Hyperfine Sublevel Correlation Spectroscopy Research Articles

A primer in pulse EPR-based hyperfine spectroscopy for NMR spectroscopists

The theoretical foundations of NMR and EPR are very similar. Nevertheless, the fields differ significantly in practical aspects. This primer is an attempt to bridge some of the gaps between the fields, and at the same time it highlights some applications of pulse EPR. Basic EPR pulse sequences to detect nuclei in the vicinity of unpaired electrons are introduced. An emphasis is placed on explaining the concept to researchers familiar with NMR. The theoretical background as well as examples are given for electron-nuclear double resonance (ENDOR), electron-spin echo envelope modulation (ESEEM), including hyperfine sublevel correlation spectroscopy (HYSCORE), and electron double resonance (ELDOR)-detected NMR.

HYSCORE Spectroscopy to Resolve Electron–Nuclear Structure of Vanadyl Porphyrins in Asphaltenes from the Athabasca Oil Sands In Situ Conditions

The purpose of this work is to analyze the electron–nuclear interactions of the vanadyl-porphyrin (VP) complexes in oil asphaltenes. Asphaltenes from the Athabasca oil sands were studied by HYperfine Sublevel CORrelation Spectroscopy (HYSCORE) electron paramagnetic resonance (EPR). It makes it possible to resolve and interpret complex hyperfine spectra of intrinsic VP with strong and weak hyperfine interactions between the electron magnetic moment and various nuclear spins (1H, 14N, 51V). The main parameters of spin-Hamiltonian for the VP spin system are determined. The axially symmetric structure of the VP complexes is revealed, and the local nuclear environment of the paramagnetic center is investigated. The results can be used for the study of asphaltene electron–nuclear structure and asphaltene aggregates with the aim of elucidating asphaltenes’ transformation(s) under the influence of external treatment.

Understanding the p-doping of spiroOMeTAD by tris(pentafluorophenyl)borane

The solid-state organization of photoabsorber, hole and electron transporting layers, and interfaces between them plays an important role in governing the performance and stability of emerging optoelectronic devices such as perovskite solar cells (PSCs). The molecular organic semiconductor (OSC) 2,2′,7,7′-tetrakis [N,N-di(4-methoxyphenyl)amino]-9,9′-spirobifluorene (spiroOMeTAD) is a promising hole-transporting material (HTM) for PSCs, which is p-doped by molecular dopants to augment the charge carrier mobility. Here, the p-type doping of spiroOMeTAD by tris(pentafluorophenyl)borane (BCF) is investigated by a combination of techniques including optical spectroscopy, X-ray diffraction, Fourier transform infrared (FTIR), solid-state (ss)NMR, and electron paramagnetic resonance (EPR) spectroscopy. BCF molecules interact with traces of water molecules to form BCF-water complexes. Optical spectroscopy analysis suggests that the BCF/BCF-water complexes oxidize spiroOMeTAD molecules and facilitate p-type doping of spiroOMeTAD molecules. The different distributions of BCF and BCF-water molecules in doped spiroOMeTAD are characterized by FTIR and 11B NMR spectroscopy. An NMR crystallography approach which combines two-dimensional (2D) ssNMR and crystallography modeling is employed to unravel the packing interactions in spiroOMeTAD, and this analysis is extended to probe the morphological and structural changes in spiroOMeTAD:BCF blends. The hyperfine interactions are characterized by 2D hyperfine sub-level correlation (HYSCORE) spectroscopy. In this way, insight into the complex spiroOMeTAD:BCF blend morphology is obtained and compared for different dopant concentrations. Molecular-level analysis of doped HTMs enabled by this study has much wider relevance for further investigation, for example, chemical design and interfacial engineering of p-type doped HTMs for stable and efficient hybrid perovskite photovoltaics.

Insight into the structure of black coatings of ancient Egyptian mummies by advanced electron magnetic resonance of vanadyl complexes.

Ancient Egyptian mummies from the Late Period to the Greco-Roman Period were covered by a black coating consisting of complex and heterogeneous mixtures of conifer resins, wax, fat and oil with variable amounts of bitumen. Natural bitumen always contains traces of vanadyl porphyrin complexes that we used here as internal probes to explore the nanoscale environment of V ions in these black coatings by electron nuclear double resonance (ENDOR) and hyperfine sub-level correlation spectroscopy (HYSCORE). Four types of vanadyl porphyrin complexes were identified from the analysis of N hyperfine interactions. Three types (referred to as VO-P1, VO-P2 and VO-P3) are present in natural bitumen from the Dead Sea, among which VO-P1 and VO-P2 are also present in black coatings of mummies. The absence of VO-P3 in mummies, which is replaced by another complex, VO-P4, may be due to its transformation during preparation of the black matter for embalming. Analysis of H hyperfine interaction shows that bitumen and other natural substances are intimately mixed in these black coatings, with aggregate sizes of bitumen increasing with the bitumen content but not exceeding a few nanometres.

EPR characterization of the heme domain of a self-sufficient cytochrome P450 (CYP116B5)

CYP116B5 is a self-sufficient cytochrome P450 (CYP450) with interesting catalytic properties for synthetic purposes. When isolated, its heme domain can act as a peroxygenase on different substrates of biotechnological interest. Here, by means of continuous wave and advanced EPR techniques, the coordination environment of iron in the isolated CYP116B5 heme domain (CYP116b5hd) is characterized. The ligand-free protein shows the characteristic EPR spectrum of a low-spin (S = 1/2) FeIII-heme with [gz = 2.440 ± 0.005, gy = 2.25 ± 0.01, gx = 1.92 ± 0.01]. These g-values reflect an electronic ground state very similar to classical P450 monooxygenases rather than P450 peroxygenases. Binding of imidazole results in g-values very close to the ones reported for CYP152 peroxygenases. The detection of hyperfine interactions through HYperfine Sub-level CORrElation (HYSCORE) Spectroscopy experiments, shows that this is due to a nitrogen-mediated axial coordination. This work adds a piece of experimental evidence to the research, aimed at elucidating the features that distinguish the classical P450 enzymes from peroxygenases. It shows that the electronic environment of heme iron of CYP116B5 in the resting state is similar to the classical P450 monooxygenases. Therefore, it is not the critical factor that confers to CYP116B5hd its peroxygenase-like activity, suggesting a crucial role of the protein matrix.

Mapping the Pathway to Organocopper(II) Complexes Relevant to Atom Transfer Radical Polymerization.

The rare organocopper(II) complex [Cu(Me6tren)(CH2CN)]+ (Me6tren = tris(2-(dimethylamino)ethyl)amine) has emerged as an important model of potential byproducts in copper-catalyzed atom transfer radical polymerization. This complex has been generated by controlled potential electrolysis of [Cu(Me6tren)(NCMe)]2+ in the presence of BrCH2CN. Time-resolved UV-vis and continuous wave and pulse electron paramagnetic resonance (EPR) spectra identified [Cu(Me6tren)Br]+ as an intermediate. Hyperfine sublevel correlation and electron nuclear double resonance spectroscopy of samples at different timepoints reveal signals that are assigned to a C-bound cyanomethylate ligand, with distinct 14N and 1H hyperfine coupling constants in comparison with the corresponding N-bound acetonitrile and bromido complexes. The experimental EPR data are supported by density functional theory calculations to understand how the geometries of the species involved produce distinct spectroscopic signatures, and a clear picture of how this unusual organocopper(II) complex is formed has emerged.

Is Deprotonation of the Oxygen-Evolving Complex of Photosystem II during the S1 → S2 Transition Suppressed by Proton Quantum Delocalization?

We address the protonation state of the water-derived ligands in the oxygen-evolving complex (OEC) of photosystem II (PSII), prepared in the S2 state of the Kok cycle. We perform quantum mechanics/molecular mechanics calculations of isotropic proton hyperfine coupling constants, with direct comparisons to experimental data from two-dimensional hyperfine sublevel correlation (HYSCORE) spectroscopy and extended X-ray absorption fine structure (EXAFS). We find a low-barrier hydrogen bond with significant delocalization of the proton shared by the water-derived ligand, W1, and the aspartic acid residue D1-D61 of the D1 polypeptide. The lowering of the zero-point energy of a shared proton due to quantum delocalization precludes its release to the lumen during the S1→ S2 transition. Retention of the proton facilitates the shuttling of a proton during the isomerization of the tetranuclear manganese-calcium-oxo (Mn4Ca-oxo) cluster, from the "open" to "closed" conformation, a step suggested to be necessary for oxygen evolution from previous studies. Our findings suggest that quantum-delocalized protons, stabilized by low-barrier hydrogen bonds in model catalytic systems, can facilitate the accumulation of multiple oxidizing equivalents at low overpotentials.

Chromium Environment within Cr-Doped Silico-Aluminophosphate Molecular Sieves from Spin Density Studies.

X-/Q-band electron paramagnetic resonance (EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopies have been employed, in conjunction with density functional theory (DFT) modeling, to determine the location of Cr5+ions in SAPO-5 zeotype materials. The interaction of the unpaired electron of the paramagnetic Cr5+ species with 27Al could be resolved, allowing for the first detailed structural analysis of Cr5+ paramagnetic ions in SAPO materials. The interpretation of the experimental results is corroborated by DFT modeling, which affords a microscopic description of the system investigated. The EPR-active species is found to be consistent with isolated Cr5+ species isomorphously substituted in the framework at P5+ sites.

Electronic Structure of the Primary Electron Donor P700+• in Photosystem I Studied by Multifrequency HYSCORE Spectroscopy at X- and Q-Band.

The primary electron donor P700 of the photosystem I (PSI) is a heterodimer consisting of two chlorophyll molecules. A series of electron-transfer events immediately following the initial light excitation leads to a stabilization of the positive charge by its cation radical form, P700+•. The electronic structure of P700+• and, in particular, its asymmetry with respect to the two chlorophyll monomers is of fundamental interest and is not fully understood up to this date. Here, we apply multifrequency X- (9 GHz) and Q-band (35 GHz) hyperfine sublevel correlation (HYSCORE) spectroscopy to investigate the electron spin density distribution in the cation radical P700+• of PSI from a thermophilic cyanobacterium Thermosynechococcus elongatus. Six 14N and two 1H distinct nuclei have been resolved in the HYSCORE spectra and parameters of the corresponding nuclear hyperfine and quadrupolar hyperfine interactions were obtained by combining the analysis of HYSCORE spectral features with direct numerical simulations. Based on a close similarity of the nuclear quadrupole tensor parameters, all of the resolved 14N nuclei were assigned to six out of total eight available pyrrole ring nitrogen atoms (i.e., four in each of the chlorophylls), providing direct evidence of spin density delocalization over the both monomers in the heterodimer. Using the obtained experimental values of the 14N electron-nuclear hyperfine interaction parameters, the upper limit of the electron spin density asymmetry parameter is estimated as RA/Bupper = 7.7 ± 0.5, while a tentative assignment of 14N observed in the HYSCORE spectra yields RB/A = 3.1 ± 0.5.

Characterization of Cr-Hydrocarbyl Species via Pulse EPR in the Study of Ethylene Tetramerization Catalysis

The characterization of complexes involved in chromium catalysis is challenging due to the paramagnetism of Cr in its common oxidation states. Here, we demonstrate the utility of pulse electron paramagnetic resonance (pulse EPR) techniques in assigning structural features of Cr organometallic complexes relevant to ethylene tetramerization. An S = 3/2, Cr(III) bisaryl-methyl ethylene tetramerization precatalyst (1) has been selected for characterization by CW and pulse EPR spectroscopies. Using an isotopically labeled Cr-CD3 complex (1-d3), the methyl ligand was confirmed to remain bound to Cr in solution by detection of 2H couplings in X-band hyperfine sublevel correlation (HYSCORE) spectroscopy. Protonolysis of 1-d3 led to an S = 3/2, Cr(III) product (2-d3) that maintained spectroscopic features in HYSCORE for the CD3 group, indicative of retention of the Cr-alkyl bond. Following protonolysis of 1-h3 and subsequent reaction with ethylene, an S = 1/2 Cr(I) species with an ethylene-derived ligand was generated, supporting a mechanism involving this Cr oxidation state. Additionally, the pulse EPR characterization of a Cr(I) allyl-diene complex was performed for comparison. This is the first direct observation of hydrocarbyl ligands on Cr using pulse EPR methods. The methods described here are broadly applicable to Cr, first-row transition metals and other open-shell organometallic catalytic systems.

Polypyridyl-Based Copper Phenanthrene Complexes: Combining Stability with Enhanced DNA Recognition.

We report a series of copper(II) artificial metallo-nucleases (AMNs) and demonstrate their DNA damaging properties and in-vitro cytotoxicity against human-derived pancreatic cancer cells. The compounds combine a tris-chelating polypyridyl ligand, di-(2-pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu-DPA-N,N' (where N,N'=1,10-phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X-ray crystallography and continuous-wave EPR (cw-EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron-nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (Kapp ) rising from 105 to 107 m-1 with increasing extent of planar phenanthrene. Cu-DPA-DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine-cytosine (G-C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu-DPA-DPPZ, display promising anticancer activity against human pancreatic tumour cells with in-vitro results surpassing the clinical platinum(II) drug oxaliplatin.

Determining the Electronic Structure of Paramagnetic Intermediates in membrane proteins: A high-resolution 2D 1H hyperfine sublevel correlation study of the redox-active tyrosines of photosystem II

The photosynthetic reaction center, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive water oxidation. The four-electron water oxidation reaction occurs at the tetranuclear manganese‑calcium-oxo (Mn4Ca-oxo) cluster that is present in the oxygen-evolving complex (OEC) of PSII. The water oxidation reaction is facilitated by proton-coupled electron transfer (PCET) at the redox-active tyrosine residue, YZ, in the OEC which is one of the two symmetric tyrosine residues, YZ and YD, in PSII. Although YZ and YD are chemically identical, their redox properties and reaction kinetics are very different. In the present study, we apply high-resolution two-dimensional (2D) 1H hyperfine sublevel correlation (HYSCORE) spectroscopy to determine the electronic structure of YZ and YD to understand better the functional tuning of PCET at each tyrosine. Most importantly, the 2D HYSCORE measurements that are described here are applicable for the study of paramagnetic cofactors in a wide variety of membrane-bound proteins.

HYSCORE and DFT Studies of Proton-Coupled Electron Transfer in a Bioinspired Artificial Photosynthetic Reaction Center.

SummaryThe photosynthetic water-oxidation reaction is catalyzed by the oxygen-evolving complex in photosystem II (PSII) that comprises the Mn4CaO5 cluster, with participation of the redox-active tyrosine residue (YZ) and a hydrogen-bonded network of amino acids and water molecules. It has been proposed that the strong hydrogen bond between YZ and D1-His190 likely renders YZ kinetically and thermodynamically competent leading to highly efficient water oxidation. However, a detailed understanding of the proton-coupled electron transfer (PCET) at YZ remains elusive owing to the transient nature of its intermediate states involving YZ⋅. Herein, we employ a combination of high-resolution two-dimensional 14N hyperfine sublevel correlation spectroscopy and density functional theory methods to investigate a bioinspired artificial photosynthetic reaction center that mimics the PCET process involving the YZ residue of PSII. Our results underscore the importance of proximal water molecules and charge delocalization on the electronic structure of the artificial reaction center.

The Mechanism of Substrate Delivery and Activation in the Solar Water Oxidation Reaction of Photosystem II

The solar water-splitting protein complex, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in Nature by using light energy to drive a catalyst capable of oxidizing water. The water oxidation reaction is catalyzed by the tetramanganese-calcium-oxo (Mn4Ca-oxo) cluster in the oxygen-evolving complex (OEC) of PSII which cycles through five light-driven charge-storage or S-state intermediates (S0-S4) as it accumulates charge equivalents to split water. However, a detailed mechanism of the reaction remains elusive as it requires knowledge of the binding and activation of substrate water molecules in the higher S-state intermediates of the OEC. In particular, the binding of substrate in the S2 to S3 state transition of the OEC that leads to O-O bond formation is poorly understood because of the inability of conventional methods to probe water molecules. We are developing state-of-the-art two-dimensional (2D) hyperfine sublevel correlation spectroscopy methods for high-resolution proton crystallography of the S state intermediates of the water oxidation reaction. In this presentation, we will describe ongoing studies in our laboratory that employ small molecule analogs and site-directed mutagenesis of PSII to elucidate the mechanism of the delivery and binding of substrate water at the Mn4Ca-oxo cluster in the S2 and S3 states the the OEC. These studies have important implications on the mechanistic models for water oxidation in the OEC which is important as it is a promising blueprint for the development of artificial catalysts for water splitting that can generate clean and renewable energy from sunlight. †This study is supported by the Photosynthetic Systems Program, Office of Basic Energy Sciences, United States Department of Energy (DE-FG02-07ER15903 (KVL) and DE-FG02-05ER1564 (GWB)).

Multiple drug binding modes in Mycobacterium tuberculosis CYP51B1

The Mycobacterium tuberculosis (Mtb) genome encodes 20 different cytochrome P450 enzymes (CYPs), many of which serve essential biosynthetic roles. CYP51B1, the Mtb version of eukaryotic sterol demethylase, remains a potential therapeutic target. The binding of three drug fragments containing nitrogen heterocycles to CYP51B1 is studied here by continuous wave (CW) and pulsed electron paramagnetic resonance (EPR) techniques to determine how each drug fragment binds to the heme active-site. All three drug fragments form a mixture of complexes, some of which retain the axial water ligand from the resting state. Hyperfine sublevel correlation spectroscopy (HYSCORE) and electron-nuclear double resonance spectroscopy (ENDOR) observe protons of the axial water and on the drug fragments that reveal drug binding modes. Binding in CYP51B1 is complicated by the presence of multiple binding modes that coexist in the same solution. These results aid our understanding of CYP-inhibitor interactions and will help guide future inhibitor design.

Structure and dynamics of catalytically competent but labile paramagnetic metal-hydrides: the Ti(iii)-H in homogeneous olefin polymerization.

Metal hydride complexes find widespread application in catalysis and their properties are often understood on the basis of the available crystal structures. However, some catalytically relevant metal hydrides are only spontaneously formed in situ, cannot be isolated in large quantities or crystallised and their structure is therefore ill defined. One such example is the paramagnetic Ti(iii)-hydride involved in homogeneous Ziegler–Natta catalysis, formed upon activation of CpTi(iv)Cl3 with modified methylalumoxane (MMAO). In this contribution, through a combined use of electron paramagnetic resonance (EPR), electron-nuclear double resonance (ENDOR) and hyperfine sublevel correlation (HYSCORE) spectroscopies we identify the nature of the ligands, their bonding interaction and the extent of the spin distribution. From the data, an atomistic and electronic model is proposed, which supports the presence of a self-assembled ion pair between a cationic terminal Ti-hydride and an aluminate anion, with a hydrodynamic radius of ca. 16 Å.

Two-dimensional 67Zn HYSCORE spectroscopy reveals that a Zn-bacteriochlorophyll aP' dimer is the primary donor (P840) in the type-1 reaction centers of Chloracidobacterium thermophilum.

Chloracidobacterium (C.) thermophilum is a microaerophilic, chlorophototrophic species in the phylum Acidobacteria that uses homodimeric type-1 reaction centers (RC) to convert light energy into chemical energy using (bacterio)chlorophyll ((B)Chl) cofactors. Pigment analyses show that these RCs contain BChl aP, Chl aPD, and Zn2+-BChl aP' in the approximate ratio 7.1 : 5.4 : 1. However, the functional roles of these three different Chl species are not yet fully understood. It was recently demonstrated that Chl aPD is the primary electron acceptor. Because Zn2+-(B)Chl aP' is present at low abundance, it was suggested that the primary electron donor might be a dimer of Zn2+-BChl aP' molecules. In this study, we utilize isotopic enrichment and high-resolution two-dimensional (2D) 14N and 67Zn hyperfine sublevel correlation (HYSCORE) spectroscopy to demonstrate that the primary donor cation, P840+, in the C. thermophilum RC is indeed a Zn2+-BChl aP' dimer. Density functional theory (DFT) calculations and the measured electron-nuclear hyperfine parameters of P840+ indicate that the electron spin density on P840+ is distributed nearly symmetrically over two Zn2+-(B)Chl aP' molecules as expected in a homodimeric RC. To our knowledge this is the only example of a photochemical RC in which the Chl molecules of the primary donor are metallated differently than those of the antenna.

Adsorption of Olefins at Cupric Ions in Metal-Organic Framework HKUST-1 Explored by Hyperfine Spectroscopy.

Metal-organic frameworks (MOFs) represent a promising platform for gas storage and separation. In this work, adsorption of olefins in Zn-doped HKUST-1 metal-organic framework is explored with hyperfine spectroscopy. By means of electron-nuclear double resonance and hyperfine sublevel correlation spectroscopy, we detect the interaction between the electron spins of the Cu2+ sites of the MOF and the 1H nuclear spins of adsorbed C2H4 and C4H8. Further analysis of the measured spectra allows us to precisely locate the protons in the vicinity of the Cu2+ ions and thereby establish ensemble-averaged structural models of the olefin molecules adsorbed at the open metal sites of HKUST-1.

Elucidating the Role of Zinc-Bacteriochlorophyll A' in the Primary Photochemistry of Chloroacidobacterium thermophilum Reaction Centers

Chloracidobacterium (Cab.) thermophilum is a microaerophilic, chlorophototrophic species in the phylum of Acidobacteria that contains homodimeric Type I reaction centers (RC). Photosynthetic RCs in bacteria convert light energy into chemical energy using (bacterio)chlorophyll ((B)Chl) cofactors. In addition to participating in energy transfer in the light-harvesting complexes, the (B)Chl molecules also act as the primary electron donor and primary electron acceptor in Type I RCs. Previously, reversed-phase high-performance liquid chromatography of Cab. thermophilum RCs revealed the presence of (B)Chl a, Chl a, and Zn2+-(B)Chl a' in a ratio of about 12.8:8.0:2.0. Moreover, it was shown that Cab. thermophilum uses chlorosomes and FMO for light harvesting, and synthesizes (B)Chl c in addition to (B)Chl a, Chl a, and Zn2+-(B)Chl a'. It has been demonstrated that Chl a is the primary electron acceptor in Cab. thermophilum RC and since Zn2+-(B)Chl a' is only present in the RC, it was suggested that it could function as the primary electron donor. In this study, we utilize high-resolution, two-dimensional (2D) 14N and 67Zn hyperfine sublevel correlation (HYSCORE) spectroscopy to determine the electronic and geometric structure of the primary donor cation, P840+, in the Cab. thermophilum RC. Additionally, we perform density functional theory (DFT) calculations to determine the electron spin-density distribution and electron-nuclear hyperfine coupling parameters of P840+ for comparison with the parameters that are obtained from the experimental 2D HYSCORE measurements. Our studies indicate that that the primary donor, P840, of the Cab. RC is comprised of Zn2+-sBChl a' molecules.

Salen Complexes as Fire Protective Agents for Thermoplastic Polyurethane: Deep Electron Paramagnetic Resonance Spectroscopy Investigation.

The contribution of copper complexes of salen-based Schiff bases N, N'-bis(salicylidene)ethylenediamine (C1), N, N'-bis(4-hydroxysalicylidene)ethylenediamine (C2), and N, N'-bis(5-hydroxysalicylidene)ethylenediamine (C3) to the flame retardancy of thermoplastic polyurethane (TPU) is investigated in the context of minimizing the inherent flammability of TPU. Thermal and fire properties of TPU are evaluated. It is observed that fire performances vary depending upon the substitution of the salen framework. Cone calorimetry [mass loss calorimetry (MLC)] results show that, in TPU at 10 wt % loading, C2 and C3 reduce the peak of heat release rate by 46 and 50%, respectively. At high temperature, these copper complexes undergo polycondensation leading to resorcinol-type resin in the condensed phase and thus acting as intumescence reinforcing agents. C3 in TPU is particularly interesting because it delays significantly the time to ignition (MLC experiment). In addition, pyrolysis combustion flow calorimetry shows reduction in the heat release rate curve, suggesting its involvement in gas-phase action. Structural changes of copper complexes and radical formation during thermal treatment as well as their influence on fire retardancy of TPU in the condensed phase are investigated by spectroscopic studies supported by microscopic and powder diffraction studies. Electron paramagnetic resonance (EPR) spectroscopy was fully used to follow the redox changes of Cu(II) ions as well as radical formation of copper complexes/TPU formulations in their degradation pathways. Pulsed EPR technique of hyperfine sublevel correlation spectroscopy reveals evolution of the local surrounding of copper and radicals with a strong contribution of nitrogen fragments in the degradation products. Further, the spin state of radicals was investigated by the two-dimensional technique of phase-inverted echo-amplitude detected nutation experiment. Two different radicals were detected, that is, one monocarbon radical and an oxygen biradical. Thus, the EPR study permits to deeply investigate the mode of action of copper salen complexes in TPU.