A beta-lactone route to chiral gamma-substituted alpha-amino acids: application to the concise synthesis of (S)-alpha-azidobutyro lactone and a natural amino acid

[reaction: see text] Beta-lactones are useful synthetic intermediates allowing access to a number of functional arrays. In this report, enantiomerically pure 4-trichloromethyl-2-oxetanone is shown to be a versatile amino acid synthon leading to a variety of gamma-substituted alpha-amino acid precursors. The utility of this methodology was demonstrated by the concise synthesis of a protected homoserine equivalent, alpha-azidobutyro lactone, and a naturally occurring alpha-amino acid from the seeds of Blighia unijugata.     

Optimization of nisin production by Lactococcus lactis

Applied Biochemistry and Biotechnology - The production of nisin by batch culture of Lactococcus lactis ATCC 11454 in MRS broth (pH 6.5), as treated in 30 assays, that were set up by a fractional...     

Developmental phases of individual mouse preimplantation embryos characterized by lipid signatures using desorption electrospray ionization mass spectrometry

Knowledge of the lipids present in individual preimplantation embryos is of interest in fundamental studies of embryology, in attempts to understand cellular pluripotency and in optimization of in vitro culture conditions necessary for the application and development of biotechnologies such as in vitro fertilization and transgenesis. In this work, the profiles of fatty acids and phospholipids (PL) in individual mouse preimplantation embryos and oocytes were acquired using an analytical strategy based on desorption electrospray ionization mass spectrometry (DESI-MS). The methodology avoids sample preparation and provides information on the lipids present in these microscopic structures. Differences in the lipid profiles observed for unfertilized oocytes, two- and four-cell embryos, and blastocysts were characterized. For a representative set of embryos (N?=?114) using multivariate analysis (specifically principal component analysis) unfertilized oocytes showed a narrower range of PL species than did blastocysts. Two- and four-cell embryos showed a wide range of PLs compared with unfertilized oocytes and high abundances of fatty acids, indicating pronounced synthetic activity. The data suggest that the lipid changes observed in mouse preimplantation development reflect acquisition of a degree of cellular membrane functional and structural specialization by the blastocyst stage. It is also noteworthy that embryos cultured in vitro from the two-cell through the blastocyst stage have a more homogeneous lipid profile as compared with their in vivo-derived counterparts, which is ascribed to the restricted diversity of nutrients present in synthetic culture media. The DESI-MS data are interpreted from lipid biochemistry and previous reports on gene expression of diverse lipids known to be vital to early embryonic development.     

Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon ca(2+) imaging

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 μM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.     

Rapid and sensitive identification of epitope-containing peptides by direct matrix-assisted laser desorption/ionization tandem mass spectrometry of peptides affinity-bound to antibody beads

A method has been developed for rapid and sensitive identification of epitope-containing peptides, based on direct MALDI-MS/MS analysis of epitope-containing peptides affinity bound to affinity beads. This technique provides sequence information of the epitope that allows unambiguous identification of the epitope either by database searching or de novo sequencing. With MALDI-MS, affinity beads with bound peptides can be placed directly on the MALDI target and analyzed. Coupling a MALDI source to an orthogonal injection quadrupole time-of-flight (QqTOF) mass spectrometer allows direct sequencing of the bound peptides. In contrast to ESI-MS/MS, elution of the affinity-bound peptides followed by additional concentration and purification steps is not required, thus reducing the potential for sample loss. Direct mass spectrometric sequencing of affinity-bound peptides eliminates the need for chemical or enzymatic sequencing. Other advantages of this direct MALDI-MS/MS analysis of epitope-containing peptides bound to the affinity beads include its sensitivity (femtomole levels) and speed. In addition, direct analysis of peptides on affinity beads does not adversely affect the high mass accuracy of a QqTOF, and database searching can be performed on the MS/MS spectra obtained. In proof-of-principle experiments, this method has been demonstrated on beads containing immobilized antibodies against phosphotyrosine, the c-myc epitope tag, as well as immobilized avidin. Furthermore, de novo sequencing of epitope-containing peptides is demonstrated. The first application of this method was with anti-FLAG-tag affinity beads, where direct MALDI MS/MS was used to determine an unexpected enzymatic cleavage site on a growth factor protein.     

Tetra­ammonium benzene‐1,2,4,5‐tetra­carboxyl­ate tetrahydrate

The anions in the title compound, 4NH₄⁺·C₁₀H₂O₈^4?·4H₂O, are held together by regular hydrogen bonds from the carboxyl­ate O atoms to the ammonium cations and water mol­ecules, forming a three‐dimensional network.     

Mono‐ and Bis(pyrrolo)tetrathiafulvalene Derivatives Tethered to C60: Synthesis,Photophysical Studies,and Self‐Assembled Monolayers

A series of mono‐ (MPTTF) and bis(pyrrolo)tetrathiafulvalene (BPTTF) derivatives tethered to one or two C₆₀ moieties was synthesized and characterized. The synthetic strategy for these dumbbell‐shaped compounds was based on a 1,3‐dipolar cycloaddition reaction between aldehyde‐functionalized MPTTF/BPTTF derivatives, two different tailor‐made amino acids, and C₆₀. Electronic communication between the MPTTF/BPTTF units and the C₆₀ moieties was studied by a variety of techniques including cyclic voltammetry and absorption spectroscopy. These solution‐based studies indicated no observable electronic communication between the MPTTF/BPTTF units and the C₆₀ moieties. In addition, femtosecond and nanosecond transient absorption spectroscopy revealed, rather surprisingly, that no charge transfer from the MPTTF/BPTTF units to the C₆₀ moieties takes place on excitation of the fullerene moiety. Finally, it was shown that the MPTTF–C₆₀ and C₆₀–BPTTF‐C₆₀ dyad and triad molecules formed self‐assembled monolayers on a Au(111) surface by anchoring to C₆₀.     

Enzyme Design from the Bottom Up: An Active Nickel Electrocatalyst with a Structured Peptide Outer Coordination Sphere

Catalytic, peptide‐containing metal complexes with a well‐defined peptide structure have the potential to enhance molecular catalysts through an enzyme‐like outer coordination sphere. Here, we report the synthesis and characterization of an active, peptide‐based metal complex built upon the well‐characterized hydrogen production catalyst [Ni(P^Ph₂N^Ph)₂]²⁺ (P^Ph₂N^Ph=1,3,6‐triphenyl‐1‐aza‐3,6‐diphosphacycloheptane). The incorporated peptide maintains its β‐hairpin structure when appended to the metal core, and the electrocatalytic activity of the peptide‐based metal complex (≈100,000 s^?1) is enhanced compared to the parent complex ([Ni(P^Ph₂N^APPA)₂]²⁺; ≈50,500 s^‐1). The combination of an active molecular catalyst with a structured peptide provides a scaffold that permits the incorporation of features of an enzyme‐like outer‐coordination sphere necessary to create molecular electrocatalysts with enhanced functionality.     

Influence of metal loading and humic acid functional groups on the complexation behavior of trivalent lanthanides analyzed by CE-ICP-MS

The complexation behavior of Aldrich humic acid (AHA) and a modified humic acid (AHA-PB) with blocked phenolic hydroxyl groups for trivalent lanthanides (Ln) is compared, and their influence on the mobility of Ln(III) in an aquifer is analyzed. As speciation technique, capillary electrophoresis (CE) was hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). For metal loading experiments 25 mg L⁻¹ of AHA and different concentrations (c_Ln(Eu+Gd) = 100–6000 μg L⁻¹) of Eu(III) and Gd(III) in 10 mM NaClO₄ at pH 5 were applied. By CE-ICP-MS, three Ln-fractions, assumed to be uncomplexed, weakly and strongly AHA-complexed metal can be detected. For the used Ln/AHA-ratios conservative complex stability constants log β_LnAHA decrease from 6.33 (100 μg L⁻¹ Ln³⁺) to 4.31 (6000 μg L⁻¹ Ln³⁺) with growing Ln-content. In order to verify the postulated weaker and stronger humic acid binding sites for trivalent Eu and Gd, a modified AHA with blocked functional groups was used. For these experiments 500 μg L⁻¹ Eu and 25 mg L⁻¹ AHA and AHA-PB in 10 mM NaClO₄ at pH-values ranging from 3 to 10 have been applied. With AHA-PB, where 84% of the phenolic OH-groups and 40% of the COOH-groups were blocked, Eu complexation was significantly lower, especially at the strong binding sites. The log β-values decrease from 6.11 (pH 10) to 5.61 at pH 3 (AHA) and for AHA-PB from 6.01 (pH 7) to 3.94 at pH 3. As a potential consequence, particularly humic acids with a high amount of strong binding sites (e.g. phenolic OH- and COOH-groups) can be responsible for a higher metal mobility in the aquifer due to the formation of dissolved negatively charged metal-humate species.     

Chemical Synthesis, 3D Structure,and ASIC Binding Site of the Toxin Mambalgin‐2

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid‐sensing ion channels (ASICs). The 57‐residue polypeptide mambalgin‐2 (Ma‐2) was synthesized by using a combination of solid‐phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three‐finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma‐2 on wild‐type and mutant ASIC1a receptors allowed us to identify α‐helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma‐2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure–activity relationship (SAR) studies and further development of this promising analgesic peptide.