CD Stretching Modes are Sensitive to the Microenvironment in Ionic Liquids.

Knowledge of the structure of the electrical double layer in ionic liquids (IL) is crucial for their applications in electrochemical technologies. We report the synthesis and applicability of an imidazolium-based amphiphilic ionic liquid with a perdeuterated alkyl chain for studies of electric potential-dependent rearrangements, and changes in the microenvironment in a monolayer on a Au(111) surface. Electrochemical measurements show two states of the organization of ions on the electrode surface. In situ IR spectroscopy shows that the alkyl chains in imidazolium cations change their orientation depending on the adsorption state. The methylene-d2 stretching modes in the perdeuterated IL display a reversible, potential-dependent appearance of a new band. The presence of this mode also depends on the anion in the IL. Supported by quantum chemical calculations, this new mode is assigned to a second νas (CD2 ) band in alkyl-chain fragments embedded in a polar environment of the anions/solvent present in the vicinity of the imidazolium cation and electrode. It is a measure of the potential-dependent segregation between polar and nonpolar environments in the layers of an IL closest to the electrode.

Metabolites of the Anaerobic Degradation of n-Hexane by Denitrifying Betaproteobacterium Strain HxN1.

The constitutions of seven metabolites formed during anaerobic degradation of n-hexane by the denitrifying betaproteobacterium strain HxN1 were elucidated by comparison of their GC and MS data with those of synthetic reference standards. The synthesis of 4-methyloctanoic acid derivatives was accomplished by the conversion of 2-methylhexanoyl chloride with Meldrum's acid. The ß-oxoester was reduced with NaBH4 , the hydroxy group was eliminated, and the double bond was displaced to yield the methyl esters of 4-methyl-3-oxooctanoate, 3-hydroxy-4-methyloctanoate, (E)-4-methyl-2-octenoate, and (E)- and (Z)-4-methyl-3-octenoate. The methyl esters of 2-methyl-3-oxohexanoate and 3-hydroxy-2-methylhexanoate were similarly prepared from butanoyl chloride and Meldrum's acid. However, methyl (E)-2-methyl-2-hexenoate was prepared by Horner-Wadsworth-Emmons reaction, followed by isomerization to methyl (E)-2-methyl-3-hexenoate. This investigation, with the exception of 4-methyl-3-oxooctanoate, which was not detectable in the cultures, completes the unambiguous identification of all intermediates of the anaerobic biodegradation of n-hexane to 2-methyl-3-oxohexanoyl coenzyme A (CoA), which is then thiolytically cleaved to butanoyl-CoA and propionyl-CoA; these two metabolites are further transformed according to established pathways.

Electrochemical-Induced Ring Transformation of Cyclic α-(ortho-Iodophenyl)-ß-oxoesters.

Cyclic α-(ortho-iodophenyl)-ß-oxoesters were converted in a ring-expanding transformation to furnish benzannulated cycloalkanone carboxylic esters. The reaction sequence started by electrochemical reduction of the iodoarene moiety. In a mechanistic rationale, the resulting carbanionic species was adding to the carbonyl group under formation of a strained, tricyclic benzocyclobutene intermediate, which underwent carbon-carbon bond cleavage and rearrangement of the carbon skeleton by retro-aldol reaction. The scope of the reaction sequence was investigated by converting cyclic oxoesters with different ring sizes yielding benzocycloheptanone, -nonanone and -decanone derivatives in moderate to good yields. Furthermore, acyclic starting materials and cyclic compounds carrying additional substituents on the iodophenyl ring were submitted to this reaction sequence. The starting materials for this transformation are straightforwardly obtained by conversion of ß-oxoesters with phenyliodobis(trifluoroacetate).

Electrochemical Activation of Self-Assembled Monolayers for the Binding of Effectors.

A self-assembled monolayer (SAM) on gold was prepared from a diaminoterephthalate (DAT) derivative as functional molecule and 1-decanthiol as a backfiller. The DAT derivative is N-protected by a tert-butyloxycarbonyl (Boc) group and is anchored to the gold surface via a liponic acid as a stable anchor group. The terminal DAT moiety exhibits interesting effector properties such as fluorescence and electrochemical activity. Irreversible oxidation of the monolayer at 0.4 V (Hg|Hg2SO4) in 0.1 M HClO4 triggers deprotection of the DAT group and subsequent chemical reactions, during which 10% of the DAT groups of the original SAM are transformed to a new surface-bound, quasi-reversible redox couple with a formal potential of 0.0 V (Hg|Hg2SO4) and a standard rate constant of 8 s-1 in 0.1 M HClO4. Immersion of the mixed SAM in 0.1 M HClO4 at open circuit potential or oxidation in 0.1 M H2SO4 did not produce this surface-bound redox couple. The monolayers were thoroughly characterized by X-ray photoelectron spectroscopy (XPS) and polarization modulation infrared reflection absorption spectroscopy (PM IRRAS) after the different preparation steps indicating only minor changes in the overall composition of the monolayer, in particular, the preservation of the heteroatoms. The new redox couple is likely a diimine, in agreement with its ability to bind nucleophiles such as anilines by conjugate addition that could be followed by multicycle voltammetry and XPS. The DAT effector group is especially interesting because it can also report the binding reaction by changed electrochemical and fluorescence signals.

Metabolites of the anaerobic degradation of diethyl ether by denitrifying betaproteobacterium strain HxN1.

The constitutions of five metabolites formed during co-metabolic, anaerobic degradation of diethyl ether by the denitrifying betaproteobacterium Aromatoleum sp. strain HxN1 were elucidated by comparison of mass spectrometric and gas chromatographic data with those of synthetic reference standards. Furthermore, the absolute configurations of two stereogenic centers in the metabolites were established. Based on these results a degradation pathway for diethyl ether by Aromatoleum sp. HxN1 analogous to that of n-hexane is proposed. Synthesis of both enantiomers of methyl (E)-4-ethoxy-2-pentenoate was accomplished by etherification of ethyl (R)- or (S)-lactate, followed by hydrolysis of the ester group and reduction to furnish 2-ethoxy-1-propanol. The primary alcohol was converted by a Swern oxidation followed by a Horner-Wadsworth-Emmons reaction to methyl (E)-4-ethoxy-2-pentenoate that was finally hydrogenated to methyl 4-ethoxypentanoate. Methyl (S)-4-ethoxy-3-oxopentanoate was prepared by conversion of (S)-2-ethoxypropanoyl chloride with Meldrum's acid. Reduction of the resulting ß-oxoester with NaBH4 or baker's yeast gave both diastereoisomers of methyl 4-ethoxy-3-hydroxypentanoate. The stereocenter at C-3 of the main diastereoisomer produced with baker's yeast was determined by Mosher ester analysis to be (R)-configurated. Dimethyl 2-(1-ethoxyethyl)succinate was prepared by Michael addition of nitroethane to diethyl maleate, followed by conjugate addition of sodium ethanolate, hydrolysis and esterification with diazomethane.

Synthesis of Annulated Indolines by Reductive Fischer Indolization.

Cyclic ß-oxoesters and arylhydrazine derivatives were converted at ambient temperature under modified Fischer indolization conditions to furnish annulated indolines with quaternary bridgehead carbon center. Reaction conditions were Brønsted acidic (with CF3 CO2 H), but also reductive (with Et3 SiH). The latter reduced intermediate iminium ions under formation of the 2,3-dihydroindole product constitutions. Racemic products (13 examples) were obtained as single diastereoisomers with relative cis-configuration, which was proved in two cases by single-crystal X-ray structure analysis. Conversion of 4-bromo-1-indanone-2-carboxylate and 4-bromophenylhydrazine gave indeno[1,2-b]indolines with either 1-bromo, 8-bromo, or 1,8-dibromo substitution, which were further diversified by Suzuki coupling reactions with several arylboronic acids (nine examples).

Synthesis of Benzo[b]azocin-2-ones by Aryl Amination and Ring-Expansion of α-(Iodophenyl)-ß-oxoesters.

Transformation of ß-oxoesters with PhI(OCOCF3 )2 leads to α-(ortho-iodophenyl)-ß-oxoesters. These materials are the starting point for the synthesis of 6-carboxybenzo[b]azocin-2-ones by a sequence of aryl amination and ring transformation. This reaction sequence starts with copper-catalyzed formation of N-alkyl anilines from the iodoarenes and primary amines in the presence of K3 PO4 as stoichiometric base. The intermediate products underwent ring transformation by addition of the nitrogen into the carbonyl group of the cycloalkanone, furnishing benzo-annulated eight-membered ring lactams. Under the same reaction conditions, the cyclohexanone and cycloheptanone derivatives gave no aminated products, but ring-transformed to benzofuran derivatives. The title compounds of this investigation contain two points for further diversification (the lactam nitrogen and the carboxylate function), thus, the suitability of this compound class as a scaffold was proven by appropriate functionalizations. The first series of derivatizations of the scaffold was initiated by hydrogenolytic debenzylation of N-benzyl derivative to provide the NH-congener, which could be deprotonated with LDA and alkylated at nitrogen to give further examples of this compound class. Secondly, the ester function was submitted to saponification and the resulting carboxylic acid could be amidated using HATU as coupling reagent to furnish different amides.

Stereochemical Insights into the Anaerobic Degradation of 4-Isopropylbenzoyl-CoA in the Denitrifying Bacterium Strain pCyN1.

The constitutions and absolute configurations of two previously unknown intermediates, (1S,2S,4S)-2-hydroxy-4-isopropylcyclohexane-1-carboxylate and (S)-3-isopropylpimelate, of anaerobic degradation of p-cymene in the bacterium Aromatoleum aromaticum pCyN1 are reported. These intermediates (as CoA esters) are involved in the further degradation of 4-isopropylbenzoyl-CoA formed by methyl group hydroxylation and subsequent oxidation of p-cymene. Proteogenomics indicated 4-isopropylbenzoyl-CoA degradation involves (i) a novel member of class I benzoyl-CoA reductase (BCR) as known from Thauera aromatica K172 and (ii) a modified ß-oxidation pathway yielding 3-isopropylpimeloyl-CoA analogously to benzoyl-CoA degradation in Rhodopseudomonas palustris. Reference standards of all four diastereoisomers of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate as well as both enantiomers of 3-isopropylpimelate were obtained by stereoselective syntheses via methyl 4-isopropyl-2-oxocyclohexane-1-carboxylate. The stereogenic center carrying the isopropyl group was established using a rhodium-catalyzed asymmetric conjugate addition. X-ray crystallography revealed that the thermodynamically most stable stereoisomer of 2-hydroxy-4-isopropylcyclohexane-1-carboxylate is formed during p-cymene degradation. Our findings imply that the reductive dearomatization of 4-isopropylbenzoyl-CoA by the BCR of A. aromaticum pCyN1 stereospecifically forms (S)-4-isopropyl-1,5-cyclohexadiene-1-carbonyl-CoA.

Diaminoterephthalate-α-lipoic acid conjugates with fluorinated residues.

Two bifunctional diaminoterephthalate (DAT) fluorescence dyes were prepared in a three-step sequence including one deprotection reaction. One functional unit is α-lipoic acid (ALA) for binding the dye to gold surfaces. It was introduced to the DAT scaffold by an amidation reaction. The other functional unit is a para-(trifluoromethyl)benzyl group for facile detection of the surface-bound material by X-ray photoelectron spectroscopy (XPS). This residue was introduced by reductive amination of the DAT scaffold with the respective benzaldehyde derivative. In one compound (60% yield over three steps) the ALA unit is directly bound to the DAT as a relatively electron-withdrawing amide. In solution (CH2Cl2), this material shows strong fluorescence (quantum yield 57% with emission at 495 nm, absorption maximum at 420 nm). The other compound (57% yield over three steps) possesses a propylene spacer between the ALA and the DAT units for electronic decoupling, thus, bathochromic shifts are observed (absorption at 514 nm, emission at 566 nm). The quantum yield is, however, lower (4%). Self-assembled monolayers on a gold surface of both compounds were prepared and characterized by high-resolution XPS of the C 1s, O 1s, S 2p, N 1s and F 1s emissions. The high signal-to-noise ratios of the F 1s peaks indicated that trifluoromethylation is an excellent tool for the detection of surface-bound materials by XPS.

Orthogonally Protected Diaminoterephthalate Scaffolds: Installation of Two Functional Units at the Chromophore.

The 2,5-diaminoterephthalate structural motif is a powerful chromophore with remarkable fluorescence properties. Containing two carboxylate and two amino functions, it defines a colored molecular scaffold which allows for orthogonal functionalization with different functional units. Therefore, different applications in life sciences and materials science could be addressed. In this study, the two amino functions were alkylated by reductive amination with side chains carrying amino (orthogonally protected as Boc or Alloc) and carboxylate functions (orthogonally protected as tBu or allyl ester). After sequential deprotections, functional units were introduced by amidation reactions. As three examples, the chromophore was coupled with retinoic acid and fullerene C60 in order to obtain a triad for studying photoinduced electron transfer processes. Furthermore, cyclooctyne and azide moieties were introduced as functional units, allowing for ligation by click reactions. These two clickable groups were applied in combination with maleimide units which are reactive toward thiol residues. The latter dyes define so-called "turn on" probes, since the fluorescence quantum yields increased by one order of magnitude upon reaction with the molecular target.

Bifunctional Diaminoterephthalate Fluorescent Dye as Probe for Cross-Linking Proteins.

Diaminoterephthalates are fluorescent dyes and define scaffolds, which can be orthogonally functionalized at their two carboxylate residues with functional residues bearing task specific reactive groups. The synthesis of monofunctionalized dyes with thiol groups for surface binding, an azide for click chemistry, and a biotinoylated congener for streptavidin binding is reported. Two bifunctionalized dyes were prepared: One with an azide for click chemistry and a biotin for streptavidin binding, the other with a maleimide for reaction with thiol and a cyclooctyne moiety for ligation with copper-free click chemistry. In general, the compounds are red to orange, fluorescent materials with an absorption at about 450 nm and an emission at 560 nm with quantum yields between 2-41 %. Of particular interest is the maleimide-functionalized compound, which shows low fluorescence quantum yield (2 %) by itself. After addition of a thiol, the fluorescence is "turned on"; quantum yield 41 %.

Formation of δ-Lactones with anti-Baeyer-Villiger Regiochemistry: Investigations into the Mechanism of the Cerium-Catalyzed Aerobic Coupling of ß-Oxoesters with Enol Acetates.

The cerium-catalyzed, aerobic coupling of ß-oxoesters with enol acetates and dioxygen yields δ-lactones with a 1,4-diketone moiety. In contrast to the Baeyer-Villiger oxidation (BVO), where the higher substituted residue migrates; in the case of this oxidative C-C coupling reaction, the less substituted alkyl residue undergoes a 1,2-shift. An endoperoxidic oxycarbenium ion comparable to the Criegee intermediate in the BVO is proposed as a reaction intermediate and submitted to conformational analysis by computational methods. As a result, the inverse regiochemistry is explained by a primary stereoelectronic effect. A Hammett analysis using different donor- and acceptor-substituted enol esters provides support for the oxycarbenium ion being the crucial intermediate in the rate determining step of the conversion. An overall mechanism is suggested with a radical chain reaction for the formation of endoperoxides from ß-oxoesters, enol acetates and dioxygen with a cerium(IV) species as initiating reagent.

Cerium-catalyzed, oxidative synthesis of annulated, tetrasubstituted dihydrofuran-derivatives.

Densely functionalized, annulated dihydrofuran derivatives are prepared by a cerium-catalyzed aerobic oxidation reaction. The operationally simple transformation is environmentally and economically benign, since the precatalyst CeCl3·7H2O is non-toxic and inexpensive and the oxidant is simply dioxygen from air. Starting materials are ß-oxoesters and silylenolethers, and the latter are derived from acetoacetate or acetylacetone. The reaction sequence is performed in one flask and consists of α-oxidation and Mukaiyama aldol reaction. Apart from tetrahydrocyclopenta[b]furan derivatives one example of a tetrahydrofuro[3,4-b]furan and one tetrahydro-3aH-furo[2,3-c]pyrrole derivative are prepared.

Mapping Calcium-Sensitive Regions in the Neuronal Calcium Sensor GCAP2 by Site-Specific Fluorescence Labeling.

Myristoylation of most neuronal calcium sensor proteins, a group of EF-hand calcium-binding proteins mainly expressed in neuronal tissue, can have a strong impact on protein dynamics and functional properties. Intracellular oscillations of the free Ca(2+) concentration can trigger conformational changes in Ca(2+) sensors. The position and possible movements of the myristoyl group in the photoreceptor cell-specific Ca(2+) sensor GCAP2 are not well-defined but appear to be different from those of the highly homologous cognate GCAP1. We designed and applied a new group of diaminoterephthalate-derived fluorescent probes to label GCAP2 at a covalently attached 12-azido-dodecanoic acid (a myristoyl substitute) and at cysteine residues in critical positions. Fluorescence emission of dye-labeled GCAP2 decreased when going from low (10(-9) M) to high [Ca(2+)] (10(-3) M), reaching a half-maximal effect of fluorescence emission at 0.44 ± 0.07 µM. The modified acyl group can therefore monitor changes in the protein conformation during binding and dissociation of Ca(2+) in the physiological range of free [Ca(2+)]. However, fluorescence quenching studies showed that the dye-acyl chain was shielded from the quencher by an adjacent polypeptide region. Further probing three cysteine positions (C35, C111, and C131) by dye labeling revealed that all positions were also sensitive to a change in [Ca(2+)], but only one (C131) was sensitive to a change in [Mg(2+)]. We suggest a scenario during illumination of the photoreceptor cell in which Ca(2+) dissociates first from low and medium affinity binding sites. These steps are sensed by dyes in cysteines at positions 35 and 111. Release of Ca(2+) from high affinity sites is sensed by regions adjacent to the dye-labeled fatty acid and involves the critical conformational change leading to activating guanylate cyclase.

Formation of δ-Lactones by Cerium-Catalyzed, Baeyer-Villiger-Type Coupling of ß-Oxoesters, Enol Acetates, and Dioxygen.

Formation of δ-lactones is observed when cyclopentanone-2-carboxylates are converted in a cerium-catalyzed reaction with α-aryl vinyl acetates under oxidative conditions. The products of this transformation possess a 1,4-dicarbonyl constitution together with a quaternary carbon center. Atmospheric oxygen is the oxidant in this process, which can be regarded as ideal from economic and ecological points of view. Further advantages of this new C-C coupling and oxidation reaction are its operational simplicity and the application of nontoxic and inexpensive CeCl3·7 H2O as precatalyst. This so far unprecedented reaction is proposed to proceed via 1,2-dioxane derivatives, which decompose under formation of an oxycarbenium cation in a Baeyer-Villiger-type pathway. This mechanistic picture is supported by the observation that electron-rich (donor substituted or heteroaromatic) enol esters give higher yields than electron deficient congeners. Apart from 1,4-diketones and α-hydroxylated ß-oxoesters formed as byproducts, the yields of δ-lactones range from moderate to good (up to 74%).

A robust synthesis of 7,8-didemethyl-8-hydroxy-5-deazariboflavin.

The biosynthetic precursor of redox cofactor F420, 7,8-didemethyl-8-hydroxy-5-deazariboflavin, was prepared in four steps from 6-chlorouracil, 2-chloro-4-hydroxybenzaldehyde and bis-isopropylidene protected D-ribose. The latter aldehyde was transformed to the corresponding protected ribitylamine via the oxime, which was submitted to reduction with LiAlH4. Key advantage compared to previous syntheses is the utilization of a polyol-protective group which allowed the chromatographic purification of a key-intermediate product providing the target compound with high purity.

Bifunctional diaminoterephthalate scaffolds as fluorescence turn-on probes for thiols.

The fluorescent diaminoterephthalate scaffold was equipped by amidation with three types of reactive functions: thiols for metal-surface binding, alkynes for click reactions, and maleimides for ligation with proteins. Starting from a succinyl succinate derivative with two orthogonally cleavable ester functions, three monoamides (38-57% yield over three steps) and two bisamides (19 and 25% yield over five steps) were prepared. Although alkyne and thiol derivatized compounds showed reasonable luminescence behavior (Φ≈1-4%), the fluorescence was quenched by the maleimide moiety. It was turned on (10- to 20-fold increase of fluorescence quantum yield) by conjugate addition of thiols.

A Geomimetic Approach to the Formation and Identification of Fossil Sterane Biomarkers in Crude Oil: 18-nor-D-homo-Androstane and 5α,14ß-Androstane.

A diazonium ion derived from 18-aminoandrostane rearranged upon decomposition by a carbonium and a carbenium ion to furnish a mixture of a cyclopropanated compound and two D-homo-androstenes. Hydrogenation of this mixture gave the saturated hydrocarbons, 18-nor-D-homo-androstane and 5α,14ß-androstane, which are both fossil sterane biomarkers in Neoproterozoic crude oil. The so far unknown constitution and configuration as well as the geochemical genesis were established by this experiment. The starting material for this investigation, 18-aminoandrostane, was prepared in twelve steps from androstan-17-one (12.5% overall yield) with a Barton reaction as the key step.

Diaminoterephthalate turn-on fluorescence probes for thiols--tagging of recoverin and tracking of its conformational change.

Diaminoterephthalates with a maleimide moiety were synthesized and used as fluorescence dyes for sensing thiols. Whereas these "NiWa Blue" dyes showed no emission, the conjugate addition of a thiol to the maleimide group turned on a fluorescence at about 400 nm when irradiating the dye at 338 nm. The neuronal-calcium sensor protein recoverin possesses a single cysteine residue at position 39, which reacts with NiWa Blue, and is therefore labeled by a fluorophore with an emission at about 440 nm. In the absence of Ca(2+), irradiation at 280 nm of a tryptophan residue in close proximity to Cys-bound NiWa Blue lead to strong FRET, which was detected by emission of the dye at 440 nm. In the presence of Ca(2+), the protein holds a conformation with distal Trp and Cys residues, thus FRET of irradiated Trp to Cys-bound NiWa Blue was significantly weakened.

Formation of δ-Lactones by Cyanide Catalyzed Rearrangement of α-Hydroxy-ß-oxoesters.

δ-Valerolactone derivatives are formed by cyanide-catalyzed ring-transformation of cyclic α-hydroxy-ß-oxoesters. This unprecedented reaction defines a new synthetic methodology, and the products are obtained in up to quantitative yields. Several alkyl substitutions as well as different ester residues are tolerated. Furthermore, benzo- and heteroarene-annulated starting materials are converted without problems. As an additional benefit, the starting materials are straightforwardly accessed by cerium-catalyzed aerobic α-hydroxylation of readily available ß-oxoesters.