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Nature abounds with ingenious nanoscopic machines employed to carry out all of the requisite tasks that collectively contribute to the molecular basis of life. This thesis focuses primarily on a sub-set known as "molecular walkers" which can perambulate along intracellular molecular motorways carrying out such essential tasks as vesicle transport and muscle contraction. A summary of these incredible natural motors is presented in Chapter I along with a review of the artificial small-molecule mimics reported to date. When elucidating a set of design principles for synthetic analogues, inspiration is taken from the mechanism of the biological bipedal motor protein kinesin with a focus on potential strategies to enable directional walking. Transition metal-ligand chemistry is utilised as one such strategy in Chapter II through the governance of walker-track interactions in the design, synthesis and operation of a bimetallic molecular biped. A palladium(II) moiety is selectively and intramolecularly stepped between pyridine-derivative binding sites in the track using a thermal stimulus in the presence of a coordinating solvent. Acid-base manipulations facilitate directional stepping by means of an energy ratchet mechanism allowing the track to do work on the biped unit and ultimately drive it away from equilibrium. The potential of malleable transition metal binding-event energetics is explored further in Chapter III with the design and synthesis of a platinum(II)-complexed [2]rotaxane. Thermodynamic and kinetic stimuli are investigated as means to mediate selective shuttling of a Pt-complexed macrocycle between two ligand binding sites in the thread. The substitution pattern of the ligands and the kinetic stability of the metal-ligand bonds afford exceptional metastability to the co-conformers of the molecule in the absence of an external stimulus providing the possibility for long-term information storage. In Chapter IV, a novel macrocycle is used to demonstrate the chemical orthogonality of acid-mediated hydrazone exchange with respect to the palladium(II) stepping mechanism described in Chapter II and show that two such motifs can be independently addressed within a single molecule. These linkages are then utilised as mutually exclusive chemo-selective switches to individually operate opposing feet in an unprecedented first-generation small-molecule walker-track system.

This thesis reports the efforts towards the design and synthesis of a small molecule walker that would potentially move along the track directionally by exploiting the secondary interactions between the track and the walker. This thesis also reports the synthesis and operation of a light driven molecular shuttle featuring a novel acylpyridyl hydrazone station. Chapter One describes the biological walkers which are the source of inspiration towards the synthetic walkers, characteristics of a walker, previously described small molecule walkers and recent progress on the synthesis of molecular shuttles that operate under variety of different stimuli. Chapter Two describes the design and synthetic efforts towards a molecular walker that has the potential to operate directionally along the track by exploiting secondary interactions between the walker and the track namely the hydrogen bonding interactions introduced by subtle incorporation of excellent hydrogen bond donor/acceptor squaramides. This chapter briefly mentions the hydrogen bonding capabilities of squaramides on which the directional operation relies. Optimization of critical reactions and attempted strategies for the assembly of the whole machine is described as well. Chapter Three describes the synthesis and operation of 1- and 2- station [2]-rotaxanes that operate under light irradiation. 2- station [2]-rotaxane that function as a light driven molecular shuttle presents remarkable positional fidelity with high efficacy. The bistable acyl pyridyl station is incorporated as a photo active station upon which light irradiation alters the binding affinities towards the macrocycle. Series of rotaxanes featuring different amide based stations were synthesized to determine the best non-photo active station.

In Nature sophisticated molecular machines are responsible for the synthesis of essential biomolecules and natural biopolymers. Inspired by these natural prototypes, scientist have begun to developed artificial systems that are able to perform complex synthetic tasks. Selected state-of-the-art examples, are presented and categorised according to their synthetic function: systems, which can distinguish between substrates and can also be regulated to produce different products, systems that selectively conduct multistep cascade reactions in mixtures of different reactants and systems that operate in a processive and sequence-specific fashion (Introduction).Following these examples, inspired by the ribosome, the synthesis and operation of the first artificial small-molecule machines based on a rotaxane structure capable of performing sequence-specific synthesis of a tri- and tetrapeptide from a molecular template is described. These machines operate through native chemical ligation (NCL), using a macrocyclic cysteine catalyst that iteratively removes proteinogenic amino acids from the strand and transfers them to a peptide elongation site. Successful operation on small scale generated milligram quantities of the peptides with a single sequence, determined by tandem mass spectrometry, corresponding to the original order of the amino acid building blocks on the strand (Chapter 1). Based on these first prototypes the system was extended to molecular machines operating on polymeric tracks. In this context the limits of the NCL mechanism were explored and the concept of the machine operation was investigated on a model system containing a polystyrene backbone with multiple leucine units. Here, machines with an average number of up to five L-leucyl groups were successfully operated. These initial studies represent the groundwork for the operation on longer polymeric systems containing sequences of amino acids (Chapter 2). Machines operating through a native chemical ligation (NCL) mechanism are restricted by a number of limitations: the rate of the reaction, the length and structure of the synthesised peptide, the cleavage of the product and finally the fact that peptide synthesis occurs in a reversed fashion to ribosomal synthesis (from C to N terminus). To overcome those limitations the development towards a 2nd generation molecular machine based on a transacylation catalyst was envisioned. Since this type of catalyst operates via a series of transacylation steps, the size of the transition state is kept the same throughout the operation, allowing access to longer peptides with fewer structural restrictions. Model systems using a thiophenol catalyst and a 1,2,4-triazol catalyst have been investigated (Chapter 3).

The work described in this thesis is inspired by natural-occurring molecules that are used throughout biology to perform specific, highly-selective tasks. This thesis illustrates the design, synthesis and investigation of novel molecular devices based on acyl hydrazones for the synthesis of a small molecule walker and light-driven molecular shuttles. Chapter One outlines a general overview of the design and synthesis of molecular devices, including molecular walkers and molecular shuttles. Some of the most important examples of walking molecules (both natural and synthetic) are described in detail, along with a comprehensive introduction of molecular shuttles and their synthetic mimics. Examples of stimuli-responsive molecular shuttles that have been developed are highlighted throughout the chapter. Chapter Two describes the design and synthetic progress towards a molecular walker, as well as detailing the optimisation of the synthetic steps achieved thus far. In this chapter, most of the work presented is based on the design and optimisation of the synthesis of an acyl hydrazone-based molecular walker, which will be able to walk directionally and repetitively along its conjugate track when the conditions are changed. A novel acyl hydrazone pyridine moiety is introduced to the system to achieve a high directional bias during the walking process. First, the concept and basis of the design is explained and further, the synthesis of the walker system is discussed in detail. Chapter Three illustrates the synthesis and operation of 1- and 2- station [2]-rotaxanes which exhibit all the requirements for a light-driven molecular shuttle. The effect of a new photo switchable binding station, an acyl pyridyl hydrazone, on the shuttling process is investigated by comparing the positional distribution of the macrocycle between the acyl hydrazone station and the succinamide-ester station, while the acyl hydrazone undergoes photo- and thermal isomerisation. The successful synthesis of this molecular architecture is described along with its operation, demonstrating high positional integrity and efficiency during the shuttling process.

Sequence-specific synthesis is essential to life. In nature, information-rich polymers such as polynucleotides, polypeptides and polysaccharides are responsible for virtually all vital processes. As opposed to nature’s optimised approach towards sequence control employing sophisticated molecular machines such as ribosomes and nucleotide polymerases, the synthetic chemist’s toolbox for the generation of highly ordered monomeric sequences is limited in scope. In this thesis, the realisation of the first artificial small-molecule machines capable of synthesising peptides, translating information that is encoded in a molecular strand, is described. The chemical structure of such machines is based on a rotaxane architecture: a molecular ring threaded onto a molecular axle. The ring carries a reactive arm, a thiolate group that iteratively removes amino acids from the strand that block the path of the macrocycle. The acyl monomers are transferred to a peptide-elongation site through native chemical ligation, thereby translating the information encoded in the track into a growing peptide strand. The synthesis is demonstrated with ~1018 molecular machines acting in parallel; this process generates milligram quantities of a peptide with a single sequence as confirmed by tandem mass spectrometry. Chapter I describes previous strategies that have been employed to realise sequence specific synthesis and gives an overview about relevant literature in the field. Chapter II describes the concept, previous work and model studies which lay the ground work for the more advanced machines. The first generation design of a molecular machine based on transacylation catalysis as well as the second generation design based on native chemical ligation are discussed. The successful operation of a single-barrier rotaxane capable of elongating its reactive arm by a single amide bond formation and subsequent self-immolation is described. Chapter III describes the first small-molecule molecular machine capable of sequence-specific assembly of a tripeptide. The sequence-integrity of the operation product is determined by tandem mass spectrometry and comparison with an authentic sample. Chapter IV describes a novel synthetic approach towards highly complex molecular machines. Using this rotaxane elongation strategy, a molecular machine with four aminoacyl monomers on the strand is reported. The successful operation afforded the expected product resulting from four amide bond forming events without any detectable sequence scrambling.

Protein secretion is a selective and regulated process that is essential in all organisms. In bacteria the preprotein translocase SecA, either free in the cytosol or associated with the SecYEG translocon, recognizes and binds most post-translational secretory proteins containing an N-terminal signal sequence. In Gram-negative bacteria, the molecular chaperone SecB binds many of the preproteins to keep them in a translocation-competent state. Subsequently, SecB delivers the preproteins to the translocon-associated SecA, which binds the signal sequence and also interacts with mature regions of the preprotein. After the preprotein/SecA/SecYEG complex has formed, the energy derived from ATP hydrolysis by SecA coupled with the proton motive force drives the insertion of the preprotein through the translocon pore. During the translocation reaction, the conformation of SecA dramatically changes from an inactive closed form (c-SecA) to one more active and open states. The various crystal structures of SecA have provided many structural details about c-SecA. The recent low resolution crystal structure of a fragment of SecA bound to SecYEG (Zimmer et al., 2008) has provided a starting point for structural analysis of the active and open conformation of SecA. Previous work in our laboratory demonstrated that an N-terminal proteolytic fragment of SecA, SecA64, is an activated form of SecA that with higher affinity signal peptides better than c-SecA (Triplett et al., 2001). To correlate the SecA64 results with full-length SecA, we determined that SecA in the presence of low concentrations of urea has an enhanced ATPase activity similar to translocation level, which is comparable to what was observed with SecA64. Analysis by CD and Trp fluorescence indicates the presence of an intermediate at 2.2 M urea at 22ºC (termed u-SecA). Using limited proteolysis, we determined that u-SecA is in an protease-sensitive conformation that mimics the translocation-active form of SecA. These structural rearrangements occur primarily in the C-terminal one-third of the protein. Next, we sought to understand the signal sequence interactions with c-SecA and translocation-active u-SecA. Using a photoactivatable cross-linking approach along with limited proteolysis, two-dimensional gels, and domain mapping with region-specific antibodies, the signal sequence-binding site was mapped to the interface of NBF II, PPXD, and HSD. The site is the same in both forms of SecA but in our data suggests u-SecA that the binding groove as expanded.

No description available.

Bacterial cells were once treated as membrane-enclosed bags of cytoplasm: a homogeneous, undifferentiated suspension in which polymers (proteins, nucleic acids, etc.) and small molecules diffused freely to interact with each other. Biochemical studies have determined the molecular mechanisms underlying the biological processes of metabolism, replication and transcription-translation, etc. However, recent advancements in optical techniques armed with fluorescent tags for proteins and nucleic acids have increased our ability to peer into the interior of live bacterial cells. This has revealed an organized layout of multi-protein complexes, or molecular machines, dedicated to specific functions at defined sub-cellular locations; the timing of their assembly and/or rates of their activity being determined by available nutrition and environmental signals from the niche occupied by the organism. In the present study, we have attempted to identify the intracellular location and organization of the molecular machines assembled for protein synthesis (ribosomes), DNA replication (replisomes) and cell division (divisome) in different bacteria. We have used the model system Escherichia coli as well as Helicobacter pylori and mycobacterial strains (Mycobacterium marinum and Mycobacterium smegmatis), which grow at different rates and move to dormancy late into stationary phase Bacterial nucleoid plays a major role in organizing the location and movement of active ribosomes, replisomes and placement of divisome. While the active ribosomes appear to follow the dynamic folds of the bacterial nucleoid during cell growth in E. coli, inactive ribosomes appear to accumulate near the periphery. The replisome in H. pylori was visualized as a sharp, single focus upon SSB and DnaB co-localization in growing helical rods but disassembled into diffused fluorescence when the cells attained non-replicative coccoid stage. Our investigation into mycobacterial life-cycle revealed unique features such as an absence of a dedicated mid-cell site for divisome assembly and endosporulation upon entry into stationary phase. In brief, we present the cell cycle-dependent subcellular organization of molecular machines in bacteria.

In the emerging aield of molecular machines, a molecular ratchet is a chemical system that allows the positional displacement of a submolecular component of be captured and directionally released. In information ratchets, the track over which a Brownian particle is to be transported is able to respond to the particle’s position. By raising energetic barriers to translation selectively behind the particle, it is possible to move the particle in a forward direction. This Thesis describes the development of a series of chemically-­‐driven information ratchets based on rotaxane architectures. Acylation of the rotaxane thread presents an impassible kinetic barrier to macrocycle shuttling. The incorporation of chiral centres into the thread allows the macrocycle’s position to have an effect on the kinetics of acylation in a chiral environment, with the result that the macrocycle is transported by successive acylation reactions in a direction speciaied by the handedness of a chiral. In Chapter One the physical principles of molecular motors are examined. It is shown that molecular motors are a subset of the much broader class of “triangular” reactions investigated by Onsager in 1931. Progress in the exciting aield of artiaicial chemical ratchets and motors is reviewed, and the deep connections between molecular motors and the cyclic reaction networks postulated to explain the origin of biological homochirality are explored. Chapter Two describes the synthesis and operation of a three-­‐compartment rotaxane information ratchet in which the macrocycle can be transported along a thread in either direction depending on the handedness of a chiral catalyst. Internal mechanisms of operation are elucidated by treating the system as a hidden Markov process. Chapter Three describes the synthesis and operation of a second-­‐generation three-­‐compartment information ratchet. A comparison between this system and that of the previous chapter sheds light on the complicated trade-­‐offs between kinetics and thermodynamics when these molecular ratchets are operated. In Chapter Four the ongoing efforts to construct extended information ratchets, incorporating many repeat units, are described. The synthesis of a aive-­‐ compartment information ratchet proved unexpectedly difaicult owing to problems of solubility. A four-­‐compartment rotaxane was easier to synthesise. Preliminary aindings suggest that an information ratchet mechanism is operating in this four-­‐compartment system.