New method for peptide purification based on selective removal of truncation peptide impurities after SPPS with orthogonal capping

Peptide purification by high-performance liquid chromatography (HPLC) is associated with high solvent consumption, relatively large effort and lack of efficient parallelization. As an alternative, many catch-and-release (c&r) purification methods have been developed over the last decades to enable the efficient parallel purification of peptides originating from solid-phase peptide synthesis (SPPS). However, with one exception, none of the c&r systems has been widely established in industry and academia until today. Herein, we present an entirely new chromatography-free purification concept for peptides synthesized on a solid support, termed reactive capping purification (RCP). The RCP method relies on the capping of truncation peptides arising from incomplete coupling of amino acids during SPPS with a reactive tag. The reactive tag contains a masked functionality that, upon liberation during cleavage from the resin, enables straightforward purification of the peptide by incubation with a resin-bound reactive moiety. In this work, two different reactive tags based on masked thiols were developed. Capping with these reactive tags during SPPS led to effective modification of truncated sequences and subsequent removal of the latter by chemoselective reaction with a maleimide-functionalized solid support. By introducing a suitable protecting group strategy, the thiol-based RCP method described here could also be successfully applied to a thiol-containing peptide. Finally, the purification of a 15-meric peptide by the RCP method was demonstrated. The developed method has low solvent consumption, has the potential for efficient parallelization, uses readily available reagents, and is experimentally simple to perform.     

The challenge of estimating kelp production in a turbid marine environment

Coastal kelp forests produce substantial marine carbon due to high annual net primary production (NPP) rates, but upscaling of NPP estimates over time and space remains difficult. We investigated the impact of variable underwater photosynthetically active radiation (PAR) and photosynthetic parameters on photosynthetic oxygen production of Laminaria hyperborea, the dominant NE-Atlantic kelp species, throughout summer 2014. Collection depth of kelp had no effect on chlorophyll a content, pointing to a high photoacclimation potential of L. hyperborea towards incident light. However, chlorophyll a and photosynthesis versus irradiance parameters differed significantly along the blade gradient when normalized to fresh mass, potentially introducing large uncertainties in NPP upscaling to whole thalli. Therefore, we recommend a normalization to kelp tissue area, which is stable over the blade gradient. Continuous PAR measurements revealed a highly variable underwater light climate at our study site (Helgoland, North Sea) in summer 2014, reflected by PAR attenuation coefficients (K_d) between 0.28 and 0.87 m⁻¹. Our data highlight the importance of continuous underwater light measurements or representative average values using a weighted K_d to account for large PAR variability in NPP calculations. Strong winds in August increased turbidity, resulting in a negative carbon balance at depths >3–4 m over several weeks, considerably impacting kelp productivity. Estimated daily summer NPP over all four depths was 1.48 ± 0.97 g C · m⁻² seafloor · d⁻¹ for the Helgolandic kelp forest, which is in the range of other kelp forests along European coastlines.     

Palladium and phosphomolybdovanadate catalyzed olefin oxidation to carbonyls

A new homogeneous catalyst system has been developed for the oxidation of olefins to carbonyls — ethylene to acetaldehyde and higher olefins to ketones. The catalyst system was first developed for the oxidation of ethylene to acetaldehyde in Wacker-type acetaldehyde plants. The aqueous catalyst solution has three key components. A palladium(II) catalyst oxidizes the olefin to the carbonyl, which is analogous to the Wacker system but with only a fraction of its palladium. Keggin phosphomolybdovanadates of the general formula PMo_(12–x) V _x O ₄₀ ^(3+x)– provide a dioxygen-reversible vanadium(V)/vanadium(IV) redox agent for palladium(O) reoxidation, which is analogous to the copper(II)/copper(I) chlorides in the Wacker system. Chloride at centimolar concentrations, lacking in earlier reported palladium and polyoxometalate catalyst systems, is essential to maintain stable palladium(II) catalyst activity. Kinetic characterization and reaction engineering provided ethylene and oxygen reaction rates comparable to those obtained with the Wacker catalyst. A new, efficient method of preparing aqueous phosphomolybdovanadate solutions was developed for laboratory and large-scale production. This paper describes the catalyst system and its reactions with emphasis on the polyoxometalate chemistry.     

Geographic and between-generation variation in the parasitoid communities associated with an invading gallwasp,Andricus quercuscalicis (Hymenoptera: Cynipidae)

The knopper gallwasp Andricus quercuscalicis Burgsdorf 1783 (Hymenoptera: Cynipidae) has invaded western and northern Europe from southern and eastern Europe over the last 400 years. A. quercuscalicis has two alternating generations, which differ in phenology, structure, and host oak species. This study describes geographic variation in the community in the tiny catkin galls of the sexual generation on Turkey oak, Quercus cerris, and compares the patterns obtained with those in the community attacking the alternate agamic generation. As predicted from considerations of parasitoid recruitment to the communities of invading phytophagous insects (Cornell and Hawkins 1993), in its native range the sexual generation shows (1) higher parasitoid community species richness, (2) higher total mortality due to parasitoid attack and (3) a higher ratio of specialist to generalist parasitoid species than is evident in the invaded range. Counter to predictions, there is no indication that parasitoid community richness in the invaded range has increased with time since the arrival of the new host. Higher host mortality in the native range is due principally to a single specialist, Aulogymnus obscuripes Mayr 1877 (Hymenoptera: Eulophidae), and is not distributed evenly among parasitoid species which attack the gall-former only in this area. This contrasts with the community in Britain, where three principal generalist parasitoids cause approximately equal mortalities. The agamic gall contains a taxonomically and structurally diverse guild of parasitoid and inquiline species, associated with the changing resource provided by a large, long-lived, complex gall. In contrast, the sexual community includes a taxonomically and structurally narrow guild, associated with a resource which is structurally simple, small in size and short-lived. No parasitoid species attacks the gall-former in both generations. Surprisingly, in spite of these differences in the nature of the gall resource in the two generations, over their entire range (native and invaded) the parasitoid guilds of the two are equally species rich.     

Subunit E of the vacuolar H+-ATPase of Hordeum vulgare L.: cDNA cloning,expression and immunological analysis†

A tonoplast protein of 31 kDa apparent molecular mass (TpP 31) was isolated from two-dimensional gels. Amino acid sequences were determined from LysC endoproteinase-peptide fragments. Using degenerate oligonucleotides, a corresponding cDNA clone of 1034 bp was isolated from a barley leaf cDNA library. It encodes for subunit E of the vacuolar H⁺-ATPase, the first one identified in plants so far. The open reading frame extends over 681 bp, encoding a gene product of 227 amino acids and a calculated molecular weight of 26 228 g mol^?1. Northern and Western blot analysis indicates constitutive expression of subunit E in all plant organs with only small effects of salt stress. Localization of TpP 31 at the tonoplast was confirmed in fractions of purified vacuolar membrane obtained by free-flow electrophoresis. Immunoprecipitation of newly synthesized ³⁵S-labelled membrane proteins with anti-TpP 31 gave two additional bands with apparent molecular masses of about 53 and 62 kDa. Gel filtration after mild solubilization showed co-purification of TpP 31 with the 55 kDa subunit of the H⁺-ATPase. Both results provide evidence beyond the sequence homology that TpP 31 is a structural component of the vacuolar H⁺-ATPase.     

The N-terminal extension of the ADP/ATP translocator is not involved in targeting to plant mitochondria in vivo

The mitochondrial ADP/ATP translocator, also called adenine nucleotide translocase (ANT), is synthesized in plants with an N-terminal extension which is cleaved upon import into mitochondria. In contrast, the homologous proteins of mammals or fungi do not contain such a transient amino terminal presequence. To investigate whether the N-terminal extension is needed for correct intracellular sorting in vivo , translational fusions were constructed of the translocator cDNA—with and without presequence—with the β-glucuronidase ( gus ) reporter gene. The distribution of reporter enzymatic activity in the subcellular compartments of transgenic plants and transformed yeast cells was subsequently analysed. The results show that: (i) the plant translocator presequence is not necessary for the correct localization of the ANT to the mitochondria; (ii) the mitochondrial targeting information contained in the mature part of the protein is sufficient to overcome, to some extent, the presence of plastid transit peptides; and (iii) the presequence alone is not able to target a passenger protein to mitochondria in vivo .     

Biotechnological production of unnatural L-amino acids from D,L-5-monosubstituted hydantions. II. L-α- and L-β-naphthylalanine

Summary Resting cells ofArthrobacter sp. (DSM 3745) with the ability to form L-tryptophan from D,L-5-(3-indolylmethy)hydantoin were used for the bioconversion of D,L-5-- and D,L-5--naphthylmethylhydantoin (D,L-5-- and D,L-5--NMH) to the corresponding L-amino acids. Under the optimal reaction conditions of pH 9.7 and 40°C specific productivities of 0.2 (-naphtylalanine) and 0.6 (-naphtylalanine) mM amino acid x g cell dry mass^–1 x h^–1 were obtained in a 0.1 M Na₂CO₃/NaHCO₃-buffer in a strirred bioreactor.