Kinetics of the Gas-Phase Reaction of Hydroxyl Radicals with Dimethyl Methylphosphonate (DMMP) over an Extended Temperature Range (273-837 K).

The kinetics of the reaction of hydroxyl radical (OH) with dimethyl methylphosphonate (DMMP, (CH3O)2CH3PO) (reaction 1) OH + DMMP → products (1) was studied at the bath gas (He) pressure of 1 bar over the 295-837 K temperature range. Hydroxyl radicals were produced in the fast reaction of electronically excited oxygen atoms O(1D) with H2O. The time-resolved kinetic profiles of hydroxyl radicals were recorded via UV absorption at around 308 nm using a DC discharge H2O/Ar lamp. The reaction rate constant exhibits a pronounced V-shaped temperature dependence, negative in the low temperature range, 295-530 K (the rate constant decreases with temperature), and positive in the elevated temperature range, 530-837 K (the rate constant increases with temperature), with a turning point at 530 ± 10 K. The rate constant could not be adequately fitted with a standard 3-parameter modified Arrhenius expression. The data were fitted with a 5-parameter expression as: k1 = 2.19 × 10-14(T/298)2.43exp(15.02 kJ mol-1/RT) + 1.71 × 10-10exp(-26.51 kJ mol-1/RT) cm3molecule-1s-1 (295-837 K). In addition, a theoretically predicted pressure dependence for such reactions was experimentally observed for the first time.

Disproportionation Channel of the Self-reaction of Hydroxyl Radical, OH + OH → H2O + O, Revisited.

The rate constant of the disproportionation channel 1a of the self-reaction of hydroxyl radicals OH + OH → H2O + O (1a) was measured at ambient temperature as well as over an extended temperature range to resolve the discrepancy between the IUPAC recommended value (k1a = 1.48 × 10-12 cm3 molecule-1 s-1, discharge flow system, Bedjanian et al. J. Phys. Chem. A 1999, 103, 7017) and a factor of ca. 1.8 higher value by pulsed laser photolysis (2.7 × 10-12 cm3 molecule-1 s-1, Bahng et al. J. Phys. Chem. A 2007, 111, 3850, and 2.52 × 10-12 cm3 molecule-1 s-1, Altinay et al. J. Phys. Chem. A 2014, 118, 38). To resolve this discrepancy, the rate constant of the title reaction was remeasured in three laboratories using two different experimental techniques, namely, laser-pulsed photolysis-transient UV absorption and fast discharge flow system coupled with mass spectrometry. Two different precursors were used to generate OH radicals in the laser-pulsed photolysis experiments. The experiments confirmed the low value of the rate constant at ambient temperature (k1a = (1.4 ± 0.2) × 10-12 cm3 molecule-1 s-1 at 295 K) as well as the V-shaped temperature dependence, negative at low temperatures and positive at high temperatures, with a turning point at 427 K: k1a = 8.38 × 10-14 × (T/300)1.99 × exp(855/T) cm3 molecule-1 s-1 (220-950 K). Recommended expression over the 220-2384 K temperature range: k1a = 2.68 × 10-14 × (T/300)2.75 × exp(1165/T) cm3 molecule-1 s-1 (220-2384 K).

Pressure-Dependent Kinetics of the Reaction between CH3O2 and OH: TRIOX Formation.

Reaction of methyl peroxy radicals with hydroxyl radicals (1) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy at 296 K over the 1-100 bar pressure range (bath gas He): CH3O2 + OH → CH3O + HO2 (1a), CH3O2 + OH → CH2OO + H2O (1b), and CH3O2 + OH → CH3OOOH (TRIOX) (1d). Channel 1a is the dominant channel under ambient conditions, while the contribution of channel 1b is less than 5% at 1 bar and 296 K. Channel 1c is strongly pressure-dependent and becomes dominant at high pressures. The measured branching ratio of channel 1c is α1c = 0.87 ± 0.20 at 100 bar and 296 K. The chain termination channel 1c forming important product TRIOX is experimentally evaluated over an extended pressure range (1-100 bar) for the first time. The stabilization channel 1c might play a role at ambient pressures and low temperatures as well as high pressures at ambient and elevated temperatures.

Kinetics and Thermodynamics of DNA Processing by Wild Type DNA-Glycosylase Endo III and Its Catalytically Inactive Mutant Forms.

Endonuclease III (Endo III or Nth) is one of the key enzymes responsible for initiating the base excision repair of oxidized or reduced pyrimidine bases in DNA. In this study, a thermodynamic analysis of structural rearrangements of the specific and nonspecific DNA-duplexes during their interaction with Endo III is performed based on stopped-flow kinetic data. 1,3-diaza-2-oxophenoxazine (tCO), a fluorescent analog of the natural nucleobase cytosine, is used to record multistep DNA binding and lesion recognition within a temperature range (5-37 °C). Standard Gibbs energy, enthalpy, and entropy of the specific steps are derived from kinetic data using Van't Hoff plots. The data suggest that enthalpy-driven exothermic 5,6-dihydrouracil (DHU) recognition and desolvation-accompanied entropy-driven adjustment of the enzyme-substrate complex into a catalytically active state play equally important parts in the overall process. The roles of catalytically significant amino acids Lys120 and Asp138 in the DNA lesion recognition and catalysis are identified. Lys120 participates not only in the catalytic steps but also in the processes of local duplex distortion, whereas substitution Asp138Ala leads to a complete loss of the ability of Endo III to distort a DNA double chain during enzyme-DNA complex formation.

Kinetics of the Reaction of CH3O2 Radicals with OH Studied over the 292-526 K Temperature Range.

Reaction of methyl peroxy radicals with hydroxyl radicals, CH3O2 + OH → CH3O + HO2 (1a) and CH3O2 + OH → CH2OO + H2O (1b) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 292-526 K temperature range and pressure 1 bar (bath gas He). Hydroxyl radicals were generated in the reaction of electronically excited oxygen atoms O((1)D), produced in the photolysis of N2O at 193.3 nm, with H2O. Methyl peroxy radicals were generated in the reaction of methyl radicals, CH3, produced in the photolysis of acetone at 193.3 nm, and subsequent reaction of CH3 with O2. Temporal profiles of OH were monitored via transient absorption of light from a DC discharge H2O/Ar low-pressure resonance lamp at ca. 308 nm. The absolute intensity of the photolysis light was determined by accurate in situ actinometry based on the ozone formation in the presence of molecular oxygen. The overall rate constant of the reaction is k1a+1b = (8.4 ± 1.7) × 10(-11)(T/298 K)(-0.81) cm(3) molecule(-1) s(-1) (292-526 K). The branching ratio of channel 1b at 298 K is less than 5%.

Reaction CH3 + CH3 → C2H6 Studied over the 292-714 K Temperature and 1-100 bar Pressure Ranges.

Reaction of recombination of methyl radicals, CH3 + CH3 → C2H6 (1) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 292-714 K temperature and 1-100 bar pressure ranges (bath gas He), very close to the high-pressure limit. Methyl radicals were produced by photolysis of acetone at 193.3 nm or in the reaction of electronically excited oxygen atoms O((1)D), produced in the photolysis of N2O at 193.3 nm, with CH4, and subsequent reaction of OH with CH4. Temporal profiles of CH3 were recorded via absorption at 216.36 and 216.56 nm using a xenon arc lamp and a spectrograph. The absolute intensity of the photolysis light inside the reactor was determined by an accurate in situ actinometry based on the ozone formation in photolysis of N2O/O2/N2 mixtures. The rate constant of reaction 1 in the high-pressure limit has a negative temperature dependence: k1,inf = (5.66 ± 0.43) × 10(-11)(T/298 K)(-0.37) cm(3) molecule(-1) s(-1) (292-714 K).

Thermodynamics of the DNA damage repair steps of human 8-oxoguanine DNA glycosylase.

Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van't Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves.

Simple no-chromatography procedure for amine-reactive Eu3+ luminescent chelates optimal for bioconjugation.

Lanthanide ions luminescence has long life time enabling highly sensitive detection in time-gated mode. The synthesis of reactive lanthanide probes for covalent labeling of the objects of interest is cumbersome task due to the large size of the probes, complex multi-step procedures and the presence of sensitive groups, which often prevents introduction of reactive cross-linking functions optimal for conjugation. We suggest simple synthetic protocol for luminescent europium chelates based on serendipitous reaction yielding acylating compounds, whose reactivity is comparable to that of commonly used N-hydroxysuccinimide (NHS) esters. The probes react with proteins at pH 7.0 within several minutes at ambient temperature displaying high coupling efficiency. The resulting conjugates survive electrophoretic separation under denaturing conditions, which makes the labels useful in proteomic studies that rely on high detection sensitivity.

New quinolone-based thiol-reactive lanthanide luminescent probes.

Luminescent lanthanide ion complexes are distinguished by unique light emitting properties that enable both highly sensitive detection of lanthanide labels attached to biomolecules and contrast imaging of various micro objects (cells, nanoparticles, etc.). Previously, we synthesized amine-reactive cs124-based luminescent lanthanide chelates with improved brightness and metal retention. Here we report the synthesis of new thiol-reactive derivatives of the same compounds including bromoacetamido-, and maleimido- forms of cs124 and cs124CF3 fluorophores. Maleimido-compounds displayed exceptional reactivity instantaneously coupling to thiols at physiological conditions at micromolar probes concentrations. Surprisingly, they displayed strong quenching by adjacent maleimido-group, which was completely eliminated after reaction with thiols, thereby enabling their simple detection by monitoring the light emission of the reaction mixture. This reaction can be used for hyper-sensitive determination of biologically important sulphydryl compounds (e.g. glutathione, co-enzyme A, etc.) in time-resolved mode.

Kinetics of the gas phase reaction CH3 + HO2.

Reaction of methyl radicals with hydroperoxy radicals, CH3 + HO2 → products (1) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy at 295 K and 1 bar (He). Photolysis of N2O/H2O2/CH4/H2O/He mixtures at 193.3 nm (ArF excimer laser) was used to simultaneously produce methyl radicals and hydroperoxy radicals in reactions of electronically excited oxygen atoms O((1)D) with CH4 and OH radicals with H2O2, respectively. Temporal profiles of CH3 and HO2 were recorded via absorption at 216.4 and 224 nm. The absolute intensity of the photolysis light inside the reactor was determined by an accurate in situ actinometry based on the ozone formation in the presence of molecular oxygen. The measured rate constant of reaction 1 is k1 = (3.7 ± 1.8 × 10(-11) cm(3) molecule(-1) s(-1) (295 K, 1 bar, He).

Gas phase kinetics and equilibrium of allyl radical reactions with NO and NO2.

Allyl radical reactions with NO and NO(2) were studied in direct, time-resolved experiments in a temperature controlled tubular flow reactor connected to a laser photolysis/photoionization mass spectrometer (LP-PIMS). In the C(3)H(5) + NO reaction 1 , a dependence on the bath gas density was observed in the determined rate coefficients and pressure falloff parametrizations were performed. The obtained rate coefficients vary between 0.30-14.2 × 10(-12) cm(3) s(-1) (T = 188-363 K, p = 0.39-23.78 Torr He) and possess a negative temperature dependence. The rate coefficients of the C(3)H(5) + NO(2) reaction 2 did not show a dependence on the bath gas density in the range used (p = 0.47-3.38 Torr, T = 201-363 K), and they can be expressed as a function of temperature with k(C(3)H(5) + NO(2)) = (3.97 ± 0.84) × 10(-11) × (T/300 K) (-1.55±0.05) cm(3) s(-1). In the C(3)H(5) + NO reaction, above 410 K the observed C(3)H(5) radical signal did not decay to the signal background, indicating equilibrium between C(3)H(5) + NO and C(3)H(5)NO. This allowed the C(3)H(5) + NO ⇄ C(3)H(5)NO equilibrium to be studied and the equilibrium constants of the reaction between 414 and 500 K to be determined. With the standard second- and third-law analysis, the enthalpy and entropy of the C(3)H(5) + NO ⇄ C(3)H(5)NO reaction were obtained. Combined with the calculated standard entropy of reaction (ΔS°(298) = 137.2 J mol(-1)K(-1)), the third-law analysis resulted in ΔH°(298) = 102.4 ± 3.2 kJ mol(-1) for the C(3)H(5)-NO bond dissociation enthalpy.

Disproportionation channel of self-reaction of hydroxyl radical, OH + OH → H2O + O, studied by time-resolved oxygen atom trapping.

The disproportionation channel of the self-reaction of hydroxyl radicals, OH + OH → H(2)O + O (1a) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 298-414 K temperature and 3-10 bar pressure ranges (bath gas He). To distinguish channel 1a from the recombination channel 1b, OH + OH → H(2)O(2) (1b), time-resolved trapping of oxygen atoms, produced in channel 1a, was used. The ozone produced in the reaction of oxygen atoms with molecular oxygen was measured using strong UV absorption at 253.7 nm. The results of this study (k(1a) = (1.38 ± 0.20) × 10(-12) (T/300)(-0.76) confirm the IUPAC recommended value of Bedjanian et al. (J. Phys. Chem. A1999, 103, 7017-7025), as well as the negative temperature dependence over the temperature range studied, and do not confirm the ca. 1.8 higher value obtained in the most recent study of Bahng et al. (J. Phys. Chem. A2007, 111, 3850-3861). The V-shaped temperature dependence of k(1a) based on combined current and previous studies in the temperature range of 233-2380 K is k(1a) = (5.1 exp(-T/190 K) + 0.30(T/300 K)(1.73)) × 10(-12) cm(3) molecule(-1) s(-1).

Highly bright avidin-based affinity probes carrying multiple lanthanide chelates.

Lanthanide ion luminescence has a long lifetime enabling highly sensitive detection in time-gated mode. The sensitivity can be further increased by using multiple luminescent labels attached to a carrier molecule, which can be conjugated to an object of interest. We found that up to 30 lanthanide chelates can be attached to avidin creating highly bright constructs. These constructs with Eu(3+) chelates display synergistic effect that enhance the brightness of heavily modified samples, while the opposite effect was observed for Tb(3+) chelates thereby significantly reducing their light emission. This undesirable quenching of Tb(3+) luminophores was completely suppressed by the introduction of an aromatic spacer between the chelate and the protein attachment site. The estimated detection limit for the conjugates is in the 10(-14)-10(-15) M range. We demonstrated a high sensitivity for the new probes by using them to label living cells of bacterial and mammalian origin.

Reaction CH3 + OH studied over the 294-714 K temperature and 1-100 bar pressure ranges.

Reaction of methyl radicals with hydroxyl radicals, CH(3) + OH → products (1) was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 294-714 K temperature and 1-100 bar pressure ranges (bath gas He). Methyl radicals were produced by photolysis of acetone at 193.3 nm. Hydroxyl radicals were generated in reaction of electronically excited oxygen atoms O((1)D), produced in the photolysis of N(2)O at 193.3 nm, with H(2)O. Temporal profiles of CH(3) were recorded via absorption at 216.4 nm using xenon arc lamp and a spectrograph; OH radicals were monitored via transient absorption of light from a dc discharge H(2)O/Ar low pressure resonance lamp at ca. 308 nm. The absolute intensity of the photolysis light inside the reactor was determined by an accurate in situ actinometry based on the ozone formation in the presence of molecular oxygen. The results of this study indicate that the rate constant of reaction 1 is pressure independent within the studied pressure and temperature ranges and has slight negative temperature dependence, k(1) = (1.20 ± 0.20) × 10(-10)(T/300)(-0.49) cm(3) molecule(-1) s(-1).

Thermodynamics of the multi-stage DNA lesion recognition and repair by formamidopyrimidine-DNA glycosylase using pyrrolocytosine fluorescence--stopped-flow pre-steady-state kinetics.

Formamidopyrimidine-DNA glycosylase, Fpg protein from Escherichia coli, initiates base excision repair in DNA by removing a wide variety of oxidized lesions. In this study, we perform thermodynamic analysis of the multi-stage interaction of Fpg with specific DNA-substrates containing 7,8-dihydro-8-oxoguanosine (oxoG), or tetrahydrofuran (THF, an uncleavable abasic site analog) and non-specific (G) DNA-ligand based on stopped-flow kinetic data. Pyrrolocytosine, highly fluorescent analog of the natural nucleobase cytosine, is used to record multi-stage DNA lesion recognition and repair kinetics over a temperature range (10-30°C). The kinetic data were used to obtain the standard Gibbs energy, enthalpy and entropy of the specific stages using van't Hoff approach. The data suggest that not only enthalpy-driven exothermic oxoG recognition, but also the desolvation-accompanied entropy-driven enzyme-substrate complex adjustment into the catalytically active state play equally important roles in the overall process.

Reaction OH + OH studied over the 298-834 K temperature and 1-100 bar pressure ranges.

Self-reaction of hydroxyl radicals, OH + OH → H(2)O + O (1a) and OH + OH → H(2)O(2) (1b), was studied using pulsed laser photolysis coupled to transient UV-vis absorption spectroscopy over the 298-834 K temperature and 1-100 bar pressure ranges (bath gas He). A heatable high-pressure flow reactor was employed. Hydroxyl radicals were prepared using reaction of electronically excited oxygen atoms, O((1)D), produced in photolysis of N(2)O at 193 nm, with H(2)O. The temporal behavior of OH radicals was monitored via transient absorption of light from a dc discharge in H(2)O/Ar low-pressure resonance lamp at ca. 308 nm. The absolute intensity of the photolysis light was determined by accurate in situ actinometry based on the ozone formation in the presence of molecular oxygen. The results of this study combined with the literature data indicate that the rate constant of reaction 1a, associated with the pressure independent component, decreases with temperature within the temperature range 298-414 K and increases above 555 K. The pressure dependent rate constant for (1b) was parametrized using the Troe expression as k(1b,inf) = (2.4 ± 0.6) × 10(-11)(T/300)(-0.5) cm(3) molecule(-1) s(-1), k(1b,0) = [He] (9.0 ± 2.2) × 10(-31)(T/300)(-3.5±0.5) cm(3) molecule(-1) s(-1), F(c) = 0.37.

New cross-linking quinoline and quinolone derivatives for sensitive fluorescent labeling.

A variety of contemporary analytical platforms, utilized in technical and biological applications, take advantage of labeling the objects of interest with fluorescent tracers-compounds that can be easily and sensitively detected. Here we describe the synthesis of new fluorescent quinoline and quinolone compounds, whose light emission can be conveniently tuned by simple structural modifications. Some of these compounds can be used as sensitizers for lanthanide emission in design of highly sensitive luminescent probes. In addition, we also describe simple efficient derivatization reactions that allow introduction of amine- or click-reactive cross-linking groups into the fluorophores. The reactivity of synthesized compounds was confirmed in reactions with low molecular weight nucleophiles, or alkynes, as well as with click-reactive DNA-oligonucleotide containing synthetically introduced alkyne groups. These reactive derivatives can be used for covalent attachment of the fluorophores to various biomolecules of interest including nucleic acids, proteins, living cells and small cellular metabolites. Obtained compounds are characterized using NMR, steady-state fluorescence spectroscopy as well as UV absorption spectroscopy.

Branching ratios in the hydroxyl reaction with propene.

The branching ratios for the reactions of attachment of hydroxyl radical to propene and hydrogen-atom abstraction were measured at 298 K over the buffer gas pressure range 60-400 Torr (N(2)) using a subatmospheric pressure turbulent flow reactor coupled with a chemical ionization quadrupole mass spectrometer. Isotopically enriched water H(2)(18)O was used to produce (18)O-labeled hydroxyl radicals in reaction with fluorine atoms. The ß-hydroxypropyl radicals formed in the attachment reactions 1a and 1b , OH + C(3)H(6) → CH(2)(OH)C(•)HCH(3) (eq 1a ) and OH + C(3)H(6) → C(•)H(2)CH(OH)CH(3) (eq 1b ), were converted to formaldehyde and acetaldehyde in a sequence of secondary reactions in O(2)- and NO-containing environment. The (18)O-labeling propagates to the final products, allowing determination of the branching ratio for the attachment channels of reaction 1. The measured branching ratio for attachment is ß(1b) = k(1b)/(k(1a) + k(1b)) = 0.51 ± 0.03, independent of pressure over the 60-400 Torr pressure range. An upper limit on the hydrogen-abstraction channel, OH + C(3)H(6) → H(2)O + C(3)H(5) (eq 1c ), was determined by measuring the water yield in reactions of OH and OD radicals (produced via H(D) + NO(2) → OH(OD) + NO reactions) with C(3)H(6) as k(1c)/(k(1a) + k(1b) + k(1c)) < 0.05 (at 298 K, 200 Torr N(2)).

Luminescent probes for ultrasensitive detection of nucleic acids.

Novel amino-reactive derivatives of lanthanide-based luminescent labels of enhanced brightness and metal retention were synthesized and used for the detection of cDNA oligonucleotides by molecular beacons. Time-resolved acquisition of the luminescent signal that occurs upon hybridization of the probe to the target enabled the avoidance of short-lived background fluorescence, markedly enhancing the sensitivity of detection, which was less than 1 pM. This value is about 50 to 100 times more sensitive than the level achieved with conventional fluorescence-based molecular beacons, and is 10 to 60 times more sensitive than previously reported for other lanthanide-based hybridization probes. These novel luminescent labels should significantly enhance the sensitivity of all type of nucleic acid hybridization probes, and could dramatically improve the detection limit of other biopolymers and small compounds that are used in a variety of biological applications.

Rate constants and hydrogen isotope substitution effects in the CH3 + HCl and CH3 + Cl2 reactions.

The kinetics of the CH3 + Cl2 (k2a) and CD3 + Cl2 (k2b) reactions were studied over the temperature range 188-500 K using laser photolysis-photoionization mass spectrometry. The rate constants of these reactions are independent of the bath gas pressure within the experimental range, 0.6-5.1 Torr (He). The rate constants were fitted by the modified Arrhenius expression, k2a = 1.7 x 10(-13)(T/300 K)(2.52)exp(5520 J mol(-1)/RT) and k2b = 2.9 x 10(-13)(T/300 K)(1.84)exp(4770 J mol(-1)/RT) cm(3) molecule(-1) s(-1). The results for reaction 2a are in good agreement with the previous determinations performed at and above ambient temperature. Rate constants of the CH3 + Cl2 and CD3 + Cl2 reactions obtained in this work exhibit minima at about 270-300 K. The rate constants have positive temperature dependences above the minima, and negative below. Deuterium substitution increases the rate constant, in particular at low temperatures, where the effect reaches ca. 45% at 188 K. These observations are quantitatively rationalized in terms of stationary points on a potential energy surface based on QCISD/6-311G(d,p) geometries and frequencies, combined with CCSD(T) energies extrapolated to the complete basis set limit. 1D tunneling as well as the possibility of the negative energies of the transition state are incorporated into a transition state theory analysis, an approach which also accounts for prior experiments on the CH3 + HCl system and its various deuterated isotopic substitutions [Eskola, A. J.; Seetula, J. A.; Timonen, R. S. Chem. Phys. 2006, 331, 26].