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Light-Induced Oxidation of Tryptophan and Histidine. Reactivity of Aromatic N-Heterocycles toward Triplet-Excited Flavins
Abstract
Mechanisms of flavin-mediated photooxidation of electron-rich amino acids tryptophan and histidine were investigated for aqueous solutions. Indole, representing the tryptophan side chain in proteins, reacted at nearly diffusion controlled rates (k approximately 2.7 x 10(9) L mol(-1) s(-1) at 293 K) with the triplet-excited flavin state, but reactions of imidazole (and histidine) were significantly slower (k < 2.0 x 10(8) L mol(-1) s(-1)) as determined by laser flash photolysis. Oxidation rates of derivates were invariably susceptible to electronic factors affecting incipient radical cation stability, while no primary kinetic hydrogen/deuterium isotope effect was observed for imidazole. Thus reaction by electron transfer was proposed in contrast to a direct hydrogen abstraction. Unlike indole compounds, imidazole derivatives suffered from the presence of a basic imino nitrogen (=N-), which caused the rate constant of histidine free base (k approximately 1.8 x 10(8) L mol(-1) s(-1)) to drop considerably upon protonation. Complexation of the imino nitrogen with transition metals provoked changes in reactivity, as rate constants decreased after addition of Zn(2+) (k of 4-methylimidazole, as histidine model, decreased from 9.0 x 10(8) L mol(-1) s(-1) in the absence to 4.1 x 10(8) L mol(-1) s(-1) in the presence of ZnCl(2)). The pyrrole nitrogen (-NH-) was not directly involved in complexation reactions, but its electron density increased upon interaction with hydrogen bond-accepting anions and resulted in higher rate constants (k of 4-methylimidazole increased from 9.0 x 10(8) L mol(-1) s(-1) to 2.0 x 10(9) L mol(-1) s(-1) after addition of NaOAc). The high rate constants were in agreement with a large thermodynamical driving force, as calculated from oxidation peak potentials determined electrochemically. After oxidation, resulting radical cations were readily deprotonated and trapped by 2-methyl-2-nitrosopropane, as detected by electron paramagnetic resonance spectroscopy. Indole-derived spin adducts were attributed to selective trapping of C(3)-centered radicals, whereas spin adducts with imidazole-derivatives arose from both carbon and nitrogen-centered imidazolyl radicals.
... With DNA, light can induce oxidative damage and protein-crosslinking (10,17). In proteins, light can induce several reactions including oxidation (18)(19)(20)(21)(22)(23)(24), fragmentation (16,25) and crosslinking (7,14,19,(25)(26)(27)(28)(29)(30)(31). These reactions can either occur by direct light absorption of a few amino acids (e.g. ...
... These reactions can either occur by direct light absorption of a few amino acids (e.g. tryptophan, phenylalanine, histidine, tryosine and cysteine) (17,22,24,(31)(32)(33)(34)(35), via photosensitization by external chromophores (4,7,(36)(37)(38) or by reaction with photo-generated reactive oxygen species such as singlet oxygen ( 1 O 2 ) (25,(39)(40)(41)(42)(43). Due to the low absorbance of natural amino acids at wavelengths above 300 nm, 1 O 2 is responsible for most lightinduced reactions in proteins. In both pathways, tryptophan (Trp) can either directly absorb light or react with lightgenerated 1 O 2 to generate a series of light-induced (16,25). ...
... In both pathways, tryptophan (Trp) can either directly absorb light or react with lightgenerated 1 O 2 to generate a series of light-induced (16,25). Therefore, the photoreactions of Trp have received considerable attention and many of its photoreaction mechanisms are now quite well-understood (18,21,22,33,39,42,44,45). It has been shown that Trp can undergo a variety of different photochemical reactions that lead to stable products with significantly stronger absorbance of light, especially in the near UV region (λ >300 nm). ...
Purpose:
Light is known to induce histidine (His) oxidation and His-His crosslinking in proteins. The crosslinking is resulted from the nucleophilic attack of a His to a photooxidized His from another protein. The goal of this work is to understand if covalent buffer adducts on His residues can be generated by light through similar mechanisms in nucleophilic buffers such as Tris and His.
Methods:
A model protein (DNase) was buffer exchanged into nucleophilic buffers before light exposure. Photogenerated products were characterized by tryptic peptide mapping with mass spectrometry (MS) analysis. Several buffer adductions on His residues were identified after light exposure. To understand the influencing factors of such reactions, the levels of adducts were measured for six nucleophilic buffers on all His residues in DNase.
Results:
The levels of adducts were found to correlate with the solvent accessibility of the His residue. The levels of adducts also correlate with the structure of the nucleophile, especially the steric restrictions of the nucleophile. The levels of adducts can be higher than that of other His photoreaction products, including photooxidation and crosslinking.
Conclusions:
In nucleophilic buffers, light can induce covalently-linked adducts to His residues.
... Oxidation of His mainly occurs via type II photo-oxidation mechanism. Thus, His is oxidized via 1 O 2 resulting in a cycloaddition, in which the imidazole ring reacts with 1 O 2 to form 2,4-or 2,5-EP intermediates [187,[385][386][387]. Irradiation of free and protein-bound His yields a broad variety of different His oxidation products, including crosslinking products [9,187,383], such as His-His crosslinks [187] as well as ring opened products, such as Asp, Asn and 2-Oxo-His [388,389]. ...
... Oxidation of His mainly occurs via type II photo-oxidation mechanism. Thus, His is oxidized via 1 O2 resulting in a cycloaddition, in which the imidazole ring reacts with 1 O2 to form 2,4-or 2,5-EP intermediates [187,[385][386][387]. Irradiation of free and protein-bound His yields a broad variety of different His oxidation products, including crosslinking products [9,187,383], such as His-His crosslinks [187] as well as ring opened products, such as Asp, Asn and 2-Oxo-His [388,389]. ...
UV and ambient light-induced modifications and related degradation of therapeutic proteins are observed during manufacturing and storage. Therefore, to ensure product quality, protein formulations need to be analyzed with respect to photo-degradation processes and eventually protected from light exposure. This task usually demands the application and combination of various analytical methods. This review addresses analytical aspects of investigating photo-oxidation products and related mediators such as reactive oxygen species generated via UV and ambient light with well-established and novel techniques.
... Exploring the utility of our side chain diversication method for other peptides beyond Ac-G-P-Dha-F-NH 2 , we examined H 2 N-G-Dha-H-W-S-Y-G-M-R-P-K-CO 2 H, a Dha-containing peptide which also contains common amino acids found in peptides and proteins that would most likely interfere with our photochemical transformation (Scheme 1C). Flavin photocatalysts are known to oxidatively modify C-terminal amino acids via decarboxylation, 32 as well as directly oxidize tyrosine (Y), 33 tryptophan (W), 33,34 histidine (H), 34 and methionine (M) 35 residues. Moreover, radicals are known to modify histidine, tyrosine, and tryptophan amino acids. ...
... Exploring the utility of our side chain diversication method for other peptides beyond Ac-G-P-Dha-F-NH 2 , we examined H 2 N-G-Dha-H-W-S-Y-G-M-R-P-K-CO 2 H, a Dha-containing peptide which also contains common amino acids found in peptides and proteins that would most likely interfere with our photochemical transformation (Scheme 1C). Flavin photocatalysts are known to oxidatively modify C-terminal amino acids via decarboxylation, 32 as well as directly oxidize tyrosine (Y), 33 tryptophan (W), 33,34 histidine (H), 34 and methionine (M) 35 residues. Moreover, radicals are known to modify histidine, tyrosine, and tryptophan amino acids. ...
Most peptide drugs contain non-proteinogenic amino acids (NPAAs), born out through extensive structure-activity relationship (SAR) studies using solid-phase peptide synthesis (SPPS). Synthetically laborious and expensive to manufacture, NPAAs also can have poor coupling efficiencies allowing only a small fraction to be sampled by conventional SPPS. To gain general access to NPAA-containing peptides, we developed a first-generation platform that merges contemporary flavin photocatalysis with parallel synthesis to simultaneously make, purify, quantify, and even test up to 96 single-NPAA peptide variants via the unique combination of boronic acids and a dehydroalanine residue in a peptide. We showcase the power of our newly minted platform to introduce NPAAs of diverse chemotypes-aliphatic, aromatic, heteroaromatic-directly into peptides, including 15 entirely new residues, and to evolve a simple proteinogenic peptide into an unnatural inhibitor of thrombin by non-classical peptide SAR.
... L-tryptophan is one of the nine amino acids essential to humans (Mustafa et al., 2018). It is used for food preservation due to its antioxidant properties (Huvaere and Skibsted, 2009), also being used as a supplement in animal feed (Favero Neto and Giaquinto, 2020). In addition, it can be applied as a pharmaceutical (Porter et al., 2005). ...
... L-tryptophan is one of the nine amino acids essential to humans, being also essential for animals, plants, and some bacteria (Mustafa et al., 2018). In the food industry, it is used for food preservation due to its antioxidant properties (Huvaere and Skibsted, 2009) and as a supplement in animal feed (Favero Neto and Giaquinto, 2020). It can also be applied as a pharmaceutical since it promotes good results in treating psychological diseases like depression (Porter et al., 2005), anxiety (van Veen et al., 2009), and sleep disorder (Sangsopha et al., 2019). ...
Lately, wastewater treatment plants are much often being designed as wastewater-resource factories inserted in circular cities. Among biological treatment technologies, aerobic granular sludge (AGS), considered an evolution of activated sludge (AS), has received great attention regarding its resource recovery potential. This review presents the state-of-the-art concerning the influence of operational parameters on the recovery of alginate-like exopolysaccharides (ALE), tryptophan, phosphorus, and polyhydroxyalkanoates (PHA) from AGS systems. The carbon to nitrogen ratio was identified as a parameter that plays an important role for the optimal production of ALE, tryptophan, and PHA. The sludge retention time effect is more pronounced for the production of ALE and tryptophan. Additionally, salinity levels in the bioreactors can potentially be manipulated to increase ALE and phosphorus yields simultaneously. Some existing knowledge gaps in the scientific literature concerning the recovery of these resources from AGS were also identified. Regarding industrial applications, tryptophan has the longest way to go. On the other hand, ALE production/recovery could be considered the most mature process if we take into account that existing alternatives for phosphorus and PHA production/recovery are optimized for activated sludge rather than granular sludge. Consequently, to maintain the same effectiveness, these processes likely could not be applied to AGS without undergoing some modification. Therefore, investigating to what extent these adaptations are necessary and designing alternatives is essential.
... The formation of pink-to red-colored complexes between monoclonal antibodies and hydroxocobalamine was realized, where hydroxocobalamine was generated photo-chemically from cyanocobalamine in the cell culture media (138,139). Riboflavin is a prominent photo-sensitizer during UV A exposure, reported to induce amino acid and protein oxidation (140)(141)(142). Pterins have generally been shown to induce protein photodegradation (143)(144)(145). ...
... The aromatic side chain of His is a prominent target for oxidation via multiple mechanisms, including peroxidedependent (93), metal-catalyzed (95,97,149,150) and photoinduced (98,99,142) reactions. Especially, the reaction of singlet oxygen with His generates peroxides, which decompose into a series of molecular products (99). ...
Purpose:
Therapeutic proteins are sensitive to photo-degradation by UV A and visible light. As none of the essential amino acids exhibits significant absorption in the UV A and visible light regions, the underlying mechanisms of photo-degradation induced by UV A and visible light are not well understood. This review addresses potential mechanisms, by which protein structure, oxidative modifications or impurities can promote the photo-degradation of therapeutic proteins during the exposure to UV A and visible light.
... Regarding the interaction between 3 Rf* and OD, the increase of 3 k q with increasing solvent polarity and the appearance of a band centred at 570 nm in the transient absorption spectra of Rf in the presence of OD, corresponding to the semireduced form of Rf, constitute clear evidences of the participation of radical ionic species in the mechanism of deactivation of 3 Rf* by OD. Electron transfer reactions, with the concomitant production of the Rf radical anion and the radical cation of the substrates, have been reported for a great variety of compounds (phenol, imidazole, amine and indole derivatives) [32,36,[48][49][50][51][52][53]. Additionally, Huvaere et al. [49] investigated the deactivation of 3 Rf* by imidazole derivatives, and through primary kinetic isotope effects they proposed that the interaction between 3 Rf* and imidazole derivatives occurs via electron transfer mechanism. ...
... Electron transfer reactions, with the concomitant production of the Rf radical anion and the radical cation of the substrates, have been reported for a great variety of compounds (phenol, imidazole, amine and indole derivatives) [32,36,[48][49][50][51][52][53]. Additionally, Huvaere et al. [49] investigated the deactivation of 3 Rf* by imidazole derivatives, and through primary kinetic isotope effects they proposed that the interaction between 3 Rf* and imidazole derivatives occurs via electron transfer mechanism. In a previous paper we studied the intermediates formed after the reaction of the sulfate radicals with several imidazoline derivatives (naphazoline, tetrahydrozoline, oxymetazoline, xylometazoline and 2methyl-2-imidazoline) through flash-photolysis and compared the experimental absorption spectra to those obtained from theoretical calculations. ...
The imidazoline-based ophthalmic drugs oxymetazoline and xylometazoline are widely used as ocular decongestants in pharmaceutical preparations. In this paper, the degradation of these drugs and the model compound 2-methyl-2-imidazoline, was studied in the presence of the vitamin B2 (Riboflavin) and visible light. The photogenerated Riboflavin electronically excited triplet state interacts with oxymetazoline and xylometazoline and as a result different free radicals and reactive oxygen species are produced. These species interact with the drugs in further steps, producing their degradation. Oxymetazoline is more easily photo-degradable than xylometazoline towards reactive oxygen species. Particularly, oxymetazoline reacts four orders of magnitude faster than xylometazoline with singlet oxygen. This fact is due to the presence of an OH-group in the benzene ring of oxymetazoline, increasing the oxidability of the drug. The degradation of xylometazoline by reactive oxygen species becomes more important as its concentration increases. This finding should warn against long-time treatments with xylometazoline. An eventual local accumulation of the drug may cause adverse effects in the ocular organ in the presence of Riboflavin. In parallel, the present results advise for a moderate precaution in relation to light exposure after topical application of the imidazoline derivatives oxymetazoline and xylometazoline.
... Moreover, water addition at C-5 directly or via epoxide formation leads to 4,5-dihydroxy-2-oxo-His that may further decompose to Asp, Asn, and urea via the 4-hydroxy-2,5-dioxo-His intermediate. His oxidation induces peptide aggregation to a significant extent (Agon et al., 2006;Castaño et al., 2015;Davies, 2016;Huvaere and Skibsted, 2009;Lei et al., 2017). ...
Peptides are small polymers composed of 40 or fewer amino acids and are an increasingly important class of drugs. Therapeutic peptides are less immunogenic and more economical than biologics while offering greater safety, selectivity, efficacy, and specificity than small molecule drugs. Despite this, they are challenging to mold in a stable formulation due to their susceptibility to degradation. By understanding the degradation behavior of such peptide drugs, researchers and pharmaceutical manufacturers can design safe, effective, and stable peptide formulations. From a scientific standpoint, forced degradation studies are an indispensable tool to forecast the stability of any molecule during its development phase. Being structurally diverse, the degradation of peptide drugs is different from the small molecules. This review provides a practical summary of strategies adopted to perform the stress stability testing for different peptide therapeutics including a selection of stress conditions, degradation products formed, and an analytical methodology used for the characterization of degradation products. Hence, it will help in developing a protocol for performing forced degradation studies on peptide therapeutics of interest. In-depth discussions of the different peptide degradation mechanisms are also included, along with preventive measures. The information presented here in the form of case studies on the degradation profile of existing peptide drugs will help in designing more resistant peptide drugs by chemical modifications, as well as aid in the advancement of generic peptide drug product development. In brief, this review presents the way of controlling peptide degradation from synthesis to formulation development based on their constituted amino acids.
... Specific tryptophan residues undergo oxidation in this protein triggering protein repair mechanisms (Dogra et al., 2019a). It has been shown that the aminoacids trypthophan and histidine easily suffer photooxidation (Huvaere and Skibsted, 2009). Recent studies have shown accumulation of tryptophan and phenylalanine under several environmental conditions as high light, drought, and temperature stress in Embryophyta (Galili et al., 2016). ...
The characterization of the molecular mechanisms, such as high light irradiance resistance, that allowed plant terrestralization is a cornerstone in evolutionary studies since the conquest of land by plants played a pivotal role in life evolution on Earth. Viridiplantae or the green lineage is divided into two clades, Chlorophyta and Streptophyta, that in turn splits into Embryophyta or land plants and Charophyta. Charophyta are used in evolutionary studies on plant terrestralization since they are generally accepted as the extant algal species most closely related to current land plants. In this study, we have chosen the facultative terrestrial early charophyte alga Klebsormidium nitens to perform an integrative transcriptomic and metabolomic analysis under high light in order to unveil key mechanisms involved in the early steps of plants terrestralization. We found a fast chloroplast retrograde signaling possibly mediated by reactive oxygen species and the inositol polyphosphate 1-phosphatase (SAL1) and 3′-phosphoadenosine-5′-phosphate (PAP) pathways inducing gene expression and accumulation of specific metabolites. Systems used by both Chlorophyta and Embryophyta were activated such as the xanthophyll cycle with an accumulation of zeaxanthin and protein folding and repair mechanisms constituted by NADPH-dependent thioredoxin reductases, thioredoxin-disulfide reductases, and peroxiredoxins. Similarly, cyclic electron flow, specifically the pathway dependent on proton gradient regulation 5, was strongly activated under high light. We detected a simultaneous co-activation of the non-photochemical quenching mechanisms based on LHC-like stress related (LHCSR) protein and the photosystem II subunit S that are specific to Chlorophyta and Embryophyta, respectively. Exclusive Embryophyta systems for the synthesis, sensing, and response to the phytohormone auxin were also activated under high light in K. nitens leading to an increase in auxin content with the concomitant accumulation of amino acids such as tryptophan, histidine, and phenylalanine.
... Many studies investigated the thermal stability of L-Trp in solution Friedman & Cuq, 1988;Lee & Rogers, 1988) or its photochemical behaviour (Asquith & Rivett, 1971;Boreen, Edhlund, Cotner, & Mcneill, 2008;Huvaere & Skibsted, 2009;Kanner & Fennema, 1987;Lee & Rogers, 1988;Schäfer, Goddinger, & Höcker, 1997;Thomas et al., 2013). Degradation mechanisms have been proposed in literature (e.g. ...
L−Tryptophan (L−Trp) is an amino acid important in nutrition, and mainly provided by food supplements. However, it is known to be unstable under light irradiation, which is an issue for the nutrition and feed industry. In the present study, the photostability of L−Trp was studied in acidic aqueous solutions under air and under an inert atmosphere, N2. The photodegradation was followed using UV−visible and fluorescence spectroscopy after photolysis. Moreover, molecular orbitals and bond dissociation energies calculations, and electron spin resonance spectroscopy were performed. From all these results, a photodegradation occurring through a free radical pathway was suggested. Interestingly, several antioxidants were tested to improve the photostability of L−Trp, especially during irradiation under air, since the L−Trp was evidenced to be much less stable under air than under N2. The results showed that sodium benzoate or EDTA were not efficient, but antioxidants such as chlorogenic acid, ascorbic acid or potassium sorbate improved significantly the photostability of L−Trp in acidic solutions.
... It has been shown that His residues can be the target of light-induced modifications, such as oxidation and crosslinking [26][27][28][29]. These reactions are proposed to be initiated by photo-generated singlet oxygen ( 1 O 2 ), that first leads to the oxidation of His residues [28,[30][31][32]. ...
Purpose
Histidine (His) undergoes light-induced reactions such as oxidation, crosslinking and addition. These reactions are initiated by singlet oxygen (¹O2) to generate His photo-oxidation products, which are subject to nucleophilic attack by a non-oxidized His residue from another protein or by nucleophilic buffer components such as Tris and His. This report aims to identify light-induced His-adducts to a monoclonal antibody (mAb-1) due to the reaction of His molecules in the buffer with the photooxidized His residues under ICH light conditions. Since polysorbate-20 (PS-20) is a commonly used excipient in biotherapeutics formulation, it is also important to study the impact of PS-20 concentration on protein photostability.
Results
We identified and characterized light-induced His-adducts of mAb-1 by LC-MS/MS. We showed that the levels of light-induced His-adducts generally correlate with the solvent accessibility of His residues in the protein. In addition, the presence of PS-20 at concentrations commonly used in protein drug formulations can significantly increase the levels of light-induced His-adducts.
Conclusions
Since His residues are present in a conserved region in the Fc domain, and may be present in the complementarity-determining region (CDR), the impact on the biological functions of the His-adducts observed here should be further studied to evaluate the risk of their presence.
... The data are consistent with the reference. 3 The oxidation potential of N-Ph-indole 2n was determined as: 2n (E p ox = 1.03 V vs Ag/AgNO 3 , 1.33 V vs SCE) ( Figure S3). The reduction potential of NHPI imidate ester 1a was determined as: 1a (E p re = -1.61 ...
N-Hydroxyphthalimide (NHPI) imidate esters were designed and used as amidyl radical precursors in the visible light photocatalyzed C-H amidation of heteroarenes for the first time. The reactions can be conducted under simple and mild conditions with a broad scope of electron-rich heteroarenes and NHPI esters, affording the desired amidation products in moderate to good yields. Moreover, the mechanistic paradigm has been clarified on the basis of Stern-Volmer analysis and the measurement of redox potentials
... The reaction mechanism of photo-induced histidine-histidine cross-linking has previously been described in inter and intra chain covalent bonds within mAb aggregates. [31][32][33] In the reaction histidine is first oxidised to 2-oxo-histidine, which is initiated by photogenerated 1 O 2 . Our data supports that histidine is similarly oxidised in the amino acid cross-links we identify here. ...
The correct choice of formulation buffer is a critical aspect of drug development and is chosen primarily to improve the stability of a protein therapeutic and protect against degradation. Amino acids are frequently incorporated into formulation buffers. In this study we have identified and characterized light induced cross-links between the side chain of histidine residues in an IgG4 monoclonal antibody and different amino acids commonly used in formulation buffers. These reactions have the potential to impact the overall product quality of the drug. The structure of each cross-link identified was elucidated using high performance liquid chromatography (HPLC) hyphenated to tandem mass spectrometry (MS/MS) with higher energy collisional dissociation (HCD). Furthermore, we speculate on the role of amino acids in formulation buffers and their influence on mAb stability. We theorize that whilst the adduction of formulation buffer amino acids could have a negative impact on product quality, it may protect against other pathways of photo-degradation.
... The hydroxypyrroloindoles 88 and 89 were obtained by a 1 O 2 [2 + 2] cycloaddition and deoxygenation of dioxetane by dimethylsulfide, followed by nucleophilic attack at C 2 by the amide nitrogen, and not through a 1 O 2 ene reaction. Many other examples of tryptophan and indole photooxidations have been investigated [91][92][93][94]. ...
Photocatalysis has been explored in numerous practical catalytic reactions under light irradiation, especially in the synthesis of valuable products and the photodegradation of various pollutants. This chapter summarizes the main photocatalysts that have been explored in the synthesis of nonaromatic N‐containing heterocyclic rings, highlighting the main results. It describes the photocatalytic systems with ruthenium and iridium polypyridyl complexes, including synthesis of pyrrolidines induced by visible light, synthesis of maleimide derivatives, synthesis of tetrahydroimidazole derivatives, synthesis of gliocladin C, and synthesis of fused pyrrolidines. The chapter discusses photocatalytic systems with porphyrin derivatives, including synthesis of oxindoles and oxazolidinone, synthesis of natural products, synthesis of dioxazoles, and synthesis of imines. It provides a discussion on photochemistry in combination with Bronsted acids. The use of different dyes as photocatalysts, with adequate photophysical and photochemical features, provides new approaches to the synthesis of nonaromatic N‐containing heterocycles.
... The essential vitamin riboflavin is also a well-known photosensitizer, and has been shown to undergo type I photochemical reactions with Trp [153], forming the riboflavin radical anion and the Trp radical cation. In aerobic conditions, the photosensitizing action of riboflavin can promote the oxidative degradation of Trp via both type I and type II photosensitized oxidation [154]. The reaction rate of these photosensitization reactions is known to increase with increasing temperature, oxygen concentration, and pH [153,155,156]. ...
Tryptophan is one of the essential mammalian amino acids, and is thus a required component in human nutrition, animal feeds, and cell culture media. However, this aromatic amino acid is highly susceptible to oxidation and is known to degrade into multiple products during manufacturing, storage and processing. Many physical and chemical processes contribute to the degradation of this compound, primarily via oxidation or cleavage of the highly reactive indole ring. The central contributing factors are reactive oxygen species, such as singlet oxygen, hydrogen peroxide, and hydroxyl radicals; light and photosensitizers; metals; and heat. In a multi-component mixture, tryptophan also commonly reacts with carbonyl-containing compounds, leading to a wide variety of products. The purpose of this review is to summarize the current state of knowledge regarding the degradation and interaction products of tryptophan in complex liquid solutions and in proteins. For the purposes of context, a brief summary of the key pathways in tryptophan metabolism will be included, along with common methods and issues in tryptophan manufacturing. The review will focus on the conditions that lead to tryptophan degradation, the products generated in these processes, their known biological effects, and methods which may be applied to stabilize the amino acid.
... Polyphenols from herbs and teas protect vitamin E in meat products during cooking and storage. Zinc may protect proteins against oxidation through coordinating to imidazole withdrawing electron density (10) . ...
... The hydroxypyrroloindoles 88 and 89 were obtained by a 1 O 2 [2 + 2] cycloaddition and deoxygenation of dioxetane by dimethylsulfide, followed by nucleophilic attack at C 2 by the amide nitrogen, and not through a 1 O 2 ene reaction. Many other examples of tryptophan and indole photooxidations have been investigated [91][92][93][94]. ...
Renowned authors from 14 countries have contributed to this book. It consists of 24 chapters on the state-of-the-art of the synthesis of nonaromatic nitrogen heterocycles, including recent developments in the most widely used reactions and also in new ones. Enantioselective synthesis and catalysis, and topics such as photocatalysis, microwave-assisted synthesis, ionic liquids and others, are also covered. The first 14 chapters after the introduction are dedicated to the synthesis of rings having one specific size, namely three-, four-, five-, six- and seven-membered rings, single or fused to other rings, in order of increasing ring size. Some concentrate on specific reactions such as cycloaddition reactions, hydroamination and dearomatization reactions, others inspect the many ways possible to obtain a specific ring. Later chapters cover topics such as the reactions of oximes and the reactions of nitrones, enantioselective radical reactions, the utilization of microwaves in synthesis, biocatalysis and photocatalysis. Sustainable procedures for obtaining nitrogen-containing heterocyclic salts are discussed, and the final chapter considers non-covalent interactions and how they can influence the synthesis of N-containing heterocycles, as well as catalysis and materials design.
... Flavin-mediated oxidation of Trp and other amino acids has been previously described on peptides; however, a direct link to protein structural perturbations as a result of exposure to photoreactive species has not been established. 12,16 More recently, covalent modification of photoexposed recombinant antibodies in the presence of cyanocobalamin (vitamin B12) in culture media has been observed. 17 While the exposure resulted in a pink coloration of process intermediates and final drug substance, no impact to binding or potency was reported. ...
During the manufacturing of protein biologics, product variability during cell culture production and harvest needs to be actively controlled and monitored to maintain acceptable product quality. To a large degree, variants that have previously been described are covalent in nature and are easily analyzed by a variety of techniques. Here, we describe a non-covalent posttranslational modification of recombinantly expressed antibodies, containing variable domain tryptophans, that are exposed to culture media components and ambient laboratory light. The modified species, designated as conformer, can be monitored by hydrophobic interaction chromatography and often exhibits reduced potency. We studied conformer formation and identified key elements driving its accelerated growth using an IgG2 monoclonal antibody. Conformer is a result of a non-covalent interaction of the antibody with riboflavin, an essential vitamin added to many production cell culture formulations. Chemical and physical factors that influence the impact of riboflavin are identified and methods for process control of this product quality attribute are addressed in order to prevent loss of antibody potency and potential safety issues. Identifying therapeutic antibody drug candidates with the potential to form conformers can be performed early in development to avoid this undesirable product quality propensity.
... Oxidation of His occurs mainly via type II photo-oxidation or MCO mechanisms (Davies and Truscott 2001;Schöneich 2000). In the first case, the reaction follows a [2 + 4] cycloaddition mechanism, in which the imidazole ring of His reacts with 1 O 2 and forms 2,4-or 2,5-endoperoxide intermediates, depending on the pH and, consequently, on the protonation state of the imidazole ring (Huvaere and Skibsted 2009;Liu et al. 2014a). At basic pH, the reaction pathway involves the formation of a 2,4-endoperoxide intermediate, which rapidly converts into 4-hydroxy-2oxo-His (Scheme 5a). ...
Peptides and proteins are preponderantly emerging in the drug market, as shown by the increasing number of biopharmaceutics already approved or under development. Biomolecules like recombinant monoclonal antibodies have high therapeutic efficacy and offer a valuable alternative to small-molecule drugs. However, due to their complex three-dimensional structure and the presence of many functional groups, the occurrence of spontaneous conformational and chemical changes is much higher for peptides and proteins than for small molecules. The characterization of biotherapeutics with modern and sophisticated analytical methods has revealed the presence of contaminants that mainly arise from oxidation- and elimination-prone amino-acid side chains. This review focuses on protein chemical modifications that may take place during storage due to (1) oxidation (methionine, cysteine, histidine, tyrosine, tryptophan, and phenylalanine), (2) intra- and inter-residue cyclization (aspartic and glutamic acid, asparagine, glutamine, N-terminal dipeptidyl motifs), and (3) β-elimination (serine, threonine, cysteine, cystine) reactions. It also includes some examples of the impact of such modifications on protein structure and function.
... In these cases, we rationalized the decreased yields could be attributed to deleterious oxidation of these residues over the C-terminal carboxylate. With respect to Lys and His, we hypothesized that decreasing the pH of the buffer should ameliorate oxidative side reactivity by increasing the equilibrium concentrations of ammonium and imidazolium, respectively, as these species should be resistant to oxidation 37 . Indeed, lowering the pH to 3.5 using a caesium formate buffer resulted in 65% yield for the Lys derivative (Table 1, entry 2, fourth column) and 70% yield for the His derivative (entry 14). ...
The advent of antibody–drug conjugates as pharmaceuticals has fuelled a need for reliable methods of site-selective protein modification that furnish homogeneous adducts. Although bioorthogonal methods that use engineered amino acids often provide an elegant solution to the question of selective functionalization, achieving homogeneity using native amino acids remains a challenge. Here, we explore visible-light-mediated single-electron transfer as a mechanism towards enabling site- and chemoselective bioconjugation. Specifically, we demonstrate the use of photoredox catalysis as a platform to selectivity wherein the discrepancy in oxidation potentials between internal versus C-terminal carboxylates can be exploited towards obtaining C-terminal functionalization exclusively. This oxidation potential-gated technology is amenable to endogenous peptides and has been successfully demonstrated on the protein insulin. As a fundamentally new approach to bioconjugation this methodology provides a blueprint toward the development of photoredox catalysis as a generic platform to target other redox-active side chains for native conjugation.
... Calcium, accordingly, makes iron less damaging to epithel in the intestines during food digestion; whether a similar effect is observed for calcium and heme pigments remains to be investigated. It is, however, known that zinc as a Lewis acid binds to heterocyclic nitrogen acting as an antioxidant, an effect which could also be important in protecting against heme-iron radical formation (59). Similar to calcium, during digestion, zinc could protect against iron induced oxidative damage. ...
Insufficient uptake of essential metals leads to serious malnutrition, which is a worldwide problem. Low bioavailability of iron and calcium may lead to anemia and osteoporosis, respectively, even in individuals with a high dietary intake. For iron, fractionation of meat proteins was studied in order to increase iron bioavailability from other meal components, and uptake of iron was found to increase with minimal risk of increasing oxidative damage. Calcium binding to peptides was found to prevent formation of insoluble calcium salts otherwise hampering absorption particularly in combination with calcium hydroxycarboxylates, entailing spontaneous supersaturation. Based on a review of results from different strategies available for increasing bioavailability, safe iron fortification is suggested to be supported by calcium, with modulation of iron as a prooxidant.
... × 10 8 M −1 s −1 and 3 k Q Tyr = 1.8(±0.8) × 10 9 M −1 s −1 ] as studied in this work (data not shown), in accordance with previous reported values (Cardoso et al., 2004;Huvaere and Skibsted, 2009). As GA is a heteropolysaccharide containing about 2% polypeptide (Mahendran et al., 2008), the quenching effect of 3 Rf* by GA can be associated with the presence of amino acid residues, such as His and Tyr, in the protein moiety of GA. ...
Microcapsules (MC) made with gum arabic (GA) as shell material without and with β-carotene (βc) as core material were prepared by the spray-drying technique. The effect of these MC on the photodegradation of riboflavin (Rf) in whole milk by fluorescent daylight lamp irradiation was evaluated at a storage temperature of 4°C. The additions of 1.37 mg/mL of MC without βc (MC-GA) and with 0.54 μg/mL of βc (MC-βc-GA) decreased the apparent first-order rate constant of Rf photodegradation by approximately 26 and 30%, respectively. A systematic kinetic and mechanistic analysis of the results indicates that the global protective effect of the MC is mainly due to the combination of quenching of the electronically excited triplet state of Rf and scavenging of the photogenerated reactive oxygen species, such as singlet molecular oxygen, superoxide radical anion and hydroxyl radical. A minor contribution to the photoprotective effect can be also associated with the inner-filter effect exerted by the MC, which partially blocks the direct excitation of Rf. These results allow us to conclude that photodegradation of Rf in milk can be considerably reduced by the addition of small amounts of MC, avoiding large losses in the nutritional value of milk.
... The hydrogen transfer reaction was further evaluated by assessing the deuterium kinetic isotope effect, because the exchangeable O-H protons in PPB could be replaced by deuterons from the large molar excess of D 2 O in the solution [33,34]. Experiments were performed in N 2 -saturated H 2 O and D 2 O solutions with lower irradiation energy, to produce the PPB degradation kinetics curve and some formation products (Figure 7). ...
The kinetics and mechanisms of ultraviolet photochemical transformation of propylparaben (PPB) were studied. Specific kinetics scavenging experiments coupled with quantum yield determinations were used to distinguish the roles of various reactive species induced by self-sensitized and direct photolysis reactions, and the excited triplet state of PPB (3PPB*) was identified as the most important species to initiate the photochemical degradation of PPB in aquatic environments. The computational results of time-resolved absorption spectra proved that 3PPB* is a highly reactive electron acceptor, and a head-to-tail hydrogen transfer mechanism probably occurs through electron coupled with proton transfer. Physical quenching by, or chemical reaction of 3PPB* with, O2 was confirmed as a key step affecting the initial PPB transformation pathways and degradation mechanisms. The transformation products were identified and the toxicity evolutions of PPB solution during photochemical degradation under aerobic and anaerobic conditions were compared. Results indicated that anaerobic conditions were more likely than aerobic conditions to lead to the elimination and detoxification of PPB, but less likely to lead to PPB mineralization. Environ Toxicol Chem © 2014 SETAC
Reduction of transient carnosine (β-alanyl-L-histidine) radicals by L-tryptophan, N-acetyl tryptophan, and the Trp-Gly peptide in neutral and basic aqueous solutions was studied using the technique of time-resolved chemically induced dynamic nuclear polarization (TR CIDNP). Carnosine radicals were generated in the photoinduced reaction with triplet excited 3,3',4,4'-tetracarboxy benzophenone. In this reaction, carnosine radicals with their radical center at the histidine residue are formed. Modeling of CIDNP kinetic data allowed for the determination of pH-dependent rate constants of the reduction reaction. It was shown that the protonation state of the amino group of the non-reacting β-alanine residue of the carnosine radical affects the rate constant of the reduction reaction. The results were compared to those obtained previously for the reduction of histidine and N-acetyl histidine free radicals and to newly obtained results for the reduction of radicals derived from Gly-His, a homologue of carnosine. Clear differences were demonstrated.
Exchanging the ribose backbone of an oligonucleotide for a peptide can enhance its physiologic stability and nucleic acid binding affinity. Ordinarily, the eneamino nitrogen atom of a nucleobase is fused to the side chain of a polypeptide through a new C−N bond. The discovery of C−C linked nucleobases in the human transcriptome reveals new opportunities for engineering nucleopeptides that replace the traditional C−N bond with a non‐classical C−C bond, liberating a captive nitrogen atom and promoting new hydrogen bonding and π‐stacking interactions. We report the first late‐stage synthesis of C−C linked carba‐Nucleopeptides (cNPs) using aqueous Rhodamine B photoredox catalysis. We prepare brand‐new cNPs in batch, in parallel, and in flow using three long‐wavelength photochemical setups. We detail the mechanism of our reaction by experimental and computational studies and highlight the essential role of diisopropylethylamine as a bifurcated two‐electron reductant.
Exchanging the ribose backbone of an oligonucleotide for a peptide can enhance its physiologic stability and nucleic acid binding affinity. Ordinarily, the eneamino nitrogen atom of a nucleobase is fused to the side chain of a polypeptide through a new C−N bond. The discovery of C−C linked nucleobases in the human transcriptome reveals new opportunities for engineering nucleopeptides that replace the traditional C−N bond with a non‐classical C−C bond, liberating a captive nitrogen atom and promoting new hydrogen bonding and π‐stacking interactions. We report the first late‐stage synthesis of C−C linked carba‐Nucleopeptides (cNPs) using aqueous Rhodamine B photoredox catalysis. We prepare brand‐new cNPs in batch, in parallel, and in flow using three long‐wavelength photochemical setups. We detail the mechanism of our reaction by experimental and computational studies and highlight the essential role of diisopropylethylamine as a bifurcated two‐electron reductant.
High-resolution photoelectron imaging and photodetachment spectroscopy of cryogenically cooled pyrrolide and imidazolide anions are used to probe the electronic structure and spectroscopy of pyrrolyl and imidazolyl radicals. The high-resolution data allow the ground state vibronic structures of the two radicals to be completely resolved, yielding accurate electron affinities of 2.1433 ± 0.0008 eV and 2.6046 ± 0.0006 eV for pyrrolyl and imidazolyl radicals, respectively. Fundamental frequencies for eight vibrational modes of pyrrolyl and ten vibrational modes of imidazolyl are measured, including several nonsymmetric Franck-Condon-forbidden modes. Two electronic excited states are also observed for the two radicals, displaying diffuse spectral features in both systems. The observations of nonsymmetric vibrational modes in the ground states and the diffuse excited state features provide strong evidence for vibronic couplings between the ground state and the two close-by excited states. The 2-pyrrolide isomer is also observed as a minor species from the electrospray ionization source and the electron affinity of 2-pyrrolyl is measured to be 1.6690 ± 0.0030 eV along with five vibrational frequencies. Even though the HOMOs of both pyrrolide and imidazolide anions are p orbitals, photodetachment spectroscopy reveals completely different threshold behaviors for the two anions: a d-wave-dominated spectrum for pyrrolide and an s-wave-dominated spectrum for imidazolide. The current study provides a wealth of electronic and spectroscopic information, which is ideal to compare with more accurate vibronic coupling calculations for these two important radicals, as well as interesting information about the photodetachment dynamics of the two anions.
Riboflavin (RF, vitamin B2) is an essential vitamin and has been considered as a promising natural photosensitizer for photodynamic therapy (PDT). However, further exploration of RF in antitumor application was limited by its poor cellular uptake. In this study, using cell-penetrating peptides Arg8, (Cha-Arg)3 and small molecule triphenylphosphine (TPP) as delivery compounds, three RF conjugates were prepared to increase the accumulation of RF in cells, termed as Arg8-RF, (Cha-Arg)3-RF and TPP-RF, respectively. Compared with TPP-RF and Arg8-RF, (Cha-Arg)3-RF exhibited better cell internalization and stronger cytotoxicity against HeLa cells upon exposure to blue light. Further researches proved that (Cha-Arg)3-RF generated reactive oxygen species (ROS) under irradiation, which could indiscriminately destroy endogenous proteins and mitochondria, ultimately inducing cell death. This work provides a new approach to explore RF as a natural photosensitizer for antitumor photodynamic therapy.
β-Carboline alkaloid harmaline (HA) is a candidate drug molecule that has been proven to have broad and significant biological activity. Herein, the effects of HA on the riboflavin (RF)-sensitized photooxidation under aerobic conditions were studied for the first time. The photooxidation reaction of HA catalyzed by RF is triggered by UV light at 365 nm and shows a time-dependent stepwise reaction process. Seven transformed products, including five undescribed compounds, oxoharmalines A-E (1-4 and 7), and two known compounds, N-(2-(6-Methoxy-2-oxoindolin-3-yl)ethyl)acetamide (5) and harmine (6), were isolated and identified from the reaction system, following as the gradual oxidation mechanisms. The rare polymerization and dehydrogenation processes in radical-mediated photocatalytic reactions were involved in the process. The transformed products 2-7 exhibited significant neuroprotective activity in a model of H2O2-introduced injury in SH-SY5Y cells, which suggested that the products of the interaction between HA and vitamins may be beneficial to health.
Irradiation from the sun has played a crucial role in the origin and evolution of life on the earth. Due to the presence of ozone in the stratosphere most of the hazardous irradiation is absorbed, nonetheless UVB, UVA, and visible light reach the earth's surface. The high abundance of proteins in most living organisms, and the presence of chromophores in the side chains of certain amino acids, explain why these macromolecules are principal targets when biological systems are illuminated. Light absorption triggers the formation of excited species that can initiate photo-modification of proteins. The major pathways involve modifications derived from direct irradiation and photo-sensitized reactions. In this review we explored the basic concepts behind these photochemical pathways, with special emphasis on the photosensitized mechanisms (type 1 and type 2) leading to protein oxidation, and how this affects protein structure and functions. Finally, a description of the photochemical reactions involved in some human diseases, and medical applications of protein oxidation are presented.
Singlet molecular oxygen (1O2) has been associated with a number of physiological processes. Despite the recognized importance of 1O2‐mediated protein modifications, little is known about the role of this oxidant in cross‐link formation and protein aggregation. Thus, using lysozyme as a model, the present study, sought to investigate the involvement of 1O2 in cross‐link formation. Lysozyme was photochemically oxidized in the presence of Rose Bengal or chemically oxidized using [18O]‐labeled 1O2 released from thermolabile endoperoxides. It was concluded that both 1O2 generating systems induce lysozyme crosslinking and aggregation. Using SDS‐PAGE and nano‐scale liquid chromatography coupled to electrospray ionization mass spectrometry the results clearly demonstrated that 1O2 is directly involved in the formation of covalent cross‐links involving the amino acids Histidine, lysine and Tryptophan.
Oxidation is one of the deterioration reactions of proteins in food, whose importance does not lag behind that of others such as Maillard, lipation or protein‐polyphenol reactions. While research on protein oxidation has led to an accurate perception on the processes and consequences in physiological systems, knowledge on specific implications of protein oxidation in food or the role of “oxidized” dietary protein for the human body is comparatively scarce. Food protein oxidation can occur during the whole processing axis, from primary production to intestinal digestion. The present review wants to summarize the current knowledge and mechanisms of food protein oxidation from a chemical, technological and nutritional‐physiological viewpoint and give a comprehensive classification of the individual reactions. Different analytical approaches will be compared, and the relationship between oxidation of food proteins and oxidative stress in vivo will be critically evaluated.
Oxidation is one of the deterioration reactions of proteins in food, whose importance does not lag behind that of others such as Maillard, lipation or protein‐polyphenol reactions. While research on protein oxidation has led to an accurate perception on the processes and consequences in physiological systems, knowledge on specific implications of protein oxidation in food or the role of “oxidized” dietary protein for the human body is comparatively scarce. Food protein oxidation can occur during the whole processing axis, from primary production to intestinal digestion. The present review wants to summarize the current knowledge and mechanisms of food protein oxidation from a chemical, technological and nutritional‐physiological viewpoint and give a comprehensive classification of the individual reactions. Different analytical approaches will be compared, and the relationship between oxidation of food proteins and oxidative stress in vivo will be critically evaluated.
UV-induced oxidation of 2-(1'H-indol-2'-yl)-[1,5]naphthyridine acetonitrile solution in the presence of air leads to formation of 2-(1,5-naphthyridin-2-yl)-4H-3,1-benzoxazin-4-one as a major product and N-(2-formylphenyl)-1,5-naphthyridine-2-carboxamide as a minor one. The probable reaction mechanisms are different for the two photoproducts and may involve both, the reaction with singlet oxygen generated by the excited substrate or the reaction of the excited substrate with ground state oxygen molecule. Electronic absorption and IR spectra indicate that both photoproducts are formed as mixtures of syn and anti rotameric forms. The obtained results indicate an efficient and easy method of synthesis of molecules with benzoxazinone structure.
Time-resolved chemically induced dynamic nuclear polarization (CIDNP) and transient absorption (TA) were applied to reveal the branching ratio of the singlet and triplet recombination channels in the reaction of short-lived radicals of carboxy benzophenones and of aromatic amino acids histidine, tryptophan, and tyrosine in neutral aqueous solition. It was established that the share of triplet recombination increases with increasing number of carboxylic groups: no triplet recombination was found for 4-carboxy benzophenone, whereas ~13% of radicals of 4,4'-dicarboxy benzophenone (DCBP) and ~27% of radicals of 3,3',4,4'-tetracarboxy benzophenone (TCBP) react with histidine radicals from triplet state of radical pairs. The main idea is that the protonated (π,π*) triplet state of TCBP or DCBP is populated via back electron transfer from the ketyl radical of TCBP or DCBP to the radical of amino acid. The protonated triplet state of ketone decays with the formation of a methastable hydroxylated product, which is detected by TA. Taking into account triplet recombination provides excellent coincidence between experimental data and simulated CIDNP kinetics.
The mechanism of photo-oxidation of tryptophan (Trp) sensitized by riboflavin (RF) was examined employing high concentrations of Trp and RF, with a high intensity 365 nm light emitting diode (LED) source under N2, 20% and 100% O2 atmospheres. Dimerization of Trp was a major pathway under the N2 atmosphere, though this occurred with a low yield (DφTrp = 5.9 x 10-3), probably as a result of extensive back electron transfer reactions between RF●- and Trp(H)●+. The presence of O2 decreased the extent of this back electron transfer reaction, and the extent of Trp dimerization. This difference is attributed to the formation of O2●- (generated via electron transfer from RF●- to O2) which reacts rapidly with Trp● leading to extensive consumption of the parent amino acid and formation of peroxides and multiple other oxygenated products (N-formylkynurenine, alcohols, diols) of Trp, as detected by LC-MS. Thus, it appears that the first step of the Type 1 mechanism of Trp photo-oxidation, induced by this high intensity 365 nm light source, is an electron transfer reaction between the amino acid and 3RF, with the presence of O2 modulating the subsequent reactions and the products formed, as a result of O2●- formation. These data have potential biological significance as LED systems and RF-based treatments have been proposed for the treatment of pathological myopia and keratitis.
In the last decade, visible‐light photoredox catalysis emerged as a powerful strategy enabling novel transformations in organic synthesis. Owing to the mild reaction conditions (i.e. room temperature, use of visible light) and to the high functional group tolerance, photoredox catalysis could represent an ideal strategy for chemoselective biomolecule modification. Indeed, a recent trend in photoredox catalysis is its application to the development of novel methodologies for amino acid modification. In this review, we provide an up‐to‐date overview of photocatalytic methodologies for the modification of single amino acids, peptides and proteins. The advantages offered by photoredox catalysis and its suitability in the development of novel biocompatible methodologies are described. In addition, a brief consideration on the current limitations of photocatalytic approaches, as well as future challenges to be addressed are discussed.
Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in crosslink generation. However, the factors that influence the reactivity of His in proteins, especially the intrinsic factors are little known. Here we used rhDNase which only forms His-His dimers after light treatment to determine the factors that influence the light-induced reactivity of His. This system allowed us to fully characterize the light-induced dimer and rank the reactivities of the His residues in this protein. The reactivities of these His residues were correlated with solvent accessibility-related parameters both by crystal structure-based calculations of solvent-accessible surface area and by hydrogen-deuterium exchange (HDX) experiments. Through this correlation, we demonstrate that the photo-reactivity of His is determined by both solvent accessibility and structural flexibility. This new insight can explain the highly complex chemistry of light-induced aggregation and help predict the aggregation propensity of protein under light treatment.
Protein oxidation in meat has, through the last decades, received increasing attention from the meat industry and meat scientists. Consumers have also noted negative effects of protein oxidation following changes in packaging practices in the meat industry in many countries. Compared to lipid oxidation, the degradation of proteins by oxidants seems to be more complex and to produce an even greater multiplicity of reaction products. While oxidation processes related to lipids, proteins, and heme proteins may individually be relatively well understood, the coupling between degradation processes in meat has not been investigated in detail. Understanding protein oxidation in biological systems, however, seems to hold the key to also understanding the connection between degradation of hydrophilic and lipophilic meat components, since certain protein oxidation products may be active at water–lipid interfaces in meat (Skibsted 2011a).
The sun is the ultimate source of energy. However, besides acting as a boon, it has some effects which are not less than a bane for living organisms, which are due to the depletion of ozone layer because of continuous human interventions. The biological effects arising due to ultraviolet radiation, whether from UV-A or UV-B, are instigated by absorption, by important biologically molecules like nucleic acids, proteins, and so on. Therefore, in this review, we will be discussing each and every aspect related to UV radiation, beginning from its action spectra to its effects at the cellular level.
Aggregates of amyloidogenic peptides are involved in the pathogenesis of several degenerative disorders. Herein, we report an Ir(III) complex, Ir-1, as a chemical tool for oxidizing amyloidogenic peptides upon photoactivation and subsequently modulating their aggregation pathways. Ir-1 was rationally designed based on multiple characteristics, including (i) photoproperties leading to excitation by low-energy radiation; (ii) generation of reactive oxygen species responsible for peptide oxidation upon photoactivation under mild conditions; (iii) relatively easy incorporation of a ligand on the Ir(III) center for specific interactions with amyloidogenic peptides. Our biochemical and biophysical investigations illuminate that the oxidation of representative amyloidogenic peptides (i.e., amyloid-β, α-synuclein, and human islet amyloid polypeptide) is promoted by light-activated Ir-1, which alters their conformations and aggregation pathways. Additionally, their potential oxidation sites are identified as methionine, histidine, or tyrosine residues. Overall, our studies of Ir-1 demonstrate the feasibility of devising metal complexes as chemical tools suitable for elucidating the nature of amyloidogenic peptides at the molecular level as well as controlling their aggregation.
This Review describes singlet oxygen ((1)O2) in the organic synthesis of targets on possible (1)O2 biosynthetic routes. The visible-light sensitized production of (1)O2 is not only useful for synthesis; it is extremely common in nature. This Review is intended to draw a logical link between flow and batch reactions-a combination that leads to the current state of (1)O2 in synthesis.
The formation and chemistry of flavin–indole charge transfer (CT) complexes has been studied using a model cationic flavin. The ability to form a CT complex is sensitive to indole structure as gauged by spectroscopic, kinetics and crystallographic studies. Single crystals of sufficient quality of a flavin-indole CT complex, suitable for X-ray diffraction, have been grown, allowing solid-state structural analysis. When CT complex formation is conducted in d4-methanol, an efficient and synthetically useful C-3 indole deuteration is observed.
The phenolic amino acid tyrosine (Tyr) was found more efficient in regenerating ß-carotene (ß-Car) from the radical cation (ß-Car•+) than tryptophan (Trp) in presence of base for conditions, where the reduction potentials for Trp and Tyr are comparable. Electron transfer from Tyr in 4:1 chloroform/methanol to ß-Car•+ in the presence of excess base, (CH3)4N+OH-, had a rate close to diffusion control and a second order rate constant in agreement with the Marcus theory for electron transfer when compared to plant phenols. A maximum of 40% ß-Car was regenerated for ten times excess of Tyr as studied by 532 nm laser flash photolysis followed by transient absorption spectroscopy in the visible and near-infrared regions. The non-regenerated fraction of ß-Car is assigned to secondary degradation processes. For Trp, the rate constant for regeneration of ß-Car•+ was one order of magnitude smaller compared to Tyr and slower than expected from Marcus theory by comparison with plant phenols.
Anilines and phenols are structurally similar compound classes that both are susceptible to oxidation by excited state triplet sensitizers, but undergo oxidation by different mechanisms. To gain an understanding of the factors that control the rate of oxidation of anilines and phenols by triplet excited states, a kinetic study was performed on the oxidation of substituted anilines and phenols by methylene blue. The rate constants of one-electron transfer from anilines to triplet state methylene blue and their dependence on the reaction free energy are well fit to a Sandros-Boltzmann model. The observed rate constants are also well modeled when using aniline oxidation potentials derived computationally. For phenols, the proton-coupled electron transfer rate constants were found to correlate primarily with O-H bond dissociation free energy and secondarily with phenol pKa. Rate constants for phenols could be modeled using computed bond dissociation free energies. These results provide a basis for predicting aniline and phenol oxidation rates, which could be valuable, for example, in assessing the likely persistence and fate of aniline- and phenol-based aqueous environmental pollutants.
The photooxidation reaction between 3,3',4,4'-benzophenone tetracarboxylic acid (TCBP) and l-histidine (His) has been investigated in neutral aqueous solution using the technique of chemically induced dynamic nuclear polarization (CIDNP). Relative values of C-13 isotropic hyperfine couplings in the TCBP and His radicals were obtained from the C-13-time-resolved CIDNP spectrum, recorded during the photoreaction of TCBP with His at natural abundance of the magnetic isotope C-13. Good agreement was found for the hyperfine coupling constants of the TCBP ketyl radical calculated using methods of density functional theory, and those obtained from the C-13-time-resolved CIDNP spectrum. The mechanism of the quenching reaction of triplet-excited TCBP by His in neutral aqueous solution was established. H-1 CIDNP field dependencies for the photoreaction of TCBP with His were obtained and the g-factor of the histidyl radical was found.
The redox equilibrium method was used to determine accurate oxidation potentials in acetonitrile for 40 benzene and biphenyl derivatives containing various heteroatom substituents. These compounds include methoxy substituted benzenes, aromatic amines, substituted acetanilides, and methoxy-substituted biphenyls. The redox equilibrium method allowed oxidation potentials to be determined with high precision (less than or equal to +/- 6 mV). While most of the relative oxidation potentials follow well-established chemical trends, interestingly, the oxidation potentials of substituted N methylacetanilides were found to be higher than those of the corresponding acetanilides. Density functional theory calculations provided insight into the origin of these surprising results in terms of the preferred conformations of the amides vs. their cation radicals.
Flavonoids, ubiquitously present in plant kingdom, preserve food and beverages at ppm level with minor perturbation of sensory impressions, are safe, and possibly attribute positive health effects. Flavonoids should be further exploited for protection of food and beverages against light-induced quality deterioration through: (i) direct absorption of photons as inner filters protecting sensitive food components; (ii) deactivation of (triplet-)excited states of sensitizers like chlorophyll and riboflavin; (iii) quenching of singlet oxygen from Type II photosensitization; and (iv) scavenging of radicals formed as reaction intermediates in Type I photosensitization. For absorption of light, combinations of flavonoids, as found in natural co-pigmentation, facilitate dissipation of photon energy to heat thus averting photodegradation. For protection against singlet oxygen and triplet sensitizers, chemical quenching gradually decreases efficiency hence the pathway to physical quenching should be optimized through product formulation. The feasibility of these protection strategies is further supported by kinetic data that are becoming available, allowing for calculation of threshold levels of flavonoids to prevent beer and dairy products from going off. On the other hand, increasing understanding of the interplay between light and matrix physicochemistry, for example the effect of aprotic microenvironments on phototautomerization of compounds like quercetin, opens up for engineering better light to heat converting channels in processed food to eventually prevent quality loss.
The essential amino acid L-tryptophan can be produced by a condensation reaction between indole and L-serine, catalyzed by B. subtilis with tryptophan synthase activity. Application of the tryptophan is widespread in the biotechnology domain and is sometimes added to feed products as a food fortifier.
The optimum concentration of the Iranian cane molasses was determined by measuring the amount of biomass after growth in 1 to 30 g/mL of molasses. The maximum amount of biomass was obtained in 10 g/mL molasses. Chromatographic methods, TLC and HPLC, were used to assay the amount of tryptophan produced in the presence of precursors of tryptophan production (indole and serine) and/or molasses.
Our results indicate the importance of the Iranian cane molasses not only as carbon source, but also as a source of precursors for tryptophan production.
This report evaluates the potential of cane molasses as an economical source for tryptophan production by B. subtilis, hence eliminating the requirement for additional serine and indole as precursors.
The oxidation of indole-3-acetic acid by horseradish peroxidase was studied using the spin traps t-nitrosobutane and 5,5-dimethyl-1-pyrroline N-oxide to trap free radical intermediates. The major free radical metabolite of indole acetic acid was unambiguously determined by the use of indole-3-[2,2-2H2]acetic acid to be the skatole carbon-centered free radical. In the presence of oxygen, superoxide was also trapped.
Proteolytic degradation of oxidatively damaged [3H] bovine serum albumin [( 3H]BSA) was studied during incubation with cell-free erythrocyte extracts and a wide variety (14) of purified proteases. [3H]BSA was pretreated by exposure (60Co radiation) to the hydroxyl radical (.OH), the superoxide anion radical (O2-), or the combination of .OH + O2- + oxygen. Treated (and untreated) samples were dialyzed and then incubated with erythrocyte extract or proteases for measurements of proteolytic susceptibility (release of acid-soluble counts). Both .OH and .OH + O2- + caused severalfold increases in proteolytic susceptibility (with extract and proteases), but O2- alone had no effect. Proteolytic susceptibility reached a maximum at 15 nmol of .OH/nmol of BSA and declined thereafter. In contrast, proteolytic susceptibility was still increasing at an .OH + O2-/BSA molar ratio of 100 (50% .OH + 50% O2-). Degradation in erythrocyte extracts was conducted by a novel ATP- and Ca2+-independent pathway, with maximal activity at pH 7.8. Inhibitor profiles indicate that this pathway may involve metalloproteases and serine proteases. Comparisons of proteolytic susceptibility with multiple modifications to BSA primary, secondary, and tertiary structure revealed a high correlation (r = 0.98) with denaturation/increased hydrophobicity by low concentrations of .OH. Covalent aggregation reactions (BSA cross-linking) may explain the declining proteolytic susceptibility observed at .OH/BSA molar ratios greater than 20. Protein denaturation may also have caused the increased proteolytic susceptibility induced by .OH + O2- + O2, but no simple correlation could be obtained. Results with .OH + O2- + O2 appear to have been complicated by direct BSA fragmentation reactions involving (.OH-induced) protein radicals and oxygen. These data indicate a direct and quantitative relationship between protein damage by oxygen radicals and increased proteolytic susceptibility. Oxidative denaturation may exemplify a simple, yet effective inherent mechanism for intracellular proteolysis.
Proteins which have been exposed to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical and oxygen (.OH + O2- + O2) exhibit altered primary structure and increased proteolytic susceptibility. The present work reveals that alterations to primary structure result in gross distortions of secondary and tertiary structure. Denaturation/increased hydrophobicity of bovine serum albumin (BSA) by .OH, or by .OH + O2- + O2 was maximal at a radical/BSA molar ratio of 24 (all .OH or 50% .OH + 50% O2-). BSA exposed to .OH also underwent progressive covalent cross-linking to form dimers, trimers, and tetramers, partially due to the formation of intermolecular bityrosine. In contrast, .OH + O2- + O2 caused spontaneous BSA fragmentation. Fragmentation of BSA produced new carbonyl groups with no apparent increase in free amino groups. Fragmentation may involve reaction of (.OH-induced) alpha-carbon radicals with O2 to form peroxyl radicals which decompose to fragment the polypeptide chain at the alpha-carbon, rather than at peptide bonds. BSA fragments induced by .OH + O2- + O2 exhibited molecular weights of 7,000-60,000 following electrophoresis under denaturing conditions, but could be visualized as hydrophobic aggregates in nondenaturing gels (confirmed with [3H]BSA following treatment with urea or acid). Combinations of various chemical radical scavengers (mannitol, urate, t-butyl alcohol, isopropyl alcohol) and gases (N2O, O2, N2) revealed that .OH is the primary species responsible for alteration of BSA secondary and tertiary structure. Oxygen, and O2- serve only to modify the outcome of .OH reaction. Furthermore, direct studies of O2- + O2 (in the absence of .OH) revealed no measurable changes in BSA structure. The process of denaturation/increased hydrophobicity was found to precede either covalent cross-linking (by .OH) or fragmentation (by .OH + O2- + O2). Denaturation was half-maximal at a radical/BSA molar ratio of 9.6, whereas half-maximal aggregation or fragmentation occurred at a ratio of 19.4. Denaturation/hydrophobicity may hold important clues for the mechanism(s) by which oxygen radicals can increase proteolytic susceptibility.
Exposure of proteins to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical (.OH + O2-) causes gross structural modification. Such modified proteins can undergo spontaneous fragmentation or can exhibit substantial increases in proteolytic susceptibility. In the present study, with the representative protein bovine serum albumin (BSA), we report that alterations to primary structure underlie such gross structural modifications. All amino acids in BSA were susceptible to modification by both .OH and .OH + O2- +O2), although tryptophan, tyrosine, histidine, and cysteine were particularly sensitive. At a radical/BSA molar ratio (nmol of radicals/nmol of BSA) of 10, we observed an average 9-10% destruction of amino acids; whereas at a ratio of 100, the average loss was 45%. Decreasing tryptophan fluorescence provided a useful index of amino acid loss and exhibited a clear dose dependence with .OH or with .OH + O2- (+O2). Linear production of the biphenol bityrosine was observed with .OH treatment. In contrast, .OH + O2- (+O2) induced only a limited bityrosine production rate which reached an early plateau. Studies with various chemical scavengers (t-butyl alcohol, isopropyl alcohol, mannitol, urate) and gasses (N2O, N2, O2, air) revealed that .OH is the primary radical responsible for all amino acid modifications, but that O2- and O2 can further transform the products of .OH reactions. Thus, O2-/O2 can potentiate .OH-dependent destruction of many amino acids (e.g. tryptophan) while inhibiting production of bityrosine by reacting with tyrosyl (phenoxyl) radicals. No amino acid loss or bityrosine production occurred with exposure to O2- (+O2) alone. Amino acid modifications caused both by .OH alone and by .OH + O2- (+O2) progressively affected the overall electrical charge of BSA. In a pH range of 3.7-6.2, some 16 new isoelectric focusing bands were induced by .OH, and some eight new bands were induced by .OH + O2- (+O2). The alterations to primary structure observed provide the key to an understanding of the link between oxidative modification and increased proteolytic susceptibility.
A flux of hydroxyl radicals generated by gamma-irradiation can fragment monoamine oxidase in the membrane of submitochondrial particles. This fragmentation can be inhibited by mannitol and in addition is more extensive in monoamine oxidase preparations that have been depleted of lipid. This latter observation is consistent with the higher yields of fragmentation induced by hydroxyl radicals in soluble proteins in the absence of added lipids. In the absence of oxygen, gamma-irradiation of submitochondrial particles leads to cross-linking reactions. A flux of hydroperoxyl radicals also causes fragmentation, whereas one of superoxide is virtually inactive in this respect. The irradiation of submitochondrial particles leads in addition to the accumulation of products of lipid peroxidation. When these irradiated preparations are exposed to ferrous or cupric salts a further fragmentation of monoamine oxidase ensues, especially at acid pH. These transition-metal-catalysed reactions do not occur with irradiated preparations depleted of lipid, and the post-irradiation protein modifications are concomitant with further lipid peroxidation. The data indicate roles for lipid radicals in both fragmentation and cross-linking reactions of proteins in biological membranes. These reactions may have an important bearing on control of protein activity and of protein turnover in membranes.
In the reaction between hydrogen peroxide and metmyoglobin, the heme iron is oxidized to its ferryl-oxo form and the globin to protein radicals, at least one of which reacts with dioxygen to form a peroxyl radical. To identify the residue(s) that forms the oxygen-reactive radical, we utilized electron spin resonance (ESR) spectroscopy and the spin traps 2-methyl-2-nitrosopropane and 3,5-dibromo-4-nitrosobenzenesulfonic acid (DB-NBS). Metmyoglobin radical adducts had spectra typical of immobilized nitroxides that provided little structural information, but subsequent nonspecific protease treatment resulted in the detection of isotropic three-line spectra, indicative of a radical adduct centered on a tertiary carbon with no bonds to nitrogen or hydrogen. Similar isotropic three-line ESR spectra were obtained by spin trapping the oxidation product of tryptophan reacting with catalytic metmyoglobin and hydrogen peroxide. High resolution ESR spectra of DBNBS/.trp and of the protease-treated DBNBS/.metMb were simulated using superhyperfine coupling to a nitrogen and three non-equivalent hydrogens, consistent with a radical adduct formed at C-3 of the indole ring. Oxidation of tryptophan by catalytic metMb and hydrogen peroxide resulted in spin trap-inhibitable oxygen consumption, consistent with formation of a peroxyl radical. The above results support self-peroxidation of a tryptophan residue in the reaction between metMb and hydrogen peroxide.
— The UV photolysis of tryptophan (Trp) and Trp-containing peptides in aerated aqueous solutions has been studied by ESR and spin-trapping techniques using f-nitrosobutane as the spin-trap. The photolysis of Trp alone at 290 nm gave rise to the addition of the spin-trap to carbon 3 of the indole ring. A large ESR signal from the hydronitroxide spin-adduct was also observed revealing the formation of hydrated electrons. Generally, the photolysis of Trp-containing dipeptides generated the deamination radical of the N-terminal amino acid followed by addition to the spin-trap. In the case of lysyl-Trp, a deamination radical from the side chain of lysine was proposed. A sensitization experiment with Trp as sensitizer and glycine (Gly) as substrate led to the generation of the deamination radical of Gly. Most of the observed free radicals resulting from the photolysis of Trp-containing peptides can be explained in terms of hydrated electrons reacting with the carbonyl group followed by deamination of the N-terminus.
Fluorescence quenching rate constants, kq, ranging from 106 to 2 × 1010 M−1 sec−1, of more than 60 typical electron donor‐acceptor systems have been measured in de‐oxygenated acetonitrile and are shown to be correlated with the free enthalpy change, ΔG 23, involved in the actual electron transfer processin the encounter complex and varying between + 5 and −60 kcal/mole. The correlation which is based on the mechanism of adiabatic outer‐sphere electron transfer requires ΔG ≠23, the activation free enthalpy of this process to be a monotonous function of ΔG 23 and allows the calculation of rate constants of electron transfer quenching from spectroscopic and electrochemical data.A detailed study of some systems where the calculated quenching constants differ from the experimental ones by several orders of magnitude revealed that the quenching mechanism operative in these cases was hydrogen‐atom rather than electron transfer.The conditions under which these different mechanisms apply and their consequences are discussed.
Reversible one-electron-transfer reactions involving the tryptophan cation and neutral radicals were investigated by pulse radiolysis. The one-electron-reduction potential of the neutral tryptophan radical at pH 7 was determined to be E{sub 7} = 1.01 {plus minus} 0.03 V by using the bis(1,4,7-triazacyclononane)nickel(III/II) redox couple with E = 0.95 V as a standard. The value obtained with promethazine (E = 0.89 V) as a standard at pH 6 was E{sub 6} = 1.11 V. From these measurements, a mean value E{sub 7} = 1.03 V results, in agreement with some earlier determinations (1.05 and 1.08 V) obtained in experiments with inorganic redox standards. The kinetics and energetics of the reactions of the tryptophan neutral and cation radicals with selected electron donors were also investigated.
The technique of pulse radiolysis has been used to demonstrate that all histidine free radicals (designated HisNsbd+) produced by oxidation of histidine by Br2˙− radical anions can oxidise the water soluble vitamin E analogue, Trolox C (k = 1.0 ± 0.2 × 109 d mol−1 s−1 at pH 6.95). It has also been shown that His˙+ radicals can react with tryptophan in electron transfer equilibria involving both His˙+ and Trp˙+ species over the pH range 6.4–9.0. The ΔE values [E(Trp˙+/Trp) − E(His˙+/His)] range from −140 to −161 mV and indicate an E7(His˙+/His) value of 1170 mV [based on E7(Trp˙+/Trp) = 1015 mV at pH 7]. The effect of pH on E(His˙+/His) was accounted for by assuming that His˙+ can deprotonate to yield a bi-allylic free radical, designated His (−H+)˙. The pKa for this dissociation was estimated to be in the range 5–7. The implications of the relatively high reduction potential for His˙+ in its possible participation in the mechanism of action of non-heme metalloenzymes is discussed.
The
use of time-resolved 337 nm and 248 nm laser flash photolysis with transient absorbance detection has shown direct evidence of electron transfer from dGMP to the triplet state of riboflavin (3RF*). dGMP
was used as a DNA model system in order to study the damaging potential of photoexcited riboflavin. The evidence
obtained was that: (1) formation of radical anion of riboflavin (RF•−/RFH•) matched the decay of
3RF*; (2) the decay of 3RF* was pseudo-first-order with the concentrations of dGMP, the bimolecular reaction rate constant was determined to be 6.6 × 108 dm3 mol−1 s−1; (3) after the complete decay of 3RF*, the
transient absorption spectra of the deprotonated radical cation of dGMP [dGMP(-H)•] was observed in aerated condition
for the first time; (4) the addition of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) to the experimental system resulted in the formation of the TMPD radical cation via
scavenging dGMP radical cation; and (5) the free energy change (ΔG) from dGMP to 3RF* was calculated to be −43.7 kJ mol−1 with
the Rehm–Weller equation, which indicates that photo-oxidation of dGMP attacked by triplet state of riboflavin is
thermodynamically possible. Electron-transfer from dGMP to the oxidized radicals of riboflavin is found,
which may be another pathway of DNA damage in vivo and in vitro. The direct observation of oxidized guanine radical has provided unambiguously direct initial
proof
for a photosensitization process
of dGMP, with riboflavin as photosensitizer.
The He(I) photoelectron spectra for the aza-derivatives of benzofuran (4 and 7), benzothiophen (8) and indole (3a, 3b, 6a, 9b), and combined He(I) and He(II) spectra of the 1,2-benzisothiazole (5), 1,2,3-benzothiadiazole (11) and benzotriazole (9a) have been obtained and assigned by a combination of heteroatom substituent effects and ab initio molecular orbital calculations. The variations in lone pair levels (LPNLPO, LPS) and π-levels, between these and the monocyclic compounds are discussed.
ESR spectra have been obtained for the fluorenyl and indenyl radicals, and their isotropic proton hfcc have been compared with the predictions of semi-empirical MO calculations.
Ab initio calculations at the B3LYP/6-31G(d) level of theory were carried out on selected cyclic hydrogen-bonded (H-bonded) dimers of glycine and alanine as models for β-sheets and on the αC-centered radicals derived from them. The structures mirrored the cycles found in the H-bonded network of parallel and antiparallel β-sheet secondary structure, and were otimized both with and without enforcement of constraints on the Φ,Ψ torsion angles. Transition structures for the migration of an H atom from an αC site to another αC site or to an S atom were located. It was found that the presence of a H-bonded strand of a β-sheet has little effect on the αC−H bond dissociation enthalpy (BDE) of glycine but raises the BDE of other residues by a significant amount. The parallel β-sheet structure and Φ,Ψ angles lead to a significant increase in BDE, relative to the random coil structure, due to loss of captodative stabilization. The antiparallel β-sheet structure and Φ,Ψ angles do not lead to a significant increase in BDE. All residues incorporated in β-sheet secondary structure, with the exception of glycine, are protected from oxidative damage because the αC−H bond is internal to the sheet and inaccessible to oxidizing radicals. Glycine is susceptible to oxidative damage because it has a second αC−H bond which is exposed. Among residues in secondary structures, only glycine is susceptible to damage by weak oxidants such as thiyl radicals and superoxide, provided it is in an antiparallel β-sheet. Radical damage may propagate readily from one strand to another above the β-sheet, but not within the β-sheet. β-Sheet structure narrows the difference between the glycyl αC−H BDE and S−H BDE and facilitates interstrand H atom transfer between the glycyl αC site and the S atom of cysteine.
The free-radical intermediates and the stable products formed on one-electron oxidation of indole-3-acetic acid (IAA) in aqueous solution were investigated. The dibromine radical anion generated radiolytically reacted with IAA to yield the IAA radical cation. In acid solution, the latter decays by a first-order process (k = 1.8 x 10(4) s(-1)) to yield carbon dioxide and the skatole radical. At pH 7 it deprotonates (pK(a) = 5.09 +/- 0.02), giving the indolyl radical, which decays only by a bimolecular process (rate constant of first-order reactions <100 s(-1)) which yields indole-3-carbinol as one of the products. Under steady-state irradiation at pH 7 the free radicals had a sufficient lifetime to allow the small fraction of radical cation present in equilibrium to undergo decarboxylation, consistent with the observed formation of carbon dioxide. Glutathione reacted with the indolyl radical, regenerating IAA, and with the skatole radical to yield skatole. In the presence of oxygen the skatole radical is rapidly converted to a peroxyl radical, which appears to decay only by bimolecular reactions with indole-3-aldehyde as one of the products. No evidence was found for the reaction of the peroxyl radical with glutathione or for elimination of superoxide.
Electrochemical oxidation of 1-methylindole, 2-methylindole and 3-methylindole on a platinum anode in acetonitrile containing NaClO4 has been studied. In any case no polymeric deposit on the working electrode was obtained. The identification of the obtained soluble products is described and the mechanisms of formation are discussed. Analogously to the case of unsubstituted indole, electrooxidized 1–3 react through positions 2 and 3. The non polymerization of 1 is explained with the formation of a tetramer which is oxidized to a stable radical cation and dication without undergoing further coupling reactions.
Imidazole has been studied extensively in enzyme chemistry. It is used as an initiator in enzyme reactions. We report the electrochemical and chemical oxidation of imidazole, its 2-methyl and 4-methyl derivatives, benzimidazole, and 1-methylimidazole.
Model systems of the tryptophan radical, present in several biological systems such as DNA photolyase, cytochrome c peroxidase, and mutated ribonucleotide reductase, have been investigated using gradient-corrected density functional theory (DFT). We report calculated spin densities and complete hyperfine tensors for all atoms, as well as potential energy and β-proton hyperfine tensor curves for the rotation about the C3−Cβ bond. Effects of hydrogen bonding to the N1 nitrogen (neutral radical) or N1−H hydrogen (cationic radical) are investigated, as is the C3 dioxygen adduct (peroxide) radical. Throughout comparisons are made to experimental data and previous theoretical studies of tryptophan model systems. The calculations support the earlier assignments of neutral Trp radicals present in mutant Y122F Escherichia coli ribonucleotide reductase at low temperatures, but most likely charged radical cations in DNA photolyase and yeast cytochrome c peroxidase. Although the calculations give clear-cut answers to, for example, the geometric arrangements, they are unable to resolve all of the ambiguities regarding the Trp radicals in the above systems, primarily due to lack of sufficient well-resolved data to compare with.
Ab initio calculations have been carried out on 3-methylindole, and the cation and neutral radicals of 3-methylindole, using density functional theory (DFT), the Becke3−Lee−Yang−Parr functional, and the 6-31G*, 6-31+G*, 3-21G*, and TZ2P basis sets. Optimized geometries, vibrational frequencies, and for the radicals, atomic spin densites are calculated. The latter are compared to experimental spin densities recently determined for the tryptophan-191 radical of compound ES of the enzyme cytochrome-c-peroxidase. The DFT spin densities for the cation radical of 3-methylindole are in excellent agreement with the data for the tryptophan-191 radical, which supports the conclusion that the tryptophan radical is a cation radical. The results are compared to calculations using second-order Møller−Plesset theory (MP2) and the 6-31G** basis set. The MP2 spin densities are in significantly worse agreement with the experimental spin densities.
We have measured the reaction rate constants of the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) with a number of small alkyl and σ parent radicals in dilute aqueous solution using in situ radiolysis time-resolved electron spin resonance spectroscopy. Unsubstituted alkyl parent radicals (methyl, ethyl, propyl, and 1-methylethyl (2-propyl)) had rate constants ranging from 5.6 × 106 to 1.6 × 107 M-1 s-1. Electron-releasing α-hydroxyalkyl radicals (hydroxymethyl, 1-hydroxyethyl, 1-hydroxypropyl, and 1-hydroxy-1-methylethyl (2-hydroxy-2-propyl)) reacted more rapidly than the unsubstituted radicals with rate constants of (2.2−6.8) × 107 M-1 s-1, while the electron-withdrawing carboxymethyl radical was slower (4.4 × 106 M-1 s-1). The bulky 2-hydroxy-2-methylpropyl radical reacted with DMPO, but with a rate constant smaller than 106 M-1 s-1. σ radicals such as sulfite anion and carboxyl anion were trapped quickly, with rate constants of 1.2 × 107 and 6.6 × 107 M-1 s-1, respectively. These results show that the zwitterionic structure of DMPO results in sensitivity to polar effects in the parent radical-spin trap encounter complex, while steric effects are also influential in the reaction of DMPO with bulky alkyl radicals. The rate constants for the reaction of DMPO with the radicals studied herein are, in general, an order of magnitude slower than the same radicals reacting with the nitroso spin trap 2-methyl-2-nitrosopropane.
The ω-technique in the simple l.c.a.o. theory is applied to a variety of organic compounds. A comparison of the method with Pople's s.c.f. method shows that the use of the ω-parameter provides for electron repulsion effects in an empirical manner. Such a procedure is required when dealing with cations; for example, in the simple l.c.a.o. theory the ionization potentials of methyl, allyl and benzyl radicals should be identical. The experimental values which vary over a 2 e.v. range are well reproduced by the ω-technique. The method is applied to several organic radicals of current interest, including cyclopentadienyl and tropylium. Agreement with experimental results, where available, is excellent. With aromatic hydrocarbons, however, including examples of the non-alternant hydrocarbon type, the simple l.c.a.o. procedure gives a good correlation of ionization potentials; the ω-technique provides no advantage with such systems alone. Heteroatoms can be treated given two additional parameters characteristic of the heteroatom. A methyl group is treated as a pseudo-heteroatom donating two electrons to the π-system with parameter values differing slightly from those previously employed. Good agreement between calculated and experimental ionization potentials was obtained for a large number of methyl substituted hydrocarbons and radicals. Experimental potentials were used for chlorine, nitrogen and oxygen compounds to determine empirically the appropriate parameter values for those elements. The derived values are compared with those used previously in other applications in the literature. The ω-technique is extended to calculations of acetylenic and allenic systems. A simple model of an alkane is presented which requires only one additional disposable parameter. The derived value of zero for this parameter is used with the ω-technique to give good agreement between calculated and experimental ionization potentials for a variety of alkanes.
ESR equipment has been developed for directly observing labile free radicals that are present at high temperatures. A pressurized fluid is heated as it slowly flows through the microwave cavity of the spectrometer. Temperatures to 566°C and pressures to 140 kg/cm² have been used. Radicals are identified from well-resolved hyperfine structure. Spectra of cyanoisopropyl from the decomposition of azobisisobutyronitrile in solution at 155 to 185°C and hydroxyisopropyl from 2% di-tert-butyl peroxide in isopropyl alcohol at 173 to 258°C are reported. Indenyl is reported from indene in benzene at 533°C and in tetralin at 471°C. 1,2-diphenylethyl has been observed from solutions of 1,2-diphenylethane in benzene from 460 to 560°C. For dilute solutions of 1,2-diphenylethane in toluene the benzyl radical is obtained, while for more concentrated solutions a mixture of radicals is present in the following equilibrium for which K = 10 +- 3 at 560°C and 105 kg/cm²; CâHâCHâCHâCâHâ + CâHâCHâ in equilibrium CâHâCHCHâCâHâ + CâHâCHâ. Diphenylmethyl is obtained from the dissociation of tetraphenylethane as a dilute solution in benzene and in 50:50 benzene-diphenylmethane. ESR parameters are given in the latter solvent at 420°C. The formation of diphenylmethyl from diphenylmethane as a consequence of impurities has been examined, and diphenylmethane that has been initially air oxidized will form diphenylmethyl upon heating. Cumyl is obtained from cumene at 560°C. ..cap alpha..-methylstyrene is a final product but not dicumyl. Dicumyl in benzene solution at 560°C decomposes to give disproportionation products cumene and ..cap alpha..-methylstyrene. Hyperfine couplings and g values are given and compared with room temperature values, and a discussion involving kinetic parameters is presented.
A mechanistic investigation has been of the thermolysis of several endoperoxides of anthracenes and naphthalenes which produce molecular oxygen and the parent aromatic species quantitatively. Qualitative thermochemical measurements in the solid state indicate that in all the cases studied, the reactions were endothermic. This situation appears to be valid in solution also. Clean first-order kinetics were observed for these thermolyses. Activation parameters were derived from the temperature dependence of the first-order rate constants. The primary yields of singlet molecular oxygen (¹Oâ) from the several endoperoxides were determined, and a correlation was discovered between the A factors (..delta..S/sup + +/ values) for thermolysis and the yield of ¹Oâ. It was found that high A factors (positive ..delta..S/sup + +/ values) correlated with relatively low yields of ¹Oâ, and that low A factors (slightly negative or near zero ..delta..S/sup + +/ values) correlated with nearly quantitative yields of ¹Oâ. These two results are interpreted in terms of a diradical mechanism which leads to low yield of ¹Oâ and a concerted mechanism which leads to quantitative yields of ¹Oâ, respectively. This interpretation is consistent with the observation of a magnetic field effect on the yield of ¹Oâ from endoperoxides whose thermolyses proceed with positive ..delta..S/sup + +/ values and the absence of a magnetic field effect on the yield of ¹O/sub i/ endoperoxides whose thermolyses proceed with near zero ..delta..S/sup + +/ values. Further support for the occurrence of a diradical mechanism is available from the demonstration of a special ¹â·O isotope effect on the thermolysis of an endoperoxide which is postulated to undergo thermolysis principally via a diradical intermediate. The thermolysis of endoperoxides which decompose mainly by a diradical mechanism yields triplet molecular oxygen that is selectively enriched in ¹â·O.
Electron spin echo envelope modulation spectroscopy was used to study the nuclear quadrupole interactions of the remote 14N of substituted imidazoles coordinated to Cu(II)-diethylenetriamine. Substitution at the carbon adjacent to the remote nitrogen only slightly altered the quadrupole coupling parameters, while alkylation at the remote nitrogen altered them significantly. By applying a modified Townes-Dailey model, we were able to relate the change of quadrupole coupling parameters of the remote nitrogen to the change of electron occupancy in the sp2 hybrid orbital that is external to the imidazole ring and to relate this to N-H or N-C bond polarization. These studies suggest that the variation of quadrupole coupling constants previously observed in Cu(II) proteins may arise from the variations in hydrogen bonding at the remote nitrogen of the coordinated histidine imidazole side chain.
Radicals produced by the reaction of OH with pyrrole, 2-pyrrolecarboxylic acid, N-methylpyrrole, N-methyl-2-pyrrolecarboxylic acid, imidazole, 2-methylimidazole, 4,5-imidazoledicarboxylic acid, isoxazole, pyrazole, 3,5-pyrazoledicarboxylic acid, maleimide, and N-ethylmaleimide have been studied by the in situ radiolysis steady-state esr technique. In all cases OH has been found to add to a carbon rather than to abstract H. With pyrroles addition takes place at the position adjacent to the nitrogen and with imidazoles addition to both positions 2 and 5 has been found. In contrast to the furans the OH adducts do not undergo ring opening in alkaline solutions. In the case of pyrrole, imidazole, and 2-methylimidazole the OH adducts have been found to undergo a base-catalyzed water elimination involving the OH and the H from NH. In the carboxy derivatives water elimination is a slow process and was not detected. In the latter radicals a small splitting by the OH proton has been observed in neutral solution but disappeared at pH >10 for imidazoles and >11 for pyrroles. The disappearance of the OH proton splitting is a result of rapid proton exchange. Furthermore, dissociation of the OH proton occurs with pK = 12 for the 4,5-imidazoledicarboxylate adduct and pK = 13.5 for the adducts of 2-pyrrolecarboxylate and N-methyl-2-pyrrolecarboxylate. Hydroxyl radicals add to isoxazole at the 5 position adjacent to the oxygen rather than that adjacent to the nitrogen. Addition to pyrazole also takes place at the 5 position, next to the NH group, and in both cases of isoxazole and pyrazole comparable allylic type radicals are observed. With 3,5-pyrazoledicarboxylic acid, however, OH adds to the 4 position probably because the 5 position is sterically hindered by the carboxyl group.
The one-electron reduction potential of riboflavine has been determined at several pH values between 6 and 12 using the pulse radiolysis technique. Solutions containing riboflavine and either duroquinone or 9,10-anthraquinone-2-sulfonic acid were irradiated under reducing conditions to produce their semiquinoidic radicals. Electron transfer from one of the radicals to the parent molecule of the other then took place until equilibrium was achieved. Spectrophotometric measurement of radical concentrations at equilibrium enabled determination of the equilibrium constant and redox potential. The reduction potential of riboflavine at pH 7 was found to be E71 = -0.292 ± 0.005 V. The dependence of the reduction potential of riboflavine on pH was found to be in agreement with the dependence expected from the known pK values for riboflavine and its semireduced form.
Pulsed laser photolysis at 347nm has been used to study the transient spectroscopy of alloxazine, lumichrome, lumiflavin, and riboflavin in acidic (pH 2.2) aqueous solution and in ethanol. Intersystem crossing quantum yields (φISC) were determined by a modification of the comparative laser excitation method which utilizes the variation of the triplet yield with intensity in conjunction with a kinetic model for the various photophysical and photochemical processes occurring during the pulse. Fluorescence quantum yields and lifetimes are also reported. Correction for quenching of the excited singlet state by H+ ions shows that, in neutral aqueous solution, intersystem crossing for flavins is an efficient process (φISC˜ 0.7) which, in conjunction with fluorescence, accounts for the fate of all absorbed photons. For alloxazine (φISC˜ 0.45) and lumichrome (φISC˜ 0.7) the results are more difficult to interpret owing to interconversion between alloxazine and isoalloxazine structures in the singlet excited state. For all four compounds, the quantum yield of products derived from the singlet excited state is estimated as ˜0.04. There is evidence of biphotonic product formation at high laser energies. In ethanol, where φISC for lumichrome is about twice that of lumiflavin, internal conversion between the excited singlet and ground states appears to be a significant process. Complete triplet-triplet absorption spectra in the region 260–750nm are reported. For lumichrome at pH 2.2 there is spectral evidence for isomeric triplet states which appear to be in equilibrium.
Abstract— Primary photochemical processes in aqueous solution have been characterised for FMN. The influence of pH on these processes is attributed to protonation of the neutral triplet and not to the presence of a dimeric species as postulated earlier. Second order rate constants for reaction between the neutral triplet and some naturally occurring amino acids are reported.
The oxidation of tryptophan and indole-3-acetic acid (IAA) by the dibromine radical anion or peroxidase from horseradish in aqueous solution was investigated and compared, especially with respect to the involvement of oxygen and superoxide. Using EPR with spin-trapping, the tryptophanyl radical, generated by either method was found to react with oxygen, although this reaction is too slow to be observed by pulse radiolysis (k < 5 × 106dm3mol−1s−1). No superoxide results from this reaction, thus excluding an electron-transfer mechanism and suggesting the formation of a tryptophan peroxyl radical, possibly in a reversible process. These observations imply that in proteins where the tryptophanyl radical exists as a stable species it must either have its reactivity modified by the protein environment or be inaccessible to oxygen. The related molecule IAA is oxidized by either peroxidase or Br2•− to a radical cation that decarboxylates to yield a skatolyl radical. The latter reacts with oxygen to give a peroxyl radical that does not release superoxide. However, O2•− is formed during the peroxidase-catalyzed oxidation of indoleacetic acid. This supports the hypothesis that the peroxidase can act in an oxidase cycle involving ferrous enzyme and compound III, with superoxide as a product.
This review presents current knowledge on light-induced effects on packaged cheeses. As research in this area is somewhat limited and involves highly non-standardized light exposure conditions, the review includes, if deemed necessary, effects observed in other dairy products. Most of these effects may be explained by general lipid oxidation mechanisms combined with knowledge on the spectral balance between the singlet oxygen quencher, β-carotene, and the sensitizer, riboflavin. As determined by Lambert-Beer's law, β-Carotene absorbs light in a concentration-dependent manner, which would otherwise be absorbed by riboflavin, thereby inducing quality changes. Consequently, these processes may be prevented by total exclusion of light and by storage in an oxygen-free atmosphere. Unfortunately, quality changes are apparent at residual oxygen levels even as low as 0.5% in headspace, which is often considered an acceptable residual oxygen level in industry. For a given product composition, spectral distribution and photon flux of the light source determine the extent of quality changes, since photochemical processes have limited temperature dependence, in contrast to the consecutive lipid autoxidation process. Hence, precautionary measures include changes of light source and targeted prevention of photon flux relative to the cheese by use of creative packaging. In order to optimize packaging and display conditions, a substantial need exists for analytical methods, which reflect the sensory perception of the consumer. Once established, optimization to include marketing and consumer aspects will harbor no major obstacles.
We report the results of a comparative investigation of two-laser resonant photoionization mass spectrometry (MS) and conventional one-laser resonant photoionization MS of several aromatic molecules in a supersonic jet. The use of a separate, independently tunable laser to photoionize excited-state molecules is shown to provide enhanced parent ion production compared with the one-laser technique. By use of two lasers in the photoionization process, isomer discrimination can be simplified by selectively exploiting differences in ionization potentials and excited-state lifetimes. In particular, we have investigated the two-laser photoionization of phenanthrene, anthracene, pyrene, aniline, and several methylindole isomers under supersonic expansion conditions. The added dimensions of employing two lasers in the ionization are discussed.
Boron-doped diamond (BDD) is a promising electrode material for use in the spectro-electrochemical study of redox proteins and, in this investigation, cyclic voltammetry was used to obtain quasi-reversible electrochemical responses from two blue copper proteins, parsley plastocyanin and azurin from Pseudomonas aeruginosa. No voltammetry was observed at the virgin electrodes, but signals were observed if the electrodes were anodised, or abraded with alumina, prior to use. Plastocyanin, which has a considerable overall negative charge and a surface acidic patch which is important in forming a productive electron transfer complex with its redox partners, gave a faradaic signal at pre-treated BDD only in the presence of neomycin, a positively charged polyamine. The voltammetry of azurin, which has a small overall charge and no surface acidic patch, was obtained identically in the presence and absence of neomycin. Investigations were also carried out into the voltammetry of two site-directed mutants of azurin, M64E azurin and M44K azurin, each of which introduce a charge into the protein's surface hydrophobic patch. The oxidizing and cleaning effects of the BDD electrode pre-treatments were studied electrochemically using two inorganic probe ions, Fe(CN)63− and Ru(NH3)63+, and by X-ray photoelectron spectroscopy (XPS). All of the electrochemical results are discussed in relation to the electrostatic and hydrophobic contributions to the protein/diamond electrochemical interaction.
The triplet-triplet absorption spectra in aqueous solution of the acid (3LfH2+), the neutral (3LfH) and the basic (3Lf-) forms of lumiflavin (6,7,9-trimethylisoalloxazine) were measured by flash photolysis. The pKa values of the corresponding protolytic equilibria of the lumiflavin triplet were found to be 4·4 5 ± 0·1 and 9·8 ± 0·15.
. Irradiation of Dulbecco's modified Eagle's tissue culture medium with “Daylight,”“Special Blue,” or “Bilirubin” fluorescent light produces photoproducts lethal to human cells. Killing is abolished when (1) riboflavin, (2) tryptophan and tyrosine, or (3) riboflavin, tryptophan and tyrosine are deleted from medium prior to irradiation with any of the above fluorescent lamps. Toxic photoproducts are also formed when buffered salt solutions containing (a) riboflavin and tryptophan, (b) riboflavin and tyrosine, or (c) riboflavin, tryptophan and tyrosine are exposed to any of these light sources.
Equilibrium constants for addition of Lewis bases to TPPFeCl in chloroform and several other solvents have been measured by visible spectral techniques. The equilibria observed are: TPPFeCl + 2B ⇌ [TPPFeB2]+Cl- (β2), where the product is an ion pair, and in some cases: TPPFeCl + B ⇌ TPPFeClB(K1), where the product may either be the six-coordinate or the five-coordinate [TPPFeB]+Cl- ion pair. Increased solvent polarity causes β2 to increase for B = N-methylimidazole more than for B = imidazole. The hydrogen bonding capabilities of N-H as compared with N-R imidazoles appear to stabilize the product of reaction 1 by about 3 log units in β2. This suggests that Fe-NIm, bond strength in heme proteins may be significantly strengthened by weakening of the N-H bond of the histidine and further increased if this N-H proton can be transferred to some adjacent basic protein residue. The steric effect of addition of a methyl group to the 2 carbon of imidazole is approximately equal to the effect on β2 of the loss of N-H⋯Cl- hydrogen bonding. The basicity of B also has a dramatic effect upon β2 within each class of B (imidazoles, pyridines). The addition of two B was also investigated as a function of para substituent, X, on the four phenyl rings of TPPFeCl for B = N-methylimidazole. A Hammett relationship between log β2 and σX is observed, with ρ -0.39. The sign of ρ is opposite those observed for the reactions of (p-X)TPPM complexes (M = Ni2+, VO2+, Co2+). This is because the product of the reaction contains a positively charged center (Fe), which is stabilized by electron-donating groups on the porphyrin ring.
In order to examine the electrostatic forces in globular proteins, pKa values and their ionic strength dependence of His residues of hen egg white lysozyme (HEWL) and human ly−sozyme (HUML) were measured, and they were compared with those calculated numerically.
pKa values of His 30 residues in HEWL, HUML, and short oligopeptides were determined from chemical shift changes of His side chains by 1H−nmr measurements. The associated changes in pKa values in HEWL and HUML were calculated by solving the Poisson-Boltzmann equations numerically for macroscopic dielectric models. The calculated pKa changes and their ionic strength dependence agreed fairly well with the observed ones.
The contribution from each residue and each α-helix dipole to the pKa values and their ionic strength dependence was analyzed using Green's reciprocity theorem. The results indicate that (1) the pKa of His residues are largely affected by surrounding ionized and polar groups; (2) the ionic strength dependence of the pKa values is determined by the overall charge distributions and their accessibilities to solvent; and (3) α-helix dipoles make a significant contribution to the pKa when the His residue is close to the helix terminus and not fully exposed to the solvent.
When rat lens homogenate or its soluble protein fractions are irradiated in the presence of riboflavin, a photo-adduct is obtained between this vitamin and the lens proteins. Irradiation of these proteins in the presence of riboflavin also leads to a modification in the chromatographic elution pattern with an increase in the high-molecular-weight fraction. In an aging study with rats, it was shown that the proportion of the high-molecular-weight protein fraction significantly increased with age, whereas the proportion of the low-molecular-weight protein fraction concomitantly decreased. It is postulated that aging produces an increase in the accessibility of the tryptophan residues of the lens proteins, as established by iodide fluorescence quenching experiments.
— The lumiflavin-sensitized photooxygenation of indole in aqueous solutions has been investigated by means of steady light photolysis and flash photolysis. The semiquinone of lumiflavin and the half-oxidized radical of indole were formed by the reaction between triplet lumiflavin and indole (3.7 times 109M-1 s-1). The semiquinone anion radical of lumiflavin reacted with oxygen to form superoxide radical. The triplet state of lumiflavin also reacted with oxygen forming singlet oxygen, 1O2. But the reaction between 1O2 and indole (7 times 107 M_l s_1; estimated from steady light photolysis using Rose Bengal as a sensitizer) was far less efficient than the reaction between indole and triplet lumiflavin. The quantum yield of the lumiflavin-sensitized photooxygenation of dilute indole via radical processes was much higher than that via1O2 processes, though appreciable 1O2 was formed.
Characteristic chemiluminescence emission of singlet (1 delta g) molecular oxygen at 1268 nm is reported from a Haber-Weiss reaction. The reaction consists of mixing aqueous hydrogen peroxide with a solution of potassium superoxide, solubilized by 18-crown-6 ether in carbon tetrachloride or in dry acetonitrile at room temperature. Since the discovery of the enzyme superoxide dismutase by J.M. McCord and I. Fridovich [(1968) J. Biol. Chem. 243, 5733-5760], the identity of the reactive oxidant in superoxide-generating systems in biology has remained a chemical mystery. The results presented here suggest strongly that the reactive species is singlet oxygen generated via the Haber-Weiss reaction and not, as usually assumed, the hydroxyl radical, .OH, generated by the same reaction.