Synthesis of alpha-aspartyl-containing cyclic peptides

alpha-Aspartyl-containing cyclic pentapeptides were synthesised in high yields using a strategy that maintained fluorenylmethyl protection on the aspartic acid side chain during chain assembly, resin cleavage and cyclisation of the linear precursors. Tetra-n-butylammonium fluoride treatment of the fluorenylmethyl-protected cyclic peptides catalysed imide formation, whereas piperidine-induced deprotection resulted in good yields of the target cyclic peptides.

Validation of a manual headspace gas chromatography method for determining volatile compounds in biological fluids

Background: We report the validation of a method for the determination of acetaldehyde, acetone, methanol, and ethanol in biological fluids using manual headspace sample introduction and an acetonitrile internal standard. Method: This method uses a capillary column (I = 30 m, I.D. = 0.25 mm, dF = 0.25 mu m) installed in a gas chromatography-flame ionization detector (GC-FID) apparatus with a run time of 7.5 minutes. Results: Analysis of the retention times and the resolution of the analyte peaks demonstrated excellent separation without widening of the peaks. Precision and accuracy were good (interassay precision < 15% and recovery between 85% and 115%) in both blood and urine. Conclusion: The method was linear (r > 0.09) over the analytical measurement range (AMR) of each analyte.

Correlation between serum and fecal concentrations of reproductive steroids throughout gestation in goats

Non-invasive techniques such as the measurement of fecal steroids are now widely used to monitor reproductive hormones in captive and free-ranging wild-life. These methods offer great advantages and deserve to be used in domestic animals. The aim of the present study was to determine the endocrine profile of dairy goats throughout pregnancy by the quantification of fecal progestins and estrogens and assess its con-elation with serum concentrations. Blood and fecal samples were collected weekly from I I adult, multiparous goats, from mating through pregnancy and 2 weeks post-partum. The extraction of estradiol and progesterone fecal metabolites was performed by dilution in ethanol. The radioimmunoassay (RIA) in solid phase was used to quantify serum 17 beta-estradiol (estradiol) and progesterone, as well as their fecal metabolites. The mean concentrations of both fecal and serum estradiol started to increase between weeks 7 and 11, reached peak values near parturition and then decreased sharply (range: 19.8 +/- 5.8 ng/g of feces to 608.6 +/- 472.4 ng/g of feces and 0.007 +/- 0.005 ng/ml to 0.066 +/- 0.024 ng/ml). An increase in both fecal and blood progestagens occurred in the second week, mean concentrations remained greater until week 20, and then decreased in the last week of gestation and 2 weeks post-partum (range: 108.8 +/- 43.6 ng/g of feces to 3119.5 +/- 2076.9 ng/g of feces and 0. 12 +/- 0.04 ng/ml to 13.10 +/- 4.29 ng/ml). The changes in blood and fecal hormone concentrations were analyzed and compared throughout gestation for each single goat, for each breed and for the whole group. Results indicated that matched values of serum and fecal hormone concentrations were correlated (r = 0.79; p < 0.001 for progesterone and r = 0.84;p < 0.001 for estradiol mean concentrations in the whole group). Regression analysis showed that logarithmic model allows significant prediction of serum from fecal concentrations with an R-2 = 0.729 (y = 0.013 1n x - 0.021) for estradiol and R-2 = 0.788 (y = 3.835 1n x - 18.543) for progesterone. Neither fecal nor serum concentrations were affected by the breed but a significant effect of the number of fetuses on progestin concentrations was found. Therefore, the profiles of progesterone and estradiol fecal metabolites reflect the serum concentrations of the same hormones in pregnant goats. (C) 2006 Elsevier B.V. All rights reserved.

Total chemical synthesis and chemotactic activity of human S100A12 (EN-RAGE)

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)binding protein), a new member of the S100 family of EF-hand calcium-binding proteins, was chemically synthesised using highly optimised 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate/tert-butoxycarbonyl in situ neutralisation solid-phase chemistry. Circular dichroism studies indicated that CaCl2 decreased the helical content by 27% whereas helicity was marginally increased by ZnCl2. The propensity of S100A12 to dimerise was examined by electrospray ionisation time-of-flight mass spectrometry which clearly demonstrated the prevalence of the non-covalent homodimer (20 890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

Extraction and purification of the zwitterions cylindrospermopsin and deoxycylindrospermopsin from Cylindrospermopsis raciborskii

The hepatotoxin cylindrospermopsin (CYN) has been isolated from the cyanobacterium Cylindrospermopsis raciborskii (C. raci.). Efforts to study this toxin have been hampered by the time-consuming requirement to extract it from cultures of the organism. It is usually extracted from lyophilized cells collected from a laboratory culture. Our preliminary work suggested far more of the toxin is available in solution in the culture media than in the cells collected. We have therefore investigated the use of commercially available solid phase extraction sorbents to extract CYN from culture media in which C. raci. has been grown. A range of reverse phase and ion-exchange sorbents were tested across a range of pHs for their ability to retain CYN without success. Subsequently, graphitized carbon cartridges were found to retain CYN strongly. Elution with 5% formic acid in methanol allowed the CYN to be regained for final purification by HPLC. Deoxy-CYN, an analog of CYN can also be extracted using this procedure. (C) 2001 John Wiley & Sons, Inc.

Carbohydrate-based templates for synthetic vaccines and drug delivery

Methyl tetra-O-allyl, and tetra-O-[2-(tetrahydro-2H-pyranyl)oxy.-3-oxapentyl glucosides, and tetra-O-(cyanoethyl)galactosyl azide were converted into derivatives containing linkers with terminal carboxylic acid functionalities at the anomeric position and bearing four arms with phthaloyl- or BOC-protected terminal amino groups. These molecules were suitable for use in solid-phase peptide synthesis and for the preparation of dendrimers, containing multiple copies of peptides. (C) 2001 Elsevier Science Ltd. All rights reserved.

Three-dimensional structure of RTD-1, a cyclic antimicrobial defensin from rhesus macaque leukocytes

Most mammalian defensins are cationic peptides of 29-42 amino acids long, stabilized by three disulfide bonds. However, recently Tang et al. (1999, Science 286, 498-502) reported the isolation of a new defensin type found in the leukocytes of rhesus macaques. In contrast to all the other defensins found so far, rhesus theta defensin-1 (RTD-1) is composed of just 18 amino acids with the backbone cyclized through peptide bonds. Antibacterial activities of both the native cyclic peptide and a linear form were examined, showing that the cyclic form was 3-fold more active than the open chain analogue [Tang et al. (1999) Science 286, 498-502]. To elucidate the three-dimensional structure of RTD-1 and its open chain analogue, both peptides were synthesized using solid-phase peptide synthesis and tert-butyloxycarbonyl chemistry. The structures of both peptides in aqueous solution were determined from two-dimensional H-1 NMR data recorded at 500 and 750 MHz. Structural constraints consisting of interproton distances and dihedral angles were used as input for simulated-annealing calculations and water refinement with the program CNS. RTD-1 and its open chain analogue oRTD-1 adopt very similar structures in water. Both comprise an extended beta -hairpin structure with turns at one or both ends. The turns are well defined within themselves and seem to be flexible with respect to the extended regions of the molecules. Although the two strands of the beta -sheet are connected by three disulfide bonds, this region displays a degree of flexibility. The structural similarity of RTD-1 and its open chain analogue oRTD-1, as well as their comparable degree of flexibility, support the theory that the additional charges at the termini of the open chain analogue rather than overall differences in structure or flexibility are the cause for oRTD-1's lower antimicrobial activity. In contrast to numerous other antimicrobial peptides, RTD-1 does not display any amphiphilic character, even though surface models of RTD-1 exhibit a certain clustering of positive charges. Some amide protons of RTD-1 that should be solvent-exposed in monomeric beta -sheet structures show low-temperature coefficients, suggesting the possible presence of weak intermolecular hydrogen bonds.

Synthesis of an analog of the thyroid hormone-binding protein transthyretin via regioselective chemical ligation

Transthyretin is an essential protein responsible for the transport of thyroid hormones and retinol in human serum and is also implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases, Here we report the solid phase synthesis of the monomeric unit of a transthyretin analog (equivalent to 127 amino acids) using t-Boc chemistry and peptide ligation and its folding to form a functional 54-kDa tetramer, The monomeric unit of the protein was chemically synthesized in three parts (positions 1-51, 54-99, and 102-127) and ligated using a chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of transthyretin's native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, transthyretin antibody recognition, and thyroid hormone binding. Other folding products included a high molecular weight aggregate as well as a transient dimeric species. This represents one of the largest macromolecules chemically synthesized to date and demonstrates the potential of protein chemical synthesis for investigations of protein-ligand interactions.

Quantitative affinity chromatography

The objective of this review is to summarize developments in the use of quantitative affinity chromatography to determine equilibrium constants for solute interactions of biological interest. Affinity chromatography is an extremely versatile method for characterizing interactions between dissimilar reactants because the biospecificity incorporated into the design of the affinity matrix ensures applicability of the method regardless of the relative sizes of the two reacting solutes. Adoption of different experimental strategies, such as column chromatography, simple partition equilibrium experiments, solid-phase immunoassay, and biosensor technology, has led to a situation whereby affinity chromatography affords a means of characterizing interactions governed by an extremely broad range of binding affinities-relatively weak interactions (binding constants below 10(3) M-1) through to interactions with binding constants in excess of 10(9) M-1. In addition to its important role in solute separation and purification, affinity chromatography thus also possesses considerable potential for investigating the functional roles of the reactants thereby purified. (C) 2001 Elsevier Science B.V. All rights reserved.

The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

An efficient Fmoc strategy for the rapid synthesis of peptide para-nitroanilides

A new strategy has been developed for the rapid synthesis of peptide para-nitroanilides (pNA). The method involves derivatization of commercially available tritylchloride resin (TCP-resin) with 1,4-phenylenediamine, subsequent coupling with desired amino acids by the standard Fmoc protocol, and oxidation of the intermediate para-aminoanilides (pAA) with Oxone(R). This procedure allows easy assembly of the desired para-aminoanilides (pAA) on standard resin and efficient oxidation and purification of the corresponding para-nitroanilides (pNA). The method allows easy access to any desired peptide para-nitroanilides, which are useful substrates for the characterization and study of proteolytic enzymes.

Computation of the linear elastic properties of random porous materials with a wide variety of microstructure

A finite-element method is used to study the elastic properties of random three-dimensional porous materials with highly interconnected pores. We show that Young's modulus, E, is practically independent of Poisson's ratio of the solid phase, nu(s), over the entire solid fraction range, and Poisson's ratio, nu, becomes independent of nu(s) as the percolation threshold is approached. We represent this behaviour of nu in a flow diagram. This interesting but approximate behaviour is very similar to the exactly known behaviour in two-dimensional porous materials. In addition, the behaviour of nu versus nu(s) appears to imply that information in the dilute porosity limit can affect behaviour in the percolation threshold limit. We summarize the finite-element results in terms of simple structure-property relations, instead of tables of data, to make it easier to apply the computational results. Without using accurate numerical computations, one is limited to various effective medium theories and rigorous approximations like bounds and expansions. The accuracy of these equations is unknown for general porous media. To verify a particular theory it is important to check that it predicts both isotropic elastic moduli, i.e. prediction of Young's modulus alone is necessary but not sufficient. The subtleties of Poisson's ratio behaviour actually provide a very effective method for showing differences between the theories and demonstrating their ranges of validity. We find that for moderate- to high-porosity materials, none of the analytical theories is accurate and, at present, numerical techniques must be relied upon.

Quantification and stability of everolimus (SDZ RAD) in human blood by high-performance liquid chromatography-electrospray tandem mass spectrometry

We report here a validated method for the quantification of a new immunosuppressant drug, everolimus (SDZ RAD), using HPLC-tandem mass spectrometry. Whole blood samples (500 mul) were prepared by protein precipitation, followed by C-18 solid-phase extraction. Mass spectrometric detection was by selected reaction monitoring with an electrospray interface operating in positive ionization mode. The assay was linear from 0.5 to 100 mug/l (r(2) > 0.996, n = 9). The analytical recovery and inter-day imprecision, determined using whole blood quality control samples (n = 5) at 0.5, 1.2, 20.0, and 75.0 mug/l, was 100.3-105.4% and less than or equal to7.6%, respectively. The assay had a mean relative recovery of 94.8 +/- 3.8%. Extracted samples were stable for up to 24 h. Fortified everolimus blood samples were stable at -80 degreesC for at least 8 months and everolimus was found to be stable in blood when taken through at least three freeze-thaw cycles. The reported method provides accurate, precise and specific measurement of everolimus in blood over a wide analytical range and is currently supporting phase 11 and III clinical trials. (C) 2002 Elsevier Science B.V. All rights reserved.

A lipophilic adjuvant carrier system for antigenic peptides

A lipoamino acid based synthetic peptide, (Lipid Core Peptide, LCP) derived from the conserved region of group A streptococci (GAS) was evaluated as potential candidate in a vaccine to prevent GAS-associated diseases, including rheumatic heart disease and post-streptococcal acute glomerulonephritis. Multiple copies of a peptide sequence from the bacterial surface M protein were incorporated into a lipid core and it was used to immunize mice with and without the application of adjuvant. The LCP construct had significantly enhanced immunogenicity compared with the monomeric peptide epitope. Furthermore, the peptides incorporated into the LCP system generated antibodies without the use of any conventional adjuvant.

Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.