Combining NMR spectroscopy and organic synthesis : from small building blocks to large biomolecules

Detailed knowledge of the structure of bio molecules with atomic resolution is essential for the understanding of their function. Moreover the identification and quantification of their dynamic processes are important, as they are the origin of molecular functionalities. Different spectroscopic methods like X-ray crystallography (limited to structure elucidation, no dynamics), mass spectroscopy, electron spin resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy can deliver this detailed structural information. Especially NMR spectroscopy has found its application in the identification of dynamic processes. This thesis was focused on the characterization of dynamic processes, the structure elucidation of natural products available in nanograms and the synthesis of lanthanide chelating tags for the study of protein-ligand and protein-protein interactions in solution. The thesis is divided into three chapters addressing a specific task of the mentioned topic. A: The influences of different donors and linkers on lanthanide chelating tags were investigated. A 4S-Tetramethylcyclen was used as enantiomerically pure core molecule. The side chains were based on enantiopure lactic acid derivatives. The carboxylic acids of lactic acid were transformed into different functional groups like thiols, nitrogens, and carbonyl. These different donor groups are expected to change the magnetic susceptibility tensor of the lanthanide cation in complex with the synthesized tags. Special protection protocols for the introduction of heteroatoms are presented. On this basis also two new linkers were introduced. These linkers bind in a selective way to cysteins on a protein surface as thioethers. The alpha-bromoketones are very selective for the coupling to cysteins, nevertheless they have a tendency to hydrolyse under Lewis acidic conditions. Vinylsulfones require harsher tagging conditions but they are much more stable against hydrolysis. All new tags were attached to proteins and tested as PCS reagents in protein NMR spectroscopy. B: The effects of para-substituents on the rotation barrier of 2,2«-propyl and 2,2«-butyl-bridged biphenyls were studied by dynamic NMR spectroscopy and dynamic HPLC measurements. Gibbs free activation energies of the rotation about the central biphenyl bond were estimated by variable temperature 1H-NMR experiments for the propyl bridged biphenyl. The resulting data were correlated to Hammett-parameters ?P as a measure of electron donor and acceptor strength. It was demonstrated that the electronic effects influence the activation barrier significantly, whereas sterics had only minor influence. Rate constants were calculated from line shape analysis and analysed by Eyring plots to calculate the entropic and enthalpic contributions. Thermodynamic data for the butyl-bridged biphenyls were directly obtained from dynamic HPLC chromatograms. DFT calculations delivered different transition states for the two series of biphenyls. The calculation of the activation parameters showed a similar trend and therefore the model is validated. The differences in the enthalpic and entropic contributions between HPLC, NMR and DFT calculations are method dependent, which was proven by changes of the solvent in NMR experiments that led to alteration of these contributions. C: The identification and total synthesis of a novel methylated lipid antigen (mLPA) was performed. The presented mLPA shows potent inhibition properties against human leukaemia. A combination of extraction protocols, activation essays and HPLC-MS measurements were used to identify the different antigen molecules from cell extracts. Three different candidates were identified. MS-MS experiments delivered structural insights, which were further confirmed by NMR spectroscopy and allowed the characterization of two of these structures. Total syntheses of the identified structures were performed in 6 linear steps. The high biological activity of the synthesized structures corroborated the identity of the active molecule

Racemisation dynamics of torsion angle restricted biphenyl push-pull cyclophanes

The thermodynamics of the atropisomerisation of torsion angle restricted, axial chiral biphenyl-based push-pull cyclophanes were studied. Using 1H NMR coalescence measurements the rotation barrier around the central C–C bond was determined to be 50 kJ mol−1 for the propyl-bridged biphenyl derivative 1b, displaying only a negligible solvent dependence. By protonation of the piperidinyl nitrogen as electron donor, the free energy ΔG‡(T) of the rotation barrier increased, indicating that the tendency of the push-pull system to planarise may be considered as a driving force for the atropisomerisation. For the more restricted butyl-bridged cyclophane 1c a rotation barrier of ΔG‡(T) = 90 kJ mol−1 was measured using dynamic chromatography. The difference in the free energy of rotation around the central C–C bond probably reflects the crowdedness of the transition states

Through the Maze: Cross-Coupling Pathways to a Helical Hexaphenyl "Geländer" Molecule

This paper highlights a new concept on how to induce chirality in a hexaphenyl Geländer-type system. Bridging a terphenyl backbone with a considerably longer benzyl ether oligomer enforces a continuous twist of the molecule, while preventing an achiral meso form. By highlighting cross-coupling strategies and explored synthetic pathways, this report aims to serve as an Ariadne`s thread for the synthesis of precisely functionalized, complex polyaromatic systems. The synthetic challenges and considerations required to access the designed target are outlined and solutions to each step of the assembly are presented. Encountered isomerizations are discussed as much as synthetic tools to access highly functionalized intermediates with multiorthogonal moieties. A strong focus is made on the employment of Suzuki–Miyaura protocols for the targeted connection of polyaromatic fragments and ultimately the desired oligomeric structure

Atropisomerization of di-para-substituted propyl-bridged biphenyl cyclophanes

The influence of electron donors and electron acceptors of variable strength in the 4 and 4' position of 2 and 2' propyl-bridged axial chiral biphenyl cyclophanes on their atropisomerization process was studied. Estimated free energies [capital Delta]G[double dagger](T) of the rotation around the central biphenyl bond which were obtained from 1H-NMR coalescence measurements were correlated to the Hammett parameters [sigma]p as a measure for electron donor and acceptor strength. It is demonstrated that the resulting nice linear correlation is mainly based on the influence of the different substituents on the [small pi]-system of the biphenyl cyclophanes. By lineshape analysis the rate constants were calculated and by the use of the Eyring equation the enthalpic and entropic contributions were evaluated. Density functional theory calculations show a planar transition state of the isomerization process and the calculated energy barriers based on this reaction mechanism are in good agreement with the experimentally obtained free energies

Inducing Axial Chirality in a "Geländer" Oligomer by Length Mismatch of the Oligomer Strands

Helical molecules are not only esthetically appealing due to their structural beauty, they also display unique physical properties as a result of their chirality. We describe herein a new approach to “Geländer” oligomers by interlinking two oligomer strands of different length. To compensate for the dimensional mismatch, the longer oligo(benzyl ether) oligomer wraps around the oligophenyl backbone. The new “Geländer” oligomer 1 was assembled in a sequence of functional-group transformations and cross-coupling steps followed by final cyclizations based on nucleophilic substitution reactions, and was fully characterized, including X-ray diffraction analysis. The isolation of pure enantiomers enabled the racemization process to be studied by circular dichroism spectroscopy

A novel self-lipid antigen targets human T cells against CD1c+ leukemias

T cells that recognize self-lipids presented by CD1c are frequent in the peripheral blood of healthy individuals and kill transformed hematopoietic cells, but little is known about their antigen specificity and potential antileukemia effects. We report that CD1c self-reactive T cells recognize a novel class of self-lipids, identified as methyl-lysophosphatidic acids (mLPAs), which are accumulated in leukemia cells. Primary acute myeloid and B cell acute leukemia blasts express CD1 molecules. mLPA-specific T cells efficiently kill CD1c(+) acute leukemia cells, poorly recognize nontransformed CD1c-expressing cells, and protect immunodeficient mice against CD1c(+) human leukemia cells. The identification of immunogenic self-lipid antigens accumulated in leukemia cells and the observed leukemia control by lipid-specific T cells in vivo provide a new conceptual framework for leukemia immune surveillance and possible immunotherapy