Ruth M. Gschwind

Identification of (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate as a major activator for human γδ T cells in Escherichia coli

The gcpE and lytB gene products control the terminal steps of isoprenoid biosynthesis via the 2‐C‐methyl‐D‐erythritol 4‐phosphate pathway in Escherichia coli. In lytB‐deficient mutants, a highly immunogenic compound accumulates significantly, compared to wild‐type E. coli, but is apparently absent in gcpE‐deficient mutants. Here, this compound was purified from E. coli ΔlytB mutants by preparative anion exchange chromatography, and identified by mass spectrometry, 1H, 13C and 31P NMR spectroscopy, and NOESY analysis as (E)‐4‐hydroxy‐3‐methyl‐but‐2‐enyl pyrophosphate (HMB‐PP). HMB‐PP is 104 times more potent in activating human Vγ9/Vδ2 T cells than isopentenyl pyrophosphate.

Hydrogel-based drug delivery systems: Comparison of drug diffusivity and release kinetics

Hydrogels are extensively studied as matrices for the controlled release of macromolecules. To evaluate the mobility of embedded molecules, these drug delivery systems are usually characterized by release studies. However, these experiments are time-consuming and their reliability is often poor. In this study, gels were prepared by step-growth polymerization of poly(ethylene glycol) (PEG) and loaded with fluoresceine isothiocyanate (FITC) labeled dextrans. Mechanical testing and swelling studies allowed prediction of the expected FITC-dextran diffusivity. The translational diffusion coefficients (D) of the incorporated FITC-dextrans were measured by fluorescence recovery after photobleaching (FRAP) and pulsed field gradient NMR spectroscopy. Because the determined values of D agreed well with those obtained from release studies, mechanical testing, FRAP, and pulsed field gradient NMR spectroscopy are proposed as alternatives to release experiments. The applied methods complemented each other and represented the relative differences between the tested samples correctly. Measuring D can therefore be used to rapidly evaluate the potential of newly developed drug delivery systems.

The Elusive Enamine Intermediate in Proline‐Catalyzed Aldol Reactions: NMR Detection, Formation Pathway, and Stabilization Trends

The detection and characterization of intermediates in organic reactions is crucial for the understanding of mechanisms and the rational optimization of reaction conditions. However, especially in the rapidly expanding field of asymmetric organocatalysis, mechanistic studies are scarce compared to new synthetic applications. Therefore, organocatalysis was characterized as still being “in its exploratory discovery phase before it can become contemplating”. Among the different organocatalytic activation modes and the wide range of identified general concepts, Brønsted acid and Lewis base catalysis have proven to be broadly applicable. After the proline-catalyzed aldol reactions (both origin and prototype for asymmetric aminocatalysis), secondary amines are preferentially employed to activate substrates via iminium or enamine intermediates. The generally accepted mechanism of enamine catalysis is based upon experimental and theoretical studies that suggest a central enamine intermediate in the proline-catalyzed reactions. To the best of our knowledge, such enamine intermediates have never been detected in situ; only product enamines or dienamines, and dienamine intermediates have been reported—for different catalysts. In contrast, putative enamine intermediates were synthesized, isolated, and characterized, 26–28] and recently an enamine intermediate was observed in the crystal structure of an aldolase antibody. So far, in situ NMR spectroscopic approaches have only resulted in the detection of the isomeric oxazolidinones, 30–34] supposedly resulting from an “unwanted and rate-diminishing parasitic equilibrium”, 35] which was believed to be responsible for the inability to observe the enamines. In fact, equilibria involving oxazolidinones have been reported 32–34] and their energetic preference has been calculated. An alternate mechanistic model of prolinecatalyzed aldol reactions that attributes a pivotal role to the predominant oxazolidinones has been proposed, and indeed such oxazolidinones have successfully been used as “soluble proline catalysts”. The detection of enamine intermediates in prolinecatalyzed aldol reactions is the missing piece of evidence for the commonly accepted mechanism of enamine catalysis. Moreover, the structural characterization of key enamine intermediates, the elucidation of their formation, and their stabilization are important for a better understanding of and the control of organocatalytic reactions, which could in turn present new options in accelerating and controlling enaminecatalyzed reactions. Herein we present our real-time NMR studies that detail the first detection and structural characterization of enamine intermediates in proline-catalyzed aldol reactions. In addition, their direct formation from oxazolidinones is evidenced in the solvent dimethylsulfoxide (DMSO). Moreover, the influences of the carbonyl species, its substitution pattern, as well as the effect of the solvent and its water content upon the detectable enamine concentration are demonstrated. The self-aldolization of propionaldehyde (1; c = 50 mm), catalyzed by 20 mol% l-proline, in [D6]DMSO at 300 K (Figure 1a) was used as a model reaction in our enamine studies. The reaction was conducted within an NMR tube and monitored by one-dimensional H NMR spectra (Figure 1b). Two diastereomeric aldol dimers 2 a and 2b, and the condensation product 3 were observed, which is in accord with previous studies. 38] In addition, three intermediate species were detected, each of which disappeared at identical rates (Figure 1c). By employing 100 mol % l-proline (which led to an acceleration of the reaction and the predominant formation of the condensation product 3, possibly through a Mannich-like mechanism ), we successfully increased the total amount of the intermediates from about 8% to 25–30% without changing their relative ratios (see the Supporting Information). This increased amount of the intermediates allowed unambiguous identification and full characterization of these species as the enamine 5 and the two diastereomeric oxazolidinones 4a and 4b (in a ratio of about 3.8:1) by using real-time homoand heteronuclear two-dimensional NMR spectroscopy during the reaction (see the Supporting Information for the complete NMR assignments). The detection of the ene unit of 5 is rather straightforward due to characteristic H chemical shifts and multiplet patterns (Figure 2b). The connection of the ene unit to the proline [*] M. B. Schmid, Dr. K. Zeitler, Prof. Dr. R. M. Gschwind Institut f r Organische Chemie, Universit t Regensburg Universit tsstraße 31, 93053 Regensburg (Germany) Fax: (+ 49)941-943-4617 E-mail: ruth.gschwind@chemie.uni-regensburg.de

Highly diastereoselective Csp3–Csp2 Negishi cross-coupling with 1,2-, 1,3- and 1,4-substituted cycloalkylzinc compounds

Stereoselective functionalizations of organic molecules are of great importance to modern synthesis. A stereoselective preparation of pharmaceutically active molecules is often required to ensure the appropriate biological activity. Thereby, diastereoselective methods represent valuable tools for an efficient set-up of multiple stereocentres. In this article, highly diastereoselective Csp3 Negishi cross-couplings of various cycloalkylzinc reagents with aryl halides are reported. In all cases, the thermodynamically most-stable stereoisomer was obtained. Remarkably, this diastereoselective coupling was successful not only for 1,2-substituted cyclic systems, but also for 1,3- and 1,4-substituted cyclohexylzinc reagents. The origin of this remote stereocontrol was investigated by NMR experiments and density functional theory calculations. A detailed mechanism based on these experimental and theoretical data is proposed. Highly diastereoselective Negishi cross-coupling reactions between 2-, 3- and 4-substituted cycloalkylzinc reagents and aryl iodides are described. In all cases, the thermodynamically most stable diastereomers of the cross-coupling products were obtained. NMR spectroscopy and density functional theory calculations were performed in order to rationalize the observed stereoselectivities.

Organocuprates and Diamagnetic Copper Complexes: Structures and NMR Spectroscopic Structure Elucidation in Solution

A key role of copper is generally accepted in various scientific fields, for example, in organic synthesis for the formation of C-C bonds, in superconductors, and in biological oxygenation processes. The structure elucidation of copper complexes in solution and the characterization of the electronic structure and bonding at Cu sites greatly benefit chemists and structural biologists in systems such as organometallic copper compounds (for selected articles and reviews see refs 1–8), copper proteins, amyloid related peptides, or copper model systems relevant to organocopper chemistry and biological systems. This is because detailed structural information is often essential for understanding the mechanisms that afford the enormous synthetic and biological potential of Cu(I)/Cu(III) and Cu(I)/Cu(II) redox systems. In solution, the spectroscopic methods, applied in structure elucidation processes, differ substantially between Cu(I)/Cu(III) compounds and systems containing Cu(II). This is due to the different magnetic properties of Cu(II) compared to Cu(I) and square planar Cu(III) complexes. Because Cu(II) is paramagnetic, electron spin resonance (ESR) spectroscopy is mainly applied to the solution state structure determination of Cu(II) containing systems. On the other hand, high resolution NMR spectroscopy is the method of choice for diamagnetic Cu(I) and Cu(III) compounds. For large proteins, paramagnetic NMR spectroscopy forms the link between the two methods and has been performed with great success. In protein structure elucidation, the marginal deviations between solid state and solution structures often allow a direct transfer of information from X-ray crystallographical data. In contrast, the radii of Cu(II) containing organometallic compounds and chiral copper complexes are usually so small that paramagnetic NMR is hardly feasible because of extreme line broadening effects. Therefore, the absence of Cu(II) is of great importance for the successful application of high resolution NMR spectroscopy of diamagnetic complexes as narrow line widths are indispensible. For example, in Cu NMR spectroscopy, the broadenings of the copper signals are 2% when 1% of the copper is present as copper(II), whereas an increase in the copper(II) content to 9% results in a line broadening effect of 870%. In addition, the solvent effects on the structures and reactivities of organocopper compounds are legendary. Therefore, for organocopper compounds in solution, the existence or dominance of a structure cannot be inferred directly from crystal structures and the solution aggregation numbers and aggregate sizes must be determined independently. Considering the different spectroscopic approaches for paramagnetic, diamagnetic, biomacromolecular, and organometallic systems, a comprehensive coverage of all these methods is far beyond the scope of this article. To address copper compounds, which are important for applications in organic synthesis, this review focuses on the NMR spectroscopic structure elucidation and the resulting structures of small diamagnetic Cu(I) and Cu(III) organocuprates and copper complexes in solution. At first, the principles and the application range of Cu NMR in solution is described. Due to the large quadrupole moments of Cu and Cu, the scope of Cu NMR in solution is limited to some highly symmetric complexes with * To whom correspondence should be addressed. E-mail: ruth.gschwind@ chemie.uni-regensburg.de. Phone: ++49 +941 943 4625. Fax: ++49 +941 943 4617. Chem. Rev. 2008, 108, 3029–3053 3029

Automated backbone assignment of labeled proteins using the threshold accepting algorithm

The sequential assignment of backbone resonances is the first step in the structure determination of proteins by heteronuclear NMR. For larger proteins, an assignment strategy based on proton side-chain information is no longer suitable for the use in an automated procedure. Our program PASTA (Protein ASsignment by Threshold Accepting) is therefore designed to partially or fully automate the sequential assignment of proteins, based on the analysis of NMR backbone resonances plus Cβ information. In order to overcome the problems caused by peak overlap and missing signals in an automated assignment process, PASTA uses threshold accepting, a combinatorial optimization strategy, which is superior to simulated annealing due to generally faster convergence and better solutions. The reliability of this algorithm is shown by reproducing the complete sequential backbone assignment of several proteins from published NMR data. The robustness of the algorithm against misassigned signals, noise, spectral overlap and missing peaks is shown by repeating the assignment with reduced sequential information and increased chemical shift tolerances. The performance of the program on real data is finally demonstrated with automatically picked peak lists of human nonpancreatic synovial phospholipase A2, a protein with 124 residues.

The relation between ion pair structures and reactivities of lithium cuprates

From Li+ well-solvating solvents or complex ligands such as THF, [12]crown-4, amines etc., lithium cuprates R2CuLi(*LiX) crystallise in a solvent-separated ion pair (SSIP) structural type (e.g. 10). In contrast, solvents with little donor qualities for Li+ such as diethyl ether or dimethyl sulfide lead to solid-state structures of the contact ion pair (CIP) type (e.g. 11). 1H,6Li HOESY NMR investigations in solutions of R2CuLi(*LiX) (15, 16) are in agreement with these findings: in THF the SSIP 18 is strongly favoured in the equilibrium with the CIP 17, and in diethyl ether one observes essentially only the CIP 17. Salts LiX (X=CN, Cl, Br, I, SPh) have only a minor effect on the ion pair equilibrium. These structural investigations correspond perfectly with Bertzs logarithmic reactivity profiles (LRPs) of reactions of R2CuLi with enones in diethyl ether and THF: the faster reaction in diethyl ether is due to the predominance of the CIP 17 in this solvent, which is the reacting species; in THF only little CIP 17 is present in a fast equilibrium with the SSIP 18. A kinetic analysis of the LRPs quantifies these findings. Recent quantum-chemical studies are also in agreement with the CIP 17 being the reacting species. Thus a uniform picture of structure and reactivity of lithium cuprates emerges.

Distinct conformational preferences of prolinol and prolinol ether enamines in solution revealed by NMR

Enamines, which are key intermediates in organocatalysis derived from aldehydes and prolinol or Jorgensen–Hayashi-type prolinol ether catalysts, were investigated conformationally in different solvents by means of NMR spectroscopy, in order to provide an experimental basis for a better understanding of the origin of stereoselection. For all of the enamines studied, surprisingly strong conformational preferences were observed. The enamines of the diarylprolinol (ether) catalysts were found to exclusively exist in the s-trans conformation due to the bulkiness of the pyrrolidine α-substituent. For prolinol enamines, however, a partial population of the s-cis conformation in solution was also evidenced for the first time. In addition, for all of the enamines studied, the pyrrolidine ring was found to adopt the down conformation. Concerning the exocyclic C–C bond, the sc-exo conformation, stabilized by CH/π interactions, is exclusively observed in the case of diarylprolinol ether enamines. In contrast, diarylprolinol enamines adopt the sc-endo conformation, allowing for an OH⋯N hydrogen bond and a CH/π interaction. A rapid screening approach for the different conformational enamine features is presented and this was applied to show their generality for various catalysts, aldehydes and solvents. Thus, by unexpectedly revealing the pronounced conformational preferences of prolinol and prolinol ether enamines in solution, our study provides the first experimental basis for discussing the previously controversial issues of s-cis/s-trans and sc-endo/sc-exo conformations. Moreover, our findings are in striking agreement with the experimental results from synthetic organic chemistry. They are therefore expected to also have a significant impact on future theoretical calculations and synthetic optimization of asymmetric prolinol (ether) enamine catalysis.

Stabilization of Tetrahedral P4 and As4 Molecules as Guests in Polymeric and Spherical Environments

Chemistry as a science has originated from the exploration and handling of native elements such as sulfur or noble metals that allowed the formulation of the crucial concept of a chemical element. Despite great achievements of inorganic chemistry in the last century, the structural features of some simple substances are still not explicitly clear. It is remarkable that modern X-ray crystallography succeeded in crystal structure determination of proteins containing several thousands of atoms, yet it still faces obstacles in the characterization of some elementary compounds. For example, the extremely high aggressiveness of fluorine gas that was discovered in 1886 impeded its structural characterization for 78 years. Another barrier is the instability and chemical reactivity of allotropic modifications such as O3, whose crystal structure was not revealed until 2001, or the molecular allotropes of phosphorus and arsenic. The high dynamic motion of tetrahedral P4 molecules in white phosphorus led to a complete disorder of the cubic a-P4 phase under ambient conditions. To overcome this problem and obtain a convincing X-ray structural determination, single crystals of the ordered b-P4 phase have to be grown at temperatures below 77 8C. Arsenic exists in three allotropic modifications of which yellow arsenic, consisting of As4 tetrahedral molecules, is the most toxic and the least stable one. It can be obtained in a time-consuming synthesis by heating gray arsenic to 750 8C. The emerging As4 is taken away by a constant flow of a carrier gas and can be discharged into a hot solvent. In contrast to white phosphorus, yellow arsenic cannot be stored as a solid. It is surprisingly poorly soluble in common organic solvents, and it readily polymerizes under ambient conditions to gray arsenic, especially when exposed to light or X-rays. Hence, until now no solid-state structure of yellow arsenic is known. Moreover, traces of gray arsenic accelerate the polymerization of As4 even in solution. Yet, only scarce facts regarding its reactivity or coordination behavior are known. One of the ways to stabilize such unstable molecules is to include them as a guest in a molecular container or polymeric matrix. Oxidation of P4 in air was shown to be prevented by inclusion into the cavity of a supramolecular arrangement of a tetranuclear iron complex. Moreover, the co-crystallization of P4 in the lattice of solid C60 was reported. [8] Recently, Fujita et al. presented an elegant method for the X-ray structural characterization of organic compounds, only available in nanogram scale, based on their inclusion into singlecrystalline, porous 3D coordination polymers. Furthermore, our group succeeded in the stabilization of the unstable paramagnetic 16-electron complex, [Cp*Cr(h-As5)], embedded as a guest in the giant [Cu20Cl20{Cp*Fe(h -P5)}12] molecule (Cp* = h-C5Me5). [10] We reasoned that the use of host molecules could not only enhance the stability of the E4 (E = P, As) molecules, especially of As4, but would also decrease their molecular motion in the solid state. We have reported that the system [Cp*Fe(h-P5)] and Cu -halides forms either polymeric structures or large fullerene-like spherical molecules capable of encapsulating guest molecules and, thus perhaps, the E4 tetrahedra themselves. Herein we present the synthesis and X-ray molecular and crystal structure of polymeric host compounds that contain intact E4 tetrahedra as guests. Furthermore we show that [Ag(h-As4)2] [pftb] (pftb = {Al(OC(CF3)3)4}) [5e] can be utilized for the release of As4 as remarkably light-stable and highly concentrated solutions, making it an ideal storage medium for yellow arsenic. Finally these As4 solutions, as well as solutions of P4, were used to build up spherical macromolecules containing intact E4 tetrahedra as guest molecules. In the presence of P4 or As4, the reaction of CuCl with [Cp*Fe(h-P5)] leads to the formation of the isostructural compounds [Cu2Cl2{Cp*Fe(h -P5)}2]1·(P4)n (1) and [Cu2Cl2{Cp*Fe(h-P5)}2]1·(0.75As4)n (2), in which the tetrahedral voids are filled by perfectly adjusted E4 molecules. Surprisingly, the crystals of 1 and 2 are lightand air-stable for days and are insoluble in common solvents. Crystal-structure analysis reveals that in 1 all the voids are totally occupied by P4, while in 2 the As4 molecules statistically occupy 75 % of the available sites (Figure 1) probably a result of the low and rapidly decreasing concentration of As4 in the reaction medium. The E4 tetrahedra are fixed between the polymeric chains by four pairs of E···P(P5) intermolecular contacts of 3.98 and 4.00 in 1 and 3.98 and 4.04 in 2 (Figure 1), together with [*] Dr. C. Schwarzmaier, Dr. A. Schindler, C. Heindl, S. Scheuermayer, Dr. M. Neumeier, Prof. Dr. R. Gschwind, Prof. Dr. M. Scheer Universit t Regensburg 93040 Regensburg (Germany) E-mail: Manfred.Scheer@chemie.uni-regensburg.de

NMR Investigations on the Proline-Catalyzed Aldehyde Self-Condensation: Mannich Mechanism, Dienamine Detection, and Erosion of the Aldol Addition Selectivity

The proline-catalyzed self-condensation of aliphatic aldehydes in DMSO with varying amounts of catalyst was studied by in situ NMR spectroscopy. The reaction profiles and intermediates observed as well as deuteration studies reveal that the proline-catalyzed aldol addition and condensation are competing, but not consecutive, reaction pathways. In addition, the rate-determining step of the condensation is suggested to be the C-C bond formation. Our findings indicate the involvement of two catalyst molecules in the C-C bond formation of the aldol condensation, presumably by the activation of both the aldol acceptor and donor in a Mannich-type pathway. This mechanism is shown to be operative also in the oligomerization of acetaldehyde with high proline amounts, for which the first in situ detection of a proline-derived dienamine was accomplished. In addition, the diastereoselectivity of the aldol addition is evidenced to be time-dependent since it is undermined by the retro-aldolization and the competing irreversible aldol condensation; here NMR reaction profiles can be used as a tool for reaction optimization.