Hydrogen Bonding Shuts Down Tunneling in Hydroxycarbenes: A Gas-Phase Study by Tandem-Mass Spectrometry, Infrared Ion Spectroscopy, and Theory.

Hydroxycarbenes can be generated and structurally characterized in the gas phase by collision-induced decarboxylation of α-keto carboxylic acids, followed by infrared ion spectroscopy. Using this approach, we have shown earlier that quantum-mechanical hydrogen tunneling (QMHT) accounts for the isomerization of a charge-tagged phenylhydroxycarbene to the corresponding aldehyde in the gas phase and above room temperature. Herein, we report the results of our current study on aliphatic trialkylammonio-tagged systems. Quite unexpectedly, the flexible 3-(trimethylammonio)propylhydroxycarbene turned out to be stable─no H-shift to either aldehyde or enol occurred. As supported by density functional theory calculations, this novel QMHT inhibition is due to intramolecular H-bonding of a mildly acidic α-ammonio C-H bonds to the hydroxyl carbene's C-atom (C:···H-C). To further support this hypothesis, (4-quinuclidinyl)hydroxycarbenes were synthesized, whose rigid structure prevents this intramolecular H-bonding. The latter hydroxycarbenes underwent "regular" QMHT to the aldehyde at rates comparable to, e.g., methylhydroxycarbene studied by Schreiner et al. While QMHT has been shown for a number of biological H-shift processes, its inhibition by H-bonding disclosed here may serve for the stabilization of highly reactive intermediates such as carbenes, even as a mechanism for biasing intrinsic selectivity patterns.

Titanium Salalen Catalyzed Enantioselective Benzylic Hydroxylation.

The titanium complex of the cis-1,2-diaminocyclohexane (cis-DACH) derived Berkessel-salalen ligand is a highly efficient and enantioselective catalyst for the asymmetric epoxidation of terminal olefins with hydrogen peroxide ("Berkessel-Katsuki catalyst"). We herein report that this epoxidation catalyst also effects the highly enantioselective hydroxylation of benzylic C-H bonds with hydrogen peroxide. Mechanism-based ligand optimization identified a novel nitro-salalen Ti-catalyst of the highest efficiency ever reported for asymmetric catalytic benzylic hydroxylation, with enantioselectivities of up to 98 % ee, while overoxidation to ketone is marginal. The novel nitro-salalen Ti-catalyst also shows enhanced epoxidation efficiency, as evidenced by e.g. the conversion of 1-decene to its epoxide in 90 % yield with 94 % ee, at a catalyst loading of 0.1 mol-% only.

Synthetic α-Helical Peptides as Potential Inhibitors of the ACE2 SARS-CoV-2 Interaction.

During viral cell entry, the spike protein of SARS-CoV-2 binds to the α1-helix motif of human angiotensin-converting enzyme 2 (ACE2). Thus, alpha-helical peptides mimicking this motif may serve as inhibitors of viral cell entry. For this purpose, we employed the rigidified diproline-derived module ProM-5 to induce α-helicity in short peptide sequences inspired by the ACE2 α1-helix. Starting with Ac-QAKTFLDKFNHEAEDLFYQ-NH2 as a relevant section of α1, a series of peptides, N-capped with either Ac-ßHAsp-[ProM-5] or Ac-ßHAsp-PP, were prepared and their α-helicities were investigated. While ProM-5 clearly showed a pronounced effect, an even increased degree of helicity (up to 63 %) was observed in sequences in which non-binding amino acids were replaced by alanine. The binding affinities of the peptides towards the spike protein, as determined by means of microscale thermophoresis (MST), revealed only a subtle influence of the α-helical content and, noteworthy, led to the identification of an Ac-ßHAsp-PP-capped peptide displaying a very strong binding affinity (KD =62 nM).

Synthesis and Biological Evaluation of Water-Soluble Esterase-Activated CO-Releasing Molecules Targeting Mitochondria.

Due to the beneficial effects of carbon monoxide as a cell-protective and anti-inflammatory agent, CO-releasing molecules (CORMs) offer some promising potential applications in medicine. In this context, we synthesized a set of acyloxy-cyclohexadiene-Fe(CO)3 complexes, all displaying a N-methyl-pyridinium triflate moiety in the ester side chain, as mitochondria-targeting esterase-triggered CORM prodrugs. Whereas the compounds in which the acyloxy substituent is attached to the 2-position of the diene-Fe(CO)3 unit (A series) spontaneously release CO upon dissolution in phosphate buffer, which remarkably is partly suppressed in the presence of porcine liver esterase (PLE), the 1-substituted isomers (B series) show the expected PLE-induced release of CO (up to 3 equiv.). The biological activity of Mito-CORMs 2/3-B and their isophorone-derived analogs 2/3-A', which also displayed PLE-induced CO release, was assessed by using human umbilical vein endothelial cells (HUVEC). Whereas Mito-CORMs 2/3-B were not cytotoxic up to 500 µM (MTT assay), Mito-CORMs 2/3-A' caused significant toxicity at concentrations above 50 µM. The anti-inflammatory potential of both Mito-CORM variants was demonstrated by concentration-dependent down-regulation of the pro-inflammatory markers VCAM-1, ICAM-1 and CXCL1 as well as induction of HO-1 in TNFα-stimulated human umbilical vein endothelial cells (HUVECs; western blotting and qPCR). Energy phenotyping by seahorse real-time cell metabolic analysis, revealed opposing shifts of metabolic potentials in cells treated either with Mito-CORMs 2/3-B (increased mitochondrial respiration and glycolytic activity) or Mito-CORMs 2/3-A' (suppressed mitochondrial respiration and increased glycolytic activity). Thus, the Mito-CORMs represent valuable tools for the safe and targeted delivery of CO to mitochondria as a subcellular compartment to induce positive anti-inflammatory effects with only minor shifts in cellular energy metabolism. Also, due to their water solubility, these compounds provide a promising starting point for further pharmacological studies.

Design and synthesis of a tetracyclic tripeptide mimetic frozen in a polyproline type II (PP2) helix conformation.

A synthesis of the new tetracyclic scaffold ProM-19, which represents a XPP tripeptide unit frozen in a PPII helix conformation, was developed. As a key building block, N-Boc-protected ethyl (1S,3S,4R)-2-azabicyclo[2.2.1]hept-5-ene-2-carboxylate was prepared through a diastereoselective aza-Diels-Alder reaction and subsequent hydrogenolytic removal of the chiral N-1-phenylethyl substituent under temporary protection of the double bond through dihydroxylation and reconstitution by Corey-Winter olefination. The target compound Boc-[ProM-19]-OMe was then prepared via subsequent peptide coupling and Ru-catalyzed ring-closing metathesis steps employing (S)-N-Boc-allylgylcine and cis-5-vinyl-proline methyl ester as additional building blocks. In addition, Ac-[2-Cl-Phe]-[Pro]-[ProM-19]-OMe was prepared by solution phase peptide synthesis as a potential ligand for the ena-VASP EVH1 domain.

Phenanthroindolizidine Alkaloids Isolated from Tylophora ovata as Potent Inhibitors of Inflammation, Spheroid Growth, and Invasion of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC), representing the most aggressive form of breast cancer with currently no targeted therapy available, is characterized by an inflammatory and hypoxic tumor microenvironment. To date, a broad spectrum of anti-tumor activities has been reported for phenanthroindolizidine alkaloids (PAs), however, their mode of action in TNBC remains elusive. Thus, we investigated six naturally occurring PAs extracted from the plant Tylophora ovata: O-methyltylophorinidine (1) and its five derivatives tylophorinidine (2), tylophoridicine E (3), 2-demethoxytylophorine (4), tylophoridicine D (5), and anhydrodehydrotylophorinidine (6). In comparison to natural (1) and for more-in depth studies, we also utilized a sample of synthetic O-methyltylophorinidine (1s). Our results indicate a remarkably effective blockade of nuclear factor kappa B (NFκB) within 2 h for compounds (1) and (1s) (IC50 = 17.1 ± 2.0 nM and 3.3 ± 0.2 nM) that is different from its effect on cell viability within 24 h (IC50 = 13.6 ± 0.4 nM and 4.2 ± 1 nM). Furthermore, NFκB inhibition data for the additional five analogues indicate a structure-activity relationship (SAR). Mechanistically, NFκB is significantly blocked through the stabilization of its inhibitor protein kappa B alpha (IκBα) under normoxic as well as hypoxic conditions. To better mimic the TNBC microenvironment in vitro, we established a 3D co-culture by combining the human TNBC cell line MDA-MB-231 with primary murine cancer-associated fibroblasts (CAF) and type I collagen. Compound (1) demonstrates superiority against the therapeutic gold standard paclitaxel by diminishing spheroid growth by 40% at 100 nM. The anti-proliferative effect of (1s) is distinct from paclitaxel in that it arrests the cell cycle at the G0/G1 state, thereby mediating a time-dependent delay in cell cycle progression. Furthermore, (1s) inhibited invasion of TNBC monoculture spheroids into a matrigel®-based environment at 10 nM. In conclusion, PAs serve as promising agents with presumably multiple target sites to combat inflammatory and hypoxia-driven cancer, such as TNBC, with a different mode of action than the currently applied chemotherapeutic drugs.

syn-Selective Epoxidation of Chiral Terminal Allylic Alcohols with a Titanium Salalen Catalyst and Hydrogen Peroxide.

In the Sharpless asymmetric epoxidation of chiral secondary allylic alcohols, one substrate enantiomer is predominantly converted to the anti-epoxy alcohol. We herein report the first highly syn-selective epoxidation of terminal allylic alcohols using a titanium salalen complex as catalyst, at room temperature, and aqueous hydrogen peroxide as oxidant. With enantiopure terminal allylic alcohols as substrates, the epoxy alcohols were obtained with up to 98 % yield and up to >99 : 1 dr (syn). Catalyst loadings as low as 1 mol % can be applied without eroding the syn-diastereoselectivity. Modification of the allylic alcohol to an ether does not affect the diastereoselectivity either [>99 : 1 dr (syn)]. Inverting the catalyst configuration leads to the anti-product, albeit at lower dr (ca. 20 : 1). The synthetic potential is demonstrated by a short, gram-scale preparation of a tetrahydrofuran building block with three stereocenters, involving two titanium salalen catalyzed epoxidation steps.

Enantioselective Cleavage of Cyclobutanols Through Ir-Catalyzed C-C Bond Activation: Mechanistic and Synthetic Aspects.

The Ir-catalyzed conversion of prochiral tert-cyclobutanols to ß-methyl-substituted ketones proceeds under comparably mild conditions in toluene (45-110 °C) and is particularly suited for the enantioselective desymmetrization of ß-oxy-substituted substrates to give products with a quaternary chirality center with up to 95 % ee using DTBM-SegPhos as a chiral ligand. Deuteration experiments and kinetic isotope effect measurements revealed major mechanistic differences to related RhI -catalyzed transformations. Supported by DFT calculations we propose the initial formation of an IrIII hydride intermediate, which then undergoes a ß-C elimination (C-C bond activation) prior to reductive C-H elimination. The computational model also allows the prediction of the stereochemical outcome. The Ir-catalyzed cyclobutanol cleavage is broadly applicable but fails for substrates bearing strongly coordinating groups. The method is of particular value for the stereo-controlled synthesis of substituted chromanes related to the tocopherols and other natural products.

Total Synthesis of (+)-Erogorgiaene and the Pseudopterosin A-F Aglycone via Enantioselective Cobalt-Catalyzed Hydrovinylation.

Due to their pronounced bioactivity and limited availability from natural resources, metabolites of the soft coral Pseudopterogorgia elisabethae, such as erogorgiaene and the pseudopterosines, represent important target molecules for chemical synthesis. We have now developed a particularly short and efficient route towards these marine diterpenes exploiting an operationally convenient enantioselective cobalt-catalyzed hydrovinylation as the chirogenic step. Other noteworthy C-C bond forming transformations include diastereoselective Lewis acid-mediated cyclizations, a Suzuki coupling and a carbonyl ene reaction. Starting from 4-methyl-styrene the anti-tubercular agent (+)-erogorgiaene (>98 % ee) was prepared in only 7 steps with 46 % overall yield. In addition, the synthesis of the pseudopterosin A aglycone was achieved in 12 steps with 30 % overall yield and, surprisingly, was found to exhibit a similar anti-inflammatory activity (inhibition of LPS-induced NF-κB activation) as a natural mixture of pseudopterosins A-D or iso-pseudopterosin A, prepared by ß-D-xylosylation of the synthetic aglycone.

Enantioselective Total Synthesis and Structural Revision of Dysiherbol†A.

A 12-step total synthesis of the natural product dysiherbol A, a strongly anti-inflammatory and anti-tumor avarane meroterpene isolated from the marine sponge Dysidea sp., was elaborated. As key steps, the synthesis features an enantioselective Cu-catalyzed 1,4-addition/enolate-trapping opening move, an Au-catalyzed double cyclization to build up the tetracyclic core-carbon skeleton, and a late installation of the C5-bridgehead methyl group via proton-induced cyclopropane opening associated with spontaneous cyclic ether formation. The obtained pentacyclic compound (corresponding to an anhydride of the originally suggested structure for dysiherbol A) showed identical spectroscopic data as the natural product, but an opposite molecular rotation. CD-spectroscopic measurements finally confirmed that both the constitution and the absolute configuration of the originally proposed structure of (+)-dysiherbol A need to be revised.

Synthesis of the 8,19-Epoxysteroid Eurysterol†A.

We report the first chemical synthesis of eurysterol A, a cytotoxic and antifungal marine steroidal sulfate with a unique C8-C19 oxy-bridged cholestane skeleton. After C19 hydroxylation of cholesteryl acetate, used as an inexpensive commercial starting material, the challenging oxidative functionalization of ring B was achieved by two different routes to set up a 5α-hydroxy-7-en-6-one moiety. As a key step, an intramolecular oxa-Michael addition was exploited to close the oxy-bridge (8ß,19-epoxy unit). DFT calculations show this reversible transformation being exergonic by about -30 kJ mol-1 . Along the optimized (scalable) synthetic sequence, the target natural product was obtained in only 11 steps in 5 % overall yield. In addition, an access to (isomeric) 7ß,19-epoxy steroids with a previously unknown pentacyclic ring system was discovered.

Pd-Catalyzed Asymmetric N-Allylation of Amino Acid Esters with Exceptional Levels of Catalyst Control: Stereo-Divergent Synthesis of ProM-15 and Related Bicyclic Dipeptide Mimetics.

A general and powerful method for the stereo-controlled Pd-catalyzed N-allylation of amino acid esters is reported, as a previously largely unsolved synthetic challenge. Employing a new class of tartaric acid-derived C2 -symmetric chiral diphosphane ligands the developed asymmetric amination protocol allows the conversion of various amino acid esters to the N-allylated products with highest levels of enantio- or diastereoselectivity in a fully catalyst-controlled fashion and predictable configuration. Remarkably, the in situ generated catalysts also exhibit outstanding levels of activity (ligand acceleration). The usefulness of the method was demonstrated in the stereo-divergent synthesis of a set of new conformationally defined dipeptide mimetics, which represent new modular building blocks for the development of peptide-inspired bioactive compounds.

ω-Quinazolinonylalkyl aryl ureas as reversible inhibitors of monoacylglycerol lipase.

The serine hydrolase monoacylglycerol lipase (MAGL) is involved in a plethora of pathological conditions, in particular pain and inflammation, various types of cancer, metabolic, neurological and cardiovascular disorders, and is therefore a promising target for drug development. Although a large number of irreversible-acting MAGL inhibitors have been discovered over the past years, there are only few compounds known so far which inhibit the enzyme in a reversible manner. Therefore, much effort is put into the development of novel chemical entities showing reversible inhibitory behavior, which is thought to cause less undesired side effects. To explore a wide range of chemical structures as MAGL binders, we have applied a virtual screening approach by docking small molecules into the crystal structure of human MAGL (hMAGL) and envisaged a library of 45 selected compounds which were then synthesized. Biochemical investigations included the determination of the inhibitory potency on hMAGL and two related hydrolases, i.e. human fatty acid amide hydrolase (hFAAH) and murine cholesterol esterase (mCEase). The most promising candidates from theses analyses, i.e. three ω-quinazolinonylalkyl aryl ureas bearing alkyl spacers of three to five methylene groups, exhibited IC50 values of 20-41 µM and reversible, detergent-insensitive behavior towards hMAGL. Among these compounds, the inhibitor 1-(3,5-bis(trifluoromethyl)phenyl)-3-(4-(4-oxo-3,4-dihydroquinazolin-2-yl)butyl)urea (96) was selected for further kinetic characterization, yielding a dissociation constant Ki = 15.4 µM and a mixed-type inhibition with a pronounced competitive component (α = 8.94). This mode of inhibition was further supported by a docking experiment, which suggested that the inhibitor occupies the substrate binding pocket of hMAGL.

Triple-Helix-Stabilizing Effects in Collagen Model Peptides Containing PPII-Helix-Preorganized Diproline Modules.

Collagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross-linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline-mimicking modules (ProMs), which were preorganized in a PPII-helix-type conformation by a functionalizable intrastrand C2 bridge. Results of CD-based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main-chain preorganization, ring-flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.

Intermediates of N-Heterocyclic Carbene (NHC) Dimerization Probed in the Gas Phase by Ion Mobility Mass Spectrometry: C-H⋠⋠⋠:C Hydrogen Bonding Versus Covalent Dimer Formation.

N-Heterocyclic carbenes (NHCs, :C) can interact with azolium salts (C-H+ ) by either forming a hydrogen-bonded aggregate (CHC+ ) or a covalent C-C bond (CCH+ ). In this study, the intramolecular NHC-azolium salt interactions of aromatic imidazolin-2-ylidenes and saturated imidazolidin-2-ylidenes have been investigated in the gas phase by traveling wave ion mobility mass spectrometry (TW IMS) and DFT calculations. The TW IMS experiments provided evidence for the formation of these important intermediates in the gas phase, and they identified the predominant aggregation mode (hydrogen bond vs. covalent C-C) as a function of the nature of the interacting carbene-azolium pairs.

Total Synthesis of α-Tocopherol through Enantioselective Iridium-Catalyzed Fragmentation of a Spiro-Cyclobutanol Intermediate.

A conceptionally new strategy for the asymmetric (2R-selective) synthesis of α-tocopherol (vitamin E) was developed. In the stereocontrolled key step, a prochiral spiro[chromane-2,3'-cyclobutanol] unit is effectively desymmetrized under C-C bond activation in an unprecedented iridium-catalyzed transformation using (S)-DTBM-SegPhos as a chiral ligand (e.r. 97:3). To complete the synthesis, the side chain was attached through Ru-catalyzed cross-metathesis employing an alkene derived from (R,R)-hexahydrofarnesol. To suppress epimerization during the final hydrogenation, PtO2 had to be used as a catalyst instead of Pd/C. In an alternative approach (employing a propargyl-substituted spiro-cyclobutanol), the side chain was constructed prior to the Ir-catalyzed ring fragmentation (>99:1 d.r.) through enyne cross-metathesis (using an alkene derived from (R)-dihydrocitronellal) followed by Cr-catalyzed 1,4-hydrogenation and (diastereoselective) Pfaltz hydrogenation of the resulting triple-substituted olefin. The work demonstrates the potential of iridium catalysis for enantioselective C-C bond activation.

Chiral Phosphine-Phosphite Ligands in Asymmetric Gold Catalysis: Highly Enantioselective Synthesis of Furo[3,4-d]-Tetrahydropyridazine Derivatives through [3+3]-Cycloaddition.

The AuI -catalyzed reaction of 2-(1-alkynyl)-2-alken-1-ones with azomethine imines regio- and diastereoselectively affords furo[3,4-d]tetrahydropyridazines in a tandem cyclization/intermolecular [3+3]-cycloaddition process under mild conditions. By employing a chiral gold catalyst (prepared in situ from a Taddol-derived phosphine-phosphite ligand, Me2 SAuCl, and AgOTf) high yields and enantioselectivities (up to 94 % yield, up to 96 % ee) are obtained. The method provides an efficient modular route to substituted heterotricyclic furan derivatives and can be easily scaled up (using catalyst loads of only 0.15 mol %).

Determination of inter-ionic and intra-ionic interactions in a monofluorinated imidazolium ionic liquid by a combination of X-ray crystallography and NOE NMR spectroscopy.

We report the very first application of a Transient 1D 1 H{19 F} NOE NMR experiment in neat ionic liquids. In comparison with classical 2D HOESY NMR spectroscopy, a substantial reduction in measurement time is gained with comparable quality and information content of the spectra. In combination with classical X-ray crystallography, we have applied this technique for the determination of inter-ionic distances (i.e. probabilities of presence) utilizing an ionic liquid containing a monofluorinated imidazolium cation. Copyright © 2017 John Wiley & Sons, Ltd.

Keto-Enol Thermodynamics of Breslow Intermediates.

Breslow intermediates, first postulated in 1958, are pivotal intermediates in carbene-catalyzed umpolung. Attempts to isolate and characterize these fleeting amino enol species first met with success in 2012 when we found that saturated bis-Dipp/Mes imidazolidinylidenes readily form isolable, though reactive diamino enols with aldehydes and enals. In contrast, triazolylidenes, upon stoichiometric reaction with aldehydes, gave exclusively the keto tautomer, and no isolable enol. Herein, we present the synthesis of the "missing" keto tautomers of imidazolidinylidene-derived diamino enols, and computational thermodynamic data for 15 enol-ketone pairs derived from various carbenes/aldehydes. Electron-withdrawing substituents on the aldehyde favor enol formation, the same holds for N,N'-Dipp [2,6-di(2-propyl)phenyl] and N,N'-Mes [2,4,6-trimethylphenyl] substitution on the carbene component. The latter effect rests on stabilization of the diamino enol tautomer by Dipp substitution, and could be attributed to dispersive interaction of the 2-propyl groups with the enol moiety. For three enol-ketone pairs, equilibration of the thermodynamically disfavored tautomer was attempted with acids and bases but could not be effected, indicating kinetic inhibition of proton transfer.

Design and Stereoselective Synthesis of ProM-2: A Spirocyclic Diproline Mimetic with Polyproline Type II (PPII) Helix Conformation.

With the aim of developing polyproline type II helix (PPII) secondary-structure mimetics for the modulation of prolin-rich-mediated protein-protein interactions, the novel diproline mimetic ProM-2 was designed by bridging the two pyrrolidine rings of a diproline (Pro-Pro) unit through a Z-vinylidene moiety. This scaffold, which closely resembles a section of a PPII helix, was then stereoselectively synthesized by exploiting a ruthenium-catalyzed ring-closing metathesis (RCM) as a late key step. The required vinylproline building blocks, that is, (R)-N-Boc-2-vinylproline (Boc=tert-butyloxycarbonyl) and (S,S)-5-vinylproline-tert-butyl ester, were prepared on a gram scale as pure stereoisomers. The difficult peptide coupling of the sterically demanding building blocks was achieved in good yield and without epimerization by using 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU)/N,N-diisopropylethylamine (DIPEA). The RCM proceeded smoothly in the presence of the Grubbs II catalyst. Stereostructural assignments for several intermediates were secured by X-ray crystallography. As a proof of concept, it was shown that certain peptides containing ProM-2 exhibited improved (canonical) binding towards the Ena/VASP homology 1 (EVH1) domain as a relevant protein interaction target.