Optimized Coordination of Uranyl in Engineered Calmodulin Site 1 Provides a Subnanomolar Affinity for Uranyl and a Strong Uranyl versus Calcium Selectivity.

As an alpha emitter and chemical toxicant, uranium toxicity in living organisms is driven by its molecular interactions. It is therefore essential to identify main determinants of uranium affinity for proteins. Others and we showed that introducing a phosphoryl group in the coordination sphere of uranyl confers a strong affinity of proteins for uranyl. In this work, using calmodulin site 1 as a template, we modulate the structural organization of a metal-binding loop comprising carboxylate and/or carbonyl ligands and reach affinities for uranyl comparable to that provided by introducing a strong phosphoryl ligand. Shortening the metal binding loop of calmodulin site 1 from 12 to 10 amino acids in CaMΔ increases the uranyl-binding affinity by about 2 orders of magnitude to log KpH7 = 9.55 ± 0.11 (KdpH7 = 280 ± 60 pM). Structural analysis by FTIR, XAS, and molecular dynamics simulations suggests an optimized coordination of the CaMΔ-uranyl complex involving bidentate and monodentate carboxylate groups in the uranyl equatorial plane. The main role of this coordination sphere in reaching subnanomolar dissociation constants for uranyl is supported by similar uranyl affinities obtained in a cyclic peptide reproducing CaMΔ binding loop. In addition, CaMΔ presents a uranyl/calcium selectivity of 107 that is even higher in the cyclic peptide.

Mechanism-Driven Development of Group 10 Metal-Catalyzed Decarbonylative Coupling Reactions.

Transition-metal-catalyzed cross-coupling reactions are widely used in both academia and industry for the construction of carbon-carbon and carbon-heteroatom bonds. The vast majority of cross-coupling reactions utilize aryl (pseudo)halides as the electrophilic coupling partner. Carboxylic acid derivatives (RC(O)X) represent a complementary class of electrophiles that can engage in decarbonylative couplings to produce analogous products. This decarbonylative approach offers the advantage that RC(O)X are abundant and inexpensive. In addition, decarbonylative coupling enables both intramolecular (between R and X of the carboxylic acid derivative) as well as intermolecular bond-forming reactions (in which an exogeneous nucleophile is coupled with the R group derived from RC(O)X). In these intermolecular reactions, the X-substituent on the carboxylic acid can be tuned to facilitate both oxidative addition and transmetalation, thus eliminating the need for an exogeneous base. This Account details our group's development of a diverse variety of base-free decarbonylative coupling reactions catalyzed by group 10 metals. Furthermore, it highlights how catalyst design can be guided by stoichiometric organometallic studies of these systems.Our early studies focused on intramolecular decarbonylative couplings that transform RC(O)X to the corresponding R-X with extrusion of CO. We first identified Pd and Ni monodentate phosphine catalysts that convert aryl thioesters (ArC(O)SR) to the corresponding thioethers (ArSR). We next expanded this reactivity to fluoroalkyl thioesters, using readily available fluoroalkyl carboxylic acids as the fluoroalkyl (RF) source. A Ni-phosphinoferrocene catalyst proved optimal, and the large bite angle bidentate ligand was necessary to promote the challenging RF-S bond-forming reductive elimination step.We next pursued intramolecular decarbonylative couplings of aroyl halides. Palladium-based catalysts bearing dialkylbiaryl ligands (e.g., BrettPhos) were identified as optimal for converting aroyl chlorides (ArC(O)Cl) to aryl chlorides (ArCl). These ligands were selected based on their ability to facilitate the key C-Cl bond-forming reductive elimination step of the catalytic cycle. In contrast, all attempts to convert aroyl fluorides [ArC(O)F)] to aryl fluorides (ArF) were unsuccessful with either Pd- or Ni-based catalysts. Organometallic studies of the Ni-system show that C(O)-F oxidative addition and CO deinsertion proceed smoothly, but the resulting nickel(II) aryl fluoride intermediate fails to undergo C-F bond-forming reductive elimination.In contrast to its inertness to reductive elimination, this nickel(II) aryl fluoride proved highly reactive toward transmetalation. The fluoride ligand serves as an internal base, such that no additional base is required. We leveraged this "transmetalation active" intermediate to achieve base-free Ni-catalyzed intermolecular decarbonylative coupling reactions between aroyl fluorides and boron reagents to access both biaryl and aryl-boronate ester products. By tuning the electrophile, transmetalating reagent, and catalyst, this same approach also proved applicable to base-free intermolecular decarbonylative fluoroalkylation (between difluoromethylacetyl fluoride and arylboronate esters) and aryl amination (between phenol esters and silyl amines).Moving forward, a key goal is to identify catalyst systems that enable more challenging bond constructions via this manifold. In addition, CO inhibition remains a major issue leading to the requirement for high temperatures and high catalyst loadings. Identifying catalysts that are resistant to CO binding and/or approaches to remove CO under mild conditions will be critical for making these reactions more practical and scalable.

Copper-catalyzed direct decarboxylative fluorosulfonylation of aliphatic carboxylic acids.

Sulfonyl fluorides are emerging as key structural motifs in organic synthesis, medicinal chemistry, and materials science. Herein we report two efficient and complementary methods for direct decarboxylative fluorosulfonylation of carboxylic acids by the merging of copper catalysis with different N-centered HAT regents. A wide range of structurally diverse sulfonyl fluorides was readily accessed from primary, secondary, and tertiary carboxylic acids in a single step under mild conditions.

Carbodiphosphorane-Stabilized Parent Dioxophosphorane: A Valuable Synthetic HO2P Source.

Introducing a small phosphorus-based fragment into other molecular entities via, for example, phosphorylation/phosphonylation is an important process in synthetic chemistry. One of the approaches to achieve this is by trapping and subsequently releasing extremely reactive phosphorus-based molecules such as dioxophosphoranes. In this work, electron-rich hexaphenylcarbodiphosphorane (CDP) was used to stabilize the least thermodynamically favorable isomer of HO2P to yield monomeric CDP·PHO2. The title compound was observed to be a quite versatile phosphonylating agent; that is, it showed a great ability to transfer, for the first time, the HPO2 fragment to a number of substrates such as alcohols, amines, carboxylic acids, and water. Several phosphorous-based compounds that were generated using this synthetic approach were also isolated and characterized for the first time. According to the initial computational studies, the addition-elimination pathway was significantly more favorable than the corresponding elimination-addition route for "delivering" the HO2P unit in these reactions.

Direct analysis of naphthenic acids in produced water and crude oil by NH2-surface-modified wooden-tip electrospray ionization mass spectrometry.

This work describes the surface coating of wooden toothpicks with amino groups (NH2) for electrospray ionization mass spectrometry (MS) analysis of naphthenic acids (NAs) in produced water samples and crude oil fractions. NH2 was introduced into the cellulosic material through a silanization reaction using aminopropyltriethoxysilane. An NH2-modified toothpick was inserted into the analyte extraction sample and was subsequently used as an electrospray emitter for MS analysis. The extraction conditions were optimized by analyzing NAs (benzoic acid, 1-naphthoic acid, decanoic acid, 3,5-dimethyladamantane-1-carboxylic acid, and 3,5-dimethyladamantane-1-acetic acid) in pure water, and the best condition was using 5 min of extraction time with the samples under agitation. Modified and unmodified wooden toothpicks were compared, and the intensities of all NAs were higher when using the modified substrates than when using the unmodified ones. Limit of detection (LOD), limit of quantification (LOQ), linearity, precision, and recovery were determined by analyzing decanoic acid in seawater samples. The LOD and LOQ were 2 and 5 µg mL-1, respectively, and a linear correlation (R2 = 0.9927) was obtained with concentrations ranging from 5 to 250 µg mL-1. Precision values ranged from 6 to 13% and recoveries from 89 to 106%. The technique was also employed to analyze three produced water samples, in which decanoic acid was semi-quantified, and the concentrations ranged from 10 to 13 µg mL-1. High abundances of acidic compounds of class O2 with DBEs (double bond equivalents) ranging from 1 to 3 and carbon numbers going from 8 to 12 were detected in the produced water samples. The results suggest that the modification of wooden toothpicks with NH2 might offer a significant advancement in the knowledge of cheap substrates that can improve the sensitivity of analysis of NAs in water samples.

Characterization of chemical constituents and metabolites in rat plasma after oral administration of San Miao Wan by ultra-high performance liquid chromatography tandem Q-Exactive Orbitrap mass spectrometry.

San Miao Wan (SMW), composed of Phellodendri Chinensis Cortex, Atractylodis Lanceae Rhizoma and Achyranthis Bidentatae Radix, is widely used for the treatment of gout, hyperuricemia and other diseases. In the present study, an overall identification strategy based on ultra-high performance liquid chromatography tandem Q-Exactive Orbitrap mass spectrometry (UPLC-Q-Exactive Orbitrap/MS) method was established to characterize the multiple chemical constituents of SMW and its metabolites in rat plasma after oral administration of SMW. A total of 76 constituents including alkaloids, organic acids, lactones, terpenes, saponins, sterones and others types of components were identified in the extract of SMW. After the oral administration of SMW, 47 prototype constituents and 66 metabolites were identified in rat plasma samples. The related metabolic pathways mainly involved reduction, demethylation, hydroxylation, methylation and glucuronide conjunction. The proposed method could be a useful approach to identify the chemical constituents of SMW and its metabolic components. Our study provide a universal strategy for the analysis of the components and metabolites of the traditional Chinese medicine prescription (TCP) extracts and plasma after administration using UPLC-Q-Exactive Orbitrap/MS method. It will assist with clarifying the substance basis of effective components in SMW. It also provides a rapid method for overall analysis of chemical constituents and metabolites of SMW.

Metabolic analysis of unripe papaya (Carica papaya L.) to promote its utilization as a functional food.

Papaya (Carica papaya L.) is widely cultivated in tropical and subtropical countries. While ripe fruit is a popular food item globally, the unripe fruit is only consumed in some Asian countries. To promote the utilization of unripe papaya based on the compositional changes of biological active metabolites, we performed liquid chromatography-Orbitrap-mass spectrometry-based analysis to reveal the comprehensive metabolite profile of the peel and pulp of unripe and ripe papaya fruits. The number of peaks annotated as phenolics and aminocarboxylic acids increased in the pulp and peel of ripe fruit, respectively. Putative carpaine derivatives, known alkaloids with cardiovascular effects, decreased, while carpamic acid derivatives increased in the peel of ripe fruit. Furthermore, the functionality of unripe fruit, the benzyl glucosinolate content, total polyphenol content, and proteolytic activity were detectable after heating and powder processing treatments, suggesting a potential utilization in powdered form as functional material.

Discovery of CYR715: A novel carboxylic acid-containing soluble guanylate cyclase stimulator.

Soluble guanylate cyclase (sGC) is a clinically validated therapeutic target in the treatment of pulmonary hypertension. Modulators of sGC have the potential to treat diseases that are affected by dysregulation of the NO-sGC-cGMP signal transduction pathway. This letter describes the SAR efforts that led to the discovery of CYR715, a novel carboxylic acid-containing sGC stimulator, with an improved metabolic profile relative to our previously described stimulator, IWP-051. CYR715 addressed potential idiosyncratic drug toxicity (IDT) liabilities associated with the formation of reactive, migrating acyl glucuronides (AG) found in related carboxylic acid-containing analogs and demonstrated high oral bioavailability in rat and dose-dependent hemodynamic pharmacology in normotensive Sprague-Dawley rats.

Aggregation and weak gel formation by pectic polysaccharide homogalacturonan.

This study presents a novel model of homogalacturonan (HG) based on the dissipative particle dynamics (DPD). The model was applied to investigate the mechanism of self-aggregation of low-methoxylated homogalacturonan in aqueous solutions in the absence of cations. The coarse-grained model provided new insights into the structural features of HG aggregates and networks in aqueous solutions. Depending on the properties and concentration of polysaccharides, two major patterns of self-assembly were observed for HG - ellipsoidal aggregates and a continuous three-dimensional network. Simulations showed that a decrease in the degree of dissociation of HG results in a higher rate of self-aggregation, as well as facilitating the formation of larger assemblies or thicker nanofilaments depending on the type of final self-assembly. Simulations of polysaccharides of different chain lengths suggested the existence of a structural threshold for the formation of a spatial network for HG consisting of less than 35 GalA units.

Novel 4-Oxo-4,10-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3-carboxylic Acid Derivatives as HIV-1 Integrase Inhibitors: Synthesis, Docking Studies, Molecular Dynamics Simulation and Biological Activities.

BACKGROUND: HIV-1 integrase (IN) has been considered as an important target for the development of novel anti-HIV-1 drugs. OBJECTIVE: The aim of this study was to design novel groups of HIV IN inhibitors. METHODS: In this study, we presented a novel series of 4-oxo-4,10-dihydrobenzo[4,5]imidazo[1,2- a]pyrimidine-3-carboxylic acid derivatives by structural modification of N-arylindole ß-diketoacids as a well-known group of IN inhibitors. RESULTS: Based on in-vitro anti-HIV-1 activity in a cell-based assay, compounds 5, 6a and 6k displayed moderate to good inhibitory activity with EC50 values of 4.14, 1.68 and 0.8 µM, respectively. However, integrase inhibition assay showed that most of the analogues did not have significant effects against integrase enzyme except compound 5 with an IC50 value of 45 µM. Our results indicated that compound 6k was the best one among synthesized compounds with an EC50 of 0.8 µM and SI of 175. Docking and molecular dynamics simulation studies were also performed to provide some insights into the probable mechanism of tested compounds. CONCLUSION: These findings suggest that 4-oxo-4,10-dihydrobenzo[4,5]imidazo[1,2-a]pyrimidine-3- carboxylic acid derivatives may consider as promising lead compounds for the development of new anti-HIV-1 drugs.

In vitro anti-leishmanial activity of Prunus armeniaca fractions on Leishmania tropica and molecular docking studies.

Prunus armeniaca (L.) is a member of the Rosaceae, subfamily Prunoideae, shows anticancer, antitubercular, antimutagenic, antimicrobial, antioxidant, and cardioprotective activities. Here we fractionated the leaves extract of this highly medicinally important plant for antileishmanial activity. In the current study, the leaves extract was fractionated and characterized using column and thin layer chromatography by n-hexane, ethyl acetate, and methanol solvents. Twelve fractions were isolated and subjected for evaluation of their cytotoxicity and in vitro antileishmanial activity against promastigotes and amastigotes of Leishmania tropica. Among all fractions used, the fraction (F7) exhibited the strongest antileishmanial activity. The bioactive fraction was further characterized by spectroscopy (FTIR, UV-Vis), and GC-MS analysis. The in silico docking was carried out to find the active site of PTR1. All derived fractions exhibited toxicity in the safety range IC50 > 100 µg/ml. The fraction (F7) showed significantly the highest antipromastigotes activity with IC5011.48 ± 0.82 µg/ml and antiamastigotes activity with IC50 21.03 ± 0.98 µg/ml compared with control i.e. 11.60 ± 0.70 and 22.03 ± 1.02 µg/ml respectively. The UV-Vis spectroscopic analysis revealed the presence of six absorption peaks and the FTIR spectrum revealed the presence of alkane, aldehyde, carboxylic acid, thiols, alkynes, and carbonyls compounds The GC-MS chromatogram exhibited the presence of nine compounds: (a) benzeneethanol, alpha, beta dimethyl, (b)carbazic acid, 3-(1 propylbutylidene)-, ethyl ester, (c)1, 2-benzenedicarboxylic acid, diisooctyl ester, (d)benzeneethanamine a-methyl, (e)2aminononadecane, (f)2-heptanamine-5-methyl, (g)cyclobutanol, (h)cyclopropyl carbine, and (i)nitric acid, nonyl ester. Among all compounds, the 1, 2-benzenedicarboxylic acid, diisooctyl ester bound well to the PTR1 receptor. Fraction (F7) showed acceptable results with no cytotoxicity. However, in vivo studies are required in the future.

A carboxylic acid isostere screen of the DHODH inhibitor Brequinar.

Dihydroorotate dehydrogenase (DHODH) enzymatic activity impacts many aspects critical to cell proliferation and survival. Recently, DHODH has been identified as a target for acute myeloid differentiation therapy. In preclinical models of AML, the DHODH inhibitor Brequinar (BRQ) demonstrated potent anti-leukemic activity. Herein we describe a carboxylic acid isostere study of Brequinar which revealed a more potent non-carboxylic acid derivative with improved cellular potency and good pharmacokinetic properties.

Synthesis, Characterization, and Biologic Activity of New Acyl Hydrazides and 1,3,4-Oxadiazole Derivatives.

Starting from isoniazid and carboxylic acids as precursors, thirteen new hydrazides and 1,3,4-oxadiazoles of 2-(4-substituted-phenoxymethyl)-benzoic acids were synthesized and characterized by appropriate means. Their biological properties were evaluated in terms of apoptosis, cell cycle blocking, and drug metabolism gene expression on HCT-8 and HT-29 cell lines. In vitro antimicrobial tests were performed by the microplate Alamar Blue assay for the anti-mycobacterial activities and an adapted agar disk diffusion technique for other non-tubercular bacterial strains. The best antibacterial activity (anti-Mycobacterium tuberculosis effects) was proved by 9. Compounds 7, 8, and 9 determined blocking of G1 phase. Compound 7 proved to be toxic, inducing apoptosis in 54% of cells after 72 h, an effect that can be predicted by the increased expression of mRNA caspases 3 and 7 after 24 h. The influence of compounds on gene expression of enzymes implicated in drug metabolism indicates that synthesized compounds could be metabolized via other pathways than NAT2, spanning adverse effects of isoniazid. Compound 9 had the best antibacterial activity, being used as a disinfectant agent. Compounds 7, 8, and 9, seemed to have antitumor potential. Further studies on the action mechanism of these compounds on the cell cycle may bring new information regarding their biological activity.

Enzymatic C-H activation of aromatic compounds through CO2 fixation.

The direct C-H carboxylation of aromatic compounds is an attractive route to the corresponding carboxylic acids, but remains challenging under mild conditions. It has been proposed that the first step in anaerobic microbial degradation of recalcitrant aromatic compounds is a UbiD-mediated carboxylation. In this study, we use the UbiD enzyme ferulic acid decarboxylase (Fdc) in combination with a carboxylic acid reductase to create aromatic degradation-inspired cascade reactions, leading to efficient functionalization of styrene through CO2 fixation. We reveal that rational structure-guided laboratory evolution can expand the substrate scope of Fdc, resulting in activity on a range of mono- and bicyclic aromatic compounds through a single mutation. Selected variants demonstrated 150-fold improvement in the conversion of coumarillic acid to benzofuran + CO2 and unlocked reactivity towards naphthoic acid. Our data demonstrate that UbiD-mediated C-H activation is a versatile tool for the transformation of aryl/alkene compounds and CO2 into commodity chemicals.

Antiparasitic Constituents of Beilschmiedia louisii and Beilschmiedia obscura and Some Semisynthetic Derivatives (Lauraceae).

The MeOH/CH2Cl2 (1:1) extracts of the roots and leaves of Beilschmiedia louisii and B. obscura showed potent antitrypanosomal activity during preliminary screening on Trypanosoma brucei brucei. Phytochemical investigation of these extracts led to the isolation of a mixture of two new endiandric acid derivatives beilschmiedol B (1) and beilschmiedol C (2), and one new phenylalkene obscurene A (3) together with twelve known compounds (4-15). In addition, four new derivatives (11a-11d) were synthesized from compound 11. Their structures were elucidated based on their NMR and MS data. Compounds 5, 6, and 7 were isolated for the first time from the Beilschmiedia genus. Additionally, the NMR data of compound 4 are given here for the first time. The isolates were evaluated for their antitrypanosomal and antimalarial activities against Tb brucei and the Plasmodium falciparum chloroquine-resistant strain Pf3D7 in vitro, respectively. From the tested compounds, the mixture of new compounds 1 and 2 exhibited the most potent antitrypanosomal activity in vitro with IC50 value of 4.91 µM.

Isolation, Absolute Configuration, and Biological Activities of Chebulic Acid and Brevifolincarboxylic Acid Derivatives from Euphorbia hirta.

Twenty new chebulic acid and brevifolincarboxylic acid derivatives, including eight optically pure or achiral compounds (1-7 and 14) and six pairs of enantiomers (8a/8b-13a/13b), along with nine known analogues (15-23), were isolated from an EtOH extract of the aerial parts of Euphorbia hirta. The absolute configurations of the new compounds were assigned based on single-crystal X-ray diffraction analysis and comparison of the experimental and calculated ECD data. Racemic or scalemic mixtures of 8-13 were isolated, and their enantiomers were analyzed by chiral-phase HPLC-ECD measurements. Compound 12 possesses an unprecedented 2H-cyclopenta[de]chromene-2,5(4H)-dione scaffold. Compounds 12, 20, and 23 displayed moderate inhibitory effects against lipopolysaccharide-induced nitric oxide production in BV-2 microglial cells, while all the isolates exhibited significant DPPH radical scavenging activities with EC50 values of 2.2-15.8 µM.

Modified multiwall carbon nanotubes display either phytotoxic or growth promoting and stress protecting activity in Ocimum basilicum L. in a concentration-dependent manner.

Carbon-based materials including multiwall carbon nanotubes (MWCNTs) have been recently implicated in a number of reports dealing with their potential use in agriculture, leading to contradictory findings. In this study, MWCNTs were successfully functionalized with carboxylic acid groups (MWCNTs-COOH) in order to increase water dispersion. Hydroponically cultured sweet basil (Ocimum basilicum L.) seedlings were subjected to four concentrations (0, 25, 50 and 100 mg L-1) of MWCNTs-COOH under three salt stress levels (0, 50 and 100 mM NaCl). An array of agronomic, physiological, analytical and biochemical parameters were evaluated in an attempt to examine the potential use of MWCNTs in plants under optimal and abiotic stress conditions. Application of MWCNTs-COOH at optimum concentration (50 mg L-1) could ameliorate the negative effects of salinity stress by increasing chlorophyll and carotenoids content and inducing non-enzymatic (i.e. phenolic content) and enzymatic antioxidant components (i.e. ascorbate peroxidase (APX), catalase (CAT) and guaiacol peroxidase (GP) activity). Furthermore, MWCNTs-COOH treatments under optimal conditions induced plant growth, while a significant increase (P ≤ 0.01) was recorded in essential oil content and compound profile. On the other hand, biochemical and epifluorescence microscopy evidence suggested that high dosage (100 mg L-1) of MWCNTs-COOH leads to toxicity effects in plant tissue. Overall, the positive response of plants to low concentrations of MWCNTs-COOH under control and abiotic stress conditions renders them as potential novel plant growth promoting and stress protecting agents, opening up new perspectives for their use in agriculture.

Design of an Automated Reagent-Dispensing System for Reaction Screening and Validation with DNA-Tagged Substrates.

Laboratory automation strategies have vast potential for accelerating discovery processes. They enable higher efficiency and throughput for time-consuming screening procedures and reduce error-prone manual steps. Automating repetitive procedures can for instance support chemists in optimizing chemical reactions. Particularly, the technology of DNA-encoded libraries (DELs) may benefit from automation techniques, since translation of chemical reactions to DNA-tagged reactants often requires screening of multiple reaction parameters and evaluation of large numbers of reactants. Here, we describe a portable, automated system for reagent dispensing that was designed from open source materials. The system was validated by performing amide coupling of carboxylic acids to DNA-linked amine and a micelle-mediated Povarov reaction to DNA-tagged hexahydropyrroloquinolines. The latter reaction required accurate pipetting of multiple components including different solvents and a surface-active reagent. Analysis of reactions demonstrated that the robotic system achieved high accuracy comparable to experimentation by an experienced chemist with the potential of higher throughput.

Organic acids produced during fermentation and sensory perception in specialty coffee using yeast starter culture.

Volatile and non-volatile compounds in coffee directly affect the beverage's quality. This study aimed to demonstrate how the organic acids and volatile profiles were impacted by coffee fermentation using four starter cultures (Meyerozyma caribbica (CCMA 0198), Saccharomyces cerevisiae (CCMA 0543), Candida parapsilosis (CCMA0544), and Torulaspora delbrueckii (CCMA 0684)) inoculated in two varieties of coffee (Bourbon Amarelo and Canário Amarelo) using natural and pulped natural processing methods and sensory perception. Real-time PCR (qPCR) was used to verify the dynamic behavior of yeast populations. Organic acids were detected using high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) was used to detected volatile compounds. Sensory analysis was performed on the roasted coffee. Citric, malic, succinic, lactic, oxalic, isobutyric, and propionic acids and 105 volatile compounds were detected. At the beginning of fermentation, treatments with natural processing presented higher number of volatiles compounds. After fermentation, the main compounds groups were acids, alcohols, and aldehydes. The perception of sensory attribute (fruity, nutty, cocoa) varied with the coffee variety, type of processing, and type of inoculum. The use of yeasts is an alternative for sensorial differentiation of coffee variety Canário Amarelo and Bourbon Amarelo. The stainless-steel containers showed good results for coffee fermentation.

LC-MS/MS assay coupled with carboxylic acid magnetic bead affinity capture to quantitatively measure cationic host defense peptides (HDPs) in complex matrices with application to preclinical pharmacokinetic studies.

Synthetic host defense peptides (HDP) are a new class of promising therapeutic agents with potential application in a variety of diseases. RP-182 is a 10mer synthetic HDP design, which selectively reduces M2-like tumor associated macrophages via engagement with the cell surface lectin receptor MRC1/CD206 and is currently being developed as an innate immune defense regulator to improve anti-tumor immunity in immunologically cold tumors. Herein, we describe a sensitive and specific liquid chromatography (LC) coupled to quadrupole electron spray tandem mass spectrometry method to measure positively charged HDPs and HDP peptide fragments in complex biological matrices. Carboxylic acid magnetic beads were used as an affinity-capturing agent to extract the positively charged RP-182 from both mouse plasma and tissue homogenates. Beads were eluted with 0.1% (v/v) formic acid and chromatographic separation was achieved on a Waters 2.1 × 100 mm, 3.5 µm XSelect Peptide CSH C18 column with a Vanguard pre-column of the same phase. MS/MS was performed on a Thermo TSQ Quantiva triple quadrupole mass spectrometer operating in Selected Reaction Monitoring (SRM) mode fragmenting the plus three parent ion 458.9+3 and monitoring ions 624.0+2, 550.5+2, and 597.3+1 for RP-182 and 462.4+3 > 629.1+2, 555.5+2, and 607.3+1 for isotopic RP-182 standard. The assay had good linearity ranging from 1 ng to 1000 ng in mouse plasma with the lower limit of detection for RP-182 at 1 ng in mouse plasma with good intra- and inter-sample precision and accuracy. Recovery ranged from 66% to 77% with minimum matrix effects. The method was successfully applied to an abbreviated pharmacokinetic study in mice after single IP injection of RP-182. The method was successfully tested on a second HDP, the 17mer D4E1, and the cationic human peptide hormone ghrelin suggesting that it might be a general sensitive method applicable to quantifying HDP peptides that are difficult to extract.