Resonance Raman Spectroscopy Research Articles

Ruthenium(v) terminal arylimido corroles: isolation, spectroscopic characterization and reactivity.

Terminal Ru(v)-imido species are thought to be as reactive to group transfer reactions as their Ru(v)-oxo homologues, but are less studied. With the electron-rich corrole ligand, relatively stable and isolable Ru(v)-arylimido complexes [Ru(tBu-Cor)(NAr)] (H3(tBu-Cor) = 5,15-diphenyl-10-(p-tert-butylphenyl)corrole, Ar = 2,4,6-Me3C6H2 (Mes), 2,6-(iPr)2C6H3 (Dipp), 2,4,6-(iPr)3C6H2 (Tipp), and 3,5-(CF3)2C6H3 (BTF)) can be prepared from [Ru(tBu-Cor)]2 under strongly reducing conditions. This type of Ru(v)-monoarylimido corrole complex with S = ½ was characterized by high-resolution ESI mass spectrometry, X-band EPR, resonance Raman spectroscopy, magnetic susceptibility, and elemental analysis, together with computational studies. Under heating/light irradiation (xenon lamp) conditions, the complexes [Ru(tBu-Cor)(NAr)] (Ar = Mes, BTF) could undergo aziridination of styrenes and amination of benzylic C(sp3)-H bonds with up to 90% product yields.

ODMR active bright sintered detonation nanodiamonds obtained without irradiation

We present the results of study the structure and composition of microcrystalline diamonds obtained by high-pressure high temperature sintering of detonation nanodiamond particles. Using optical detected magnetic resonance method, photoluminescence spectroscopy and Raman spectroscopy we found sintering of detonation nanodiamond significantly differ from initial detonation nanodiamonds and can be compared to high quality diamonds. Monocrystals of diamonds obtained by the method of oriented attachment have dimensions of up to tens of microns, possess the habitus of high-quality diamonds, and do not contain metal catalysts in the lattice structure. In those crystals, the presence of optically active nitrogen impurities in the crystal lattice is observed. In particular, there is a bright nitrogen-vacancy defects. They are characterized by optical detected magnetic resonance method, which shows that spin properties of the obtained single crystals correspond to high-quality natural diamonds and surpass synthetic diamonds obtained from graphite in the presence of metal catalysts, followed by irradiation and annealing to obtain nitrogen-vacancy defects optical defects in the diamond lattice. The presence of nitrogen-vacancy defects defects and the high-quality of the crystal structure of sintering of detonation nanodiamond allows us to consider them as potential candidates in quantum magnetometry. For this purpose, the possibility of a simple way to improve the AFM probe by fixing a microcrystalline sintering of detonation nanodiamond particle on its tip is demonstrated. Keywords: detonation nanodiamond, HPHT sintering, ODMR, single crystalline, photoluminescence, defects.

Towards therapeutic drug monitoring of antibiotic levels - analyzing the pharmacokinetics of levofloxacin using DUV-resonance Raman spectroscopy.

Therapeutic drug monitoring (TDM) plays an important role in clinical practice. Here, pharmacokinetics has a decisive influence on the effective antibiotic concentration during treatment. Moreover, different kinetics exist for different administration forms. Accordingly, adjusting the correct concentration depends, in addition to gender, age, weight, clinical picture, etc., on the dosage form of the antibiotic. This study investigates the capability of deep UV resonance Raman spectroscopy (DUV-RRS) to simulate the pharmacokinetics of fluoroquinolone levofloxacin after two different administration forms (intravenous and oral). Three different pre-processing methods were applied, and the best agreement with the simulation was achieved using the extended multiplicative scatter correction. The resulting spectra were used for partial least squares (PLS) regression and ordinary least squares (OLS) regression. The kinetic parameters were compared with the simulated data, with PLS showing the best performance for intravenous administration and a comparable result to OLS for oral administration, while the errors are smaller. The acquired results show the potential of DUV-RRS in combination with PLS regression as a promising supportive method for TDM.

Active site architecture of coproporphyrin ferrochelatase with its physiological substrate coproporphyrin III: Propionate interactions and porphyrin core deformation.

Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV-vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies.

Time-resolved spectroscopic mapping of vibrational energy flow in proteins: Understanding thermal diffusion at the nanoscale.

Vibrational energy exchange between various degrees of freedom is critical to barrier-crossing processes in proteins. Hemeproteins are well suited for studying vibrational energy exchange in proteins because the heme group is an efficient photothermal converter. The released energy by heme following photoexcitation shows migration in a protein moiety on a picosecond timescale, which is observed using time-resolved ultraviolet resonance Raman spectroscopy. The anti-Stokes ultraviolet resonance Raman intensity of a tryptophan residue is an excellent probe for the vibrational energy in proteins, allowing the mapping of energy flow with the spatial resolution of a single amino acid residue. This Perspective provides an overview of studies on vibrational energy flow in proteins, including future perspectives for both methodologies and applications.

Substrate-Dependent Conformational Switch of the Noncubane [4Fe-4S] Cluster in Heterodisulfide Reductase HdrB

The noncubane [4Fe-4S] cluster identified in the active site of heterodisulfide reductase (HdrB) displays a unique geometry among Fe-S cofactors found in metalloproteins. Here we employ resonance Raman (RR) spectroscopy and density functional theory (DFT) calculations to probe structural, electronic, and vibrational properties of the noncubane cluster in HdrB from a non-methanogenic Desulfovibrio vulgaris (Dv) Hildenborough organism. The immediate protein environment of the two neighboring clusters in DvHdrB is predicted using homology modeling. We demonstrate that in the absence of substrate, the oxidized [4Fe-4S]3+ cluster adopts a "closed" conformation. Upon substrate coordination at the "special" iron center, the cluster core translates to an "open" structure, facilitated by the "supernumerary" cysteine ligand switch from iron-bridging to iron-terminal mode. The observed RR fingerprint of the noncubane cluster, supported by Fe-S vibrational mode analysis, will advance future studies of enzymes containing this unusual cofactor.

Electrochemical and Structural Study of the Buried Tryptophan in Azurin: Effects of Hydration and Polarity on the Redox Potential of W48.

Tryptophan serves as an important redox-active amino acid in mediating electron transfer and mitigating oxidative damage in proteins. We previously showed a difference in electrochemical potentials for two tryptophan residues in azurin with distinct hydrogen-bonding environments. Here, we test whether reducing the side chain bulk at position Phe110 to Leu, Ser, or Ala impacts the electrochemical potentials (E°) for tryptophan at position 48. X-ray diffraction confirmed the influx of crystallographically resolved water molecules for both the F110A and F110L tyrosine free azurin mutants. The local environments of W48 in all azurin mutants were further evaluated by UV resonance Raman (UVRR) spectroscopy to probe the impact of mutations on hydrogen bonding and polarity. A correlation between the frequency of the ω17 mode─considered a vibrational marker for hydrogen bonding─and E° is proposed. However, the trend is opposite to the expectation from a previous study on small molecules. Density functional theory calculations suggest that the ω17 mode reflects hydrogen bonding as well as local polarity. Further, the UVRR data reveal different intensity/frequency shifts of the ω9/ω10 vibrational modes that characterize the local H-bonding environments of tryptophan. The cumulative data support that the presence of water increases E° and reveal properties of the protein microenvironment surrounding tryptophan.

Oxygen versus Sulfur Coordination in Cobalt Superoxo Complexes: Spectroscopic Properties, O2 Binding, and H-Atom Abstraction Reactivity.

A five-coordinate, disiloxide-ligated cobalt(II) (S = 3/2) complex (1) was prepared as an oxygen-ligated analogue to the previously reported silanedithiolate-ligated CoII(Me3TACN)(S2SiMe2) (J. Am. Chem. Soc., 2019, 141, 3641-3653). The structural and spectroscopic properties of 1 were analyzed by single-crystal X-ray diffraction, electron paramagnetic resonance (EPR), and NMR spectroscopies. The reactivity of 1 with dioxygen was examined, and it was shown to bind O2 reversibly in a range of solvents at low temperatures. A cobalt(III)-superoxo complex, CoIII(O2·-)(Me3TACN)((OSi2Ph)2O) (2), was generated, and was analyzed by UV-vis, EPR, and resonance Raman spectroscopies. Unlike its sulfur-ligated analogue, complex 2 can thermally release O2 to regenerate 1. Vibrational assignments for selective 18O isotopic labeling of both O2 and disiloxide ligands in 2 are consistent with a 6-coordinate, Co(η1-O2·-)("end-on") complex. Complex 2 reacts with the O-H bond of 4-methoxy-2,2,6,6-tetramethylpiperidin-1-ol (4-MeO-TEMPOH) via H-atom abstraction with a rate of 0.58(2) M-1 s-1 at -105 °C, but it is unable to oxidize phenol substrates. This bracketed reactivity suggests that the O-H bond being formed in the putative CoIII(OOH) product has a relatively weak O-H bond strength (BDFE ∼66-74 kcal mol-1). These thermodynamic and kinetic parameters are similar to those seen for the sulfur-ligated Co(O2)(Me3TACN)(S2SiMe2), indicating that the differences in the electronic structure for O versus S ligation do not have a large impact on H-atom abstraction reactivity.

Excited-state resonance Raman spectroscopy probes the sequential two-photon excitation mechanism of a photochromic molecular switch.

Some diarylethene molecular switches have a low quantum yield for cycloreversion when excited by a single photon, but react more efficiently following sequential two-photon excitation. The increase in reaction efficiency depends on both the relative time delay and the wavelength of the second photon. This paper examines the wavelength-dependent mechanism for sequential excitation using excited-state resonance Raman spectroscopy to probe the ultrafast (sub-30fs) dynamics on the upper electronic state following secondary excitation. The approach uses femtosecond stimulated Raman scattering (FSRS) to measure the time-gated, excited-state resonance Raman spectrum in resonance with two different excited-state absorption bands. The relative intensities of the Raman bands reveal the initial dynamics in the higher-lying states, Sn, by providing information on the relative gradients of the potential energy surfaces that are accessed via secondary excitation. The excited-state resonance Raman spectra reveal specific modes that become enhanced depending on the Raman excitation wavelength, 750 or 400nm. Many of the modes that become enhanced in the 750nm FSRS spectrum are assigned as vibrational motions localized on the central cyclohexadiene ring. Many of the modes that become enhanced in the 400nm FSRS spectrum are assigned as motions along the conjugated backbone and peripheral phenyl rings. These observations are consistent with earlier measurements that showed higher efficiency following secondary excitation into the lower excited-state absorption band and illustrate a powerful new way to probe the ultrafast dynamics of higher-lying excited states immediately following sequential two-photon excitation.

Antibiotic Susceptibility Testing with Raman Biosensing.

Antibiotics guard us against bacterial infections and are among the most commonly used medicines. The immediate consequence of their large-scale production and prescription is the development of antibiotic resistance. Therefore, rapid detection of antibiotic susceptibility is required for efficient antimicrobial therapy. One of the promising methods for rapid antibiotic susceptibility testing is Raman spectroscopy. Raman spectroscopy combines fast and contactless acquisition of spectra with good selectivity towards bacterial cells. The antibiotic-induced changes in bacterial cell physiology are detected as distinct features in Raman spectra and can be associated with antibiotic susceptibility. Therefore, the Raman-based approach may be beneficial in designing therapy against multidrug-resistant infections. The surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RRS) additionally provide excellent sensitivity. In this review, we present an analysis of the Raman spectroscopy-based optical biosensing approaches aimed at antibiotic susceptibility testing.

Identifying antibiotics based on structural differences in the conserved allostery from mitochondrial heme-copper oxidases

Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.

The combination of resonance Raman spectroscopy and site directed mutagenesis to study the diverse aspects of heme protein structure and function

This review provides examples illustrating the powerful combination of resonance Raman spectroscopy and site-directed mutagenesis to investigate the structure-function relationship in structurally different heme proteins with diverse physiological functionality. The selective mutation of key amino acid residues gives rise to distinct spectroscopic fingerprints, as a result of the subtle alterations of the heme pocket environment. This review includes, but it is not limited to, the study of: i) the interactions between bound exogenous ligands with distal residues, ii) the effects of hydrogen bonds between the proximal residues and the surrounding cavity, iii) the interaction between the peripheral substituents of the heme group with the protein matrix with the concomitant effect on specific biological processes.

Abstract 302: Esophageal Saturation Monitoring More Accurately Detects Severe Tissue Hypoxia Than Near-infrared Spectroscopy

Introduction: Balance between oxygen delivery and demand is typically assessed based on venous oxyhemoglobin saturation (SvO2), which requires repeated blood sampling from a central location. Alternative, non-invasive methods such as near-infrared spectroscopy (NIRS) demonstrate poor sensitivity to tissue hypoxia. Methods: We explored the possibility of measuring regional tissue oxyhemoglobin saturation in the esophagus (EsSO2) using a new, linear probe using resonance Raman spectroscopy (RRS, 420 nm excitatory light, A) to quantify the fraction of oxyhemoglobin. The RRS probe was passed from the mouth into the mid-esophagus in Yorkshire swine (n=7, weight 11.2-13.4 kg). Cerebral (cNIRS) and splanchnic NIRS (sNIRS, Somanetics) were continuously recorded. Swine underwent complete asphyxia by endotracheal tube occlusion for 12 minutes, during which time they were resuscitated using CPR and drugs. EsSO2, cNIRS, sNIRS were compared with SO2 based on arterial blood gas co-oximetry (SaO2). For the purpose of analysis, SaO2 was considered functionally equivalent to SvO2 due to the obligate intrapulmonary (veno-arterial) shunting in this physiology. Results: During asphyxia, SO2 rapidly declined to nearly 0%. This was reflected in EsSO2, but cNIRS and sNIRS decreased to a lesser extent (B). EsSO2 exhibited a strong correlation with SO2 (r 2 =0.63, p<0.001) compared to sNIRS (r 2 =0.53, p<0.001) and cNIRS (r 2 =0.06, p=0.106, C). Defining SO2 <30% as the presence of critical hypoxia, EsSO2 had a higher sensitivity than both the sNIRS and cNIRS (D). EsSO2 was <30 in 100% (33/33) of timepoints in which critical hypoxia was present, significantly more sensitive than rSO2 based on cNIRS (which was <30 in 58% (19/33) of timepoints) and sNIRS (<30 in 24% (8/33) of timepoints). Conclusion: EsSO2 accurately detects critical hypoxic states, more so than cNIRS or sNIRS, and may represent an alternative, continuous, less invasive approach to central venous monitoring.

The divergent pH dependence of substrate turnover in dehaloperoxidases A and B

The pH-dependent peroxidase activity in both dehaloperoxidases A and B was studied by a kinetic assay, stopped flow spectroscopy, resonance Raman spectroscopy, and high-performance liquid chromatography at pH 5.0, 6.0, and 7.0. At pH 7.0, both isozymes follow the peroxidase ping-pong kinetic model derived from the three-step reaction scheme using the steady-state approximation. However, deviation from standard saturation behavior is observed at pH < 6.0 and [TCP] > 0.7 mM, owing to multiple processes: a) self-inhibition of TCP by internal binding; b) oxidation of the product by a pH- and concentration-dependent secondary reaction; and c) formation of an inactive species known as compound RH in the absence of oxidizable substrate. Although DHP-A and DHP-B differ by only 5 amino acids, they show a complete trend reversal in their observed peroxidase kinetics and product yields. Although at pH 7.0 DHP-B had higher TCP oxidation activity than DHP-A as reported previously, as pH was lowered, DHP-A appeared to have a higher peroxidase activity than DHP-B. This is an unprecedented result. However, the fact that there are multiple processes contributing to both kinetics and yield of TCP oxidation complicates interpretation of these data. Deactivation via compound RH and self-inhibition are pH dependent reactions that compete with substrate oxidation. Compound RH formation was observed to be rapid at low pH. A complete set of control experiments were conducted to differentiate the various contributions to the observed enzyme kinetics.

Ultrafast Time-Resolved Spectroscopic Study on the Photophysical and Photochemical Reaction Mechanisms of ortho-Methylbenzophenone in Selected Solutions.

The photophysical and photochemical reaction pathways of ortho-methylbenzophenone (o-MeBP) in different solutions were investigated by employing femtosecond to nanosecond transient absorption and nanosecond time-resolved resonance Raman spectroscopy methods. In pure acetonitrile, neutral or pH 1 aqueous solutions, o-MeBP exhibit similar excited-state evolutions upon excitation in which o-MeBP will experience excitation to an excited state then undergo intersystem crossing and solvent arrangement followed by 1,5 hydrogen atom transfer processes to form the first singlet excited state, triplet state (n, π*), biradical intermediates, and enol form transients, respectively. However, in a pH 0 acidic solution, the protonation of o-MeBP will form the cation biradical intermediate that facilitates radical coupling to generate a benzocyclobutanol product, which causes a dramatic reduction of the lifetime of the enol form transients. In contrast, in sodium bicarbonate solution, the biradical intermediate may be quenched by the bicarbonate ion to construct a C-C bond and form the carboxylic acid that causes a fast decay of biradical intermediate. These results demonstrate that the photophysical and photochemical reaction pathways of o-MeBP are pH-dependent in aqueous solution which may be very useful for the capture of CO2 capture by photoexcitation of aromatic ketones.

Stoichiometry of reactions of ozone and hypochlorous acid with lignin and hexenuronic acid and its chlorination

The stoichiometry of ozone and hypochlorous acid reactions with lignin and hexenuronic acid (HexA) was measured in bleaching experiments of Eucalyptus sp. kraft pulp. The progress of the reactions was followed by UV Resonance Raman spectroscopy that can quantify lignin and HexA based on the Raman scattering intensities of the carbon–carbon double bond in HexA and the aromatic ring in lignin. Here, one mol of ozone converted 0.16 mol of lignin (C9 monomer units) and 0.28 mol of HexA, whereas 1 mol of hypochlorous acid converted 0.09 mol of lignin and 0.23 mol of HexA. The use of a tertiary amine catalyst with the hypochlorous acid treatments did not affect these stoichiometries. The stoichiometric ratios showed that ozone was more efficient in oxidizing lignin than hypochlorous acid, while both electrophiles reacted with HexA to a similar extent. HexA reaction by hypochlorous acid was concluded to involve initial electrophilic chlorination of the carbon–carbon double bond, contributing to significant organochlorine (OX) formation in the pulp. Evidence on this was the linear correlation between the initial HexA content and OX (0.59 mol OX per mol HexA) and the high OX content in the xylan extracted from the bleached pulp. The 2D NMR HSQC and TOCSY spectra of the isolated xylans showed the disappearance of HexA signals after the treatment with hypochlorous acid and the appearance of a new spin system, yet to be fully identified.

Use of polymers as wavenumber calibration standards in deep-UVRR

Deep-UV resonance Raman spectroscopy (UVRR) allows the classification of bacterial species with high accuracy and is a promising tool to be developed for clinical application. For this attempt, the optimization of the wavenumber calibration is required to correct the overtime changes of the Raman setup. In the present study, different polymers were investigated as potential calibration agents. The ones with many sharp bands within the spectral range 400–1900 cm−1 were selected and used for wavenumber calibration of bacterial spectra. Classification models were built using a training cross-validation dataset that was then evaluated with an independent test dataset obtained after 4 months. Without calibration, the training cross-validation dataset provided an accuracy for differentiation above 99 % that dropped to 51.2 % after test evaluation. Applying the test evaluation with PET and Teflon calibration allowed correct assignment of all spectra of Gram-positive isolates. Calibration with PS and PEI leads to misclassifications that could be overcome with majority voting. Concerning the very closely related and similar in genome and cell biochemistry Enterobacteriaceae species, all spectra of the training cross-validation dataset were correctly classified but were misclassified in test evaluation. These results show the importance of selecting the most suitable calibration agent in the classification of bacterial species and help in the optimization of the deep-UVRR technique.

Lutein/β-carotene ratio in extra virgin olive oil: An easy and rapid quantification method by Raman spectroscopy

Carotenoids play an important role in the stability, freshness, and nutritional value of extra-virgin olive oil (EVOO). However, the carotenoid content in EVOO changes over time as a function of olive ripening and degrading events. A reliable quality marker is the ratio between the two most abundant carotenoids, namely lutein and β-carotene, since the second degrades more rapidly. Thus, to obtain a fast quantification of the lutein/β-carotene ratio in olive oil could deserve a certain interest. Resonant Raman spectroscopy is a rapid and non-destructive technique, widely applied for food chemical characterization. In this work, using high-performance liquid chromatography and UV–vis absorption spectroscopy as calibration techniques, we present a reliable method to assess the lutein/β-carotene ratio in EVOO using a single Raman spectrum. The novel approach deserves several methodological and applicative interests, since it would allow rapid, on-site screening of EVOO quality and authenticity, especially if implemented as a portable system.

5f covalency from x-ray resonant Raman spectroscopy

X-ray resonant Raman spectroscopy (XRRS), a variant of resonant inelastic x-ray scattering, has been used to investigate the two prototype systems, UF4 and UO2. Both are U5f2 and each is an example of 5f localized, ionic behavior and 5f localized, covalent behavior, respectively. From the M5 XRRS measurements, the 5f band gap in each can be directly determined and, moreover, a clear and powerful sensitivity to 5f covalency emerges.

Resonance Raman spectroscopy of MoS2 monolayers treated with nitrogen plasma

The creation of defects in two-dimensional (2D) materials is crucial for tuning their optical and electrical properties. Molybdenum disulfide is the most promising 2D material, with applications ranging from optoelectronics to high-performance Li-S batteries. Hence, it is possible to tune its properties for desirable applications by introducing defects into the monolayer structure. In this study the presence of defects is probed by resonance Raman spectroscopy The results show that the Raman spectra of the MoS2 monolayers gradually change as the nitrogen plasma treatment time increases. The changes in the longitudinal acoustic LA(M) intensity, and second-order 2LA(M), and 2LA(K) intensity and full width at half maximum variations observed in the resonance Raman spectra were used as figures of merit to characterize the MoS2 samples with respect to the density of defects. Photoluminescence spectroscopy and atomic force microscopy were used to characterize the samples.