Bruce F. Milne

Dermacozines, a new phenazine family from deep-sea dermacocci isolated from a Mariana Trench sediment.

Dermacoccus abyssi sp. nov., strains MT1.1 and MT1.2 are actinomycetes isolated from Mariana Trench sediment at a depth of 10 898 m. Fermentation using ISP2 and 410 media, respectively, lead to production of seven new oxidized and reduced phenazine-type pigments, dermacozines A-G (1-7), together with the known phenazine-1-carboxylic acid (8) and phenazine-1,6-dicarboxylic acid (9). Extensive use was made of 1D and 2D-NMR data, and high resolution MS to determine the structures of the compounds. To confirm the structure of the most complex pentacyclic analogue (5) we made use of electronic structure calculations to compare experimental and theoretical UV-Vis spectra, which confirmed a novel structural class of phenazine derivatives, the dermacozines. The absolute stereochemistry of dermacozine D (4) was determined as S by a combination of CD spectroscopy and electronic structure calculations. Dermacozines F (6) and G (7) exhibited moderate cytotoxic activity against leukaemia cell line K562 with IC(50) values of 9 and 7 microM, respectively, while the highest radical scavenger activity was observed for dermacozine C (3) with an IC(50) value of 8.4 microM.

Synoxazolidinones A and B: novel bioactive alkaloids from the ascidian Synoicum pulmonaria.

Bioassay-guided fractionation of the sub-Arctic ascidian Synoicum pulmonaria collected off the Norwegian coast led to the isolation of a novel family of brominated guanidinium oxazolidinones named synoxazolidinones A and B (1 and 2). The backbone of the compounds contains a 4-oxazolidinone ring rarely seen in natural products. The structure of the compounds was determined by spectroscopic methods. The synoxazolidinones exhibited antibacterial and antifungal activities.

Absorption spectrum of the firefly luciferin anion isolated in vacuo.

The excited-state physics of the firefly luciferin anion depends on its chemical environment, and it is therefore important to establish the intrinsic behavior of the bare ion. Here we report electronic absorption spectra of the anion isolated in vacuo obtained at an electrostatic ion storage ring and an accelerator mass spectrometer where ionic dissociation is monitored on a long time scale (from 33 μs and up to 3 ms) and on a short time scale (0-3 μs), respectively. In the ring experiment the yield of all neutrals (mainly CO(2)) as a function of wavelength was measured whereas in the single pass experiment, the abundance of daughter ions formed after loss of CO(2) was recorded to provide action spectra. We find maxima at 535 and 265 nm, and that the band shape is largely determined by the sampling time interval, which is due to the kinetics of the dissociation process. Calculations at the TD-B3LYP/TZVPP++ level predict maximum absorption at 533 and 275 nm for the carboxylate isomer in excellent agreement with the experimental findings. The phenolate isomer lies higher in energy by 0.22 eV, and also its absorption maximum is calculated to be at 463 nm, which is far away from the experimental value. Our data serve to benchmark future theoretical models for bioluminescence from fireflies.

Spontaneity in the patellamide biosynthetic pathway.

Post-translationally modified ribosomal peptides are unusual natural products and many have potent biological activity. The biosynthetic processes involved in their formation have been delineated for some, but the patellamides represent a unique group of these metabolites with a combination of a macrocycle, small heterocycles and d-stereocentres. The genes encoding for the patellamides show very low homology to known biosynthetic genes and there appear to be no explicit genes for the macrocyclisation and epimerisation steps. Using a combination of literature data and large-scale molecular dynamics calculations with explicit solvent, we propose that the macrocyclisation and epimerisation steps are spontaneous and interdependent and a feature of the structure of the linear peptide. Our study suggests the steps in the biosynthetic route are heterocyclisation, macrocyclisation, followed by epimerisation and finally dehydrogenation. This study is presented as testable hypothesis based on literature and theoretical data to be verified by future detailed experimental investigations.

On the Influence of Water on the Electronic Structure of Firefly Oxyluciferin Anions from Absorption Spectroscopy of Bare and Monohydrated Ions in Vacuo

A complete understanding of the physics underlying the varied colors of firefly bioluminescence remains elusive because it is difficult to disentangle different enzyme-lumophore interactions. Experiments on isolated ions are useful to establish a proper reference when there are no microenvironmental perturbations. Here, we use action spectroscopy to compare the absorption by the firefly oxyluciferin lumophore isolated in vacuo and complexed with a single water molecule. While the process relevant to bioluminescence within the luciferase cavity is light emission, the absorption data presented here provide a unique insight into how the electronic states of oxyluciferin are altered by microenvironmental perturbations. For the bare ion we observe broad absorption with a maximum at 548 ± 10 nm, and addition of a water molecule is found to blue-shift the absorption by approximately 50 nm (0.23 eV). Test calculations at various levels of theory uniformly predict a blue-shift in absorption caused by a single water molecule, but are only qualitatively in agreement with experiment highlighting limitations in what can be expected from methods commonly used in studies on oxyluciferin. Combined molecular dynamics simulations and time-dependent density functional theory calculations closely reproduce the broad experimental peaks and also indicate that the preferred binding site for the water molecule is the phenolate oxygen of the anion. Predicting the effects of microenvironmental interactions on the electronic structure of the oxyluciferin anion with high accuracy is a nontrivial task for theory, and our experimental results therefore serve as important benchmarks for future calculations.

Analysis of the Structure and Electrophysiological Actions of Halitoxins: 1,3 Alkyl-pyridinium Salts from Callyspongia ridleyi

Abstract. We have chemically characterized a preparation of halitoxins, (1,3 alkyl-pyridinium salts) isolated from the marine sponge Callyspongia ridleyi. At concentrations of 50 and 5 μg/ml the halitoxin preparation caused irreversible membrane potential depolarization, decreased input resistance and inhibited evoked action potentials when applied to cultured dorsal root ganglion neurones. Under whole cell voltage clamp the halitoxins produced an increase in cation conductance that was attenuated by replacing sodium with N-methyl-d-glucamine. Fura-2 fluorescence ratiometric calcium imaging was used to directly measure calcium flux into neurones after exposure to halitoxins. Calcium influx, evoked by the halitoxins, persisted when the neurones were bathed in medium containing the voltage-activated calcium channel antagonists cadmium and nickel. Experiments on undifferentiated F-11 cells showed little or no calcium influx in response to depolarizing concentrations of potassium and indicated that halitoxins evoked massive calcium influx in the absence of voltage-activated calcium channels. The halitoxins also produced transient increases in intracellular calcium when F-11 cells were bathed in calcium-free medium suggesting that the toxins could release calcium from intracellular stores. The pore-forming action of the halitoxins was identified when the toxins were applied to artificial lipid bilayers composed of phosphatidylcholine and cholesterol. Halitoxins evoked channel-like activity in the lipid bilayers, with estimated unitary conductances of between 145pS and 2280pS, possibly indicating that distinct channels could be produced by the different components in the preparation of halitoxins.

Metal binding of Lissoclinum patella metabolites. Part 2: Lissoclinamides 9 and 10

Abstract Studies on the Thz, Thn and Oxn containing cyclic peptides, lissoclinamides 9 ( 9 ) and 10 ( 10 ) isolated from the Indo-Pacific ascidian (seasquirt) Lissoclinum patella have delineated their metal binding selectivity. MS and CD competition studies show that lissoclinamide 10 ( 10 ) shows selectivity for Cu 2+ in the presence of an excess of Zn 2+ whereas lissoclinamide 9 ( 9 ) is less selective for Cu 2+ . Comparison of the solution state conformations derived from nOe restrained molecular dynamics and additional Monte–Carlo conformational searches suggested binding environments for the Cu 2+ which confirmed the MS measurements and suggested a reason for the selectivity in the case of lissoclinamides 9 and 10.

Bioheterojunction Effect on Fluorescence Origin and Efficiency Improvement of Firefly Chromophores

We propose the heterojunction effect in the analysis of the fluorescence mechanism of the firefly chromophore. Following this analysis, and with respect to the HOMO−LUMO gap alignment between the chromophore’s functional fragments, three main heterojunction types (I, II, and I*) are identified. Time-dependent density functional theory optical absorption calculations for the firefly chromophore show that the strongest excitation appears in the deprotonated anion state of the keto form. This can be explained by its high HOMO−LUMO overlap due to strong bioheterojunction confinement. It is also found that the nitrogen atom in the thiazolyl rings, due to its larger electronegativity, plays a key role in the emission process, its importance growing when the HOMO and LUMO overlap at its location. This principle is applied to enhance the chromophore’s fluorescence efficiency and to guide the functionalization of molecular optoelectronic devices.

Computational study of molecules with high intrinsic hyperpolarizabilities.

In the current manuscript we present the results of a computational study on a series of chromophores with enhanced intrinsic hyperpolarizability. The high hyperpolarizability values of these molecules were previously reported and were achieved by making use of aromatic moieties in order to modulate the aromatic stabilization energy along the conjugated bridge between the donor and the acceptor. Calculations were performed using semiempirical, DFT, and TDDFT methods, and the results reproduce the trend determined experimentally for the first hyperpolarizability values. Several calculation schemes were used, and the best agreement was achieved when long-range Hartree-Fock exchange corrections and solvent effects are included in the DFT calculations. The long-range corrections proved to be especially important for the azobenzene derivatives, which otherwise have their hyperpolarizability overestimated considerably in the DFT calculations. The results are also analyzed within the framework of a two-level model, which correctly reproduces the trend in the hyperpolarizabilities of the molecules under study.

Unraveling the Intrinsic Color of Chlorophyll

The exact color of light absorbed by chlorophyll (Chl) pigments, the light-harvesters in photosynthesis, is tuned by the protein microenvironment, but without knowledge of the intrinsic color of Chl it remains unclear how large this effect is. Experimental first absorption energies of Chl a and b isolated in vacuo and tagged with quaternary ammonium cations are reported. The energies are largely insensitive to details of the tag structure, a finding supported by first-principles calculations using time-dependent density functional theory. Absorption is significantly blue-shifted compared to that of Chl-containing proteins (by 30-70 nm). A single red-shifting perturbation, such as axial ligation or the protein medium, is insufficient to account even for the smallest shift; the largest requires pigment-pigment interactions.