Development and validation of LC-MS method for the determination of heptaethylene glycol monomethyl ether in benzonatate bulk drugs.

A simple and isocratic reverse-phase liquid chromatography with mass spectrometric method has been developed and validated for the determination of heptaethylene glycol monomethyl ether in benzonatate drug substance. Benzonatate is an oral antitussive drug used to relieve and suppress cough in patients older than 10 years. The presence of residual heptaethylene glycol monomethyl ether in the benzonatate drug substance affects the safety, strength, purity and quality of the drug substance. The subject compound separation was achieved using 0.1% formic acid and acetonitrile (50:50 v/v) at a flow rate of 0.3 ml/min. The Suplex PKB-100 250 × 4.6 mm, 5 µm LC column was used for a better peak shape. Detection was carried out at an m/z value of 341. The linearity curve showed a correlation of coefficient of >0.999. The precision and intermediate precision (RSD) were <7.30. The accuracy values were >90% for all levels. The developed method was validated as per International Conference on Harmonization guidelines and found to be a novel, specific and sensitive analytical method for determination of components of interest.

NMR Study of Intercalates and Grafted Organic Derivatives of H2La2Ti3O10.

The protonated perovskite-like titanate H2La2Ti3O10 has been used to produce organic-inorganic hybrids with simple organic molecules: methylamine, methanol, monoethanolamine, and n-butylamine. The optimal pathways for the preparation of such hybrids are summarized. Solid-state NMR, combined with thermal analysis, Raman, and IR spectroscopy, has been applied to determine the bonding type in the obtained organic-inorganic hybrids. It has been found that, in the methanolic hybrid, the organic residues are covalently bound to the inorganic matrix. In contrast, in the methylamine and n-butylamine hybrids, the organic molecules are intercalated into the inorganic matrix in cationic forms. The structure of the monoethanolamine hybrid is composite and includes both the covalently bound and intercalated organic species.

Strategy for Isolation, Preparation, and Structural Analysis of Chondroitin Sulfate Oligosaccharides from Natural Sources.

The structure of chondroitin sulfate oligosaccharides (CSOs), especially their sulfation pattern, has been found to be closely related with many biological pathways and diseases. However, detailed functional analysis such as their interaction with glycan binding proteins (GBPs) has been lagging, presumably due to the unavailability of well-defined, diverse structures. Besides challenging chemical and enzymatic synthesis, this is also due to the challenges in their purification at the isomer level and structural analysis owing to their instability, structural complexity, and low mass spectrometry detection sensitivity. Herein, we first used recycling preparative HPLC to separate and purify shark CS tetrasaccharide component labeled by a bifunctional fluorescent linker 2-amino-N-(2-aminoethyl)benzamide (AEAB) at the isomer level. Then, each isomer was derivatized through a multistage procedure including N-acetylation, carboxyl amidation, permethylation, and desulfation with silylating reagent. Structural analysis of each derivatized isomer was performed with ESI-MSn in positive ion mode. A total of 16 isomers of CSO-AEAB were isolated, with a minimum mass component of 0.007 mg and a maximum mass component of 17.53 mg, of which 10 isomers (>90 µg) were structurally analyzed. This preparation and structure analysis of CSOs lay the foundation for further study of the structure-activity relationship of CSOs.

In Situ Differentiation of Multiplex Noncovalent Interactions Using SERS and Chemometrics.

Probing changes of noncovalent interactions is crucial to study the binding efficiencies and strengths of (bio)molecular complexes. While surface-enhanced Raman scattering (SERS) offers unique molecular fingerprints to examine such interactions in situ, current platforms are only able to recognize hydrogen bonds because of their reliance on manual spectral identification. Here, we differentiate multiple intermolecular interactions between two interacting species by synergizing plasmonic liquid marble-based SERS platforms, chemometrics, and density functional theory. We demonstrate that characteristic 3-mercaptobenzoic acid (probe) Raman signals have distinct peak shifts upon hydrogen bonding and ionic interactions with tert-butylamine, a model interacting species. Notably, we further quantify the contributions from each noncovalent interaction coexisting in different proportions. As a proof-of-concept, we detect and categorize biologically important nucleotide bases based on molecule-specific interactions. This will potentially be useful to study how subtle changes in biomolecular interactions affect their structural and binding properties.

Development of ion pairing LC-MS/MS method for itaconate and cis-aconitate in cell extract and cell media.

Accumulation of Immune Responsive Gene 1(IRG1) in macrophage induced by lipopolysaccharide (LPS) and interferon gamma (IFN-γ) leads to production of itaconate by decarboxylation of cis-aconitate. The biology associated with IRG1 and itaconate is not fully understood. A rapid and sensitive method for measurement of itaconate will benefit the study of IRG1 biology. Multiple HPLC and derivatization methods were tested. An ion pairing LC-MS/MS method using tributylamine/formic acid as ion pairing agents and a HypercarbTM guard column we proposed demonstrated better peak shape and better sensitivity for itaconate. The current protocol allows baseline separation of itaconate, citraconate, and cis-aconitate without derivatization and direct analysis of analytes in 80% methanol/water solution to avoid the dry-down step. It provides the limit of quantitation (LOQ) of 30 pg itaconate on column with a 4.5-minute run time. This method is validated for measurement of itaconate and cis-aconitate in RAW264.7 cell extract and cell media in a 96-well plate format. We applied this method to successfully measure the increase of itaconate and the decrease of cis-aconitate in RAW cell extract and cell media after LPS/IFN-γ treatment.

Efficacy evaluation and mechanism study on inhibition of breast cancer cell growth by multimodal targeted nanobubbles carrying AMD070 and ICG.

To construct targeted nanobubbles carrying both small-molecule CXCR4 antagonist AMD070 and light-absorbing material indocyanine green (ICG), and to study their in vitro multimodal imaging, as well as their mechanism and efficacy of inhibition of breast cancer cell growth. Nanobubbles carrying AMD070 and ICG (ICG-TNBs) were constructed by carbodiimide reaction and mechanical oscillation. The physical characteristics and in vitro multimodal imaging were determined. The binding potential of ICG-TNBs to human breast cancer cells were observed by laser confocal microscopy. CCK-8 and flow cytometry were used to analyze the role of ICG-TNBs + US in inhibiting proliferation and inducing apoptosis of tumor cells. Flow cytometry and Western blotting are used to analyse the ROS generation and molecular mechanisms. ICG-TNBs had a particle size of 497.0 ± 29.2 nm and a Zeta potential of -8.05 ± 0.73 mV. In vitro multimodal imaging showed that the image signal intensity of ICG-TNBs increased with concentration. Targeted binding assay confirmed that ICG-TNBs could specifically bind to MCF-7 cells (CXCR4 positive), but not to MDA-MB-468 cells (CXCR4 negative). CCK-8 assay and flow cytometry analysis showed that ICG-TNBs + US could significantly inhibit the growth of MCF-7 breast cancer cells and promote their apoptosis. Flow cytometry and Western blotting showed that ICG-TNBs + US could significantly raise generation of ROS, reduce the expression of CXCR4, inhibit phosphorylation of Akt, and increase the expression of Caspase3 and Cleaved-caspase3. This indicated that ICG-TNBs could effectively inhibit and block the SDF-1/CXCR4 pathway, thus leading to the apoptosis of MCF-7 cells. ICG-TNBs can specifically bind to CXCR4 positive breast cancer cells, furthermore inhibit growth and promote apoptosis of breast cancer cells combined with ultrasonic irradiation by blocking the SDF-1/CXCR4 pathway. This study introduces a novel concept, method and mechanism for integration of targeted diagnosis and treatment of breast cancer.

n-Butylamine for Improving the Efficiency of Untargeted Mass Spectrometry Analysis of Plasma Metabolite Composition.

A comparative study of the impact of n-butylamine and traditionally used additives (ammonium hydroxide and formic acid) on the efficiency of the electrospray ionization (ESI) process for the enhancement of metabolite coverage was performed by direct injection mass spectrometry (MS) analysis in negative mode. Evaluation of obtained MS data showed that n-butylamine is one of the most effective additives for the analysis of metabolite composition in ESI in negative ion mode (ESI(-)) The limitations of the use of n-butylamine and other alkylamines in the analysis of metabolic composition and a decontamination procedure that can reduce MS device contamination after their application are discussed. The proposed procedure allows the performance of high-sensitivity analysis of low-molecular-weight compounds on the same MS device in both polarities.

An Explanation about the Use of (S)-Citronellal as a Chiral Derivatizing Agent (CDA) in 1H and 13C NMR for Sec-Butylamine, Methylbenzylamine, and Amphetamine: A Theoretical-Experimental Study.

A chiral derivatizing agent (CDA) with the aldehyde function has been widely used in discriminating chiral amines because of the easy formation of imines under mild conditions. There is a preference for the use of cyclic aldehydes as a CDA since their lower conformational flexibility favors the differentiation of the diastereoisomeric derivatives. In this study, the imines obtained from the reaction between (S)-citronellal and the chiral amines (sec-butylamine, methylbenzylamine, and amphetamine) were analyzed by the nuclear Overhauser effect (NOE). Through NOE, it was possible to observe that the ends of the molecules were close, suggesting a quasi-folded conformation. This conformation was confirmed by theoretical calculations that indicated the London forces and the molecular orbitals as main justifications for this conformation. This conformational locking explains the good separation of 13C NMR signals between the diastereomeric imines obtained and, consequently, a good determination of the enantiomeric excess using the open chain (S)-citronellal as a CDA.

Application of Butylamine as a Conjugative Reagent to On-Column Derivatization for the Determination of Antioxidant Amino Acids in Brain Tissue, Plasma, and Urine Samples.

(1) Antioxidants are involved in body protection mechanisms against reactive oxygen species. Amino acids such as glutathione (GSH) and N-acetylcysteine (NAC) are known to be involved in providing protection against oxidative lethality. A quick and simple method for the determination of NAC and GSH in various biological matrices such as urine, plasma, and homogenates of brain tissues has been developed and described in this work. (2) The assay is based on reversed phase high performance liquid chromatography with spectrofluorimetric detection and on-column derivatization. Butylamine and o-phthaldialdehyde have been used as derivatization reagents. Since o-phthaldialdehyde constitutes a part of the mobile phase, the derivatization reaction and chromatographic separation occur simultaneously. (3) Linearity in the detector response for NAC in human urine was observed in the range of 5-200 nmol mL-1, and NAC and GSH in the brain tissue homogenates were observed in the range of 0.5-5 nmol mL-1 and 0.5-15 nmol mL-1, respectively. Human plasma linearity ranges covered 0.25-5.00 nmol mL-1 and 0.5-15 nmol mL-1 for NAC and GSH, respectively. The LODs for NAC and GSH were 0.01 and 0.02 nmol mL-1 while the LOQs were 0.02 and 0.05 nmol mL-1, respectively. The usefulness of the proposed method was proven through its application to real samples.

Synthesis and in vitro antitumor activity of novel acylspermidine derivative N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide (AAHD) against HepG2 cells.

Naturally occurring polyamines like Putrescine, Spermidine, and Spermine are polycations which bind to the DNA, hence stabilizing it and promoting the essential cellular processes. Many synthetic polyamine analogues have been synthesized in the past few years, which have shown cytotoxic effects on different tumours. In the present study, we evaluated the antiproliferative effect of a novel, acylspermidine derivative, (N-(4-aminobutyl)-N-(3-aminopropyl)-8-hydroxy-dodecanamide) (AAHD) on HepG2 cells. Fluorescence staining was performed with nuclear stain (Hoechst 33342) and acridine orange/ethidium bromide double staining. Dose and the time-dependent antiproliferative effect were observed by WST-1 assays, and radical scavenging activity was measured by ROS. Morphological changes such as cell shrinkage & blebbing were analyzed by fluorescent microscopy. It was found that AAHD markedly suppressed the growth of HepG2 cells in a dose- and time-dependent manner. It was also noted that the modulation of ROS levels confirmed the radical scavenging activity. In the near future, AAHD can be a promising drug candidate in chalking out a neoplastic strategy to control the proliferation of tumour cells. This study indicated that AAHD induced anti-proliferative and pro-apoptotic activities on HCC. Since AAHD was active at micromolar concentrations without any adverse effects on the healthy cells (Fibroblasts), it is worthy of further clinical investigations.

Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides.

This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation.

An electrochemical biosensor based on novel butylamine capped CZTS nanoparticles immobilized by uricase for uric acid detection.

Quaternary chalcopyrite, i.e., Cu2ZnSnS4 (CZTS) nanoparticles films have been proposed as a novel matrix system for enzyme-based electrochemical biosensors providing a non-toxic, low-cost alternative for the fabrication of bioelectrodes. The easy tuneability of the band gap of CZTS by varying the cation ratio and size of nanoparticles facilitate to impart desirable electrical properties in the material. Butylamine capped spherical CZTS nanoparticles of size 15-16 nm and band gap 2.65 eV have been synthesized by colloidal hot injection technique. The films of CZTS onto ITO substrates are deposited using dip coating technique, and uricase enzyme have been immobilized onto CZTS films using EDC-NHS binding chemistry. Electrochemical analyses of this bioelectrode revealed that the uricase/CZTS/ITO/glass electrode exhibits good linearity over a wide range of 0-700 µM uric acid concentration with a limit of detection (LOD) of 0.066 µM. The low value of 0.13 × 10-4 M of Michaelis-Menten constant (Km) indicate the enhanced affinity of immobilized enzyme (uricase) towards uric acid. Thus, the present report confirms the promising application of the p-type CZTS thin film matrix for the realization of an electrochemical biosensor.

Isotope Labeling Strategies for Acylcarnitines Profile in Biological Samples by Liquid Chromatography-Mass Spectrometry.

Acylcarnitines are closely related to many metabolic diseases and likely to be good biomarkers for clinical diagnosis. Studies on acylcarnitines may help deepen the understanding of their functions associated with disease processes. However, the diversity of their structures and lack of commercial standards make them difficult to be fully detected. In this work, a highly efficient isotope labeling strategy was developed to detect acylcarnitines in biological samples using liquid chromatography-mass spectrometry (LC-MS). A pair of reagents with or without deuterium labeling tags containing both a positive charge and a primary amine group were synthesized, which were used to label the carboxyl group of acylcarnitines. The reaction was performed under mild conditions to avoid hydrolysis of acylcarnitines. The reaction products had two positive charges, forming a double charge peak in MS spectra and with mass-to-charge ratio difference (Δ m/ z) of 4.0251 Da between the light- and heavy-labeled products. The feature made it possible to distinguish signals of acylcarnitines from other carboxyl metabolites with Δ m/ z of 8.0502 Da between their light- and heavy-labeled products. On the basis of the characteristics and the tandem mass spectrometry (MS/MS) spectra, a total of 108 acylcarnitines were discovered and identified in urine samples. Our established approach will be very helpful for the studies of diseases associated with acylcarnitines metabolism.

Interaction between 3,4­dichlorophenyl­propenoyl­sec.­butylamine (3,4­DCPB), an antiepileptic drug, and cytochrome P450 in rat liver microsomes and recombinant human enzymes in vitro.

The compound 3,4­dichlorophenyl­propenoyl­sec.­butylamine (3,4­DCPB) is an antiepileptic drug. The purpose of the present research was to identify cytochrome P450 (CYP450) responsible for the metabolism of 3,4­DCPB and evaluate the effects of 3,4­DCPB on the activities of CYP450 enzymes. 3,4­DCPB was incubated with rat liver microsomes (RLMs) plus six CYP450 enzyme-specific inhibitors, or six recombinant human CYP450 enzymes (rhCYP450s). The concentrations of 3,4­DCPB and six CYP450 enzyme-activities probe drugs were detected by high-performance liquid chromatographic (HPLC). The results showed that the prototype of 3,4­DCPB was metabolized by multiple CYP450 enzymes into three metabolites, and the predominant isoforms were CYP2D6 (metabolite M1), CYP1A2 (M2), CYP2C19 and CYP3A4 (M3), respectively., in the presence of ß-NADPH (1 mM) in RLMs or rhCYP450s. Compared with the control (PB-), phenobarbital pre-treatment (PB+) significantly enhanced levels (all of p < 0.01) of hydroxylmethytobutamide (CYP2C9), 4­hydroxy­mephenytoin (CYP2C19), acetaminophen (CYP1A2), 6­hydroxychlorzoxazone (CYP2E1) and oxidized nifedipine (CYP3A4), respectively, in spite of dextrophan (CYP2D6) was not markedly enhanced in RLMs. Conversely, the inhibitory ratios of 3,4­DCPB (16 µg/mL, 59 µM) on the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6 were 97.6%, 59.0%, 53.5% and 36.5%, respectively. However, CYP2E1 (both of PB- and PB+) and CYP3A4 (PB+) were not inhibited by 3,4­DCPB in RLMs. In conclusion, the present study showed that 3,4­DCPB was metabolized by multiple CYP450 enzymes. 3,4­DCPB inhibited the activities of CYP2C9, CYP2C19, CYP1A2 and CYP2D6, rather than that CYP2E1 and CYP3A4 enzymes, suggesting that the different effects of 3,4­DCPB on the CYP450 enzymes might influence metabolic drug-drug interaction in antiepileptics therapy.

Facile synthesis of amine-functional reduced graphene oxides as modified quick, easy, cheap, effective, rugged and safe adsorbent for multi-pesticide residues analysis of tea.

Amine-functional reduced graphene oxide (amine-rGO) with different carbon chain length amino groups were successfully synthesized. The graphene oxides (GO) reduction as well as amino grafting were achieved simultaneously in one step via a facile solvothermal synthetic strategy. The obtained materials were characterized by X-ray diffraction, Raman spectroscopy, Fourier-transform infrared spectrometry and X-ray photoelectron spectroscopy to confirm the modification of GO with different amino groups. The adsorption performance of catechins and caffeine from tea acetonitrile extracts on different amine functional rGO samples were evaluated. It was found that tributylamine-functional rGO (tri-BuA-rGO) exhibited the highest adsorption ability for catechins and caffeine compared to GO and other amino group functional rGO samples. It was worth to note that the adsorption capacity of catechins on tri-BuA-rGO was 11 times higher than that of GO (203.7mgg-1 vs 18.7mgg-1). Electrostatic interaction, π-π interaction and surface hydrophilic-hydrophobic properties of tri-BuA-rGO played important roles in the adsorption of catechins as well as caffeine. The gravimetric analysis confirmed that the tri-BuA-rGO achieved the highest efficient cleanup preformance compared with traditional dispersive solid phase extraction (dSPE) adsorbents like primary-secondary amine (PSA), graphitized carbon black (GCB) or C18. A multi-pesticides analysis method based on tri-BuA-rGO is validated on 33 representative pesticides in tea using gas chromatography coupled to tandem mass spectrometry or high-performance liquid chromatography coupled with tandem mass spectrometry. The analysis method gave a high coefficient of determination (r2>0.99) for each pesticide and satisfactory recoveries in a range of 72.1-120.5%. Our study demonstrated that amine functional rGO as a new type of QuEChERS adsorbent is expected to be widely applied for analysis of pesticides at trace levels.

Comparison of air samplers for determination of isocyanic acid and applicability for work environment exposure assessment.

Isocyanic acid (ICA) is one of the most abundant isocyanates formed during thermal decomposition of polyurethane (PUR), and other nitrogen containing polymers. Hot-work, such as flame cutting, forging, grinding, turning and welding may give rise to thermal decomposition of said polymers potentially forming significant amounts of ICA. A newly launched dry denuder sampler for airborne isocyanates using di-n-butylamine (DBA) demonstrated build-up of background ICA-DBA over time. Build-up of background ICA-DBA was not observed when stored at inert conditions (Ar atmosphere) for 84 days. Thus, freshly prepared denuders were used. The sampling efficiency of ICA using freshly prepared denuder samplers (0.2 L min-1), impinger + filter samplers (0.5 L min-1) using DBA and 1-(2-methoxyphenyl) piperazine (2MP)-impregnated filter cassette samplers (1 L min-1) was investigated. PTR-MS measurements of ICA were used as a quantitative reference. Dynamically generated standard ICA atmospheres covered the range 5.6 to 640 ppb at absolute humidities (AH) 4.0 and 16 g m-3. Recovered ICA was found to be 73-115% (denuder), 89-115% (impinger + filter) and 62-100% (2MP filter cassette). The method limit of detection (LOD) was equal to an amount of ICA of 24 ng (denuder), 8.9 ng (impinger + filter) and 9.4 ng (2MP filter cassette). The PTR-MS LOD for ICA was 1.8 and 2.8 ppb in atmospheres with an AH of 4 and 16 g m-3. Denuder samplers were used for personal (n = 176) and stationary (n = 31) air sampling during hot-work at six industrial sites (n = 23 workers). ICA was detected above method LOD in 66% and 58% of the personal and stationary samples, respectively. ICA workroom air concentrations were determined to be 1.8-320 ppb (median 12 ppb) (personal samples), and 1.5-44 ppb (median 6.6 ppb) (stationary samples).

Synthesis, characterization, and application of a novel multifunctional stationary phase for hydrophilic interaction/reversed phase mixed-mode chromatography.

A novel multifunctional stationary phase based on silica gel was synthesised starting from L- isoleucine and 4-phenylbutylamine and evaluated as a hydrophilic interaction/reversed-phase mixed-mode stationary phase for high-performance liquid chromatography (HPLC). The prepared stationary phase was characterized by elemental analysis, infrared spectroscopy (IR), scanning electron microscopy (SEM), Brunauer, Emmett and Teller (BET) and solid-state 13C nuclear magnetic resonance (NMR). The mechanisms involved in the chromatographic separation are multi-interaction, including hydrophobic, π-π, hydrogen-bonding, dipole-dipole and ion-dipole interactions. Based on these interactions, successful separation could be achieved among several aromatic compounds having different polarities under both hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP) condition. Nucleotides/nucleosides were separated in the HILIC mode. The effects of different separation conditions, such as pH value, mobile-phase content, column temperature, buffer concentration and flow rate, on the separation of nucleotides/nucleosides in HILIC mode were investigated. The seven nucleotides/nucleosides were separated within 22min, while six of them were separated within 10min by isocratic elution. To determine the influence of the new multifunctional stationary phase under the RP condition, a number of moderately and weakly polar and nonpolar compounds, such as 10 substituted anilines and eight substituted phenols were separated successfully under the RP condition within 14 and 15min, respectively. Additionally, nine mixtures of polar/nonpolar test compounds were simultaneously separated within 19min, while seven of them were separated within 12min, under HILIC/RP mixed-mode conditions. Chromatographic parameters, such as the retention factor and peak asymmetry factor, were calculated for all of the analytes, while the theoretical plate number was calculated for analytes separated by isocratic elution. Compared to traditional C18 and commercial HILIC columns, the new stationary phase exhibited both HILIC and RPLC performance, and the scope of analyte separation was thus enlarged.

PEGylation with the thiosuccinimido butylamine linker significantly increases the stability of haloalkane dehalogenase DhaA.

Haloalkane dehalogenase (HLD) can catalyze the hydrolytic dehalogenation of halogenated compounds. However, HLD suffers from the poor stability to resist the environmental stress. PEGylation is an effective approach to enhance the stability of enzymes. The linker is an important stabilization factor of PEGylation. Thus, the linkers of the PEGylated HLD were optimized to improve the stability of HLD in the present study. The PEGylated haloalkane dehalogenase DhaAs with methylamine (Ml), carbamate (Cm) and thiosuccinimido butylamine (Tb) linkers were prepared, respectively. The effects of the Ml, Cm and Tb linkers on the stability of the PEGylated DhaAs were investigated under different environmental stresses. Among the three linkers, the Tb linker showed the highest efficacy to improve the stability of the PEGylated DhaA. The Tb linker significantly increased the thermal stability of the PEGylated DhaA by slowing its structural unfolding, and the pH stability of the PEGylated DhaA by slowing the protonation process. In addition, the PEGylated DhaA with the Tb linker showed the maximum resistance to high ionic strength (1M NaCl) and organic solvent (40% DMSO). PEGylation with the Tb linker is of general interest to effectively improve the stability of proteins, particularly the protein with poor stability.

Functional Characterization of a Novel Series of Biased Signaling Dopamine D3 Receptor Agonists.

Dopamine receptors play an integral role in controlling brain physiology. Importantly, subtype selective agonists and antagonists of dopamine receptors with biased signaling properties have been successful in treating psychiatric disorders with a low incidence of side effects. To this end, we recently designed and developed SK609, a dopamine D3 receptor (D3R) selective agonist that has atypical signaling properties. SK609 has shown efficacy in reversing akinesia and reducing L-dopa-induced dyskinesia in a hemiparkinsonian rats. In the current study, we demonstrate that SK609 has high selectivity for D3R with no binding affinity on D2R high- or low-affinity state when tested at a concentration of 10 µM. In addition, SK609 and its analogues do not induce desensitization of D3R as determined by repeated agonist treatment response in phosphorylation of ERK1/2 functional assay. Most significantly, SK609 and its analogues preferentially signal through the G-protein-dependent pathway and do not recruit ß-arrestin-2, suggesting a functional bias toward the G-protein-dependent pathway. Structure-activity relationship (SAR) studies using analogues of SK609 demonstrate that the molecules bind at the orthosteric site by maintaining the conserved salt bridge interactions with aspartate 110 on transmembrane 3 and aryl interactions with histidine 349 on transmembrane 6, in addition to several hydrophobic interactions with residues from transmembranes 5 and 6. The compounds follow a strict SAR with reference to the three pharmacophore elements: substituted phenyl ring, length of the linker connecting phenyl ring and amine group, and orientation and hydrophobic branching groups at the amine among SK609 analogues for efficacy and functional selectivity. These features of SK609 and the analogues suggest that biased signaling is an inherent property of this series of molecules.

Highly sensitive fluorescence detection of heparin based on aggregation-induced emission of a tetraphenylethene derivative.

Tetraphenylethene derivatives were reported for sensitive "turn on" detection of heparin (Hep) based on the typical aggregation-induced emission (AIE). In the present strategy, three derivatives of tetraphenylethene (TPE) were designed，synthesized and compared the sensitivities for the detection of Hep. Furthermore, the (Z)-4,4'-(((1,2-diphenylethene-1,2-diyl) bis(4,1-phenylene))bis (oxy))bis(butan-1-aminium) (Z-TPE-5) was the most sensitive and chosen as the probe for Hep detection. Since Hep could be employed as a medium for inducing the aggregation of positively charged Z-TPE-5, the designed Z-TPE-5 shows excellent fluorescence response to Hep through multiple electrostatic interactions with a detection limit of 1.53ng/mL, which is far below than most of the reported method for the detection of Hep. Due to the stronger affinity between Hep and protamine, Hep preferred to bind with it instead of Z-TPE-5 after the addition of protamine, so the fluorescence could be reduced. In comparison to prior studies, this developed strategy here not only simplifies the preparation procedure of the fluorescent probes but also can be applied in sensitive determination of Hep with good accuracy. Moreover, the detection of Hep with Z-TPE-5 was not interfered by Hep analogues, such as chondroitin sulfate and hyaluronic acid, as well as in the matrix of human serum.