Marc-Olivier Ebert

Blue Luminescence of Ripening Bananas

As revealed in the last two decades, chlorophyll breakdown in senescent leaves appears to occur by a largely common and well-controlled catabolic path, which rapidly furnishes non-fluorescent, colorless chlorophyll catabolites (NCCs) as “final” products.[1,2] Recently NCCs detected in ripe apples and pears were found to be the same as those in degreened leaves, suggesting chlorophyll catabolism in leaf senescence and fruit ripening to be similar (Scheme 1).[3,4]

Conformational and configurational analysis in the study and synthesis of chlorinated natural products.

The first detailed study of the J-based configuration analysis method in chlorinated hydrocarbons and chlorohydrins is presented along with the development of a spectroscopic database that facilitates configurational assignment of these structures. The data are generated through the investigation of model structures in solution by NMR spectroscopic methods and in the solid state by X-ray crystallography. Consequently, complete conformational analysis of trichlorinated hexane-1,2- and -1,3-diols is presented. The investigations in chlorinated systems for the first time attest to the relevance, reliability, and accuracy of the spectroscopic approach in configurational assignment, which had been otherwise developed for polyketides. During the synthesis of the various molecules that constitute the database and exemplify the various possible stereochemical patterns, a number of observations were made that underscore the unique features of these chlorinated systems. Thus, certain diastereomeric subclasses of 4,5-dichloro-2,3-epoxyhexane-1-ols display a propensity to undergo ring-opening reactions at C-3 with concomitant inversion of configuration at the neighboring C-Cl at C4, implicating the intermediacy of chloronium ions. The observations of positional and stereochemical scrambling in polychlorinated hydrocarbons underscore the necessity of a spectroscopic database that enables rapid, reliable configurational assignment of chlorinated natural products and intermediates en route to these.

Redox-switchable resorcin[4]arene cavitands: molecular grippers.

Diquinone-based resorcin[4]arene cavitands that open to a kite and close to a vase form upon changing their redox state, thereby releasing and binding guests, have been prepared and studied. The switching mechanism is based on intramolecular H-bonding interactions that stabilize the vase form and are only present in the reduced hydroquinone state. The intramolecular H-bonds were characterized using X-ray, IR, and NMR spectroscopies. Guests were bound in the closed, reduced state and fully released in the open, oxidized state.

5-Fluoro pyrimidines: labels to probe DNA and RNA secondary structures by 1D 19F NMR spectroscopy

19F NMR spectroscopy has proved to be a valuable tool to monitor functionally important conformational transitions of nucleic acids. Here, we present a systematic investigation on the application of 5-fluoro pyrimidines to probe DNA and RNA secondary structures. Oligonucleotides with the propensity to adapt secondary structure equilibria were chosen as model systems and analyzed by 1D 19F and 1H NMR spectroscopy. A comparison with the unmodified analogs revealed that the equilibrium characteristics of the bistable DNA and RNA oligonucleotides were hardly affected upon fluorine substitution at C5 of pyrimidines. This observation was in accordance with UV spectroscopic melting experiments which demonstrated that single 5-fluoro substitutions in double helices lead to comparable thermodynamic stabilities. Thus, 5-fluoro pyrimidine labeling of DNA and RNA can be reliably applied for NMR based nucleic acid secondary structure evaluation. Furthermore, we developed a facile synthetic route towards 5-fluoro cytidine phosphoramidites that enables their convenient site-specific incorporation into oligonucleotides by solid-phase synthesis.

Donor–Acceptor (D–A)‐Substituted Polyyne Chromophores: Modulation of Their Optoelectronic Properties by Varying the Length of the Acetylene Spacer

A series of donor-acceptor-substituted alkynes, 2 a-f, was synthesized in which the length of the π-conjugated polyyne spacer between the N,N-diisopropylanilino donor and the 1,1,4,4-tetracyanobuta-1,3-diene (TCBD) acceptor was systematically changed. The effect of this structural change on the optoelectronic properties of the molecules and, ultimately, their third-order optical nonlinearity was comprehensively investigated. The branched N,N-diisopropyl groups on the anilino donor moieties combined with the nonplanar geometry of 2 a-f imparted exceptionally high solubility to these chromophores. This important property allowed for performing INADEQUATE NMR measurements without (13) C labeling, which, in turn, resulted in a complete assignment of the carbon skeleton in chromophores 2 a-f and the determination of the (13) C-(13) C coupling constants. This body of data provided unprecedented insight into characteristic (13) C chemical shift patterns in push-pull-substituted polyynes. Electrochemical and UV/Vis spectroscopic studies showed that the HOMO-LUMO energy gap decreases with increasing length of the polyyne spacer, while this effect levels off for spacers with more than four acetylene units. The third-order optical nonlinearity of this series of molecules was determined by measuring the rotational averages of the third-order polarizabilities (γrot ) by degenerate four-wave mixing (DFWM). These latter studies revealed high third-order optical nonlinearities for the new chromophores; most importantly, they provided fundamental insight into the effect of the conjugated spacer length in D-A polyynes, that can be exploited in the future design of suitable charge-transfer chromophores for applications in optoelectronic devices.

Quinone‐Based, Redox‐Active Resorcin[4]arene Cavitands

Since the preparation of the first quinoxaline-bridged resorcin[4]arene cavitand by Cram and co-workers in 1982, numerous derivatives with various structures and functions have been prepared and employed as switches, receptors and sensors, catalysts, and molecular hosts. The most fascinating feature of top-open resorcin[4]arene cavitands is their ability to adopt two spatially well-defined conformations: an expanded “kite” form and a contracted “vase” form. While various methods have been employed to study the conformational properties of cavitands, the most convenient one is H NMR spectroscopy. Figure 1 illustrates the vase– kite1–kite2 equilibrium of a generic cavitand, and how three situations, vase, kite with slow kite1–kite2 interconversion, and kite with fast kite1–kite2 interconversion, can be distinguished by H NMR spectroscopy. The three stimuli that have been identified for switching the cavitand between its vase and kite forms are changes in temperature, 8] pH, and metal-ion concentration. Additionally, cavitands can function as molecular grippers by binding guest molecules in their vase conformations; these binding properties have been modulated using changes in pH, metal-ion complexation, and light. To use redox processes as new stimuli for tuning cavitand properties, however, is a highly desirable, yet unreached goal. Electrochemically induced redox switching, if truly reversible, and performed on the surface of a metal electrode, could enable the application of cavitands as molecular grippers. Towards this end, we chose to investigate cavitands containing the quinone moiety as a new and easily installed redoxactive wall component and to study how changing their redox states affects their conformational and binding properties. It has been shown that cavitand wall size and solvent identity can influence the vase–kite equilibrium of resorcin[4]arene cavitands: 14] the vase conformation is preferred more strongly in cavitands with larger walls and in solvents such as tetrahydrofuran, benzene, and toluene than in cavitands with smaller walls and in chlorinated solvents (CD2Cl2, CDCl3, (CDCl2)2). We therefore prepared a series of quinoid cavitands with different wall sizes (ox-1 a–c, Figure 2) and investigated their conformational preferences in one member of each solvent class (for the synthesis of all reported compounds, see the Supporting Information, Section 2). The H NMR spectra (298 K, 500 MHz) of cavitand ox-1b in CD2Cl2 and [D8]THF are shown in Figure 2. In both solvents, the cavitand is present in the kite conformation: the methine protons (*) are located at 4.25 ppm in CD2Cl2 and at 4.43 ppm in [D8]THF, respectively. The main difference between the two solvents, however, is that in CD2Cl2, two sharp signals are observed for each of the bowl protons (~ and !), while in [D8]THF, these signals are close to coalescence. This indicates that the kite1–kite2 interconversion is faster in [D8]THF than in CD2Cl2. Thus, [D8]THF not only stabilizes the vase conformation more strongly but also the vaselike transition state for the kite1–kite2 interconversion. The preference of cavitand ox-1b for the kite conformation was Figure 1. Vase–kite1–kite2 equilibrium of a generic cavitand and summary of characteristic H NMR features of three situations: vase, kite with slow kite1–kite2 interconversion, and kite with fast kite1–kite2 interconversion.

Biosynthesis of the proteasome inhibitor syringolin A: the ureido group joining two amino acids originates from bicarbonate

BackgroundSyringolin A, an important virulence factor in the interaction of the phytopathogenic bacterium Pseudomonas syringae pv. syringae B728a with its host plant Phaseolus vulgaris (bean), was recently shown to irreversibly inhibit eukaryotic proteasomes by a novel mechanism. Syringolin A is synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase and consists of a tripeptide part including a twelve-membered ring with an N-terminal valine that is joined to a second valine via a very unusual ureido group. Analysis of sequence and architecture of the syringolin A synthetase gene cluster with the five open reading frames sylA-sylE allowed to formulate a biosynthesis model that explained all structural features of the tripeptide part of syringolin A but left the biosynthesis of the unusual ureido group unaccounted for.ResultsWe have cloned a 22 kb genomic fragment containing the sylA-sylE gene cluster but no other complete gene into the broad host range cosmid pLAFR3. Transfer of the recombinant cosmid into Pseudomonas putida and P. syringae pv. syringae SM was sufficient to direct the biosynthesis of bona fide syringolin A in these heterologous organisms whose genomes do not contain homologous genes. NMR analysis of syringolin A isolated from cultures grown in the presence of NaH13CO3 revealed preferential 13C-labeling at the ureido carbonyl position.ConclusionThe results show that no additional syringolin A-specific genes were needed for the biosynthesis of the enigmatic ureido group joining two amino acids. They reveal the source of the ureido carbonyl group to be bicarbonate/carbon dioxide, which we hypothesize is incorporated by carbamylation of valine mediated by the sylC gene product(s). A similar mechanism may also play a role in the biosynthesis of other ureido-group-containing NRPS products known largely from cyanobacteria.

On the Terminal Homologation of Physiologically Active Peptides as a Means of Increasing Stability in Human Serum – Neurotensin, Opiorphin, B27‐KK10 Epitope, NPY

The terminal homologation by CH2 insertion into the peptides mentioned in the title is described. This involves replacement of the N‐terminal amino acid residue by a β2‐ and of the C‐terminal amino acid residue by a β3‐homo‐amino acid moiety (β2hXaa and β3hXaa, resp.; Fig. 1). In this way, the structure of the peptide chain from the N‐terminal to the C‐terminal stereogenic center is identical, and the modified peptide is protected against cleavage by exopeptidases (Figs. 2 and 3). Neurotensin (NT; 1) and its C‐terminal fragment NT(8–13) are ligands of the G‐protein‐coupled receptors (GPCR) NT1, NT2, NT3, and NT analogs are promising tools to be used in cancer diagnostics and therapy. The affinities of homologated NT analogs, 2b–2e, for NT1 and NT2 receptors were determined by using cell homogenates and tumor tissues (Table 1); in the latter experiments, the affinities for the NT1 receptor are more or less the same as those of NT (0.5–1.3 vs. 0.6 nM). At the same time, one of the homologated NT analogs, 2c, survives in human plasma for 7 days at 37° (Fig. 6). An NMR analysis of NT(8–13) (Tables 2 and 4, and Fig. 8) reveals that this N‐terminal NT fragment folds to a turn in CD3OH. – In the case of the human analgesic opiorphin (3a), a pentapeptide, and of the HIV‐derived B27‐KK10 (4a), a decapeptide, terminal homologation (→3b and 4b, resp.) led to a 7‐ and 70‐fold half‐life increase in plasma (Fig. 9). With N‐terminally homologated NPY, 5c, we were not able to determine serum stability; the peptide consisting of 36 amino acid residues is subject to cleavage by endopetidases. Three of the homologated compounds, 2b, 2c, and 5c, were shown to be agonists (Fig. 7 and 11). A comparison of terminal homologation with other stability‐increasing terminal modifications of peptides is performed (Fig. 5), and possible applications of the neurotensin analogs, described herein, are discussed.

NMR Solution Structure of the Duplex Formed by Self‐Pairing of α‐L‐Arabinopyranosyl‐(4′2′)‐(CGAATTCG)

The solution structure of the duplex formed by α-L-arabinopyranosyl-(4′2′)-(CGAATTCG) was studied by NMR. The resonances of all H-, P- and most C-atoms could be assigned. Dihedral angles and distance estimates derived from coupling constants and NOESY spectra were used as restraints in a simulated annealing calculation, which generated a well-defined bundle of structures for the six innermost nucleotide pairs. The essential features of the resulting structures are an antiparallel, WatsonCrick-paired duplex with a strong backbone inclination of ca. −50° and, therefore, predominant interstrand base stacking. The very similar inclination and rise parameters of arabinopyranosyl-(4′2′)-oligonucleotides and p-RNA explain why these two pentapyranosyl isomers are able to cross-pair.

Comparison of the NMR spectroscopy solution structures of pyranosyl-RNA and its nucleo-δ-peptide analogue

The design of polymers that could mimic biomolecules in their ability to form assemblies similar to ribo‐ and deoxyribonucleic acids has become an attractive field of chemical research, and NMR spectroscopy has played a vital role in the determination of the three‐dimensional structure of these newly designed nonnatural polymers. The structure of a self‐complementary octamer duplex of pyranosyl‐RNA (pRNA) has been determined by using NMR spectroscopy experimental data and an Xplor structure calculation protocol. The structure has been compared with the structure of a duplex formed by a designed nucleo‐δ‐peptide analogue of pRNA. The two duplexes assume one predominant conformation and show a high structural similarity. The conformation type of both structures agrees with those predicted based on qualitative conformational analysis and both structures show a good convergence toward the average torsion angles derived by NMR spectroscopy.