Using Diffusion NMR To Characterize Guanosine Self-Association: Insights into Structure and Mechanism Mark S. Kaucher, [a] Yui-Fai Lam, [a] Silvia Pieraccini, [b] Giovanni Gottarelli, [b] and Jeffery T. Davis* [a] Introduction While molecular self-assembly is becoming a powerful ap- proach for nanoscale synthesis, [1] characterization of supra- molecules remains a constant challenge. Single crystals of non-covalent assemblies are not always possible. Further- more, packing forces may give solid-state structures that aren)t well-populated in solution. Determination of supra- molecular solution structure can also be daunting. Mass spectrometry, [2] analytical ultracentrifugation, [3] dynamic light scattering, [4] gel permeation chromatography and vapor pressure osmometry have been used to determine sizes of supramolecular complexes. None of these techniques, how- ever, provide the atomic resolution offered by NMR spec- troscopy. Whereas standard NMR techniques are excellent at determining molecular composition, defining the sizes of high-symmetry complexes can be a problem. For example, NMR spectroscopy cannot readily distinguish a C 4 -symmet- ric tetramer from a C 6 -symmetric hexamer, nor can signal integration differentiate an AB dimer and an A 2 B 2 tetramer. One method for solving such problems is through the use of pulsed field gradient (PFG) NMR. PFG-NMR, a method for measuring diffusion rates, pro- vides information about the sizes of molecules in solution. [5] PFG-NMR, used to study self-association of natural prod- ucts, [6] peptides, [7] and proteins, [8] is also an emerging tech- nique in supramolecular chemistry. Diffusion NMR has been used to define the aggregation state of ion pairs and other organometallic assemblies. [9,10] The sizes of dendrimers, supramolecular polymers and nanoparticles have been de- termined with the technique. [11–13] Cohen and colleagues have pioneered the use of diffusion NMR in host–guest chemistry, with detailed studies of macrocyclic complexes. [14] [a] M. S. Kaucher, Dr. Y.-F. Lam, Prof. Dr. J.T. Davis Department of Chemistry and Biochemistry University of Maryland, College Park, MD 20742 (USA) Fax: (+ 1)301-314-9121 E-mail: jd140@umail.umd.edu [b] Dr. S. Pieraccini, Prof. Dr. G. Gottarelli Dipartimento di Chimica Organica “A Mangini” Universita di Bologna, Via S. Donato 15 40127 Bologna (Italy) Abstract: This paper presents results from a series of pulsed field gradient (PFG) NMR studies on lipophilic gua- nosine nucleosides that undergo cation- templated assembly in organic solvents. The use of PFG-NMR to measure dif- fusion coefficients for the different ag- gregates allowed us to observe the in- fluences of cation, solvent and anion on the self-assembly process. Three case studies are presented. In the first study, diffusion NMR confirmed for- mation of a hexadecameric G-quadru- plex [G 1] 16 ·4K + ·4pic  in CD 3 CN. Fur- thermore, hexadecamer formation from 5’-TBDMS-2’,3’-isopropylidene G 1 and K + picrate was shown to be a cooperative process in CD 3 CN. In the second study, diffusion NMR studies on 5’-(3,5-bis(methoxy)benzoyl)-2’,3’- isopropylidene G 4 showed that hier- archical self-association of G 8 -octamers is controlled by the K + cation. Evi- dence for formation of both discrete G 8 -octamers and G 16 -hexadecamers in CD 2 Cl 2 was obtained. The position of this octamer–hexadecamer equilibrium was shown to depend on the K + con- centration. In the third case, diffusion NMR was used to determine the size of a guanosine self-assembly where NMR signal integration was ambigu- ous. Thus, both diffusion NMR and ESI-MS show that 5’-O-acetyl-2’,3’-O- isopropylidene G 7 and Na + picrate form a doubly charged octamer [G 7] 8 ·2Na + ·2pic  9 in CD 2 Cl 2 . The anion)s role in stabilizing this particular complex is discussed. In all three cases the information gained from the diffu- sion NMR technique enabled us to better understand the self-assembly processes, especially regarding the roles of cation, anion and solvent. Keywords: G-quadruplex · NMR spectroscopy · self-assembly · supramolecular chemistry # 2005 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim DOI: 10.1002/chem.200400782 Chem. Eur. J. 2005, 11,164–173 164