Time-resolved Chemically Induced Dynamic Nuclear Polarization Research Articles

Time-Resolved Chemically Induced Dynamic Nuclear Polarization of Biologically Important Molecules.

In this work, we review the hyperpolarization technique named chemically induced dynamic nuclear polarization (CIDNP), focusing on the time-resolved variant of this method and its biological applications. We introduce the main principles of polarization formation in liquids at high magnetic fields, provided by the so-called spin sorting mechanism. Applications of CIDNP to studying fast reactions of short-lived free radicals of biologically important molecules are discussed, as well as the potential of the method to probe the structure and magnetic parameters of such radicals. We also explain the principles of protein CIDNP and discuss applications of time-resolved CIDNP to studies of protein structure and dynamics.

Kinetics of Reversible Protonation of Transient Neutral Guanine Radical in Neutral Aqueous Solution.

Time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP) is applied to follow transformation of the short-lived neutral guanine radical into a secondary guanine radical by its protonation, presumably at position N7. In the initial step the photoreaction of guanosine-5'-monophosphate (GMP) with triplet excited 3,3',4,4'-tetracarboxy benzophenone (TCBP) leads to formation of the neutral radical G(-H). . The evidence of the radical conversion is based on the inversion of CIDNP sign for TCBP and GMP protons on the microsecond timescale as a result of the change in magnetic resonance parameters in the pairs of TCBP and GMP radicals due to structural changes of the GMP radical. Acceleration of the CIDNP sign change upon addition of phosphate (proton donor) confirms that the radical transformation responsible for the observed CIDNP kinetics is protonation of the neutral guanine radical with formation of the newly characterized cation radical, (G.+ )'. From the full analysis of the pH-dependent CIDNP kinetics, the protonation and deprotonation behaviour is quantitatively characterized, giving pKa =8.0±0.2 of the cation radical (G.+ )'.

Relation between CIDNP formed upon geminate and bulk recombination of radical pairs.

A theoretical approach to time-resolved Chemically Induced Dynamic Nuclear Polarization (CIDNP) is proposed, which allows one to obtain the general relation between polarization formed upon recombination of geminate spin-correlated radical pairs, the so-called G-pairs, and upon recombination of radical pairs formed by encounters of free radicals in solution, the so-called F-pairs. This relation is described by a universal parameter denoted as γ. In this work, the γ value is computed for the arbitrary spin multiplicity, singlet or triplet, of the precursor of the G-pairs as well as for arbitrary recombination rate constants of radical pairs in singlet and triplet states, kS and kT, respectively. Furthermore, the treatment is extended to the situation where radicals undergo transformation resulting in different reactivity or magnetic parameters for F-pairs and G-pairs. The proposed theory enables modeling of time-resolved CIDNP data in cases where (i) both recombination channels are active and (ii) fast protonation/deprotonation of radicals changes the effective γ value.

Magnetic Resonance Characterization of One-Electron Oxidized Cyclic Dipeptides with Thioether Groups.

Photo-oxidation of seven cyclic dipeptides containing methionine, Met, and/or S-methylcysteine, Cys(Me) by electron transfer from the sulfur atom was studied in aqueous solution by time-resolved and field dependent CIDNP (chemically induced dynamic nuclear polarization). Hyperpolarized high resolution NMR spectral patterns of the starting peptides detected immediately after pulsed laser excitation show signals of all protons that are bound to carbons neighboring the sulfur atom, thus proving the involvement of sulfur-centered cation radicals. The magnetic field dependence of CIDNP shows a pronounced maximum that is determined by the g-factors and hyperfine coupling constants of the transient radical species. From simulation of the experimental data obtained for the magnetic field dependences of CIDNP, three types of radical structures were characterized: (1) a linear sulfur-centered cation radical of the methionine (Met) residue (g = 2.0107 ± 0.0010) for cyclo-(d-Met-l-Met) (trans-configuration), cyclo-(d-Met-l-Cys(Me)) (trans-configuration), and cyclo-(Gly-Met); (2) a cyclic radical (S∴O)(+) (g = 2.0088 ± 0.0010) with a two-center three-electron bond (2c-3e) structure between the sulfur atom of the Cys(Me) residue and the oxygen atom of cyclo-(d-Met-l-Cys(Me)) and cyclo-(Gly-Cys(Me)); (3) a cyclic radical (S∴S)(+) (g = 2.013 ± 0.0020) with a two-center three-electron bond structure between the two sulfur atoms of the peptides cyclo-(l-Met-l-Met), cyclo-(l-Met-l-Cys(Me)), and cyclo-(l-Cys(Me)-l-Cys(Me)). In contrast, no indication of any type of cyclic radicals with a two-center three-electron bond between sulfur and nitrogen atoms was found. In addition, the hyperfine coupling constants (HFCCs) were determined.

CIDNP study of sensitized photooxidation of S-methylcysteine and S-methylglutathione in aqueous solution

Time-resolved CIDNP (chemically induced dynamic nuclear polarization) was applied to the investigation of the photo-oxidation of S-methylcysteine, Cys(Me), and S-methylglutathione, GS(Me). It was established that the reaction of Cys(Me) with a photosensitizer, triplet excited 4-carboxybenzophenone or 3,3′,4,4′-carboxybenzophenone, occurs via electron transfer from the sulfur atom with the formation of a sulfur-centered cation radical. From the pH-dependence of nuclear polarization it is established that the pKa value of the terminal amino group of this radical is 4.3, which is much lower than that of the initial compound, pKa = 9.0. At pH below 4.3 the sulfur-centered cation radical of Cys(Me) formed as a result of triplet quenching participates in the degenerate electron exchange reaction with the parent molecule resulting in a fast decay of CIDNP in time. At pH above this pKa, CIDNP is constant in time, which is a result of fast decarboxylation of the cyclic radical with a three-electron bond between sulfur and oxygen atoms that is formed from a linear radical with an uncharged amino group. The involvement of this radical was established by the dependence of CIDNP on the external magnetic field ranging from 0.1 to 7 T. For GS(Me), the formation of the cyclic radical the indication of which is time-independent CIDNP correlates with the deprotonation of the terminal amino group of the glutamate residue of GS(Me). The formation of a cyclic radical with a three-electron bond between sulfur and nitrogen atoms is ruled out because of the absence of polarization of the CH-proton in α-position with respect to the amino group, and also by the CIDNP field dependence which is typical for an (S∴O) radical. Thus, the cyclic radical originating from one-electron removal from sulfur of GS(Me) is the 10-membered one with a three-electron bond between the sulfur atom and the oxygen atom of the carboxylic group of the glutamate residue.

Photooxidation of Histidine by 3,3′,4,4′-Benzophenone Tetracarboxylic Acid in Aqueous Solution: Time-Resolved and Field-Dependent CIDNP Study

The photooxidation reaction between 3,3′,4,4′-benzophenone tetracarboxylic acid (TCBP) and l-histidine (His) has been investigated in neutral aqueous solution using the technique of chemically induced dynamic nuclear polarization (CIDNP). Relative values of 13C isotropic hyperfine couplings in the TCBP and His radicals were obtained from the 13C-time-resolved CIDNP spectrum, recorded during the photoreaction of TCBP with His at natural abundance of the magnetic isotope 13C. Good agreement was found for the hyperfine coupling constants of the TCBP ketyl radical calculated using methods of density functional theory, and those obtained from the 13C-time-resolved CIDNP spectrum. The mechanism of the quenching reaction of triplet-excited TCBP by His in neutral aqueous solution was established. 1H CIDNP field dependencies for the photoreaction of TCBP with His were obtained and the g-factor of the histidyl radical was found.

Reduction of Guanosyl Radicals in Reactions with Proteins Studied by TR-CIDNP

Pulsed-field-gradient nuclear magnetic resonance (NMR) combined with time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP) was applied to study the reduction of guanosyl radicals in reactions with the proteins hen egg white lysozyme (HEWL) and bovine α-lactalbumin (BLA) in their native state. Guanosyl radicals were generated photochemically in the reaction of guanosine-5′-monophosphate with photosensitizer, triplet-excited 2,2′-dipyridyl. In this reaction, at pH 5 guanosyl cation radical is formed, which deprotonates to yield the neutral guanosyl radical. To minimize the contribution of the cation radical, phosphate buffer was added, which accelerates the deprotonation of guanosyl cation radical. From model simulations of CIDNP kinetics the rate constants of the reduction were found to be (3.1 ± 0.5) × 107 M−1s−1 for HEWL and (1.6 ± 0.4) × 107 M−1s−1 for BLA. Also, experiments were carried out at the conditions for denatured HEWL, i.e., at 50 °C in the presence of 10 M urea-d4. The rate constant of the reduction of guanosyl radical in this case was (3.6 ± 0.5) × 108 M−1s−1.

Reduction of Guanosyl Radical by Cysteine and Cysteine-Glycine Studied by Time-Resolved CIDNP

As a model for chemical DNA repair, reduction of guanosyl radicals in the reaction with cysteine or the dipeptide cysteine-glycine has been studied by time-resolved chemically induced dynamic nuclear polarization (CIDNP). Radicals were generated photochemically by pulsed laser irradiation of a solution containing the photosensitizer 2,2'-dipyridyl, guanosine-5'-monophosphate, and the amino acid or peptide. In neutral and basic aqueous solution, the neutral guanosyl radical is formed via electron or hydrogen atom transfer to the triplet excited dye. The rate constants for reduction of guanosyl radical were determined by quantitative analysis of the CIDNP kinetics, which are sensitive to the rates of fast radical reactions. The rate constants vary from (1.0 ± 0.3) × 10(7) M(-1) s(-1) for the thiol form of cysteine to (1.6 ± 0.2) × 10(8) M(-1) s(-1) for the thiolate anion. These values are comparable with corresponding rate constants for reduction of neutral guanosyl radical by tyrosine.

Quantitative Approach to CIDNP in Proteins with Several Polarizable Residues on the Surface

A quantitative theoretical approach to protein chemically induced dynamic nuclear polarization (CIDNP) is formulated in the present work, which is based on the theory of sterically specific chemical reactions in liquids. Kinetic equations for polarization of all CIDNP-active accessible amino acid residues are presented. Relations between the kinetic parameters and the total side-chain accessibility (TSA) values are established. Numerical calculations of the CIDNP kinetics are also shown to demonstrate how the time behavior of polarization depends on TSA. The present theoretical approach was applied to model time-resolved protein CIDNP data obtained for Tyr59 and His68 of ubiquitin in the native state. Comparison of theoretical predictions with the experimental data confirms the accuracy of the approach.

Deprotonation of Transient Guanosyl Cation Radical Catalyzed by Buffer in Aqueous Solution: TR-CIDNP Study

The reaction of deprotonation of the guanosyl cation radical formed in the photoinduced reaction of guanosine monophospate (GMP) with triplet 2,2′-dipyridyl-d8 is studied in aqueous solution by time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP). In the course of the cyclic photoreaction, spin-polarized products are generated. Their polarization patterns that reflect the properties at the radical stage are analyzed using high-resolution nuclear magnetic resonance. The identification of transient radicals contributing to the polarization kinetics is based on its sensitivity to the degenerate electron exchange reaction of transient radicals with the parent diamagnetic molecules. Degenerate electron exchange is allowed only for the cation radical and manifests itself in the fast decay of the CIDNP signal in time with the rate of decay proportional to the concentration of parent GMP molecules. Because the formation of the neutral transient radical stops the exchange, the deprotonation changes the CIDNP kinetics from a decaying to a growing one. The rate constant of deprotonation, k d, was obtained from modeling of CIDNP kinetics data with taking into consideration the difference of the CIDNP enhancement factors for neutral and cation guanosyl radicals. The value obtained at pH* 5 for k d = 1 × 106 s−1 is consistent with the proton dissociation constant of the radical (pK a = 3.9). The linear dependence of the deprotonation rate on the buffer concentration is revealed for phosphate, formate, and acetate. Deprotonation is catalyzed by the buffer to a degree that depends on the difference in pK a value of the buffer and the guanosyl cation radical in full accordance with Eigen’s model.

Time-resolved CIDNP: an NMR way to determine the EPR parameters of elusive radicals

Chemically Induced Dynamic Nuclear Polarization (CIDNP) of the diamagnetic products of radical reactions is exploited for the purpose of determination of the hyperfine coupling constants (HFCCs) of the radical intermediates. A simple proportionality relation between geminate CIDNP of a nucleus and its HFCC at the radical stage is established. The applicability range of this relation is determined: the relation is fulfilled in the case of a large difference in g-factor between the radicals involved and for the situation where the number of magnetic nuclei in the system is sufficiently large. The validity of the relation was confirmed by CIDNP experiments on radical pairs with precisely known HFCCs. Using the proportionality relation we were able to measure the HFCCs in various short-lived radicals of the amino acids histidine and tryptophan and of the S-N-centered cyclic radical of methionine derived from the methionine-glycine dipeptide in aqueous solution.

Toward the Photoinduced Reactivity of 1,5-Diphenylpenta-1,4-diyn-3-one (DPD): Real-Time Investigations by Magnetic Resonance

The reactivity of 1,5-diphenylpenta-1,4-diyn-3-one (DPD) upon photolysis in acetonitrile and propan-2-ol was followed by continuous-wave time-resolved EPR (TR-EPR) and 1H CIDNP (chemically induced dynamic nuclear polarization) spectroscopy. It was found that radical polymerizations using DPD can only be accomplished if an efficient hydrogen donor, such as propan-2-ol, is present, reflecting an analogous reaction pathway as the related benzophenone (BP). The same behavior was also established for ODPD, the bis(p-methoxy) derivative of DPD. In contrast to parent BP, where the steric hindrance of the ortho phenyl protons causes a twisted geometry, DPD and ODPD adopt an essentially planar minimum geometry which is also conserved in the corresponding radicals of type C as revealed by density functional theory calculations. This planarity is possibly one important factor for the considerable efficiency of DPD and its derivatives to act as initiators for two-photon-induced radical polymerizations.

2,5‐Dihydro‐1H‐imidazole‐Based Nitroxides as Prospective Mediators in Living Radical Polymerization

AbstractA number of spectroscopic methods were applied to obtain kinetic parameters of reactions modelling the 2,5‐dihydro‐1H‐imidazole 1‐oxide mediated living polymerization of acrylates. The homolysis rate constants of alkoxyamines based on five nitroxides were measured by EPR spectroscopy at different temperatures. The recombination rate constants kc between the corresponding alkyl radicals and the nitroxides were measured by means of laser flash photolysis. The time‐resolved chemically induced dynamic nuclear polarization (TR‐CIDNP) experiments revealed the negligible contribution of disproportionation in the recombination reaction. In addition, the thermodecomposition of alkoxyamines in the NMR probe showed the absence of intramolecular elimination of hydroxylamines from the corresponding alkoxyamines. Analysis of the kinetic parameters showed that the 2,5‐dihydro‐1H‐imidazole 1‐oxide type radicals are promising mediators for the living polymerization of acrylates and methacrylates.

Laser Flash Photolysis and CIDNP Studies of Steric Effects on Coupling Rate Constants of Imidazolidine Nitroxide with Carbon-Centered Radicals, Methyl Isobutyrate-2-yl andtert-Butyl Propionate-2-yl

Time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP) and laser flash photolysis (LFP) techniques have been used to measure rate constants for coupling between acrylate-type radicals and a series of newly synthesized stable imidazolidine N-oxyl radicals. The carbon-centered radicals under investigation were generated by photolysis of their corresponding ketone precursors RC(O)R (R = C(CH3)2-C(O)OCH3 and CH(CH3)-C(O)-OtBu) in the presence of stable nitroxides. The coupling rate constants kc for modeling studies of nitroxide-mediated polymerization (NMP) experiments were determined, and the influence of steric and electronic factors on kc values was addressed by using a Hammett linear free energy relationship. The systematic changes in kc due to the varied steric (Es,n) and electronic (sigmaL,n) characters of the substituents are well-described by the biparameter equation log(kc/M- 1s(-1)) = 3.52sigmaL,n + 0.47Es,n + 10.62. Hence, kc decreases with the increasing steric demand and increases with the increasing electron-withdrawing character of the substituents on the nitroxide.

Time-Resolved CIDNP Study of Non-Native States of Bovine and Human α-Lactalbumins

The reaction mechanism and details of the formation of CIDNP (chemically induced dynamic nuclear polarization) in the photoreactions of the aromatic dye 2,2'-dipyridyl with non-native states of bovine and human alpha-lactalbumins (BLA and HLA) in aqueous solution have been studied using the time-resolved CIDNP technique. Non-native states have been obtained at pH 2 in the presence of 0, 8, and 10 M urea-d(4) and at pH 6.7 in the presence of 10 M urea-d(4). The dependence of the geminate CIDNP spectra of the two proteins on the denaturant concentration is shown to be determined by the intrinsic reactivity of the amino acid residues toward the triplet excited dye rather than by structural changes in the proteins. Values of the proton paramagnetic relaxation times (T(1)) have been obtained from an analysis of the CIDNP kinetics. For tryptophan and tyrosine residues, the T(1) values change in opposite directions when the proteins are progressively denatured, reflecting the different internal mobilities of the two types of residues. It has been found that for both BLA and HLA the CIDNP kinetics of the non-native states formed at pH 6.7 in the presence of 10 M urea are almost identical to those at pH 2 with no urea, suggesting that the polarizable amino acid side chains have closely similar solvent accessibilities and motional properties in the two non-native states.

Reversibility of Electron Transfer in Tryptophan−Tyrosine Peptide in Acidic Aqueous Solution Studied by Time-Resolved CIDNP

Time-resolved chemically induced dynamic nuclear polarization (CIDNP) has been used to study electron transfer reactions in tryptophan-tyrosine peptide under strongly acidic conditions. It is demonstrated that a decrease in pH from 2.4 to 1.6 reduces the overall efficiency of intramolecular electron transfer from the tyrosine residue to the oxidized tryptophan residue. A detailed analysis of the CIDNP kinetics revealed that the rate constant of this reaction k(f) stays unchanged upon pH variation, whereas the rate constant of electron transfer in the opposite direction k(r) increases with decreasing pH. The values of the rate constants extracted from model simulations are as follows: k(f) = (5.5 +/- 0.5) x 10(5) s(-1); k(r) = (5.5 +/- 1.0) x 10(4) s(-1) at pH 2.4, (1.2 +/- 0.2) x 10(5) s(-1) at pH 2.0, and (3.2 +/- 0.4) x 10(5) s(-1) at pH 1.6. The pH dependence of log K = log(k(f)/k(r)) is linear and allows for the determination of the difference between the one-electron reduction potentials of the tryptophanyl and tyrosyl radicals in the peptide. The efficiency of IET in acidic aqueous solution containing 10 M urea-d(4) was estimated.

Time-Resolved CIDNP Study of Native-State Bovine and Human α-Lactalbumins

The reaction mechanism and details of the formation of CIDNP (chemically induced dynamic nuclear polarization) in the photoreactions of two aromatic dyes (2,2‘-dipyridyl, DP, and flavin mononucleotide, FMN) with bovine and human α-lactalbumins (BLA and HLA) in aqueous solution have been studied using the time-resolved CIDNP technique. With DP as the photosensitizer, polarization in BLA is observed for the protons of Trp118, His68, Tyr18, and Tyr103. The latter is not manifested in the CIDNP spectra obtained with FMN. This threshold effect on CIDNP formation indicates that accessibility is a combined property of both the residue and the dye. The nuclear spin−lattice relaxation times in the radicals formed in these reactions have been determined from the CIDNP kinetics: T1 = 60 μs for H2,4,7 of Trp118, T1 = 53 μs for H3,5 of Tyr103, T1 = 16 μs for H3,5 of Tyr18, and T1 = 10 μs for the H2 and H4 of His68. The correlation times for the side chain motion as determined from the T1 of the radicals are correlated with the accessibility of the side chains in the intact protein.

Time-Resolved CIDNP Study of Intramolecular Charge Transfer in the Dipeptide Tryptophan-Tyrosine

Time-resolved chemically induced dynamic nuclear polarization (CIDNP) and laser flash photolysis (LFP) techniques have been used to study the kinetics and mechanism of the photochemical reaction between 2,2‘-dipyridyl (DP) and the dipeptide l-tryptophan-l-tyrosine (TrpH-TyrOH) in acidic aqueous solution (pH = 3.8). Analysis of the geminate CIDNP pattern reveals that the quenching of the protonated triplet dipyridyl TDPH+ results in the formation of both tryptophan and tyrosine radicals from the dipeptide with comparative efficiency. The total quenching rate constant of triplet dipyridyl by TrpH-TyrOH was found to be (2.5 ± 0.3) × 109 M-1 s-1. The radical transformation TrpH+•→TyrO• via intramolecular electron transfer (IET) leads to an increasing tyrosyl radical concentration, growth of the tyrosine CIDNP signals, fast decay of the CIDNP signal of TrpH, and inversion of the CIDNP sign from emission to enhanced absorption for DP. The nuclear spin−lattice relaxation times of the radicals formed in the react...

Time-Resolved CIDNP and Laser Flash Photolysis Study of the Photoreactions of N-Acetyl Histidine with 2,2‘-Dipyridyl in Aqueous Solution

The reaction mechanism and details of the formation of CIDNP (chemically induced dynamic nuclear polarization) in the photoreactions of 2,2‘-dipyridyl (DP) and N-acetyl histidine (HisH) in aqueous solution have been studied using laser flash photolysis and time-resolved CIDNP techniques. The triplet state TDP reacts with protonated HisH2+ via hydrogen atom transfer with a rate constant kH = 1.2 × 108 M-1 s-1, and with deprotonated His- via electron transfer with ke = 7.5 × 109 M-1 s-1. No reaction occurs when the histidine imidazole ring is in its neutral state HisH, or when the dipyridyl triplet is protonated, TDPH+. The nuclear spin−lattice relaxation times in the radicals formed in these reactions have been determined from the CIDNP kinetics: T1 = 44 ± 9 μs for all DP protons, T1 = 196 ± 25 μs for the β-CH2 protons of HisH, and T1 = 16 ± 5 μs for the H-2 and H-4 protons of HisH. Under strongly basic conditions the CIDNP is greatly affected by degenerate electron exchange between the neutral His· radic...

Kinetics and Mechanism of the Photochemical Reaction of 2,2‘-Dipyridyl with Tryptophan in Water: Time-Resolved CIDNP and Laser Flash Photolysis Study

The mechanism of the reactions between photoexcited 2,2‘-dipyridyl and N-acetyl tryptophan has been studied by laser flash photolysis and time-resolved CIDNP (Chemically Induced Dynamic Nuclear Polarization). The transient absorption spectra obtained at different delays after the laser pulse are attributed to the triplet state of dipyridyl and to dipyridyl and tryptophan radicals. Depending on the pH of the solution, all three intermediates can be present in either protonated or deprotonated forms. It is shown that irrespective of pH the primary photochemical step is electron transfer from the tryptophan to triplet dipyridyl followed by protonation/deprotonation of the radicals so formed. The rate constant of the reaction of triplet dipyridyl with tryptophan is close to the diffusion-controlled limit and decreases slightly with increasing pH. The kinetics and the stationary value of the CIDNP are determined by the rates of radical termination, nuclear paramagnetic relaxation, and degenerate electron excha...