organogel matrix: Selective luminescence quenching by an aromatic nitro derivative In this chapter the discovery of metallo organogel formation by mixing methanolic solutions of Tb(OAc)3 and sodium deoxycholate (NaDCh) has been explored. Sensitization of Tb3+ was observed by doping micromolar quantities of 2,3-dihydroxynaphthalene (DHN). Mechanical properties of Tb3+-DCh gels were investigated by rheology at three different ratios of Tb3+ and DCh. It was observed that increasing in the Tb3+ to DCh ratio increased the mechanical property of the gels. Time delayed emission spectra were recorded with increasing concentration of DHN and luminescence increase was noticed in a linear fashion. Importance of gel matrix was demonstrated by measuring the Tb3+ luminescence at fixed concentration (5 mM) with/without DHN in the solution and gel media. Figure 2:: Schematic representation of Tb3+ sensitization by DHN. Photograph (right)) of the Tb3++-DCh (5/15 mM) gels with (a) 50 µM DHHN (b) No DHHN under UVV (365 nm). Sensitization by an electron rich chromophore created interest in us to dope relatively electron deficient compounds into the gel matrix for possible quenching off Tb3+-luminescence. Among the electron deficient analytes screened included 1,5-difluro-2,4-diinitrobenzenne (DFDNB)), 2,4 dinitrophenol (DNPPh), p-nitrobenzaldehydde (p-NB), 2,4,6-trinitrootoluene (TTNT) and 22,4,7¬trinitrofluuorenone (TTNF). Microscopy studies such as AFM, TEMM and SEMM revealed highly entangled fibrous network in the morphology of Tb3+--DCh xerogel. Solid state luminescence experiments suggested that sensiitization was observed in the xerogels and extent of sensitization was comparable to that of the gel state. Xerogel soaking studies inferred the strong adherence of the DHNN to the gel fibres. Chapter 3(B): Anion dependent structural, morphological and mechanical features of Ln(III)-Cholate gels In this chapter the counter anion influence on various aspects of hydrogels has been discussed. It has been reported from our laboratory that mixing of aqueous solutions of sodium cholate (15 mM) and various lanthanide acetates (5 mM) followed by sonication resulted in either transparent or transluscent gels. Unsurprisingly we found that aqueous solutions of lanthanide nitrates and lanthanide chlorides also formed hydrogels upon mixing with sodium cholate (Figure 33). Dried films of Tb3++-cholate and Eu3+-cholate gels prepared from their respective nitrate salts displayed birefringent structures under polarizing optical microscopy (POM). But no significant textures of any type were observed in the case of gels prepared from either chloride or acetate salts. Figure 3:: Photographs of the hydrogels prepared by mixing of aqueous solutions of various salts Tb33+ and Eu3+ with sodium cholate solutions. Scanning electron microscopic images exhibited fibrous structures for all the xerogels in the morphology. Atomic force microscopy and transmission electron microscopy measurements revealed helical morphology for xerogels prepared from nitrate salts where as flat tape-like cross linkage was observed for chloride or acetate based xerogels. Anion effect on mechanical properties was significant in the sense that gels prepared from acetate salts displayed highest mechanical strength followed by nitrate based gels which were stronger than that of chloride based gels. Titration of sodium cholate solution with various lanthanide salt solutions gave the direct evidence of thee pH variation as a function of the anions. Figure 4: TEM images of xerogels prepared from gels of nitrate salts of Tb3+ (left) and Eu3+ (right) Chapter 4: Design, synthesis a nd study of bile acid ‘click’ conjugates of perylene bisimides (PBIs) and naphthalene bisimides (NBIs) In this chapter the synthesis of novel bile acid derivatives of perylene and naphthalene bisimides is discussed. The ‘click’ chemistry procedure was used to link bile acid groups on to the chromophores. Azide derivatives of PBIs and NBIs were prepared inn 3 step methods which were coupled to propargyl esters of bile acids by following standard ‘click chemistry’ protocols to achieve the target molecules (Scheme 3). Scheme 3 The studies conducted mainly focused on Cholic acid (CA) conjugates of PBIs and NBIs. Steady state absorption and emission studies of CA conjugates were performed in 10% MeOH/DCM system. POM and fluorescence images showed red emissive aggregates in case of PBI films. TEM measurements revealed uniform aggregate sizes for both the films of PBI(CA)2 and NBI(CA)2. SEM and AFM (Fig 5) studies exhibited spherical aggregates of diameter around 100-200 nm for PBI(CA)2 films where as aggregates of diameter around 500-700 nm were observed for NBI(CA)2 films. Figure 5: AFM images and their corresponding height profiles of PBI films (left) and NBI films (right)

Chapter 1: Amphiphilic self-assembled systems as nanocarriers Nanocarriers are the nanometric size molecular assemblies that are used for the transport of small molecules into their non-solvating environments. These systems find major applications as drug delivery systems (DDS) in pharmacological research. These drug delivery systems improves solubility and stability of the drug molecules through encapsulation and also offer additional advantages like target specificity and stimuli responsive release of the drug molecules. Several types of DDS are reported in the literature, which can be prepared by a variety of processing techniques. Of these, molecular self- Chart 1: Developments in the design of amphiphilic nanocarriers assembly has attained considerable attention due to its greater tunability and control in the preparation of nanocarriers. In this chapter we discussed about the amphiphilic nanocarriers which are prepared through self-assembly of amphiphiles through hydrophobic interactions. Several developments in the area of amphiphilic nanocarriers such as di-block polymeric systems, dendritic systems and core-shell architectures are also mentioned. We also highlighted some recent developments in the design of amphiphilic nanocarriers through supramolecular interactions and advantages of such systems. Chapter 2: Bile acid derived dendrons and their application as nanocarriers Host-guest chemistry is well known for dendritic systems. To understand the influence of steric crowding, dendritic effect and importance of number of hydroxyl groups on the bile acid backbone in the host-guest chemistry of bile acid dendrons, we designed and synthesized a new series of C3 symmetric systems and studied the above-mentioned objectives through extraction of polar dyes into nonpolar media. Dye extraction experiments performed using trimeric molecules suggested that only the cholate derivatives (3 and 4) showed considerable extraction of the polar dyes into chloroform; deoxycholate derivatives did not show any extraction, thus emphasizing the importance of the number of hydroxyl groups for dye extraction in these molecular architectures. The effect of steric crowding at the core of these trimeric molecules was shown by efficient extraction of the dyes with the triethylbenzene core (4) compared to the benzene core (3). Greater influence of the aggregates in the case of triethylbenzene core on the extracted dye was also manifested in the Chart 2: Structures of the designed molecules 1-6 value of the induced circular dichroism signal. Surprisingly, a higher analogue in these molecular architectures showed lesser efficiency in dye extraction (on a per bile acid residue basis) compared to the trimers, suggesting a more compact structure for the higher analogue. This was supported by molecular modeling studies. Generality of these systems as nanocarriers for hydrophilic dyes was investigated by screening several other dyes and polar molecules, which are diverse in their structure and functionalities. All these experiments suggested a dependency of the extraction profile on the size of the dye molecule. This was also examined by dynamic light scattering studies, which showed larger size and wider distribution in the size of the aggregates in the case of larger dyes. We also demonstrated selective extraction of a single dye molecule from a blended food color (apple green) using one of the trimer (4) and demonstrated solvent dependent morphological changes in these compounds using electron microscopy. The self-assembly of these amphilic molecules at the air-water interface was studied through Langmuir monolayer studies. Chart 3: Structure of polar guest molecules (Cresol red (7). Erioglaucine (8), Eriochrome black T (9),) phenyl β-D-glucopyranoside (10) and Eosin B (11) Chapter 3: Design and synthesis of bile acid derived surfactants: Study of their aggregation and potential applications Bile acids are facially amphiphilic systems and their amphiphilicity can be improved by attaching polar groups on the bile acid back bone or by synthesizing oligomeric systems which show better self-assembly compared to their monomeric units. To study and improve the amphiphilicity of bile acids, we designed and synthesized a new tripodal surfactant system, with a phosphine oxide based central core to which the bile acids were attached through the C-3 position using click chemistry. Our molecular design also offers added advantage of studying the influence of the stereochemistry at the C-3 position on the aggregation of these molecular architectures. We synthesized trimeric systems with both cholic and deoxycholic acids attached to the central phosphine oxide core with α and β stereochemistry at the C-3 position. Aggregation of these molecules was studied by surface tension measurements, dye extraction studies and NMR. All these compounds showed aggregation at micromolar concentrations. NMR studies suggested changes in the structure of the aggregates at higher temperature and these changes were studied by DLS, which suggested thermodynamically stable monodispersed aggregates for cholic acid derivatives (13 and 15) at higher temperature. These aggregates are stable even after cooling to room temperature and with time. The aggregates of these derivatives were also characterized by atomic force microscopy. Gelation was observed in the case of α derivatives (12 and 13) in phosphate buffer (0.1 M) at pH 7.5 for both deoxy and cholic derivatives, which emphasized the influence of stereochemistry at C-3 position in these architectures. These gels were characterized by rheology experiments. Finally, the possible utility of these micellar systems as model systems to study photophysical processes was demonstrated through lanthanide sensitization experiments in these micellar solutions. Chart 4: Structure of the designed molecules Chapter 4: Synthesis of oligomeric bile acid-taurine conjugates: Study of their aggregation and efficiency in cholesterol solubilization Bile acids are bio-surfactants that are used for the emulsification of fats, vitamins etc. in our body. Bile salts also solubilize the excess cholesterol in our body through mixed micelle formation in the bile and when the bile gets saturated with cholesterol, it leads to cholesterol gallstone formation, which needs to be treated. Ursodeoxycholic acid (UDCA) is used as drug in some cases for the solubilization of (small) cholesterol gallstones, even though the efficiency to solubilize cholesterol is less for UDCA compared to the other bile acids (UDCA is less toxic than the others). So there is a need to develop new cholesterol solubilizing agents. Since oligomeric systems can aggregate better, we designed and synthesized two tetramer taurine conjugates, which differ in the spacer between the bile acid units. Since these conjugates are not soluble in water, their solubility and aggregation was studied in 10% MeOH/Water using pyrene fluorescence experiments. Aggregation studies suggested better aggregation for these molecules compared to their monomeric analogues. These aggregates were also characterized byDLS and electron microscopy. These systems were subsequently studied as nanocarriers for liphophilic dye molecules into aqueous media. Finally, the influence of oligomeric effect in cholesterol solubilization was investigated by cholesterol solubilization studied using these two tetramer taurine compounds and a control, sodium taurocholate. These studies suggested efficient solubilization of cholesterol by oligomers compared to monomeric analogues.(For structural formula pl see the abstract file)

Bile is a biofluid synthesized by liver and concentrated in the gallbladder. Interference with the bile flow may cause cholestasis. Primary sclerosing cholangitis (PSC) is an inflammatory cholestatic disorder which eventually may result in liver cirrhosis and failure. The management of PSC is controversial. The only effective treatment for end stage disease is orthotopic liver transplantation (OLT). However, cholangiocarcinoma (CC), which is the major complication of this long-lasting disease, is an absolute contraindication for the surgery. Therefore, early diagnosis of the disease can not only improve the outcome of PSC, but also facilitate the allocation of donated livers to those who can benefit from transplantation. Unfortunately, the diagnosis of CC is challenging. Endoscopic retrograde cholangiopancreatography (ERCP), the gold standard technique, is highly invasive. Non-invasive alternatives such as magnetic resonance cholangiopancreatography (MRCP) have lower accuracy. Therefore, it is essential to develop more accurate and less invasive diagnostic techniques. Magnetic resonance spectroscopy (MRS) is an evolving technique with potential to detect disease-related metabolic changes. In vitro studies have proven the capacity of MRS in the early detection of hepatopancreatobiliary (HPB) disorders based on the metabolic analysis of bile obtained invasively. An in vivo alternative has been attempted by others on human bile within the gallbladder. However, due to the poor quality of the acquired spectra, quantification of most major bile metabolites was not possible, except for choline-containing phospholipids (chol-PLs). In the current study, the quality of the in vivo 1D spectra has been greatly improved, and we have obtained the first 2D L-COSY spectra from bile within the gallbladder. Spectral data from healthy controls and PSC patients were compared. Statistically significant differences in the concentrations of chol-PLs, and glycine- and taurine-conjugated bile acids were revealed in the 1D analysis. Our 2D spectra also demonstrated potential for the detection of metabolic differences between the two groups. The success of these studies indicates a strong potential of in vivo bile MRS techniques to characterize and diagnose a wide variety of HPB disorders.
May 2014

Chapter 1. An Overview of Bile Acid Science This chapter deals with an overview of bile acid science (cholanology) compiling elevant literature review, covering bile acid chemistry, biosynthesis, bile salt evolution, physiology and medicinal values. Figure 1. (a) Digestive system; (b) enterohepatic circulation and (c) cholic acid Bile acids are the end products of cholesterol metabolism, secreted in the liver and stored in the gall bladder (Figure 1). They are normally conjugated with glycine (75%) or taurine (25%). Because of their facially amphiphilic nature, bile salts tend to form micellar aggregates in aqueous solution. They have remarkable ability to transform lamellar array of lipids into mixed micelles. All primary bile acids seem to have three features in common: (1) They are major products of cholesterol metabolism; (ii) they are secreted into the bile largely in a conjugated form and (iii) the conjugates are membrane impermeable, water soluble, amphiphilic molecules. Recent advances in molecular biology have greatly accelerated the knowledge relating to the significance of bile salts in a number of physiological functions. The new role of bile salts as pheromones and ligands for nuclear hormone receptors has been discussed. Chapter 2. Pythocholic Acid: A Major Constituent of Python’s Bile and 16α-Hydroxycholic Acid: A Minor Constituent of Avian’s Bile The first chemical synthesis of pythocholic acid (major constituent of python’s bile) and 16α-Hydroxycholic acid (a minor constituent of avian’s bile) were accomplished starting from cholic acid with overall yields of 5.0% and 5.5%, respectively. A biomimetic remote functionalization strategy was utilized as a key step to achieve the selective chlorination at C-17. Dehydrochlorination of 17-chlorosteroid resulted in the Δ16 olefin. Hydroboration-oxidation of the Δ16 olefin followed by the selective oxidation of the pentol under TEMPO mediated oxidation resulted in an ε-lactone. Hydrolysis of the lactone using 5% KOH in MeOH furnished the 16α-Hydroxycholic acid. On the other hand, selective oxidation of 7-OH of the lactone was achieved using N-bromosuccinimide in acetone/H2O to yield the 7-keto lactone. The ketolactone when subjected to the Huang-Minlon modification of the Wolf-Kishner reduction furnished pythocholic acid. Pythocholic acid showed unusual aggregation behavior and high cholesterol solubilization ability, compared to other trihydroxy bile acids. Chapter 3. 16-Epi-pythocholic acid: An Unnatural Analogue of Pythocholic Acid The synthesis of 16-epi-pythocholic acid, an unnatural analogue of pythocholic acid, was accomplished starting from cholic acid. Cholic acid was converted to Δ8-14) olefin using ZnCl2 in refluxing acetone. Methylation followed by isomerization in CHCl3 by passing dry. HCl at -78 oC resulted in the Δ14 olefin. Allylic oxidation using Na2Cr2O7.2H2O in the presence of N-hydroxysuccinimide in acetone furnished the enone. Selective reduction of the olefin using Pd/C-H2 resulted in 16-Epi-pythocholic acid the 16-keto steroid. NaBH4 reduction of this ketone in MeOH/THF (2:1 v/v) followed by hydrolysis produced the 16-OH bile acid. Analysis of spectral data confirmed that it is a 16β-epimer of pythocholic acid (3α,12α,16β-trihydroxy-5β-cholan-24-oic acid). Critical micellar concentration and cholesterol solubilization properties were studied. Chapter 4. Low Molecular Mass Organogelators Derived from Simple Esters of Cholic Acid This chapter begins with an introduction to low molecular mass organogelators and highlights their applications. Serendipitous gelation of a number of organic solvents by allyl cholate and the design of related simple esters of cholic acid are discussed. A series of simple and easily accessible esters of bile acids were prepared. Ethyl cholate and propyl cholate were found to immobilize a variety of organic solvents like benzene, toluene, xylene, mesitylene, 1,2-dichlorbenzene (DCB) and chlorobenzene (Figure 2). The morphology of the xerogels was well characterized using SEM, AFM and polarizing optical microscopy (POM) techniques, Which revealed the presence of highly entangled self-assembled 3D-fibrillar network (SAFINs). The fiber diameter was found to vary between 300-500 nm. Direct imaging of the collapse of this fibrillar network and direct observation of the evolution of nanofibers was achieved for the first time using variable temperature POM techniques. FT-IR studies, X-ray powder diffraction and variable temperature POM studies revealed the resemblance of SAFINs to the bulk solid. Formation of helical fibrillar network was observed in SEM images and the existence of chiral aggregates was confirmed by induced circular dichroism experiment using achiral Reichardt’s dye as the chromophore. Chapter 5. Ambidextrous Gelators Derived from Spacer Linked Bile Acid Derivatives Based on our observation of simple esters of cholic acid as organogelators a rational design of a series of spacer linked dimers and tripodal derivatives were carried out. Some of these molecules formed highly transparent gels in solvents like haloarenes, anisole, xylene and dibromoalkanes. These molecules also showed rapid gelation in DMF/H2O and DMSO/H2O mixtures in varying proportions of water and the co-solvent. These types of gelators are known as ambidextrous gelators. The xerogels were characterized using SEM, TEM and POM techniques and the presence of highly entangled 3D-fibrillar network (Figure 3) was observed. XRPD showed crystalline nature of bulk solid, whereas the xerogels were shown to lose their crystalline nature. (For figures and structural formula pl see the pdf file.)

Chapter 1. Supramolecular Gels and their Applications Supramolecular gels are viscoelastic materials composed of a solid like three dimensional fibrillary network that is embedded in a liquid. Supramolecular gels are derived from low molecular weight compounds (typically MW < 3000). In the 1990s, the investigations on gels were mainly focused on designing new gelator molecules. However, during the last decade, research focus shifted towards designing functional gels and their applications. As a result of extensive work in this area, gels have been found to have varied applications in the templated synthesis of inorganic nanomaterials, hybrid materials, light harvesting systems, as responsive system and sensors, and also in drug delivery, tissue engineering etc. This chapter gives an introduction to supramolecular hydrogels/organogels and relevant bile acid chemistry touching upon the gelation properties of the bile acid derivatives. Diverse applications of the supramolecular gels are also illustrated with several examples. Scheme 1. Various applications of functional supramolecular gels Chapter 2. Bile Acid derived novel Hydrogelators Part 1. Hydrogelation of Bile acid protected Amino acids and Hybrid Materials Hydrogels from low molecular weight molecules have significant importance in biomedical applications. In this chapter, we report injectable hydrogel formation from bile acid conjugates of various amino acids. Hydrogel formation was found to be dependent on multiple factors such as bile acid backbone structure, linkage between the bile acid and the amino acid, pH etc. Single crystal structures of lithocholyl phenylalanine, lithocholyl-glycine, lithocholyl-L valine and lithocholyl-L alanine were also determined. Finally, the hydrogel frameworks were utilized to produce hybrid materials with Gold and ZnO nanoparticles. Scheme 2. (a) Crystal structure of LC-LF-OH gelator molecule, (b) photograph of gel, (c) SEM and (d) AFM image of LC-LF-OH xerogel Part 2. Hydrogelation of bile acid-dipeptide conjugates and in situ synthesis of silver and gold nanoparticles in the hydrogel matrix Fabricating supramolecular hydrogels with embedded metal nanostructures are important for the design of novel hybrid nanocomposite materials for diverse applications such as bio sensing and chemo sensing platforms, catalytic and antibacterial functional materials etc. Supramolecular self-assembly of bile acid-dipeptide conjugates have led to the formation of new supramolecular hydrogels. Gelation of these molecules depends strongly on the hydrophobic character of the bile acids. Ag+ and Au3+ salts were incorporated in the hydrogels, and photo reduction and chemical reduction led to the in situ generation of Ag and Au NPs in these supramolecular hydrogels without the addition of any external stabilizing agent. The color, size and shape of silver nanoparticles formed by photo reduction depended on the amino acid residue on the side chain. Furthermore, the hydrogel-Ag nanocomposite was tested for its antimicrobial activity. Scheme 3. Bile acid based dipeptide hydrogelators and soft hybrid materials Chapter 3. Sonogels of bile salts of In(III): use in the formation of self-templated indium sulfide nanostructures In this chapter, facile hydrogel formation by Indium(III) cholate and deoxy cholate are reported. When In(III) solution was added to aqueous solutions of sodium cholate and sodium deoxy cholate and sonicated, the mixtures formed gels. The gels thus obtained were translucent/turbid and thermos irreversible. Rheological measurements showed that all of them could be classified as viscoelastic soft solids. Scanning electron microscopy and atomic force microscopy showed typical entangled three dimensional fibrous networks. The In-Ch hydrogel were further used to prepare nanostructured In2S3 in which the cholate units possibly acted as a surfactant to confine the growth of the Nano flakes. Scheme 4. In-Ch hydrogel (Photograph and SEM image of In-Ch gel) Chapter 4. Palladium-Hydrogel Nanocomposite for C-C Coupling Reactions Supported metallic nanoparticles are important composite materials owing to their enormous potential for applications in various fields. This chapter describes the in situ formation of palladium nanoparticles in a calcium-cholate (Ca-Ch) hydrogel by reduction with sodium cyan borohydride. The hydrogel matrix appeared to assist the controlled growth as well as stabilization of palladium nanoparticles. The palladium nanoparticle/Ca-Ch hydrogel hybrid was characterized by scanning and transmission electron microscopy, atomic force microscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy. Furthermore, PdNP/Ca-Ch hybrid xerogel was shown to act as an active catalyst for Suzuki reaction under aqueous aerobic conditions, up to 4 cycles. This PdNP/Ca-Ch xerogel retained its catalytic activities on storage for several months. Scheme 5. Palladium-hydrogel nanocomposite for C-C coupling reactions in water Chapter 5. Sensitization of Terbium/Europium in self-assembled cholate hydrogel: An approach towards the detection of amine vapours "Luminescent" lanthanides have intrinsic low molar absorptivity, although this problem can be addressed by complexing the lanthanide ion with suitable chelating ligands which improve the luminescence properties drastically. However the design of such systems often involves careful planning and laborious synthetic steps. It is therefore desirable to have a simpler way to sensitize lanthanides with high efficiency. It was observed in our group that trivalent lanthanides formed hydrogels on the addition of sodium cholate. This chapter describes the discovery of the several biphenyl derivatives (such as 4-biphenylcarbaxaldehyde, 4-acetylbiphenyl) for sensitization of Tb(III) and Eu(III) in lanthanide hydrogels. Sensitization of Tb(III) and Eu(III) were observed by doping was characterized by scanning and transmission electron microscopy, atomic force microscopy, X-ray diffraction, and energy-dispersive X-ray spectroscopy. Furthermore, PdNP/Ca-Ch hybrid xerogel was shown to act as an active catalyst for Suzuki reaction under aqueous aerobic conditions, up to 4 cycles. This PdNP/Ca-Ch xerogel retained its catalytic activities on storage for several months. Scheme 6. Schematic representation of the sensitization process (the arrangement of themolecules in the gel fiber is arbitrary)(For figures pl refer the abstract pdf file)