PCR-Based Strategy for Introducing CRISPR/Cas9 Machinery into Hematopoietic Cell Lines.

Acute myeloid leukemia is a complex heterogeneous disease characterized by the clonal expansion of undifferentiated myeloid precursors. Due to the difficulty in the transfection of blood cells, several hematological models have recently been developed with CRISPR/Cas9, using viral vectors. In this study, we developed an alternative strategy in order to generate CRISPR constructs by fusion PCR, which any lab equipped with basic equipment can implement. Our PCR-generated constructs were easily introduced into hard-to-transfect leukemic cells, and their function was dually validated with the addition of MYBL2 and IDH2 genes into HEK293 cells. We then successfully modified the MYBL2 gene and introduced the R172 mutation into the IDH2 gene within NB4 and HL60 cells that constitutively expressed the Cas9 nuclease. The efficiency of mutation introduction with our methodology was similar to that of ribonucleoprotein strategies, and no off-target events were detected. Overall, our strategy represents a valid and intuitive alternative for introducing desired mutations into hard-to-transfect leukemic cells without viral transduction.

A Carbon Paste Electrode Modified by Bentonite and l-Cysteine for Simultaneous Determination of Ascorbic and Uric Acids: Application in Biological Fluids.

A novel modification of a paste carbon electrode by Bentonite (Bent) and l-Cysteine (l-Cyst) was carried out for uric acid (UA) and ascorbic acid (AA) detection and quantification. Morphological and compositional characterization of the electrode surface were carried out using electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopic analysis (EDS). Cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques were used to analyze UA and AA. The obtained sensor shows a good stability, sensibility, selectivity, and regeneration ability. Accordingly, the limit of detection (LOD) is found to be 0.031 µm and 9.6 µm for UA and AA, respectively. A good linearity in the range of 0.1 to 100 µm for UA and 10 to 1000 µm for AA was obtained. The peak-to-peak separation of UA-AA (ΔEUA-AA ) was determined to be 330 mV. In addition, the sensor is applied successfully to monitor UA and AA in serum samples.

Influence of Metal Salts Addition on Physical and Electrochemical Properties of Ethyl and Propylammonium Nitrate.

In this work, we deepen in the characterization of two protic ionic liquids (PILs), ethylammonium nitrate (EAN) and propylammonium nitrate (PAN). With this aim, we determined the influence of inorganic nitrate salts addition on their physical properties and their electrochemical potential window (EPW). Thus, experimental measurements of electrical conductivity, density, viscosity, refractive index and surface tension of mixtures of {EAN or PAN + LiNO3, Ca(NO3)2, Mg(NO3)2 or Al(NO3)3} at a temperature range between 5 and 95 °C are presented first, except for the last two properties which were measured at 25 °C. In the second part, the corresponding EPWs were determined at 25 °C by linear sweep voltammetry using three different electrochemical cells. Effect of the salt addition was associated mainly with the metal cation characteristics, so, generally, LiNO3 showed the lower influence, followed by Ca(NO3)2, Mg(NO3)2 or Al(NO3)3. The results obtained for the EAN + LiNO3 mixtures, along with those from a previous work, allowed us to develop novel predictive equations for most of the presented physical properties as functions of the lithium salt concentration, the temperature and the water content. Electrochemical results showed that a general order of EPW can be established for both PILs, although exceptions related to measurement conditions and the properties of the mixtures were found.

Novel Pyridazin-3(2H)-one-Based Guanidine Derivatives as Potential DNA Minor Groove Binders with Anticancer Activity.

Novel aryl guanidinium analogues containing the pyridazin-3(2H)-one core were proposed as minor groove binders (MGBs) with the support of molecular docking studies. The target dicationic or monocationic compounds, which show the guanidium group at different positions of the pyridazinone moiety, were synthesized using the corresponding silyl-protected pyridazinones as key intermediates. Pyridazinone scaffolds were converted into the adequate bromoalkyl derivatives, which by reaction with N,N'-di-Boc-protected guanidine followed by acid hydrolysis provided the hydrochloride salts 1-14 in good yields. The ability of new pyridazin-3(2H)-one-based guanidines as DNA binders was studied by means of DNA UV-thermal denaturation experiments. Their antiproliferative activity was also explored in three cancer cell lines (NCI-H460, A2780, and MCF-7). Compounds 1-4 with a bis-guanidinium structure display a weak DNA binding affinity and exhibit a reasonable cellular viability inhibition percentage in the three cancer cell lines studied.

Aberrant Alternative Splicing in U2af1/Tet2 Double Mutant Mice Contributes to Major Hematological Phenotypes.

Mutations in splicing factors are recurrent somatic alterations identified in myelodysplastic syndromes (MDS) and they frequently coincide with mutations in epigenetic factors. About 25% of patients present concurrent mutations in such pathways, suggesting a cooperative role in the pathogenesis of MDS. We focused on the splicing factor U2AF1 involved in the recognition of the 3' splice site during pre-mRNA splicing. Using a CRISPR/Cas9 system, we created heterozygous mice with a carboxy-terminal truncated U2af1 allele (U2af1mut/+), studied the U2af1mut/+ hematopoietic system, and did not observe any gross differences in both young (12-13 weeks) and old (23 months) U2af1mut/+ mice, except for a reduction in size of approximately 20%. However, hematopoietic stem/progenitor cells lacked reconstitution capacity in transplantation assays and displayed an aberrant RNA splicing by RNA sequencing. We also evaluated U2af1mut/+ in conjunction with Tet2-deficiency. Novel double mutant U2af1mut/+Tet2-/- mice showed increased monogranulocytic precursors. Hematopoietic stem/progenitor cells were also enhanced and presented functional and transcriptomic alterations. Nonetheless, U2af1mut/+Tet2-/- mice did not succumb to MDS disease over a 6-month observation period. Collectively, our data suggest that cooperation between mutant U2af1 and Tet2 loss is not sufficient for MDS initiation in mice.

A Single-Run Next-Generation Sequencing (NGS) Assay for the Simultaneous Detection of Both Gene Mutations and Large Chromosomal Abnormalities in Patients with Myelodysplastic Syndromes (MDS) and Related Myeloid Neoplasms.

Myelodysplastic syndromes (MDS) and myelodysplastic/myeloproliferative neoplasms are clonal disorders that share most of their cytogenetic and molecular alterations. Despite the increased knowledge of the prognostic importance of genetics in these malignancies, next-generation sequencing (NGS) has not been incorporated into clinical practice in a validated manner, and the conventional karyotype remains mandatory in the evaluation of suspected cases. However, non-informative cytogenetics might lead to an inadequate estimation of the prognostic risk. Here, we present a novel targeted NGS-based assay for the simultaneous detection of all the clinically relevant genetic alterations associated with these disorders. We validated this platform in a large cohort of patients by performing a one-to-one comparison with the lesions from karyotype and single-nucleotide polymorphism (SNP) arrays. Our strategy demonstrated an approximately 97% concordance with standard clinical assays, showing sensitivity at least equivalent to that of SNP arrays and higher than that of conventional cytogenetics. In addition, this NGS assay was able to identify both copy-neutral loss of heterozygosity events distributed genome-wide and copy number alterations, as well as somatic mutations within significant driver genes. In summary, we show a novel NGS platform that represents a significant improvement to current strategies in defining diagnosis and risk stratification of patients with MDS and myeloid-related disorders.

Screen-printed electrodes-based technology: Environmental application to real time monitoring of phenolic degradation by phytoremediation with horseradish roots.

The following is a description of a simple strategy for monitoring phenolic pollutants from highly-contaminated water samples. These phenolic compounds are removed from tap water using horseradish roots (Raphanus sativus) that contain peroxidase as catalyst and H2O2 to generate hydroxyl radicals. The later (•OH) acts on the aromatic structure, causing them to degrade to non-toxic by-products. The tool used to follow up the evolution of the process is based on screen-printed carbon electrodes (SPCEs) as electrochemical sensor for simultaneous detection of hydroquinone (Epa at 0.047 V), m-cresol (Epa at 0.506 V) and 4-nitrophenol (Epa at 0.696 V) by differential pulse voltammetry (DPV). This electroanalytical methodology enables close monitoring of the situation and rapid sensor response time. Furthermore, this direct methodology works for opaque and heterogeneous samples, as tap water with chopped horseradish roots, without any treatment of samples previously to the analysis. For better knowledge of the electrodic-transfer process, the electrochemical behavior of these phenolic compounds by cyclic voltammetry (CV) is also included. This simple methodology shows a low detection limit (below to 5 µM) and an excellent selectivity (peak potential separation between them up to 200 mV or greater) in a linear range of three orders of concentration (from 1-5 µM to 1 mM) for all of the analytes studied. The DPV responses of the phenolic compounds during the phytoremediation process are simultaneously monitored by this direct, cheap, reproducible (RSD < 2.3%) and rapid DPV-SPCE electroanalytical methodology. Portable device as electrochemical sensor with this optimized and validated technology can be applied for decentralized analysis, on-site assays and rapid screening purposes. The use of the horseradish roots achieves the total elimination of phenolic pollutants in concentrations 1000 times higher than the legal limits in less than 1 h.

Analysis of SNP Array Abnormalities in Patients with DE NOVO Acute Myeloid Leukemia with Normal Karyotype.

Nearly 50% of patients with de novo acute myeloid leukemia (AML) harbor an apparently normal karyotype (NK) by conventional cytogenetic techniques showing a very heterogeneous prognosis. This could be related to the presence of cryptic cytogenetic abnormalities (CCA) not detectable by conventional methods. The study of copy number alterations (CNA) and loss of heterozygozity (LOH) in hematological malignancies is possible using a high resolution SNP-array. Recently, in clinical practice the karyotype study has been complemented with the identification of point mutations in an increasing number of genes. We analyzed 252 de novo NK-AML patients from Hospital La Fe (n = 44) and from previously reported cohorts (n = 208) to identify CCA by SNP-array, and to integrate the analysis of CCA with molecular alterations detected by Next-Generation-sequencing. CCA were detected in 58% of patients. In addition, 49% of them harbored CNA or LOH and point mutations, simultaneously. Patients were grouped in 3 sets by their abnormalities: patients carrying several CCA simultaneously, patients with mutations in FLT3, NPM1 and/or DNMT3A and patients with an amalgam of mutations. We found a negative correlation between the number of CCA and the outcome of the patients. This study outlines that CCA are present in up to 50% of NK-AML patients and have a negative impact on the outcome. CCA may contribute to the heterogeneous prognosis.

CRISPR to fix bad blood: a new tool in basic and clinical hematology.

Advances in genome engineering in the last decade, particularly in the development of programmable nucleases, have made it possible to edit the genomes of most cell types precisely and efficiently. Chief among these advances, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a novel, versatile and easy-to-use tool to edit genomes irrespective of their complexity, with multiple and broad applications in biomedicine. In this review, we focus on the use of CRISPR/Cas9 genome editing in the context of hematologic diseases and appraise the major achievements and challenges in this rapidly moving field to gain a clearer perspective on the potential of this technology to move from the laboratory to the clinic. Accordingly, we discuss data from studies editing hematopoietic cells to understand and model blood diseases, and to develop novel therapies for hematologic malignancies. We provide an overview of the applications of gene editing in experimental, preclinical and clinical hematology including interrogation of gene function, target identification and drug discovery and chimeric antigen receptor T-cell engineering. We also highlight current limitations of CRISPR/Cas9 and the possible strategies to overcome them. Finally, we consider what advances in CRISPR/Cas9 are needed to move the hematology field forward.

USH2A Gene Editing Using the CRISPR System.

Usher syndrome (USH) is a rare autosomal recessive disease and the most common inherited form of combined visual and hearing impairment. Up to 13 genes are associated with this disorder, with USH2A being the most prevalent, due partially to the recurrence rate of the c.2299delG mutation. Excluding hearing aids or cochlear implants for hearing impairment, there are no medical solutions available to treat USH patients. The repair of specific mutations by gene editing is, therefore, an interesting strategy that can be explored using the CRISPR/Cas9 system. In this study, this method of gene editing is used to target the c.2299delG mutation on fibroblasts from an USH patient carrying the mutation in homozygosis. Successful in vitro mutation repair was demonstrated using locus-specific RNA-Cas9 ribonucleoproteins with subsequent homologous recombination repair induced by an engineered template supply. Effects on predicted off-target sites in the CRISPR-treated cells were discarded after a targeted deep-sequencing screen. The proven effectiveness and specificity of these correction tools, applied to the c.2299delG pathogenic variant of USH2A, indicates that the CRISPR system should be considered to further explore a potential treatment of USH.

Effects of dietary cadmium exposure on osmoregulation and urine concentration mechanisms of the semi-desert rodent Meriones shawi.

Contamination by cadmium in the environment is of great concern because of its toxicity and threats to human and animal health. The current study was conducted to investigate the effects of a cadmium contaminated diet on the osmoregulation and urine concentration mechanisms of the semi-desert rodent Meriones shawi and eventual accumulation of this metal in vital organs such as the kidneys, which are directly implicated in water regulation. Originally, we used Differential Pulse Anodic Stripping Voltammetry (DPASV) to avoid the matrix interference due to the highly organic content in the biological samples. Our results show that Meriones shawi successfully maintained a homeostasis state and presented a special adaptation to regulate urine volume during cadmium exposure by decreasing diuresis and increasing urinary osmolality. The plasma osmolality and hematocrit remained constant throughout the experiment. The stripping signals of cadmium are linear up to 0.3-100 µg L(-1) range, with a detection limit of 0.28 µg L(-1). The DPASV technique was useful for easy, fast, selective and sensitive determination of Cd, which permits working at cellular concentration. This gives us more information about the chemical form in which Cd is introduced into the organ, as well as the intracellular Cd quantities. This study has potential importance if this valuable novel animal model, imitating human and animal environmental chronic exposure to Cd, could serve as an appropriate terrestrial biomonitor for Cd contaminated sites. These results are encouraging in the context of developing a low-cost and fast technology for the detection of pollutants and for studying the impairment caused by their effects.

Determining alpha-tocopherol distributions between the oil, water, and interfacial regions of macroemulsions: novel applications of electroanalytical chemistry and the pseudophase kinetic model.

The assumptions of the pseudophase model for chemical reactivity in homogeneous microemulsions are used to determine the distribution of alpha-tocopherol (TOC) in macroemulsions from changes in the observed rate constant (k(obs)) for reaction between 4-hexadecylarenediazonium ion (16-ArN2+) probe and TOC with increasing surfactant concentration. Two partition constants are needed to describe the distribution of TOC or other antioxidant (AO) or polar uncharged molecule between the oil and interfacial (P(O)(I)) and the water and interfacial (P(W)(I)) regions of stirred fluid emulsions. The observed rate constants are measured electrochemically. Here we report values of P(O)(I) and P(W)(I) for the distribution of TOC in octane/acidic water/C12E6 (hexaethylene glycol monododecyl ether) and octane/acidic water/C12E4 (Brij 30, tetraethylene glycol dodecyl ether) emulsions obtained by fitting two kinetic data sets with an equation based on the pseudophase model and solving two equations in two unknowns. The partition constants were used to estimate the %TOC in each region of the emulsions. In 1:1 oil:water C12E6 emulsions, at 2% volume fraction of C12E6, 73% of TOC is in the interfacial region, 26% in the octane and about 1% in the water. The distributions of TOC in C12E4 emulsions are similar. The combined electrochemical-pseudophase model approach is applicable to any AO or other compound that reacts with 16-ArN2+. The second-order rate constant, k(I), for reaction in the interfacial region of the emulsions is also estimated from the kinetic data and is about the same for both surfactants (k(I) approximately 0.1-0.2 M(-1)s(-1)) showing that the medium properties of the interfacial regions of C12E6 and C12E4 emulsions are similar. Comparison of these rate constants for a variety of AOs may provide a scale of AO efficiency that is independent of AO distribution between the oil, interfacial and aqueous regions of emulsions.

Kinetics and mechanism of the reaction between ascorbic acid derivatives and an arenediazonium salt: cationic micellar effects.

The effects of tetradecyltrimethylammonium bromide, TTAB, and hexadecyl-trimethylammonium bromide, CTAB, micellar systems on the reaction of 3-methylbenzenediazonium, 3MBD, tetrafluoroborate with ascorbic acid, VC, and with the hydrophobic derivatives 6-O-dodecyl-L-ascorbic acid, VC12, and 6-O-palmitoyl-L-ascorbic acid, VC16, were investigated at different pH values by employing a combination of UV-vis spectroscopy and high-performance liquid chromatography, HPLC, techniques. Previous studies in the absence of surfactant showed that the reaction between 3MBD and VC derivatives takes place through a rate-limiting decomposition of a transient diazo ether, DE, formed from reaction between 3MBD and the monoanion form of ascorbic acid, VC-, in a rapid preequilibrium step. In the presence of a fixed [CTAB], the kinetics of the reaction of 3MBD with VC follows a saturation kinetics similar to that observed in its absence, but for the reaction with VC12 and VC16, only the first linear portions of the saturation profiles could be obtained because k(obs) values become too large. HPLC analyses of the reaction mixtures show that no unexpected products are detected, suggesting that cationic micelles do not modify the mechanism of the reaction. Analyses of the kinetic data allowed estimations of the rate constant for the decomposition of the diazo ether and of the equilibrium constant for the formation of DE in the presence of CTAB micelles, which is approximately 6 times higher than in its absence; this suggests that CTAB micelles promote diazo ether formation. At constant [antioxidant], the variations of k(obs) for the reactions with VC, VC12, or VC16 follow bell-shaped curves, with rate enhancements of up to 2-3-fold for VC with respect to the value in the absence of surfactant. The rate maximum for the reaction of 3MBD with VC is reached at [CTAB] = 0.02 M suggesting a CTAB-induced rate increase, i.e., micellar catalysis; meanwhile the rate maximum for the reaction with VC12 and VC16, which may behave as amphiphilic compounds, is reached at [CTAB] approximately 1 x 10(-4) M, a concentration about 10 times lower than its critical micelle concentration, cmc, in pure water, but only approximately 3 times lower than the cmc of VC16, suggesting the formation of reactive CTAB-VC12 and CTAB-VC16 premicellar aggregates. Kinetic and HPLC results are consistent with the predictions of the pseudophase model and are interpreted in terms of 3MBD ions sampling in the aqueous bulk phase and the micellar effects on the different equilibrium involved. The results should contribute to a better understanding of the role of compartmentalized systems on the efficiency with which hydrophilic and hydrophobic reductants such as ascorbic acid derivatives interact with potentially mutagenic and carcinogenic ArN2+ ions.

Inhibition of the beta-cyclodextrin catalyzed dediazoniation of 4-nitrobenzenediazonium tetrafluoroborate. blocking effect of sodium dodecyl sulfate.

We have investigated the effects of sodium dodecyl sulfate, SDS, on the reaction between 4-nitrobenzenediazonium, 4NBD, ions and beta-cyclodextrin, beta-CD, under acidic conditions at T = 60 degrees C by employing a combination of spectrophotometric, chromatographic, and conductometric techniques. Previous studies under acidic conditions indicate that the secondary -OH groups of beta-CD solvate 4NBD ions, which are included in the beta-CD cavity, leading to the formation of a highly unstable transient diazo ether complex that undergoes homolytic fragmentation with an observed rate constant about 1700 times higher than that in pure aqueous acid solution (t(1/2) = 6 h at T = 60 degrees C) when [beta-CD]/[4NBD] = 40. Addition of SDS to a 4NBD/beta-CD system makes the k(obs) values decrease up to its value in a SDS micellar solution, which is similar to that in aqueous acid solution. Dediazoniation product distribution is significantly affected; the reaction between 4NBD and beta-CD ([beta-CD]/[4NBD] = 40), in the absence of SDS, proceeds exclusively through a homolytic mechanism leading to the quantitative formation of nitrobenzene, ArH, but addition of SDS turns over the mechanism by promoting the heterolytic mechanism. In addition, mixtures of 4-nitrophenol, ArOH, and ArH dediazoniation products are formed; their relative yields depend on the amount of added SDS so that at very high [SDS(T)], the heterolytic mechanism becomes the predominant one. Results are consistent with conductometric measurements showing that addition of beta-CD to an aqueous surfactant solution inhibits micelle formation and elevates CMC(app) values because CD encapsulation of surfactant monomers competes with the micellization process and are interpreted in terms of SDS monomers blocking the beta-CD cavity by forming a nonreactive complex, releasing 4NBD to the bulk solution.

Dediazoniation in SDS/BuOH/H2O reverse micelles: structural parameters, kinetics, and mechanism of the reaction.

Dediazoniation of o-methylbenzenediazonium tetrafluoroborate was investigated in SDS/BuOH/H2O (SDS = sodium dodecyl sulfate) reverse micelles, RMs, and, for comparison, in binary BuOH/H2O mixtures by employing a combination of spectrophotometric and chromatographic techniques. RMs were characterized by steady-state fluorescence; the data indicate that the aggregation number of the RMs increase upon increasing [SDS], while the radius of the water pool is mainly controlled by the amount of water in the system, and that the thickness of the interfacial region increases upon increasing the amount of BuOH in the system, in agreement with literature reports. Experimental evidence suggests that dediazoniation mainly takes place in the interfacial region of the RMs. Kinetic data show that a turnover from the heterolytic to the homolytic mechanism takes place about pH = 5; the variation of the observed rate constants, k(obs,) with pH following an S-shaped curve. At pH approximately 2, k(obs) values are insensitive to solvent composition both in RMs and in the binary mixture; however, k(obs) values in RMs are slightly lower than those in BuOH/H2O, probably due to the presence of SDS. High-performance liquid chromatography analyses of the reaction mixture indicate, in both RMs and in binary mixtures, the main dediazoniation products are the heterolytic ArOH and ArOBu, their yields depending on the composition of the system, and only small (<10%) amounts of the reduction ArH product were detected. The data at low pH are interpreted in terms of a DN + AN dediazoniation mechanism, i.e., a rate-limiting formation of an extremely reactive aryl cation that further reacts with available nucleophiles in the solvation shell.

Determining partition constants of polar organic molecules between the oil/interfacial and water/interfacial regions in emulsions: a combined electrochemical and spectrometric method.

We have developed a new approach for estimating distributions of polar additives in opaque, surfactant based, macroemulsions based on the pseudophase model for homogeneous micellar and microemulsion solutions. The distribution of a polar additive, such as an antioxidant, AO, within emulsions is expressed in terms of two partition constants, one between the oil and interfacial regions, P(O)I, and the other between the water and interfacial regions, P(W)I. To estimate values for P(O)I and P(W)I requires fitting two independent data sets with two independent mathematical relations and solving equations simultaneously for the two parameters. The experimental protocols were developed for determining the partition constants of tertbutylhydroquinone, TBHQ, in a stirred emulsion composed of octane, dilute aqueous acid, and hexaethyleneglycol monododecyl ether, C12E6. One data set was obtained by electrochemical determination of the observed rate constant, k(obs), for reaction of TBHQ with an arenediazonium ion probe as a function of C12E6 volume fraction. The second data set was obtained by determining the partition constant, P(O)W, of TBHQ between octane and water in the absence of surfactant by UV-visible spectrometry. TBHQ is almost 30 times more soluble in water than octane: P(O)W = 27.5. The values of the partition constants in the emulsion are P(O)I = 1.84 x 10(4) and P(W)I = 6.73 x 10(2). The partition constants were used to estimate the fraction of TBHQ in each region; for example, 96% of the TBHQ is located in the interfacial region at 0.02 volume fraction of C12E6. Our approach is quite general and should be applicable to any polar organic compound that reacts with the arenediazonium ion probe in emulsions composed of virtually any type of oil and surfactant. Comparisons of the rate constants for reaction of the antioxidant in the interfacial region of the emulsion, which can be obtained from the electrochemical results, may lead to a scale of antioxidant efficiency that is independent of the distribution of the antioxidant in the emulsion.

Monitoring dediazoniation product formation by high-performance liquid chromatography after derivatization.

A derivatization protocol that exploits the rapid reaction between arenediazonium ions and a suitable coupling agent followed by high-performance liquid chromatography analyses of the reaction mixture was employed to determine the product distribution, the rate constants for product formation and the association constant of 4-nitrobenzenediazonium, PNBD, ion with beta-cyclodextrin, beta-CD. The derivatization of PNBD with the coupling agent leads to the formation of a stable azo dye that prevents by-side reactions of PNBD with the solvents of the mobile phase, including water, or the metallic parts of the chromatographic system that would eventually lead to erroneous identification and quantification of dediazoniation products. The results show that in the presence of beta-CD, nitrobenzene is formed at the expense of 4-nitrophenol, which is the major product in its absence. The observed rate constants for the interaction between PNBD and beta-CD increase upon increasing [beta-CD] showing a saturation profile indicative of the formation of an inclusion complex between PNBD and beta-CD. By fitting the experimental data to a simplified Lineaweaver-Burk equation, the corresponding association constant and the maximum acceleration rate of beta-CD towards PNBD were estimated. The protocol is applicable under a variety of experimental conditions provided that the rate of the coupling reaction is much faster than that of dediazoniation.

Micellar effects on dediazoniation and on azo coupling reactions.

The effects of a sodium dodecyl sulfate, SDS, micellar solution on the coupling rates of two arenediazonium ions, ArN(2)(+), with the hydrophobic 1-naphthylamine, 1NA and N-(1-naphthyl) ethylenediamine, NED, coupling agents and with the hydrophilic Na salt of 2-naphthol-6-sulfonic acid, 2N6S, have been studied. First, we explored the micellar effects on the thermal decomposition of the arenediazonium ions. The observed rate constants are slightly depressed or increased, depending on the nature of ArN(2)(+), compared to those in pure water upon increasing [SDS]. Estimations of the corresponding association constant to the micelle indicate that a significant fraction of the arenediazonium ions are incorporated into the micelles even at low surfactant concentrations. The sulfonate group in 2N6S prevents its incorporation into the micellar aggregate due to the electrostatic barrier imposed by the micelles and, in consequence, the coupling reaction is inhibited. In contrast, when employing the naphthylamine derivatives, the observed rate constant increase rapidly up to a maximum at [SDS]