"Inherently chiral" thiophene-based electrodes at work: a screening of enantioselection ability toward a series of pharmaceutically relevant phenolic or catecholic amino acids, amino esters, and amine.

"Inherently chiral" thiophene-based electroactive oligomer films have recently been shown to exhibit outstanding chirality manifestations. One of the most exciting among them is an unprecedented enantioselection ability as electrode surfaces. In fact, in preliminary chiral voltammetry experiments, the new electrodes have been shown to both discriminate the enantiomers of chiral probes (either enantiopure or in a mixture, in terms of large differences in peak potentials) and quantify them (in terms of linear dynamic ranges in peak currents), without the need for preliminary separation steps. Such ability has now been tested on a series of chiral DOPA-related molecules, from phenolic amino acid tyrosine (together with its methyl ester) to catecholic amino acid DOPA (together with its methyl ester), to catecholamine epinephrine (adrenaline). The wide-range enantioselectivity of the new inherently chiral electrode surfaces is fully confirmed, as large peak potential differences are obtained for probe enantiomers of the whole series working in common aqueous buffers. Moreover, interesting modulating effects on enantiodiscrimination can be observed as a function of both molecular structure and pH. Graphical abstract Inherently chiral thiophene-based electrodes at work with pharmaceutically relevant probes.

Assembly of surface-confined homochiral helicates: chiral discrimination of DOPA and unidirectional charge transfer.

Surface-confined double-helical polymers are generated by dynamic covalent assembly with preservation of chirality, metal coordination environment, and oxidation state of the precursor complexes. This one-step procedure involves both in solution and solution-to-surface assembly and resulted in chiral interfaces where pairs of ligands are wrapped around arrays of metal ions. In-plane XRD experiments revealed the formation of a highly ordered structure along the substrate surface. The chirality of the surfaces is expressed by the selective recognition of 3,4-dihydroxyphenylalanine (DOPA). The CD measurements show a response of the Δ-polymer-modified quartz substrates toward D-DOPA, whereas no change was observed after treatment with L-DOPA. These coordination-based interfaces assembled on metal-oxide substrates in combination with a redox-probe, [Os(bpy)3](PF6)2, in solution can resemble the behavior of a rectifier.

Chiral electrokinetic chromatography-electrospray ionization-mass spectrometry using a double junction interface.

A double junction interface was utilized to preserve separation efficiency and alleviate ion suppression from sulfated ß-cyclodextrin (S-ß-CD) in electrokinetic chromatography-electrospray ionization-mass spectrometry. The utility of the approach was demonstrated by chiral EKC-MS analysis of dihydroxyphenylalanine and methyldihydroxyphenylalanine enantiomers using either low concentration (counter-migration mode; 0.1% S-ß-CD) or high concentration (carrier mode; 2% S-ß-CD). In the counter-migration mode, S-ß-CD anions were supplied continuously from the junction reservoir to the separation column so that the effective separation length was preserved. This interface is especially useful under carrier mode in which high concentration of S-ß-CD will migrate toward the ESI source. With the use of the double junction interface, the S-ß-CD exited the separation column will remain in the junction reservoir, whereas the analyte will flow toward the ESI source through a connecting column. As a result, no ion suppression was observed and the sensitivity was improved significantly.

Tyrosinase inhibition by water and ethanol extracts of a far eastern sea cucumber, Stichopus japonicus.

BACKGROUND: Tyrosinase plays a key role in hyperpigmentaion and enzymatic browning. The present study was aimed at investigating the inhibitory effects of water and 70% aqueous ethanol extracts of Stichopus japonicus, a sea cucumber long consumed as a tonic food and traditional medicine, on the diphenolase activity of tyrosinase. RESULTS: In the tyrosinase inhibition study, high-performance liquid chromatography completely separated L-3,4-dihydroxyphenylalanine and dopachrome from other compounds present in the extracts, and provided more reliable results than the commonly used spectrophotometry. The ethanol extract (IC(50)=0.49-0.61 mg mL(-1)) showed higher inhibitory activity than the water extract (IC(50)=1.80-1.99 mg mL(-1)). Enzyme inhibition by the extracts was reversible and of mixed type. For both extracts, the dissociation constants for binding to free enzyme were significantly smaller than those for binding to enzyme-substrate complex. Ethyl-α-D-glucopyranoside (IC(50)=0.19 mg mL(-1)), isolated for the first time from sea cucumber, and adenosine (IC(50)=0.13 mg mL(-1)), were identified as key tyrosinase inhibitors. CONCLUSION: The sea cucumber extracts were demonstrated to possess considerable inhibitory potency against the diphenolase activity of tyrosinase, suggesting that the sea cucumber may be a good source of safe and effective tyrosinase inhibitors.

New analytical portable instrument for microchip electrophoresis with electrochemical detection.

A new portable instrument that includes a high voltage power supply, a bipotentiostat, and a chip holder has been especially developed for using microchips electrophoresis with electrochemical detection. The main unit of the instrument has dimensions of 150 x 165 x 70 mm (wxdxh) and consists of a four-outputs high voltage power supply with a maximum voltage of +/-3 KV and an acquisition system with two channels for dual amperometric (DC or pulsed amperometric detection) detection. Electrochemical detection has been selected as signal transduction method because it is relatively easily implemented, since nonoptical elements are required. The system uses a lithium-ion polymer battery and it is controlled from a desktop or laptop PC with a graphical user interface based on LabVIEW connected by serial RS232 or Bluetooth. The last part of the system consists of a reusable chip holder for housing the microchips, which contain all the electrical connections and reservoirs for making the work with microchips easy. The performance of the new instrument has been evaluated and compared with other commercially available apparatus using single- and dual-channel pyrex microchips for the separation of the neurotransmitters dopamine, epinephrine, and 3,4-dihydroxy-L-phenyl-alanine. The reduction of the size of the instrument has not affected the good performance of the separation and detection using microchips electrophoresis with electrochemical detection. Moreover, the new portable instrument paves the way for in situ analysis making the use of microchips electrophoresis easier.

Enantioseparation of amino acids and alpha-hydroxy acids on ligand-exchange continuous beds by capillary electrochromatography.

A new chiral stationary phase based on continuous bed (CB) technology using L-prolinamide as a chiral selector was prepared. Its ability for enantioseparation of amino acids and alpha-hydroxy acids by ligand-exchange CEC was compared with that of a CB containing L-4-hydroxyproline as chiral selector. Preparation was done by a one-step in situ copolyermerization procedure using methacrylamide as monomer, vinylsulfonic acid as charge providing agent and piperazine diacrylamide as crosslinker. L-Prolinamide showed marked enantioselective properties for the separation of amino acids and alpha-hydroxy acids. Furthermore, we show the possibility of simultaneous enantioseparation of amino acids and alpha-hydroxy acids using the CB with L-4-hydroxyproline as chiral selector.

Antibody drug separation using thermoresponsive anionic polymer brush modified beads with optimised electrostatic and hydrophobic interactions.

Antibody drugs play an important role in biopharmaceuticals, because of the specificity for target biomolecules and reduction of side effects. Thus, separation and analysis techniques for these antibody drugs have increased in importance. In the present study, we develop functional chromatography matrices for antibody drug separation and analysis. Three types of polymers, poly(N-isopropylacrylamide (NIPAAm)-co-2-acrylamido-2-methylpropanesulfonic acid (AMPS)-co-N-phenyl acrylamide (PhAAm)), P(NIPAAm-co-AMPS-co-n-butyl methacrylate (BMA)), and P(NIPAAm-co-AMPS-co-tert-butylacrylamide (tBAAm)), were modified on silica beads through atom transfer radical polymerisation. Rituximab elution profiles were observed using the prepared beads-packed column. Rituximab adsorption at high temperature and elution at low temperature from the column were observed, as a result of the temperature-modulated electrostatic and hydrophobic interactions. Using the column, rituximab purification from contaminants was performed simply by changing the temperature. Additionally, three types of antibody drugs were separated using the column through temperature-modulated hydrophobic and electrostatic interactions. These results demonstrate that the temperature-responsive column can be applied for the separation and analysis of biopharmaceuticals through a simple control of the column temperature.

Dopa and dopamine in glusulase: possible artifact in studies on catecholamine metabolism.

Relatively high concentrations of dopa and dopamnine were found in Glusulase, an enzyme preparation widely used in studies on catecholamine metabolism. This contamination may be a source of error in some studies, particularly in those measuring the endogenous concentrations of these catechols and their metabolic products.

Chiral Analysis of Non-Protein Amino Acids by Capillary Electrophoresis.

A high number of non-protein amino acids are chiral compounds that have demonstrated to be relevant in different fields. Their determination enables to obtain valuable information related to food quality and safety and has also a high interest from a biological point of view since many of them are key compounds in metabolic pathways or are related with different pathologies.In the development of analytical methodologies to perform chiral separations, capillary electrophoresis (CE) is well-established and one of the most powerful separation techniques as a consequence of its high efficiency, short analysis time, and versatility.This chapter shows, by means of three interesting examples, the application of different CE methodologies to the chiral analysis of non-protein amino acids. The first example describes different electrokinetic chromatography (EKC)-UV methodologies based on the use of negatively charged cyclodextrins as chiral selectors to carry out the stereoselective separation of ten different non-protein amino acids of relevance from a biological or food analysis point of view. The second method illustrates the EKC-UV analysis of L-citrulline and its enantiomeric impurity in food supplements using sulfated-γ-cyclodextrin as chiral selector. The last example shows the simultaneous enantiomeric separation of 3,4-dihydroxy-DL-phenylalanine and all the other chiral constituents involved in the phenylalanine-tyrosine metabolic pathway by using an EKC-MS methodology.

Enantioseparation of dopa and related compounds by cyclodextrin-modified microemulsion electrokinetic chromatography.

A chiral microemulsion electrokinetic chromatography method has been developed for the enantiomeric separation of 3,4-dihydroxyphenylalanine (dopa), its precursors phenylalanine and tyrosine, and the structurally related substance methyldopa. The separations were achieved using an oil-in-water microemulsion, which consisted of the oil-compound ethyl acetate, the surfactant sodium dodecylsulfate (SDS), the co-surfactant 1-butanol, the organic modifier propan-2-ol and 20mM phosphate buffer pH 2.5 or 2.0 as aqueous phase. For enantioseparation sulfated beta-cyclodextrin was added. The resolution of each racemate was optimized by varying the concentration of the buffer and all components of the microemulsion. Enantioseparation could be achieved for dl-dopa, dl-phenylalanine and dl-tyrosine within 13 min with a resolution of 4.3, 3.1 and 3.3, respectively, and for methyldopa in 17 min (Rs: 1.4). The established methods allowed the detection of dopa, phenylalanine, tyrosine and methyldopa with a limit at 0.5, 1.0, 0.2 and 2.0 microg/ml.

Bioactive DOPA melanin isolated and characterised from a marine actinobacterium Streptomyces sp. MVCS6 from Versova coast.

The melanin pigment produced from Streptomyces sp., MVCS6 was isolated and dihydroxyphenyalanine (DOPA) melanin compound was biochemically identified and spectroscopically characterised (ultraviolet and FT-IR). DOPA melanin showed a promising activity as an antibacterial natural product against 12 pathogenic bacteria from hospital isolations, particularly, against Pseudomonas aeruginosa RMMH7 (inhibition zone of 18 ± 0.02 at 30 µg/disc, and MIC of 10 ± 0.02 µg/mL) and Vibrio parahaemolytics RMMH12 (inhibition zone of 15 mm ± 0.03 at 30 µg/disc, and MIC of 14 ± 0.02 µg/mL). Moreover, in vitro evaluation of reducing power (Ascorbic Acid Equivalent (160 µg/mL)), DPPH radical-scavenging (89%), NO-scavenging (72%) and lipid peroxidation activities (89.6%) were determined. Cytotoxicity of DOPA melanin against cervical cancer cell line showed a dose-response activity, and IC50 value was found to be 300 µg/mL. These results would open the way to propose Streptomyces sp. MVCS6 as a promising source of bioactive eumelanin with therapeutic potential in medicine.

Enantioselective synthesis of 6-[fluorine-18]-fluoro-L-dopa from no-carrier-added fluorine-18-fluoride.

METHODS: A trimethylammonium veratraldehyde triflate was synthesized and used as a precursor for the asymmetric synthesis of 6-[18F]fluoro-L-dopa. RESULTS: Its nucleophilic fluorination with 18F-fluoride produced by the 18O(p,n)18F nuclear reaction on enriched 18O-water led to the corresponding no-carrier-added [18F]fluoroveratraldehyde (45 +/- 5% EOB). Diiodosilane was used to prepare the corresponding [18F]fluorobenzyl iodide (36.5 +/- 5.3% EOB). Akylation of (S)-1-tert-boc-2-tert-butyl-3-methyl-4-imidazolidinone with this electrophilic agent, hydrolysis and purification by preparative high-pressure liquid chromatography made 6-[18F]fluoro-L-dopa ready for human injection, in a 23% +/- 6% decay-corrected radiochemical yield. The enantiomeric purity and the specific activity were above 96% and 1 Ci/mumole respectively. CONCLUSION: Through this procedure, starting from 250 mCi of 18F-fluoride, multimillicurie amounts (32 +/- 8.5 mCi) of no-carrier-added 6-[18F]fluoro-L-dopa are now available at the end of synthesis (90 min) with a good radiochemical purity (more than 98%).

High yield synthesis of 6-[18F]fluoro-L-dopa by regioselective fluorination of protected L-dopa with [18F]acetylhypofluorite.

Regioselective fluorination of a completely protected phosgene derivative of 3,4-dihydroxy-phenyl-L-alanine (5-(benzyl-3',4'-carbonate)-oxazolidine-2,5-dione) with gaseous 18F-labeled acetylhypofluorite and [18F]F2 in acetonitrile is described. Fluorination with [18F]acetylhypofluorite yields 6-[18F]fluoro-L-dopa with 95% radiochemical purity; fluorination of the same substrate with [18F]F2 yields a mixture of all three structural isomers in a ratio of 70:16:14 for 6-, 5-, and 2-fluoro compounds. Radiochemical yield, relative to [18F] acetylhypofluorite, measured at the end of the synthesis, is (21 +/- 4)% (N = 8). The synthesis requires approximately 40 min (50 min if HPLC was done) and yields the final radiopharmaceutical in a two-step procedure. The specific activity of the final product was approximately 763 mCi/mmol at the end of a 40-min synthesis when 30-min irradiation was used.

The separation of epinephrine from norepinephrine and dopamine from DOPA on Sephadex G-10.

Epinephrine and norepinephrine were separated by acid elution through a Sephadex G-10 column with high recovery (better than 90%), excellent reproducibility, and little overlap (less than 10%). Once packed, the columns could be re-used indefinitely. Total elution time was about six hours and the columns could be left untended since the gravity flow stops automatically once the level of the eluant reaches the gel bed. The resulting dilution was five-fold. 3,4-Dihydroxyphenylalanine (DOPA) was completely separated from 3,4-dihydroxyphenylethylamine (dopamine) by the same procedure.

Aspects of 6-[18F]fluoro-L-DOPA preparation: precursor synthesis, preparative HPLC purification and determination of radiochemical purity.

A modified method for the synthesis of the intermediate product N-Boc-3,4-di(Boc-O)-6-iodo-L-phenylalanine ethyl ester of the [18F]FDOPA precursor preparation was developed. With the application of bis-(trifluoroacetoxy)-iodobenzene for the iodination step with elemental iodine the yield of the intermediate can be increased from 12% to 50-60%. By replacing silica-gel-based RP HPLC column by a polymer-based column for semi-preparative purification of [18F]FDOPA from the reaction mixture the radiochemical purity of the final product can be increased up to >99%. For the determination of the radiochemical impurity [18F]fluoride a HPLC method using a column with polymer-based RP material was introduced.

Catalytic enantioselective synthesis of 18F-fluorinated alpha-amino acids under phase-transfer conditions using (S)-NOBIN.

We describe a new method for the asymmetric synthesis of [(18)F]fluorinated aromatic alpha-amino acids (FAA) under phase transfer conditions using achiral glycine derivative NiPBPGly and (S)-NOBIN as a novel substrate/catalyst pair. The key alkylation step proceeds under mild conditions. Substituted [(18)F]fluorobenzylbromides were prepared using nucleophilic [(18)F]fluoride and were used as alkylation agents. Two important FAA, 2-[(18)F]fluoro-L-tyrosine (2-FTYR) and 6-[(18)F]fluoro-L-3,4-dihydroxyphenylalanine (6-FDOPA), were synthesized with an ee of 92 and 96%, respectively. The total synthesis time was 110-120 min and radiochemical yields (d.c.) were 25+/-6% for 2-FTYR and 16+/-5% for 6-FDOPA.

Enantioseparation of 3,4-dihydroxyphenylalanine and 2-hydrazino-2-methyl-3-(3,4-dihydroxyphenyl)propanoic acid by capillary electrophoresis using cyclodextrins.

The enantiomeric separations of 3,4-dihydroxyphenylalanine (dopa) and 2-hydrazino-2-methyl-3-(3,4-dihydroxyphenyl)propanoic acid (carbidopa) by capillary electrophoresis were studied using several native, neutral and anionic cyclodextrins as chiral additives and uncoated fused-silica capillaries. The effect of the type and concentration of the cyclodextrin added to 20 mM phosphate buffer (pH 2.5) on enantioseparation and migration times was studied. A high resolution value of 15.63 was obtained for dopa enantiomers with a buffer containing 20 mM single isomer, heptakis(2,3-diacetyl-6-sulfato)-beta-cyclodextrin. The enantiomers of carbidopa were separated using 20 mM carboxymethyl-beta-cyclodextrin as a chiral resolving agent. Both methods allowed the determination of 0.1% of the D-enantiomer (second migrating) in the presence of the L-enantiomer (first migrating) of dopa and carbidopa with a good precision. These methods also gave good results in terms of precision for both peak area, migration time, linearity and accuracy.

Robotic synthesis of 6-[18F]fluoro-L-dopa.

The aim of this study was to develop a method of semi-automated 6-[18F]fluoro-L-dopa (6-[18F]FDOPA) synthesis using a robotic system (Scanditronix Anatech RB III, Uppsala, Sweden). [18F]Fluorine was produced via 20Ne(d,alpha)18F using a Scanditronix MC17F cyclotron (Uppsala). The radiosynthesis was performed by the Scanditronix Anatech RB III robotic system. On average, a typical run produced 16-19 mCi of 6-[18F]FDOPA at end of synthesis (EOS) after 2 h irradiation of the F2/neon gas target. The total synthesis time was 110 min. The retention time of 6-[18F]FDOPA (the radio peak) was 8.2 min, which was consistent with the 6-[19F]FDOPA ultraviolet peak. The radiochemical purity was greater than 97%. A robotic, semi-automated method for 6-[18F]FDOPA radiosynthesis is therefore feasible. The radiation burden for the operator can be reduced as much as possible. Sufficient activity of 6-[18F]FDOPA could be obtained for positron emission tomography studies of dopaminergic function.

Separation of aromatic acids, DOPA, and methyl-DOPA by capillary electrophoresis with dendrimers as buffer additives.

Polyamidoamine starburst dendrimers with terminal carboxylate groups are used as a pseudo-stationary phase in electrokinetic chromatography for separating the selected aromatic amino acids phenylalanine, phenylglycine, homophenylalanine, and tyrosine and the catecholamines 3-(3,4-dihydroxyphenyl)alanine and 3-(3,4-dihydroxyphenyl)-2-methylalanine at different pH levels. A significant difference in analyte selectivity is observed between dendrimers of different generations and at different pH levels. At pH 7.0, the utility of the dendrimers for separating these analytes is limited by relatively low selectivity and a noisy baseline. However, good separation and selectivity are obtained with low generation dendrimers (G0.5 and G1.5) at low pH. Strong association of the solute with dendrimers is observed for high generations (G2.5, G3.5, and G5.5).

Efficient synthesis of the 18F-labelled 3-O-methyl-6-[18F]fluoro-L-DOPA.

The 18F-labelled amino acid derivative 3-O-methyl-6-[18F]fluoro-L-DOPA ([18F]OMFD) is a potential radiotracer for imaging tumour tissue using positron emission tomography. The precursor N-formyl-3-O-methyl-4-O-boc-6-trimethyl-stannyl-L-DOPA-ethyl ester enables direct electrophilic radiofluorination by stereoselective destannylation. After partial hydrolysis, optimized HPLC purification and sterile filtration, the [18F]OMFD obtained with high radiochemical purity and ready for use. The total synthesis time is about 50 min and the radiochemical yield achieved is 20-25% (decay corrected, related to [18F]F(2)). It was demonstrated that [18F]OMFD could be produced routinely and reliably for clinical use. [18F]FDOPA-preparation devices can be used with minor modifications.