Biosynthesis of a new skyllamycin in Streptomyces nodosus: a cytochrome P450 forms an epoxide in the cinnamoyl chain.

Activation of a silent gene cluster in Streptomyces nodosus leads to synthesis of a cinnamoyl-containing non-ribosomal peptide (CCNP) that is related to skyllamycins. This novel CCNP was isolated and its structure was interrogated using mass spectrometry and nuclear magnetic resonance spectroscopy. The isolated compound is an oxidised skyllamycin A in which an additional oxygen atom is incorporated in the cinnamoyl side-chain in the form of an epoxide. The gene for the epoxide-forming cytochrome P450 was identified by targeted disruption. The enzyme was overproduced in Escherichia coli and a 1.43 Å high-resolution crystal structure was determined. This is the first crystal structure for a P450 that forms an epoxide in a substituted cinnamoyl chain of a lipopeptide. These results confirm the proposed functions of P450s encoded by biosynthetic gene clusters for other epoxidized CCNPs and will assist investigation of how epoxide stereochemistry is determined in these natural products.

Proton-Induced Spin State Switching in an FeIII Complex.

Reversible proton-induced spin state switching of an FeIII complex in solution is observed at room temperature. A reversible magnetic response was detected in the complex, [FeIII (sal2 323)]ClO4 (1), using Evans' method 1 H NMR spectroscopy which indicated cumulative switching from low-spin to high-spin upon addition of one and two equivalents of acid. Infrared spectroscopy suggests a coordination-induced spin state switching (CISSS) effect, whereby protonation displaces the metal-phenoxo donors. The analogous complex, [FeIII (4-NEt2 -sal2 323)]ClO4 (2), with a diethylamino group on the ligand, was used to combine the magnetic change with a colorimetric response. Comparison of the protonation responses of 1 and 2 reveals that the magnetic switching is caused by perturbation of the immediate coordination sphere of the complex. These complexes constitute a new class of analyte sensor which operate by magneto-modulation, and in the case of 2, also yield a colorimetric response.

A convenient synthetic route to (2S,4S)-methylproline and its exploration for protein engineering of thioredoxin.

4-Substituted prolines, especially 4-fluoroprolines, have been widely used in protein engineering and design. Here, we report a robust and stereoselective approach for the synthesis of (2S,4S)-methylproline starting from (2S)-pyroglutamic acid. Incorporation studies with both (2S,4R)- and (2S,4S)-methylproline into the Trx1P variant of the model protein thioredoxin of E. coli show that the stereochemistry of the 4-methyl group might be a key determinator for successful incorporation during ribosomal synthesis of this protein.

Design and Synthesis of Pyrrolidinyl Ferrocene-Containing Ligands and Their Application in Highly Enantioselective Rhodium-Catalyzed Olefin Hydrogenation.

Herein, we report the design and synthesis of a series of chiral pyrrolidine-substituted ferrocene-derived ligands. The proficiency of this novel structural motif was demonstrated in the Rh-catalyzed asymmetric hydrogenation of dehydroamino acid esters and α-aryl enamides. The products were obtained with full conversions and excellent levels of enantioselectivities of up to >99.9% ee and 97.7% ee, respectively, using a BINOL-substituted phosphine-phosphoaramidite ligand which possesses planar, central, and axial chirality elements.

Dynamics of a Molecular Rotor Exhibiting Local Directional Rotational Preference within Each Enantiomer.

Directional internal rotation in molecular systems, generally controlled by chirality, is known to occur in natural and artificial systems driven by light or fueled chemically, but spontaneous directional molecular rotation is believed to be forbidden. We have designed a molecular rotor, whereby ferrocene and triptycene linked by a methylene bridge provide two rotational degrees of freedom. On the basis of experimental observations, in conjunction with computational data, we show that the two different modes of rotation are strongly coupled and the spatial orientation of the bistable ferrocene moiety controls the barrier to its own rotation about the triptycene axis. It is proposed that the barrier to clockwise 120° rotation across each individual triptycene blade is lower in the M-enantiomer and for counterclockwise 120° rotation, it is lower in its P-counterpart. These findings demonstrate the possibility of locally preferred thermal directional intramolecular rotation for each dynamically interconverting enantiomer.

Dynamic Cross-Exchange in Halophosphonium Species: Direct Observation of Stereochemical Inversion in the Course of an SN 2 Process.

The complex fluxional interconversions between otherwise very similar phosphonium bromides and chlorides R3 PX+ X- (R=Alk, Ar, X=Cl or Br) were studied by NMR techniques. Their energy barriers are typically ca. 11 kcal mol-1 , but rise rapidly as bulky groups are attached to phosphorus, revealing the importance of steric factors. In contrast, electronic effects, as measured by Hammett analysis, are modest (ρ 1.46) but still clearly indicate negative charge flow towards phosphorus in the transition state. Most significantly, detailed analysis of the exchange pathways unequivocally, and for the first time in any such process, shows that nucleophilic attack of the nucleophilic anion on the tetrahedral centre results in inversion of configuration.

Two Independent Orthogonal Stereomutations at a Single Asymmetric Center: A Narcissistic Couple.

The energy barriers in our recently discovered Walden-type inversion of chlorophosphonium salts are similar to those for Cope rearrangements of caged cyclic hydrocarbons. Therefore, we have designed a molecular system that integrates the two processes, thereby producing the first embodiment of a chemical species that can undergo two entirely different and independent stereomutation mechanisms at the same nominal asymmetric center. Thus, the energy barrier to the rearrangement of 9-phenyl-9-phosphabarbaralane oxide, which is easily prepared by a new high-yielding synthesis, was found to be roughly 11 kcal mol-1 . This value is in contrast to the parent barbaralane (7.3 kcal mol-1 ) but in good agreement with our computational results for the rearrangement barriers. Crucially, in the corresponding chlorophosphonium derivative, two stereomutations occur simultaneously: a fast Cope rearrangement (barrier ≈12 kcal mol-1 ) and a slow Walden-type inversion of the phosphorus center (barrier ≈21 kcal mol-1 ). The computational model also revealed a relationship between the Cope rearrangement barrier and the bridgehead distance. The phenomenon of two independent and geometrically orthogonal stereomutations at a single asymmetric center provided important general insights into reaction pathway bifurcation, microscopic reversibility, and dynamic stereochemistry. This first example of coexisting alternative mechanisms that involve covalent bonds may encourage the design of new types of dynamic molecular structures.

Biotransformation of fluorophenyl pyridine carboxylic acids by the model fungus Cunninghamella elegans.

1. Fluorine plays a key role in the design of new drugs and recent FDA approvals included two fluorinated drugs, tedizolid phosphate and vorapaxar, both of which contain the fluorophenyl pyridyl moiety. 2. To investigate the likely phase-I (oxidative) metabolic fate of this group, various fluorinated phenyl pyridine carboxylic acids were incubated with the fungus Cunninghamella elegans, which is an established model of mammalian drug metabolism. 3. 19F NMR spectroscopy established the degree of biotransformation, which varied depending on the position of fluorine substitution, and gas chromatography-mass spectrometry (GC-MS) identified alcohols and hydroxylated carboxylic acids as metabolites. The hydroxylated metabolites were further structurally characterised by nuclear magnetic resonance spectroscopy (NMR), which demonstrated that hydroxylation occurred on the 4' position; fluorine in that position blocked the hydroxylation. 4. The fluorophenyl pyridine carboxylic acids were not biotransformed by rat liver microsomes and this was a consequence of inhibitory action, and thus, the fungal model was crucial in obtaining metabolites to establish the mechanism of catabolism.

Degenerate nucleophilic substitution in phosphonium salts.

Rates and energy barriers of degenerate halide substitution on tetracoordinate halophosphonium cations have been measured by NMR techniques (VT and EXSY) using a novel experimental design whereby a chiral substituent ((s)Bu) lifts the degeneracy of the resultant salts. Concomitantly, a viable computational approach to the system was developed to gain mechanistic insights into the structure and relative stabilities of the species involved. Both approaches strongly suggest a two-step mechanism of formation of a pentacoordinate dihalophosphorane via backside attack followed by dissociation, resulting in inversion of configuration at phosphorus. The experimentally determined barriers range from <9 kcal mol(-1) to nearly 20 kcal mol(-1), ruling out a mechansm via Berry pseudorotation involving equatorial halides. In all cases studied, epimerization of chlorophosphonium chlorides has a lower energy barrier (by 2 kcal mol(-1)) than the analogous bromo salts. Calculations determined that this was due to the easier accessibility in solution of pentacoordinate dichlorophosphoranes when compared to analogous dibromophosphoranes. In line with the proposed associative mechanism, bulky substituents slow the reaction in the order Me < Et < (i)Pr < (t)Bu. Bulky substituents affect the shape of the reaction energy profile so that the pentacoordinate intermediate is destabilized eventually becoming a transition state. The magnitude of the steric effects is comparable to that of the same substituents on substitution at primary alkyl halides, which can be rationalized by the relatively longer P-C bonds. The reaction displays first-order kinetics due to the prevalence of tight- or solvent-separated ion pairs in solution. Three-dimensional reaction potential energy profiles (More O'Ferrall-Jencks plots) indicated a relatively shallow potential well corresponding to the trigonal bipyramid intermediate flanked by two transition states.

Substituent effects on spin state in a series of mononuclear manganese(III) complexes with hexadentate Schiff-Base ligands.

Eleven new mononuclear manganese(III) complexes prepared from two hexadentate ligands, L1 and L2, with different degrees of steric bulk in the substituents are reported. L1 and L2 are Schiff bases resulting from condensation of N,N'-bis(3-aminopropyl)ethylenediamine with 3-methoxy-2-hydroxybenzaldehyde and 3-ethoxy-2-hydroxybenzaldehyde respectively, and are members of a ligand series we have abbreviated as R-Sal2323 to indicate the 323 alkyl connectivity in the starting tetraamine and the substitution (R) on the phenolate ring. L1 hosts a methoxy substituent on both phenolate rings, while L2 bears a larger ethoxy group in the same position. Structural and magnetic properties are reported in comparison with those of a previously reported analogue with L1, namely, [MnL1]NO3, (1e). The BPh4(-) and PF6(-) complexes [MnL1]BPh4, (1a), [MnL2]BPh4, (2a), [MnL1]PF6, (1b'), and [MnL2]PF6, (2b), with both ligands L1 and L2, remain high-spin (HS) over the measured temperature range. However, the monohydrate of (1b') [MnL1]PF6·H2O, (1b), shows gradual spin-crossover (SCO), as do the ClO4(-), BF4(-), and NO3(-) complexes [MnL1]ClO4·H2O, (1c), [MnL2]ClO4, (2c), [MnL1]BF4·H2O, (1d), [MnL2]BF4·0.4H2O, (2d), [MnL1]NO3, (1e), and [MnL2]NO3·EtOH, (2e). The three complexes formed with ethoxy-substituted ligand L2 all show a higher T1/2 than the analogous complexes with methoxy-substituted ligand L1. Analysis of distortion parameters shows that complexes formed with the bulkier ligand L2 exhibit more deformation from perfect octahedral geometry, leading to a higher T1/2 in the SCO examples, where T1/2 is the temperature where the spin state is 50% high spin and 50% low spin. Spin state assignment in the solid state is shown to be solvate-dependent for complexes (1b) and (2e), and room temperature UV-visible and NMR spectra indicate a solution-state spin assignment intermediate between fully HS and fully low spin in 10 complexes, (1a)-(1e) and (2a)-(2e).

Synthesis of a conformationally constrained δ-amino acid building block.

Conformationally restricted amino acids are important components in peptidomimetics and drug design. Herein, we describe the synthesis of a novel, non-proteinogenic constrained delta amino acid containing a cyclobutane ring, cis-3(aminomethyl)cyclobutane carboxylic acid (ACCA). The synthesis of the target amino acid was achieved in seven steps, with the key reaction being a base induced intramolecular nucleophilic substitution. A small library of dipeptides was prepared through the coupling of ACCA with proteinogenic amino acids.

Different rearrangement behaviour of the cation or anion derived from the Diels-Alder adduct of 9-ferrocenylanthracene and 1,4-benzoquinone: ring-opening or paddlewheel formation.

Prototropic rearrangement of the Diels-Alder adduct (3a) of 9-ferrocenylanthracene and 1,4-benzoquinone potentially furnishes 9-ferrocenyl-1,4-dihydroxytriptycene (3b) incorporating a C(2v) symmetrical paddlewheel moiety. However, reaction of 3a with HBF(4) unexpectedly yields instead 9-ferrocenyl-10-(2,5-dihydroxyphenyl)anthracene (4) via cleavage of the C9-C12 bond to generate initially a ferrocenyl-stabilized cation. Treatment of 3a with sodium hydride and iodomethane yields 1,4-dimethoxy-9-ferrocenyltriptycene (3c) in high yield but, surprisingly, also leads to fission of the C9-C12 bond resulting, after methylation, in the formation of 9-hydroxy-9-ferrocenyl-10-(2-hydroxy-5-methoxyphenyl)dihydroanthracene (12), which readily dehydrates on silica to form 9-ferrocenyl-10-(2-hydroxy-5-methoxyphenyl)anthracene (8). The X-ray crystal structures of 3a, 3c and 4 are reported.

Impact of dynamic sub-populations within grafted chains on the protein binding and colloidal stability of PEGylated nanoparticles.

Polyethylene glycol grafting has played a central role in preparing the surfaces of nano-probes for biological interaction, to extend blood circulation times and to modulate protein recognition and cellular uptake. However, the role of PEG graft dynamics and conformation in determining surface recognition processes is poorly understood primarily due to the absence of a microscopic picture of the surface presentation of the polymer. Here a detailed NMR analysis reveals three types of dynamic ethylene glycol units on PEG-grafted SiO2 nanoparticles (NPs) of the type commonly evaluated as long-circulating theranostic nano-probes; a narrow fraction with fast dynamics associated with the chain ends; a broadened fraction spectrally overlapped with the former arising from those parts of the chain experiencing some dynamic restriction; and a fraction too broad to be observed in the spectrum arising from units closer to the surface/graft which undergo slow motion on the NMR timescale. We demonstrate that ethylene glycol units transition between fractions as a function of temperature, core size, PEG chain length and surface coverage and demonstrate how this distribution affects colloidal stability and protein uptake. The implications of the findings for biological application of grafted nanoparticles are discussed in the context of accepted models for surface ligand conformation.

Molecular dials: hindered rotations in mono- and diferrocenyl anthracenes and triptycenes.

The syntheses, X-ray crystal structures, and molecular dynamics of 9-ferrocenylanthracene, 3, 9,10-diferrocenylanthracene, 4, 9-ferrocenyltriptycene, 7, and 9,10-diferrocenyltriptycene, 8, are reported. At 193 K, 3 exhibits C(s) symmetry via oscillation of the ferrocenyl only about the anthracene plane; at higher temperatures, complete rotation about the C(9)-ferrocenyl linkage becomes evident with a barrier of 10.6 kcal mol(-1). At 193 K, the ferrocenyls in 4 give rise to syn (C(2v)) and anti (C(2h)) rotamers that also interconvert at room temperature. In the corresponding triptycyl systems, 7 and 8, these rotational barriers increase to 17 kcal mol(-1); 9,10-diferrocenyltriptycene exists as slowly interconverting meso and racemic rotamers, in which the ferrocenyl moieties are, respectively, eclipsed (C(2v)) or staggered (C2). 2D-EXSY NMR data recorded with different mixing times indicate clearly that these interconversions proceed in a stepwise manner, for example, rac→meso→rac, thus behaving as a set of molecular dials.

Design and synthesis of a tetradentate '3-amine-1-carboxylate' ligand to mimic the metal binding environment at the non-heme iron(II) oxidase active site.

Non-heme iron(II) oxidases (NHIOs) catalyse a diverse array of oxidative chemistry in Nature. As part of ongoing efforts to realize biomimetic, iron-mediated C-H activation, we report the synthesis of a new 'three-amine-one-carboxylate' ligand designed to complex with iron(II) and mimic the NHIO active site. The tetradentate ligand has been prepared as a single enantiomer in nine synthetic steps from N-Cbz-L-alanine, pyridine-2,6-dimethanol and diphenylamine, using Seebach oxazolidinone chemistry to control the stereochemistry. X-Ray crystal structures are reported for two important intermediates, along with variable temperature NMR experiments to probe the hindered interconversion of conformational isomers of several key intermediates, 2,6-disubstituted pyridine derivatives. The target ligand and an N-Cbz-protected precursor were each then complexed with iron(II) and tested for their ability to promote alkene dihydroxylation, using hydrogen peroxide as the oxidant.

Syntheses, X-ray crystal structures and reactivity of fluorenylidene- and dibenzosuberenylidene-allenes: convenient precursors to dispirotetracenes, di-indenotetracenes and 2-phenyl-11bH-dibenz[cd,h]azulene.

3,3-(Biphenyl-2,2'-diyl)-1-alpha,alpha,alpha-trifluoro-p-tolyl-allene, 9, sequentially forms head-to-tail, 12, cis-tail-to-tail, 13, and trans tail-to-tail, 14, 1,2-dialkylidene-cyclobutane dimers, each of which has been characterised by X-ray crystallography. Thermolysis at 180 degrees C yields the dispirotetracene, 15, and di-indenotetracene, 16; the latter compound forms an air-stable Diels-Alder adduct, 17, with N-methylmaleimide. In contrast, the dibenzo[a,d]cycloheptenylidene-allenes, (C(14)H(10))C=C=C(Br)Ph, 20a, and (C(14)H(10))C=C=C(H)Ph, 21a, do not dimerise under relatively mild conditions. However, protonation of the bromo-allene, 20a, and subsequent addition of hydride, provide a facile entry to the difficultly accessible bowl-shaped dibenz[cd,h]azulene framework, as in 30, that had not previously been structurally characterised. Among others, the X-ray crystal structures of 12, 13, 14, 17, 20a, 21a and 30 are reported.

A molecular paddlewheel with a sliding organometallic latch: syntheses, X-ray crystal structures and dynamic behaviour of [Cr(CO)3{eta(6)-2-(9-triptycyl)indene}], and of [M(CO)3{eta(5)-2-(9-triptycyl)indenyl}] (M = Mn, Re).

In [eta(6)-2-(9-triptycyl)-indene]tricarbonylchromium (2a), the indenyl-chromium moiety is linked directly to the axis of the three-bladed triptycene paddlewheel. However, the molecular structure of 2a reveals that there is no steric interaction between these components, and the paddlewheel is free to rotate. Accordingly, its NMR spectrum indicates the full equivalence of the blades of the triptycene. Deprotonation of the indene induces a haptotropic shift of the organometallic fragment from the six-membered to the five-membered ring of the indene and, in the sodium [eta(5)-2-(9-triptycyl)-indenyl]tricarbonylchromium salt (3a), so formed, rotation of the three-bladed molecular paddlewheel is now blocked by the bulky tripod. NMR data for 3a, and also for the isostructural eta(5)-Mn(CO)(3) and eta(5)-Re(CO)(3) complexes, 3b and 3c, respectively, reveal a 2:1 splitting of the blades of the triptycyl moiety, thus breaking its original threefold symmetry. The X-ray crystal structures of the chromium complex, 2a, and of the manganese and rhenium complexes, 3b and 3c, provide pictures of the system in both its "ON" and "OFF" states, whereby the M(CO)(3) tripod has moved about 2 A towards the triptycene, thus blocking its rotation. Comparison of the rotational barriers in 2-(9-triptycyl)indene (1) and its complexes 2 and 3, suggests that rotation of the paddlewheel can be slowed by a factor of approximately 10(8).

Directing self-assembly in solution towards improved cooperativity in Fe(iii) complexes with amphiphilic tridentate ligands.

An amphiphilic iron(iii) complex with a tridentate Schiff-base ligand was prepared by condensation of a hexadecyloxy functionalised salycylaldehyde with a diamine followed by complexation with FeCl2 and anion methathesis with NaClO4. The complex shows spin crossover both in the solid state and solution. However in solution self-assembly and consequently aggregation of individual molecules form concentration dependent particles with sizes of 300 nm for higher concentrations, or 5 nm for lower concentrations. Aggregate formation was confirmed by NANO-flex 180° DLS Size, scan-rate dependent cyclic voltammetry and scanning electron microscopy. Molecular simulations were used to investigate the self-assembly of the complex in solution, including the role of residual water molecules. The simulations showed the self-assembly of reverse micelle-like structures when a small water cluster is inserted in solution, whereas no large aggregates formed in dehydrated environments. The perchlorate anions were found near the metal centres, stabilizing the aggregates around the water pool. Simulations of pre-assembled structures further showed the lack of stability of large aggregates in the absence of water. The larger aggregates promoted efficient communication between the iron(iii) centres and the compound displayed spin crossover in solution at around 220 K with a 10 K hysteresis window, as measured by NMR and SQUID magnetometry.

[Co2 (CO)6 (Alkynyl)] Complexes of Dibenzosuberyl and Dibenzosuberenyl Carbocations: Dibenzotropylium or Dibenzoheptafulvene?

The reaction of dibenzo[a,d]cycloheptan-5-one (dibenzosuberone) and dibenzo[a,d]cyclohept-10-en-5-one (dibenzosuberenone) with aryl- or trimethylsilylacetylides led to the formation of the corresponding alkynyldibenzosuberols and alkynyldibenzosuberenols. Treatment with dicobalt octacarbonyl and then with bis(diphenylphosphino)methane (dppm) furnished the corresponding [Co2 (CO)4 (dppm)(alkynol)] clusters 25 and 29. Upon protonation with HBF4 at 203 K to generate the relevant cobalt-stabilised cations, the dibenzosuberyl system 30 exhibited fluxionality such that the cation migrated between cobalt centres. Variable-temperature 31 P NMR spectroscopy revealed a barrier of approximately 12.5 kcal mol-1 . In contrast, in the supposedly aromatic [Co2 (CO)4 (dppm)(dibenzosuberenyl)]+ cation (31), which would be expected to have less need of cobalt stabilisation, the barrier was too high to be measured experimentally, but is certainly in excess of 16 kcal mol-1 . These data were rationalised by DFT calculations on the structures and energies of the relevant ground states and transition states, which suggested that the nonplanar alkynyldibenzosuberenyl moiety in 31 is better regarded as a neutral dibenzoheptafulvene coordinated to a cationic alkynyl-dicobalt cluster. The question of the bonding of both aromatic and antiaromatic cations to alkyne-dicobalt clusters is considered, and it is proposed that their stabilities, when complexed, parallel the inversion of (4n+2) π and 4n π systems seen under photochemical conditions.

First ever observation of the intermediate of phosphonium salt and ylide hydrolysis: P-hydroxytetraorganophosphorane.

P-Hydroxytetraorganophosphorane, the long-postulated intermediate in phosphonium salt and ylide hydrolysis, has been observed and characterised by low temperature NMR, finally definitively establishing its involvement in these reactions. The results require modification of the previously accepted mechanism for ylide hydrolysis: P-hydroxytetraorganophosphorane is generated directly by 4-centre reaction of ylide with water.