Solving a 3D structural puzzle

Abstract

NMR (nuclear magnetic resonance) spektroskopi er et alsidigt og vigtigt værktøj i analytisk kemi, der ofte er involveret i strukturopklaring af organiske molekyler såvel som i mange andre forskningsområder. Denne afhandling omhandler metoder til at opnå rummelig information om molekylære strukturer ved hjælp af NMR spektroskopi.3D strukturer af nye natur- og syntetiske stoffer blev således undersøgt ved hjælp af NOE (nuclear Overhauser effekt) korrelationer og tre-bindings J-kobling konstanter. NOE korrelationer kan bruges til at bestemme afstande mellem atomer i de organiske strukturer, og J-koblingskonstanter kan give information of den dihedrale vinkel i H-X-X-H systemer (hvor X oftest er C, N, O). For naturstofferne, inklusivt et stof med et hidtil ukendt to-rings-system, ledte NMR data til, at alle relative stereocentre blev bestemt. NOE og J-koblinger resulterede også i ny viden om en gruppe af potentielle anti-cancer stoffer, kaldet azumamiderne, hvor et naturstof og syntetiske analoger blev undersøgt. Der blev identificeret forskelle i det konformatielle rum mellem de forskellige strukturer, hvilket blev relateret til deres anti-cancer aktivitet. I et andet projekt blev interaktionen mellem to peptider undersøgt, via viden om deres respektive struktur i vand fra afstande mellem atomer. De to peptiderne er af særlig interesse, da de selektivt interagerer med hinanden i vandig opløsning, ligesom f.eks. proteiner i celler. Derudover blev det undersøgt, i hvor høj grad proton-par i organiske strukturer roterer og bevæger sig identisk, hvilket er en antagelse, der gøres for at kunne finde afstande fra NOE korrelationer. Det undersøgtes også om det var muligt a korrigere afstandene, fra viden om proton-pars relative rotationshastigheder i organiske solventer.To nye NMR eksperimenter, kaldet S3 (spin-state selective) HMBC, blev udviklet til at måle henholdsvis proton-proton og proton-kulstof J-koblingskonstanter i organiske strukturer, hvor de to koblende atomer er adskilt af op til flere kemiske bindinger (nJCH og n+1JHH, n=2-4). De to NMReksperimenter er baseret på to eksisterende eksperimenter, der kombineredes sådan, at det var muligt at måle proton-proton koblingskonstanter mellem en proton bundet til et kulstof og en proton, der kobler til dette kulstof. En ny pulssekvens blev tilføjet dette første S3 HMBC eksperiment, hvilket gjorde det muligt også at måle proton-kulstof koblingskonstanterne. De målte koblingskonstanter fra begge sekvenser korrelerede i meget høj grad med teoretisk bestemte koblingskonstanter, selv når disse var meget små. Dette medfører derfor muligheden for øget strukturel viden.Residuale dipolære koblinger (RDCs), en forholdsvis nyligt introduceret parameter for små molekyler, og er baseret på vinklen mellem en internukleær vektor og det magnetiske felt fra NMR spektrometret. Her er få internukleære vektorer ofte et problem for små molekyler. Det blev vist at det homo-nukleare S3 HMBC kunne benyttes til at bestemme adskillige RDCs mellem protoner, og derved blev flere internukleære vektorer bestemt. For at kunne aflæse RDCs bruges såkaldte alignment media, hvor et overskud af en bestemt orientering af molekylerne i NMR prøven skabes. Nye alignment media blev syntetiseret, og disse muliggjorde bestemmelsen af enantiomerer (spejlede molekyler) ud fra deres aflæste RDCs. Endeligt blev en ny beregningsmetode af RDCs fra 3D strukturer testet. Metoden fungerede klart bedre end standardmetoden SVD, når fleksible strukturer blev undersøgt, hvilket blev vist for flere molekyler.Nuclear magnetic resonance (NMR) spectroscopy is a versatile tool in analytical chemistry, highly suitable for structural elucidation of organic molecules – as well as multiple other areas of research. The subjects covered within this thesis all concern methods which allow a shift from covalent tospatial structural information using NMR spectroscopy. Experimental distances from nuclear Overhauser effect (NOE) correlations, and dihedral anglesfrom 3JHH-coupling constants, were used to obtain 3D structural information for several natural and synthetic compounds. The stereochemistry of novel natural compounds was determined, including that of a bicyclic non-ribosomal peptide (a novel structural motif), a steroid and several polyketides.Structural insights were gained for potential anti-cancer agents; the azumamides, including synthetic analogues. Differences in the conformational space of solution state compounds were identified experimentally between structural analogues, and compared to the in vitro potency of the compounds. The structures of two peptides that exhibited a high degree of molecular recognition were investigated, resulting in the elucidation of a possible mode of interaction. Also a major assumption in the calculation of distances from NOEs, the assumption of equal rotational correlation times between proton pairs, was investigated for molecules in organic solvents.Two spin-state selective (S3) HMBC experiments were developed for measurements of homonuclear and hetereonuclear long-range coupling constants, respectively. The new NMR experiments were based on two existing experiments, the multiplicity edited HMBC and the HAT HMBC, which were combined to obtain S3 editing of long-range homonuclear coupling constants. The output of the first S3 HMBC experiment was HMBC type spectra with nJCH correlated crosspeaks, from which n+1JHH-coupling constants were sign-selectively determined with high accuracy. Very small coupling constants, including previously unreported coupling constants from strychnine, were extracted, with all experimental values correlating very well to theoretical coupling constants from DFT calculation. A pulse segment was developed to change the polarization of the CH-H pairs in the homonuclear S3 HMBC, to gain S3 edited nJCH-coupling constants in the cross-peaks. While only determining coupling constants to methine carbons, the extracted experimental coupling constants correlated very well to theoretical coupling constants, thus extending the S3 HMBC methodology to include both n+1JHH- and nJCH-coupling constants. Residual dipolar couplings (RDCs) are a relatively late addition to the small molecular NMR community, where alignment media are used to obtain anisotropic samples, which allows for RDCs to be extracted. The number of internuclear vectors for the correlation of RDCs to 3D structures is often limited for small molecules. Homonuclear RDCs were extracted by use of the homonuclear S3 HMBC that correlated well to alignment tensors from 1DCH-coupling constants, thus increasing the number of inter-nuclear vectors. The topic of enantiodiscrimination by RDC measurements of rigid organic molecules was also investigated, and new alignment media were developed to allow slight discrimination of enantiomers by stretched polymers. Finally a new method of back-calculation of RDCs from 3D structures was developed and tested, which copes better with multiple conformersthan the commonly used SVD methodology. The approach thus resulted in good conformer populations for several small molecules, including multiple cinchona alkaloids