Fourier Transform Electron Paramagnetic Resonance Research Articles

Spectroscopic Investigation of a Metal-Metal-Bonded Fe6 Single-Molecule Magnet with an Isolated S = 19/2 Giant-Spin Ground State.

The metal-metal-bonded molecule [Bu4N][(HL)2Fe6(dmf)2] (Fe6) was previously shown to possess a thermally isolated spin S = 19/2 ground state and found to exhibit slow magnetization relaxation below a blocking temperature of ∼5 K [J. Am. Chem. Soc. 2015, 137, 13949-13956]. Here, we present a comprehensive spectroscopic investigation of this unique single-molecule magnet (SMM), combining ultrawideband field-swept high-field electron paramagnetic resonance (EPR) with frequency-domain Fourier-transform terahertz EPR to accurately quantify the spin Hamiltonian parameters of Fe6. Of particular importance is the near absence of a 4th-order axial zero-field splitting term, which is known to arise because of quantum mechanical mixing of spin states on account of the relatively weak spin-spin (superexchange) interactions in traditional polynuclear SMMs such as the celebrated Mn12-acetate. The combined high-resolution measurements on both powder samples and an oriented single crystal provide a quantitative measure of the isolated nature of the spin ground state in the Fe6 molecule, as well as additional microscopic insights into factors that govern the quantum tunneling of its magnetization. This work suggests strategies for improving the performance of polynuclear SMMs featuring direct metal-metal bonds and strong ferromagnetic spin-spin (exchange) interactions.

Photochemical Spin Dynamics of the Vitamin B12 Derivative, Methylcobalamin.

Derivatives of vitamin B12 are six-coordinate cobalt corrinoids found in humans, other animals, and microorganisms. By acting as enzymatic cofactors and photoreceptor chromophores, they serve vital metabolic and photoprotective functions. Depending on the context, the chemical mechanisms of the biologically active derivatives of B12-methylcobalamin (MeCbl) and 5'-deoxyadenosylcobalamin (AdoCbl)-can be very different from one another. The extent to which this chemistry is tuned by the upper axial ligand, however, is not yet clear. Here, we have used a combination of time-resolved Fourier transform-electron paramagnetic resonance (FT-EPR), magnetic field effect experiments, and spin dynamic simulations to reveal that the upper axial ligand alone only results in relatively minor changes to the photochemical spin dynamics of B12. By studying the photolysis of MeCbl, we find that, similar to AdoCbl, the initial (or "geminate") radical pairs (RPs) are born predominantly in the singlet spin state and thus originate from singlet excited-state precursors. This is in contrast to the triplet RPs and precursors proposed previously. Unlike AdoCbl, the extent of geminate recombination is limited following MeCbl photolysis, resulting in significant distortions to the FT-EPR signal caused by polarization from spin-correlated methyl-methyl radical "f-pairs" formed following rapid diffusion. Despite the photophysical mechanism that precedes photolysis of MeCbl showing wavelength dependence, the subsequent spin dynamics appear to be largely independent of excitation wavelength, again similar to AdoCbl. Our data finally provide clarity to what in the literature to date has been a confused and contradictory picture. We conclude that, although the upper axial position of MeCbl and AdoCbl does impact their reactivity to some extent, the remarkable biochemical diversity of these fascinating molecules is most likely a result of tuning by their protein environment.

Broadband W-band Rapid Frequency Sweep Considerations for Fourier Transform EPR.

A multi-arm W-band (94 GHz) electron paramagnetic resonance spectrometer that incorporates a loop-gap resonator with high bandwidth is described. A goal of the instrumental development is detection of free induction decay following rapid sweep of the microwave frequency across the spectrum of a nitroxide radical at physiological temperature, which is expected to lead to a capability for Fourier transform electron paramagnetic resonance. Progress toward this goal is a theme of the paper. Because of the low Q-value of the loop-gap resonator, it was found necessary to develop a new type of automatic frequency control, which is described in an appendix. Path-length equalization, which is accomplished at the intermediate frequency of 59 GHz, is analyzed. A directional coupler is favored for separation of incident and reflected power between the bridge and the loop-gap resonator. Microwave leakage of this coupler is analyzed. An oversize waveguide with hyperbolic-cosine tapers couples the bridge to the loop-gap resonator, which results in reduced microwave power and signal loss. Benchmark sensitivity data are provided. The most extensive application of the instrument to date has been the measurement of T1 values using pulse saturation recovery. An overview of that work is provided.

Fourier-transform electron spin resonance with bandwidth-compensated chirp pulses

Electron spin echo experiments using chirp pulses at X-band around 9GHz have been performed with a home-built spectrometer based on an arbitrary waveform generator. Primary echoes without phase dispersion were obtained by employing the Böhlen–Bodenhausen scheme with the refocusing pulse being half as long as the coherence-generating pulse. To account for physical bandwidth limitation by the resonator, the instantaneous sweep rate of the chirps was adapted to the spectrometer’s frequency response function, which can be recorded from the sample under study within a few minutes. Such bandwidth-compensated chirp pulses are experimentally shown to achieve an almost uniform excitation bandwidth that exceeds the resonator bandwidth.This uniform excitation allows for computing frequency-domain spectra by Fourier-transformation (FT) of the echo signal. For a nitroxide in dilute solid solution with a spectral width of 200MHz, the FT EPR spectrum agrees remarkably well with a field-swept echo-detected EPR spectrum. The overall spectral perturbation for operation far beyond the resonator bandwidth was characterized by acquiring a 700MHz wide spectral range of a copper (II) EPR spectrum with nearly uniform amplitude with excitation and refocusing pulses of 200 and 100ns, respectively.Furthermore, peculiarities were observed in solid-state FT EPR spectra of disordered systems. To understand these peculiarities two-dimensional data sets were acquired that correlate the FT EPR spectrum to inversion recovery or nuclear modulation. The echo envelope modulation experiments reveal echo decay rates increased by enhanced instantaneous diffusion and passage-specific effects in the nuclear modulations. The latter effect can be suppressed by nuclear modulation averaging. Apparent longitudinal relaxation times for a given subset of orientations are influenced by nuclear modulation effects. Proper extraction of orientation-dependent relaxation times thus requires an experimental setup that minimizes the modulations.

Fourier Transform EPR Spectroscopy of Trityl Radicals for Multifunctional Assessment of Chemical Microenvironment

AbstractPulse techniques in electron paramagnetic resonance (EPR) allow for a reduction in measurement times and increase in sensitivity but require the synthesis of paramagnetic probes with long relaxation times. Here it is shown that the recently synthesized phosphonated trityl radical possesses long relaxation times that are sensitive to probe the microenvironment, such as oxygenation and acidity of an aqueous solution. In principle, application of Fourier transform EPR (FT‐EPR) spectroscopy makes it possible to acquire the entire EPR spectrum of the trityl probe and assess these microenvironmental parameters within a few microseconds. The performed analysis of the FT‐EPR spectra takes into consideration oxygen‐, proton‐, buffer‐, and concentration‐induced contributions to the spectral shape, therefore enabling quantitative and discriminative assessment of pH, pO2, and concentrations of the probe and inorganic phosphate.

Fourier Transform EPR Spectroscopy of Trityl Radicals for Multifunctional Assessment of Chemical Microenvironment

Pulse techniques in electron paramagnetic resonance (EPR) allow for a reduction in measurement times and increase in sensitivity but require the synthesis of paramagnetic probes with long relaxation times. Here it is shown that the recently synthesized phosphonated trityl radical possesses long relaxation times that are sensitive to probe the microenvironment, such as oxygenation and acidity of an aqueous solution. In principle, application of Fourier transform EPR (FT-EPR) spectroscopy makes it possible to acquire the entire EPR spectrum of the trityl probe and assess these microenvironmental parameters within a few microseconds. The performed analysis of the FT-EPR spectra takes into consideration oxygen-, proton-, buffer-, and concentration-induced contributions to the spectral shape, therefore enabling quantitative and discriminative assessment of pH, pO2, and concentrations of the probe and inorganic phosphate.

X-band rapid-scan EPR of samples with long electron spin relaxation times: a comparison of continuous wave, pulse and rapid-scan EPR

X-band room temperature spectra obtained by rapid-scan, continuous wave, field-swept echo-detected and Fourier transform electron paramagnetic resonance (FTEPR) were compared for three samples with long electron spin relaxation times: amorphous hydrogenated silicon (T1 = 11 μs, T2 = 3.3 μs), 0.2% N@C60 solid (T1 = 120–160 μs, T2 = 2.8 μs) and neutral single substitutional nitrogen centres (NS0) in diamonds (T1 = 2300 μs, T2 = 230 μs). For each technique, experimental parameters were selected to give less than 2% broadening of the lineshape. For the same data acquisition times, the signal-to-noise for the rapid-scan spectra was one-to-two orders of magnitude better than for continuous wave or field-swept echo-detected spectra. For amorphous hydrogenated silicon, T2* (∼ 10 ns) is too short to perform FTEPR. For 0.2% N@C60, the signal-to-noise ratio for rapid scan is about five times better than for FTEPR. For NS0 the signal-to-noise ratio is similar for rapid scan and FTEPR.

Evaluation of partial k-space strategies to speed up time-domain EPR imaging

Narrow-line spin probes derived from the trityl radical have led to the development of fast in vivo time-domain EPR imaging. Pure phase-encoding imaging modalities based on the single-point imaging scheme have demonstrated the feasibility of three-dimensional oximetric images with functional information in minutes. In this article, we explore techniques to improve the temporal resolution and circumvent the relatively short biological half-lives of trityl probes using partial k-space strategies. There are two main approaches: one involves the use of the Hermitian character of the k-space by which only part of the k-space is measured and the unmeasured part is generated using the Hermitian symmetry. This approach is limited in success by the accuracy of numerical estimate of the phase roll in the k-space that corrupts the Hermiticy. The other approach is to measure only a judicially chosen reduced region of k-space (a centrosymmetric ellipsoid region) that more or less accounts for >70% of the k-space energy. Both of these aspects were explored in Fourier transform-EPR imaging with a doubling of scan speed demonstrated by considering ellipsoid geometry of the k-space. Partial k-space strategies help improve the temporal resolution in studying fast dynamics of functional aspects in vivo with infused spin probes.

Dynamics and Binding Affinity of Spin-Labeled Stearic Acids in β-Lactoglobulin: Evidences from EPR Spectroscopy and Molecular Dynamics Simulation

β-Lactoglobulin (β-LG) is a member of the lipocalin protein family involved in the transport of fatty acids and other small hydrophobic molecules. The main binding site is at a central cavity, referred to as "calyx", formed by the protein β-barrel sandwich. Continuous-wave and pulsed Fourier transform electron spin resonance (cw- and FT-EPR) spectroscopy and molecular dynamics (MD) simulation were combined to investigate the interaction of fatty acids with bovine β-LG. Stearic acid bearing the nitroxide label at different positions, n, along the acyl chain (n-SASL, n = 5, 7, 10, 12, 16) were used. The EPR data show that the protein affinity for SASL decreases on going from n = 5 to 16. This behavior is due to the accommodation of the SASL in the protein calyx, which is hampered by steric hindrance of the doxyl ring for n ≥ 10, as evidenced by MD data. Conformation and dynamics of 5-SASL are similar to those of the unlabeled stearate molecule. 5-SASL in the protein binding site undergoes librational motion of small amplitude on the nanosecond time scale at cryogenic temperature and rotational dynamics with correlation time of 4.2 ns at physiological temperature. The results highlight the dynamical features of fatty acids/β-LG interaction.

Pulsed electron paramagnetic resonance spectroscopy powered by a free-electron laser

Electron paramagnetic resonance (EPR) spectroscopy interrogates unpaired electron spins in solids and liquids to reveal local structure and dynamics; for example, EPR has elucidated parts of the structure of protein complexes that other techniques in structural biology have not been able to reveal. EPR can also probe the interplay of light and electricity in organic solar cells and light-emitting diodes, and the origin of decoherence in condensed matter, which is of fundamental importance to the development of quantum information processors. Like nuclear magnetic resonance, EPR spectroscopy becomes more powerful at high magnetic fields and frequencies, and with excitation by coherent pulses rather than continuous waves. However, the difficulty of generating sequences of powerful pulses at frequencies above 100 gigahertz has, until now, confined high-power pulsed EPR to magnetic fields of 3.5 teslas and below. Here we demonstrate that one-kilowatt pulses from a free-electron laser can power a pulsed EPR spectrometer at 240 gigahertz (8.5 teslas), providing transformative enhancements over the alternative, a state-of-the-art ∼30-milliwatt solid-state source. Our spectrometer can rotate spin-1/2 electrons through π/2 in only 6 nanoseconds (compared to 300 nanoseconds with the solid-state source). Fourier-transform EPR on nitrogen impurities in diamond demonstrates excitation and detection of EPR lines separated by about 200 megahertz. We measured decoherence times as short as 63 nanoseconds, in a frozen solution of nitroxide free-radicals at temperatures as high as 190 kelvin. Both free-electron lasers and the quasi-optical technology developed for the spectrometer are scalable to frequencies well in excess of one terahertz, opening the way to high-power pulsed EPR spectroscopy up to the highest static magnetic fields currently available.

W-band frequency-swept EPR

This paper describes a novel experiment on nitroxide radical spin labels using a multiarm EPR W-band bridge with a loop-gap resonator (LGR). We demonstrate EPR spectroscopy of spin labels by linear sweep of the microwave frequency across the spectrum. The high bandwidth of the LGR, about 1 GHz between 3 dB points of the microwave resonance, makes this new experiment possible. A frequency-tunable yttrium iron garnet (YIG) oscillator provides sweep rates as high as 1.8 × 10 5 GHz/s, which corresponds to 6.3 kT/s in magnetic field-sweep units over a 44 MHz range. Two experimental domains were identified. In the first, linear frequency sweep rates were relatively slow, and pure absorption and pure dispersion spectra were obtained. This appears to be a practical mode of operation at the present level of technological development. The main advantage is the elimination of sinusoidal magnetic field modulation. In the second mode, the frequency is swept rapidly across a portion of the spectrum, and then the frequency sweep is stopped for a readout period; FID signals from a swept line oscillate at a frequency that is the difference between the spectral position of the line in frequency units and the readout position. If there is more than one line, oscillations are superimposed. The sweep rates using the YIG oscillator were too slow, and the portion of the spectrum too narrow to achieve the full EPR equivalent of Fourier transform (FT) NMR. The paper discusses technical advances required to reach this goal. The hypothesis that trapezoidal frequency sweep is an enabling technology for FT EPR is supported by this study.

Stochastic Liouville equation studies of FT-EPR spectra of singlet–triplet mixing photo-chemically generated radical pair system

A stochastic Liouville equation (SLE) was numerically solved to obtain pulsed Fourier-transform (FT) EPR spectra on a radical pair system created in a photo-induced chemical reaction. Numerical calculations were applied to the photo-chemical reaction of deuterated acetone and 2-propanol at low temperatures. In this reaction system, the antiphase structures of the EPR signals, so called spin-correlated radical pair (SCRP) signals of two identical isopropyl ketyl radicals and spin-polarized free isopropyl ketyl radicals were observed by FT-EPR and continuous wave time-resolved (CW TR) EPR techniques. In the present work, FT-EPR spectra of the antiphase structure signals of the radical pair themselves as well as spin-polarized free radical signals were simulated. Additionally, rising behavior of free radical signals polarized by the radical pair mechanism (RPM) was also clarified. Furthermore two-dimensional (2D) FT-EPR nutation spectra were simulated in the both cases with and without the radical pairs by the use of SLE. In these simulations, strong DC components in the nutation frequency dimension, were well reproduced as was obtained in experiments. It was shown that relaxation during the microwave pulse was essential for the appearance of the DC components.

Oxidation of Cyclic Dipeptides by Photoinduced H-Atom Abstraction. A Laser Flash FT EPR and Optical Spectroscopy Study

Laser flash photolysis with the Fourier transform electron paramagnetic resonance (FT EPR) and optical spectroscopy detection methods on the nanosecond time scale have been employed in order to investigate the oxidation mechanism of cyclic dipeptides glycine, alanine, and sarcosine anhydrides initiated by SO4*- or 9,10-anthraquinone-2,6-disulfonate (2,6-AQDS) triplet in oxygen free aqueous solutions. A direct hydrogen abstraction from the ring C-H position of an anhydride by both oxidants is proposed as the primary reaction, rather then an electron transfer from nitrogen followed by (alpha)C-H deprotonation. The overall second-order rate constants for the reaction with SO4*- were determined to be 7.2 x 10(7) M(-1) s(-1), 1.2 x 10(8) M(-1) s(-1), and 5.2 x 10(8) M(-1) s(-1) for glycine anhydride, alanine anhydride, and sarcosine anhydride, respectively. The rate constants for 2,6-AQDS triplet as oxidizing species are about two times lower. The radical intermediate products derived from cyclic dipeptides observed on the microsecond time scale were assigned to the general structure of piperazine-2,5-dione-3-yl radical. These are spin polarized by the mechanisms of chemically induced dynamic electron polarization (CIDEP). For SO4*- as the oxidant the spectra are exhibiting an E/A* polarization pattern originating partially from F-pairs of two piperazine-2,5-dione-3-yl radicals.

Time-Resolved FT EPR and Optical Spectroscopy Study on Photooxidation of Aliphatic α-Amino Acids in Aqueous Solutions; Electron Transfer from Amino vs Carboxylate Functional Group

Using time-resolved Fourier transform electron paramagnetic resonance, FT EPR, and optical spectroscopy, the photooxidation of glycine, alpha-alanine, alpha-aminoisobutyric acid, and model compounds beta-alanine, methylamine and sodium acetate, by excited triplets of anthraquinone-2,6-disulfonate dianion was studied in aqueous solutions in the pH range 5-13. Anthraquinone radical trianions showing strong emissive spin-polarization (CIDEP) were formed, indicating fast electron transfer from the quenchers to the spin-polarized quinone triplet as the primary reaction. None of the primary radicals formed upon one-electron oxidation of quenchers could be detected at the nanosecond time scale of FT EPR measurements because of their very fast transformation into secondary products. The latter were identified to be decarboxylated alpha-aminoalkyl radicals for alpha-amino acids anions and zwitterions, beta-aminoalkyl radicals for beta-alanine zwitterions, and methyl radicals for acetate anions; corresponding aminyl radicals were the first EPR detectable products from beta-alanine anions and methylamine. Thus, anthraquinone-2,6-disulfonate triplet can take an electron from both NH(2)- and -CO(2)(-) functional groups forming aminium ((+*)NH(2)-) and acyloxyl (-CO(2)(*)) radicals, respectively. Aminium radicals derived from beta-alanine anions and CH(3)-NH(2) stabilize by deprotonation into aminyl radicals, whereas these derived from alpha-amino acids anions are known to suffer ultrafast decarboxylation (tau approximately 10 ps). Analysis of the polarization patterns revealed that decarboxylation from acyloxyl radicals are considerably slower (ns < tau < 0.1 micros). Therefore, in the case of alpha-amino acids, the isoelectronic structures NH(2)-CR(2)-CO(2)(*) and (+*)NH(2)-CR(2)-CO(2)(-) probably do not constitute resonance mesomeric forms of one and the same species and the decarboxylation of aminium radicals is not preceded by the intramolecular carboxylate to amino group electron transfer. Absolute triplet quenching rate constants at zero ionic strength were in the range of 2 x 10(8) to 2 x 10(9) M(-1) s(-1) for R-NH(2) and 2 x 10(7) to 10(8) M(-1) s(-1) for R-CO(2)(-) type of electron donors, reflecting in principle their standard reduction potentials. The strengths of acids: (+)NH(3)-(*)CH(2), (+)NH(3)-(*)C(CH(3))H, and (+)NH(3)-(*)C(CH(3))(2), pK(a) <4, >6, and >7, respectively, were found to be remarkably strongly dependent on alpha-C substitution. The conjugate bases of these alpha-aminoalkyl radicals reduce anthraquinone-2,6-disulfonate dianion ground state with k(sec) = 3 x 10(9) M(-1) s(-1).

Integration of digital signal processing technologies with pulsed electron paramagnetic resonance imaging

The integration of modern data acquisition and digital signal processing (DSP) technologies with Fourier transform electron paramagnetic resonance (FT-EPR) imaging at radiofrequencies (RF) is described. The FT-EPR system operates at a Larmor frequency ( L f) of 300 MHz to facilitate in vivo studies. This relatively low frequency L f, in conjunction with our ∼10 MHz signal bandwidth, enables the use of direct free induction decay time-locked subsampling (TLSS). This particular technique provides advantages by eliminating the traditional analog intermediate frequency downconversion stage along with the corresponding noise sources. TLSS also results in manageable sample rates that facilitate the design of DSP-based data acquisition and image processing platforms. More specifically, we utilize a high-speed field programmable gate array (FPGA) and a DSP processor to perform advanced real-time signal and image processing. The migration to a DSP-based configuration offers the benefits of improved EPR system performance, as well as increased adaptability to various EPR system configurations (i.e., software configurable systems instead of hardware reconfigurations). The required modifications to the FT-EPR system design are described, with focus on the addition of DSP technologies including the application-specific hardware, software, and firmware developed for the FPGA and DSP processor. The first results of using real-time DSP technologies in conjunction with direct detection bandpass sampling to implement EPR imaging at RF frequencies are presented.

Effects of finite pulse width on two-dimensional Fourier transform electron spin resonance

Two-dimensional (2D) Fourier transform ESR techniques, such as 2D-ELDOR, have considerably improved the resolution of ESR in studies of molecular dynamics in complex fluids such as liquid crystals and membrane vesicles and in spin labeled polymers and peptides. A well-developed theory based on the stochastic Liouville equation (SLE) has been successfully employed to analyze these experiments. However, one fundamental assumption has been utilized to simplify the complex analysis, viz. the pulses have been treated as ideal non-selective ones, which therefore provide uniform irradiation of the whole spectrum. In actual experiments, the pulses are of finite width causing deviations from the theoretical predictions, a problem that is exacerbated by experiments performed at higher frequencies. In the present paper we provide a method to deal with the full SLE including the explicit role of the molecular dynamics, the spin Hamiltonian and the radiation field during the pulse. The computations are rendered more manageable by utilizing the Trotter formula, which is adapted to handle this SLE in what we call a “Split Super-Operator” method. Examples are given for different motional regimes, which show how 2D-ELDOR spectra are affected by the finite pulse widths. The theory shows good agreement with 2D-ELDOR experiments performed as a function of pulse width.

A Closer Look at Photochemical Reactions of Transition‐Metal Compounds by Time‐Resolved EPR

AbstractFor Abstract see ChemInform Abstract in Full Text.

Direct detection and time-locked subsampling applied to pulsed electron paramagnetic resonance imaging

The application of direct time-locked subsampling (TLSS) to Fourier transform electron paramagnetic resonance (FT-EPR) spectroscopy at radio frequencies (rf) is described. With conventional FT-EPR spectroscopy, the high Larmor frequencies (L(f)) often necessitate the use of intermediate frequency (IF) stages to down convert the received free induction decay (FID) signal to a frequency that can be acquired with common data acquisition technology. However, our research focuses on in vivo studies, and consequently utilizes a FT-EPR system with a L(f) of 300 MHz. This relatively low frequency L(f), in conjunction with the advent of bandpass sampling analog-to-digital conversion and signal processing technologies, has enabled us to omit the IF stage in our FT-EPR system. With this in mind, TLSS techniques have been developed to directly sample the 300 MHz FID signal at a sampling rate of 80 MHz providing a signal bandwidth of 20 MHz. The required modifications to the data acquisition and processing system specific to this application are described. Custom software developed to control the EPR system setup, acquire the signals, and post process the data, is outlined. Data was acquired applying both coherent averaging and stochastic excitation sequences. The results of these experiments demonstrate digital down conversion of the 300 MHz FID signal to quadrature baseband. Direct FID TLSS eliminates many noise sources common in EPR systems employing traditional analog receiver techniques, such as the IF mixer stage in single channel systems, and the quadrature baseband mixer stage in dual channel systems.

A closer look at photochemical reactions of transition-metal compounds by time-resolved EPR

A review is given of applications of time-resolved electron paramagnetic resonance (TREPR) in the field of photochemistry of transition-metal compounds. The two main TREPR techniques used in these studies are described. A brief overview is given of chemically induced dynamic electron polarization mechanisms that can affect TREPR spectra and that can give insights into the mechanism of photochemical reactions. Following these background sections, experimental results are presented. The discussion focuses in particular on the Fourier-transform EPR studies of photoinduced metal-alkyl bond homolysis reactions of a series of transition-metal (Co, Ru, Re, Pt) complexes carried out by the authors.

Interactions of Dendrimers with Selected Amino Acids and Proteins Studied by Continuous Wave EPR and Fourier Transform EPR

Interactions of polyamidoamine dendrimers, termed Gn, where n indicates the generation (=number of amidoamine layers), at different protonation levels with selected amino acids and proteins have been investigated by means of continuous wave electron paramagnetic resonance (cw-EPR) and pulsed-EPR (electron spin-echo = ESE) analyses. A low-generation dendrimer (G2) and a high-generation one (G6) were labeled with nitroxides for the EPR measurements. Gly, Glu, Arg, and Leu were selected as representative of neutral(zwitterionic)-polar, acidic, basic, and low-polar amino acids, respectively. The water-soluble proteins alpha-chymotrypsin and albumin were selected on the basis of a basic and an acidic isoelectric point, respectively. The cw-EPR spectra were analyzed by computing the line shapes to extract information about the dendrimer-biomolecule interactions. In general, dendrimers at a high protonation level interact stronger with amino acids than those at a low level of protonation. However, even for highly protonated dendrimers, a synergistic effect between hydrophilic and hydrophobic interactions promoted the formation of stable Gn-amino acid adducts, as demonstrated by the enhanced interactions with Leu. As expected from acid-base interactions, stable adducts were formed between Arg and highly protonated dendrimers and between Glu and low level protonated dendrimers. The relatively strong dendrimer interactions with the protein chymotrypsin and the poor interactions of dendrimers with albumin demonstrated that the protonated amino groups of the dendrimers are predominantly involved in the interactions with these proteins and indicated a significant role in the interactions with the dendrimers of the hydrophobic external residues of chymotrypsin. Computer-aided analysis of the ESE experiments was consistent with the cw-EPR results and supported the conclusion of a partial complexation of the nitroxides of the dendrimer with Leu and alpha-chymotrypsin.