Aminobenzohydrazide based colorimetric and ‘turn-on’ fluorescence chemosensor for selective recognition of fluoride

Chemosensors based on aminobenzohydrazide Schiff bases bearing pyrene/anthracene as fluorophores have been designed and synthesized for F⁻ ion recognition. The addition of fluoride ions to the receptors causes a dramatically observable colour change from pale yellow to brown/red. ¹H NMR studies confirm that the F⁻ ion facilitates its recognition by forming hydrogen bond with hydrogens of amide and amine groups. Moreover these sensors have also been successfully applied to detection of fluoride ion in commercial tooth paste solution.     

Expansion of time window for mass spectrometric measurement of amide hydrogen/deuterium exchange reactions

Backbone amide hydrogen exchange rates can be used to describe the dynamic properties of a protein. Amide hydrogen exchange rates in a native protein may vary from milliseconds (ms) to several years. Ideally, the rates of all amide hydrogens of the analyte protein can be determined individually. To achieve this goal, monitoring of a wider time window is critical, in addition to high sequence coverage and high sequence resolution. Significant improvements have been made to hydrogen/deuterium exchange mass spectrometry methods in the past decade for better sequence coverage and higher sequence resolution. On the other hand, little effort has been made to expand the experimental time window to accurately determine exchange rates of amide hydrogens. Many fast exchanging amide hydrogens are completely exchanged before completion of a typical short exchange time point (10–30 s) and many slow exchanging amide hydrogens do not start exchanging before a typical long exchanging time point (1–3 h). Here various experimental conditions, as well as a quenched‐flow apparatus, are utilized to monitor cytochrome c amide hydrogen exchange behaviors over more than eight orders of magnitude (0.0044–1 000 000 s), when converted into the standard exchange condition (pH 7 and 23°C). Copyright © 2010 John Wiley & Sons, Ltd.     

Site-specific amide hydrogen exchange in melittin probed by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

Electron capture dissociation (ECD) has been proposed to be a non-ergodic process, i.e. to provide backbone dissociation of gas-phase peptides faster than randomization of the imparted energy. One potential consequence could be that ECD can fragment deuterated peptides without causing hydrogen scrambling and thereby provide amino acid residue-specific amide hydrogen exchange rates. Such a feature would improve the resolution of approaches involving solution-phase amide hydrogen exchange combined with mass spectrometry for protein structural characterization. Here, we explore this hypothesis using melittin, a haemolytic polypeptide from bee venom, as our model system. Exchange rates in methanol calculated from consecutive c-type ion pairs show some correlation with previous NMR data: the amide hydrogens of leucine 13 and alanine 15, located at the unstructured kink surrounding proline 14 in the melittin structure adopted in methanol, appear as fast exchangers and the amide hydrogens of leucine 16 and lysine 23, buried within the helical regions of melittin, appear as slow exchangers. However, calculations based on c-type ions for other amide hydrogens do not correlate well with NMR data, and evidence for deuterium scrambling in ECD was obtained from z*-type ions.     

Intramolecular migration of amide hydrogens in protonated peptides upon collisional activation

Presently different opinions exist as to the degree of scrambling of amide hydrogens in gaseous protonated peptides and proteins upon collisional activation in tandem mass spectrometry experiments. This unsettled controversy is not trivial, since only a very low degree of scrambling is tolerable if collision-induced dissociation (CID) should provide reliable site-specific information from (1)H/(2)H exchange experiments. We have explored a series of unique, regioselectively deuterium-labeled peptides as model systems to probe for intramolecular amide hydrogen migration under low-energy collisional activation in an orthogonal quadrupole time-of-flight electrospray ionization (Q-TOF ESI) mass spectrometer. These peptides contain a C-terminal receptor-binding sequence and an N-terminal nonbinding region. When the peptides form a receptor complex, the amide hydrogens of the interacting sequences are protected against exchange with the solvent, while the amide hydrogens of the nonbinding sequences exchange rapidly with the solvent. We have utilized such long-lived complexes to generate peptides labeled with deuterium in either the binding or nonbinding region, and the expected regioselectivity of this labeling was confirmed after pepsin proteolysis. CID of such deuterated peptides, [M + 2H](2+), yielded fragment ions (b- and y-ions) having a deuterium content that resemble the theoretical values calculated for 100% scrambling. Thus, complete randomization of all hydrogen atoms attached to nitrogen and oxygen occurs in the gaseous peptide ion prior to its dissociation.     

A new method for the accurate determination of the isotopic state of single amide hydrogens within peptides using Fourier transform ion cyclotron resonance mass spectrometry

A new method is presented to accurately determine the probability of having a deuterium or hydrogen atom on a specific amide position within a peptide after deuterium/hydrogen (D/H) exchange in solution. Amide hydrogen exchange has been proven to be a sensitive probe for studying protein structures and structural dynamics. At the same time, mass spectrometry in combination with physical fragmentation methods is commonly used to sequence proteins based on an amino acid residue specific mass analysis. In the present study it is demonstrated that the isotopic patterns of a series of peptide fragment ions obtained with capillary-skimmer dissociation, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer, can be used to calculate the isotopic state of specific amide hydrogens. This calculation is based on the experimentally observed isotopic patterns of two consecutive fragments and on the isotopic binomial distributions of the atoms in the residue constituting the difference between these two consecutive fragments. The applicability of the method is demonstrated by following the sequence-specific D/H exchange rate in solution of single amide hydrogens within some peptides.     

Hydrogen atom scrambling in selectively labeled anionic peptides upon collisional activation by MALDI tandem time-of-flight mass spectrometry

We have previously shown that peptide amide hydrogens undergo extensive intramolecular migration (i.e., complete hydrogen scrambling) upon collisional activation of protonated peptides (Jørgensen et al. J. Am. Chem. Soc. 2005, 127, 2785–2793). The occurrence of hydrogen scrambling enforces severe limitations on the application of gas-phase fragmentation as a convenient method to obtain information about the site-specific deuterium uptake for proteins and peptides in solution. To investigate whether deprotonated peptides exhibit a lower level of scrambling relative to their protonated counterparts, we have now measured the level of hydrogen scrambling in a deprotonated, selectively labeled peptide using MALDI tandem time-of-flight mass spectrometry. Our results conclusively show that hydrogen scrambling is prevalent in the deprotonated peptide upon collisional activation. The amide hydrogens (¹H/²H) have migrated extensively in the anionic peptide, thereby erasing the original regioselective deuteration pattern obtained in solution.     

Studying protein-carbohydrate interactions by amide hydrogen/deuterium exchange mass spectrometry

Protein-carbohydrate interactions play a significant role in biological processes. Presented here is the novel application of amide hydrogen/deuterium exchange mass spectrometry (amide exchange-MS) to the study of the interaction between a protein and its carbohydrate substrate. The degree of deuterium incorporation into hen egg lysozyme was monitored with and without substrate to verify that a carbohydrate can provide sufficiently stable protection of the amide hydrogen atoms in a protein's backbone from exchange with deuterated solvent. The substrate protected a number of amide hydrogens from exchange, implying that protein-carbohydrate binding systems will be compatible with amide exchange-MS. Endopolygalacturonase-II (EPG-II) from Aspergillus niger, a pectin-degrading enzyme, was chosen as the first carbohydrate-binding system to be extensively studied using quenched amide exchange-MS. Monitoring the changes in deuterium incorporation of EPG-II in the presence and absence of an oligomer of galacturonic acid implied the location of substrate binding. This study demonstrates the ability of amide exchange-MS to investigate protein-carbohydrate interactions.     

Improving foldamer synthesis through protecting group induced unfolding of aromatic oligoamides

The hydrogen bond rigidified backbones of aromatic oligoamides are temporarily interrupted by replacing the amide hydrogens with the acid-labile 2,4-dimethoxybenzyl (DMB) group, which allows the efficient preparation of long folding oligomers that, upon removal of the DMB groups, fold into multiturn helices. [structure: see text]     

Far‐ and Mid‐Infrared Spectroscopic Analysis of the Substrate‐Induced Structural Dynamics of Respiratory Complex I

The catalytic activity of the respiratory NADH:ubiquinone oxidoreductase (complex I) is based on conformational reorganizations. Herein we probe the effect of substrates on the conformational flexibility of complex I by means of ¹H/²H exchange kinetics at the level of the amide proton in the mid‐infrared spectral range (1700–1500 cm^?1). Slow, medium, and fast exchanging domains are distinguished that reveal different accessibilities to the solvent. Whereas amide hydrogens undergo rapid exchange with the solvent in an open structure, hydrogens experience much slower exchange when they are involved in H‐bonded structures or when they are sterically inaccessible for the solvent. The results indicate a structure that is more open in the presence of both NADH and quinon. Complementary information on the overall internal hydrogen bonding of the protein was probed in the far infrared (300–30 cm^?1), a spectral range that includes a continuum mode of the hydrogen bonding signature.     

Efficient fluoride-selective fluorescent host: experiment and theory

A new naphthalene derivative containing a urea group at the 1,8-position of naphthalene was synthesized and showed a unique absorption and fluorescence peak with fluoride ion. Calculations suggested that a new peak was attributed to the increased anionic character of urea nitrogen due to the strongly charged hydrogen bonding between fluoride and amide protons of the urea. The fluoride selectivity among halides (F(-), Cl(-), Br(-)) comes from the fact that the fluoride approaches much closer to the amide protons than other halides and resides in the cavity with fast dynamics. The nature of electronic transitions that were analyzed from the calculations by the collective electronic oscillator method also supports the anionic nature of the complex between host and fluoride.     

Anion receptors: a new class of amide/quaternized amine macrocycles and the chelate effect

A new class of tetraamide macrocyclic receptors for anions with two quaternized amine functionalities exhibited higher affinities for anions compared with the corresponding neutral amides. In two crystal structures of halide complexes of the prototypes with phenyl and pyridine spacers, the anions are held by hydrogen bonding with the amide hydrogens. The pyridine analogues display higher affinities in general than the phenyl systems, a phenomenon which is attributed to the anion version of the chelate effect.     

Novel bile acid-based cyclic bisimidazolium receptors for anion recognition

[structure: see text] Novel deoxycholic acid-based cyclic receptors, 3 and 4, containing two imidazolium groups and m-xylene and p-xylene as spacers have been synthesized. These receptors bind anions through hydrogen bonds utilizing two imidazolium (C-H)(+) and inwardly directed methylene hydrogens of both acetyl groups. Receptor 3 shows a moderate selectivity for fluoride ion whereas receptor 4 shows high affinity and selectivity for chloride ion in CDCl(3).     

Inter- and intra-molecular migration of peptide amide hydrogens during electrospray ionization

The isotopic exchange of amide hydrogens in proteins in solution strongly depends on the surrounding protein structure, thereby allowing structural studies of proteins by mass spectrometry. However, during electrospray ionization (ESI), gas phase processes may scramble or deplete the isotopic information. These processes have been investigated by on-line monitoring of the exchange of labile deuterium atoms in homopeptides with hydrogens from a solvent suitable for ESI. The relative contribution of intra- and inter-molecular exchange in the gas phase could be studied from their distinct influence on the well-characterized exchange processes in the spraying solution. The deuterium content of individual labile hydrogens was assessed from the isotopic patterns of two consecutive collision-induced dissociation fragments, as observed with a 9.4 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Results demonstrate that gas phase exchange in the high-pressure region between the capillary and the skimmer cause substantial depletion of the isotopic information of penta-phenylalanine and penta-aspartic acid. For penta-alanine and hexa-tyrosine, the amide hydrogens located close to the N-terminus are depleted from deuterium during mass analysis. Amide hydrogens located close to the C-terminus still retain the information of the isotopic state in solution, but they are redistributed by intra-molecular exchange of the amide hydrogens with the C-terminal hydroxyl group.     

Regional stability changes in oxidized and reduced cytochrome c located by hydrogen exchange and mass spectrometry

Amide hydrogen exchange rates are highly sensitive to protein structure and may, therefore, be used to detect and characterize structural changes in proteins. Specific regions within folded proteins undergoing structural change can often be identified if localized amide hydrogen exchange rates are determined by nuclear magnetic resonance (NMR). The ability to measure localized amide hydrogen exchange rates by proteolytic fragmentation followed by mass spectrometric analysis opens the possibility to also identify localized structural changes in proteins by mass spectrometry. If successful, this approach offers considerable advantage over NMR in speed, sensitivity, protein solubility, and ability to study large proteins. This possibility has been investigated by determining the amide hydrogen exchange rates in oxidized and reduced cytochrome c by protein fragmentation/mass spectrometry. The fundamental difference in these forms of cytochrome c is the oxidation state of the iron, which other studies have shown results in only minor structural changes in the protein. In the present study, the largest differences in hydrogen exchange rates were found for peptide amide hydrogens located distant from the Nand C-termini, indicating that the structure in these regions is most affected by the oxidation state of the iron. These results are consistent with previous studies of oxidized and reduced cytochrome c, suggesting that hydrogen exchange and mass spectrometry may be generally useful for locating subtle changes in protein structure.     

Electron and hydrogen transfer in small hydrogen fluoride anion clusters

A new stable structure has been found for the anion clusters of hydrogen fluoride. The ab initio method was used to optimize the structures of the (HF)(3)(-), (HF)(4)(-), (HF)(5)(-), and (HF)(6)(-) anion clusters with an excess "solvated" electron. Instead of the well-known "zig-zag" (HF)(n)(-) structure, a new form, (HF)(n-1)F(-)···H, was found with lower energy. In this new form, the terminal hydrogen atom in the (HF)(n)(-) chain is separated from the other part of the cluster and the inner hydrogens transfer along the hydrogen bonds toward the outside fluoride. The negative charge also transfers from the terminal HF molecule of the chain to the center fluoride atoms. The (HF)(n)(-) clusters for n = 4, 5, and 6 have not yet been observed experimentally. These results should assist in the search for these systems and also provide a possible way to study the proton and electron transfer in some large hydrogen bonding systems.     

FT–IR microspectroscopy study of blends of ester functionalized ethylene–propylene copolymer with (vinylidene fluoride-hexafluoropropene) elastomer

An ethylene—propylene (EPR) copolymer functionalized with (1,2-dicarboethoxy)ethyl groups has been blended with a vinylidene fluoride—hexafluropropene elastomer (NML). The existence of intermolecular interactions involving mainly the carbonyl groups of the side chains of the functionalized copolymer and the methylene hydrogens of the fluoroelastomer through hydrogen bonding was shown by means of FT—IR spectroscopy. The structure and composition of some microdomains in the mixture was examined by means of FT—IR microspectroscopy.     

Synthesis of organogelling, fluoride ion-responsive, cholesteryl-based benzoxazole containing intra- and intermolecular hydrogen-bonding sites

A cholesteryl-based 2-(2′-hydroxyphenyl)benzoxazole (HPB) derivative 3 linked with an amide bond was prepared through an efficient synthetic pathway. The HPB, amide, and cholesteryl groups play important roles in constructing the supramolecular gel structure. UV-vis and fluorescence spectroscopy also showed that HPB and amide groups, which provide intra- and intermolecular hydrogen bonding, respectively, also contribute the recognition of fluoride anions.     

The thermal decomposition of fluorinated esters. III. Esters without β hydrogen atoms

The kinetics of the gas-phase decomposition of methyl trifluoroacetate and 2,2,2-trifluoroethyl trifluoroacetate, two esters without β hydrogen atoms, have been investigated, and a comparative study was carried out on methyl acetate. All these compounds are thermally much more stable than fluorinated esters with β hydrogens, decomposing at temperatures some 150°C above the latter by a completely different mechanism involving partly heterogeneous radical chains. The marked difference in behavior between the two types of fluorinated ester confirms that none of them decomposes by hydrogen fluoride elimination, but that those with β hydrogen atoms follow the normal ester decomposition route. The fluorinated esters examined here decompose in a manner generally similar to methyl acetate, but the presence of fluorine in the molecule brings about an extreme sensitivity to surface conditions.     

Energetics and structural elucidation of mechanisms for gas phase H/D exchange of protonated peptides

Hydrogen/deuterium exchange reactions involving protonated triglycine and deuterated ammonia (ND(3)) have been examined in the gas phase using a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Ab initio and density functional theory (DFT) calculations have been carried out to model the exchanges and to obtain energetics and vibrational frequencies for molecules involved in the proposed exchange mechanisms. Structural optimization and frequency calculations have been performed at the B3LYP level of theory with the 6-311+G(d,p) basis set. Transition states have been calculated at the same level of theory and basis set as above using the QST2 and QST3 methods. Single-point energy calculations have been performed at the MP2/6-311+G(d,p) level. Six labile sites of protonated triglycine were found to undergo H/D exchange. Of these six labile hydrogens, two are amide, three are ammonium, and one is carboxyl. Detailed mechanisms for each of these transfers are proposed. Qualitative onium ion and tautomer mechanisms for the exchanges of ammonium and amide hydrogens, respectively, using semiempirical calculations were suggested in previous studies by Beauchamp et al. As shown by the current ab initio and DFT calculations completed during this study, the mechanisms proposed in that study are notionally correct; however, the tautomer mechanisms are shown here to be the result of the fact that a second stable isomer of protonated triglycine exists in which the amide1 carbonyl oxygen is protonated. The exchange of the carboxyl hydrogen is found to proceed via a transition state resembling an ammonium ion interacting with a carboxylate moiety via two hydrogen bonds. The current work thus provides significant mechanistic and structural detail for a considerably more in-depth understanding of the processes involved in gas phase H/D exchange of peptides.     

A Non-Covalent Dimer Formation of Quaternary Ammonium Cation with Unusual Charge Neutralization in Electrospray-Ionization Mass Spectrometry

Specific and nonspecific non-covalent molecular association of biomolecules is characteristic for electrospray-ionization mass spectrometry analysis of biomolecules. Understanding the interaction between two associated molecules is of significance not only from the biological point of view but also gas phase analysis by mass spectrometry. Here we reported a formation of non-covalent dimer of quaternary ammonium denatonium cation with +1 charge detected in the positive ion mode electrospray ionization mass spectrometry analysis of denatonium benzoate. Hydrogen deuterium exchange of amide and carbon-bonded hydrogens revealed that charge neutralization of one denatonium cation is the consequence of amide hydrogen dissociation. DFT (Density Functional Theory) calculations proved high thermodynamic stable of formed dimer stabilized by the short and strong N..H-N hydrogen bond. The signal intensity of the peak characterizing non-covalent dimer is low intensity and does not depend on the sample concentration. Additionally, dimer observation was found to be instrument-dependent. The current investigation is the first experimental and theoretical study on the quaternary ammonium ions dimer. Thus the present study has great significance for understanding the structures of the biomolecules as well as materials.