CH3O Substitution Effect Revisited in the Vibrationally Resolved Laser-Induced Fluorescence Spectra of Methoxycyclohexoxy Radicals.

The oxy-substituted alkoxy radicals have attracted wide attention due to the increasing application of oxygenated volatile organic compounds as fuel additives and solvents. Direct detection of these intermediate radicals is desired for measuring the reaction rate and investigating the oxidation mechanism of organic compounds in the atmosphere. A charge-transfer excited state induced by CH3O substitution was identified in our previous study of 3-methoxy-1-propoxy radical [Xue, J. Phys. Chem. Chem. Phys. 2021, 23, 2586]. As the C-C bonds of chain alkoxy radicals can freely rotate, further studies are needed to understand the mechanism of this long-range charge-transfer effect. In this work, vibrational-resolved laser-induced fluorescence (LIF) spectra of 3- and 4-methoxycyclohexoxy radicals were obtained under jet-cooled conditions. A large red-shift of ∼454 cm-1 of the origin band was observed when the CH3O substituent moved from the δ site to the γ site of the cyclohexoxy radical. The LIF spectra are assigned to 3-cis (e, e) and 4-trans (e, e) conformers, respectively, with the assistance of structural optimization and electron excitation studies conducted at the CAM-B3LYP/6-311++G(d,p) level of theory. Natural transition orbital analysis reveals that the intramolecular charge transfer from the C-O-C p orbital to the radical O p orbital in 3-methoxycyclohexoxy has a strong effect on the radical CO σ → O p excitation and hence results in a spectral change. On the other hand, the spectral effect of CH3O substitution almost vanishes at δ carbon. The results propose a through-bond interaction between CH3O and radical CO groups.

Intramolecular charge transfer excitation induced by CH3O substitution in the 3-methoxy-1-propoxy radical.

The oxy-substituted alkoxy radicals are generated from the oxidation of ethers. Their degradation path affects ozone production and the formation of the secondary organic aerosol in the atmosphere. In this work, three alkoxy radicals with methoxy (CH3O) substitution at ß, γ, and δ carbon are studied using laser-induced fluorescence (LIF) spectroscopy and theoretical calculation methods. A charge transfer (CT) excited state induced by the CH3O substitution is identified to be because of the intramolecular electron transfer from the C-O-C p orbital to the radical O p orbital. Comparison of the structure and CT transition strength between GGt and TTt conformers of the 3-methoxy-1-propoxy radical (CH3OCH2CH2CH2O) suggests that this long-range charge transfer effect is mainly a through-bond interaction. The CT excited state of CH3OCH2CH2CH2O has a conical intersection with the CO σ → O p excited state, which, hence, changes the LIF spectrum of the radical. Only the decomposition product HCHO was observed in the LIF spectrum of ß substituted radical CH3OCH2CH2O. For δ substituted radical CH3OCH2CH2CH2CH2O, the substitution effect on the radical stability is negligible and its LIF spectrum is close to that of unsubstituted alkoxy radicals. The results provide information for understanding the degradation chemistry of oxygenated hydrocarbon molecules in the atmosphere.

Direct observation of the intermediate radical in the photodissociation of 1,3-cyclohexane dinitrite.

A two-step photodissociation mechanism was proposed in the literature for dinitrites in the absence of direct evidence of the intermediate species. In this work, photodissociation dynamics of cis and trans 1,3-cyclohexane dinitrites are investigated by laser-induced fluorescence (LIF) spectroscopy and theoretical calculation methods. Observation of the fluorescence spectra of the 3-nitrosooxy cyclohexoxy radical provides direct experimental evidence that the intermediate species exists. The results indicate that photodissociation of dinitrites indeed follows a two-step mechanism, i.e. one of the O-NO bonds of the molecule breaks first upon 355 nm laser photolysis and generates an alkoxy radical (RO) plus NO; the alkoxy radical further dissociates in the secondary dissociation step and produces small fragments such as vinoxy etc.

Energetic switch of the proline effect in collision-induced dissociation of singly and doubly protonated peptide Ala-Ala-Arg-Pro-Ala-Ala.

Suppression of the selective cleavage at N-terminal of proline is observed in the peptide cleavage by proteolytic enzyme trypsin and in the fragment ion mass spectra of peptides containing Arg-Pro sequence. An insight into the fragmentation mechanism of the influence of arginine residue on the proline effect can help in prediction of mass spectra and in protein structure analysis. In this work, collision-induced dissociation spectra of singly and doubly charged peptide AARPAA were studied by ESI MS/MS and theoretical calculation methods. The proline effect was evaluated by comparing the experimental ratio of fragments originated from cleavage of different amide bonds. The results revealed that the backbone amide bond cleavage was selected by the energy barrier height of the fragmentation pathway although the strong proton affinity of the Arg side chain affected the stereostructure of the peptide and the dissociation mechanism. The thermodynamic stability of the fragment ions played a secondary role in the abundance ratio of fragments generated via different pathways. Fragmentation studies of protonated peptide AACitPAA supported the energy-dependent hypothesis. The results provide an explanation to the long-term arguments between the steric conflict and the proton mobility mechanisms of proline effect.

Photodissociation dynamics of dinitrite at 355 nm: initiation of a reactive pathway.

Dinitrites are effective nitrosating reagents in preparative chemistry and their decomposition products are involved in the atmospheric reaction of volatile organic compounds with nitrogen oxides (NOx). In this work, photodissociation dynamics of five alkyl dinitrites were investigated by detecting the LIF spectra of the dissociation products and theoretical calculations. The results showed that the C-C bond connecting the two nitrosooxy (-ONO) functional groups was the weakest bond in vicinal dinitrites and aldehydes were the dissociation products. For dinitrites with two ONO groups separated by a CH2 group, vinoxy and 1-methylvinoxy radicals were detected as the fragments via photodissociation, indicating a different mechanism compared to thermal decomposition. This observation demonstrated that a new reactive pathway could be initiated by photolysis of dinitrites.

Methyl substitution effect on the jet-cooled laser-induced fluorescence spectrum of cyclohexoxy radical.

Understanding the structure and properties of cyclohexoxy radical and its substitutes is important because of their presence in combustion processes, in atmospheric chemistry, and as intermediates in the hydrocarbon reactions. In this work, jet-cooled laser-induced fluorescence (LIF) spectra of five dimethyl substituted cyclohexoxy radicals are obtained for the first time. The correlation between the spectral variations and the radical structural changes is studied with the assistance of theoretical calculations at the B3LYP/6-31+G(d) and CASSCF/6-31+G(d) levels. The results show that the spectral characters of the dimethylcyclohexoxy radicals and their dissociation kinetics are predominantly affected by the methyl substitution position related to the C-O group. The spectral effect of the two methyl groups will add up if they locate on asymmetric carbons of the cyclohexoxy ring. Methyl substitution on ß carbon weakens the six-member ring of cyclohexoxy and results in unimolecular dissociation via ß C-C bond cleavage on the methyl group side and forms vinoxy variants. This study clearly shows that the LIF spectra can be used to identify cyclohexoxy and the isomers of its methyl substitutes. The results will help to understand the photochemistry of cyclic hydrocarbons in the atmospheric and combustion processes.

The role of basic residues in the fragmentation process of the lysine rich cell-penetrating peptide TP10.

Selective cleavage effect of basic residues in the fragmentation of short peptides has been studied intensively. In contrast, the role of basic residues in the degradation of large peptides, such as cell-penetrating peptides, is largely unknown. In this work, the fragmentation of a 21 residues cell-penetrating peptide TP10 containing four lysine residues was studied by collision-induced dissociation mass spectrometry and computation methods. The influence of lysine residues on amide bond cleavage and fragmentation products was investigated. The results revealed that the selective cleavage effect of lysine residue did not present when the adjacent lysine residues in TP10 were both protonated. The localized high positive charge density might be the reason of preventing the mobile proton from migrating to the amide bonds in this part of the peptide. In contrast, the mobile proton preferred to reside in the N-terminal part of TP10 which had less positive charge. This preference gave more information of the peptide sequence in the mass spectrometry study and was helpful for stabilizing the C-terminal part of TP10, in which the basic lysine residues were preserved and crucial to the cell-penetrating process.

Time-Resolved Fluorescence Anisotropy Study of the Interaction Between DNA and a Peptide Truncated from the p53 Protein Core Domain.

Time-resolved fluorescence anisotropy spectroscopy was applied to study the interaction between a peptide truncated from the binding site of tumor suppressor p53 protein and the DNAs covalently labeled with 6-carboxyfluorescein (FAM) dye. Fluorescence intensity quenching and changes of anisotropy decay lifetime were monitored when FAM labeled DNA formed complex with the peptide. The results demonstrated that the sequence of DNA could not define the binding specificity between the peptide and DNA. But the anisotropy decay of FAM can be used to examine the binding affinity of the peptide to DNA. The fluorescent dynamics of FAM can also be used to represent the rigidity of the complex formed between the peptide and DNA.

Laser-induced fluorescence of isobutoxy in competition with ground state decomposition.

Spectroscopic detection is an important method to monitor alkoxy radicals in atmospheric photochemistry studies. In this work, we report the first observation of the laser-induced fluorescence (LIF) spectra of isobutoxy, 2-methyl-1-butoxy, and 3-methyl-1-butoxy in supersonic jet-cooled condition. Ground state unimolecular decomposition and isomerization as well as excited state relaxation dynamics of isobutoxy were discussed in combination with the theoretical calculations. Analysis of the experimental and theoretical results showed that methyl substitution on the ß carbon of the alkoxy radicals changed the LIF spectra of alkoxy radicals significantly. The competition between the ground state reactions and the photoexcitation process depended strongly on the radical structure and hence affected the involvement of alkoxy radicals in the photochemical reactions in the upper troposphere. This study will help to understand the dynamic role of alkoxy radicals in the atmosphere.

Selective cleavage enhanced by acetylating the side chain of lysine.

Selective cleavage is of great interest in mass spectrometry studies as it can help sequence identification by promoting simple fragmentation pattern of peptides and proteins. In this work, the collision-induced dissociation of peptides containing internal lysine and acetylated lysine residues were studied. The experimental and computational results revealed that multiple fragmentation pathways coexisted when the lysine residue was two amino acid residues away from N-terminal of the peptide. After acetylation of the lysine side-chain, b(n)+ ions were the most abundant primary fragment products and the Lys(Ac)-Gly amide bond became the dominant cleavage site via an oxazolone pathway. Acetylating the side-chain of lysine promoted the selective cleavage of Lys-Xxx amide bond and generated much more information of the peptide backbone sequence. The results re-evaluate the selective cleavage due to the lysine basic side-chain and provide information for studying the post-translational modification of proteins and other bio-molecules containing Lys residues.

Steady-state and time-resolved fluorescence of tetramethylrhodamine attached to DNA: correlation with DNA sequences.

Time-resolved fluorescence as well as steady-state absorption and fluorescence were detected in order to study the interactions between tetramethylrhodamine (TAMRA) and DNA when TAMRA was covalently labeled on single- and double-stranded oligonucleotides. Fluorescence intensity quenching and lifetime changes were characterized and correlated with different DNA sequences. The results demonstrated that the photoinduced electron transfer interaction between guanosine residues and TAMRA introduced a short lifetime fluorescence component when guanosine residues were at the TAMRA-attached terminal of the DNA sequences. The discrepancy of two-state and three-state models in previous studies was due to the DNA sequence selection and sensitivity of techniques used to detect the short lifetime component. The results will help the design of fluorescence-based experiments related to a dye labeled probe.

Vibrationally resolved LIF spectrum of tertiary methylcyclohexoxy radical.

Cyclohexoxy radical and its substitutes are intermediates of the combustion reaction in automobile engines, and hence play an important role in the atmospheric chemistry. Spectroscopic and conformational studies can provide convenient methods to monitor these species. In this work, we report the observation of the laser induced fluorescence (LIF) excitation spectrum of 1-methylcyclohexoxy, a tertiary ring alkoxy radical, in supersonic jet condition. The spectrum was assigned preliminarily to the chair-axial and chair-equatorial conformers of 1-methylcyclohexoxy. No C-O stretch progression was observed in 1-methylcyclohexoxy spectrum, which was different from t-butoxy. The short lifetime of excited state 1-methylcyclohexoxy and increased C-O bond length suggested a less stable excited state induced by the increased steric repulsion via methyl substitution on the alkoxy carbon. Dissociation rather than H transfer was suggested as the major nonradiative relaxation process, which competed with the fluorescence path.

A LabVIEW-Based Virtual Instrument System for Laser-Induced Fluorescence Spectroscopy.

We report the design and operation of a Virtual Instrument (VI) system based on LabVIEW 2009 for laser-induced fluorescence experiments. This system achieves synchronous control of equipment and acquisition of real-time fluorescence data communicating with a single computer via GPIB, USB, RS232, and parallel ports. The reported VI system can also accomplish data display, saving, and analysis, and printing the results. The VI system performs sequences of operations automatically, and this system has been successfully applied to obtain the excitation and dispersion spectra of α-methylnaphthalene. The reported VI system opens up new possibilities for researchers and increases the efficiency and precision of experiments. The design and operation of the VI system are described in detail in this paper, and the advantages that this system can provide are highlighted.

Conformation and spectroscopy study of cycloheptoxy radical.

Alkoxy radicals are important intermediates in the formation of tropospheric ozone. The spectroscopic identification and characterization of these species are important for understanding their chemistry in the atmosphere. In this work, we report the observation of the laser induced fluorescence (LIF) excitation spectrum of cycloheptoxy radical. The spectrum was assigned preliminary to the lowest energy twist-chair conformer (TC-i) of cycloheptoxy. The whole picture of the interconversions at ground state between different conformers of cycloheptoxy radicals was described by density functional theory calculations. The results revealed that despite the ring strain, the seven-membered ring alkoxy radical could exist in the supersonic jet-cooled condition. The decomposition and the low energy barrier pseudorotation between twist-chair conformers might be the reason of the much quieter spectrum of cycloheptoxy compared with the LIF spectrum of cyclohexoxy.

Selective cleavage of protonated penetratin and its substitutes under low-energy collision-induced dissociation condition.

An understanding of the dissociation of penetratin is important for improving its metabolic stability and cargo-delivery efficiency. In this study, we describe the selective cleavage of the K15-K16 amide bond of penetratin under the low-energy collision-induced dissociation condition in mass spectrometry. A variety of penetratin substitutes have been studied in which key basic amino acids have been substituted within the sequence. The calculated structure indicates that an alpha-helix structure prevents the fragmentation of the central peptide domain and the side chain of lysine is involved in the proton translocation process.

Dissociation of protonated peptides containing adjacent arginines.

The dissociation of protonated peptides containing adjacent arginines has been studied by electrospray ionization-tandem mass spectrometry (ESI MS/MS) and theoretical calculations. The experimental results show that singly protonated peptides cleave at the Arg-Arg amide bond and generate the y(1) ion when adjacent arginines are the C-terminal residues. The major cleavage occurs at the C-terminal amide bond and produces the b(n-1) ion when adjacent arginines are not the C-terminal residues. The diketopiperazine and oxazolone fragmentation pathways of protonated NRR (Asn-Arg-Arg) have been investigated at the B3LYP/6-31G(d) and B3LYP/6-31++G(d,p) levels of theory. The geometries and energies of transition state species and hydrogen-bonding interaction are also discussed.

High resolution spectra and conformational analysis of 2-butoxy radical.

We have recorded five high resolution (200 MHz), rotationally resolved, vibrational bands of the B-X electronic transition of 2-butoxy. Two bands of the 2-butoxy spectrum have been rotationally analyzed and assigned to two different geometrical conformers of the molecule. The analyses allow the determination of the six experimental rotational constants defined by the geometry of the species in the ground (X) and excited (B) electronic states and also four spin-rotation constants for the X electronic state of the conformers. Comparison of the experimental rotational constants with the results of ab initio computations provides unambiguous conformational assignment of these bands. This approach can be extended to assign two other spectral bands to the third 2-butoxy conformer.

Jet-cooled laser spectroscopy of the cyclohexoxy radical.

The laser-induced fluorescence and laser-excited dispersed fluorescence spectra of the cyclohexoxy radical has been observed under two sets of free-jet-cooling conditions, characterized by rotational temperatures of approximately 1 and 100 K. Although five conformers of cyclohexoxy are possible, it appears that all presently observed spectral bands can be accounted for by a single one. All cold spectral bands are assigned to the B-X electronic transition of the cyclohexoxy radical. Transitions to both a' and a" B state vibrational levels are observed and allowed due to a substantial pseudo-Jahn-Teller effect in the X state. Hot bands are also observed, which we attributed to transitions to the B state from the low-lying A electronic state. Analysis of the spectra yields vibrational frequencies for the X, A, and B states as well as the energy separations of their vibrationless levels.