Jimmy Muldoon

NMR spectroscopy reveals cytochrome c-poly(ethylene glycol) interactions.

In vitro protein studies are typically performed on samples that are composed almost entirely of water. However, the cell interior is a heterogeneous “crowded” solution of small molecules, proteins, nucleic acids and membranes. At a concentration of 300–400 gL , the macromolecular content of the cell influences the kinetics and thermodynamics of protein folding, ligand binding and protein–protein interactions through excluded volume effects. Therefore, in order to build realistic models of protein structure and function, it is necessary to study proteins in vivo or under “crowded” conditions that mimic the cellular environment. The necessity for in vivo protein characterisation is being addressed by the development of in-cell NMR spectroscopy. While “biologically inert” proteins are largely unaffected by the crowded cell interior, the disordered protein FlgM was shown to gain structure inside Escherichia coli cells. A similar gain in structure occurred in vitro in the presence of crowding agents. Artificially crowded environments can be created by using sugars, proteins or polymers such as Ficoll, dextran and poly(ethylene glycol) (PEG). Such sample conditions are accessible by NMR spectroscopy, and the effects of macromolecular crowding on protein structure and dynamics have been investigated. Related NMR studies of macromolecular confinement have been performed by using polyacrylamide gels, reverse micelles, sol–gels and agarose gels. Generally, crowding/confinement tends to accelerate protein folding, promotes self-association and stabilises protein structure. 5,8–13] Given that macromolecular crowding can enhance protein association, the use of crowding agents is likely to facilitate the structural characterisation of weak protein interactions. We are interested in using NMR spectroscopy to study the effects of macromolecular crowding on the transient interactions of redox proteins. Saccharomyces cerevisiae cytochrome c (cyt c) in PEG-containing solutions was chosen for initial studies. PEG– protein interactions are usually repulsive, and volume exclusion results in preferential hydration of the protein surface. The repulsive interactions can be reduced by minimising (through conformation changes, precipitation or crystallisation) the protein surface area exposed to the solvent. The effect of PEG on protein solutions is not limited to volume exclusion. Although highly water soluble, PEG is hydrophobic in nature and can interact with hydrophobic proteins. An interesting example of this type of interaction is found in the crystal structure of the odorant-binding protein from Anopheles, in which a hydrophobic channel is occupied by a PEG molecule (PDB code: 2erb). The study of protein–PEG mixtures is further underlined by the growing importance of PEGylated-protein therapeutics. When modified by the covalent attachment of a PEG chain, proteins are less susceptible to proteolysis and have reduced immunogenicity. We report here the interaction of cyt c with PEG as revealed by H,N correlation spectroscopy. For comparison, experiments were performed on cyt c embedded in agarose gels. Similar PEG-induced effects were observed for both reduced and oxidised cyt c, and therefore this report focuses on the ACHTUNGTRENNUNGresults for reduced cyt c.N-labelled cyt c was studied in the presence of different sizes and concentrations of PEG. Samples containing up to 300 gL 1 of PEG were used to mimic the ACHTUNGTRENNUNGintracellular macromolecular content. Figure 1A illustrates a region of the H,N correlation spectrum of cyt c and the spectral changes associated with the presence of increasing concentrations of PEG 8000. The majority of cyt c resonances demonstrated small changes in line width, increasing on average by 25–35% at 200 and 300 gL 1 PEG. Compared to the approximately twofold line-width increases for cyt c bound to cyt c peroxidase, and cyt b5 encapsulated in sol–gel, [14] this indicates that the rotational correlation time (tc) of cyt c is weakly influenced by PEG. Resonance broadening was greater for a number of amides found in flexible loops, including Gly34, which was broadened beyond detection. Considering that loops are prone to conformation changes, the resonance broadening suggests that, in the presence of PEG, the exchange between different conformations is slow on the NMR timescale. In addition to line broadening, concentration-dependent chemical-shift perturbations of the order of 0.1 (H) and 0.3 (N) ppm were observed. Figure S1 in the Supporting Information gives a plot of the averaged H and N shifts for each backbone amide. Mapping these perturbations onto the crystal structure of cyt c reveals that the majority of the shifts surround the exposed haem edge (Figure 1B) with Gln16 and Lys79 standing out as most strongly affected. Note that Lys79 lies flat on the protein surface and thus contributes to the hydrophobic patch around the haem (Figure S2). Similar results were found for PEG 3350, 8000 and 20000; this indicates that the molecular weight of PEG does not affect its propensity to bind cyt c. Surprisingly, the chemical-shift-perturbation map of cyt c in the presence of PEG is qualitatively similar to the binding maps for cyt c in complex with cyt c peroxidase, and the nonphysiological partner cyt f. In particular, the down-field [a] Dr. P. B. Crowley, K. Brett UCD School of Biomolecular and Biomedical Science Conway Institute, University College Dublin Belfield, Dublin 4 (Ireland) Fax: (+ 353) 1-716 6701 E-mail : peter.crowley@ucd.ie [b] Dr. J. Muldoon UCD Centre for Synthesis and Chemical Biology University College Dublin Belfield, Dublin 4 (Ireland) Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.

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.

Mechanistic insight into the formation of tetraarylazadipyrromethenes.

The tetraarylazadipyrromethene chromophore class has gained increasing attention in the past decade for a diverse set of scientific interests and applications. The most direct synthetic route available for their generation is heating of 4-nitro-1,3-diarylbutan-1-ones with an ammonium source in an alcohol solvent. Despite the practical simplicity, the reaction pathway(s) for these conversions are lengthy and unclear. To gain insight into the steps involved, (15)N labeling experiments with MS and NMR analysis were utilized for conversion of 4-nitro-1,3-diphenylbutan-1-one 1 into tetraphenylazadipyrromethene 2 with (15)NH(4)OAc. To permit examination of later stages of the reaction sequence to 2, the (15)N-labeled potential intermediate 3,5-diphenyl-1H-pyrrol-2-amine 10 was synthesized. A study of the dimerization pathway utilizing (15)N-labeled 10 revealed an unprecedented nitrogen rearrangement in the final stages of the pathway involving a ring-opening/closing of a pyrrole ring. Utilizing (15)N labeling experiments we have shown that 2,4-diphenylpyrrole 8 can also react under the reaction conditions with 3,5-diphenyl-2H-pyrrol-2-imine 7 (from oxidation of 10) to produce 2. Overall in the conversion of 1 into 2, two related pathways are ongoing concurrently; the first involves a dimerization of 3,5-diphenyl-2H-pyrrol-2-imine 7, and the other a reaction of 7 with 2,4-diphenylpyrrole 8.

Restricted rotation in 9-phenyl-anthracenes: a prediction fulfilled.

The calculated phenyl rotation barrier in 9-phenylanthracene has been reported as ~21 kcal mol(-1), but experimental verification of this barrier is limited by its intrinsic symmetry. V-T NMR indicated the barrier to interconversion of the syn (C(2v)) and anti (C(2h)) rotamers of 9,10-bis(3-fluorophenyl)anthracene to be ~21 kcal mol(-1). Likewise, the V-T NMR spectra of 9-(1-naphthyl)-10-phenylanthracene reveal that the rotational barrier of the unsubstituted phenyl ring is at least 21 kcal mol(-1).

Iridium-mediated isomerization-cyclization of bicyclic Pauson-Khand derived allylic alcohols.

Treatment of 2-(toluene-4-sulfonyl)-2,3,4,4a,5,6-hexahydro-1H-[2]pyrindin-6-ol 10, accessed from the diastereoselective Luche reduction of a Pauson-Khand derived bicylic cyclopentenone, with a catalytic amount of (1,5-cyclooctadiene)(pyridine)(tricyclohexylphosphine)iridium(I) hexafluorophosphate 1 (Crabtrees catalyst) under a hydrogen atmosphere resulted in the formation of 4-(toluene-4-sulfonyl)-2-oxa-4-azatricyclo[5.2.1.0(3,8)]decane 12 as a single diastereoisomer. This process is likely to proceed via an initial Ir(I)-mediated isomerization of the alkene to form an N-sulfonyl enamine 11, followed by cyclization. Evidence to support this came when, after short reaction periods, 11 was isolated, characterized spectroscopically, and on resubmission to the reaction conditions formed 12.

A Ferrocenyl Kaleidoscope: Slow Interconversion of Six Diastereo‐atropisomers of 2,6‐Di‐tert‐butyl‐9,10‐diferrocenyltriptycene

The title triptycene, 6, has been isolated as the product of 9,10-cycloaddition of benzyne to 9,10-diferrocenyl-2,6-di-tert-butylanthracene, 5, whose X-ray crystal structure is reported. Each ferrocenyl unit in 6 has access to the same three non-equivalent molecular environments, and their rotations relative to the molecular paddlewheel give rise to six slowly interconverting atropisomers. Their dynamic behaviour in solution is a challenging NMR puzzle that can be successfully solved by taking advantage of the recently described very large diamagnetic anisotropy of the ferrocenyl moiety, together with the C2 symmetry of particular atropisomers. Application of one- and two-dimensional NMR techniques over a range of temperatures together, with a detailed analysis of the homo- and heteronuclear correlations in 6, resulted in unequivocal mapping of the 99 (1)H and 162 (13)C positions in the six interconverting systems. Variable-temperature 2D-EXSY measurements revealed that, while the stability of the atropisomers is almost identical, they are separated by energy barriers which the ferrocenyls must overcome in the course of their interconversions. The heights of two different rotational barriers have been identified and these experimental findings are in good agreement with DFT calculations.

Mysterious Decomposition of Alkoxyphosphonium Chlorides: Postulated Involvement of the HCl2 Anion and Its Capture in the Solid State

P-Alkoxyphosphonium (AP) chlorides were generated by reacting P-chlorophosphonium chlorides with alcohols. Their well-known spontaneous Arbuzov-type collapse leading to phosphine oxides was studied and its rate found to be dependent on a number of factors in an unexpected fashion: it is inversely proportional to the initial concentration and it shows strong dependence on the acidity of the media but is not very sensitive to the presence of base. To explain these observations, we evoke a self-inhibition model with the formation of the less nucleophilic hydrodichloride anion HCl2 in solution. Detailed analysis of the kinetic data yields the association constant (K=3×102  m-1 ) of the putative HCl2 species in chloroform. Experimental observations for the collapse of highly enriched diastereomeric alkoxyphosphonium (DAP) chlorides are fully analogous to the achiral AP also implying the involvement of HCl2 anions. Moreover, crystallisation of a highly enriched DAP salt derived from (-)-menthol furnished, for the first time, crystals of individual (RP )-DAP hydrodichloride as confirmed by X-ray diffractometry. Importantly, the P-configuration and detailed conformation of the DAP moiety is in good agreement with DFT-level computational results. The thermal collapse of (RP )-DAP⋅HCl2 proceeds with complete retention of the P-configuration furnishing the phosphine oxide of exceptional enantiomeric purity.