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)

In this thesis, a polypeptide binder concept is illustrated. By conjugation to a set of sixteen polypeptides, a small binding molecule can evolve into a polypeptide binder with increased affinity and selectivity. The concept was applied to 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) and acetazolamide (AZM) for development of high affinity binders targeting human C-reactive protein (CRP) and human carbonic anhydrase (HCA) II and IX respectively. In addition, peptididic macrocycles were developed as inhibitors of lysine specific demethylase 1 (LSD1). CRP is a well-known biomarker of inflammation in humans and binders recognizing it are therefore of large interest as medical diagnostics. Until now, phosphocholine (PCh) and derivatives are the only known small molecule binders for CRP, but they have low μM affinity and bind CRP in a Ca2+ dependent manner. The small molecule DQ was designed as a CRP binder that is structurally unrelated to PCh. Its polypeptide conjugate, 4-C25l22-DQ, was demonstrated as a strong, Ca2+ independent binder for CRP, and had an affinity approximately three orders of magnitude higher than DQ itself. HCA IX is a protein that is interesting for diagnosis of cancer. AZM is a small molecule inhibitor of HCAs with a dissociation constant of 38 nM for HCA II and 3 nM for HCA IX. Interestingly, polypeptide conjugate 4-C10L17-AZM displayed stronger binding to both HCA II (KD 4 nM) and HCA IX (KD 90 pM). This result provided evidence that the binder concept can be applied also for small molecules which already have high affinity for their protein receptors. LSD1 is an enzyme that regulates the methylation of Lys 4 of histone 3 via a PPI-like interaction and which is of therapeutic interest in certain cancers. Based on the structures of two peptidic ligands bound to LSD1, we sequentially prepared truncated, mono-substituted and macroyclic peptides in order to develop reversible inhibitors of LSD1. Some stapled cyclic peptides bound to LSD1 with 10-fold higher affinity than the corresponding linear parent peptide. Changing the staple into a lactam further improved the binding potency and the best lactams inhibited the enzymatic activity of LSD1 at low μM Ki values.

The thesis entitled ‘Studies on the Chemistry of Carbonates and Carbamates’ comprises of seven chapters. Chapter 1 The reactivity of propargyloxycarbonyl (Poc) derivatives of amines and alcohols with various sulphur nucleophiles is addressed in this chapter. The chapter is divided into three different parts. Part 1: The difference in reactivity of propargyloxycarbonyl (Poc) derivatives of amines and alcohols with benzyltriethylammonium tetrathiomolybdate [(PhCH2NEt3)2MoS4, 1] is studied in detail and the results are discussed. It has been shown that amino alcohols can be protected as their diPoc derivatives using 2 equiv of propargyloxycarbonyl chloride (2). The selective deprotection of the O-Poc group using 1 equiv of 1 without affecting the N-Poc group is achieved (Scheme 1). Scheme 1 Part 2: The reactivity of propargyloxycarbonyl derivatives of various alcohols, phenols and primary and secondary amines with benzyltriethylammonium tetrathiomolybdate (1) is compared with the reactivity of these Poc derivatives with other sulphur nucleophiles such as sodium thiophenoxide, lithium sulphide, hydrogen sulphide and ammonium sulphide. The study reveals that tetrathiomolybdate (1) is the best sulphur nucleophile for the deprotection of Poc group. Poc derivatives of primary amines cyclized to the corresponding 4-methylene-2-oxazolidinones when treated with other sulphur nucleophiles (Scheme 2). Scheme 2 Part 3: The reaction between different propargyloxycarbonyl derivatives of alcohols and benzyltriethylammonium tetrathiomolybdate (1) is studied. It is found that propargyloxycarbonyl derivatives can be made more reactive towards tetrathiomolybdate by substituting the propargyl system with electron withdrawing substituents. Chapter 2 The application of propargyloxycarbonyl group for the protection of the side chain hydroxyl groups of serine, threonine and tyrosine is discussed. The O-Poc derivatives are shown to be stable to a variety of acidic and basic conditions and the applications of these derivatives in solution phase peptide synthesis is addressed. The easy and effective deprotection of the O-Poc group provides a new strategy for the synthesis of peptides bearing the hydroxy amino acid residues: serine, threonine and tyrosine. Scheme 3 Chapter 3 Development of a novel C2-symmetric protecting group for amines and amino acids is described in this chapter. But-2-ynyl-1,4-bisoxycarbonyl chloride (BbcCl, 3) is synthesized from 1,4-dihydroxybut-2-yne and used as a reagent for protecting amines as biscarbamates (Scheme 4). These biscarbamates (Bbc derivatives) are deblocked using benzyltriethylammonium tetrathiomolybdate (1) to get the amines back. Scheme 4 The orthogonal stability of the Bbc group with Boc, Cbz and Fmoc groups is established. It is also shown that Bbc group can be deblocked to the corresponding amines using resin-bound tetrathiomolybdate. The application of Bbc protected amino acids in solution phase peptide synthesis is demonstrated (Scheme 5). Scheme 5 Chapter 4 The simultaneous protection and activation of amino acids using various pentafluorophenyl carbonates is described in two parts. Part 1: A very efficient and high yielding method for the simultaneous protection of the amino group and activation of carboxylic acid group using propargyl pentafluorophenyl carbonate (PocOPfp, 4) is discussed. Treating amino acids with 2 equiv of 4 protects the amino group as a propargyl carbamate and activates the carboxylic acid group as a pentafluorophenyl ester (Scheme 6). Scheme 6 Part 2: The generality of the methodology developed for the simultaneous protection and activation of amino acids using PocOPfp (4) is studied with five different pentafluorophenyl carbonates viz. AlocOPfp, CbzOPfp, BocOPfp, EocOPfp and TrocOPfp. The studies reveal that the effectiveness of the methodology depends on the nature of the pentafluorophenyl carbonates and on the nature of the amino acids. Sterically bulky pentafluorophenyl carbonates such as BocOPfp reacted slowly with amino acids while electron deficient pentafluorophenyl carbonates such as TrocOPfp reacted faster and gave the N-protected active esters in very good yields. Amino acids bearing longer aliphatic side chains reacted better than the other amino acids. Chapter 5 The chapter describes results of the detailed studies on the base catalyzed cyclization of N-alkyl and N-aryl-O-propargyl carbamates to the corresponding 4-alkylidene-2-oxazolidinones. The effect of various bases and solvents on these cyclization reactions is studied systematically to design the most suitable conditions. The best results were obtained using catalytic amount of LiOH in DMF. The cyclization reactions of N-aryl-O-propargyl carbamates were faster than the cyclization of N-alkyl-O-propargyl carbamates. The effect of substitutions on the propargyl group in these reactions is studied by preparing various substituted propargyl carbamate derivatives from the corresponding amines and propargyl chloroformates (Scheme 7). Scheme 7 Chapter 6 An efficient procedure for the synthesis of dehydroalanine and dehydroamino butyric acid derivatives from the preformed carbonate derivatives of serine and threonine respectively, by treating with K2CO3 in DMF is discussed in this chapter. The reaction proceeds stereoselectively through a trans E2-elimination pathway to give only the Z-isomer of dehydroamino butyric acid derivatives from the carbonate derivatives of threonine. The methodology offers an easy access to dehydropeptides and proceeds without racemization of other stereogenic centers present in the peptide (Scheme 8). Scheme 8 Chapter 7 This chapter describes the use of propargyloxycarbonyl derivatives of lysine as an efficient tool for the synthesis of peptide conjugates using a click chemistry approach. The Cu(I) catalyzed cycloaddition reaction between azides and alkynes is employed in the synthesis of conjugates of lysine. Peptides bearing an Nε-Poc Lysine residue can be synthesized using traditional strategies and these peptides can be easily conjugated with azide derivatives of sugars and amino acids (Scheme 9). Scheme 9 The efficiency of the method is demonstrated by carrying out more than one click reaction in one pot using di and tri-propargyl derivatives of lysine. A dendritic core (6) is prepared from a tri-propargyl derivative (5) of lysine and an azide derived from leucine (Scheme 10). Scheme 10 The abbreviations used in the thesis are consistent with those reported in J. Org. Chem. 2007, 71, 23A. Less common abbreviations are defined, the first time they are mentioned in the thesis.

Platinum-based anticancer drugs, such as cisplatin, carboplatin, and oxaliplatin, have been approved for clinical use worldwide for decades. Despite their enormous success, their widespread application is hindered by either cross-resistance or toxic side effects, including nephrotoxicity and neurotoxicity. The need to overcome these drawbacks has stimulated the search for new platinum-based drugs. This dissertation will start with the accidental discovery of cisplatin, followed by an introduction of other platinum-based anticancer agents, including the action mechanism, general structures, and development history. Picoplatin is a newer generation of platinum-based anticancer agent. The bulky 2-methylpyridine as a non-leaving group on picoplatin could reduce the detoxification effect caused by thiol-containing species, such as glutathione and metallothionein, thus may grant picoplatin the ability to overcome cisplatin resistance. A convenient synthesis route for picoplatin derivatives has been developed. 11 new picoplatin derivatives have been designed by varying the bulkiness of the non-leaving amine group. All complexes have been characterized by different instrumentations, including MS, 1H NMR, 13C NMR, 195Pt NMR, HMQC, X-ray crystallography, and elemental analysis. Different bioassays, such as DNA binding, cell viability, and cellular accumulation, have been applied to evaluate their efficacy on cisplatin-sensitive ovarian cancer cell line A2780 and cisplatin-resistant ovarian cancer cell line A2780cis. The newly designed picoplatin derivatives show comparable efficacy with that of picoplatin and less resistance compared with cisplatin. The study of picoplatin derivatives laid the foundation toward the research of bifunctional platinum-based anticancer agents by incorporating histone deacetylase (HDAC) inhibition. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) are a pair of important enzymes in epigenetic regulation. They work in harmony to acetylate and deacetylate histone lysine residues, resulting in a more relaxed or more condensed chromatin structure, respectively. HDAC has been found to be overexpressed in some cancer cells. Since 2006, 5 HDAC inhibitors (HDACi) have entered clinical use for cancer treatment. 19 new HDACi with additional coordination sites on the phenyl cap have been designed, synthesized, and evaluated. A few of the new HDACi show comparable or even better HDAC inhibition than that of Vorinostat (SAHA, the first FDA approved HDACi). A logical design would involve the installation of HDACi on the platinum center as a non-leaving group ligand. When the bifunctional drug reaches the cancer cell, the synergistic effect could be maintained as the relaxed chromatin structure makes DNA more susceptible to be attacked by the platinum centers, thus increase the anticancer activity and possibly selectivity toward cancer cells. 6 Pt-HADCi conjugates have been designed and synthesized. Dual functions of the new Pt-HDACi have been confirmed by DNA electrophoresis assay and HDAC inhibition assay. One of the Pt-HDACi (CF-101) shows comparable cytotoxicity with cisplatin and less resistance, which could be used as the lead compound for further structural modification and in vivo studies.

Therapeutically restricted sites present a formidable barrier in medicine. Herein, chemical strategies to overcome two restricted sites, HIV reservoirs and intracellular bacteria, will be discussed. First, cellular and anatomical HIV reservoirs, such as those in the brain, limit HIV eradication using currently known therapeutic regimes. HIV therapies are unable to localize in the brain, in part, due to high expression of efflux transporters, such as P-glycoprotein (P-gp), at the BBB, because many of these therapies are P-gp substrates. In an effort to overcome therapeutically restricted HIV sanctuaries, a dimerized combination HIV therapy was designed to act two-fold. First, the dimeracts as a P-gp inhibitor allowing therapeutic access to restricted sites. Second, the dimeractsas a prodrug, which once in the reducing environment of the cell, may release monomeric HIV therapies. The dual conjugate, Abacavir-S2-Darunavir, was shown to potently inhibit P-gp across two separate cell lines, was able to regenerate the component monomers in a reducing environment and contained modest anti-HIV activity.<div><br><div>Further, mammalian cells create sanctuary sites for bacteria to grow and proliferate, because many common antibiotic therapies are unable to cross the mammalian cell membrane. Therefore, these pathogens are able to proliferate without therapeutic constraint. Here, a chemical strategy was developed to deliver a dual antibiotic therapy inside mammalian cells in an effort to clear these intracellular pathogens. First, a new synthetic strategy was developed for facile synthesis of dual conjugates, composedof an aminoglycoside and a cell penetrating peptide (CPP) linked with a reversible disulfide tether, using kanamycin and the known CPP Arg8as a model system. Next, this synthetic methodology was expanded for use with theaminoglycoside tobramycin and theknown broad-spectrum antibiotic and cell penetrating peptide, P14LRR, once again linked via the reversible disulfide tether (TobP14). Two distinct isomers of TobP14 were synthesized, isolated, and fully characterized by 2D NMR. The TobP14 isomers were shown to be an effective antibiotic across various Gram positive and negative pathogens such as MRSA, S. epidermidis, P. aeruginosa, and A. baumannii. Further, the isomers effectively releasedthe monomeric therapies (tobramycin and P14-SH) in a reducing environment and werenontoxic to mammalian cells up to 16 μM. Finally, the dual conjugate isomers significantly reduce two different strains of intracellular A. baumanniiwithin macrophages.<br><div><br></div><div><br></div></div></div>

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第20569号 / 医科博第79号 / 新制||医科||6(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 齊藤 博英, 教授 江藤 浩之, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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no / The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are a class of DNA minor groove binding agents that react covalently with guanine bases, preferably at Pu-G-Pu sites. A series of three fluorescent PBD¿coumarin conjugates with different linker architectures has been synthesized to probe correlations between DNA binding affinity, cellular localization and cytotoxicity. The results show that the linker structure plays a critical role for all three parameters. Graphical abstract A series of three fluorescent PBD¿coumarin conjugates with different linker architectures has been synthesized to probe correlations between DNA-binding affinity, cellular localization and cytotoxicity.

Yes / The synthesis of nanoparticles of conjugates of caffeic acid-modified chitosan with ruthenium arene complexes is described. The chemical structure and physical properties of the nanoparticles were characterised by electronic absorption spectroscopy (UV-vis), Fourier transform infrared spectroscopy (FT-IR), 1H NMR spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), X-ray powder diffraction (XRD), and circular dichroism (CD) analysis. The multi-spectral results revealed that caffeic acid is covalently bound to chitosan and chelates to {Ru(p-cymene)Cl}+. The DLS studies indicated that the Ru–caffeic acid modified chitosan nanoparticles are well-defined and of nanometre size. Such well-defined nanocomposites of chitosan and metal complexes might find a range of applications, for example in drug delivery. / We thank the National Natural Science Foundation of China (Project No. 21571154), the Jiangsu Overseas Research & Training Program for University Prominent Young & Middle-aged Teachers and Presidents, Leverhulme Trust (Early Career Fellowship No. ECF-2013-414 to NPEB), the ERC (Grant No. 247450 to PJS), EPSRC (EP/F034210/1 to PJS) and Science City (AWM/ERDF) for support, and EU COST Action CM1105 for stimulating discussions.

Immunotoxins (ITx) represent a new, alternate class of therapeutic agent. ITx is made when the active part of a toxin is conjugated with the binding portion of an antibody that recognizes a cancer-specific antigen. The antibody component makes ITx highly specific, as it will only bind to cells displaying the correct surface antigen. This characteristic lowers the chance of nonspecific cell damage, which causes many of the severe side effects of other chemotherapeutics. The ITx we use is a conjugate of saporin toxin. Saporin is a ribosomal inhibiting protein derived from the plant Saponaria officinales, which kills the cell by inhibiting protein synthesis. ITx enters the cancer cell by binding to the cellular marker it is specific for on the cell surface. From there, it is endocytosed, compartmentalized in an endosome, and eventually escapes to the cytosol where its ribosomal target is located. Increasing the rate of escape to the cytosol is the key to increasing cell death. The mechanism by which saporin escapes the endosome and enters the cytosol is poorly understood. Two potential mechanisms involving the rupture of the endocytic vesicle were examined. Through experiments using large unilamellar vesicles as endosomal mimics, we have been able to characterize the mechanism by which saporin works to burst the endosomal membrane through RET and calcein release. Understanding this process is the key to producing more effective immunotoxin sensitizing drugs.