Assessing the correlation of microscopy‐based and volumetry‐based measurements for resin swelling in a range of potential greener solvents for SPPS

The degree of resin swelling in a particular solvent system is one of the critical parameters for solid‐phase peptide synthesis (SPPS) and for solid‐phase synthesis in general. Methods used for measuring the degree of resin swelling include microscopy‐based and volumetry‐based methods. This study describes and compares the use of both methods for a number of commercially available resins commonly used in SPPS, with a range of solvents, which have been identified in the literature as ‘greener’ than DCM, DMF and NMP. The results were analysed by statistical methods, and a significant correlation between the two distinct methods has been demonstrated for the first time. The results will likely be used, in conjunction with other literature methods, to help in choosing both the resin and solvent system for greener SPPS, as well as for continuous flow SPPS, which is of growing importance.     

Complexation of labile aluminium species by chelating resins Iontosorb--a new method for Al environmental risk assessment

The utilization of chelating ion-exchange by the method based on binding strength and kinetic discrimination for aluminium fractionation was studied. Two chelating cellulose resins, Iontosorb Oxin (IO) and Iontosorb Salicyl (IS), were used for the determination of quickly reacting labile aluminium species. The possibilities of aluminium fractionation on these chelating resins were investigated by a solid phase extraction technique. The study of the pH (2.5-6.0) influence on the Al complexation by both resins indicates that at low pH the IS has lower sorption capacity but better adsorptive kinetic properties than IO. The optimal resin complexation time for reactive Al species was experimentally found after aluminium sorption study at pH 4.0 in synthetic solutions containing some inorganic and organic ligands, which simulate the composition of analysed acid soil and water samples. The negative influence of sulphate and iron on the Al complexation by IS resin was found and investigated. The flame atomic absorption spectrometry was used for the aluminium quantification.     

Determination of uric acid in urine, saliva and calcium oxalate renal calculi by high-performance liquid chromatography/mass spectrometry

A very simple and direct method for determination of uric acid, in various biological matrices, based on high-performance liquid chromatography and mass spectrometry is described. Chromatographic separations were performed with a stationary phase Zorbax Sax Column, an anion exchange resin, with 50% sodium citrate 1 mM at pH 6.5 and 50% acetonitrile as mobile phase delivered at a flow rate of 1 ml/min. The detector counted negative ions by monitoring m/z 167.1, which corresponds to the urate anion. The method does not use an internal standard but quality control samples were used. Intra-day precision ranged between 1.1 and 1.5%, whereas inter-day precision was between 1.3 and 2.8% (n=5) working with some selected standards. Recovery tests of added standard have been successfully performed in urine and saliva samples, thus showing an appropriate accuracy of the method. The limit of quantitation found was 70 microg/l. Different urine and saliva samples were analyzed using an alternative analytical methodology based on an enzymatic reaction and photometric detection at 520 nm, resulting both methods comparable at a 95% confidence level. The method has been also applied to the determination of trace amounts of uric acid in the core of some selected calcium oxalate renal calculi.     

Neurotrophic peptide aldehydes: solid phase synthesis of fellutamide B and a simplified analog

A combination of solid phase and solution phase synthetic methods have been used to complete the total synthesis of the neurotrophic lipopeptide aldehyde fellutamide B (2). The beta-hydroxy aliphatic tail was prepared by regioselective reductive opening of a cyclic sulfate, and later coupled to a solid phase resin. The synthetic compound was then examined in cytotoxicity and nerve growth factor (NGF) induction assays. A simplified analog of fellutamide B also showed activity.     

Solid phase synthesis of an amphiphilic peptide modified for immobilisation at the C-terminus

The solid phase peptide synthesis (SPPS) of the amphiphilic peptide Ac-(Leu-Ala-Arg-Leu)(3)-linker, which is modified at the C-terminus with 1,8-diamino-3,6-dioxaoctane as linker moiety, has been investigated. Two different approaches that allow for the synthesis of C-terminally modified, side-chain protected peptides were examined. The solid phase peptide synthesis using aliphatic safety-catch resin followed by activation and aminolysis with the mono-Boc protected linker was compared with the synthesis on 1,8-diamino-3,6-dioxaoctane loaded 2-chlorotrityl resin.     

Fast Diffusion of Multivalent Ions Facilitated by Concerted Interactions in Dual‐Ion Battery Systems

Sluggish solid‐phase diffusion has been an essential issue in developing intercalation electrode materials using multivalent ions. Compared to monovalent Li ions, the diffusion of multivalent ions is still not well understood. Here, combining first‐principles calculations with electrochemical experiments, it is shown that the diffusion of divalent Mg ions is significantly facilitated in Li–Mg dual‐ion systems, and the activation energy is remarkably reduced by the concerted interactions of the preceding Li ions and following Mg ions. Thus, making dual‐ion systems is a promising way to construct high‐energy‐density, rechargeable batteries with multivalent ions. This work will provide a new perspective on solid‐phase diffusion that is typically a rate‐controlling process in battery systems and fuel cell devices.     

Synthesis of a shark repellent peptide toxin, Pardaxin (16-33) on a highly flexible polymer support: CLPSER

A high swelling resin, CLPSER has been developed and utilized for the solid phase synthesis of Pardaxin, which is an 18-residue peptide. The resin was characterized by gel phase (13)C NMR, IR and SEM. The utility of the new polymer support in polypeptide synthesis was further established by the comparative synthesis of pardaxin with commercially available Merrifield resin. The MALDI TOF MS, amino acid analysis and the HPLC revealed the superior quality of CLPSER.     

Pseudomonas aeruginosa immobilized multiwalled carbon nanotubes as biosorbent for heavy metal ions

Pseudomonas aeruginosa immobilized multiwalled carbon nanotubes has been used as biosorbent for the solid phase extraction of some heavy metal ions in environmental samples. Cobalt(II), cadmium(II), lead(II), manganese(II), chromium(III) and nickel(II) ions have been selected as analytes for the presented study, due to their important negative and positive roles in human life. In order to investigate quantitative biosorption conditions of the analytes, the influences of pH of the aqueous solution, eluent type, eluent volume, samples volume, etc. were examined. The effects of alkaline, earth alkaline and some transitions metals on the biosorption of analyte ions on P. aeruginosa immobilized multiwalled carbon nanotubes were also investigated. The presented biosorption procedure was applied to the determination of analytes in tomato leaves, bovine liver, boiled wheat, canned fish, black tea, lichen and natural water samples.     

Synthesis of a thymine-functionalized nucleoamino acid for the solid phase assembly of cationic nucleopeptides

In this work, we report the synthesis of a thymine-functionalized nucleoamino acid suitable for the solid phase synthesis of nucleopeptides. The monomer was obtained in solution starting from commercial compounds and after NMR (¹H and ¹³C) and ESIMS (positive ions) characterization it was used for the assembly of a cationic nucleopeptide obtained by sequentially introducing underivatized l-lysine units and nucleoamino acid monomers. After detachment from the resin, performed in acidic conditions, the oligomer was purified by HPLC and characterized by LC-ESIMS (positive ions) which confirmed the identity of the thymine-based nucleopeptide. The cationic nucleobase-containing peptide, well soluble in water, was studied by CD spectroscopy which allowed us to exclude any helical pre-organization of the nucleopeptide in the experimental conditions used. Furthermore, CD behavior of the oligomer at different temperatures was also studied as described in this work.     

Determination of the immunosuppressant mycophenolic acid in human serum by solid-phase microextraction coupled to liquid chromatography

A solid phase microextraction (SPME)-HPLC-UV method for the determination of the immunosuppressant mycophenolic acid (MPA) in human serum samples was developed for the first time. The procedure, that employed a carbowax/templated resin (Carbowax/TPR-100) as fiber coating, required a very simple sample pretreatment, an isocratic elution, and provides an highly selective extraction. The linear range was 0.2-100 microg x ml(-1). Recovery was practically unchanged (63+/- 4%) passing from 0.2 to 100 microg x ml(-1) level. Within-day and between-days coefficient of variation ranged from 5.9 to 6.5% and from 8.8 to 9.2%, respectively. A detection limit of 0.05 microg x ml(-1) was estimated in spiked serum. The method was successfully applied to the determination of MPA in serum of a patient under mycophenolate mophetil ester (MMF) therapy, as demonstrated by the relevant concentration-time profiles.     

Comparative evaluation of four trityl-type amidomethyl polystyrene resins in Fmoc solid phase peptide synthesis.

Four trityl-type (i.e. non-substituted trityl-, o-Cl-trityl-, o-F-trityl- and p-CN-trityl-) amidomethyl polystyrene resins were evaluated comparatively, in terms of the stability of the trityl-ester bond in slightly acidic dichloromethane solutions, and the p-CN-trityl-amidomethyl polystyrene resin was found to be the most stable of them. The above resins were applied, in parallel with Wang benzyl-type resin, well known for its stability in mild acidic conditions, to the Fmoc solid phase synthesis of the 43-amino acid residue long bioactive peptide thymosin beta-4. Independent of their differences in acid sensitivity, the resins seemed to function equally well under the conditions used, since pure thymosin beta-4 was obtained with a final yield of approximately 30% from each resin. The trityl-type amidomethyl polystyrene resins were also applied, in parallel with the Wang resin, to the Fmoc solid phase synthesis of a bioactive peptide containing proline at its C-terminus, i.e. the N-terminal tetrapeptide of thymosin beta-4, AcSDKP. In this case, the best yield (87%) was obtained with the o-Cl-trityl-amidomethyl polystyrene resin, which may be the resin of choice, of those studied, for the Fmoc solid phase peptide synthesis.     

Determination of a new bisphosphonate, YM175, in plasma, urine and bone by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method for the determination of disodium dihydrogen(cycloheptylamino)methylenebisphosphonate monohydrate (YM175) in plasma, urine and bone is described. Plasma obtained in high-dose animal studies is pretreated by Method A, a simple method using 1 ml of plasma, which is based on deproteinization of plasma followed by coprecipitation of the drug with calcium phosphate and removal of excess calcium ions by AG 50W-X8 resin. Plasma obtained in lower-dose clinical studies is treated by Method B, a more sensitive method using 10 ml of plasma, which is based on solid-phase extraction using a Sep-Pak C₁₈ cartridge coupled with Method A. Urine and bone are treated similarly to Method B. The chromatographic system consists of a mobile phase at pH 11, an alkali-stable column and an electrochemical detector operating in the oxidation mode. The determination limit is 5 ng/ml for Method A and 0.5 ng/ml for Method B in plasma, 1 ng/ml in urine, and 25 ng/g in bone.     

Double-receptor sandwich supramolecule sensing method for the determination of ATP based on uranyl-salophen complex and aptamer

In this study, we report a double-receptor sandwich supramolecule sensing method for the determination of adenosine triphosphate (ATP). One receptor is a uranyl-salophen complex which can bind the triphosphate group in ATP selectively, and another is an anti-adenosine aptamer which is a single-stranded oligonucleotide and can recognize the adenosine group in ATP specifically. The uranyl-salophen complex was immobilized on the surface of amino-silica gel particles and used as the solid phase receptor of ATP. The anti-adenosine aptamer was labeled with a fluorescent group and used as the labeled receptor of ATP. In the procedure of ATP detection, ATP was first combined with the solid phase receptor and then conjugated with the labeled receptor to form a sandwich-type supramolecule. The conditions of fabricating solid phase receptor and the influence of manifold variables on the determination were studied. The experimental results demonstrate that the method has a number of advantages such as high selectivity, high sensitivity, good stability and low cost. Under optimal conditions, the linear range for detection of ATP is 0.2-5.0 nmol/mL with a detection limit of 0.037 nmol/mL. The proposed method was successfully applied for the determination of ATP in real samples with the recoveries of 96.8-103.3%.     

Hydrolytic activity of lipase on anion-exchange solid phase column after separation and electrotransfer by non-denaturing electrophoresis

This study reports the initial separation of phospholipase C-alpha from porcine retina using non-denaturing two-dimensional gel electrophoresis (2-DE). Detection was by negative staining and then its hydrolytic activity was estimated using alpha-naphthyl acetate in a 2-DE gel. A spot of phospholipase C-alpha separated by 2-DE was excised. It was then electrophoretically transferred to an anion-exchange solid phase column after 40 mg, equal to dry weight of the solid resin from the cartridge (Accell Plus QMA, Waters Corporation), was packed into a disposable 1 ml syringe to make an anion-exchange solid phase column. Phosphatidylcholine was hydrolyzed in the anion-exchange solid phase column containing phospholipase C-alpha. The results indicated that a column with hydrolytic activity could be produced once lipases separated by non-denaturing 2-DE were transferred to the solid phase column.     

Analysis of carbohydrates and amino acids in vegetable waste waters by ion chromatography

High-performance anion exchange chromatography coupled with pulsed amperometric detection was used for the quantitative determination of total and free sugars in olive oil mill waste waters (OMWW). Automated amino acid ion chromatography was employed to analyse total and free amino acids in the same OMWW. Sugars were analysed in samples pre-purified by means of a three-step purification procedure involving: (i) methanol precipitation of OMWW; (ii) dialysis of the obtained solid and liquid fractions; and (iii) chromatographic purification on RP18 phase followed by Amberlite resin. The amino acids were determined directly in samples obtained from the first two steps performed for sugar analysis. The analysis carried out with the reported methodologies allowed the quantitative determination of total sugars and amino acids and the differentiation between their free and bound forms. The sugars determined were arabinose, fructose, galactose, glucose, rhamnose, xylose, galacturonic and glucuronic acids, and the amino acids were Asp, Glu, Thr, Ser, Pro, Gly, Ala, Val, Met, Ile, Leu, Tyr, Phe, Lys, His, Arg and Cys. Asn, Gin, and Trp were not detected. The technological, biotechnological and environmental advantages arising from this analytical methodology applied to OMWW are briefly discussed.     

Determination of sodium nifurstyrenate and nitrovin residues in edible food by liquid chromatography-tandem mass spectrometry after ultrasound-assisted extraction

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of nitrovin and sodium nifurstyrenate residues in muscle and liver of swine and chicken and in muscle of fish. Sample preparation procedure includes ultrasound-assisted extraction with acetonitrile, defatting with n-hexane and final clean-up with solid phase extraction (SPE) on Oasis HLB cartridges. The analytes were detected in multiple reaction monitoring (MRM) under negative scan mode acquiring two diagnostic product ions for sodium nifurstyrenate and under positive mode for nitrovin. The averaged decision limits (CCα; α 1%) ranged 0.09-0.26 μg/kg while the detection capability (CCβ; β 5%) was 0.33-0.97 μg/kg in the tissues. Reasonable recoveries (71-110%) spiked in muscle and liver showed excellent relative standard deviation (RSD). The validated method was simple, rapid, sensitive, and complied with the regulations for the determination of nitrovin and sodium nifurstyrenate residues in food matrices.     

Synthesis and mass spectrometric characterization of a metal-affinity decapeptide: copper-induced conformational changes

A decapeptide with high affinity toward heavy metal ions (RCHQYHHNRE) has been prepared by Fmoc strategy using TGR resin as solid support. The model peptide provides a simple system that can be used for a systematic study of the impact of different metal ions on peptide secondary structure on a molecular level; histidine residues were incorporated into the peptide in a sequence similar to beta-amyloid peptide (Abeta1-40) to generate possible complexation sites for Cu (2+) ions. The peptide secondary structure, as investigated by circular dichroism, and self-assembled nanostructures were observed to depend strongly on the presence of copper and sodium dodecyl sulfate (SDS). Atomic force microscopy (AFM) revealed also that copper and SDS affected slightly the Abeta1-40 nanostructures. An explanation for the effect of metal ions and SDS on the self-assembly of peptides was proposed. The extensive beta-sheet formation may further promote peptide self-assembly into longer fibers.     

Synthesis, conformation, and biological activity of the carbohydrate-free vespulakinin 1

Synthesis of the carbohydrate-free heptadecapeptide corresponding to the amino acid sequence of vespulakinin 1 was achieved by the continuous flow solid phase procedure on 4-hydroxymethyl-phenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin, as well as by a combination of solid phase and solution syntheses. Preformed Fmoc-amino acid symmetrical anhydrides (Boc derivative for the N-terminal residue) were used for amine acylation in the continuous flow method. Serine and threonine were side chain protected as tert.-butyl ethers and the 4-methoxy-2, 3, 6,-trimethyl-benzenesulfonyl group was used for masking the guanidino function of arginine residues. After cleavage from the resin the final peptide was purified by ion exchange chromatography and characterized by amino acid analysis, high voltage electrophoresis, and RP-HPLC analysis. Alternatively, the protected N-terminal octapeptide, Fmoc-Thr(But)-Ala-Thr(But)-Thr(But)-Arg(Mtr)-Arg-(Mtr)-Arg(Mtr)-Gly-OH was prepared on 4-hydroxymethyl-3-methoxyphenoxyacetyl-norleucyl derivatized Kieselguhr-supported polydimethylacrylamide resin and the C-terminal nonapeptide H-Arg(NO2)-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-(NO2)-OBzl was synthesized in solution through the fragment condensation method. The two fragments were coupled by the DCC-HOBt procedure and the resulting heptadecapeptide was deblocked and purified. The conformational features of the synthesized peptides are reported. Preliminary pharmacological experiments indicated that carbohydrate-free vespulakinin 1 is more potent than bradykinin in lowering rat blood pressure.     

Hydrolytic activity of lipase on anion-exchange solid phase column after separation and electrotransfer by non-denaturing electrophoresis

This study reports the initial separation of phospholipase C-alpha from porcine retina using non-denaturing two-dimensional gel electrophoresis (2-DE). Detection was by negative staining and then its hydrolytic activity was estimated using α-naphthyl acetate in a 2-DE gel. A spot of phospholipase C-alpha separated by 2-DE was excised. It was then electrophoretically transferred to an anion-exchange solid phase column after 40 mg, equal to dry weight of the solid resin from the cartridge (Accell™ Plus QMA, Waters Corporation), was packed into a disposable 1 ml syringe to make an anion-exchange solid phase column. Phosphatidylcholine was hydrolyzed in the anion-exchange solid phase column containing phospholipase C-alpha. The results indicated that a column with hydrolytic activity could be produced once lipases separated by non-denaturing 2-DE were transferred to the solid phase column.     

1,2-Dimethylimidazole-4-sulfonyl chloride, a novel derivatization reagent for the analysis of phenolic compounds by liquid chromatography electrospray tandem mass spectrometry: application to 1-hydroxypyrene in human urine

A novel derivatization method employing 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) to improve the mass spectrometric response for phenolic compounds in liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS) and tandem mass spectrometry (LC-ESI-MS/MS) is described. Several environmentally relevant compounds, including chloro-, aryl- and alkylphenols, steroidal estrogens, and hydroxy-polycyclic aromatic hydrocarbons (OHPAHs), were selected to evaluate this technique. A facile derivatization procedure employing DMISC results in dimethylimidazolesulfonyl (DMIS) derivatives that are stable in aqueous solution. These DMIS derivatives produced intense [M+H](+) ions in positive-ion LC-ESI-MS. The product ion spectra of the [M+H](+) ions of simple phenols were dominated by ions representing the DMIS and dimethylimidazole moieties, whereas product ion spectra of the DMIS derivatives of OHPAHs with three or more fused aromatic rings showed prominent ArO(+) ions, the relative intensity of which increased with the number of rings. The DMIS derivatives of the selected phenolic compounds showed excellent chromatographic properties. To substantiate the utility of derivatization with DMISC, an analytical method employing enzyme hydrolysis, solid phase extraction, derivatization with DMISC, and analysis by LC-ESI-MS/MS with multiple reaction monitoring for determination in human urine of 1-hydroxypyrene, a widely used biomarker for the assessment of human exposure to PAHs, was developed and validated.