Ligand Binding Constants to Lithium Hexamethyldisilazide Determined by Diffusion-Ordered NMR Spectroscopy.

We report the direct measurement of ligand-binding constants of organolithium complexes using a 1H NMR/diffusion-ordered NMR spectroscopy (DOSY) titration technique. Lithium hexamethyldisilazide complexes with ethereal and ester donor ligands (THF, diethyl ether, MTBE, THP, tert-butyl acetate) are characterized using 1H NMR and X-ray crystallography. Their aggregation and solvation states are confirmed using diffusion coefficient-formula weight correlation analysis, and the 1H NMR/DOSY titration technique is applied to obtain their binding constants. Our work suggests that steric hindrance of ethereal ligands plays an important role in the aggregation, solvation, and reactivity of these complexes. It is noteworthy that diffusion methodology is utilized to obtain binding constants.

Conformational Polymorphism of Lithium Pinacolone Enolate.

A metastable, polymorphic hexameric crystal structure of lithium pinacolone enolate (LiOPin) is reported along with three preparation methods. NMR-based structural characterization implies that the lithium pinacolate hexamer deaggregates to a tetramer in toluene but retains mainly the hexameric structure in nonaromatic hydrocarbon solvents such as cyclohexane. Moreover, the presence of a small amount of lithium aldolate (LiOA) dramatically influences the aggregation state of LiOPin by forming a mixed aggregate with a 3:1 ratio (LiOPin3·LiOA).

Perfluoroalkyl Grignard Reagents: NMR Study of 1-Heptafluoropropylmagnesium Chloride in Solution.

We report on the generation of a perfluoroalkyl Grignard reagent ((F)RMgX) by exchange reaction between a perfluoroalkyl iodide ((F)R-I) and a Grignard reagent (RMgX). (19)F NMR was applied to monitor the generation of n-C3F7MgCl. Additional NMR techniques, including (19)F COSY, NOESY, and pulsed gradient spin-echo (PGSE) diffusion NMR, were invoked to assign peaks observed in (19)F spectrum. Schlenk equilibrium was observed and was significantly influenced by solvent, diethyl ether, or THF.

Lithium pinacolone enolate solvated by hexamethylphosphoramide.

We report the crystal structure of a substoichiometric, HMPA-trisolvated lithium pinacolone enolate tetramer (LiOPin)4·HMPA3 abbreviated as T3. In this tetramer one HMPA binds to lithium more strongly than the other two causing a reduction in spatial symmetry with corresponding loss of C3 symmetry. A variety of NMR experiments, including HMPA titration, diffusion coefficient-formula weight (D-FW) analysis, and other multinuclear one- and two-dimensional NMR techniques reveal that T3 is the major species in hydrocarbon solution when more than 0.6 equiv of HMPA is present. Due to a small amount of moisture from HMPA or air leaking into the solution, a minor complex was identified and confirmed by X-ray diffraction analysis as a mixed aggregate containing enolate, lithium hydroxide, and HMPA in a 4:2:4 ratio, [(LiOPin)4·(LiOH)2·HMPA4], that we refer to as pseudo-T4. A tetra-HMPA-solvated lithium cyclopentanone enolate tetramer was also prepared and characterized by X-ray diffraction, leading to the conclusion that steric effects dominate the formation and solvation of the pinacolone aggregates. An unusual mixed aggregate consisting of pinacolone enolate, lithium diisopropyl amide, lithium oxide, and HMPA in the ratio 5:1:1:2 is also described.

Diffusion Coefficient-Formula Weight (D-FW) Analysis of (2)H Diffusion-Ordered NMR Spectroscopy (DOSY).

We report extension of the D-FW analysis using referenced (2)H DOSY. This technique was developed in response to limitations due to peak overlay in (1)H DOSY spectra. We find a corresponding linear relationship (R(2) > 0.99) between log D and log FW as the basis of the D-FW analysis. The solution-state structure of THF solvated lithium diisopropyl amide (LDA) in hydrocarbon solvent was chosen to demonstrate the reliability of the methodology. We observe an equilibrium between monosolvated and disolvated dimeric LDA complexes at room temperature. Additionally we demonstrate the application of the (2)H D-FW analysis using a compound with an exchangeable proton that is readily labeled with (2)H. Hence, the (2)H DOSY D-FW analysis is shown to provide results consistent with the (1)H DOSY method, thereby greatly extending the applicability of the D-FW analysis.

Influence of steric factors on chiral lithium amide aggregates.

The solution structures of three mixed aggregates dissolved in toluene-d8 consisting of the lithiated amides derived from (S)-N-isopropyl-1-((triisopropylsilyl)oxy)propan-2-amine, (R)-N-(1-phenyl-2-((triisopropylsilyl)oxy)ethyl)propan-2-amine, or (S)-N-isobutyl-3-methyl-1-((triisopropylsilyl)oxy)butan-2-amine and n-butyllithium are characterized by various NMR experiments including diffusion-ordered NMR spectroscopy with diffusion coefficient-formula weight correlation analyses (D-FW) and other one- and two-dimensional NMR techniques. We report that steric hindrance of R1 and R2 groups of the chiral lithium amide controls the aggregation state of the mixed aggregates. With a less hindered R2 group, lithium (S)-N-isopropyl-1-((triisopropylsilyl)oxy)propan-2-amide forms mostly a 2:2 ladder-type mixed aggregate with n-butyllithium. Increase of steric hindrance of the R1 and R2 groups suppresses the formation of the 2:2 mixed aggregate and promotes formation of a 2:1 mixed aggregate. We observe that lithium (S)-N-isobutyl-3-methyl-1-((triisopropylsilyl)oxy)butan-2-amide forms both a 2:2 mixed aggregate and a 2:1 mixed trimer with n-butyllithium. Further increase in the steric hindrance of R1 and R2 groups results in the formation of only 2:1 mixed aggregate as observed with lithium (R)-N-(1-phenyl-2-((triisopropylsilyl)oxy)ethyl)propan-2-amide.

Chiral lithium diamides derived from linked N-isopropyl valinol or alaninol.

Four different chiral diamino diethers synthesized from N-isopropyl valinol or N-isopropyl alaninol were lithiated with n-butyllithium in tetrahydrofuran or diethyl ether. Crystal structures of the dilithiated diamino diethers were determined by X-ray diffraction. Three dilithiated diamino diethers including (2S,2'S)-1,1'-(butane-1,4-diylbis(oxy))bis(N-isopropylpropan-2-amine) 7, (2S,2'S)-1,1'-(pentane-1,5-diylbis(oxy))bis(N-isopropylpropan-2-amine) 8, and (2S,2'S)-1,1'-(heptane-1,7-diylbis(oxy))bis(N-isopropyl-3-methylbutan-2-amine) 9 are dimers, whereas dilithiated (2S,2'S)-1,1'-(pentane-1,5-diylbis(oxy))bis(N-isopropyl-3-methylbutan-2-amine) 10 is a monomer. The lithium atoms in all crystal structures adopt a nonequivalent coordination protocol and exist in two different environments in which one of the lithium atoms is tetra-coordinated while the other one is tri-coordinated. The solution structures of the dilithiated diamino diethers are also characterized by a variety of NMR experiments including diffusion-ordered NMR spectroscopy (DOSY) with diffusion coefficient-formula (D-FW) weight correlation analyses and other one- and two-dimensional NMR techniques.

Characterization of cyclopentyllithium and cyclopentyllithium tetrahydrofuran complex.

The solid-state structures of unsolvated, hexameric cyclopentyllithium and tetrameric cyclopentyllithium tetrahydrofuran solvate were determined by single-crystal X-ray diffraction. Cyclopentyllithium easily crystallized in hydrocarbon solvents. Solution-state structural analyses of cyclopentyllithium and cyclopentyllithium-tetrahydrofuran complexes in toluene-d8 were also carried out by diffusion-ordered NMR spectroscopy with diffusion coefficient-formula weight correlation analyses and other one- and two-dimensional NMR techniques. The solution-state studies suggest that unsolvated cyclopentyllithium exists as hexamer and tetramer equilibrating with each other. Upon solvation with tetrahydrofuran, cyclopentyllithium exists mostly as a tetrahydrofuran tetrasolvated tetramer.

Mixed aggregates of an alkyl lithium reagent and a chiral lithium amide derived from N-ethyl-O-triisopropylsilyl valinol.

The crystal structure of a mixed aggregate containing lithiated (S)-N-ethyl-3-methyl-1-(triisopropylsilyloxy)butan-2-amine derived from (S)-valinol and cyclopentyllithium is determined by X-ray diffraction. The mixed aggregate adopts a ladder structure in the solid state. The ladder-type mixed aggregate is also the major species in a toluene-d8 solution containing an approximately 1:1 molar ratio of the lithiated chiral amide to cyclopentyllithium. A variety of NMR experiments including diffusion-ordered NMR spectroscopy (DOSY) with diffusion coefficient-formula (D-FW) weight correlation analyses and other one- and two-dimensional NMR techniques allowed us to characterize the complex in solution. Solution state structures of the mixed aggregates of n-butyl, sec-butyllithium, isopropyllithium with lithiated (S)-N-ethyl-3-methyl-1-(triisopropylsilyloxy)butan-2-amine are also reported. Identical dimeric, ladder-type, mixed aggregates are the major species at a stoichiometric ratio of 1:1 lithium chiral amide to alkyllithium in toluene-d8 solution for all of the different alkyllithium reagents.

Isotopically enriched 13C diffusion-ordered NMR spectroscopy: analysis of methyllithium.

We report the development of isotopic-labeled (13)C diffusion-ordered NMR spectroscopy (DOSY) NMR with diffusion coefficient-formula weight (D-FW) analysis and its application in characterizing the aggregation state of methyllithium aggregates and complexes with several widely used diamines. Commercially available (13)C-labeled benzene and several easily synthesized (13)C-labeled compounds using (13)C-labeled iodomethane as the isotopic source are developed as internal references for diffusion-formula weight analysis (D-FW). The technique greatly expands the applicability of DOSY D-FW analysis to a much wider variety of compounds because of isotopic labeling. These results reveal that methyllithium exists as a tetrasolvated tetramer in diethyl ether and exclusively as bis-solvated dimers with chelating diamines.

Crystal structure and solution state characterization of lithium (S)-(1-(bis(2-methoxyethyl)amino)-3-methylbutan-2-yl)(methyl)amide.

The solid state structure of lithiated (S)-N(1),N(1)-bis(2-methoxyethyl)-N(2),3-dimethylbutane-1,2-diamine, which is a chiral amide base synthesized from (S)-valine was determined by single-crystal X-ray diffraction. The complex in solution state is also characterized by a variety of NMR experiments including diffusion-ordered NMR spectroscopy (DOSY) with diffusion coefficient-formula weight correlation analyses and other one- and two-dimensional NMR techniques by dissolving the crystal in toluene-d8. The crystallography and NMR results suggest that the chiral amide is dimeric in both solid and solution states.

Identification of a Vitamin-D Receptor Antagonist, MeTC7, which Inhibits the Growth of Xenograft and Transgenic Tumors In Vivo.

Vitamin-D receptor (VDR) mRNA is overexpressed in neuroblastoma and carcinomas of lung, pancreas, and ovaries and predicts poor prognoses. VDR antagonists may be able to inhibit tumors that overexpress VDR. However, the current antagonists are arduous to synthesize and are only partial antagonists, limiting their use. Here, we show that the VDR antagonist MeTC7 (5), which can be synthesized from 7-dehydrocholesterol (6) in two steps, inhibits VDR selectively, suppresses the viability of cancer cell-lines, and reduces the growth of the spontaneous transgenic TH-MYCN neuroblastoma and xenografts in vivo. The VDR selectivity of 5 against RXRα and PPAR-γ was confirmed, and docking studies using VDR-LBD indicated that 5 induces major changes in the binding motifs, which potentially result in VDR antagonistic effects. These data highlight the therapeutic benefits of targeting VDR for the treatment of malignancies and demonstrate the creation of selective VDR antagonists that are easy to synthesize.

Characterization of dimeric chiral lithium amide structures derived from N-isopropyl-O- triisopropylsilyl valinol.

The dimeric structure is characterized for a chiral amide base complex consisting of an (S)-N-isopropyl-O-triisopropylsilyl valinol ligand and lithium. The complex is characterized by a variety of NMR techniques, including multinuclear one- and two-dimensional NMR experiments and diffusion-ordered NMR spectroscopy (DOSY) as well as diffusion coefficient-formula weight (D-fw) correlation analyses. Spartan calculations are presented which support the structural assignment. This structural characterization leads to an explanation of the behavior and the reactivity of these complexes in solution.

Synthesis, characterization, and reaction of a ketone-derived 1,4-dienolate compound.

The tetrahydrofuran tetrasolvated dimeric lithium dienolate derived from 2,2,7,7-tetramethyloctan-3,6-dione is characterized in the solid state by X-ray diffraction analysis and in solution by diffusion NMR. This dienolate was reacted with tropanone to yield two new products that are also described.

Characterization of reactive intermediates by multinuclear diffusion-ordered NMR spectroscopy (DOSY).

Nuclear magnetic resonance (NMR) is the most powerful and widely utilized technique for determining molecular structure. Although traditional NMR data analysis involves the correlation of chemical shift, coupling constant, and NOE interactions to specific structural features, a largely overlooked method introduced more than 40 years ago, pulsed gradient spin-echo (PGSE), measures diffusion coefficients of molecules in solution, thus providing their relative particle sizes. In the early 1990s, the PGSE sequence was incorporated into a two-dimensional experiment, dubbed diffusion-ordered NMR spectroscopy (DOSY), in which one dimension represents chemical shift data while the second dimension resolves species by their diffusion properties. This combination provides a powerful tool for identifying individual species in a multicomponent solution, earning the nickname "chromatography by NMR". In this Account, we describe our efforts to utilize DOSY techniques to characterize organometallic reactive intermediates in solution in order to correlate structural data to solid-state crystal structures determined by X-ray diffraction and to discover the role of aggregate formation and solvation states in reaction mechanisms. In 2000, we reported our initial efforts to employ DOSY techniques in the characterization of reactive intermediates such as organolithium aggregates. Since then, we have explored DOSY experiments with various nuclei beyond (1)H, including (6)Li, (7)Li, (11)B, (13)C, and (29)Si. Additionally, we proposed a diffusion coefficient-formula weight relationship to determine formula weight, aggregation number, and solvation state of reactive intermediates. We also introduced an internal reference system to correlate the diffusion properties of unknown reactive intermediates with known inert molecular standards, such as aromatic compounds, terminal olefins, cycloolefins, and tetraalkylsilanes. Furthermore, we utilized DOSY to interpret the role of aggregation number and solvation state of organometallic intermediates in the reactivity, kinetics, and mechanism of organic reactions. By utilizing multinuclear DOSY methodologies at various temperatures, we also correlated solid-state X-ray structures with those in solution and discovered new reactive complexes, including a monomeric boron enolate, a product-inhibition aggregate, and a series of intermediates in the vinyl lithiation of allyl amines. As highlighted by our efforts, DOSY techniques provide practical and feasible NMR procedures and hold the promise of even more powerful insights when extended to three-dimensional experiments.

Formula weight prediction by internal reference diffusion-ordered NMR spectroscopy (DOSY).

Formula weight (FW) information is important to characterize the composition, aggregation number, and solvation state of reactive intermediates and organometallic complexes. We describe an internal reference correlated DOSY method for calculating the FW of unknown species in different solvents with different concentrations. Examples for both the small molecule (DIPA) and the organometallic complex (aggregate 1) yield excellent correlations. We also found the relative diffusion rate is inversely proportional to the viscosity change of the solution, which is consistent with the theoretical Stokes-Einstein equation. The accuracy of the least-squares linear prediction r(2) and the percentage difference of FW prediction are directly related to the density change; greater accuracy was observed with decreasing density. We also discuss the guidelines and other factors for successful application of this internal reference correlated DOSY method. This practical method can be conveniently modified and applied to the characterization of other unknown molecules or complexes.

Biosynthesis of the sesquiterpene botrydial in Botrytis cinerea. Mechanism and stereochemistry of the enzymatic formation of presilphiperfolan-8beta-ol.

Presilphiperfolan-8beta-ol synthase, encoded by the BcBOT2 gene from the necrotrophic plant pathogen Botrytis cinerea, catalyzes the multistep cyclization of farnesyl diphosphate (2) to the tricyclic sesquiterpene alcohol presilphiperfolan-8beta-ol (3), the preursor of the phytotoxin botrydial, a strain-dependent fungal virulence factor. Incubation of (1R)-[1-(2)H]farnesyl diphosphate (2b) with recombinant presilphiperfolan-8beta-ol synthase gave exclusively (5R)-[5alpha-(2)H]-3b, while complementary incubation of (1S)-[1-(2)H]FPP (2c) gave (5S)-[5beta-(2)H]-3c. These results established that cyclization of farnesyl diphosphate involves displacement of the diphosphate group from C-1 with net inversion of configuration and ruled out the proposed intermediacy of the cisoid conformer of nerolidyl diphosphate (9) in the cyclization. While not a mandatory intermediate, (3R)-nerolidyl diphosphate was shown to act as a substrate surrogate. Cyclization of [13,13,13-(2)H(3)] farnesyl diphosphate (2d) gave [14,14,14-(2)H(3)]-3d, thereby establishing that electrophilic attack takes place exclusively on the si face of the 12,13-double bond of 2. The combined results provide a detailed picture of the conformation of enzyme-bound farnesyl diphosphate at the active site of presilphiperfolan-8beta-ol synthase.

13C INEPT diffusion-ordered NMR spectroscopy (DOSY) with internal references.

13C INEPT Diffusion-ordered NMR spectroscopy (DOSY) with an internal reference system was developed to study the aggregation state of THF-solvated LDA dimeric complex. Six components are clearly identified in the diffusion dimension, and their DOSY-generated 13C INEPT spectrum slices agree extremely well with their respective INEPT spectra. The correlation between log D and log FW of the linear least-squares fit to reference points of all components is exceptionally high: (r = 0.9985).

Microcoil NMR spectroscopy: a novel tool for biological high throughput NMR spectroscopy.

Microcoil NMR spectroscopy is based on the increase of coil sensitivity for smaller coil diameters (approximately 1/d). Microcoil NMR probes deliver a remarkable mass-based sensitivity increase (8- to 12-fold) when compared with commonly used 5-mm NMR probes. Although microcoil NMR probes are a well established analytical tool for small molecule liquid-state NMR spectroscopy, after spectroscopy only recently have microcoil NMR probes become available for biomolecular NMR spectroscopy. This chapter highlights differences between commercially available microcoil NMR probes suitable for biomolecular NMR spectroscopy. Furthermore, it provides practical guidance for the use of microcoil probes and shows direct applications for structural biology and structural genomics, such as optimal target screening and structure determination, among others.

Aggregation and Solvation of n-Butyllithium.

Solution characterizations and ligand binding constants were determined for n-butyllithium in hydrocarbon and ethereal solvents using diffusion-ordered NMR. In hydrocarbon solvents, n-butyllithium exists primarily as an octamer at -40 °C and deaggregates to a hexamer when the temperature is increased. In the presence of THF or diethyl ether, n-butyllithium exists predominantly as a tetra-solvated tetramer and deaggregates to a tetra-solvated dimer in the presence of a large excess or neat THF. The ligand binding constants for the tetra-solvated tetramers were measured using 1H NMR/DOSY titration.