Russell Hopson

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-8β-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.

Genome mining in Streptomyces clavuligerus: expression and biochemical characterization of two new cryptic sesquiterpene synthases.

Two presumptive terpene synthases of unknown biochemical function encoded by the sscg_02150 and sscg_03688 genes of Streptomyces clavuligerus ATCC 27074 were individually expressed in Escherichia coli as N-terminal-His₆-tag proteins, using codon-optimized synthetic genes. Incubation of recombinant SSCG_02150 with farnesyl diphosphate (1, FPP) gave (-)-δ-cadinene (2) while recombinant SSCG_03688 converted FPP to (+)-T-muurolol (3). Individual incubations of (-)-δ-cadinene synthase with [1,1-²H₂]FPP (1a), (1S)-[1-²H]-FPP (1b), and (1R)-[1-²H]-FPP (1c) and NMR analysis of the resulting samples of deuterated (-)-δ-cadinene supported a cyclization mechanism involving the intermediacy of nerolidyl diphosphate (4) leading to a helminthogermacradienyl cation 5. Following a 1,3-hydride shift of the original H-1(si) of FPP, cyclization and deprotonation will give (-)-δ-cadinene. Similar incubations with recombinant SSCG_03688 supported an analogous mechanism for the formation of (+)-T-muurolol (3), also involving a 1,3-hydride shift of the original H-1(si) of FPP.

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).

Internally referenced diffusion coefficient-formula weight (D-FW) analysis of 31P diffusion-ordered NMR spectroscopy (DOSY).

The development of (31)P DOSY NMR with diffusion coefficient-formula weight (D-FW) analysis is reported. Commercially available trialkyl phosphine internal references were used in a model system to establish the molecular weight of a phosphorous containing organolithium compound. The feasibility of (31)P DOSY D-FW studies is established. This extension of DOSY D-FW analysis expands its applicability to solution structure studies of a wide variety of compounds.

6Li diffusion-ordered NMR spectroscopy (DOSY) and applications to organometallic complexes.

The development of (6)Li diffusion-ordered NMR spectroscopy (DOSY) is reported. This technique is applied to (6)Li organometallic complexes. (6)Li DOSY provides a facile means of identification of peaks in the (6)Li spectrum, as well as evidence of mixed aggregates based on relative diffusion coefficients. (6)Li data is correlated to (1)H diffusion experiments through (6)Li{(1)H} HOESY and/or (1)H{(6)Li} HMBC experiments to obtain formula weight information of Li aggregates.

Aggregation Studies of Complexes Containing a Chiral Lithium Amide and n-Butyllithium

A system consisting of a chiral lithium amide and n-BuLi in tol-d(8) solution was investigated with (1)H and (13)C INEPT DOSY, (6)Li and (15)N NMR, and other 2D NMR techniques. A mixed 2:1 trimeric complex was identified as the major species as the stoichiometry approached 1.5 equiv of n-BuLi to 1 equiv of amine compound. (1)H and (13)C INEPT DOSY spectra confirmed this lithium aggregate in the solution. The formula weight of the aggregate, correlated with diffusion coefficients of internal references, indicated the aggregation number of this complex. Plots of log D(rel) vs log FW are linear (r > 0.9900). (6)Li and (15)N NMR titration experiments also corroborated these results. These NMR experiments indicate that this mixed aggregate is the species that is responsible for asymmetric addition of n-BuLi to aldehydes.

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.

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.

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.