Strategies towards selective functionalization of polycationic amphiphilic cyclodextrins (paCDs): engineering novel non-viral gene carriers by tailoring self-assembling and DNA-condensing capabilities

University of Sevilla Spanish National Research Council Department of Organic Chemistry Institute for Chemical Research Strategies towards Selective Functionalization of Polycationic Amphiphilic Cyclodextrins (paCDs): Engineering Novel Non-viral Gene Carriers by Tailoring Self-assembling and DNA-condensing Capabilities Iris Pflueger 2013 University of Sevilla Spanish National Research Council Department of Organic Chemistry Institute for Chemical Research Strategies towards Selective Functionalization of Polycationic Amphiphilic Cyclodextrins (paCDs): Engineering Novel Non-viral Gene Carriers by Tailoring Self-assembling and DNA-condensing Capabilities Memoria presentada por Iris Pflueger, MSc. para optar al grado de Doctor en Química This work was performed by Iris Pflueger at the Institute for Chemical Research (IIQ, CSIC – University of Sevilla) under the supervision of Dr. Juan M. Benito Hernández, Tenured Scientist at CSIC, and under the tutorship of Dr. Carmen Ortiz Mellet, Professor of Organic Chemistry at the University of Sevilla. This work has not previously been accepted in substance for any degree and is not being concurrently submitted in candidature of any degree. Carmen Ortiz Mellet Juan Manuel Benito Hernández This Thesis is dedicated to my parents for their endless love, support and encouragement. Acknowledgements i Acknowledgements viii Ph.D. Thesis - Iris Pflueger 3. Assessment of self-assembling and pH buffering capabilities of cyclen/cyclam-grafted paCDs in aqueous environment ..................................... 119 4. Assessment of DNA-condensing capabilities of cyclen/cyclam-grafted paCDs . 124 5. Gene transfer capabilities towards COS-7 and HeLa cell lines ......................... 129 Chapter 4 - Solid-phase assisted selective functionalization of multivalent scaffolds. Application to selective monofunctionalization of cyclodextrins ...... 133 1. Strategies towards selective functionalization of cyclodextrins: scope and limitations ............................................................................................................ 134 2. Implementation of the Catch-and-Release concept for regioselective monofunctionalization of multivalent scaffolds ................................................... 143 Conclusions ................................................................................................................... 163 Experimental Part .......................................................................................................... 167 1. General Methods ................................................................................................ 168 1.1. General Methods for Synthesis in Solution Phase ...................................... 170 1.2. General Methods for Solid-Phase Synthesis .............................................. 171 1.3. General Methods for Characterization of paCDs ........................................ 176 1.4. General Methods for Characterization of CDplexes .................................... 178 1.5. Evaluation of the Gene Transfer Capabilities of CDplexes ......................... 182 2. Starting Materials ................................................................................................ 183 3. New Compounds ................................................................................................ 184 3.1. New Compounds synthesized in Chapter 2 ................................................ 184 3.2. New Compounds synthesized in Chapter 3 ................................................ 214 3.3. New Compounds synthesized in Chapter 4 ................................................ 248 References ..................................................................................................................... 261 List of Abbreviations ix List of Abbreviations A A Absorbance A. U. Arbitrary unit AAV Adeno-associated virus Ac Acetyl AD Adamantane AM-PS Aminomethylated polystyrene AMP Adenosine-5'-monophosphate AMS (Aminomethyl)polystyrene anh. anhydrous aq. aqueous ASGPr Asialoglycoprotein receptors ATP Adenosine-5'-triphosphate av. average B BCA Bicinchoninic acid BHK-21 Baby hamster kidney fibroblast cell line Bn Benzyl BNL-CL2 A murine embryonic hepatocyte cell line Boc tert-Butoxycarbonyl bp Base pair BPA Bis(pyridyl)amine bPEI Branched PEI Bu Butyl But Butanoyl C CAC Critical aggregation concentration CD Cyclodextrin x Ph.D. Thesis - Iris Pflueger CDE Clathrin-dependent endocytosis CDP Linear polymers with alternating CD and cationic segments CDplex paCD-DNA complex chol Cholesterol CIE Clathrin-independent endocytosis conc. concentrated COS-7 A fibroblast-like cell line derived from African Green Monkey, Cercopithecus aethiops, kidney tissue, carrying the SV40 genetic material COSY Correlation spectroscopy ctDNA Calf thymus DNA CuAAC Copper-catalyzed azide-alkyne cycloaddition D δ Chemical shift DABCO 1,4-Diazabicyclo[2.2.2]octane DBF Dibenzofulvene DBU 1,8-Diazobicyclo[5.4.0]undec-7-ene DC-Chol 3b-[N-(N',N'-Dimethylaminoethane)-carbamoyl] cholesterol DCM Dichloromethane DETA Diethylenetriamine DIBAL-H Diisobutylaluminium hydride DIC Diisopropylcarbodiimide dios Diosgenin DIPEA Diisopropylethylamine DLS Dynamic light scattering DMAP 4-(Dimethylamino)pyridine DMEM Dulbecco’s modified Eagle’s medium DMF N,N-Dimethylformamide DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid List of Abbreviations xi DOGS Dioctadecylamidoglycylspermine DOPE 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine DOSK Dioleoyl succinyl kanamycin A DOSP Dioleoyl succinyl paromomycin DOSPA 2,3-Dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N- dimethyl-1-propanaminium trifluoroacetate DOTA N 1 ,N 4 ,N 7 ,N 10 -cyclentetraacetic acid DOTAM N 1 ,N 4 ,N 7 ,N 10 -cyclentetraacetic acid amide DOTAP N-[1-(2,3,Dioleoyloxypropyl)]-N,N,N-trimethylammonium chloride DOTMA N-[1-(2,3,Dioleyloxypropyl)]-N,N,N-trimethylammonium chloride DVB Divinylbenzene E EDA Ethylenediamine EDTA Ethylenediaminetetraacetic acid ELS Evaporative light scattering EMA European Medicines Agency ENA Ethylene-bridged nucleic acids eq Equivalent ESI-MS Electrospray ionization mass spectrometry Et Ethyl F FCS Fetal calf serum FDA Food and Drug Administration Fmoc 9-Fluorenylmethoxycarbonyl FTIR Fourier transform infrared spectroscopy H H9c2 Embryonic rat cardiomyoblast cell line HA Haemagglutinin xii Ph.D. Thesis - Iris Pflueger HeLa Human epithelioid cervix carcinoma cell line HEPES 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid HepG2 Human epithelial-like liver hepatocellular carcinoma cell line Hex Hexanoyl HIV Human immunodeficiency virus HMQC Heteronuclear multiple quantum correlation HPβCD Hydroxypropyl-β-cyclodextrin HPLC High performance liquid chromatography I I Intensity IR Infrared K K a Acid dissociation constant L LC Liquid Chromatography LG Leaving group LNA Locked nucleic acid LNP Lipid nanoparticle lPEI Linear PEI LPLD Lipoprotein lipase deficiency LS Light scattering M m/z Mass-to-charge ratio M3-PALS Mixed mode measurement-phase analysis light scattering Me Methyl MeβCD Methyl-β-cyclodextrin MP Macropinocytosis MRI Magnetic resonance imaging MS Mass spectrometry MSNT 1-(2-Mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazol List of Abbreviations xiii µw Microwave N N/P Number of protonable amino groups (N) of the vector per phosphate (P) of DNA NIS N-Iodosuccinimide NMR Nuclear magnetic resonance O OEI Oligoethyleneimine P paCD Polycationic amphiphilic cyclodextrin PAMAM Poly(amidoamine) PBS Phosphate buffered saline PDI Polydispersity index pDMAEMA Poly[2-(dimethylamino)ethyl methacrylate] pDNA Plasmid DNA PEG Polyethylene glycol PEGA Poly[acryloyl-bis(aminopropyl)polyethylene glycol] PEI Polyethyleneimine PET Positron emission tomography PG Protecting group PGAA Polyglycoamidoamine pGaCD Glycosylated paCD Ph Phenyl PL-AMS Aminomethylated polystyrene commercialized by Polymer Laboratories PLL Poly-L-lysine PMO Phosphorodiamidate morpholino oligomer (Morpholinos) PNA Peptide nucleic acid PP i Pyrophosphate Pr Propanoyl xiv Ph.D. Thesis - Iris Pflueger PS Polystyrene PTG Py Pyridine R RAFT Radical addition-fragmentation chain transfer RISC RNA-induced silencing complex RNA Ribonucleic acid RNAi RNA interference RPC Polycationic copper chelator RP-HPLC Reversed phase HPLC rt Room temperature S SAR Structure-activity relationship SCID Severe combined immunodeficiency SEC Size exclusion chromatography shRNA Small hairpin RNA siRNA Small interfering RNA SPECT Single photon emission computed tomography T t ½ Reaction half-time TACN 1,4,7-Triazacyclononane TAE Buffer containing Tris, acetic acid and EDTA TBTA Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine TBTU O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate TCR T-cell receptor TEM Transmission electron microscopy Tf Transferrin TFA Trifluoroacetic acid THF Tetrahydrofuran List of Abbreviations xv TLC Thin layer chromatography TOCSY Total correlation spectroscopy TPP Triphenylphosphine Tr Trityl t r Retention time TREN Tris(2-aminoethyl)amine Tris 2-Amino-2-hydroxymethyl-propane-1,3-diol Ts Tosyl U UV Ultraviolet C HAPTER 1 - G ENERAL I NTRODUCTION 8 Ph.D. Thesis - Iris Pflueger 2. Viral vs. Non-viral Gene Carriers The simplest way of gene delivery consists on direct treatment of the target with naked nucleic acids, but due to their low bioavailability, a number of techniques have been developed. These can be roughly classified into two main groups: physical methods and carrier-assisted ones. The formers exploit mechanical or electric stimuli to promote uptake of the therapeutic nucleic acid into the target cell or tissue. Physical methods include microinjection, 25 bio-ballistic transfection (gene gun), 26 electroporation, 27 ultrasounds, or pressure gradients, 28 among others (Figure 4). 29 Evidently, the nature of most of these techniques makes them quite unsound for in vivo application, though they have demonstrated certain success ex vivo. 30 Figure 4. Physical methods used in gene therapy. 2.1. Viral-based gene carriers Vector-assisted techniques, conversely, aim at mimicking the capabilities of natural viruses to transport nucleic acids into different targets. A successful vector should (a) Chapter 1 9 provide nucleic acid protection, (b) enable cellular uptake of its payload and delivery to its subcellular target, and (c) promote its biological action (e.g. gene transcription). Along millions of years, viruses have evolved the most fascinating mechanisms to fulfill these tasks and their capabilities were immediately recognized at the nascence of the gene therapy paradigm. Indeed, it is not surprising the recombinant viruses (devoid of replicating capabilities) were the first nucleic acid carrier candidates investigated to deliver nucleic acids at the right spot. 31,32 Unfortunately, engineering of non-replicating viruses still is nowadays an unmet challenge. Only relatively simple viruses such as retroviruses, 33 adenoviruses, 34 adeno-associated viruses (AAV), 35 the poxvirus, 36 the vaccinia virus, 37 or the simplex herpes 38 can be reliably modified (Figure 5). Figure 5. Some viral vectors used in gene therapy. Despite therapeutically relevant levels of gene expression have been achieved in different organs such as kidney, 39 ovary, 40 eye, 41 and muscle tissues, 42 and over 70% of ongoing gene therapy clinical trials (approximately 1500, Figure 6) are assessing the performance of viral vectors, serious risks and limitations still exist concerning the broad applicability. 43 On one hand, viral vectors are potentially capable of recombining with replication-competent viruses. On the other, they can also induce immunogenic or inflammatory response, or eventually, undesired insertion on the host genome. 43,44 10 Ph.D. Thesis - Iris Pflueger Moreover, large scale manufacturing of even the simplest viral carriers still is far from obvious. In fact, due to these drawbacks, only one viral-vector-based gene therapy treatment has received so far the green light from the European Medicines Agency (EMA): Glybera® (UniQure), an AAV vector encoding the human lipoprotein lipase gene indicated for the treatment of lipoprotein lipase deficiency (LPLD), a very rare inherited condition that is associated with pancreatic inflammation. 45 The FDA has not approved any formulation yet. The more relaxed Chinese authorities granted Shenzhen SiBionoGenTech the use of Gendicine, a modified adenovirus vector encoding the p53 tumor suppressor gene for treating head and neck cancer in 2003. 46 Two years later, Sunway Biotech gained approval in China for H101, a recombinant oncolytic adenovirus that targets p53-deficient tumor cells. 47 Western authorities questioned both decisions due to a lack of available information on the two therapies. 48 Figure 6. Vectors used in gene therapy clinical trials. 7 Very recently, Naldini and co-workers published the results of two clinical trials using lentiviral vectors for stem cell gene therapy in patients with Wiskott-Aldrich Syndrome, 49 an inherited disease that disables the immune system, and a methachromatic leukodystrophy, 50 a metabolic disease. Three years after the start of the clinical trials, disease progression stopped in all six patients and some of them showed no symptoms for 18 to 32 month following the therapy. 51 Even after fairly long-term follow up, the Adenovirus 23.2% (n = 453) Retrovirus 19.4% (n = 378) Naked/Plasmid DNA 18% (n = 351) Vaccinia virus 7.8% (n = 152) Lipofection 5.7% (n = 112) Adeno-associated virus 5.1% (n = 99) Poxvirus 4.9% (n = 95) Lentivirus 3.2% (n = 62) Herpes simplex virus 3.1% (n = 60) Other categories 6.3% (n = 123) Unknown 3.3% (n = 64) Chapter 1 11 therapy appears to be safe and effective, as the risk of the corrected gene inserting itself next to the cancer-activating one could be reduced. 52 However, despite the progress achieved in the treatment of challenging diseases such as muscular dystrophy, 42 HIV, 53 or cancer, 54 (two thirds of investigational gene therapies are focused at cancer treatment), gene therapy is immature and clinical success is still distant. 2.2. Non-viral gene carriers Alternatively, non-viral-based vectors have gathered momentum. In the era of nanotechnologies, synthetic materials can be designed and constructed using bottom-up approaches to tailor the desired functional features in terms of bioavailability and biocompatibility, nucleic acid complexation, protecting and packing capabilities, or membrane-crossing and targeting abilities. As the understanding of the self-assembling processes involved in aggregate formation between nucleic acids and synthetic vectors increases, the range of designed biomaterials for this purpose open wider (literally hundreds). 55,56 Most of these non-viral nucleic acid vectors fall within the category of cationic lipids 57 or polymers 58 (Figure 7 and Figure 8, respectively), featuring functional groups that electrostatically neutralize nucleic acids and cooperatively promote compaction into colloidal nanoparticles (termed lipo- and polyplexes, respectively) with increased metabolic stability and membrane permeability. Nanoparticle stability and transfection efficiency largely depend on the structural features of the cationic vector, the type of nucleic acid to be delivered and the relative formulation ratio. 12 Ph.D. Thesis - Iris Pflueger Figure 7. Illustrative examples of cationic lipids used as non-viral vectors. Non-viral vectors are, in principle, less prone to elicit the immune system response. Moreover, they are more flexible than viral particles regarding the type and size of the payload and far easier to produce. However, they still may trigger inflammatory response and their delivery efficiency and selectivity, despite few exceptions, 2 are far from that of their viral counterparts. This is not surprising considering that most of the first generation non-viral candidates were not purposely conceived for such task, but just off-the-shelf materials, such as poly- L -lysine (PLL) or polyethylenenimine (PEI), for which gene delivery capabilities were incidentally discovered. Their densely charged nature does not usually furnish them with the optimal biocompatibility and cytotoxicity profiles. 59 Chapter 1 13 Figure 8. Illustrative examples of cationic polymers used as non-viral vectors. In the last decade, the efforts in field have been focused on furnishing these off-the- self non-viral carriers with the functional elements that viruses exploit to overcome the physiological barriers to improve their inherently poor efficiency. That is, elaborating artificial viruses (Figure 9). 58b,60 Either covalent or supramolecular chemical manipulation of these carriers has been implemented to interfere in and improve their performance in several folds: 61 • improving nucleic acid condensation and protection in the extracellular milieu, 62 • enhancing cellular uptake by specific mechanisms by, for instance, grafting cell surface receptor complementary ligands, 63 • promoting endosome release by including membrane destabilizing agents 64 or pH-sensitive architectures, 65 • exploiting intracellular active transport machineries to selectively move through the cytoplasm, 66 • targeting nuclear membrane pores by using nuclear localization antennae, 67 14 Ph.D. Thesis - Iris Pflueger • or improving biocompatibility and bioavailability (eventually also biodegradability) in order to prevent interferences with the immune system or undesired unspecific interactions in the biological milieu 68 by, for instance, shielding carriers with polyethylene glycol (PEG) chains. 69,70 Figure 9. Schematic representation of the components of an artificial virus for nucleic acid delivery. Cationic lipids are amphiphilic molecules consisting on a charged head group linked to a hydrophobic domain that self-assemble in aqueous media in the presence of nucleic acids into more or less defined liposomes. Their use in non-viral gene delivery dates back from 1987, when Felgner described an improvement of two orders of magnitude in gene expression efficiency by using DOTMA (Figure 7) as compared to other non-viral carriers. 71 This milestone inspired a number of contributions focused on assessing structure-activity relationships (SAR). 72 Nucleic acid condensation by cationic lipids is supposed to be a two-step process initiated by an initial electrostatic interaction between cationic heads and the anionic polyphosphate backbone. Then, entropy-driven desolvation by clustering of the hydrophobic domains produces aggregate compaction. It has been suggested that, once internalized into cells, cationic lipids in lipoplexes interact with anionic lipids of the endosome, causing destabilization of the endosomal membrane (Figure 10). 73 Chapter 1 15 Figure 10. Illustration of the proposed mechanism for endosomal escape of cationic lipids. 74 The length of the lipophilic tail(s), nature of the cationic head group (size and charge), the linker between both domains, or their susceptibility to changes in the biological media have been shown to exert major roles in their performance. 75 Several generations of cationic lipids have evolved improving the performance of the formers. For instance, N-[1- (2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate (DOTAP, Figure 7) has been successfully applied to correct lung endothelial dysfunction via antisense oligodeoxynucleotide delivery in mice. 76 Huang have shown that the use of cholesterolbased cationic lipids feature reduced toxicity, allowing them to be the first cationic lipids entering clinical trials in humans. 77 The well-defined molecular structure of cationic lipids has allowed the rational fine-tuning of their self-assembling capabilities. As an illustrative case, Behr combined the enhanced nucleotide binding ability of polyamines within the cationic lipid framework in dioctadecylamido-glycylspermine (DOGS, Figure 7). 78 The improved gene transfer capability of DOGS was attributed to the enhanced DNA compaction and protection achieved by multipoint cationic lipid-nucleic acid interaction. 79 16 Ph.D. Thesis - Iris Pflueger Figure 11. Example of a neutral lipopolythiourea described by Herscovici and Leblond. 80 Figure 12. Structure of some lipidic aminoglycoside derivatives (dioleylsuccinyl kanamycin A, DOSK, and dioleylsuccinylparomomycin, DOSP) and a representative Cryo-TEM micrograph of their siRNA complexes (Reproduced from ref. 81b). Other synthetic alternatives have been reported to modulate cationic lipid-nucleic acid condensation. For instance, though the vector-nucleic acid interaction in lipoplexes is essentially electrostatic, recently Herscovici and Leblond 80 have shown that neutral lipopolythioureas (Figure 11) may promote DNA condensation and compaction by cooperative phosphate-thiourea H-bonding and hydrophobic clustering. Probably due to their neutral character, these series of carriers exhibit remarkably low toxicity, though their gene transfer efficiency is comparatively lower than that reported for structurally related cationic lipids. 81 The diversity of cationic lipids architecture has been further extended exploiting nucleic acid binding epitopes such as complementary Chapter 1 17 oligonucleotides, 82 peptides 83 or carbohydrates. 81a As illustrative example, Pitard and coworkers have designed a series of cationic lipids inspired in the structure of RNA-binding aminoglycosides (Figure 12), their lipoplexes exhibiting enhanced colloidal stability as compared with conventional cationic lipids. Remarkably, lipoplex particle features and performance could be related to lipid aminoglycoside structure and siRNA binding capabilities, finely illustrating the potential of rational design in nucleic acid delivery. 81b In a seminal contribution, Zuber and Behr designed a novel family of environmental sensitive non-viral vectors consisting undimerizable cationic lipids programmed to protect more efficiently the nucleic acid payload in the extracellular milieu while facilitating intracellular release (Figure 13). Once formulated with DNA, their thiolated lipids undergo air-mediated oxidation producing more stable lipoplexes. In the glutathione-rich intracellular environment, disulfide tethers are reduced, facilitating plasmid release. 84 Figure 13. Dimerizable polycationic lipids reported by Zuber and co-workers. 84 Cationic polymers, conversely, have been around much longer. Their nucleic acid binding capabilities were already noticed in the 70s’, 85 but it was not until Behr reported the outstanding gene carrier features polyethyleneimine (PEI, Figure 8) 86 that their development was initiated. Upon formulation with nucleic acids, cationic polymers form positively charged complexes (polyplexes) by purely electrostatic interactions. Cationic 24 Ph.D. Thesis - Iris Pflueger intracellular barriers for gene delivery. 123 In a number of cases, lactosylated carriers appeared to induce faster escape from the late endosome/lysosome than other glycosylated counterparts and more efficiently mediated nuclear import. 124 Though these observations are still a matter of debate, recent publications by the same group 125 and others 126 highlight the potential benefits of controlling of intracellular trafficking as a prerequisite for efficient non-viral gene delivery. 3.2. De novo designed carbohydrate polymers Efficiency and toxicity of polymeric vectors can be largely (and unexpectedly) affected by subtle structural changes, for example in counterion nature, 127 branching degree 128 or size, 129 which may lead even by batch-to-batch disparities. This undesired variability has been overcome to a large extent by the implementation of novel polymerization techniques, such as controlled polycondensation, radical polymerization and click chemistry reactions, which offer superior control over functional features. 130 Through the judicious choice of monomer structure and polymerization strategies a number of relevant parameters regarding gene delivery capabilities can be tailored. In this context, the de novo design of polymeric gene carriers from carbohydrate-derived building blocks has been very appealing, given the structural diversity and availability of simple saccharides. This section surveys a number of examples that illustrate the nucleic acid delivery capabilities of synthetic carbohydrate-based polymers. One of the most representative examples of the potential of synthetic glycopolymers at tailoring non-viral gene vectors is the case of polyglycoamidoamines (PGAAs). 131 The first insight into the gene delivery capabilities of PGAAs were almost simultaneously reported by Reineke 132 and Guan 133 (Figure 16). In both cases the diversity, availability and biocompatibility of carbohydrates and their readily conversion to polymerizable building blocks was the main motivation. Reineke and co-workers focused on imitating the structure of linear PEI polymers by co-polymerizing their carbohydrate building blocks with a set of short oligoethyleneimine (OEI) chains. Guan’s design aimed at mimicking Chapter 1 25 the structure of poly- L -lysine (PLL) by condensing carbohydrate bricks with lysine segments. Both studies revealed significantly reduced cytotoxicity of the resulting PGAAs as compared to PEI and PLL, respectively, while maintained comparable pDNA transfection efficiencies. Figure 16. Schematic representation of the synthesis of polyglycoamidoamines (A) and building blocks used by Reineke’s 132 (B) and Guan’s 133 (C) groups. Reineke and co-workers have thoroughly analyzed the influence of architectural issues of their PEI-inspired PGAAs on nucleic acid complexation, nanoparticle topology and stability, cell uptake, toxicity and gene expression efficiency. 134 By screening a library of cationic PGAAs, synthesized from a range of linear OEIs and esterified aldaric acid and lactone co-monomers (Figure 17), they identified vector candidates that mediated high gene expression levels in several cell lines (HeLa, H9c2(2-1), HepG2, or BHK-21). pDNA transfer efficiency peaked for the polymers containing the longer OEI chains tested. 135 Transfection efficiency compares favorably to that achieved with PEI, but with a less toxic profile. They hypothesized that the iterative interruption of OEI charge density 26 Ph.D. Thesis - Iris Pflueger by carbohydrate blocks was at the origin of the reduced toxicity as compared to PEI, though their rapid hydrolysis in biological media might also account for this merit. 136 O N H O A O OH MeO OH H N OH OH O B N H N H OHO OH OH N H OHO OH OH H N N Hn av. 11-12 H O OH MeO OH H N OH OH OH N N Hn av. 11-14 H D1, n = 1 D2, n = 2 D3, n = 3 D4, n = 4 M1, n = 1 M2, n = 2 M3, n = 3 M4, n = 4 O OH MeO OH H N OH OH OH N N Hn av. 11-14 H O MeO OH N OH OH H N N Hn av. 11-12 H G1, n = 1 G2, n = 2 G3, n = 3 G4, n = 4 T1, n = 1 T2, n = 2 T3, n = 3 T4, n = 4 glucarate-based PGAAs mannarate-based PGAAs galacarate-based PGAAs tartrate-based PGAAs N H N H H N H N av. 12-14 H DS, = glucarate TS, = tartrate spermine-based PGAAs O O MeO OH OH Figure 17. General structure of PGAAs studied by Reineke and co-workers (A) with indication of their structural diversity (B). PGAAs with longer OEI segments did not significantly influenced cell uptake and overall transgene expression, but significantly improved polyplex stability in serumcontaining media. Unfortunately, increased toxicity was also observed. 137 Polymers with longer inter-amine spacers (e.g. spermine) also exhibited increased toxicity, highlighting a critical relationship between charge density distribution and biocompatibility. 138 The nature of the saccharide segment, though modestly, also influenced polymer performance, apparently by altering the buffering capabilities of the system. Since the response of polyplexes at the increase of pH in the endosomes closely relates to the pDNA release dynamics, it should be expected certain folding differences upon pDNA- Chapter 1 27 PGAA complex formation. Overall, PGAA G4, combining pentaethylenehexaamine and galactarate segments (Figure 17), was identified as the best performing candidate. PGAA G4, shown to induce long lasting transgene expression (over 7 days) without appreciable cytotoxicity, 139 was recently licensed to Techulon to be commercialized as Glycofect TM . 140 Scheme 2. Synthesis of trehalose-based cationic “click” polymers. 142 Alternatively, carbohydrate-based cationic polymers with amidine 141 or triazole linking tethers 142 have also been explored. Though polyamidines usually exhibit severe cytotoxicity, Reineke and Davis observed that this could be overcome by intercalating saccharidic moieties (e.g. trehalose) between the cationic elements while maintaining the gene transfer capabilities. 141 The closer the amidine and the carbohydrate units are located in the polymer chain, the more efficiently the cytotoxicity is reduced. Trehalose, moreover, seems to impart additional favorable properties to polymeric gene vectors, such as shielding aggregation of the resulting polyplexes with proteins and lipids. 143 To fully benefit from this, Reineke and co-workers have devised a strategy to build a new family of cationic trehalose-embedding polymers that exploits the Cu(I)-catalyzed azidealkyne cycloaddition (CuAAC) reaction. 144 By coupling diazido-functionalized trehalose units with a diverse set of α,ω-dipropargylated OEIs (Scheme 2), a series of polymers were obtained that allowed the systematic investigation of architectural effects (OEI size 28 Ph.D. Thesis - Iris Pflueger and polymer length) on pDNA complexation, nanoparticle properties, cellular uptake and transgene expression. Transfection efficiencies that paralleled that of JetPEI-based polyplexes were achieved using trehalose “click” polymers with 3-4 ethylenediamine segments. 142a Dynamic light scattering (DLS) revealed that polyplexes formulated with larger polymers exhibited the best tolerance to serum-containing media, but displayed higher toxicity. 142b This unfavorable feature was alleviated by the fact that “click” polymers required far lower N/P ratios, as compared to PGAAs, to achieve similar gene expression levels. Scheme 3. Schematic synthesis of statistical vs. diblock copolymers (A) illustrated with the cationic glycopolymers synthesized by Ahmed and Narain (B). 146 Chapter 1 29 Ahmed and Narain have recently used radical addition-fragmentation chain transfer (RAFT) polymerization to synthesize a library of carbohydrate-pendant cationic polymers, where carbohydrate and cationic units can be either statistically distributed (random polymers) or segregated into separate blocks (diblock polymers, Scheme 3). 145 Precise control on chain length, monomer ratio and polydispersity allowed the authors to rationalize the large influence that polymer architecture exerts on gene delivery capabilities. Statistical copolymers with high molecular weight produced superior gene expression with lower toxicity as compared to the corresponding diblock copolymers, both in the presence or absence of serum. 146 On the other hand, diblock copolymers resulted in nanoparticles that were shielded against non-specific interactions in a similar way of pegylated cationic polymers. However, current examples of diblock glycopolymers do not perform as other synthetic glycopolymers, the control over polymer structure open new opportunities in the field of non-viral gene delivery. Targeted delivery for instance, where non-specific interactions have to be shielded at the same time that specific uptake is promoted, might be one of those channels. 3.3. Cyclodextrins in gene delivery The above evidences point to significant advantages of using non-viral carriers that include carbohydrates in their structure. 102 Indeed this is not just restricted to naturally occurring carbohydrate polymers or glycocoated cationic vectors. In the last decade, a growing awareness has been gathered on the remarkable features of cyclodextrins (CDs) for such purpose. First identified from bacterial digests of starch by Villiers 147 at the fall of 19 th century and latter isolated by Schardinger during the first decades of the past century, 148 CDs largely remained as chemical curiosities for over half a century. By the mid-20 th century, Freudenberg and co-workers unveiled their macrocyclic structure, composed of α(1→4)-linked glucopyranose units featuring a basket-shaped topology in which glucose hydroxyls orient to the outer space flanking the upper and lower rims, while methinic protons (H-5 and H-3) point to the inner cavity (Figure 18), 149 and reported 30 Ph.D. Thesis - Iris Pflueger on their ability to form inclusion complexes with hydrophobic guests. 150 This observation represented a milestone that certainly switched the course of CD research history and steered it for the second half to the century. Indeed, the academic and (bio)technological interest for cyclodextrins has been dominated by this unique feature. 151 Figure 18. General structure of cyclodextrins (CDs). CDs have long occupied a prominent position in most pharmaceutical laboratories as “off-the-shelve” tools to manipulate the pharmacokinetics and dynamics of a broad range of active principles (e.g. drugs). 152 But a body of evidence also indicates that CDs might be useful for formulation improvement on biomacromolecule delivery in general 153 and in gene therapy in particular. 1,154 In the following paragraphs, selected examples are described to illustrate the significant contribution of CDs to the field of non-viral-mediated gene therapy. The potential of native CDs as transfection enhancers, ascribed at least in part to their ability to host cell membrane components such as cholesterol in their hydrophobic cavity, has been long known and exploited to improve the gene delivery capabilities of polycationic lipidic or polymeric non-viral vectors. 155 However, the major breakthrough in the field was reported by Davis and co-workers. They conceived a class of cationic polymers based on the controlled condensation of bifunctional CD monomers, such as bis-(C-6)-cysteaminylated βCD, and cationic co-monomers, in order to yield linear chains with alternating CD and cationic segments (CDP, A in Figure 19). 2,156 Electrostatically- Chapter 1 31 driven complexation of the resulting cationic CDPs and negatively charged pDNA (~5 kpb) rendered nanometric complexes (polyCDplexes; 100-150 nm) featuring in vitro cell transfection efficiency comparable to that obtained with PEI and Lipofectamine™, while preserving a reduced toxicity. Figure 19. Schematic representation of the transferrin-targeted nucleic acid-CDP nanoparticles (RONDEL™) developed by Davis and co-workers. 2 This milestone was followed by a series of reports in which the effect of structural modifications of CDPs on the gene delivery capabilities were investigated. 141,157 Most interestingly, supramolecular host-guest CD chemistry could be put forward to further decorate the nanoparticle surface. Thus, inclusion of the adamantane (AD) moiety of ADPEG conjugates into CD cavities of polyCDplexes could prevent from non-specific 32 Ph.D. Thesis - Iris Pflueger interactions with biological components 158 and also biorecognizable ligands could be installed at the distal end of these PEG chains to target the polyCDplexes to specific tissues (B in Figure 19). 159 Using transferrin (Tf) as the peripheral ligand, whose receptor is known to be up-regulated in malignant cells, the resulting polyCDplexes were shown to selectively target and efficiently transfect different tumor tissues in vivo in mammals (murine 160 and primates). 161 This tripartite therapeutic concept (CDP as nucleic acid complexing element/ AD-PEG as surface shielding element / AD-PEG-Tf as targeting ligand) is named RONDEL™. In June 2008, CALAA-01 (Arrowhead Research), a version of RONDEL™ loaded with a specific oligonucleotide sequence (siRNA) that inhibits tumor growth via RNA interference (RNAi), entered phase I clinical trials to determine its safety and efficacy towards solid tumors refractory to standard-of-cure therapies. 162 Figure 20. Examples of molecularly well-defined non-amphiphilic polycationic CDs assayed as gene vectors. At the light of these thrilling results, research on polycationic polymeric gene vectors incorporating CDs has flourished, including linear, 163 dendritic, 119,121a,164 star-shaped, 165 or rotaxanized structures. 166 However, despite a significant success, the above systems still suffer from the inconveniences associated to polydispersity. Alternatively, attempts to overcome this limitation were made designing monodisperse polycationic clusters taking advantage of the molecular scaffolding capabilities of CDs. Using strategies for the selective chemical manipulation of CD topology, O’Driscoll and Darcy reported the first examples of monodisperse cyclodextrin-centered polycationic clusters intended for DNA complexation (A in Figure 20) 167 and demonstrated their ability to promote transfection in Chapter 1 33 vitro. Their work inspired other architectural proposals to enhance nucleic acid binding (e.g. guanidinylated, 168 oligoethyleneimine-grafted, 169 and histidine/lysine-decorated 170 CDs, B, C and D in Figure 20, respectively). The highly hydrophilic character of these materials does not help self-organization of the monomers at the surface of the oligonucleotide chain, however. Consequently, relatively large excesses are necessary to achieve full protection from the environment. Notwithstanding, some of the resulting CD- nucleic acid complexes (CDplexes) exhibited remarkable transfection capabilities. The above examples illustrate the suitability of CDs as molecular platforms for the incorporation of cationic elements with a precise spatial orientation and the potential of the resulting monodisperse polycations as gene vectors. Most interestingly, the rim anisotropy of the basket-shaped CD structure further allows accessing compounds with segregated cationic and lipophilic domains for which, according to the ”facial amphiphilicity” concept, 171 biomimetic self-assembly and gene delivery properties could be expected. Darcy and co-workers first tested this hypothesis by preparing heterogeneous βCD derivatives bearing thioalkyl chains at the primary rim and amineterminated oligoethylene branches of variable lengths at the secondary hydroxyl positions (A in Figure 21). The resulting polycationic amphiphilic CDs (paCDs) were shown to entrap pDNA by forming CDplexes that transfected COS-7 and human hepatocellular liver carcinoma (HepG2) cells with efficiencies and toxicity profiles comparable to Lipofectamine 2000™-based lipoplexes. 172 Figure 21. Examples of amphiphilic polycationic CDs assayed as gene vectors. 40 Ph.D. Thesis - Iris Pflueger 1. Structure-activity relationships in the rational design of novel gene carriers A prerequisite to better understanding and consequently improving transfection efficiency of non-viral gene delivery systems is to get a deeper insight into how nucleic acid-carrier interactions and assemblies are established, the cell uptake, endosomal escaping and intracellular trafficking mechanisms involved in the process. Hence, elucidation of structure-activity relationships (SARs) is a crucial parameter for the rational design of efficient novel gene carriers. Structure-activity correlations for DNA carrier systems are difficult to establish due to intricate cellular events involved in the multistep transfection process. Generally, there is a great difference between in vitro and in vivo experiments. Furthermore, different cell lines behave very differently when subjected to the same series of transfection experiments with a certain vector. Another difficulty is derived from the essentially polydisperse nature of many of the most prominent non-viral gene carriers, which considerably hampers inferring structure–activity links. Although there are exceptional cases, such as CD-based polymers described by Davis,2 this is a problem that breaks down many promising designs. To overcome these drawbacks, monodisperse multifunctional molecular vectors have emerged as a new generation of gene delivery systems. Chemical tailoring and systematic structural modification facilitate a deeper insight into their structure-activity relationship. As illustrated in the preceding chapter, preorganized macrocyclic structures such as CDs could be very helpful for this task, since they can be furnished with functional elements endowing them with self-assembling capabilities in the presence of nucleic acids. In our laboratories, we have succeeded to produce a series of amphiphilic CDs to investigate the role of structural features such as architectural design (skirt 174,180b vs. jellyfish like), 178 CD scaffold size, 179 density and display of the cationic cluster, 177 type and length of the connecting tethers (Figure 23), 180a and the presence of selective targeting Chapter 2 41 elements 183 on their self-assembling, cellular uptake, 182 and transgene expressing capabilities have been assessed in vitro and in vivo. 181 Figure 23. paCDs under investigation in SAR studies with indication of the modified structural elements. SAR analysis on these families taught that the “skirt”-type arrangement in combination with multiple aminoethylthiourea segments presented advantages in terms of synthetic straightforwardness and architectural flexibility, and moreover, resulted in improved gene transfer efficiencies. However, there remain a number of questions to be addressed, in special those regarding the influence of paCD structure on CDplex formation and dissociation. Towards this goal, in this Ph.D. Thesis a series of monodisperse paCDs with a precise arrangement of functional elements are prepared and used to insight on how their structural features determine their physicochemical properties and, ultimately, whether these features can be related to their DNA delivery capabilities. In particular, this chapter comprises: • the synthesis of library of paCDs featuring subtle differences in their hydrophilic/hydrophobic balance, • the assessment of self-assembling capabilities in aqueous environment by means of critical aggregation concentration measurements, 42 Ph.D. Thesis - Iris Pflueger • the determination of pH buffering capabilities of paCD aggregates, • the assessment of DNA-paCD complex (CDplex) formation and dissociation dynamics, • and the determination of their pDNA delivery capabilities towards COS-7 cells. Chapter 2 43 2. Synthesis of paCDs differing in their hydrophilic/hydrophobic balance Exploiting in-house-developed synthetic methodologies, a semi-convergent, modular strategy towards C 7 -symmetric βCD derivatives differing in their hydrophobic/hydrophilic balance has been implemented for the preparation of two sub-collections of paCDs, namely (i) the linear heptacationic family and (ii) the branched 21-cationic subset, respectively, each of them containing members differing in secondary rim acyl chain lengths (Figure 24). Figure 24. Structure of polycationic CD library members. The first part of the synthetic pathway comprised the incorporation of a cysteaminyl segment into the primary βCD face and acylation of the fourteen secondary hydroxyl 44 Ph.D. Thesis - Iris Pflueger groups by employing different acid anhydrides as reagents. In principle, two alternative routes can be presented, differing in the order of functionalization of the two βCD faces (Scheme 5). Although both can provide polycationic amphiphilic paCDs 18-21 in only four steps from commercial βCD, they differ significantly in their performance and efficiency. Route A Route B Scheme 5. Synthesis of compounds 18-21 (1 st generation paCDs). Reagents and conditions: a) hexanoic anhydride, DMAP, Py, 16 h, 72%; b) BocHN(CH 2 ) 2 SH, Cs 2 CO 3 , DMF, 60 ºC, 48 h, 70%; c) BocHN(CH 2 ) 2 SH, Cs 2 CO 3 , DMF, 60 ºC, 48 h, 99%; d) 1:1 Ac 2 O-Py, rt, 15 h (for R = CH 3 ),80%; propionic anhydride (for R = CH 2 CH 3 ), butanoic anhydride (for R = CH 2 CH 2 CH 3 ), or hexanoic anhydride (for R = (CH 2 ) 4 CH 3 ), DMAP, DMF, 70 ºC, 24 h, 73-78%; c) e) i, 1:1 TFA-DCM, 2 h; ii, HCl, 99%. The synthesis of CD derivative 21 was explored using both synthetic pathways. Starting from heptakis(6-bromo-6-deoxy)cyclomaltoheptaose 11, obtained from commercial βCD according to the procedure described by Defaye, 175,184 acylation of the secondary hydroxyl groups using hexanoic anhydride and DMAP was accomplished. When DMF was used as solvent for the acylation the reaction proceeded slowly. Increasing the temperature, in turn, led to concomitant nucleophilic substitution of the Chapter 2 45 halide at C-6. This drawback could be overcome by using pyridine instead, which allowed for a complete acylation of secondary hydroxyl groups without formation of side products (Scheme 5, route A). Subsequent functionalization of the primary face by nucleophilic substitution of halides with N-Boc-protected cysteamine in presence of cesium carbonate was achieved in good yield, although requiring purification by silica gel column chromatography. On the other hand, compound 21 could also be obtained by reversing the order of functionalization of the two CD faces (Scheme 5, route B). Nucleophilic substitution of the halide on the primary face of 11 by N-Boc-protected cysteamine was carried out to yield derivative 13 in virtually quantitative yield. Subsequent acylation of the secondary hydroxyl groups in the presence of DMAP was accomplished in DMF to smoothly furnish 17 in 73% yield. This alternative synthetic route presents considerable advantages, since it reveals a more convergent character and requires only a single chromatographic purification step, i.e. after acylation of secondary hydroxyl groups. Together with its better overall performance these factors clearly privilege this synthetic pathway. Consequently, CD derivatives 18-20 were prepared exclusively by this route. Under these reactions conditions using the corresponding acid anhydrides, i.e. propanoic anhydride (15, 73%) or butanoic anhydride (16, 78%), the formation of side products ascribed to over-acylation by the reaction of the acylating agent with carbamic nitrogen atoms was not observed in any case. Acetyls were introduced using standard conditions (1:1 mixture of Ac 2 O-Py). After purification by silica gel column chromatography, homogeneity of all acylated CD derivatives was confirmed by NMR, ESI-MS and combustion analysis (see Figures S1-S4 for NMR spectra and Figures S81- S84 for MS spectra, Supporting Information). Finally, acid hydrolysis of the carbamate groups by treatment with 1:1 TFA-DCM led to paCDs 18-21 in quantitative yield. The reason for choosing a 2-aminoethanothiol (cysteamine) segment to link the CD core with the cationic moieties is not trivial. Previous results of our research group have shown that the nucleophilicity of the amino group in βCD derivatives is significantly increased after insertion of the 3-atom (SCH 2 CH 2 ) spacer in between the nitrogen atom and the CD core, 185 which in turn should facilitate interactions with phosphate anions in 46 Ph.D. Thesis - Iris Pflueger the nucleic acid backbone. Moreover, distancing the amino groups from the CD core reduces steric hindrance that may limit reactivity with bulky electrophiles. Previous insights into paCD SAR led to the identification of the 2-aminoethylthiourea segment as a privileged phosphate-binding motif. 177 This structural element has been preserved in the architectural diversification of paCDs 18-21. Thus, two isothiocyanate synthons (22 186 and 23, 177 Figure 25) have been coupled to each CD derivative (18-21) to furnish the linear or branched paCD subsets, respectively. NHBoc N BocHN NCS BocHN NCS 22 23 Figure 25. Isothiocyanate synthons used for the elaboration of the paCD collection. O O O S O NH3Cl 7 R R O O R = CH3,18 R = CH2CH3,19 R = CH2CH2CH3,20 R = CH2(CH2)3CH3,21 O O O S O N H 7 R R O O N H NHX S O O O S O N H 7 R R O O N H N S NHX NHX R = CH324 1 R = CH2CH325 2 R = CH2CH2CH326 3 R = CH2(CH2)3CH327 4 R = CH328 5 R = CH2CH329 6 R = CH2CH2CH330 7 R = CH2(CH2)3CH331 8 b a c X = Boc X = H·HCl c X = Boc X = H·HCl Scheme 6. Synthesis of thioureido paCDs 1-8. Reagents and conditions: a) 22, Et 3 N, DCM, 15 h, 75-94%; b) 23, Et 3 N, DCM, 20 h, 62-95%; c) i, anh. TFA, 5 min; ii, HCl, 99%. Chapter 2 47 As shown in Scheme 6, Et 3 N-catalyzed coupling of isothiocyanates 22 and 23 to polyamino compounds 18-21 in DCM led to the corresponding N-Boc protected linear poly(aminoethylthioureido) CD derivatives (24, 94%; 25, 75%; 26, 84% and 27, 81%) as well as to their respective branched analogs (28, 95%; 29, 62%; 30, 74% and 31, 71%), respectively. In all cases the thiourea formation reaction was completed in a relatively short time (2 to 3 h), as evidenced by TLC and ESI-MS (see Figures S89- S96 for MS spectra, Supporting Information) of the reaction mixture, provided pH was maintained at 8-9. The formation of products from isothiocyanate self-condensation, typically associated with long reaction times or elevated temperatures, was not observed. 187 All N-Boc protected products were purified by conventional silica gel column chromatography and were obtained in remarkable final yields (62-95%) keeping in mind that coupling reactions should take place at seven positions simultaneously. Scheme 7. Synthesis of polycationic thioureido CDs 9 and 10. Reagents and conditions: a) 22 (33), 23 (34), 1:1 Py-H 2 O, 24 h; b) 24 (33), 28 (34), NaOMe, MeOH, 14 h, 98% (33), 97% (34); c) i, anh. TFA, 5 min; ii, HCl, quantitative yield in both cases. In addition to this set of compounds, the non-amphiphilic CD analogs (lacking the secondary rim acyl chains) have been synthesized. In principle, chemoselectivity of the thiourea formation reaction should allow performing the coupling without the need of protecting the secondary hydroxyls, as previously described. However, when CD derivative 32 185a was coupled to the isothiocyanates 22 and 23, products resulting from partial addition precipitated from the reaction mixture, thus preventing reaction 48 Ph.D. Thesis - Iris Pflueger completion. Alternatively, advantage was taken of CD derivative 24 prepared previously, which exhibits acetyl protected hydroxyl groups on the secondary face of the CD core and hence organic solvent solubility (Scheme 7). Zemplén transesterification of the acetyls gave N-Boc protected CD derivative 33 in excellent yield (98%). The synthesis of the corresponding branched polythioureido CD derivative 34 was performed in an analogous manner, as shown in Scheme 7. Although all other synthetic steps to obtain the collection of linear and branched polycationic amphiphilic CDs as well as their two analogs without acyl chains on the secondary face furnished the target compounds with remarkable yields and purities, final acidic hydrolysis of the carbamate protecting groups of compounds 24-31, 33 and 34 proved to be a delicate operation. In particular, two aspects have to be considered in order to prevent side reactions during acid-promoted cleavage of carbamate protecting groups. On one hand, glycosidic linkages of the βCD core are sensitive to harsh acidpromoted hydrolysis and may therefore be partially cleaved upon prolonged acidic treatment, leading to a mixture of ring opening and transglycosylation products. While CD derivatives equipped with long fatty acid ester chains seems to be unaffected, probably due to steric inaccessibility to the glycosidic bond, minimizing the reaction time is critical to obtain a homogenous products with shorter or none-acyl chains. 188 On the other hand, ester hydrolysis may take place under acidic conditions. In this regard, long fatty acid chains seem to be invulnerable, probably due to either steric crowding or the decreased rate of hydrolysis associated to longer acyl chains. 189 In any case, it was hypothesized that using anhydrous conditions in shorter reaction times should diminish the rate of hydrolysis to tolerable levels. In a first attempt, hexanoylated compounds 27 and 31 were submitted to standard Boc cleavage conditions (i.e. treatment with 1:1 DCM-TFA at rt for two hours, followed by solvent evaporation and freeze-drying from 0.1 N HCl). CDs bearing long acyl chains resisted these acid-promoted Boc cleavage conditions, as evidenced by the preserved symmetry and homogeneity of the reaction products. However, Boc group removal in compounds furnished with shorter acyl chains revealed a completely distinct scenario. For example, a similar treatment of propanoylated CD derivative 25 furnished a heterogeneous product. As revealed by ESI- Chapter 2 49 MS, carbamate groups were completely removed, and the origin of heterogeneity was attributed to partial hydrolysis of ester chains (Figure 26 ) . The repetitive spacing of 56 mass units between adjacent mass peaks clearly indicated the partial cleavage of propanoyl esters. However, no products derived from glycosidic bond cleavage were detected. Figure 26. ESI-MS spectrum evidencing partial ester hydrolysis of compound 25 when treated with 1:1 TFA-DCM at rt for 1.5 h. It is noteworthy that CD derivatives with butanoyl chains experienced ester hydrolysis far more slowly than their propanoyl analogs, highlighting the substantial influence of an additional carbon atom in the acyl chain. Softening reaction conditions (i.e. lower acid proportions and shorter reaction times) did not improve the reaction outcome. Neither the use of diluted hydrochloric acid in water nor dioxane furnished homogeneous products, though results could be slightly improved. Conversely, deprotection of amine groups under anhydrous conditions (i.e. neat anhydrous TFA at rt for 5 min, immediately followed by reaction mixture freeze-drying with a minimum amount of water) turned to be successful, as carbamate protection groups were completely removed, whereas esters (including acetyls) remained intact. Homogeneity was confirmed by ESI-MS, as depicted exemplarily for paCD 2 in Figure 27 (see Figures S97-S104 for MS spectra, Supporting Information). To facilitate handling and to provide greater stability, the crude paCD derivatives were converted into their 56 Ph.D. Thesis - Iris Pflueger The fact that these oligo(ethylene oxide) CDs 39-42 exhibited significantly different self-assembling capabilities from 6-thioalkylated CDs 35-38 highlighted the utmost role of the hydrophilic/hydrophobic balance determining supramolecular features. 196 For instance, grafting monosaccharide moieties onto the oligo(ethylene oxide) chains of amphiphilic CD derivatives 40-42 directly using glycosyl isothiocyanates 197 (Scheme 9) eventually tuned amphiphilicity to render nanoparticle formation (Figure 30). 198 In addition to non-ionic CD amphiphiles, supramolecular assemblies composed entirely of anionic 199 and cationic 200 amphiphilic CD have been described. Very recently, Darcy and co-workers 201 showed that cationic amphiphilic CDs 46 and 47, which could be obtained from oligo(ethylene oxide) amphiphiles 41 and 42 by a iodination-azidation- reduction sequence 202 (Scheme 9), feature different self-assembling properties. While compound 47 forms bilayer vesicles in aqueous environment, 46, equipped with shorter lipid chains tends to form micelles. Figure 31. Library of paCDs and their respective non-amphiphilic analogs. Chapter 2 57 The intimate link between amphiphilic CD structure and self-assembling capabilities has also been demonstrated recently in our laboratory. It has been shown that subtle modifications in the nature of the functional groups installed on the primary rim of the βCD core dramatically affect the size a surface electrostatic (ζ) potential of the resulting nanoparticles. 203 In turn, these features exert an important influence of nanoparticle stability and their molecular encapsulation and release capabilities. In a first step to correlate molecular structure of the synthesized paCDs (Figure 31) with self-assembling properties, in this section their critical aggregation concentrations (CAC) have been measured. Figure 32. Fluorescence excitation spectra of pyrene (λ em 375 nm) in water containing compound 4 at the indicated concentrations. Experimentally, the critical aggregation concentration can be estimated by correlating a suitable physical property to surfactant concentration. Earlier attempts of our research group to determine the critical aggregation concentration of amphiphilic CDs by surface tension measurements based on the Du Noüy ring method 204 failed because measurements fell below the detection limit in the low µM range. In order to measure the 0 2000 4000 6000 8000 10000 12000 310 315 320 325 330 335 340 fluorescence intensity (A. U.) wavelength (nm) 500 microM 100 microM 20 microM 4 microM 0.8 microM 0.16 microM 0.032 microM 0.0064 microM 58 Ph.D. Thesis - Iris Pflueger critical aggregation concentrations of the paCD collection synthesized in this Ph.D. Thesis, an established easy-to-use fluorescence technique based on the environment polarity-induced change in fluorescence spectra of pyrene 205 was applied. In aqueous environment, this extremely hydrophobic dye is preferentially incorporated in the interior of hydrophobic aggregates, leading to a significant change in its fluorescence spectrum. The onset of aggregate formation is detected by a shift of its fluorescence excitation spectra at an emission wavelength of 375 nm. A series of aqueous solutions of each paCD were prepared (0.256 nM to 500 µM), each of them containing 0.6 µM pyrene. After incubation of the samples, the fluorescence excitation spectra were registered at 375 nm (λ ex 310-340 nm, Figure 32) and the fluorescence intensity at 339 nm relative to 335 nm (I 339 /I 335 ) was plotted against Log[CD], yielding for each CD derivative a graphic as shown in Figure 33. Figure 33. CAC determination for compound 4 using pyrene fluorescence excitation spectra switch at λ em 375 nm. In such representation, two linear data ranges can be distinguished: (i) a section with constant I 339 /I 335 ratio corresponded to CD concentrations below CAC (sub) and (ii) another with a linear increase in I 339 /I 335 ratio associated with CD concentrations above CAC (super). By linear fitting of both data ranges, two linear equations could be obtained: 0,0 0,2 0,4 0,6 0,8 1,0 1,2 -4 -3 -2 -1 0 1 2 3 4 I339/I335 Log [c] (  _ 339 /  _ 335 ) _   = 0 . 3801 ·  (  ) + 0 . 0198 (  _ 339 /  _ 335 ) _   = 0 . 0051 ·  (  ) + 0 . 3418 Chapter 2 59      ! "#$ = % "#$ ·()+  "#$ (1)      ! "#&'( = % "#&'( ·()+  "#&'( (2) The critical aggregation concentrations were then determined from the intersection of both lines according to equation (3): )*) = 10 +,-./01+,-+ 2,-+12,-./0 (3) Table 2. Critical aggregation concentrations in aqueous environment of both linear and branched paCDs and their non-amphiphilic analogs. Acyl chain length Linear series CAC (µ µµ µM) Branched series CAC (µ µµ µM) none 9 64.7 10 43.3 Ac 1 94.4 5 66.5 Pr 2 26.4 6 49.3 But 3 5.7 7 21.3 Hex 4 0.94 8 2.79 Table 2 collects the critical aggregation concentrations in aqueous environment of both linear and branched paCDs and their non-amphiphilic analogs. The expected progressive decrease of CAC with increasing acyl chain length was observed. This was in agreement with an increasing hydrophobicity of the acyl chains and thus amphiphilicity of the CD conjugates. In general, this trend could be observed in both the linear and the branched paCD series, although it was more pronounced in the linear one. CAC dispersion for the former group ranged from 1 µM (hexanoylated paCD 4) to approximately 100 µM (acetylated paCD 1), while CAC values of branched paCDs varied from 3 µM (hexanoyl compound 8) to approximately 70 µM (acetylated compound 5). Although an increase in acyl chain length and hence in amphiphilicity of the CD conjugate resulted in a decrease of CAC, charge density also plays a major role, 60 Ph.D. Thesis - Iris Pflueger evidencing that an appropriate equilibrium between acyl chain length and number of polycationic groups is decisive for the self-aggregation tendency of paCDs. The sixmembered acyl chains of CD derivatives 4 and 8 seemed to be able to counterbalance arrays of both seven and fourteen cationic groups on the primary rim of the CD scaffolds, which led to low CACs in both cases. In contrast, the shorter acyl chain of the set of propanoyl compounds 2 and 6 apparently matched up better with a lower charge density. Hence, CAC of compound 2 is markedly lower than that of its branched analog 6. The CAC values obtained for the non-amphiphilic CD derivatives 9 and 10 are, however, unexpectedly lower than those measured for the acetylated conjugates. 1 H NMR registered in D 2 O up to low mM concentrations gave no evidence of selfaggregation behavior for compounds 9 and 10. Thereby, the observed effect must be the consequence of the molecular inclusion of the pyrene moiety in the CD cavity, which may also potentially lead to fluorescence spectral changes. 206 Lacking acyl chains on the secondary rim may clear the access to the cavity of compounds 9 and 10. The experimental setup of the fluorescence technique used to determine the CACs does not allow for distinguishing whether the shift of the fluorescence excitation spectra originates from incorporation of pyrene in the interior of micelles or from the formation of inclusion complexes between CD and pyrene. As a consequence, it has not been possible to define the contribution of inclusion complex formation to the allover fluorescence shift and the actual CACs of both derivatives 9 and 10 could not be determined although they are expected to be much higher than those of their amphiphilic analogs. In order to round investigations of self-assembling capabilities in aqueous environment out, the stability of the paCD aggregates has been studied. For such purpose, paCD 4 was chosen to be surveyed in a more detailed manner. Its pyrene-containing aqueous solutions were incubated at rt for one month and fluorescence spectra were registered at different times over that period. Measurements revealed that the CAC remained constant; hence, aggregates remained unaltered for that time. Chapter 2 61 4. Determination of pK a values and buffering capabilities of CD-scaffolded polycations It is generally recognized that the major obstacles to efficient gene delivery are cellular internalization and intracellular trafficking. 207,208 There have been noticed major disparities among the mechanisms used by non-viral carriers to overcome these hurdles. For instance, lipoplexes have been shown to enter the cells predominately via clathrindependent endocytosis (CDE). 209 PEI polyplexes, however, are internalized through several routes, being the fraction taken up via clathrin-independent (CIE) mechanisms the one that leads to gene expression. 210 Similarly to the later, in the case of CDplexes, though the largest fraction of gene complexes is taken up via CDE, the smaller fraction internalized via CIE is predominately responsible for successful transgene expression. 182 Figure 34. Schematic representation of the structure of haemagglutin (HA) at neutral pH (A) and at low pH (B). These evidences highlight the utmost relevance of endosomal escape skills in the overall efficiency of gene carriers. Understanding of the mechanisms of viral escape from endosomes is important for improving non-viral gene delivery systems. For instance, the naturally evolved strategies by viruses are incomparably more efficient than those envisioned for non-viral vectors. Haemagglutinin (HA) protein, which is pH sensitive and membrane destabilizing, helps viral vectors to disrupt the endosome efficiently and enter the cytoplasm. 211 The escape mechanism of HA and other fusion proteins is that they shift from an ionized and hydrophilic conformation to a hydrophobic and membrane-active 62 Ph.D. Thesis - Iris Pflueger conformation as the environment changes from neutral to acidic, resulting in destabilization of the endosomal membrane and its subsequent leakage (Figure 34). 212 Though still a matter of debate, this is much alike the endosomal escape mechanism responsible for the gene transfer efficiency of lipidic vectors. The interaction of cationic lipids with anionic phospholipids, a model introduced by Xu and Szoka, 213 destabilizes endosomal membrane and facilitates its rupture (Figure 35). 214 Figure 35. Proposed mechanisms of cationic lipid/nucleic acid complexes after endocytosis along the endosomal pathway: (1) cationic lipids interact with anionic lipids in endosomes by forming ionpairs; (2) the lipid bilayer is destabilized; (3) the hexagonal phase is formed; (4) the nucleic acid is released into the cytoplasm. 215 Conversely, the gene transfer efficiency of certain cationic polymers is associated to their pH buffering capabilities by virtue of the proton sponge effect. 65 Endosomal pH gradually drops from physiologic (ca. 7.5) to acidic (ca. 5-5.5) upon maturation, a pH range in which nearly half of high-molecular weight PEI nitrogens may protonate. This large buffering capacity may be accompanied by extensive swelling to compensate the Chapter 2 63 osmotic pressure and/or conformational changes in the DNA-carrier assembly that leads to endosome destabilization and subsequent bursting (Figure 36). 86 It is hypothesized that the proton sponge mechanism is not only valid for PEI but is more generally applicable for compounds containing amino groups with pK a values at or below physiological pH. Figure 36. Endosomal rupture is mediated by polymers with a buffering capacity at the endosomal pH range that can trigger (a) the proton-sponge effect and (b) polymer swelling according to the umbrella hypothesis. 65 Considering the similarities regarding the endocytic mechanisms between paCD CDplexes and PEI polyplexes, it was of interest to investigate the pH buffering capabilities of paCDs and their aggregates. In general, the pK a of each amine group in a polyamine is affected by the type of amine (primary, secondary, tertiary, or quaternary), electronic factors more than steric factors, and especially by the distance between neighboring amines. 216,217 A variety of methods for the determination of polyamine pK a values exist. The most common is potentiometric titration, which is characterized by a high accuracy although a relatively large amount of pure compound is required. 218 Alternatively, any other property directly or indirectly affected by protonation can be used as probe for pK a determination (UV absorption, electrophoretic mobility or LC, among others). 217 NMR-pH titration is also an 64 Ph.D. Thesis - Iris Pflueger excellent technique for determining pK a values as the protonation of a basic site leads to deshielding effects on the adjacent NMR-active nuclei, so the average chemical shifts of all the measureable NMR-active nuclei, as a function of pH, are expected to reflect the fractional protonation of each basic group of a molecule. NMR-pH titration offers the advantage of measuring pK a in solutions that are not pure, e.g. in the presence of biological fluids or even impurities, 219 and may allow the sequence of polyamine protonation to be determined or even followed. To determine pK a values of a set of CD-scaffolded polycations synthesized in this Ph.D. Thesis (Figure 37), and to evaluate their buffering capacity, two different techniques were combined. 1 H NMR-pH titration of non-amphiphilic polycationic CDs and their respective model compounds allowed gaining a first insight into the range of their pK a values. An advantage of this technique is that only small amounts of compounds are required, but cannot be used for self-aggregating paCDs . Alternatively, potentiometric titration was used for amphiphilic CDs instead. Figure 37. Compounds for 1 H NMR-pH titration: CD-scaffolded polycations (A) and their corresponding monovalent models (B). Chapter 2 65 Thereby, 1 H NMR-pH titration experiments were used to measure the pK a values of non-amphiphilic CDs 9 and 10, together with more simple cysteaminylated CD derivative 32 185a and the single branch models 50 and 51 (Figure 37). Compound 32 is the simplest one, only bearing cysteamine moieties, whereas both compound 9 and 10 additionally feature thiourea functionalities identified as the optimal tethers between the CD core and cationic cluster for gene delivery and expression. Keeping this element fixed, compound 10 has an increased density of primary amine groups in a dendritic display and is additionally furnished with a tertiary amine group. Structural modifications may have an influence on pK a values of the primary amines corresponding to different CD derivatives. Furthermore, tertiary amines of compound 10 may have a lower pK a than primary amines and thus enable the proton sponge effect. Model compounds 50 and 51 (Figure 37) were synthesized by reacting butylamine with isothiocyanates 22 and 23, respectively (see Experimental Section for details) to assess the effect of the multivalent display on pK a values. Figure 38. pH-Dependent chemical shifts of polycationic CD 9 (500 MHz, D 2 O-H 2 O, 298 K). 72 Ph.D. Thesis - Iris Pflueger 5. Assessment of DNA-paCD complex (CDplex) formation and dissociation Facile cellular uptake of free DNA via plasma membrane permeation is hindered by the size and negative charge of the DNA. Although several studies have shown that naked DNA can be introduced into cells the in vivo relevance of these techniques is limited. Systemic circulation of free DNA is hindered by nuclease degradation. Thus, compaction and masking the negative charge of its polyphosphate backbone constitutes a prerequisite for effective DNA delivery. Gene-carrier complex formation is known to critically determine gene transfer efficiency. The size of the complex and stability significantly depends on the type of cationic structure used, although preparation conditions including concentration of DNA, pH, type of buffer, and N/P ratio also affect size. 55 Moreover, particle size affects the extent to which nanoparticles are taken up and are able to transfect cells, as the endocytotic machinery and cell membrane have welldefined geometries and flexibility that may restrict entry of incompatibly large or small particles. 222,223 Figure 44. Two-step self-assembling mechanism of paCDs in the presence of DNA. Along with size, surface charge (ζ potential) also critically determines particle fate and transfection efficiency. It is generally believed that positively charged nanoparticles Chapter 2 73 perform better for in vitro transfections of cells through their enhanced binding to negatively charged proteoglycans on cell surfaces, 224,225 though interaction with negatively charged serum proteins, could also be a drawback. 226 It has been shown that paCDs furnish small nanoparticles (CDplexes) with narrow polydispersity upon incubation in the presence of pDNA. These CDplexes are spherical and positively charged (av. 30 mV, Figure 45) with an average size below 100 nm. CDplex formation has been hypothesized to take place through a two-step self-assembling mechanism of paCDs in the presence of pDNA, as shown in Figure 44. 177 The first step is a rapid and reversible electrostatically driven interaction between phosphates and the positively charged amine headgroups of paCDs, during which the paCDs act separately in an independent manner. Driven by desolvation, the second probably slower and essentially irreversible step involves the fusion and rearrangement of the paCDs, thus triggering extensive compactation by hydrophobic interactions. 227 Figure 45. TEM micrograph of paCD 52:pDNA CDplexes with amplification of the ultrafine structure of the particles and a schematic representation of the proposed arrangement of paCDs and the DNA double helix. Transmission electron microscopy (TEM) showed the small size (40 nm) and homogeneous distribution of CDplex formulations from thioureido-cysteaminyl paCD 52 74 Ph.D. Thesis - Iris Pflueger (Figure 45). 177 At high magnification, a snail-like ultra-structure was observed, which has been attributed to the alternating lamellar arrangements of paCDs and electron-dense regions corresponding to the pDNA molecule, similarly to that reported by Pitard and coworkers (see Figure 12, Chapter 1). 81b The spontaneous self-assembly of CDplexes may seem very simple in its concept, yet could determine their characteristics (stability, biocompatibility, membrane-crossing ability etc.) and, thereby, their transfection potential. Control of the hydrophilic/hydrophobic balance between the CD primary and secondary faces is therefore crucial for efficient pDNA complexation and nanoparticle (CDplex) formation. Among the most important parameters that are affected by the molecular architecture of paCDs are complex size and uniformity, complex overall charge, as well as its pDNA condensation and dissociation capabilities. For a better understanding of the effects of structural variations on the molecular construct in the CDplex stability and its physicochemical properties, in this Ph.D. Thesis a deep insight on CDplex formation and dissociation capabilities of paCDs differing in their hydrophobic/hydrophilic balance was accomplished. Figure 46. Schematic representation of a conventional, 90º dynamic light scattering (DLS) instrument. 228 First, the DNA-paCD nanoparticles were characterized by dynamic light scattering (DLS) in order to determine their average hydrodynamic size and ζ potential. Briefly, the DLS technique measures the time-dependent fluctuations in the intensity of scattered Chapter 2 75 light, which occurs because the particles are undergoing random Brownian motion. Analysis of these intensity fluctuations enables the determination of the distribution of diffusion coefficients of the particles, which can be converted into a size distribution using established theories (Figure 46). Figure 47. Schematic representation of zeta potential. 229 In general, the surface charges of particles dispersed in an aqueous system modify the distribution of the surrounding ions, resulting in a layer around the particle that is different to the bulk solution. The ζ potential is the potential at the point in this layer where it moves past the bulk solution (slipping plane, Figure 47). ζ Potential is one of the main features that determine particle interactions, thus a high zeta potential will confer stability, i.e. the solution or dispersion will resist aggregation. ζ Potential measurement were performed by a technique called M3-PALS, which is a combination of laser Doppler velocimetry and phase analysis light scattering (PALS) to measure particle electrophoretic mobility. The mobility measured is converted to ζ potential using established theories. 76 Ph.D. Thesis - Iris Pflueger The capability of the polycationic CDs differing in their hydrophobic/hydrophilic balance synthesized in this Ph.D. Thesis (Figure 48) to form stable CDplexes with DNA (calf thymus) was studied at N/P ratios 5 and 10. This ratio, which represents the ionic balance within the complexes, is referred to the number of protonable amino groups (N) in the polycationic CD per phosphate (P) of DNA. These N/P ratios were chosen since at these proportions the most efficient transfection has been achieved in earlier reports. 177,181 To avoid premature self-aggregation phenomena, the CD stock solutions were prepared in DMSO and further diluted with the DNA solution in a 20 mM 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at physiological pH, thereby assuring that the final DMSO content never exceeded 1% v/v. Figure 48. Library of polycationic CDs for particle size and ζ potential measurements. The DLS results showed that all assayed polycationic amphiphilic CDs as well as their non-amphiphilic analogs formed compact, ordered and stable nanoparticles with DNA, Chapter 2 77 exhibiting rather small hydrodynamic diameters as compared with the polyplexes obtained using branched polyethyleneimine (bPEI, 25 kDa), one of the most efficient commercial gene-delivery systems (60-90 nm vs. 150 nm; Figure 49). 230,231 Figure 49. Size (hydrodynamic diameters, bars, nm) and ζ potential (lines, mV) of CDplexes determined by dynamic light scattering and M3-PALS analysis, respectively. Hydrodynamic diameter measurements demonstrated that particle size scarcely depends on paCD structure. Even in the case of non-amphiphilic CDs 9 and 10, hydrodynamic diameters could be measured in the range of 70 nm, in contrast to our previous observations, 177 but in agreement with that reported for other non-amphiphilic CD-scaffolded polycations. 169 Reineke and co-workers demonstrated that structurallyrelated non-amphiphilic polycationic CD condense pDNA at N/P 5 into spherical nanoparticles in the size range of 80-130 nm. These nanoparticles exhibited near neutral to positive zeta potentials (+5 < ζ < +15 mV). In particular, non-amphiphilic CD derivatives 9 and 10 exhibited similar hydrodynamic diameters to their hexanoylated counterparts (ca. 70 nm). While this result may sound counterintuitive, their hydrodynamic diameters do not reflect how nanoparticles are -70 -50 -30 -10 10 30 50 70 90 0 25 50 75 100 125 91015263748 ζ ζ ζ ζ (mV), lines Size (nm), bars N/P ratio of 5 N/P ratio of 10 78 Ph.D. Thesis - Iris Pflueger compacted and similar values could still reflect quite different behaviors for any case. The proposed two-step compaction process cannot be operating for the formation of this nonamphiphilic nanoparticles. Additionally, narrow populations of cationic nanoparticles were observed in each individual experiment, thus indicating rather monodisperse nanoparticles (Figure 50). Such remarkable behavior (low particle size and monodispersity) has only been reported for a monomolecular condensation process that occurs upon mixing DNA with dimerizable, polycationic detergents. 84a,232,233 Figure 50. Particle size distribution by volume determined by DLS for paCD 8 at N/P 5 (diameter in nm). M3-PALS measurements at both N/P ratios 5 and 10 revealed that ζ potential ranked in the range 30-70 mV, amenable values for cell-based assays. ζ potential values did not depend on the N/P ratio, which indicated that complex stoichiometry does not significantly change upon an increase of the N/P ratio. We hypothesize that excess CD does not incorporate into the CDplex nanostructure. Previous results of our research group indicated that nanoparticles of non-amphiphilic CDs exhibited zeta potential values close to neutrality even at high N/P ratio, although these CDplexes were not stable. 177 In sharp contrast, ζ potential values of CDplexes of non-amphiphilic CD derivatives 9 and 10 were 0 2 4 6 8 10 12 14 1 10 100 1000 10000 Volume (%) Size (d.nm) Size Distribution by Volume Chapter 2 79 clearly in a positive range (ca. 40 and 50 mV, respectively, for both N/P ratios), thus perfectly fitting within the scope of paCDs. Results reported by Reineke and coworkers 169 support these findings, since they also demonstrated that non-amphiphilic CDs were capable of binding and compacting pDNA at N/P 5 into spherical nanoparticles with slightly positive zeta potentials. Altogether, DLS experiments pointed out that all polycationic CD derivatives 1-10 were able to form nanoparticles with DNA, regardless of their hydrophilic/hydrophobic balance. All these CDplexes featured remarkably similar hydrodynamic diameters, ζ potentials and relatively narrow size distributions. These results were contrasted using agarose gel electrophoresis shift assays. Thus, paCD:ctDNA complexes were formulated in the same manner as described before for different N/P ratios in the range of 0.5 to 20. Naked ctDNA was used for comparative purposes. A decrease of DNA migration up to complete inhibition could be observed at different N/P ratios for all tested CD derivatives. Moreover, if DNA is efficiently compacted and protected in the CDplexes, it becomes inaccessible to the GelRed™ (Biotium), a fluorescent intercalating agent used as staining reagent, as demonstrated by the absence of fluorescent staining in the corresponding lanes (Figure 51). At N/P ratios higher than 1, DNA charge neutralization and migration were achieved with all assayed polycationic CDs, which was indicated by the absence of “free” mobile plasmid. However, still an intense GelRed™ stain is visible in most of the cases. At N/P ratios higher than 1, however, paCDs bearing acyl chains of different length showed essentially different behaviors, thereby evidencing the subtle differences between nanoparticles with respect to amphiphilicity. From N/P ratio 2 and up, linear amphiphilic CDs 2, 3 and 4, as well as branched analogs 6, 7 and 8 were capable to efficiently compact and protect DNA in their CDplexes, which was indicated by the absence of fluorescent staining in the corresponding lanes. On the contrary, the CDplexes formulated with both acetylated (1 and 5) and non-amphiphilic (9 and 10) derivatives did not compact and protect DNA efficiently, since it remained accessible to GelRed™ in the whole range of N/P values, including even high N/P ratio 20. 80 Ph.D. Thesis - Iris Pflueger Figure 51. Protection of DNA from GelRed™ intercalation at different N/P ratios (0.5-20) by paCDs 1-8 and their non-amphiphilic analogs 9 and 10. Chapter 2 81 These results clearly evidenced that CD derivatives that bear longer acyl chains cover the DNA surface more efficiently. However, no remarkable difference between paCDs featuring either four-membered or six-membered acyl chains was detected. When comparing the linear and the branched subseries among one another, it has to be kept in mind that equal N/P ratios do not correspond to equimolecular ratios. To put it differently, for the preparation of CDplexes with a given N/P ratio the number of moles of linear CD derivatives required triples that of branched CDs. This is a considerable factor because therapeutic doses of non-viral gene delivery systems are related to issues of immunogenicity and toxicity. Additionally, for paCDs 4 and 6 gel electrophoresis shift assays were accomplished using luciferase-encoding pDNA for CDplex formation. Whereas ctDNA used so far consists of 13000-20000 base pairs (13-20 kbp), luciferase-encoding pDNA (later used for later transfection experiments) is only 5739 bp long. Hence, the question rose whether CDplexes formulated with either ctDNA or pDNA would have similar characteristics. Indeed, the results obtained for both paCDs in gel electrophoresis shift assays using luciferase-encoding pDNA were very similar to those obtained for ctDNA. Figure 52. Schematic representation of the experiment designed for fluorescence tracking of CDplex formation and dissociation. 88 Ph.D. Thesis - Iris Pflueger Figure 56. Luciferase-encoding gene transfer efficiency (A) and cell viability (B) in COS-7 cells treated with CDplexes formed with paCDs 1-8 and their non-amphiphilic counterparts 9 and 10 at N/P 2, 5 and 10. Naked pDNA and JetPEI polyplexes were used as negative and positive controls, respectively. Despite their amphiphilic structure, the acetylated and propanoylated paCDs featured a similar behavior to the non-amphiphilic CDs. Luciferase expression increases as 0 2 4 6 8 10 pDNA JetPEI 8 4 7 3 6 2 5 1 9 10 Luc expression / log[fg Luc/mg protein] N/P 2 N/P 5 N/P 10 A 0 25 50 75 100 125 150 175 200 pDNA JetPEI 8 4 7 3 6 2 5 1 9 10 cell viability / % N/P 2 N/P 5 N/P 10 B Chapter 2 89 compared to naked pDNA ranged between 1 to 2 orders of magnitude. Again heptacationic paCDs 1 and 2 at the highest N/P ratio (10) exhibited the largest efficiencies but also considerable toxicities. In any case, in the best performing conditions, luciferase expression resulted more than 3 orders of magnitude lower than that achieved with JetPEI. Though acetylated and propanoylated paCDs have been shown to efficiently condense and compact DNA at these N/P ratios, the stability of their CDplexes is largely compromised in competing media, as illustrated previously. We hypothesize that the reason for such poor performance may lay on the fact that serumcontaining media could accelerate the dissociation of these particles, though other reasons may be considered. For instance, it has been shown that fusogenic capabilities of lipoplexes critically determine their cell membrane-binding and cell uptake abilities. 210b These capabilities in paCDs equipped with short acyl chains are probably far from optimal. The experiments with hexanoylated and butanoylated paCDs, however, revealed a quite distinct scenario. Both series exhibited much higher transgene expression efficiencies, which might be linked to the higher stability of their CDplexes (see preceding sections), and an N/P-dependent performance, the highest efficiencies generally achieved at N/P 10. In contrast to previously reported observations in serum-devoid media, 177 heptacationic paCDs resulted more efficient than 21-cationic ones. Even considering the paCD-pDNA mass instead of N/P ratio, heptacationic paCDs resulted more efficient than their branched counterparts. The branched paCDs 7 and 8, regardless of the acyl chain length, featured gradual transgene expression efficiency enhancement upon N/P increase and remarkably low cytotoxicities even at N/P 10. Interestingly, the CDplexes formulated with the corresponding heptacationic conjugates 3 and 4 resulted better performing even at the lowest N/P ratio (2). In this case, efficiency increased only mildly with N/P and was handicapped due to cytotoxicity at N/P 10 (as also observed for heptacationic CDs with shorter acyl chains). A remarkable efficiency decrease was observed for CDplexes formulated with paCD 8 in the presence of serum. paCD 8 has been previously reported to surpass JetPEI efficiency towards COS-7 cells, 177 though in 10% serum-containing media, efficiency drops by 3 orders of magnitude. It is also noticeable that the performance of its 90 Ph.D. Thesis - Iris Pflueger heptacationic analog is only mildly affected by the presence of serum. And, the dramatic performance differences between butanoylated and propanoylated paCDs, which may arise from the comparatively distinct stability of their CDplexes in biological media, were even more astonishing. Altogether, these experiments led to the identification of the butanoylated heptacationic CD 3 as the best performing candidate, exhibiting luciferase expression levels almost equaling that of JetPEI but with a slightly better cytotoxic profile (43% vs. 19% cell viability for 3 and JetPEI, respectively, at their best N/P ratio). A slightly lower but virtually non-toxic performance was achieved at N/P 5 with the same compound. These results also highlight that, despite paCD 3 is not the most efficient at DNA complexation, dynamics and kinetics of CDplex formation play a critical role in overall performance. Unfortunately, performance cannot be anticipated so far, due to the complex balance of supramolecular features that should be taken into account. But the reported results can be considered as a useful tool to experimentally tune up the carrier capabilities towards a particular target. C HAPTER 3 - D ESIGN OF CDSCAFFOLDED CYCLIC POLYAMINE CLUSTERS : E NGINEERING OLIGONUCLEOTIDE BINDING PATTERNS IN NON - VIRAL GENE DELIVERY 92 Ph.D. Thesis - Iris Pflueger 1. Novel DNA binding motifs at improving the efficiency of artificial gene carriers A large array of cationic epitopes has been explored as nucleic acid binding elements. The topic has been comprehensively reviewed very recently. 72a Some of the most commonly used are depicted in Figure 57. Amines with different substitution and pH- dependent ionization degree are by far the most frequently used. Quaternary ammonium groups are permanently charged at any pH and consequently devoid of buffering capability. 86 Amines and ammonium groups are rarely found alone, but combined in the most bewildering way to enhance their nucleic acid binding abilities and displayed onto polymeric or lipidic platforms aimed at zipping oligonucleotides through multipoint concerted interactions. Figure 57. Illustrative examples of some commonly used of cationic headgroups for non-viral gene carrier design. Aimed at exploring the effect of alternative cationic headgroups on paCD-based gene carriers, we have turned our attention to cyclic oligoamines, such as cyclen (1,4,7,10- tetraazacyclododecane) and cyclam (1,4,8,11-tetraazacyclotetradecane), featuring quite Chapter 3 93 unique DNA-binding. 239 Cyclen and cyclam are known to form stable complexes with transition metal ions. 240 Cyclen and its simple N-functionalized derivatives, such as DOTA 241 (N 1 ,N 4 ,N 7 ,N 10 -cyclentetraacetic acid) and DOTAM 242 (N 1 ,N 4 ,N 7 ,N 10 - cyclentetraacetamide, Figure 58) are widely used in the construction of a wide variety of functional molecules such as contrast agents for magnetic resonance imaging (MRI), 243 single photon emission computed tomography (SPECT) 244 and positron emission tomography (PET), 245 fluorescent 246 and luminescent 247 probes and metal sensors. 248 Many studies have also revealed that both cyclen 249 and its metal complexes 249b,250 may behave as artificial nucleases, featuring DNA-cleavage abilities, and protease-like catalytic activity. 251 Macrocyclic polyamines, e.g. cyclen, cyclam or bicyclam AMD3100 252 (1,1′-[1,4-phenylenebis(methylene)]bis(1,4,8,11-tetraazacyclotetradecane), Figure 58) have also exhibited antitumor and anti-HIV activity. 253 NH HN HNNH cyclen N N N N R O O R O R O R NH NH cyclam HN HN NH NH HN N AMD3100 NH N HN HN DOTA, R = OH DOTAM, R = NH2 Figure 58. Structures of cyclen, its derivatives DOTA and DOTAM, cyclam and bicyclam AMD3100. Metal complexes of cyclam such as AMD3100- 64 Cu have been shown to be suitable PET probes for tracking cancer cells in vivo. 254 Furthermore, ADM3100-based lipidic and polycationic conjugates have been exploited as cancer cell-targeted non-viral gene vectors. 252,255 Several factors may account for the unique oligonucleotide binding capabilities of cyclic oligoamines, such as cyclen and cyclam. The preorganization of the cyclic scaffolds may influence the phosphate binding capacity in a positive manner. The cyclen moiety is composed of repeated ethylenediamine units, comparable to those in PEI, while cyclam alternates ethylenediamine and propylenediamine segments. The cyclic arrangement of 94 Ph.D. Thesis - Iris Pflueger amino groups in macrocyclic oligoamines exerts an important influence on their pK a values (Figure 59). 256,257 Figure 59. pK a values of cyclic oligoamines. NH2NH2 HNNH2N O O O O .5 CF3COO NH2NH2 HN NH2N N .5 CF3COO N NH2NH2 HNNH2N .5 CF3COO N R NH2NH2 HNNH2N N .5 CF3COO O NH2HN H2NNH2 .4 CF3COO N H N O N H H N R O O N O NH O O NH2HN H2N NH2 .4 CF3COO N H N O N H H NO O O N O NH O O R NH2NH2 HNNH2N .5 CF3COO OO O O O NH2NH2 NNH2 .3 CF3COO H NON H R O O O R = , a b cholesterol (chol) diosgenin (dios) c dfor R = chol efor R = dios ffor R = chol gfor R = dios hfor R = chol ifor R = dios jfor R = chol kfor R = dios l Figure 60. Molecular structures of lipids with cationic cyclen headgroups as gene vectors synthesized by Yu and co-workers. 239a,259,260 Chapter 3 95 Considering these features, it is somehow surprising that the use of cyclic oligoamines in gene delivery has being barely investigated. To the best of our knowledge, until 2008 only a single example of cyclen-based gene delivery system was reported, i.e. a dendritic polymer with a cyclen core. 258 It was only five years ago when application of cyclen as DNA binding motif in gene vectors got rolling. Yu and co-workers 239b were the first to describe a cationic lipid with a protonated cyclen headgroup (Figure 60a), yet this imidazolium salt group-containing gene carrier showed very poor transfection efficiency. To overcome this drawback, the same research group made a considerable effort and synthesized over the years a series of lipids bearing cyclen as cationic headgroup 239a,259,260 (Figure 60b-l). By systematic modifications of both the hydrophobic tail 259 and the functional linking group 239a,260 gene delivery efficacies could be improved dramatically and transfection efficiencies of most cyclen-based cationic lipids surpassed that of Lipofectamine 2000™, thus exhibiting similar or slightly lower cytotoxicities than the lipoplexes prepared from Lipofectamine 2000™ and illustrating the potential of SAR in the improvement of transgene expression efficiency. Arbuthnot and co-workers 261 reported a cyclen-conjugated cholersteryl derivative containing biodegradable carbamate linkers, and lipoplexes containing this lipid proved to be non-toxic DNA and siRNA delivery systems (Figure 61). Although cyclen-based lipoplexes resulted in efficient siRNA-mediated target gene silencing, almost catching up the efficiency of Lipofectamine 2000™, their efficiency was poor for DNA transfection. Figure 61. Molecular structure of the cyclen-conjugated cholersteryl derivative reported by Arbuthnot and co-workers. 261 96 Ph.D. Thesis - Iris Pflueger Nearly at the same time, cyclen-based polymers emerged. 262 Yu and co-workers have pioneered the field of investigating cyclen-based cationic polymers for gene delivery. Initially, a cyclen-based side-chain homopolymer capable of self-assembling in presence of pDNA has been prepared by Yu and co-workers, 262 however, its transfection efficiency has not been reported so far (Figure 62a). Figure 62. Molecular structures of cyclen-based cationic polymers as gene vectors synthesized by Yu and co-workers. 262,263,264,265 Recently, the same research group has reported a variety of cyclen-based polymeric vectors, including reticular cyclen-based polymer (Figure 62b), 263 linear cyclen-based polyamine (Figure 62c), 264 as well as diolglycidyl ether-bridged cyclens (Figure 62d). 265 A biodegradable disulfide-linked version of the latter has been also reported. 266 Almost all cyclen-based cationic polymers showed transfection efficiencies close to that of PEI (20 kDa or 25 kDa) at N/P ratios in the range of 5-15 but reduced cytotoxicities. In addition, Jiang and co-workers 267 synthesized chitosan grafted with two kinds of macrocyclic polyamines, i.e. cyclen and 1,4,7-triazacyclononane (TACN), at either C-2 or the C-6 position (Figure 63). They showed that, depending on the functionalization Chapter 3 97 position, physicochemical features could be tailored. Indeed, C-2-grafted chitosanes exhibited higher cytotoxicity and transfection efficiency than those grafted at C-6. Figure 63. Structures of chitosan grafted with cyclen or TACN on different positions. Figure 64. Structure of reducible polycationic chelators based on cyclam (The precise attachment position of the linker could not be determined. Any of the secondary amines are susceptible to modification). Besides, Oupický and co-workers 268 described the first comprehensive report on the SAR of cyclam-based polymeric gene vectors (Figure 64). They designed a series of bioreducible cyclam-based polycationic Cu(II) chelators (RPCs) aimed at combining gene delivery and real-time PET monitoring. The cyclam moieties in the polycations retained their ability to form complexes with Cu(II). The presence of disulfide bonds in the 104 Ph.D. Thesis - Iris Pflueger To obtain isothiocyanate-armed cyclen and cyclam derivatives for subsequent thiourea-forming reaction with CD heptaamine 21, initially a similar N-alkylation approach to that described above was used. Reaction of tri-N-Boc protected cyclen 61 with n-1- azido-7-bromoheptane 65, prepared by statistic substitution of n-1,7-dibromoheptane, under µw irradiation afforded azide 66 in good yield. Aza-Wittig type reaction of azide 66 with triphenylphosphine-carbon disulfide 277 afforded the corresponding isothiocyanate 67. Alternatively, cyclic polyamine derivatives 61 and 62 were reacted with an excess of 1,6- hexamethylenediisothiocyanate 68 278 to afford cyclen and cyclam-derived thioureas 69 and 70, both furnished with a terminal isothiocyanate group on the aliphatic spacer. These dissymmetrization reactions proceeded smoothly in excellent yields and formation of bis-cyclen derivatives was not observed, provided a sufficiently large excess of diisothiocyanate was used (3 eq). In order to obtain CD-scaffolded cyclic polyamine clusters, both the CD building blocks 54 and 21 and the armed cyclen and cyclam derivatives 63, 64, 67, 69 and 70 were then coupled by “click chemistry” methodologies (Scheme 15 and Scheme 16). The coupling of the CD core with the cyclic oligoamine blocks is not trivial, since coupling reactions are to take place at seven positions simultaneously, which requires very efficient procedures to warrant monodispersity. Figure 66. Structure of silica-supported heterogeneous catalyst Si-BPA·Cu + . The sevenfold Cu(I)-catalyzed azide-alkyne cycloaddition reaction between heptaazide 54 and alkynes 63 and 64 was accomplished using Si-BPA·Cu + as solidsupported catalyst in refluxing 3:1 t BuOH-H 2 O. Recent results had indicated the efficiency of silica-based nanoparticles incorporating bis(pyridyl)amine (BPA) Cu(I) chelating adsorbents Si-BPA·Cu + to catalyze multiple CuAAC couplings 180a,279 (Figure 66). Chapter 3 105 Scheme 15. Synthesis of cyclic oligoamine-grafted paCDs 75 and 76 by CuAAC reaction. Reagents and conditions: a) 63 (71) or 64 (72), Si-BPA·Cu + , t BuOH-H 2 O, reflux, 16 h, 97% (71) or 84% (72); b) i, 1:1 TFA-DCM, 2 h; ii, HCl, quantitative yield in both cases. Homogeneous heptatriazoles 71 and 72 were obtained in remarkable yields (97% for 71 and 84% for 72, respectively, Scheme 15) after purification by flash column chromatography. ESI-MS and NMR confirmed homogeneity of both compounds and neither ESI-MS nor elemental analysis detected traces of copper ions (Figure 67; see Figures S40 and S41 for NMR and Figures S119 and S120 for MS spectra, Supporting Information). Although NMR spectra of CD-scaffolded cyclic polyamine clusters 71 and 72 were recorded at elevated temperatures (60 ºC), line broadening due to restricted flexibility of Boc-protected cyclic polyamine segments was evident. 106 Ph.D. Thesis - Iris Pflueger 012345678 ppm Figure 67. ESI-MS (top) and 1 H NMR (bottom, 500 MHz, MeOD, 333 K) spectra of compound 71. All attempts to couple isothiocyanate derivative 67 to heptaamine 21 in a thioureaforming reaction failed to furnish the heptabranched product homogeneously. However, coupling of isothiocyanate derivatives 69 and 70, respectively, and CD building block 21, followed by purification by size exclusion chromatography, afforded monodisperse CD derivatives 73 and 74 in excellent yields, i.e. 75% for 73 and 77% for 74, respectively (Scheme 16). Chapter 3 107 Scheme 16. Synthesis of cyclic oligoamine-grafted paCDs 77 and 78 by thiourea-forming reaction. Reagents and conditions: a) Et 3 N, DCM, 40 ºC, 72 h; b) 69 (73) or 70 (74), Et 3 N, DCM, 40 ºC, 72 h, 75% (73) or 77% (74); c) i, 1:1 TFA-DCM, 2 h; ii, HCl, quantitative yield in both cases. Homogeneity of both heptathiourea derivatives was confirmed by NMR and ESI-MS, as illustrated for 74 in Figure 68 (see Figures S42 and S43 for NMR and Figures S121 and S122 for MS spectra, Supporting Information). As observed for the heptatriazole analogs, NMR spectra of compounds 73 and 74 showed broad signals even at high temperatures. Final carbamate hydrolysis in compounds 71-74 using TFA-DCM afforded the target compounds 75-78 in virtually quantitative yields after lyophilization. 108 Ph.D. Thesis - Iris Pflueger 1.01.52.02.53.03.54.04.55.05.5 ppm 4.85.05.25.4 ppm Figure 68. ESI-MS (top) and 1 H NMR (bottom, 500 MHz, MeOD, 333 K) spectra of compound 74. The set of CD-scaffolded cyclic polyamine clusters synthesized exhibits structural resemblance with the most efficient compounds studied in Chapter 2. Keeping the hydrophobic domain on the secondary rim unaltered, the polycationic head has been modified by introducing cyclic elements. The number and nature of protonable amino groups, however, differ among these compounds. Whereas branched polyamine segments contain two primary amines and one tertiary amine, cyclic polyamines possess three secondary amino groups in the case of the thiourea-tethered derivatives 77 and 78, Chapter 3 109 and an additional tertiary amino group in the case of triazole-linked paCDs 75 and 76. As anticipated by literature, theoretical calculations (MarvinSketch, ChemAxon) predict large disparities in pK a values of each amino group, which, as later demonstrated, exert important effects on the pH buffering abilities of these compounds (Figure 69). Figure 69. Calculated pK a values for acyclic and cyclic polyamine motifs. Cyclen derivative 60 has been also exploited as scaffold for further installing additional oligoamine branches. Thus, a series of isothiocyanate-armed Boc-protected (oligo)amines were prepared according to the sequence depicted in Scheme 17 . Monotritylation of a terminal amino group and Boc-protection of the remaining amines was followed by selective trityl group cleavage. In situ treatment of the resulting monoamines with thiophosgene. furnished the target isothiocyanates derived from ethylenediamine, diethylenetriamine and spermine (12, 186 85, 177 and 86, respectively) in good overall yields. 110 Ph.D. Thesis - Iris Pflueger Scheme 17. Synthesis of aliphatic linear isothiocyanate compounds 22, 186 85 177 and 86. Reagents and conditions: a) EDA (79), DETA (80), spermine (81), TrCl, DCM, 14 h, 89-95%; b) 79 (82), 80 (83), 81 (84), Boc 2 O, DCM, 15 h, 86-93%; c) 82 (22), 83 (85), 84 (86) i, 2% TFA-DCM, 1.5 h; ii, CSCl 2 , CaCO 3 , 1:1 H 2 O-DCM, 1 h, 73-85%. Scheme 18. Synthesis of cyclen-based multivalent polyamino displays 87-89. Reagents and conditions: a) i, 1:1 TFA-DCM, 2 h; ii, HCl, 99%; b) 22 (87), 85 (88), 86 (89), Et 3 N, DCM, 15 h, 96% (87), 88% (88), 68% (89). Chapter 3 111 Cyclen derivative 60 was obtained in almost quantitative yield from N-Boc protected cyclen 63 by acidic hydrolysis of carbamate groups. Coupling of the title isothiocyanates to the cyclen scaffold 60 proceeded smoothly in the presence of Et 3 N and alkyne-armed compounds 87-89 were obtained in good yields (Scheme 18 ) . Scheme 19. CuAAC reaction of CD heptaazide 54 and cyclen-based alkyne 87. Reaction conditions are summarized in Table 5. To finally obtain homogeneous paCDs furnished with extended multivalent patterns of polyamino motifs, both the amphiphilic CD building block 54 and the cyclen-based multivalent polyamino displays 87-89 were then to be coupled by triazole-forming methodology. Unfortunately, this synthetic step turned out to be tricky. Taking advantage of the reaction conditions optimized before for the preparation of cyclen and cyclam-containing paCDs 75-78, in a first attempt the reaction between CD heptaazide derivative 54 and alkyne 87 was carried out in the same conditions used to couple the Boc-protected cyclen derivative 63 (Si-BPA·Cu + in refluxing 3:1 t BuOH-H 2 O, Scheme 19; Entry 1 in Table 5), but the reaction did not run to completion as denoted by NMR or ESI-MS. Integration of characteristic 1 H NMR signals of the coupling adduct indicated a conversion of ca. 12%. Similar results were obtained for the coupling of the other two isothiocyanates 88 and 89, most probably the presence the thiourea tethers playing a role in catalyst inactivation. A variety of reaction conditions, including the use of different Cu(I) sources, solvents, reaction temperatures and times were explored, though none of them led to homogeneous conjugates. Conversion rates were estimated by integration 112 Ph.D. Thesis - Iris Pflueger of characteristic 1 H NMR signals (Table 5) after isolation of the reaction products by size exclusion chromatography. Table 5. Reaction conditions employed for CuAAC reaction of heptaazide 54 and cyclen-based alkyne 87 (1.1 eq relative to azide) shown in Scheme 19. The conversion rate was estimated by integration of characteristic 1 H NMR signals of the purified products. (For entry 5 µw irradiation was used.) Entry Solvent Catalyst T (ºC) t (h) Conversion 1 t BuOH-H 2 O 3:1 Si-BPA·Cu + (30 mg/mmol 87) 85 14 12% 2 t BuOH-H 2 O 3:1 CuI (0.2 eq relative to 87) 85 16 25% 3 anh. DMF (EtO) 3 P·CuI (0.2 eq relative to 87) 85 16 5% 4 anh. DMF (EtO) 3 P·CuI (0.2 eq relative to 87) 100 3 8% 5 t BuOH-H 2 O 3:1 CuI (0.2 eq relative to 87 ) + TBTA (0.2 eq relative to 87) 60 1 36% 6 t BuOH-H 2 O 3:1 CuI (0.2 eq relative to 87 ) + DIPEA (10 eq relative to 87) 85 15 30% Scheme 20. CuAAC reaction of model glucopyranoside 91 with cyclen derivatives 63 and 87. Reagents and conditions: CuI (0.2 eq relative to alkyne), 3:1 t BuOH-H 2 O, rt, 1 h. Chapter 3 113 To probe the origin of such impaired reactivity a series of experiments were carried out. Firstly, 6-azido-6-deoxy-2,3,4-tri-O-methyl-α-D-glucopyranoside 91 280 was used as model compound in order to study its capacity to undergo CuAAC reaction with both tri-N- Boc protected cyclen 63 and thiourea-functionalized analog 87 (Scheme 20). Figure 70. 1 H NMR spectra (500 MHz, MeOD, 323 K) of cyclen derivative 63 in absence (A) and in presence (B) of 1 eq CuI. When CuI was used in catalytic amounts, i.e. 0.2 eq relative to alkyne, coupling of derivative 63 proceeded smoothly. In the case of cyclen derivative 87 no reaction took place under the same conditions, pointing to the Cu(I) complexing ability of the thiourea groups as the origin of catalyst inactivation. However, addition of excess CuI resulted in reaction completion at a rate similar to that observed for tri-N-Boc protected cyclen 63. 1 H NMR spectra of both compounds 63 and 87 were recorded in absence and in presence of equimolar amounts of CuI (Figure 70 and Figure 71). Whereas the 1 H NMR spectrum of cyclen derivative 63 remained unaltered upon addition of CuI, in the case of thioureaderived cyclen 87 a drastic change in its 1 H NMR spectrum could be observed. This alteration clearly indicated complexation of Cu(I) ions by thiourea moieties present on the 120 Ph.D. Thesis - Iris Pflueger purposes, CACs of hexanoylated paCDs 4 and 8 with acyclic polycationic elements have been included in this table. Table 7. Critical aggregation concentrations in aqueous environment of cyclen/cyclam-grafted paCDs 75-78 and paCDs 4 and 8 with acyclic cationic elements. paCD with cyclic amines CAC (µ µµ µM) paCD with acyclic amines CAC (µ µµ µM) 75 2.92 4 0.94 76 2.85 8 2.79 77 0.54 78 0.38 All paCDs studied in this chapter are furnished with the same hexanoyl chains; therefore, possible differences in their hydrophobic/hydrophilic balance would derive exclusively from different polycationic heads. Results, however, showed that CACs of hexanoylated paCDs bearing cyclic or acyclic polycationic elements did not significantly vary, and all of them were in the range from 0.4 µM to 2.9 µM. This indicated that hexanoyl chains were able to counterbalance arrays of seven (paCD 4), 21 (paCDs 8, 77 and 78) and 28 (paCDs 75 and 76) cationic groups on the primary rim respectively, leading to low CACs in all cases. CAC values do not apparently depend on type of cyclic oligoamine (cyclen or cyclam), but on the type of tether, aggregation taking place for thiourea-linked conjugated at lower concentrations than for the corresponding triazoletethered analogs. However, it is unclear whether this is solely due to the nature of the tether or to the number of protonable nitrogen atoms in each series. Buffering capacities of cyclen/cyclam-grafted paCDs 75-78 in the pH range from 5 to 7 were evaluated by potentiometric titration, and compared to that of paCDs 3 and 7 furnished with acyclic cationic elements. The potentiometric titration studies were conducted over a pH range of 9-2. Additionally, model compounds 104-107, featuring identical cationic domains as their CD-scaffolded analogs, were synthesized (Figure 75). Their protonation profiles were determined by acid-base titration and compared to 50 and 51. Chapter 3 121 N HN H NH 3 Cl S N HN H N NH 3 Cl NH 3 Cl S 50 51 N N NNH 2 Cl N N H 2 Cl NH 2 Cl H 2 Cl N N NH 2 Cl NH 2 Cl N HN H SH N S n = 1 106 n = 2 107 n n = 1 104 n = 2 105 n n n Figure 75. Monovalent model compounds used for potentiometric acid-base titrations. Figure 76. Potentiometric titration profiles of monovalent model compounds 50, 51, 104-107. Titrations of NaCl and bPEI solutions are included for comparative purposes. The grey-shaded area represents the pH range where effective buffering ability may enhance lysis of the endosomes. The results are summarized in Figure 76. Similar to model compounds 50 and 51, whose acid-base titration curves drop down rapidly in the pH range between 7 and 5, triazole-bearing model compounds 104 and 105 apparently are devoid of buffering 0 1 2 3 4 5 6 7 8 9 10 11 12 0 0,005 0,01 0,015 0,02 0,025 pH mmol HCl 50 51 104 105 106 107 bPEI NaCl 122 Ph.D. Thesis - Iris Pflueger capabilities in this range. In contrast, thiourea-tethered models 106 and 107 showed a certain buffering capability in this pH range. The potentiometric titration profiles of cyclen/cyclam-grafted paCDs 75-78 as well as paCDs 3 and 7 furnished with acyclic cationic elements are shown in Figure 77. All paCDs exhibited buffering capacities in the pH range from 7 to 5. Whereas buffering capacities of triazole-bearing paCDs 75 and 76 resembled those of paCDs 3 and 7, cyclen/cyclam-grafted paCDs 77 and 78 furnished with thiourea linkers presented considerably improved buffering abilities in this pH range. Figure 77. Potentiometric titration profiles of cyclen/cyclam-grafted paCDs 75-78, and paCDs 3 and 7 with acyclic cationic elements. Titrations of NaCl and bPEI solutions are included for comparative purposes. The grey-shaded area represents the pH range where effective buffering ability may enhance lysis of the endosomes. To study the buffering capacities of paCDs 75-78 and their respective model compounds 104-107 in a more detailed manner, for each compound the equivalents of HCl consumed to switch pH from 7 to 5 have been quantified. A comparison of the results 0 1 2 3 4 5 6 7 8 9 10 11 12 0 0,005 0,01 0,015 0,02 0,025 pH mmol HCl 3 7 75 76 77 78 bPEI NaCl Chapter 3 123 with those obtained for paCDs 3 and 7 and their corresponding model compounds 50 and 51 is shown in Table 8. Table 8. Equivalents of HCl consumed to switch pH from 7 to 5 for paCDs 3, 7 and 75-78, their respective model compounds (50, 51, 104-107) and bPEI. Compound meq of HCl consumed 3 67 7 84 50 17 51 <17 75 67 76 34 77 168 78 118 104 <17 105 <17 106 50 107 50 bPEI 134 The buffering capacities of all paCDs in the pH range 7-5 were better than those of their respective monovalent model compounds, which may indicate that either the multivalency of CD platform or their aggregation abilities improve their buffering capacity. The more favorable protonation profiles of monovalent models 106 and 107, as compared to all other model compounds, suggest that incorporation of cyclic polyamine groups into the CD scaffold via a thiourea tether adjacent to a flexible aliphatic spacer may result in improved buffering capabilities. Indeed, among all CD derivatives studied, cyclen/cyclam-grafted paCDs 77 and 78 furnished with thiourea linkers presented the best buffering capacities, catching up with the golden standard bPEI. Although slightly lower, the buffering capabilities of triazole-bearing paCDs 75 and 76 were similar to those of paCDs 3 and 7. Regarding the type of cyclic polyamine element, i.e. cyclen vs. cyclam, the cyclen motif evidently enhances buffering capacity. For cyclic polyamines grafted to the CD scaffold via triazole or thiourea linkers, cyclen derivatives 75 and 77 showed higher buffering capacities than their cyclam-grafted analogs 76 and 78, respectively. 124 Ph.D. Thesis - Iris Pflueger 4. Assessment of DNA-condensing capabilities of cyclen/cyclam-grafted paCDs To study the influence of cyclic polyamine headgroups on DNA-condensing capabilities the CDplex formation capabilities of paCDs 75-78 were critically compared to paCDs grafted with acyclic oligoamines (Figure 78). First, the paCD-DNA nanoparticles were characterized by dynamic light scattering (DLS) in order to determine their average hydrodynamic size and ζ potential using ctDNA at N/P ratios 5 and 10. Figure 78. Library of cyclen/cyclam-grafted paCDs (75-78) and paCDs synthesized in Chapter 2 used for comparative purposes (3, 4, 7 and 8). The DLS results showed that all assayed cyclen/cyclam-grafted paCDs formed compact and stable nanoparticles with ctDNA that, with the exception of compound 75 at N/P ratio 10, exhibited rather small hydrodynamic diameters as compared with the polyplexes obtained from 25 kDa bPEI (80-100 nm vs. 150 nm; Figure 79). Chapter 3 125 Figure 79. Size (hydrodynamic diameter, bars, nm) and ζ potential (lines, mV) of CDplexes of paCDs 75-78 determined by DLS and M3-PALS analysis, respectively (for comparative purposes, CDplexes of paCDs 4 and 8 are included). Hydrodynamic diameter measurements indicated that modifications of the molecular structure of paCDs did not influence particle size significantly. Moreover, the N/P ratio did scarcely affect particle hydrodynamic diameter. Although nanoparticles of paCD derivative 75 exhibited an extraordinary large hydrodynamic diameter at N/P ratio 10, in general no substantial difference between analogous cyclen and cyclam-derived paCDs could be disclosed. Furthermore, the nature of the linking element marginally affected particle size, since paCDs 75 and 76 furnished with rigid and short methylene triazole segments and their respective analogs 77 and 78 bearing long and flexible aliphatic tethers and thiourea groups led to CDplex nanoparticles of almost the same size. These facts suggest that, despite their architectural disparity, the overall hydrophilic/hydrophobic balance is barely altered. Similar to the results obtained for acyclic oligoamines, narrow populations of cationic nanoparticles were observed in each individual experiment, thus indicating rather monodisperse nanoparticles (Figure 80). Such remarkable behavior (relatively low -70 -50 -30 -10 10 30 50 70 90 0 25 50 75 100 125 150 75 76 77 78 4 8 ζ ζ ζ ζ (mV), lines Size (nm), bars N/P ratio of 5 N/P ratio of 10 126 Ph.D. Thesis - Iris Pflueger particle size and polydispersity) makes CD-scaffolded cyclic polyamine clusters suitable candidates for artificial gene carriers. Figure 80. Particle size distribution by volume determined by DLS for ctDNA:78 CDplexes formulated at N/P 5 (diameter in nm). M3-PALS measurements at both N/P ratios 5 and 10 revealed that ζ potential of CDplexes formulated with cyclen/cyclam-grafted paCDs 75-78 ranked in the range 40-60 mV, amenable values for cell-based assays. ζ Potential values were marginally depended on the N/P ratio, thus indicating that complex stoichiometry does not change significantly upon an increase of the N/P ratio. We hypothesize that excess paCD does not incorporate into the CDplex nanostructure. ζ Potential of CDplexes formulated with paCDs 4 and 8, however, were slightly higher than those with cyclen/cyclam-grafted paCDs (50-80 mV for paCDs 4 and 8 vs. 40-60 mV for paCDs 75-78, respectively). Altogether, DLS experiments pointed out that cyclen/cyclam-grafted paCDs 75-78 were able to form stable nanoparticles with DNA. These nanoparticles all featured remarkably similar hydrodynamic diameters, positive ζ potentials and relatively narrow size distributions. Subsequently, their capabilities to compact and protect DNA were investigated by submitting the cyclen or cyclam-grafted paCDs 75-78 to agarose gel electrophoresis shift 0 2 4 6 8 10 12 14 1 10 100 1000 10000 Volume (%) Size (d.nm) Size Distribution by Volume Chapter 3 127 assays. Therefore, paCD:ctDNA complexes were formulated in the same manner as described before for different N/P ratios in the range of 1 to 10. Naked ctDNA was used for comparative purposes. Complete inhibition of DNA migration indicated complex formation, whereas absence of fluorescent staining denoted efficient compaction and protection of DNA in the CDplexes of the corresponding lanes (Figure 81). At N/P ratios 1 or higher, formation of the respective CDplexes was achieved for all assayed paCDs, which was indicated by the absence of “free” mobile plasmid. Starting from N/P ratio 2, both cyclen and cyclam grafted paCDs were in addition capable to efficiently compact and protect DNA in their CDplexes. Although cyclen and cyclam moieties exhibit different spatial orientation of the cationic amino groups, no appreciable difference in their DNA-condensing capabilities could be inferred and efficient covering of the DNA surface by both cyclen and cyclam grafted paCDs has been clearly evidenced. Figure 81. paCD-mediated protection of ctDNA from GelRed™ intercalation at different N/P ratios (1-10) for cyclen/cyclam-grafted paCDs 75-78 and paCDs with acyclic polycationic elements 3, 4, 7 and 8. 128 Ph.D. Thesis - Iris Pflueger However, when triazole bearing paCDs 75 and 76 were compared with their thiourea analogs 77 and 78, respectively, a remarkable difference became apparent. Whereas triazole paCDs 75 and 76 were only capable of efficiently compacting and protecting DNA at N/P ratios 5 or higher, thiourea derivatives 77 and 78 already turned DNA inaccessible to the GelRed™ intercalating agent at N/P ratio 2. This difference might be attributed to the superior ability of thiourea segments to complement electrostatic phosphate binding via hydrogen-bonding interactions as compared to triazole tethers. Interestingly, the additional protonable nitrogen atom in the triazole-linked series, as compared to the thiourea-tethered one, does not compensate the beneficial effects added by the thiourea moieties. The presumably low pK a value of the additional tertiary amine in the triazolederived paCDs does not seem to favor DNA compaction and protection. 256,257 Compared to the library of paCDs prepared in Chapter 2, which differ in the length of the lipophilic chain and are furnished with either 7 or 21 protonable amino groups, cyclen or cyclam-grafted paCDs exhibited DNA compactation and protection capabilities similar to those of linear and branched paCDs bearing butanoyl or hexanoyl chains on the secondary rim. At N/P ratio 1 cyclen and cyclam-derived paCDs showed slightly better compactation capabilities as they completely inhibited DNA migration, while linear paCDs 3 and 4 as well as branched paCDs 7 and 8, respectively, still demonstrated partial DNA migration. Nonetheless, regarding DNA protection, the most efficient paCDs prepared in Chapter 2 performed as well as cyclen or cyclam-derived paCDs, as all of them efficiently compacted and protected DNA at N/P ratios 2 or 5 at most. Chapter 3 129 5. Gene transfer capabilities towards COS-7 and HeLa cell lines Similarly to that reported in the preceding chapter, the gene transfer efficiency of CDplexes formed from cyclic amine-grafted paCDs was evaluated using the luciferaseencoding gene (pTG11236, pCMV-SV40-luciferase-SV40pA) as reporter towards COS-7 and HeLa cell lines. The experiments towards COS-7 cells were developed using in-house facilities to comparatively assess the performance of triazole-tethered paCDs 75 and 76 and thiourea-tethered CDs 77 and 78 (Figure 82) in the absence of serum. JetPEI and naked pDNA were included as positive and negative controls, respectively. In addition, paCD 8, furnished with a cluster of acyclic oligoamines, 177 was also included in the experiments as positive control (Figure 82). Figure 82. Library of cyclen/cyclam-grafted paCDs 75-78 and acyclic reference paCD 8 screened as pDNA carriers towards COS-7 and HeLa cell lines. 232 Ph.D. Thesis - Iris Pflueger 4,9,13-Tris(tert-butoxycarbonyl)-4,9,13-triazatridecyl-1-isothiocyanate (86). Compound 84 (229 mg, 0.23 mmol) was treated with a solution of 2% TFA in DCM (5 mL) at rt for 1.5 h. Then, the solvent was removed to yield crude 1,4,9-tris(tertbutoxycarbonyl)spermine which subsequently was suspended in a 1:1 mixture of H 2 O- DCM (5 mL). CaCO 3 (232 mg, 2.32 mmol, 10 eq) was added and the mixture was vigorously stirred for a few minutes. The suspension was then cooled to 0 ºC, CSCl 2 (27 µL, 0.35 mmol, 1.5 eq) was added and the reaction mixture was further stirred for 1 h. The organic phase was decanted, the solvent was evaporated and the resulting residue was purified by flash column chromatography (1:2 EtOAc-petroleum ether) to yield compound 86 (92 mg, 73%). R f = 0.34 (1:2 EtOAc-petroleum ether). 1 H NMR (500 MHz, CDCl 3 , Figure S50): δ = 5.29 (NHBoc), 3.56 (t, 2 H, 3 J H,H = 6.5 Hz, H- 1), 3.30 (t, 2 H, 3 J H,H = 6.6 Hz, H-10), 3.20, 3.11 (2 m, 8 H, H-3, H-4, H-7, H-8), 1.92 (m, 2 H, H-9), 1.66 (m, 2 H, H-2), 1.50 (m, 4 H, H-5, H-6), 1.47 (bs, 18 H, C(CH 3 ) 3 ), 1.44 (s, 9 H, C(CH 3 ) 3 ). 13 C NMR (125.7 MHz, CDCl 3 , Figure S50): δ = 155.8, 155.2 (CO), 130.4 (NCS), 79.6, 79.3, 78.6(C(CH 3 ) 3 ), 47.3 (C-1), 46.6, 46.0 (C-4, C-7), 43.9, 42.6 (C-3, C-8), 37.4 (C-10), 28.8 (C-2), 28.2 (C(CH 3 ) 3 ), 25.7 (C-9), 25.3 (C-5, C-6). ESI-MS (Figure S129): m/z 567.5 [M + Na] + , 583.4 [M + K] + , 543.4 [M – H] - , 579.5 [M + Cl] - . Elemental analysis calculated (%) for C 26 H 48 N 4 O 6 S: C 57.33, H 8.88, N 10.28, S 5.89; found: C 57.53, H 8.99, N 9.95, S 6.01. Experimental Part 233 1,4,7-Tris(N’-(2-tert-butoxycarbonylaminoethyl)thioureido)-10-propargyl-1,4,7,10- tetraazacyclododecane (87). To a solution of compound 60 274 (135 mg, 0.42 mmol) and Et 3 N (212 µL, 1.53 mmol, 1.2 eq) in DCM (8 mL) a solution of 2-(tertbutoxycarbonylamino)ethylisothiocyanate 22 186 (284 mg, 1.40 mmol, 1.1 eq) in DCM (2 mL) was added drop wise. The reaction mixture was stirred overnight at rt maintaining pH 9. After evaporation of the solvent under reduced pressure, the resulting residue was purified by flash column chromatography (1:1 EtOAc-petroleum ether) yielding compound 87 as a white solid (333 mg, 96%). R f = 0.66 (1:1 EtOAc-petroleum ether). 1 H NMR (400 MHz, MeOD, 333 K, Figure S51): δ = 4.04 (m, 4 H, H-3, H-5), 3.89 (m, 8 H, H-2, H-6, H-8, H-12), 3.77 (t, 2 H, 3 J H,H = 6.0 Hz, H-1’’), 3.71 (bs, 2 H, CH 2 C≡CH), 3.68 (t, 4 H, 3 J H,H = 6.0 Hz, H-1’), 3.36 (t, 2 H, 3 J H,H = 6.1 Hz, H-2’’), 3.28 (t, 4 H, 3 J H,H = 6.1 Hz, H- 2’), 2.98 (m, 4 H, H-9, H-11), 2.62 (m, 1 H, C≡CH), 1.46 (s, 27 H, C(CH 3 ) 3 ). 13 C NMR(100.6 MHz, MeOD, 333 K, Figure S51): δ = 183.0, 182.4 (CS), 157.4 (CO), 79.1 (C(CH 3 ) 3 ), 77.3 (C≡CH), 74.0 (C≡CH), 53.5 (C-9, C-11), 50.5 (C-2, C-3, C-5, C-6, C- 8, C-12), 46.0 (C-1’, C-1’’), 40.4 (CH 2 C≡CH), 39.8 (C-2’, C-2’’), 27.5 (C(CH 3 ) 3 ). ESI-MS (Figure S130): m/z 839.4 [M + Na] + , 855.4 [M + K] + , 815.3 [M – H] - , 851.2 [M + Cl] - . Elemental analysis calculated (%) for C 35 H 64 N 10 O 6 S 3 : C 51.44, H 7.89, N 17.14, S 11.77; found: C 51.64, H 7.82, N 16.84, S 11.39. 234 Ph.D. Thesis - Iris Pflueger 1,4,7-Tris[N’-(2-(N-(tert-butoxycarbonyl)-N-(2-(tert-butoxycarbonylamino)ethyl)- amino)ethyl)]thioureido-10-propargyl-1,4,7,10-tetraazacyclododecane (88). To a solution of compound 60 274 (226 mg, 0.71 mmol) and Et 3 N (355 µL, 2.56 mmol, 1.2 eq) in DCM (9 mL) a solution of 2-[N-(tert-butoxycarbonyl)-N-[2-(tertbutoxycarbonylamino)ethyl]amino]ethyl isothiocyanate 85 177 (810 mg, 2.34 mmol, 1.1 eq) in DCM (5 mL) was added drop wise. The reaction mixture was stirred overnight at rt maintaining pH 9. After evaporation of the solvent under reduced pressure, the resulting residue was purified by flash column chromatography (2:1 EtOAc-petroleum ether) yielding compound 88 as a white solid (782 mg, 88%). R f = 0.56 (2:1 EtOAc-petroleum ether). 1 H NMR (500 MHz, MeOD, 333 K, Figure S52): δ = 4.03 (m, 4 H, H-3, H-5), 3.85 (m, 8 H, H-2, H-6, H-8, H-12), 3.73 (t, 6 H, 3 J H,H = 6.1 Hz, H-1’), 3.68 (bs, 2 H, CH 2 C≡CH), 3.53 (t, 2 H, 3 J H,H = 6.1 Hz, H-2’), 3.47 (t, 4 H, 3 J H,H = 6.1 Hz, H-2’), 3.41-3.35 (m, 6 H, H-3’), 3.25, 3.24 (2 t, 6 H, 3 J H,H = 6.2 Hz, H-4’), 2.97 (m, 4 H, H-9, H-11), 2.62 (t, 1 H, 4 J H,H = 2.2 Hz, C≡CH), 1.50, 1.46 (2 s, 27 H each, C(CH 3 ) 3 ). 13 C NMR (100.6 MHz, MeOD, 333 K, Figure S52): δ = 182.9, 182.4 (CS), 156.8, 156.5 (CO), 80.1, 78.8 (C(CH 3 ) 3 ), 77.4 (C≡CH), 74.0 (C≡CH), 53.7 (C-9, C-11), 50.5 (C-2, C-3, C-5, C-6, C-8, C-12), 46.6 (C-2’, C-3’), 44.2 (C-1’), 40.7 (CH 2 C≡CH), 39.0 (C-4’), 27.6, 27.5 (C(CH 3 ) 3 ). ESI-MS (Figure S131): m/z 1268.7 [M + Na] + , 1284.6 [M + K] + . Elemental analysis calculated (%) for C 56 H 103 N 13 O 12 S 3 : C 53.95, H 8.33, N 14.61, S 7.72; found: C 53.60, H 7.99, N 14.67, S 7.84. Experimental Part 235 1,4,7-Tris[N’-(3-((4-((3-(tert-butoxycarbonyl)amino)propyl)(tert-butoxycarbonyl)- amino)butyl)(tert-butoxycarbonyl)amino)propyl)]thioureido-10-propargyl-1,4,7,10- tetraazacyclododecane (89). To a solution of compound 60 274 (27 mg, 83 µmol) and Et 3 N (42 µL, 0.30 mmol, 1.2 eq) in DCM (5 mL) a solution of compound 86 (150 mg, 0.28 mmol, 1.1 eq) in DCM (2 mL) was added drop wise. The reaction mixture was stirred overnight at rt maintaining pH 9. After evaporation of the solvent under reduced pressure, the resulting residue was purified by flash column chromatography (1:1 EtOAc-petroleum ether) yielding compound 89 as a white solid (105 mg, 68%). R f = 0.38 (2:1 EtOAc-petroleum ether). 1 H NMR (400 MHz, MeOD, 333 K, Figure S53): δ = 4.05 (m, 4 H, H-3, H-5), 3.89 (m, 8 H, H-2, H-6, H-8, H-12), 3.71 (bs, 2 H, CH 2 C≡CH), 3.68 (t, 2 H, 3 J H,H = 6.7 Hz, H-1’), 3.60 (t, 4 H, 3 J H,H = 6.7 Hz, H-1’), 3.35 (t, 2 H, 3 J H,H = 6.7 Hz, H-3’), 3.26 (m, 22 H, H-3’, H-4’, H- 7’, H-8’), 3.07 (m, 6 H, H-10’), 2.99 (m, 4 H, H-9, H-11), 2.63 (t, 1 H, 4 J H,H = 2.3 Hz, C≡CH), 1.92 (m, 2 H, H-2’), 1.85 (m, 4 H, H-2’), 1.73 (m, 6 H, H-9’), 1.56 (m, 12 H, H-5’, H-6’), 1.50 (s, 9 H, C(CH 3 ) 3 ), 1.49 (s, 18 H, C(CH 3 ) 3 ), 1.48 (s, 27 H, C(CH 3 ) 3 ), 1.46 (s, 27 H, C(CH 3 ) 3 ). 13 C NMR (100.6 MHz, MeOD, 333 K, Figure S53): δ = 182.6, 182.0 (CS), 156.9, 156.2, 156.1 (CO), 79.7, 79.6, 78.7 (C(CH 3 ) 3 ), 77.4 (C≡CH), 74.0 (C≡CH), 53.6 (C-9, C-11), 50.8 (C-2, C-3, C-5, C-6, C-8, C-12), 44.6 (C-3’, C-4’, C-7’, C-8’), 43.2 (C-1’), 40.6 (CH 2 C≡CH), 37.8 (C-10’), 29.2 (C-9’), 27.5 (C-2’), 27.5 (C(CH 3 ) 3 ), 25.6 (C-5’, C-6’). ESI-MS (Figure S132): m/z 942.4 [M + 2 Na] 2+ , 1867.0 [M + Na] + , 1843.1 [M – H] - , 1878.0 [M + Cl] - . 236 Ph.D. Thesis - Iris Pflueger Elemental analysis calculated (%) for C 89 H 166 N 16 O 18 S 3 : C 57.95, H 9.07, N 12.15; found: C 58.17, H 9.23, N 12.48. Heptakis[6-(4-(4,7,10-tris(N’-(2-tert-butoxycarbonylaminoethyl)thioureido)-1,4,7,10- tetraazacyclododecyl-1-methyl)-1H-1,2,3-triazol-1-yl)-6-deoxy-2,3-di-O-hexanoyl]- cyclomaltoheptaose (90). To a solution of heptakis(6-azido-6-deoxy-2,3-di-O- hexanoyl)cyclomaltoheptaose 54 180b (8.5 mg, 3.2 µmol) and compound 87 (20 mg, 25 µmol, 1.1 eq) in acetonitrile (0.5 mL) bis(triphenylphosphine)copper(I)iodide 93 289 (1.8 mg, 2.5 µmol, 0.1 eq relative to the alkyne) was added and the reaction mixture was heated under microwave irradiation to 85 ºC for 1 h (60 W). Then, the solvent was evaporated and the resulting residue was purified by size exclusion chromatography in MeOH to yield compound 90 (20 mg, 75%). As determined by integration of characteristic 1 H NMR signals, the average degree of substitution of heterogeneous compound 90 was estimated 85%. R f = 0.61 (9:1 DCM-MeOH). [α] D = +19.5 (c = 1.0 in MeOH). 1 H NMR (500 MHz, MeOD, 323 K, Figure S54): δ = 7.82 (bs, 6 H, CH triazole ), 5.72-4.65 (m, 35 H, H-1, H-2, H-3, H-6), 4.33-3.52 (m, 126 H, H-4, H-5, CH 2triazole , H-1’’,H-3’, H-5’, H-6’, H-8’, H-9’, H-11’), 3.52-3.10 (m, 35 H, H-2’’), 3.05-2.63 (m, 24 H, H-2’, H-12’), 2.60-2.09 (m, 28 H, CH 2 CO), 1.64 (m, 28 H, CH 2 CH 2 CO), 1.46 (bs, 149 H, C(CH 3 ) 3 ), 1.37 (m, 56 H, CH 3 CH 2 , CH 3 CH 2 CH 2 ), 0.95 (m, 42 H, CH 3 ). Experimental Part 237 Heptakis[6-(4-(4,7,10-tris(N’-(2-N-((N-tert-butoxycarbonylaminoethyl)tert-butoxy- carbonyl)amino)ethyl)thioureido)-1,4,7,10-tetraazacyclododecyl-1-methyl)-1H-1,2,3- triazol-1-yl)-6-deoxy-2,3-di-O-hexanoyl]cyclomaltoheptaose (95). To a solution of heptakis(6-azido-6-deoxy-2,3-di-O-hexanoyl)cyclomaltoheptaose 54 180b (5.6 mg, 2.1 µmol) and compound 88 (20 mg, 16 µmol, 1.1 eq) in acetonitrile (0.5 mL) bis(triphenylphosphine)copper(I)iodide 93 289 (1.2 mg, 1.6 µmol, 0.1 eq relative to the alkyne) was added and the reaction mixture was heated under microwave irradiation to 85 ºC for 1 h (60 W). Then, the solvent was evaporated and the resulting residue was purified by size exclusion chromatography in MeOH to yield compound 95 (12 mg, 50%). As determined by integration of characteristic 1 H NMR signals, the average degree of substitution of heterogeneous compound 95 was estimated 80%. R f = 0.26 (9:1 DCM-MeOH). [α] D = +15.6 (c = 0.6 in MeOH). 1 H NMR (500 MHz, MeOD, 323 K, Figure S55): δ = 8.10 (bs, 5 H, CH triazole ), 6.40-4.64(m, 35 H, H-1, H-2, H-3, H-6), 4.39-3.63 (m, 92 H, H-4, H-5, CH 2triazole , H-3’, H-5’, H-6’, H-8’, H-9’, H-11’), 3.59-3.35 (m, 101 H, H-1’’, H-2’’, H-3’’), 3.25 (m, 34 H, H-4’’), 3.06-2.66 (m, 22 H, H-2’, H-12’), 2.56-2.17 (m, 28 H, CH 2 CO), 1.63 (m, 28 H, CH 2 CH 2 CO), 1.50, 1.46 (2 bs, each 151 H, C(CH 3 ) 3 ), 1.37 (m, 56 H, CH 3 CH 2 , CH 3 CH 2 CH 2 ), 0.95 (m, 42 H, CH 3 ). 238 Ph.D. Thesis - Iris Pflueger Heptakis[6-(4-(4,7,10-tris(N’-(-(3-((4-((3-(tert-butoxycarbonyl)amino)propyl)(tertbutoxycarbonyl)amino)butyl)(tert-butoxycarbonyl)amino)propyl))thioureido)- 1,4,7,10-tetraazacyclododecyl-1-methyl)-1H-1,2,3-triazol-1-yl)-6-deoxy-2,3-di-O- hexanoyl]cyclomaltoheptaose (96). To a solution of heptakis(6-azido-6-deoxy-2,3-di-O- hexanoyl)cyclomaltoheptaose 54 180b (4.7 mg, 1.8 µmol) and compound 89 (25 mg, 13.6 µmol, 1.1 eq) in acetonitrile (0.5 mL) bis(triphenylphosphine)copper(I)iodide 93 289 (1.0 mg, 1.4 µmol, 0.1 eq relative to the alkyne) was added and the reaction mixture was heated under microwave irradiation to 85 ºC for 1 h (60 W). Then, the solvent was evaporated and the resulting residue was purified by size exclusion chromatography in MeOH to yield compound 96 (8.4 mg, 31%). As determined by integration of characteristic 1 H NMR signals, the average degree of substitution of heterogeneous compound 96 was estimated 73%. R f = 0.25 (9:1 DCM-MeOH). [α] D = +6.37 (c = 0.4 in MeOH). 1 H NMR (500 MHz, MeOD, 323 K, Figure S56): δ = 7.85 (bs, 5 H, CH triazole ), 5.70-4.81 (m, 35 H, H-1, H-2, H-3, H-6), 3.78-3.50 (m, 32 H, H-4, H-5, CH 2triazole , H-1’’, H-3’, H-5’, H-6’, H-8’, H-9’, H-11’), 3.32-3.20 (m, 122 H, H-3’’, H-4’’, H-7’’, H-8’’), 3.07 (t, 31 H, 3 J H,H = 6.4 Hz, H-10’’), 3.00-2.81 (m, 20 H, H-2’, H-12’), 2.57-2.17 (m, 28 H, CH 2 CO), 1.98-1.79 (m, 31 H, H-2’’), 1.72 (m, 31 H, H-9’’), 1.61 (m, 28 H, CH 2 CH 2 CO), 1.56 (m, 61 H, H-5’’, H-6’’), 1.49, 1.46 (2 bs, 414 H, C(CH 3 ) 3 ), 1.37 (m, 56 H, CH 3 CH 2 , CH 3 CH 2 CH 2 ), 0.95 (m, 42 H, CH 3 ). Experimental Part 239 Heptakis[6-(4-(4,7,10-tris(N’-(2-aminoethyl)thioureido)-1,4,7,10-tetraazacyclo- dodecyl-1-methyl)-1H-1,2,3-triazol-1-yl)-6-deoxy-2,3-di-O-hexanoyl]cyclomaltoheptaose henicosahydrochloride (97). Treatment of compound 90 (20 mg, 2.4 µmol) with TFA following the procedure described in the general methods, followed by freezedrying from a 0.1 N aq. HCl solution, gave compound 97 in quantitative yield (15 mg). [α] D = +18.3 (c = 0.4 in MeOH). 1 H NMR (500 MHz, MeOD, 323 K, Figure S57): δ = 7.86 (bs, 6 H, CH triazole ), 5.72-4.61 (m, 35 H, H-1, H-2, H-3, H-6), 4.42-3.59 (m, 126 H, H-4, H-5, CH 2triazole , H-1’’, H-3’, H-5’, H-6’, H-8’, H-9’, H-11’), 3.49-3.18 (m, 35 H, H-2’’), 3.07-2.63 (m, 24 H, H-2’, H-12’), 2.62-2.15 (m, 28 H, CH 2 CO), 1.65 (m, 28 H, CH 2 CH 2 CO), 1.38 (m, 56 H, CH 3 CH 2 , CH 3 CH 2 CH 2 ),0.95 (m, 42 H, CH 3 ). 240 Ph.D. Thesis - Iris Pflueger Heptakis[6-(4-(4,7,10-tris(N’-(2-(N-2-aminoethyl)aminoethyl)thioureido)-1,4,7,10- tetraazacyclododecyl-1-methyl)-1H-1,2,3-triazol-1-yl)-6-deoxy-2,3-di-O- hexanoyl]cyclomaltoheptaose dotetracontahydrochloride (98). Treatment of compound 95 (11 mg, 1.0 µmol) with TFA following the procedure described in the general methods, followed by freeze-drying from a 0.1 N aq. HCl solution, gave compound 98 in quantitative yield (8.4 mg). [α] D = +15.0 (c = 0.6 in MeOH). 1 H NMR (500 MHz, MeOD, 323 K, Figure S58): δ = 7.85 (bs, 5 H, CH triazole ), 5.66-4.68 (m, 35 H, H-1, H-2, H-3, H-6), 4.40-3.85 (m, 92 H, H-4, H-5, CH 2triazole , H-3’, H-5’, H-6’, H-8’, H-9’, H-11’), 3.78-3.40 (m, 134 H, H-1’’, H-2’’, H-3’’, H-4’’), 2.66-2.07 (m, 50 H, CH 2 CO, H-2’, H-12’), 1.65 (m, 28 H, CH 2 CH 2 CO), 1.38 (m, 56 H, CH 3 CH 2 , CH 3 CH 2 CH 2 ), 0.96 (m, 42 H, CH 3 ). Experimental Part 241 Heptakis[6-(4-(4,7,10-tris(N’-(3-((4-(3-aminopropyl)aminobutyl)aminopropyl))thioureido)-1,4,7,10-tetraazacyclododecyl-1-methyl)-1H-1,2,3-triazol-1-yl)-6-deoxy-2,3- di-O-hexanoyl]cyclomaltoheptaose trishexacontahydrochloride (99). Treatment of compound 96 (8 mg, 0.5 µmol) with TFA following the procedure described in the general methods, followed by freeze-drying from a 0.1 N aq. HCl solution, gave compound 99 in quantitative yield (6 mg). [α] D = +7.86 (c = 0.4 in MeOH). 1 H NMR (500 MHz, MeOD, 323 K, Figure S59): δ = 7.86 (bs, 5 H, CH triazole ), 5.66-4.64 (m, 35 H, H-1, H-2, H-3, H-6), 4.44-3.44 (m, 32 H, H-4, H-5, CH 2triazole , H-1’’, H-3’, H-5’, H-6’, H-8’, H-9’, H-11’), 3.28-3.10 (m, 173 H, H-3’’, H-4’’, H-7’’, H-8’’, H-10’’, H-2’, H-12’), 2.52- 1.87 (m, 151 H, CH 2 CO, H-2’’, H-5’’, H-6’’, H-9’’), 1.65 (m, 28 H, CH 2 CH 2 CO), 1.38 (m, 56 H, CH 3 CH 2 , CH 3 CH 2 CH 2 ), 0.95 (m, 42 H, CH 3 ). 1,4,7-Tris(tert-butoxycarbonyl)-10-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-1,4,7,10- tetraazacyclododecane (100). To a solution of compound 63 (50 mg, 97.9 µmol) and 248 Ph.D. Thesis - Iris Pflueger Elemental analysis calculated (%) for C 25 H 48 Cl 3 N 7 S 2 : C 48.65, H 7.84, N 15.89, S 10.39; found: C, H, N, S. 3.3. New Compounds synthesized in Chapter 4 6,6’-Diazido-6,6’-dideoxy-2,2’,3,3’,4,4’-hexa-O-methyl-α,α’-trehalose (109). To a solution of 6,6’-diazido-6,6’-dideoxy-α,α’-trehalose 108 269 (0.56 g, 1.31 mmol) in dry DMF (15 mL) under N 2 atmosphere at 0 ºC NaH (0.63 g, 15.8 mmol, 2 eq, 60% dispersion in mineral oil) and methyl iodide (0.98 mL, 15.8 mmol, 2 eq) were sequentially added. The reaction mixture was allowed to warm to rt and stirred overnight. Then, the reaction was quenched by addition of water (15 mL). The reaction mixture was then concentrated under reduced pressure and the residue was taken in DCM (15 mL) and washed with water (2 x 15 mL). The organic layer was dried over Na 2 SO 4 , filtered and evaporated to dryness. The crude product was purified by flash column chromatography (1:2 EtOAcpetroleum ether) to furnish 109 in 72% yield (0.45 g). R f = 0.41 (1:2 EtOAc-petroleum ether). [α] D = +161.8 (c = 1.0 in MeOH). 1 H NMR (500 MHz, CDCl 3 , Figure S68): δ = 5.19 (d, 2 H, J 1,2 = 3.7 Hz, H-1), 4.07 (ddd, 2 H, J 4,5 = 9.9 Hz, J 5,6b = 5.5 Hz, J 5,6a = 2.7 Hz, H-5), 3.64, 3.57, 3.48 (3 s, 6 H each, OCH 3 ), 3.51 (t, 2 H, J 2,3 = J 3,4 = 9.2 Hz, H-3), 3.45 (m, 4 H, H-6a, H-6b), 3.20 (dd, 2 H, H-2), 3.08 (dd, 2 H, H-4). 13 C NMR (125.7 MHz, CDCl 3 , Figure S68): δ = 93.2 (C-1), 82.8 (C-3), 81.6 (C-2), 80.4 (C- 4), 70.5 (C-5), 60.8, 60.7, 58.6 (OCH 3 ), 51.5 (C-6). ESI-MS (Figure S141): m/z 499.5 [M + Na] + , 515.5 [M + K] + . Experimental Part 249 Elemental analysis calculated (%) for C 18 H 32 N 6 O 9 : C 45.37, H 6.77, N 17.64; found: C 45.32, H 6.66, N 17.41. RP-HPLC: t R = 10.5 min (according to the RP-HPLC analytical procedure described in the General Methods section). N-Benzyl-4-(diphenylphosphino)benzamide (116). To a solution of 4- (diphenylphosphino)benzoic acid 112 334 (0.68 g, 2.2 mmol) in DMF (20 mL) DIPEA (0.42 mL, 2.5 mmol, 1.1 eq) and TBTU (0.72 g, 2.2 mmol, 1 eq) were sequentially added. The reaction mixture was stirred for 5 min and then benzylamine (0.24 mL, 2.2 mmol, 1 eq) was added. The reaction mixture was stirred at rt overnight. The solvent was evaporated and the residue was taken in DCM, and successively washed with 1 M aq. HCl, saturated aq. NaHCO 3 , brine and H 2 O. The organic layer was dried over Na 2 SO 4 , filtered and evaporated to dryness. The crude product was purified by flash column chromatography (1:6  1:3 EtOAc-petroleum ether) to afford 116 as a white solid (729 mg, 83%). R f = 0.55 (1:3 EtOAc-petroleum ether). 1 H NMR (500 MHz, CDCl 3 , Figure S69): δ =7.75 (d, 2 H, 3 J H,H = 7.6 Hz, CHCCONH), 7.38-7.29 (m, 17 H, Ar), 6.46 (bs, 1 H, NH), 4.66 (d, 2 H, 3 J H,H = 5.6 Hz, CH 2 ). 13 C NMR (125.7 MHz, CDCl 3 , Figure S69): δ = 167.0 (CO), 138.1 (PCCHCHCCONH), 136.4, 136.3 (CP), 134.4 (CCH 2 NH), 134.0, 133.8 (PCCH), 133.7, 133.5 (PCCHCHCCONH), 129.1 (CCONH, CHCHCCH 2 NH), 128.8 (PCCHCHCH), 128.7, 128.6 (PCCHCH), 127.9 (CHCCH 2 NH), 127.7 (CHCHCHCCH 2 NH), 126.9, 126.8 (CHCCONH), 44.2 (CH 2 ). 31 P NMR (202.5 MHz, CDCl 3 , Figure S69): δ = -6.21 (s). ESI-MS (Figure S142): m/z 396.4 [M + H] + , 418.4 [M + Na] + , 434.3 [M + K] + , 394.2 [M – H] - . 250 Ph.D. Thesis - Iris Pflueger Elemental analysis calculated (%) for C 26 H 22 NOP: C 78.97, H 5.61, N 3.54; found: C 78.48, H 5.82, N 3.42. [4-(Diphenylphosphino)phenoxy]acetic acid (118). To a solution of (4- iodophenoxy)acetic acid 117 (1.39 g, 5 mmol) and Et 3 N (1.39 mL, 10 mmol, 2 eq) in degassed MeCN (30 mL) under N 2 Pd(AcO) 2 (1.1 mg, 5 µmol, 0.1% eq) and diphenylphosphine (0.87 mL, 5 mmol, 1 eq) were sequentially added. The intensely orange-red colored reaction mixture was refluxed at 85 °C for 12 h. The volatiles were then evaporated and the residue was suspended in water (15 mL), taken to basic pH by adding KOH (0.56 g, 10 mmol, 2 eq) and washed with Et 2 O (3 x 20 mL). The aqueous layer was then acidified to pH 2 by addition of 1.8 M HCl and extracted with Et 2 O (3 x 20 mL). The collected organic phase was washed with water (15 mL), dried over Na 2 SO 4 , filtered and evaporated to dryness. The crude product was purified by flash column chromatography (1:2  2:1 EtOAc-petroleum ether) to afford 118 as a white solid (1.57 g, 93%). R f = 0.35 (1% AcOH in 2:1 EtOAc-petroleum ether). 1 H NMR (400 MHz, CDCl 3 , Figure S70): δ = 9.04 (bs, 1 H, COOH), 7.38-7.28 (m, 12 H, Ar), 6.93 (dd, 2 H, 3 J H,H = 8.8 Hz, 4 J H,P = 0.9 Hz, CHCOCH 2 COOH), 4.71 (s, 2 H, CH 2 ). 13 C NMR (100.6 MHz, CDCl 3 , Figure S70): δ = 173.6 (CO), 158.1 (COCH 2 COOH), 138.5 (PCCHCHCOCH 2 COOH), 137.5, 137.4 (CP), 135.8, 135.6 (PCCHCHCOCH 2 COOH), 133.6, 133.4 (PCCH), 128.7, 128.6, 128.5 (PCCHCH, PCCHCHCH), 114.9, 114.8 (CHCOCH 2 COOH), 64.7 (CH 2 ). 31 P NMR (162 MHz, CDCl 3 , Figure S70): δ = -7.02 (s). ESI-MS (Figure S143): m/z 335.0 [M – H] - . Elemental analysis calculated (%) for C 20 H 17 O 3 P: C 71.42, H 5.09; found: C 71.54, H 5.38. Experimental Part 251 N-Benzyl-[4-(diphenylphosphino)phenoxy]acetamide (119). To a solution of compound 118 (100 mg, 0.30 mmol) in DMF (10 mL) DIPEA (54 µL, 0.33 mmol, 1.1 eq) and TBTU (96 mg, 0.3 mmol, 1 eq) were sequentially added. The reaction mixture was stirred for 5 min and then benzylamine (33 µL, 0.3 mmol, 1 eq) was added. The reaction mixture was stirred at rt overnight. The solvent was then evaporated and the residue was taken in DCM, washed with 1 M aq. HCl, saturated aq. NaHCO 3 , brine and H 2 O. The organic layer was dried over Na 2 SO 4 , filtered and evaporated to dryness. The crude product was purified by flash column chromatography (1:3 EtOAc-petroleum ether) to yield 119 as a white solid (75 mg, 59%). R f = 0.23 (1:3 EtOAc-petroleum ether). 1 H NMR (400 MHz, CDCl 3 , Figure S71): δ = 7.37-7.29 (m, 17 H, Ar), 6.92 (d, 2 H, 3 J H,H = 8.4 Hz, CHCOCH 2 CONH), 4.59 (m, 3 H, OCH 2 CO, CONHCH a ), 4.57 (m, 1 H, CONHCH b ). 13 C NMR (75.5 MHz, CDCl 3 , Figure S71): δ = 167.9 (CO), 157.8 (COCH 2 CONH), 137.6 (PCCHCHCOCH 2 CONH), 135.9, 135.5 (CP), 133.6, 133.4 (PCCHCHCOCH 2 CONH), 132.1 (CCH 2 NH), 128.8, 128.6, 128.5, 127.7 (PCCH, PCCHCH, PCCHCHCH, CHCHCCH 2 NH, CHCCH 2 NH, CHCHCHCCH 2 NH), 115.1, 114.8 (CHCOCH 2 CONH), 67.3 (OCH 2 CONH), 43.1 (CONHCH 2 ). 31 P NMR (162 MHz, CDCl 3 , Figure S71): δ = -7.03 (s). ESI-MS (Figure S144): m/z 448.2 [M + Na] + , 464.2 [M + K] + . Elemental analysis calculated (%) for C 27 H 24 NO 2 P: C 76.22, H 5.69, N 3.29; found: C 76.00, H 5.90, N 3.46. 252 Ph.D. Thesis - Iris Pflueger Phosphine-functionalized PS resins 123 and 129. Commercially available AM-PS resin (2.0 g, 0.39 mmol·g -1 ) was placed in a plastic syringe and allowed to swell in DMF for 20 min. Fmoc-Gly-OH (0.70 g, 2.34 mmol, 3 eq) was dissolved in a minimum amount of DMF. DIPEA (0.54 mL, 3.12 mmol, 4 eq) and TBTU (0.75 g, 2.34 mmol, 3 eq) were then sequentially added to the glycine solution. The mixture was stirred for approximately 5 min until complete dissolution of TBTU. Then, the solution was added to the swollen resin and the mixture was incubated rt overnight. Disappearance of amino groups was monitored by Kaiser test. 337 The resin was then washed with DMF (6 x 2 min) and DCM (4 x 2 min). The resin was finally dried under vacuum to constant weight. Resin substitution was estimated to be close to the nominal value (0.39 mmol·g -1 ) by UV determination of the dibenzofulvene released from a portion of resin upon 2% DBU in DMF treatment according to standard procedures. Fmoc was removed by treating the resin with 20% v/v piperidine in DMF at rt for 20 min, followed by rinsing the resin with degassed DMF (6 x 2 min) and degassed DCM (4 x 2 min) under N 2 to prevent oxidation during phosphine coupling. To a solution of phosphine 118 (0.54 g, 1.63 mmol, 2.1 eq) in dry and degassed DCM under N 2 atmosphere DMAP (0.20 g, 1.63 mmol, 2.1 eq) and DIC (0.20 g, 1.63 mmol, 2.1 eq) were sequentially added. The mixture was stirred for approximately 5 min under N 2 and the solution was subsequently transferred to the resin. After incubation for 16 h, the resin was then washed under N 2 stream with dry DCM (6 x 2 min) and dried under vacuum to constant weight. The resin was stored under an inert atmosphere to prevent phosphine oxidation. Successful phosphine incorporation was confirmed by appearance of the corresponding resonance at ca. -6 ppm in the 31 P NMR spectra. Experimental Part 253 31 P NMR (121 MHz, DMF, Figure S72): δ = -7.78 (s). Minute amounts (< 5%) of oxide phosphine (signal at ca. 32 ppm) were observed. Final resin loading (due to resin mass increase) was estimated in 0.34 mmol·g -1 . For the synthesis of the diluted version of resin 129, the coupling of the initial amino acid was effected using a mixture of Fmoc-Gly-OH and Boc-Gly-OH (1:9 in a molar basis). Thus, AM-PS resin (2.0 g, 0.39 mmol·g -1 ) was treated with mixture of Fmoc-Gly-OH (68 mg, 0.23 mmol, 0.3 eq), Boc-Gly-OH (0.37 g, 2.1 mmol, 2.7 eq), DIPEA (0.54 mL, 3.12 mmol, 4 eq) and TBTU (0.75 g, 2.34 mmol, 3 eq) in DMF. After disappearance of amino groups, the resin was then washed with DMF (6 x 2 min) and DCM (4 x 2 min). Then Boc protecting groups were removed by treatment with a 1:1 mixture of TFA-DCM for 1 h and the resin was washed with DCM (4 x 2 min), 5% DIPEA in DCM and neat DCM again (4 x 2 min). Free amino groups were capped by treating the DMF-swollen resin with a 1:1 Ac 2 O-Py mixture (10 eq each) for 1 h. The resin was finally rinsed with DMF (6 x 2 min) and DCM (4 x 2 min). Phosphine installation on the diluted resin was effected as described for 123. 31 P NMR (121 MHz, DMF, Figure S73): δ = -7.78 (s). Minute amounts (< 5%) of oxide phosphine (signal at ca. 32 ppm) were observed. Final resin loading (due to resin mass increase) was estimated in 40 µmol·g -1 . Methyl 6-amino-6-deoxy-2,3,4-tri-O-methyl-α αα α-D-glucopyranoside (124). Amine 124 was obtained from methyl 6-azido-6-deoxy-2,3,4-tri-O-methyl-α-D-glucopyranoside 91 280 (35 mg, 136 µmol, 2 eq) and resin 123 (0.2 g, 0.34 mmol·g -1 ) using the catch-and-release protocol as described in procedure A (General Methods for Solid-Phase Synthesis) in 89% yield (14 mg). ESI-MS (Figure S145): m/z 236.1 [M + H] + , 258.1 [M + Na] + . 254 Ph.D. Thesis - Iris Pflueger Alternatively, compound 124 was prepared in solution as follows: To a mixture of methyl 6-azido-6-deoxy-2,3,4-tri-O-methyl-α-D-glucopyranoside 91 280 (100 mg, 0.38 mmol) and Et 3 N (106 µL, 0.77 mmol, 2 eq) in MeOH (3 mL) was added 1,3-propanedithiol (77 µL, 0.77 mmol, 2 eq). The reaction mixture was stirred at rt overnight and solvents were subsequently evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (30:2:1 MeCN-H 2 O-NH 4 OH) to yield compound 124 (88 mg, 98%). R f = 0.19 (30:2:1 MeCN-H 2 O-NH 4 OH). [α] D = +41.6 (c = 1.0 in MeOH). 1 H NMR (300 MHz, MeOD, Figure S74): δ = 4.86 (d, 1 H, J 1,2 = 3.6 Hz, H-1), 3.59, 3.54, 3.49, 3.42 (4 s, 3 H each, OCH 3 ), 3.45 (ddd, 1 H, J 4,5 = 10.4 Hz, J 5,6b = 5.7 Hz, J 5,6a = 2.7 Hz, H-5), 3.39 (m, 1 H, H-3), 3.20 (dd, 1 H, J 2,3 = 9.6 Hz, H-2), 2.98 (dd, 1 H, H-6a), 2.59 (dd, 1 H, J 3,4 = 9.0 Hz, H-4), 2.71 (dd, 1 H, J 6a,6b = 13.2 Hz, H-6b). 13 C NMR (75.5 MHz, MeOD, Figure S74): δ = 97.0 (C-1), 83.2 (C-3), 81.6 (C-4), 81.3 (C- 2), 71.0 (C-5), 59.6, 59.4, 57.4, 54.1 (OCH 3 ), 42.2 (C-6). ESI-MS (Figure S146): m/z 236.1 [M + H] + , 258.2 [M + Na] + , 471.3 [2 M + H] + . Elemental analysis calculated (%) for C 10 H 21 NO 5 : C 51.05, H 9.00, N 5.95; found: C 51.23, H 9.04, N 5.96. 6,6’-Diamino-6,6’-dideoxy-2,2’,3,3’,4,4’-hexa-O-methyl-α,α’-trehalose (125). To a solution of compound 109 (49 mg, 96 µmol) in MeOH (1.5 mL) Pd/C (5 mg, 10% w/w) was added. The reaction mixture was stirred at rt for 24 h at 2 bar hydrogen pressure. Then, the reaction mixture was filtered over celite to remove the catalyst. The solvent was evaporated yielding compound 125 (43 mg, 97%). The crude product was used without further purification. R f = 0.15 (10:1:1 MeCN-H 2 O-NH 4 OH). [α] D = +33.1 (c = 0.52 in MeOH). Experimental Part 255 1 H NMR (500 MHz, CDCl 3 , Figure S75): δ = 5.14 (d, 2 H, J 1,2 = 3.5 Hz, H-1), 3.83 (ddd, 2 H, J 4,5 = 9.6 Hz, J 5,6b = 6.1 Hz, J 5,6a = 2.8 Hz, H-5), 3.64, 3.58, 3.47 (3 s, 6 H each, OCH 3 ), 3.55 (t, 2 H, J 2,3 = J 3,4 = 9.2 Hz, H-3), 3.16 (dd, 2 H, H-2), 3.03 (m, 4 H, H-4, H-6a), 2.81 (dd, 2 H, J 6a,6b = 13.1 Hz, H-6b). 13 C NMR (125.7 MHz, CDCl 3 , Figure S75): δ = 92.8 (C-1), 83.0 (C-3), 81.8 (C-2), 80.9 (C- 4), 71.9 (C-5), 60.8, 60.5, 58.8 (OCH 3 ), 42.7 (C-6). ESI-MS (Figure S147): m/z 425.5 [M +H] + , 447.4 [M + Na] + . Elemental analysis calculated (%) for C 18 H 36 N 2 O 9 : C 50.93, H 8.55, N 6.60; found: C 50.70, H 8.24, N 6.45. RP-HPLC: t R = 5.4 min (according to the RP-HPLC analytical procedure described in the General Methods section). 6-Amino-6’-azido-6,6’-dideoxy-2,2’,3,3’,4,4’-hexa-O-methyl-α,α’-trehalose (126). Compound 126 was obtained from compound 109 (32 mg, 68 µmol, 2 eq) and resin 123 (0.1 g, 0.34 mmol·g -1 ) using the catch-and-release protocol as described in procedure A (General Methods for Solid-Phase Synthesis) in 52 % yield (8 mg). HPLC: t R = 6.3 min (according to the RP-HPLC analytical procedure described in the General Methods section). ESI-MS (Figure S148): m/z 451.4 [M + H] + , 473.4 [M + Na] + . Alternatively, compound 126 was prepared in solution by statistical reduction of a single azido group as follows: To a mixture of compound 109 (100 mg, 0.21 mmol), Et 3 N (58 µL, 0.42 mmol, 2 eq) and 1,3-propanedithiol (3 µL, 0.02 mmol, 0.1 eq) in 2-propanol-DMF (2:1, 3 mL) was added NaBH 4 (8.7 mg, 0.23 mmol, 1.1 eq). The reaction mixture was stirred at rt for 4.5 h, and then H 2 O (1 mL) was added and solvents were evaporated under reduced pressure. The resulting residue was purified by flash column chromatography (30:2:1 MeCN-H 2 O-NH 4 OH) to yield compound 126 (38 mg, 40%). 256 Ph.D. Thesis - Iris Pflueger R f = 0.36 (30:2:1 MeCN-H 2 O-NH 4 OH). [α] D = +101.2 (c = 1.0 in MeOH). 1 H NMR (500 MHz, CDCl 3 , Figure S76): δ = 5.18, 5.17 (2 d, 1 H each, J 1,2 = 3.7 Hz, H-1, H-1’), 4.09 (ddd, 1 H, J 4,5 = 9.9 Hz, J 5,6b = 5.2 Hz, J 5,6a = 2.7 Hz, H-5’), 3.84 (ddd, 1 H, J 4,5 = 12.8 Hz, J 5,6b = 5.8 Hz, J 5,6a = 2.7 Hz, H-5), 3.65, 3.64, 3.59, 3.58, 3.49, 3.48 (6 s, 3 H each, OCH 3 ), 3.53 (2 t, 1 H each, J 2,3 = J 3,4 = 9.7 Hz, H-3, H-3’), 3.47-3.42 (m, 2 H, H-6a’, H-6b’), 3.20, 3.18 (2 dd, 1 H each, H-2, H-2’), 3.10-3.01 (m, 3 H, H-4, H-4’, H-6a), 2.83 (dd, 1 H, J 6a,6b = 13.5 Hz, H-6b). 13 C NMR (125.7 MHz, CDCl 3 , Figure S76): δ = 93.1, 93.0 (C-1, C-1’), 83.0, 82.8 (C-3, C- 3’), 81.8, 81.6 (C-2, C-2’), 80.8, 80.4 (C-4, C-4’),71.9 (C-5’), 70.4 (C-5), 60.8, 60.6, 58.7 (OCH 3 ), 51.5 (C-6’), 42.6 (C-6). ESI-MS (Figure S149): m/z 451.5 [M + H] + , 473.4 [M + Na] + . Elemental analysis calculated (%) for C 18 H 34 N 4 O 9 : C 47.99, H 7.61, N 12.44; found: C 47.67, H 7.54, N 12.35. RP-HPLC: t R = 6.3 min (according to the RP-HPLC analytical procedure described in the General Methods section). 6 I -Amino-6 II-VII -hexaazido-6 I-VII -heptadeoxy-2 I-VII ,3 I-VII -tetradeca-O-methylcyclomalto- heptaose (127). Monoamine 127 was obtained from heptakis(6-azido-6-deoxy-2,3-di-O- methyl)cyclomaltoheptaose 110 180a (153 mg, 0.1 mmol, 2 eq) and resin 123 (0.15 g, 0.34 mmol·g -1 ) using the catch-and-release protocol as described in procedure A (General Methods for Solid-Phase Synthesis) in 79% yield (60 mg). HPLC: t R = 9.2min (according to the RP-HPLC analytical procedure described in the General Methods section). ESI-MS (Figure S150): m/z 1480.6 [M + H] + , 1502.5 [M + Na] + . Experimental Part 257 Alternatively, compound 127 was prepared in solution by statistical reduction of a single azido group as follows: To a solution of heptakis(6-azido-6-deoxy-2,3-di-O- methyl)cyclomaltoheptaose 110 180a (262 mg, 0.17 mmol) in 2-propanol (10 mL) under nitrogen atmosphere, Et 3 N (48 µL, 0.35 mmol, 2 eq), 1,3-propanedithiol (1.75 µL, 17 µmol, 0.1 eq) and NaBH 4 (7.3 mg, 0.19 mmol, 1.1 eq) were added, and the reaction mixture was vigorously stirred overnight. The reaction was quenched by addition of water (2 mL). The solvent was evaporated under reduced pressure and the residue was diluted with DCM (20 mL) and washed with 1 M aq. NaOH (3 x 5 mL). The organic layer was dried, filtered and concentrated. The resulting residue was purified by flash column chromatography (20:1  9:1 DCM-MeOH) to obtain the starting material and compound 127 as an amorphous white solid. Yield: 56 mg (23 %) along with unreacted starting material (126 mg, 48%). R f = 0.50 (9:1 DCM-MeOH). [α] D = +149.1 (c = 1.0 in DCM). 1 H NMR (500 MHz, MeOD, 333 K, Figure S77): δ =5.27 (d, 1H, J 1,2 = 3.7 Hz, H-1 II ), 5.22- 5.18 (m, 6 H, H-1 I,III-VII ), 3.95-3.84 (m, 11 H, H-5 II-VII , H-6a II-VI ), 3.81-3.78 (dd, 1 H, H-6a VII ), 3.77-3.74 (ddd, 1 H, H-5 I ), 3.70-3.50 (m, 20 H, H-4 I-VII , H-3 I-VII , H-6b II-VII ), 3.66 (m, 21 H, OCH 3 I-VII ), 3.55 (m, 21 H, OCH 3 I-VII ), 3.26-3.21 (m, 7 H, H-2 I-VII ), 3.19-3.16 (dd, 1 H, H- 6a I ), 3.07-3.02 (dd, 1 H, H-6b I ). 1D-TOCSY (500 MHz, MeOD, H-6a I irradiation): δ =5.21 (d, 1 H, J 1,2 = 2.8 Hz, H-1 I ), 3.75 (ddd, 1 H, J 4,5 = 8.4 Hz, J 5,6a = 2.9 Hz, J 5,6b = 5.6 Hz, H-5 I ), 3.58-3.52 (m, 2 H, H-3 I , H-4 I ), 3.22 (dd, 1 H, J 2,3 = 8.6 Hz, H-2 I ), 3.16 (dd, 1 H, H-6a), 3.04 (dd, 1 H, H-6b, J 6a,6b = 14.0 Hz). 1D-TOCSY (500 MHz, MeOD, H-2 II irradiation): δ =5.25 (d, 1 H, J 1,2 = 3.9 Hz, H-1 II ), 3.92 (ddd, 1 H, H-5 II ), 3.85 (d, 1 H, H-6a II ), 3.69 (dd, 1 H, J 6a,6b = 14.0 Hz, J 5,6b = 4.0 Hz, H-6b II ), 3.6 (t, 1 H, H-4 II ), 3.52 (t, 1 H, J 2,3 = J 3,4 = 9.1 Hz, H-3 II ), 3.23 (dd, 1 H, J 2,3 = 9.9 Hz, H-2 II ). 13 C NMR (125.7 MHz, MeOD, 333 K, Figure S77): δ = 100.6-100.4 (C-1 I-VII ), 84.0-82.1 (C- 2 I-VII ,C-3 I-VII , C-4 I-VII ), 73.8-73.2 (C-5 I-VII ), 62.8-62.4 (OCH 3 ), 60.2-59.9 (OCH 3 ), 54.1-53.9 (C-6 II-VII ), 43.9 (C-6 I ). 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