Introduction of 4-chloro-alpha-cyanocinnamic acid liquid matrices for high sensitivity UV-MALDI MS

Matrix-assisted laser desorption/ionization (MALDI) is a key ionization technique in mass spectrometry (MS) for the analysis of labile macromolecules. An important area of study and improvements in relation to MALDI and its application in high-sensitivity MS is that of matrix design and sample preparation. Recently, 4-chloro-alpha-cyanocinnamic acid (ClCCA) has been introduced as a new rationally designed matrix and reported to provide an improved analytical performance as demonstrated by an increase in sequence coverage of protein digests obtained by peptide mass mapping (PMM) (Jaskolla, T. W.; et al. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 12200-12205). This new matrix shows the potential to be a superior alternative to the commonly used and highly successful alpha-cyano-4-hydroxycinnamic acid (CHCA). We have taken this design one step further by developing and optimizing an ionic liquid matrix (ILM) and liquid support matrix (LSM) using ClCCA as the principle chromophore and MALDI matrix compound. These new liquid matrices possess greater sample homogeneity and a simpler morphology. The data obtained from our studies show improved sequence coverage for BSA digests compared to the traditional CHCA crystalline matrix and for the ClCCA-containing ILM a similar performance to the ClCCA crystalline matrix down to 1 fmol of BSA digest prepared in a single MALDI sample droplet with current sensitivity levels in the attomole range. The LSMs show a high tolerance to contamination such as ammonium bicarbonate, a commonly used buffering agent

Synthesis and Testing of Inhibitors of Dihydrodipicolinate Synthase

There are two distinct biosynthetic pathways to the essential amino acid L-lysine (A). The diaminopimelate pathway to L-lysine occurs in higher plants and bacteria. The second pathway known is the alpha-aminoadipate pathway and is found to operate in fungi and yeasts. This thesis will deal with only the diaminopimelate pathway to L-lysine and in particular with the first step, which involves the condensation of L-aspartic acid beta-semialdehyde (ASA) (B) with pyruvate (C) to form L-dihydrodipicolinate (DHDP) (D). The mechanism of formation of L-DHDP (D) was studied using electrospray mass spectrometry. The synthesis and testing of potential inhibitors of dihydrodipicolinate synthase (DHDPS) was also studied. [diagram] L-ASA is a substrate of the first enzyme of the diaminopimelate pathway to L-lysine. A former co-worker in the group, Dr D. Tudor had developed a route to 1-ASA as the trifluoroacetate salt. This route was low yielding, approximately 14% for four steps, thus a higher yielding route was developed utilising the para-methoxybenzyl (PMB) ester protecting group. This material was not suitable for use in the biochemical assay as an impurity from the deprotection stage was found to absorb strongly at the wavelength used for the our enzyme assay system. An improved procedure for the synthesis of L-ASA was developed increasing the overall yield of the procedure to approximately 48% for the same four steps. The synthesis of L-ASA as its trifluoroacetate salt allowed a number of analogues of 1-ASA to be prepared with some synthetic modification of the original route. The compounds prepared this way were alkylated derivatives of L-ASA. The alpha-methyl ASA (E) was prepared by the reaction of methyl iodide with the anion generated from treating diprotected allylglycine with lithium diisopropylamide (LDA). It proved to be a poor inhibitor but initial studies suggest that it may be a reasonably good substrate for DHDPS. A number of other derivatives were prepared including beta-methyl ASA (F) which again was a poor inhibitor but was found to be a good substrate for DHDPS (beta-methyl ASA is utilised at approximately 20% of the rate that L-ASA is consumed). A number of other derivatives and analogues of L-ASA were prepared. A number of heterocyclic compounds were prepared as analogues of DHDP and were tested for inhibitory effects with DHDPS. These compounds were prepared by a 1,3-dipolar cycloaddition of a nitrile oxide onto an alkene or alkyne. The isoxazolines produced had a general structure (G). These compounds were found to be poor inhibitors of DHDPS with none showing inhibition below 1 mM. The ring opened isoxazolines (H) were prepared as analogues of pyruvate but again they proved to be poor inhibitors of DHDPS. [diagram] An attempt to synthesise glutamic acid gamma-semialdehyde (I) starting from glutamic acid was undertaken. The compound isolated from the series of reactions was found to have cyclised and was stable as the carbinolamine (J). This route was abandoned due to the cyclisation of the product. [diagram] A number of pyridinedicarboxylic acid derivatives and analogues were prepared to test for inhibitory activity. The pyridine-2,6-dicarboxylic acid N-oxide (K) and the pyridine-2,6-dinitrile (L) showed very good inhibitory activity. These compounds were studied in detail to determine the type of inhibition they showed. The two compounds (K) and (L) were found to be non-competitive inhibitors of DHDPS. A number of other saturated and unsaturated analogues of L-DHDP were prepared and tested for inhibitory action. A study of the mechanism of DHDPS was undertaken using electrospray mass spectrometry to detect enzyme bound intermediates. The electrospray mass spectrometer was able to provide evidence for a number of pyruvate analogues bound to the enzyme as Schiff's bases. No evidence for L-ASA bound to DHDPS could be found. Further to these studies was the need to preserve stocks of DHDPS used for inhibitor testing and biotransformations. A study of the immobilisation of DHDPS on Eupergit resins was undertaken to determine the feasibility of this technique as a method of obtaining reusable DHDPS. The studies found that only up to 18% of the initial sample of DHDPS was bound to the beads. This suggests that this may not be a suitable method for the immobilisation of DHDPS, however DHDPS bound to the beads was found to have long term stability

Research Briefing: Strava-using Parkrunners: A Community Study

No abstract available

Novel antioxidant and anti-inflammatory peptides from the Siamese crocodile (Crocodylus siamensis) hemoglobin hydrolysate

Novel antioxidant and anti-inflammatory peptides were isolated from hydrolysates of Siamese crocodile (Crocodylus siamensis) hemoglobin. C. siamensis hemoglobin hydrolysates (CHHs) were obtained by pepsin digestion at different incubation times (2, 4, 6, and 8 H) at 37 °C and subjected to antioxidant and anti-inflammatory activity assessment. CHH obtained by 2-H hydrolysis (2H-CHH) showed the highest anti-inflammatory activity with respect to decreasing nitric oxide (NO) production, whereas the strongest antioxidant activity was found for 6-H hydrolysis (6H-CHH) against nitric oxide radicals. To evaluate the anti-inflammatory and antioxidant activity of individual peptide components, 2H-CHH and 6H-CHH were purified by semipreparative HPLC. Peptide fraction P57 isolated from 6H-CHH was found to exhibit the highest nitric oxide radical inhibition activity (32.0%). Moreover, purification of 2H-CHH yielded peptide fraction P16, which displayed a high efficacy in decreasing NO production of macrophage RAW 264.7 cells (83.2%) and significantly reduced proinflammatory cytokines and inflammatory mediators interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and prostaglandin-E2 (PGE2) production to about 2.0, 0.3, and 1.9 ng/mL, respectively. Using LTQ orbitrap XL mass spectrometry, active peptide sequences were identified as antioxidant KIYFPHF (KF7), anti-inflammatory SAFNPHEKQ (SQ9), and IIHNEKVQAHGKKVL (IL15). Additionally, CHHs simulated gastric and intestinal in vitro digestion positively contributed to antioxidant and anti-inflammatory activity. Taken collectively, the results of this work demonstrate that CHHs contain several peptides with anti-inflammatory and antioxidant properties, which may prove valuable as treatment or supplement against diseases associated with inflammation and oxidative stress

Encouraging independent thought and learning in first year practical classes

The transition from A-level to degree-level practical classes then to a research project, hence from dependent learner to independent researcher, is a hurdle that all students face when studying for a chemistry degree. This can be daunting so any innovations that aid this transition are of great value. At the University of Reading, the first year practical course has been redesigned to facilitate this transition by embedding independent thought and experimentation across all chemistry disciplines (introductory, organic, inorganic and physical). Examples of experiments that provide opportunities for independent student investigation, along with student perceptions of the experiments of the course, are given. Using this model for practical-class delivery, student engagement, confidence, independence and ultimately preparedness for year 2 were improved

Rapamycin carbonate esters

Certain embodiments include carbonate esters of rapamycin at position 42 that are synthesized by a lipase catalyzed regio-specific process. These compounds or a pharmaceutically acceptable salt thereof are useful in the treatment of organ and tissue transplant rejection, autoimmune disease, proliferative disorder, restenosis, cancer, or microbial infection

Res Medica, Autumn 1959, Volume 2, Number 1

TABLE OF CONTENTSSYLLABUS FOR THE 223RD SESSIONHYPOTENSIVE DRUGS: G. E. Mawer, B.ScAN OCULIST LOOKS ATENDOCRINE EXOPHTHALMOS: C. R. S. Jackson.M.A., M.D., D.O.M .S., F.R.C.S.E.RES MEDICA THE TREATMENT OF CANCER: John McKendrick,M.B., Ch.B.DOCTORS AND ARTISTS: Professor W. I. C.Morris, F.R.C.S.E.SOME ASPECTS OFRHEUMATIC FEVER: David J. ClarkABDOMINAL PAIN: Geoffrey T. Millar,M.B., Ch.B.TWO HUNDRED YEARS AGO: James A. Gray, M.B..Ch.B

Bringing 'place' back in: regional clusters, project governance, and new product outcomes

We examine new product outcomes in the context of regional clusters. Based on past research on marketing relationships, clusters, and social networks, we propose that the overall configuration of a cluster helps promote particular governance practices among its members. These practices have distinct value-creating properties, and when they are brought to bear on a specific new product development project within a cluster, they promote performance outcomes like product novelty and speed to market. Ultimately, these performance effects are reinforced by the configuration of the cluster itself. In general, we propose that new product outcomes follow from complex interactions between a cluster's macro-level configuration and its micro-level governance processes. More broadly, our framework points to the importance of geographical variables and to the role of “place” in marketing decision-making