Tryptophan Degradation

Free Radical Biology and Medicine 160 (2020) 696–718
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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed
Review Article
Reactivity and degradation products of tryptophan in solution and proteins T

Stephanie Bellmaine, Alisa Schnellbaecher, Aline Zimmer ∗
Merck Life Science, Upstream R&D, Frankfurter Strasse 250, 64293, Darmstadt, Germany
ARTICLE INFO ABSTRACT
Keywords: Tryptophan is one of the essential mammalian amino acids and is thus a required component in human nutrition,
Tryptophan animal feeds, and cell culture media. However, this aromatic amino acid is highly susceptible to oxidation and is
Degradation products known to degrade into multiple products during manufacturing, storage, and processing. Many physical and
Oxidation chemical processes contribute to the degradation of this compound, primarily via oxidation or cleavage of the
Reactivity
highly reactive indole ring. The central contributing factors are reactive oxygen species, such as singlet oxygen,
Stability
hydrogen peroxide, and hydroxyl radicals; light and photosensitizers; metals; and heat. In a multi-component
Reactive oxygen species
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.
L-Tryptophan (Trp, 1) is an aromatic amino acid (AA) that is essential several fates. It can undergo deamination to kynurenic acid (8, KNA) or
for the synthesis of proteins in mammals and is also a pleiotropic precursor undergo a series of catabolic reactions involving kynurenine hydroxylase
for many important metabolites that are known to influence cellular path and kynureninase, resulting in the formation of 3-hydroxykynurenine (9)
ways and physiological responses (Fig. 1) [1]. As an example, Trp can be and 3-hydroxyanthranilic acid (10) [13–15]. Enzymatic catalyzed ring
hydroxylated to 5-hydroxytryptophan (2) by tryptophan hydroxylase [2] opening of 3-hydroxyanthranilic acid and differing ring closing reactions
and further decarboxylated by aromatic L-amino acid decarboxylase to lead to picolinic acid (11) and quinolinic acid (12) [16], a precursor of
generate serotonin (3) [3], a key neurotransmitter involved in the regula nicotinic acid (13) and NAD/NADP [17]. Finally, Trp can be deaminated to
tion of adaptive reactions and responses to environmental changes such as indole pyruvic acid (14), which further yields indole-3-acetaldehyde (15)
sleep, cognition, feeding behavior, and body temperature [4]. Serotonin can and indole acetic acid (16) via decarboxylase and aldehyde dehydrogenase
further be metabolized by N-acetyltransferase to N-acetylserotonin (4), catalyzed reactions [18–20].
which can be methylated by hydroxyindole-O-methyltransferase to form From a structural perspective, tryptophan contains an α-amino
melatonin (5) [5], a potent antioxidant and a neurohormone involved in group, an α-carboxylic acid group and an indole side chain, which is
synchronizing the circadian rhythm [6]. Trp can be hydrolyzed by trypto responsible for its specific reactivity and susceptibility to oxidative
phanase [7] to indole, ammonia and pyruvate, the latter being a key pre cleavage. The ring nitrogen is an extremely weak nucleophile due to the
cursor for the formation of acetyl-CoA used to generate energy via the ci delocalization of its electron pair in the indole ring [21–23], but can
trate cycle. Trp can also function as precursor of the coenzymes react with very strong electrophiles (e.g. aldehydes and carbocations)
nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, under more forcing conditions (e.g. heat and prolonged reaction times)
NADP [8]. Moreover, Trp is, via the kynurenine pathway, a precursor to [24]. The hydrophobic indole ring also gives unique properties to
molecules involved in inflammation, immune response, protection against proteins and peptides through van der Waals forces that are essential to
UV radiation, and neurotransmission [4,9]. In more detail, Trp can, via the promote the protein-protein hydrophobic interactions needed to stabi
action of tryptophan 2,3-dioxygenase (also called indoleamine 2,3-dioxy lize secondary and tertiary conformational structures. For instance, the
genase), form N-formylkynurenine (6, NFK) [10], which is converted into crucial role of Trp in the stabilization and maintenance of hydrophobic
kynurenine (7, KYN) by kynurenine formamidase [11,12]. Kynurenine has clusters has been extensively illustrated for hen lysozyme [25].
∗
Corresponding author.
E-mail address: aline.zimmer@merckgroup.com (A. Zimmer).
https://doi.org/10.1016/j.freeradbiomed.2020.09.002
Received 20 May 2020; Received in revised form 6 August 2020; Accepted 2 September 2020
Available online 08 September 2020
0891-5849/ © 2020 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Bellmaine, et al. Free Radical Biology and Medicine 160 (2020) 696–718
Abbreviations GSH glutathione

KNA kynurenic acid
AA amino acids KYN kynurenine
AGE advanced glycation end products NAD nicotinamide adenine dinucleotide
AhR aryl hydrocarbon receptor NADP phosphorylated nicotinamide adenine dinucleotide
βC β-carboline NFK N-formylkynurenine
CCM cell culture medium NHE normal hydrogen electrode
CHO Chinese Hamster Ovary Oia oxindolylalanine
CYP1a1 phase I mono-oxygenase cytochrome P450 1a1 PIC 3-hydroxypyrroloindole carboxylic acid
diHβC dihydro-β-carbolines RNS reactive nitrogen species
diOia dioxindolylalanine ROS reactive oxygen species
DMEM Dulbecco's Modified Eagle's Medium RSS reactive sulfur species
EDG electron-donating groups RT room temperature
EMS eosinophilia-myalgia syndrome tetHβC tetrahydro-β-carboline
EWG electron-withdrawing groups
Fig. 1. Main metabolic pathways downstream of tryptophan including the central metabolites serotonin, melatonin, and kynurenine.
In the food industry, Trp is of interest as a dietary nutrient and is been studied extensively in food proteins due to its susceptibility to
mainly found in protein-rich food of plant- and animal-origin such as oxidation during processing and storage [24,30,31]. This is particularly
dairy products, meat, seafood, soybeans or nuts [26–29]. The AA has important since oxidized proteins are responsible for the deterioration
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of the quality of food products, including changes in color as well as off- chemical, enzymatic, or biotechnological [39]. There are about twenty
flavors and off-odors [32–34]. In the pharmaceutical industry, Trp, as standard chemical synthetic approaches [40–42], that can be divided
key building block of proteins, is of particular interest since its oxida into two major categories: those that start with precursors already
tion may lead to alterations in the quality of therapeutic proteins containing an indole ring (e.g. 3-formylindole (17, Fig. 2) [43], gra
[35,36] used to treat life threatening diseases such as cancer or auto mine [44], indole (18, Fig. 2) [45]) and those which use even more
immune diseases. Trp is also a key component of cell culture media basic precursors [46]. A key difficulty with the chemical production
(CCM) used for the biomanufacturing of these therapeutic proteins and method is that they all produce racemic DL-Trp, and thus require iso
its degradation products have been shown to impact cellular processes lation of just the biologically active L-isomer. This purification of the L-
and overall process performance [37,38]. isomer from the racemic mixture following chemical synthesis of the
The purpose of this review is to summarize the information available on core tryptophan structure can be carried out using chemical, enzymatic,
Trp stability in complex liquid solutions and in proteins, with a specific or cellular technologies. While racemic Trp can be directly resolved to
emphasis on its degradation products and their known biological effects. the L-isomer via transamination [47] with another racemically pure L-
amino acid (carried out by a Pseudomonas cell culture [48]), the isomer
purification is commonly performed with simple derivatives of Trp,
1. Production of Trp and known contaminants
such as an N-acyl, ester, or amide. The pure L-Trp can be obtained by
fractional crystallization followed by removal of the protecting group
1.1. Production of Trp
[49,50], or by selective hydrolysis of the L-isomer using either a purified
enzyme or microbial cultures [51–53].
Tryptophan is manufactured by one of three synthetic methods:
Fig. 2. Known impurities detected in commercially available tryptophan source classified by function or structure.
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Enzymatic synthesis utilizes a purified enzyme (isolated from e.g. formed via the reaction of Trp and either indole-3-methanol or indole-
Escherichia Coli) which can convert very simple precursors to Trp, 3-acetaldehyde, or as the product of the reaction between Trp, indole
producing exclusively the L-isomer. This method requires extensive and formaldehyde [64]. Since methyleneindole-Trp 30 was formed
purification from the remaining byproducts, the enzyme, other cofac mainly at acidic (< 5) and basic (> 8.5) pHs, the authors hypothesized
tors, and macromolecules that may be used to stabilize the enzyme. The that it might be generated during the downstream chromatography
two most commonly used enzymes are tryptophan synthetase, which steps requiring acidic pHs [64]. Other identified contaminants include
uses serine and indole as the precursors [54], and tryptophanase, which 2-(3-indolyl)-Trp (31) [71], its hydration product, the dihydroindole-
uses indole, pyruvic acid, and ammonium [55]. Trp conjugate 32 [72], and non-conventional adducts of Trp such as 1-
Biotechnology utilizes microorganisms (e.g. Bacillus subtilis [56], or (2-Trp)-3-(3-indole)propylene (33) or 1-(2-Trp)-1-(3-indole)propylene
Escherichia Coli [57]) or yeasts (e.g. Candida strains [58]) which en (34), for which the mechanism of formation remains unknown. The
dogenously produce L-tryptophan. These organisms are engineered to tetHβCs 26, 27 and 28 proved to be the main contaminants in com
produce increased amounts of Trp, and depending on the strain, “de mercially available Trp in amounts ranging from 10 to 13500 mg/kg.
novo” processes may be used, using generic raw materials as carbon One final group consisted of small Trp degradation products such as
and nitrogen sources (e.g. glucose, ammonium salts, urea [59]), or re indole (18) [73], 3-formylindole (17) and tryptamine (35) [71].
quire specific precursors (e.g. indole (18) [60], anthranilic acid (19, The impurities that actually correlated with some of the symptoms
Fig. 2) [61,62]). Results from multiple groups indicate that the pur of EMS were PIC (25), 2-(3-methyleneindole)Trp (30) and 3-(pheny
ification process, rather than the fermentation, governs the pattern of lamino)alanine (36), which was the only contamination product not
contaminants in biotechnologically derived Trp [63]. Trp or indole related and was likely formed from dehydroalanine and
aniline [65]. The ethylene-bridged Trp dimer 37 (EBT), the only 1-
1.2. Known contaminants in commercially available Trp substituted Trp derivative found in the study (which was probably
formed by condensation reaction from acetaldehyde and Trp), was also
Impurities present in commercially available Trp for pharmaceutical related to the onset of EMS [65,74]. Until now, no conclusive proof has
or nutritional use were investigated in the 1990s following the outbreak been published to demonstrate that any of these products is the root
of a new autoimmune disease called eosinophilia-myalgia syndrome cause leading to EMS, but these studies constitute a very interesting
(EMS). The root cause for the disease was linked to the intake of Trp library of Trp metabolites, byproducts or degradation products that
from a specific Japanese manufacturer [63–65] wherein up to 60 con may have significant effects in vitro or in vivo when Trp is used in the
taminants were detected in the product [65], but only a few were hy food or pharmaceutical industry.
pothetically correlated with the symptoms (Fig. 2). One group of con
taminants detected and identified in commercially available Trp 2. Trp reactivity and degradation in complex solutions
sources were known metabolites of the amino acid such as 5-hydro
xyTrp (2), KYN (7), NFK (6), anthranilic acid (19) [63] and very small The stability of Trp in solution may be impacted by various en
amounts of indole acetic acid (16) [66]. These compounds were most vironmental factors such as light exposure, reactive oxygen species
likely formed during the microbial production process due to the ac (ROS), and temperature. Additionally, the amino acid may react with
tivity of Trp-degrading enzymes. small molecules such as trace metals, aldehydes, keto acids, sugars, and
Another group of contaminants comprised oxidation products of Trp vitamins, leading to a wide array of products. The main chemical in
such as 4-hydroxyTrp (20), 6-hydroxyTrp (21), and 7-hydroxyTrp (22) teractions and degradation pathways are summarized hereafter with a
[63], wherein 6- and 7-substituted Trp were claimed to be more stable focus on the products generated.
than the 4-substituted derivative [67]. The diastereomers of both oxi
ndolylalanine (23, Oia) and dioxindolylalanine (24, diOia) as well as 2.1. Degradation products and pathways induced by reactive oxygen species
the 3-hydroxypyrroloindole carboxylic acid (25, PIC) were reported in (ROS)
many studies [63], indicating that the pyrrole moiety is the part of the
Trp molecule most susceptible to oxidation, especially in acidic media 2.1.1. Singlet oxygen
[68]. The detection of PIC, Oia and diOia, which are all non-physio While ground state molecular oxygen is generally unreactive, pho
logical Trp oxidation compounds, revealed a rough treatment of Trp tosensitization in solutions can generate singlet oxygen, which reacts
during the manufacturing process [66], which may occur during fer readily with Trp to form multiple oxidation products (Fig. 3) [75]. It is
mentation, downstream processing or storage [63]. commonly stated that the 3-hydroperoxytryptophan (38) is the first
A third group of contaminants corresponded to condensation pro formed product via the Schenck ene reaction [76], which can then re
ducts of Trp with carbonyls. Indeed, as will be covered in detail later, arrange to the dioxetane 39 [77,78], however the formation of the
Trp is known to react with aldehydes and ketones to form tetrahydro-β- dioxetane directly from [2 + 2] cycloaddition with singlet oxygen has
carbolines (tetHβCs) via the Pictet-Spengler reaction [24]. TetHβC-3- not been disproven [75,79,80]. In either case, these are the primary
carboxylic acid (26) and 1-methyl-tetHβC-3-carboxylic acid (27) were intermediates and decomposition of these intermediates leads to the
detected in many commercial products and are formed from the con wide variety of singlet oxygen-derived oxidation products. In most
densation of Trp with formaldehyde and acetaldehyde, respectively conditions, the major products of singlet oxygen addition are the dia
[69]. Since highly reactive aldehydes are generated during the fer stereomers of both dioxindolylalanine (24) and hydroxypyrolloindole
mentation processes, these tetHβCs may be detected in any bio 25 as well as NFK (6) [75,77,79,81]. Dioxindolylalanine can further
technologically-derived Trp [66]. The diastereomers of 1-(3-methyle degrade under neutral and alkaline conditions to diol 40 [82], while
neindole)-tetHβC-3-carboxylic acid (28), formed from the reaction NFK can either further degrade to N-formylanthranilic acid (41), un
between Trp and indole-3-acetaldehyde (15), were also detected in Trp dergo more complicated transformations to generate polymeric brown
lots. Since indole-3-acetaldehyde is a metabolite in the indole-3-acetic precipitates, or function as photosensitizer in both type I and type II
acid pathway of Trp, the product was likely formed during the fer photosensitized oxidation reactions [83]. 3-Hydroperoxytryptophan
mentation process [63]. (38) was reported to readily undergo intramolecular cyclization to form
A fourth group included 2-substituted derivatives formed by sub the hydroperoxypyrolloindole 42 [84] and while the hydroperoxide
stitution of the H on the C2 of Trp with another indole-containing seemed fairly stable upon thermal treatment (up to 37 °C) for few hours
compound [70]. This category included the diastereomers of diol 29, and at acidic pH [79,84], the exposure to trace metals such as iron and,
formed from the non-enzymatic reaction between Trp and indole-3- to a lower extent, copper [84] led to the formation of hydro
glycerol [70], and 2-(3-methyleneindole)Trp (30), which could be xypyrolloindole 25. In contrast, exposure of the hydroperoxides to
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Fig. 3. Trp degradation products formed in presence of singlet oxygen.
mildly basic conditions at high temperatures (pH 7–8, > 70 °C) favored More recent investigations using LC-MS/MS enabled the identifi
NFK formation [84]. cation of a range of minor products generated upon exposure of Trp to
hydroxyl radicals using Fenton chemistry (H2O2/Fe2+) (Fig. 4). Among
2.1.2. Oxygen-centered radicals and UV radiation the novel components were the unsaturated Trp 43, 3-methyleneindole-
Reaction of Trp with oxygen-centered radicals and UV radiation substituted Trp 44, and oxidized variants of these adducts, including
mostly form the same degradation products as they proceed via the unsaturated, hydroxylated and hydroperoxylated species 45–50 [90].
same intermediate – the tryptophan radical. Even though the 3-methylindole moiety connected at position 6 on the
Oxygen-centered radicals (e.g. hydroxyl radical, peroxyl radical, Trp cores were the proposed structures for these compounds in the
alkoxyl radical) initiate degradation by first abstracting a hydrogen study, the authors did note that the 2-, 3-, 4-, 5- or 7-linked regioi
atom from the indole core of the tryptophan to form the Trp radical somers were also theoretically possible. Interestingly, the C2-linked
[85]. Then this delocalized electron either reacts with oxygen at the C3 variant of these newly discovered 3-methyleneindoleTrp isomers (i.e. 2-
carbon (the carbon with the greatest electron density in the indole ring) (3-methylindole)tryptophan (30) Fig. 4) was previously identified as a
producing 3-hydroperoxyTrp (38), or reacts with another indole ra contamination product of commercially available Trp [64]. Regarding
dical, forming C–C bonds linking two indole-containing species to the hydroxylated and hydroperoxylated species, the authors were un
gether [86]. The literature regarding the specific products known to be able to establish the precise location of the hydroxy and hydroperoxy
generated from each particular radical and from UV light will be dis substituents [90]. A 2016 in silico computational chemistry study de
cussed in more detail below. termined that position 2 is the most favored site for addition of the
The study of the degradation of Trp by hydroxyl radicals typically hydroxyl radical in hydroxyl radical-mediated oxidation of Trp, though
involves generating these radicals by either classical Fenton chemistry added that the other regioisomers can also plausibly be formed [91].
(H2O2/Fe2+) or by using γ-radiation (e.g. 137Cs, 60Co). In the latter, the Therefore, the 2-substituted isomers of the Domingues et al. study are
yield of the hydroxyl radical can be enhanced by the inclusion of dis represented in Fig. 4 (compounds 46–50). A Trp-Trp dimer and a hy
solved N2O. However, the hydroxyl radical is clearly not the only ROS droxylated form, hydroxyTrp-Trp, were also identified in this study,
in these systems, as is evidenced by the starting reagents in the Fenton presumably resulting from the formation of indole-centered radicals
system, the catalytic nature of the process and by the discovery that [90]. As with the other dimers, the exact location of the newly formed
hydrogen peroxide is produced in γ-radiation processes [87]. Wickern covalent bonds, either the dimer-forming bond or that of the hydroxyl
et al. also noted that peroxyl radicals are produced along with hydroxyl addition, was not established, however both 3-linked dimers, Trp-Trp
radicals in the presence of oxygen, and that Trp radicals and peroxy 51 and hydroxyTrp-Trp 52, are depicted as examples in Fig. 4.
compounds are presumed to be intermediates in the radiolytic de Under anaerobic γ-irradiation at several pH values, the dominant
gradation process [67]. These caveats are worthy of note when re products are 3-indolylpropionic acid (53) and 3-indolylacetic acid (16,
viewing literature on the hydroxyl radical-mediated degradation of Trp. Fig. 1) [92], while the minor products are the same as the dominant
Nevertheless, exposure of Trp to hydroxyl radicals generates some products generated by hydroxyl radicals. The authors suggested that 3-
of the same products as singlet oxygen- or H2O2-induced degradation indolylacetic acid is formed stepwise, via the indole-3-acetaldehyde
such as NFK (6) and diOia (24), and also affords Oia (23) [88], 4-, 5-, 6- (15, Fig. 1), as a result of the action of hydrogen and hydroxyl radicals
and 7-hydroxytryptophan (20, 2, 21, 22), KYN (7), 3-hydro [92].
xykynurenine (9) [67,87,89] and the common AAs Gly, Ala, and Asp Significantly more research has been done on the effects of hydroxyl
[89]. radicals on Trp degradation as compared to the effects of the peroxyl or
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Fig. 4. Tryptophan degradation products that are formed via exposure to irradiation and hydroxyl radicals.
alkoxyl radical. As it has been well established that peroxyl radicals will ring, forming a Trp radical [99], or the photoexcited Trp can return to
autodecompose to form both hydroxyl radicals and alkoxyl radicals ground state by releasing its energy to dissolved molecular oxygen,
[93], separating the effects of each is not trivial. Fuentes-Lemus et al. generating singlet oxygen [100]. Consequently, UV radiation will de
suggested that the concentration of Trp alters which species promotes grade Trp either through the production of a Trp radical or singlet
the degradation, but did not establish the Trp degradation products for oxygen. The established degradation products are therefore the same as
each condition [94]. While it is believed that the major intermediate in the combination of the products from oxygen-centered radical and
Trp degradation by peroxyl/alkoxyl radicals is 3-hydroperoxyTrp (38) singlet oxygen degradation [99,101–103]. Even more extensive de
[95], in studies of peroxyl radical-induced degradation of Trp, the gradation has been observed from UV radiation, such as the formation
major products found for both the free amino acid and the protein- of alanine, tryptamine (35), indole (18), 3-indolylpropionic acid (53)
bound Trp were NFK (6) and KYN (7) [96]. Proteins have also been and ammonia [103,104], but such extensive degradation is more pre
shown to produce crosslinks between Trp and other AAs upon exposure valent in anaerobic conditions.
to peroxyl radicals, however formation of the hydroperoxy inter In many ROS/Trp systems, higher doses of ROS have been shown to
mediate 38 is likely the favored degradation route in proteins due to the generate colored solutions and dark-colored precipitates. These colored
movement constrains of the Trp in the protein [86]. compounds and insoluble precipitates are assumed to be polymers, but
The superoxide radical has been established as a weak reaction none have been characterized to date [67,88,89,92]. Similar color
partner for Trp, but studies into the reaction of superoxide and the Trp formation from high molecular weight degradation products has been
radical yielded contradictory results. Fang et al. reacted superoxide shown for the degradation of solutions of Trp by UV light [103,105].
with N-acetylTrp methyl ester radicals to produce the corresponding N-
acetyl, O-methyl derivatives of NFK (6), KYN (7) and PIC (25) [97],
whereas Itakura et al. demonstrated that such oxidation requires a 2.1.3. Hydrogen peroxide
metal catalyst [98]. When exposed to hydrogen peroxide, major Trp oxidation products
UV light promotes the excitation of Trp to a triplet state where one are mostly the same products that are generated by singlet oxygen or
of two processes can occur. Either an electron is ejected from the indole hydroxyl radical action, namely NFK (6), KYN (7), 5-hydroxyTrp (2)
(Fig. 1), Oia (23), diOia (24) and PIC (25) (Fig. 2) [68,106]. Kell et al.
701
also identified several additional compounds formed from hydrogen Biologic and atmospheric oxidants, such as hypochlorous acid,
peroxide treatment. KNA (8) and 3-hydroxykynurenine (9) (Fig. 1) ozone, nitric oxides, and sulfur oxides, are likely to be present at much
were detected at all pHs tested, while a range of other products were lower concentrations in such solutions, if at all. However, they have
only discernible at higher pHs or temperatures, such as xanthurenic been routinely investigated for their effects on Trp degradation, espe
acid, 3-indolylacetic acid (16), tryptamine (35), indole (18), amino cially in proteins, as they are damaging to human health. Biologic
benzoic acid (possibly anthranilic acid (19), but the isomer was not oxidants are often products of enzymatic reactions, while atmospheric
specified) and the amino acids Gly, Ala, Ser, and Asp [106]. Simat et al. oxidants are commonly produced by combustion reactions, such as
however, explicitly noted a lack of most of these oxidation products, or powerplants, manufacturing or in vehicle exhaust [107–109].
at least a level so low that it was below their level of detection (xan Hypochlorous acid (HOCl) is generated in biological systems by the
thurenic acid, aminobenzoic acid and the AAs were not tested). Several enzyme myeloperoxidase, a heme enzyme released by activated neu
hydrogen peroxide-induced degradation products exhibited further trophils [110]. While HOCl has been shown to react with Trp residues,
degradation pathways. All three of PIC, Oia and diOia produced KYN, the major reactivity is with sulfur-containing amino acids, and HOCl
while PIC and Oia also yielded NFK and diOia, respectively [68]. Simat even reacts preferentially with the backbone alpha-amino group in
et al. noted that some of these products are generated more in mild peptides or the side chains of other amino-containing AAs (e.g. Lys, His)
oxidizing conditions, while stronger oxidizing conditions degrade these [111,112]. Absorbance measurements of the oxidation products by
compounds and enhance the production of others, indicating potential Aspee et al. suggest the formation of NFK (6) and KYN (7) from hy
specific pathways of degradation and levels of compound stability. pochlorous acid-induced degradation of Trp [113], while other studies
using derivatives of Trp (e.g. melatonin, indole, 5-methoxyindole) or
Trp-containing peptides claim that hypochlorous acid action on indole
2.1.4. Biologic and atmospheric oxidants
compounds yields predominantly 2-oxo products (similar to oxindole
The oxidants that have already been covered here (singlet oxygen,
compounds 23 and 114) [114–116]. Dellegar et al. showed that for
oxygen-centered radicals, UV light, hydrogen peroxide) are likely the
indole compounds lacking an alkyl group at C3, chlorinated products
most common oxidants that will be found in any complex chemical
are also obtained (e.g. 5-methoxy-3-chloroindole (54), Fig. 5), and for
solutions containing Trp, such as nutritional and pharmaceutical pro
those with a C3-alkyl group, doubly oxidized species are also detected,
ducts. These originate from the storage conditions (light, heat) and
but no identification of these compounds was made [115].
from other chemicals commonly present in such solutions (dissolved
Ozone is a key molecule of atmospheric pollution, produced from
oxygen, unsaturated hydrocarbons that can form alkylperoxides, pho
combustion sources [109], and is generated in biological systems by
tosensitizers, transition metals).
Fig. 5. Degradation products of Trp or Trp derivatives, as generated by the biological and atmospheric oxidants hypochlorous acid, nitrogen oxides and sulfur oxides.
The starting compound which the oxidizing conditions were applied to is written in brackets above the degradation product.
702
antibody catalysis or neutrophils [117]. While it has high reactivity, it valuable nitrogen sources for biological organisms (nitrite, nitrate),
has an extremely short half-life in aqueous solutions (20–30 min), some have other biological uses (nitric oxide is produced by nitric oxide
where it decomposes into molecular oxygen. At higher pH values, it synthase and is a signaling molecule [124–126]), while others are
forms hydroxyl radicals, therefore oxidative degradation of organic considered to be primarily damaging reactive nitrogen species (RNS)
species in alkaline solutions is likely due to hydroxyl radicals rather arising from pollution (nitrogen dioxide) or reactions in biological
than ozone [118]. Due to its high reactivity, short half-life, and harm systems (peroxynitrite is formed in the mitochondria from spontaneous
less byproducts of decomposition, ozone is commonly used to sanitize reaction of nitric oxide with superoxide [127]). Many enzymes exist to
drinking water and food [118–120]. Studies into the ozone-induced convert between these forms, utilizing transition metal-based catalytic
degradation of Trp showed that the major byproducts are NFK and KYN sites [128]. Some interconversions are possible with non-enzymatic
for both the free AA and peptide-bound Trp [121–123]. Mudd et al. transition metal complexes, such as the reduction of nitrite to nitric
demonstrated that the reaction is extremely fast, and also identified oxide [128], which may be important for complex solutions containing
some minor byproducts, namely ammonia and “ninhydrin positive nitrite anions and transition metals, however such complexes are often
materials”, which are then presumably primary amines (such as other only catalytic with very specific, uncommon ligands.
amino acids), although their identities were not determined [123]. Suzuki et al. studied the reaction of N-acetylTrp with various RNS.
Nitrogen oxides are a group of species including nitric oxide (NO), Reaction with peroxynitrite produced N-Ac-6-NO2Trp (nitro compound
nitrogen dioxide (NO2), nitrous oxide (N2O), nitrite (NO2−), nitrate 55), N-Ac-1-NOTrp (nitroso compound 56), N-AcNFK (57) and (tenta
(NO3−) and peroxynitrous acid (ONOOH). Some of these species are tive identification) N-Ac-1-NO2Trp (58) (Fig. 5) [129]. Reaction in
Fig. 6. Main degradation products of kynurenine following irradiation (hν) and heat (Δ).
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nitric oxide-generating conditions produced the same nitroso and NFK under the name of its enol tautomer, 4-hydroxyquinoline (75), even
products (56 and 57), but not the nitro products, whereas application of though the keto form is the predominant species in solution [148].
a system that purportedly generates a nitrogen dioxide radical [130] Kynurenine yellow can react further, undergoing either oxidative dec
did the reverse, producing the two nitro species (55 and 58), but no arboxylation to also afford 4-quinolone [147], oxidation to KNA (8)
NFK or nitroso species. The 1-NOTrp product 56 was found to be highly [149] or, under anaerobic conditions, photolytic reduction to dihy
unstable, easily undergoing reduction back to the original N-AcTrp, and droquinolone 76 [148]. The KYN deamination product, the α,β-un
1-NO2Trp 58 was also unstable, but decomposed into a mixture of saturated carbonyl 71, can also be chemically reduced (e.g. with
starting material and the previously established oxidation products. NADH) to 4-(2-aminophenyl)-4-oxobutanoic acid (77) [150] or un
Applying the same conditions to a protein also yielded predominantly dergo an aza-Michael addition with KYN to afford the deaminated KYN-
6-NO2Trp residues. Other studies investigating the products of perox KYN adduct 78 [150]. KNA can further degrade under anaerobic pho
ynitrous acid-induced degradation of Trp also found 5- and 6-nitro Trp tolysis conditions in the presence of a photosensitizer (such as N-acet
(60 and 61, respectively) [131,132] as the main degradation products ylkynurenine) to yield 1,4-dihydroquinoline-2-carboxylic acid (79)
(Fig. 5). One study using the myeloperoxidase-hydrogen peroxide-ni [151]. UV-induced dimerization of KNA is also possible, forming
trite system to generate the nitrogen dioxide radical identified 4-, 5- compounds such as diol 80 and the C4-linked dimer 81 [151]. The
and 6-nitroTrp products (compounds 59, 60, 61; Fig. 5) [133]. Another majority of these thermal and photolytic degradation reactions were
study investigating the reaction of N-acetyltryptophanamide with a often shown to be oxygen independent, or even inhibited by the pre
wide variety of RNS provided evidence of nitration at the 7-position of sence of oxygen due to quenching of photoinduced excited states by
these Trp-like compounds (compound 62; Fig. 5), along with typical molecular oxygen. It is therefore uncertain whether these same species
nitration seen in other systems (i.e. at the 4- and 6- positions) [134]. would be generated in a system containing a high amount of ROS, or
The complexities of these RNS reactions with tryptophan has been whether other degradation pathways and products would predominate.
summarized in an earlier review [135]. The effects of nitric oxide and
peroxynitrite are especially important in the food industry, where nitric 2.2. Trp degradation mediated by photosensitizers
oxides and nitrated products/proteins can either be considered detri
mental for altering the color of meat products through reactions with While type II photosensitization reactions are mediated by singlet
key proteins, or favorable for antioxidant and antibacterial functions oxygen, type I reactions are defined by the direct reaction between the
[136–139]. excited sensitizer and the substrate, which occurs following irradiation,
The study of oxidation of proteins and amino acids by sulfur oxides and can take place in presence or absence of oxygen. Many of the direct
is in part important due to the role of sulfur dioxide as a key atmo degradation products of Trp, such as KYN (7), NFK (6) and KNA (8)
spheric pollutant [109], but is also of key value to the wine industry, (Fig. 1), are very efficient photosensitizers. As such, should any of these
where wines often have sulfites (sulfur dioxide is interchangeable with compounds catalyze the photosensitized decomposition of Trp to gen
bisulfite in aqueous solutions [140]) added as a preservative, where erate more of these same compounds, an autocatalytic system of Trp-
they remain in the solution for months during fermentation and sto degradation is initiated [148,152]. The essential vitamin riboflavin is
rage. In multiple studies looking for decomposition markers of wine also a well-known photosensitizer, and has been shown to undergo type
following storage, multiple sulfonated indole species and sulfonated Trp I photochemical reactions with Trp [153], forming the riboflavin ra
metabolites (compounds 63, 64, 65 and 66; Fig. 5) were detected, dical anion and the Trp radical cation. In aerobic conditions, the pho
presumably the result of the added sulfur dioxide [141,142]. Experi tosensitizing action of riboflavin can promote the oxidative degradation
mental work demonstrated that sulfonated compounds 63–65 can be of Trp via both type I and type II photosensitized oxidation [154]. The
generated by a mild reaction of the respective indole with a sodium reaction rate of these photosensitization reactions is known to increase
bisulfite solution. This is concordant with a study that followed the with increasing temperature, oxygen concentration, and pH
kinetics of Trp decomposition in the presence of sulfur dioxide [140]. In [153,155,156]. Under anaerobic conditions, the riboflavin radical
has also been indicated that oxidation of tryptophan by sulfite species is anion and Trp radical cation can yield multi- and polymeric structures
enhanced via radical mechanisms initiated by UV light, transition me such as Trp dimers and trimers, or riboflavin aggregates [153]. The Trp-
tals or enzymic reactions, which start with the aerobic oxidation of riboflavin covalently bonded photoproduct 82 (Fig. 7) was proposed to
sulfite to sulfite radicals [143]. In biology, it has been proposed that be formed between the N-5 of the flavin and the C-3 of the Trp indole
oxidative stress can lead to the formation of reactive sulfur species moiety [156–158]. Riboflavin is known to decompose as a result of
(RSS), such as disulfide-S-oxides and sulfenic acids [144]. Other species light absorption [159], and photodegradation products such as lumi
which may be enzymatically generated and harmful to protein residues flavin have been confirmed to be very efficient type I sensitizers for
are the sulfite, peroxymonosulfate, and sulfate radical anions [145], indoles [160], so even the photodegradation of riboflavin may promote
produced by the oxidation of bisulfite by peroxidases [146]. While one oxidative degradation of Trp. Riboflavin was also shown to be involved
study showed that Trp is degraded by a peroxidase/sulfite system in the photosensitization of Trp oxidation products, such as indole
[143], to date there are no established oxidative degradation products acetic acid (16), thus potentiating degradation reactions [161].
of Trp as a result of this serie of sulfur oxides.
2.3. Temperature and pH
2.1.5. Kynurenine decomposition
ROS can mediate the direct oxidative degradation of Trp but may In the absence of oxygen, Trp is not sensitive to temperatures up to
also target Trp degradation products, in particular KYN (7). Indeed, the 140 °C. At temperatures above 140 °C, decarboxylation and oxidative
decomposition of KYN under a variety of conditions has been ex deamination of Trp occur, forming tryptamine (35), indole-3-pyruvic
tensively investigated (Fig. 6). Heat treatment cleaves the compound in acid (14) and ammonia (Fig. 8). Tryptamine can degrade further, being
two on either side of the ketone moiety, producing either aniline (67) cleaved at one of the three bonds in the alkyl chain to form three
and 2-amino-4-oxobutanoic acid (68) or 2-aminobenzaldehyde (69) possible product pairs: 3-ethylindole (83) and ammonia, skatole (84)
and Ala (70) [24]. Both thermal and UV radiation cause a cascade of and methylamine (85), or indole (18) and ethylamine (86) [162]. The
reactions, starting with oxidizing KYN, with concomitant deamination two thermal degradation products tryptamine and indole-3-pyruvic
or decarboxylation, to afford the unsaturated acid 71 and the un acid can also condense to form the 1-(3-methyleneindole)-tetHβC 87.
saturated amine 72, respectively [147]. Both of these oxidized species The exposure of Trp to heat has also been shown to yield minimally
are unstable in these reaction conditions and cyclize to yield kynur soluble brown products that have not been characterized [24]. At
enine yellow (73) and 4-quinolone (74), which is sometimes referred to higher temperatures (150–185 °C) and particularly in strongly alkaline
704
stability of the complex depends on nature of the metal and was shown
to rise with increasing temperatures. The binding strength of transition
metal cations to Trp was classified as follows: Co(II) ~ Zn(II) < Pb
(II) < Ni(II) < Cu(II) < Fe(III) [171]. Other studies evaluated the
binding of Trp to alkali and alkaline earth metals [169]. Results showed
that the binding strength of these metals complexed with Trp increased
in the order K(I) < Na(I) < Ca(II) < Mg(II), suggesting that the
valency and the atomic radius of the metal cations are critical factors
determining the binding strength. As for many complexes, binding of
metal cations to Trp was shown to decrease the fluorescence of the AA
and change the color of the aqueous solution [172,173].
2.5. Interaction of Trp with carbonyls or aldehydes
One of the most common reactions of tryptophan with organic

compounds is the condensation reaction with aldehydes/ketones to
form tetrahydro-beta-carbolines (tetHβCs) (Fig. 9), known as the Pictet-
Spengler reaction [174,175]. The transformation is usually acid-cata
lyzed when applied in organic synthesis, but can also occur in much
milder conditions, favored at low pH and high temperatures [176–178].
The condensation can also be effected when solid components are
mixed together, such as the formation of tetHβC 91 (Fig. 10) from
Fig. 7. Trp-riboflavin photoproduct between N5 of the flavin and C3 of the
tryptophan and glucose, which occurred when the two compounds
indole.
were part of a powder cell culture medium formulation stored at ele
vated temperatures (i.e. RT or 37 °C) [37]. Following the formation of
conditions, partial or complete racemization of Trp has been observed. tetHβCs from the Pictet-Spengler reaction, oxidation can take place in
At temperatures above 200 °C, α-carbolines (88) and γ-carbolines (89) the presence of even a mild oxidant (e.g. atmospheric oxygen), forming
were detected, but in much lower quantities than β-carbolines (βC) (90) beta-carbolines (βCs) (Fig. 9) [179]. While the formation of dihydro-β-
[24]. carbolines (diHβCs) is also probable, they appear to be highly unstable
Trp also proved to be an unstable compound in solutions with and were never isolated in conditions that oxidize the tetHβCs
particularly high (> 9) or low (< 2) pHs [63]. A popular method of [179,180]. Alternative synthetic routes can produce diHβCs, and oxi
measuring the individual AA content of peptides and proteins involves dation to the fully aromatic βCs was found to take place easily in the
HCl-based hydrolysis of the peptide bonds, but these conditions were presence of atmospheric oxygen and base, via the formation of a car
shown to completely degrade the Trp residues, and thus cannot be used banion [181,182]. To form these oxidation products, one of the R
for quantification of Trp [24]. However, such acidic degradation may groups from the original carbonyl compound must also be lost (Fig. 9).
be highly condition specific and not merely a result of pH, as methane This oxidation is very common with an aldehyde precursor (R2 = H),
sulfonic acid was demonstrated to be a suitable reagent for acidic hy and also takes place easily for an alpha-keto acid precursor
drolysis of peptide bonds, without damaging the Trp residues [163]. In (R2 = CO2H), owing to the ease of oxidative decarboxylation
alkaline conditions, the presence of oxygen in solution and the presence [177,179,183,184].
of metal contaminants in the base were shown to be the main factors There has been a significant interest in the food industry in de
leading to free tryptophan degradation [164]. In oxidizing conditions tecting and identifying many of these types of βC in various foods and
known to degrade Trp, an increase in heat or pH has been found to drinks such as fruit and vegetables [185], fermented foods and bev
enhance degradation, as was shown for hydrogen peroxide-induced erages [69] or food products that have been subjected to high tem
oxidation [68,106]. perature such as roasted coffee and grilled meat or fish [186]. The βCs
are often identified via comparison with standards, which are easily
2.4. Metal ion catalyzed oxidation and interaction of Trp with metals chemically synthesized from Trp and the relevant carbonyl. Examples
of some tetHβCs that have been identified include those with the car
Metals are well-known catalyzers of oxidation reactions through the bonyl precursors of ascorbic acid (92) [187,188], pyridoxal (93) [188],
formation of hydroxyl radicals by Fenton and Fenton-like reactions, but formaldehyde (26), acetaldehyde (27) [69,189], pyruvic acid (94) and
can also catalyze reactions in the absence of hydrogen peroxide [165]. glyoxylic acid (95) [176,190] (Fig. 10). As tryptophan can easily de
As an example, Zn and Cu(II) catalyze the decarboxylation of Trp, grade into tryptamine, a metabolic intermediate in many organisms, it
producing tryptamine (35), or indole (18), pyruvic acid and ammonia is unsurprising that the tetHβCs identified in foods often include con
[166,167]. While Fe(II)/(III) can catalyze the oxidation of Trp in pure densation products between tryptamine and the corresponding car
solutions, Mn(II) was shown to oxidize Trp exclusively in the presence bonyl (e.g. compounds 96 from acetaldehyde and 97 from for
of sulfite ions, which are oxidized to sulfate via a free radical chain maldehyde [69] (Fig. 10)). However, these products could also be
mechanism involving the superoxide radical anion [168]. generated by reductive decarboxylation of the tetHβC-3-carboxylic acid
While metal cations in solution typically react with molecular formed directly from Trp. In the highly oxidative conditions afforded in
oxygen or ROS to induce Trp oxidation, metal cations can also be food preparation, this pathway seems unlikely, and this is supported by
complexed by Trp. Many experimental and theoretical studies analyzed the finding that tetHβCs lacking the 3-carboxylic acid substituent were
the complexation of tryptophan with metal cations in the gas phase, found in foods which exhibited a high level of tryptamine, and not
however only few works investigated the interactions in the solvated found in those with little to no tryptamine content [69].
state [169]. In solution, calculations predict two theoretical bidentate Generally, the formation of tetrahydro-β-carboline-3-carboxylic
Trp-metal complex structures: (a) the neutral AA with the metal cation acid compounds from tryptophan and a carbonyl produces both pos
bound to the nitrogen of the amine and the oxygen of the carbonyl sible diastereomers, often with one in excess, though there is no specific
group, and (b) the zwitterionic form with the metal cation bound to the trend and this is dependent on the starting carbonyl. It has been shown
carboxylic acid, which is the most favored structure [169–171]. The that for some carbonyls, one of the resultant diastereomers is
705
Fig. 8. Tryptophan degradation products that are formed after exclusive exposure to heat.
significantly more unstable than the other, or the two diastereomers polymerization, as there was a larger consumption of pyruvic acid
have very different physical properties. One study reported on the compared to Trp in the reaction. Another interesting example of βC
product of pyruvic acid and Trp, the tetHβC 94, claiming that one formation in very mild conditions was shown for the reaction of Trp
diastereomeric product was an unstable white product, while the other with methylglyoxal. In conditions of limited oxygen availability (dilute
diastereomeric product was yellow, stable and more water-soluble HCl, closed vial, room temperature, 10 days), the β-carboline-1,3-di
[176]. In contradiction to this, Gutsche et al. also produced the two carboxylic acid 99 was detected, while similar conditions with a shorter
diastereomers of 94, but did not mention any differences in stability or reaction time and stirring open to the atmosphere produced the dec
physical properties [190]. The same authors did experience handling arboxylated βC 100 [179] (Fig. 10). β-Carbolines norharmane (90) and
differences in the two diastereomers of tetHβC 95, the product of Trp harmane (98) were identified in numerous food products
and glyoxylic acid condensation, in which the major diastereomer [69,185,186,189] and an experiment which involved spiking fish
precipitated out of the aqueous acidic solution, and the other decom samples with the corresponding tetHβC-3-carboxylic acids 26 and 27
posed upon attempted isolation in both lyophilization and preparative and then grilling them, showed that the levels of the respective βCs
HPLC [190]. For anybody looking to detect or isolate these tetHβCs, following grilling were much higher than the controls, suggesting that
potential differences in physical properties and stability between the the tetHβCs were indeed converted to the βCs upon cooking [69].
diastereomeric products may be something to keep in mind. An interesting exception to the straightforward Pictet-Spengler re
As mentioned previously, oxidation of tetHβCs is possible in even actions of carbonyls was found in the reaction of tryptophan and 2-
mild oxidizing conditions, and the fully oxidized βC products can also oxoglutaric acid (α-ketoglutaric acid). The reaction did not produce the
be found in conditions that produce the tetHβC Pictet-Spengler pro expected tetHβC, but rather a decarboxylation product thereof (tetHβC
ducts. One example of this was in the case where the fully oxidized βC 101) [190], which was also discovered in some foodstuffs, and may be
harmane (98) was detected in an aqueous solution of methyl ester easily prepared by a reaction of succinic semialdehyde with Trp [190].
tryptophan and pyruvic acid, which had been left standing at room Previous investigations into the reactions between Trp and 2-ox
temperature overnight [176] (Fig. 10). This study also detected an oglutaric acid were also unable to isolate the Pictet-Spengler con
unidentifiable yellow product, presumed to be the result of pyruvic acid densation product [191]. This study isolated five colored compounds
706
Fig. 9. Pathway for β-carboline formation from reaction of tryptophan with aldehydes and ketones, including hypothesized dihydro-β-carboline intermediates. The
precise sequence for the transformations (i.e. condensation, oxidation, decarboxylation) is not definitive, and the schematic serves only as a conceptual example.
(blue, green, and yellow-brown), but was unable to identify them. As ribose [195]) and hexoses (e.g. glucose [177,196,197], fructose [198])
with the pyruvic acid experiments (by the same group, see above), and reaction products have been linked with browning and detrimental
excess 2-oxoglutaric acid was consumed, leading to the assumption that nutritional effects in sterilized or dehydrated milk [164]. The reaction
at least one of the products was the result of polymerization of the of Trp with glucose initially forms the Amadori product 104, and pro
ketone. However, the Pictet-Spengler product of 2-oxoglutaric acid longed or more intense heating of the mixture produces tetHβC 91, and
condensation with Trp, tetHβC 102, is known (Fig. 10). The compound interestingly, 1-acetyl-βC 100 [177]. Curiously, heating of a pure so
was isolated from Kitasatospora setae bacteria, along with the βC 103, lution of tetHβC 91 either at temperatures above 120 °C or in presence
the decarboxylated and oxidized form of the previously identified 2- of oxidants, such as oxygen or dehydroascorbic acid, did not produce
oxoglutaric acid + Trp product, tetHβC 101 [192]. The discoverers of the previously identified 1-acetyl-βC 100, but rather the oxidative
the tetHβC 102 named the compound kitasetelic acid and demonstrated decarboxylation product, pentanol-βC 105 [177].
that the compound is likely derived from tryptophan, as external In the presence of catalytic ferrous or copper sulfate, the βC nor
tryptophan supplementation was shown to increase its production. harmane (90) was produced from Trp and glucose at the low tem
Supplementation of fluorinated Trp analogues into bacterial culture perature of 40 °C [199]. More forcing reaction conditions (80 °C, pH
provided the corresponding fluorinated derivatives of both 102 and 1.0, 12 days) produced the βCs 106–109 [200]. These βCs were de
103. Rather than being formed spontaneously in situ, the authors pro tected in food products such as balsamic vinegar, pineapple or plum
pose that the Ks1B enzyme catalyzes the condensation of 2-oxoglutaric juice, or soy sauce [196] and the βCs 106 and 107 received increased
acid and tryptophan, and indeed Picter-Spenglerases are known [174]. attention recently because of their detection in human urine after to
To date no chemical synthesis of kitasetelic acid (102) has been re mato juice consumption [200]. At very acidic pHs, the mixture of
ported, although Ueda et al. demonstrated that the compound was glucose and Trp may also generate Trp-glycoside conjugates such as
stable enough to be isolated, characterized, and tested in microbial and 110 and 111, which may be formed through radical reactions [201].
cell-based assays [192]. Similar reactions products were obtained for other carbohydrates
Another aldehyde class which has been demonstrated to condense [193].
with Trp are carbohydrates (Fig. 10). However, due to the structural
features of the carbohydrates, they are prone to undergo the Maillard 3. Impact of Trp degradation products on cellular metabolism
reaction upon condensation with the primary amine in Trp, which is not
a single reaction, but rather a series of mostly undefined reactions Since Trp is readily degraded in conditions relevant for food pro
leading to undefined products which are largely responsible for the ducts or cell culture media used to produce recombinant therapeutic
browning and aromas in cooked foods. Reactions of Trp with carbo proteins, the study of the impact of Trp degradation products on cel
hydrates have been noted for both pentoses (e.g. xylose [193,194], lular metabolism is of particular interest. In 1972, Yoakum et al.
707
Fig. 10. Products generated upon reaction between Trp and carbonyl-containing compounds. (A) Tetrahydro-β-carbolines formed from tryptophan and specified
carbonyls; (B) Tetrahydro-β-carbolines formed from tryptamine and specified carbonyls; (C) Oxidized β-carbolines formed from specified carbonyls; (D) β-Carbolines
formed from 2-oxoglutaric acid and tryptophan; (E) Products formed from tryptophan and glucose.
demonstrated that irradiated Trp was toxic to mutants of salmonella process led to the identification of Trp as a root cause for impaired cell
typhimurium, but disproved the known Trp degradation products and growth [37]. To understand which molecules were responsible for the
metabolites KYN (7), serotonin (3), quinolinic acid (12) and oxindole toxic effect, the failed media lot was investigated using a LC-MS-based
(112; Fig. 11) as a source of toxicity [202]. The release of H2O2 from analytical strategy. Principle component analysis indicated a significant
the photosensitization of other AA by NFK (6) was proven, at least increase in NFK (6), a 2-fold increase in 5-hydroxyTrp (2) and a 6-fold
partially, to be involved in the toxic effect [203]. A study investigating increase in known riboflavin degradation product, lumichrome [159],
the lethal effect of near-UV irradiated Dulbecco's Modified Eagle's when the medium was exposed to light. The authors proposed that the
medium (DMEM) in human D98/AH2, mouse 3T6 and Chinese hamster
V79 cells showed that only the combinations of riboflavin and Trp or
riboflavin and Tyr was critical to induce cell death [204]. In particular,
the Trp-riboflavin adduct 82 (Fig. 7) was shown to inhibit the growth
and adhesive capability of cultured F9 teratocarcinoma cells. Cultured
cells formed aggregates and were impaired in their capacity to adopt
their usual shape. A cytotoxic effect, manifested as necrosis and de
velopment arrest, was observed in embryos in the presence of this ri
boflavin-Trp adduct [205].
In the biopharmaceutical industry, the investigation of a mal Fig. 11. Further Trp degradation products that are known for their specific
functioning CCM lot used in a monoclonal antibody manufacturing biological effect.
708
detrimental effects were the result of light exposure during solution their widespread presence in food products. While α- and γ-carbolines
preparation and storage which led to riboflavin-sensitized photo (Trp pyrolysis products, see section 2.3) have been clearly linked to
oxidation of tryptophan. A subsequent study sought to determine which mutagenic and carcinogenic activities in many cellular systems in
exact change produced the toxic effect – the loss of one media com cluding mammalian cells [212–214], βCs are considered less toxic and
ponent or the production of new toxic products [38]. Several known non-mutagenic. Indeed, harmane (98) and norharmane (90), two βCs
Trp degradation products (NFK (6), KYN (7), 5-hydroxyTrp (2), and 3- found in heat processed food, have been shown to act as a mutagens
hydroxykynurenine (9)) were supplemented into cell culture to eval exclusively in the presence of aromatic amines such as aniline or o-
uate their toxicity. NFK and to a lesser extent 3-hydroxykynurenine toluidine [199,215] and are thus called co-mutagenic substances. Si
reduced the growth rate of recombinant human embryonic kidney cells milarly, the mutagenic effects of βCs formed by the reaction between
[38], but 3-hydroxykynurenine was discounted as the source of toxicity Trp and carbohydrates or other aldehydes such as methylglyoxal were
as it was confirmed to be absent in the degraded medium, so ultimately found to be present exclusively after nitrite treatment [216].
NFK was deemed to be one of the major root causes of toxicity. Another Other cellular effects of βCs do not exhibit any connections or
interesting Trp degradation product investigated in these studies was patterns. The tetHβC 91, formed from glucose and Trp, was shown to
tetHβC 91 (Fig. 10), the product of the Pictet-Spengler chemical con inhibit muscle protein synthesis in an in vitro culture of chicken embryo
densation between Trp and glucose, which was formed when the myoblasts [217], while a product of the Maillard reaction between
powdered medium was stored at elevated temperatures (RT or 37 °C) fructose and tryptophan was shown to present a strong antiproliferative
[37]. This molecule was degraded over time in the failed lot that was effect on human gastric cancer cell lines [198]. In disease models, some
exposed to light following hydration and thus was hypothesized to be βCs have been studied for their neurotoxic properties [218] and their
required for cellular performance. However, spiking studies indicated ability to bind to serotonin, dopamine and benzodiazepine receptors
that 91 did not restore the cell culture performance of the light irra [219], but most of the βCs that showed a strong activity were sub
diated medium [38]. Another compound with a mass of 359.1 stituted (e.g. methylated) and thus formed only by specific enzymes of
(C21H17N3O3) was found in significantly larger quantities (50-fold) in the brain. Some βCs have also been established as efficient inhibitors of
the failed CCM lot exposed to light. The molecule was not identified but monoamine oxidase and monoamine uptake, but here as well methy
was thought to result from the interaction between riboflavin and Trp lated βCs or diHβCs were found to have a higher potency [220].
and was proposed as a marker defining medium expiry and thus could Finally, some tetHβCs such as 26 or 27 might exert actions as an
be used to assess overall CCM quality [206]. tioxidants and free radical scavengers [221,222] and some synergetic
Kynurenine (7), a metabolic and degradation product of Trp, was effects have been reported with some common antioxidants such as α-
shown to induce apoptosis in a human Natural Killer (NK) cell line. The tocopherol [223]. Interestingly, one study investigated the anti
treatment with antioxidants, such as N-acetylcysteine, inhibited KYN- oxidative effect of solutions formed by the interaction of Trp with
induced apoptosis, suggesting that the cell death occurs primarily carbonyls and aldehydes [187] and their results indicated that the an
through a ROS-mediated pathway [207]. Kynurenine (7), 3-hydro tioxidative capacities of the solutions composed of βCs correlated with
xykynurenine (9), 3-hydroxyanthranilic acid (10) and picolinic acid the browning of the solution. Whereas the mixture of Trp with carbo
(11) (Fig. 1) were shown to inhibit antigen-specific T cell proliferation nyls such as glyceraldehyde, glycolaldehyde, glyoxal, methylglyoxal
and to exert a cytotoxic action on CD3+ T cells, B cells and NK cells and dehydroascorbic acid showed antioxidative capacities and a brown
[207]. For these cell types, the inhibitory effect was mediated by color, the mixtures with other aldehydes, namely glucose, xylose and
blockage of the cell cycle progression in G1 phase in cells undergoing acetoin, showed neither color nor antioxidant activity [187]. This
activation [208]. clearly demonstrate that βCs may have multiple beneficial or detri
Aqueous solutions of Trp, allowed to photodegrade by the actions of mental functions in cells and thus that each carboline should be eval
laboratory fluorescent lighting or sunlight through a window, displayed uated individually.
the ability to induce phase I mono-oxygenase cytochrome P450 1a1 A holistic approach to the study of oxidative degradation in nutri
(CYP1a1), an enzyme involved in drug metabolism and synthesis of tion was recently reported, regarding the importance of oxidative de
cholesterol, steroids and other lipids [206,209]. One of the most potent gradation in infant formula. Two papers from the same team comprised
photodegradation products in the solution was 6-formylindolo[3,2-b] a complete study of the formula oxidation effects, first undertaking an
carbazole (FICZ) (113; Fig. 11) [210], although it was clearly not the elaborate characterization of the degradation products of the formulas,
only photodegradation product of Trp that induced this effect, and in and then investigating the cellular and metabolic effects of select de
fact the photodegraded Trp solution contained over 100 photooxidation gradation products [224,225]. Of the several Trp degradation products
products and over 30 inducing compounds, however these were not they identified (NFK (6), KYN (7), 3-hydroxykynurenine (9), protein
characterized [209]. The induction of CYP1a1 is mediated by binding of Trp crosslinks), they performed cellular studies on KYN and 3-hydro
these compounds to the aryl hydrocarbon receptor (AhR), which in xykynurenine, both individually and as a part of a mixture of five
itiates a cascade leading to upregulated transcription of CYP1a1. Other oxidative degradation products (the remaining arising from Tyr and
Trp degradation products such as KYN, tryptamine, indole-3-acetic acid Met). Both compounds as individuals had a detrimental effect on the
or KNA have also been reported as direct agonists or precursors to metabolic activity of 3T3-L1 cells and yet increased the metabolic ac
agonists of the AhR [209,211]. FICZ (113) was shown to be produced tivity of Caco-2 cells. Most interestingly, when detrimental cellular ef
by photodegradation of Trp in DMEM, and induction of CYP1a1 was fects displayed by the mix of compounds were evident, no similar ef
mediated by very low concentration of FICZ (around 8 pM) in a rat fects were seen for any individual compound of the set. This
hepatoma cell line, corresponding to only 0.0001% of the tryptophan demonstrates that when a negative cellular effect is seen by a mixture of
from the DMEM [206]. The same authors showed that FICZ was stable oxidative degradation products, identifying what is causing this effect
in stored medium, which has implications for CCM handling, in that at an individual compound level is certainly not trivial.
photodegradation products of Trp can arise during short light exposure, To summarize, many Trp degradation products have been linked to
during production and transport for example, but will not decompose specific biological effects in very different systems/setups. However, a
during storage, and will thus still be present during cell culture. As comprehensive characterization of the effect of Trp degradation pro
CYP1a1 is involved in cell regulatory processes such as proliferation ducts on cellular metabolism is currently missing. To be relevant, such a
and differentiation, cellular oxidation/antioxidation response or im study would have to consider that some degradations products might be
mune regulation [211], the control of its activity by Trp degradation formed in very low quantities and might not induce significant effects.
products might be extremely relevant for many cellular systems. Nevertheless, the example of FICZ, which is highly potent in the pM
Many studies sought to investigate the toxicity of carbolines due to range, indicate that such a study may generate knowledge relevant in
709
many fields, including nutrition or the understanding of physio-pa commonly detected using LC-MS, in this example by monitoring mass
thological conditions. shifts of +16, +32, +4, +32 or +20, respectively. Some studies have
also reported an oxidized (-2H) Trp residue 119 and a oxidized (-2H)
oxindolylalanine residue 120, leading to mass shifts of −2 and +14
4. Trp degradation in proteins respectively (structure and exact mass shift are presented in Fig. 12)
[227]. Trp side chain cleavage products have also been described in
Proteins are major targets for reactive oxygen species and many IgG1 exposed to light in the presence of oxygen. Trp residues were
excellent reviews summarize the current knowledge on oxidation pro reported to be converted into Gly 121, Gly hydroperoxide 122 and
ducts of the most susceptible AA residues (Trp, His, Met, Cys, and Tyr) aspartic acid 123 residues (Fig. 12) [228,229].
[31,80,226]. Similarly to free Trp, Trp protein residues are sensitive to The presence of Trp oxidation in proteins has been linked with a loss
oxidation via two-electron oxidants (e.g. HOCl, ONOOH, 1O2, O3), hy of potency and antigen-binding capabilities for antibodies or re
drogen peroxide, hydroxyl radicals or other free radicals leading to combinant proteins, in particular when oxidation occurred in the Fab.
multiple proteoforms. For example, the bioactivity was reduced by 40–50% for a subfraction
In the biopharmaceutical industry, numerous studies have shown of an IgG2 (purified using mixed mode chromatography) that contained
that recombinant proteins produced using mammalian cell culture oxidized Trp in the CDR region [230]. Modifications of Trp residues in
processes are susceptible to oxidative degradation following exposure recombinant proteins following exposure to oxidizing conditions such
to light, ROS, or metal ions. While Met is the AA residue that is most as light also proved to be responsible for a color change of the drug
preferentially oxidized in therapeutic antibodies (due to the low oc substance, which is a key critical quality attribute for the drug product
currence of free cysteine residues that are rather involved in disulfide [231]. An increase in yellow color correlated with the abundance of
bonds), Trp is reported as the second target for oxidation. The main NFK- and KYN-containing peptides. Between both oxidized forms, KYN-
modifications found in recombinant IgG, in e.g. forced degradation containing peptides showed an absorption at higher wavelengths than
studies, correspond to oxidation of Trp residues to oxindolylalanine NFK-containing peptides, supporting that KYN-containing peptides
114, dioxindolylalanine 115, KYN 116, NFK 117 or 3-hydroxyKYN 118 contribute more to the yellow/brown color than NFK-containing
residues (Fig. 12). In proteins, modified residues such as these are
Fig. 12. Structures resulting from oxidation of Trp residues in proteins and examples of crosslinks with amino acid residues or small molecules in solution. Values are
the exact mass differences that allow detection of the modifications using LC-MS.
710
peptides [207,231]. Further data indicate that the heavy chain and the protein aggregation, which is suggested as one of the root causes for
light chain of the antibody can be impacted differently by oxidation, diseases such as amyotrophic lateral sclerosis, a motor neuron disorder
most likely due to a difference in protein conformation and accessi [245–247]. Studies on identification and characterization of protein
bility. In another study dealing with the manufacturing process of a cross-links induced by oxidative reactions have been reviewed recently
fusion protein produced in Chinese Hamster Ovary (CHO) cells, an in [248].
crease in total Trp oxidation from 1.6% to 5.8% was sufficient to sig Most of the knowledge about Trp-mediated crosslinks has been
nificantly change the color of the drug substance from pale yellow to obtained through the study of proteins of the eye lens and the me
light brown [36]. chanisms which lead to cataracts. In addition to its role in focusing light
Several studies have investigated methods to reduce tryptophan onto the retina, the human lens has a significant role in the absorption
oxidation in recombinant proteins produced using mammalian cell of UV radiation to avoid photooxidative damage to the retina.
culture technologies. Since the composition of the CCM was demon Protection via UV absorption is mainly provided by protein-bound
strated to impact the oxidation of Trp directly, several composition tryptophan residues or by free tryptophan metabolites such as KYN, 3-
changes were proposed to reduce oxidation. Examples of medium hydroxykynurenine or 3-hydroxykynurenine glucosides. However,
changes include decreasing both cysteine and iron concentrations to during aging, the amount of these free Trp-derived UV filters decreases
reduce the occurrence of Fenton reactions, and increasing the con as they undergo deamination leading to α,β-unsaturated carbonyl
centration of Cu2+, Mn2+ or free Trp [35]. An increase in copper structures such as 71 (for kynurenine). Free nucleophiles present in the
concentration was linked to a decrease in intracellular oxidative reac lens, such as glutathione (GSH), can add to the electrophile 71 gen
tions via the modulation of genes involved in redox signaling [232]. In erating the GSH-KYN adduct 126 (Fig. 12) [249]. AA residues in the
another study, the mitigation strategy to reduce color formation in lens protein presenting a nucleophilic side chain, such as cysteine,
volved a decrease in iron and vitamin B12 concentrations, whereas an histidine and lysine, can also add to the oxidatively deaminated ky
increase in copper had no effect [36,233]. The addition of free me nurenine 71, leading to the covalent protein adducts 127, 128 and 129,
thionine within the final formulation of the antibody (or in formulation respectively (Fig. 12). These adducts display an increased photoactivity
buffer during forced degradation studies) was also effective at sig compared to KYN and thus are believed to promote the oxidative da
nificantly reducing the percentage of oxidized tryptophan residues, mage of the lens upon UV exposure [250].
most likely through scavenging of radicals [234,235]. Incubation of the This addition of KYN to lens proteins was reported to contribute to
protein with sodium borohydride, a reducing agent, also decreased the the increased yellow/brown pigmentation and fluorescence of the lens,
intensity of the color of the antibody product [36,207]. characteristic of cataracts. The exposure of these KYN-modified pro
Trp oxidation was also reported for endogenous mitochondria pro teins to UV light was reported to generate peroxides on Trp, Tyr and His
teins such as aconitase 2 [236], ATP synthase [237] or multiple redox residues, which led to an increase in Trp or Tyr related crosslinks. These
proteins and metalloproteins that are exposed to ROS [237]. In the crosslinks were reported as a root cause for the insoluble high mole
mitochondria, considered the major site of ROS generation (e.g. su cular weight aggregates found in patients suffering from cataracts
peroxide radical anions generated by electron leakage from the re [251]. Moreover, in the presence of UV light and the absence of oxygen,
spiratory chain), oxidation reactions are required as part of normal protein-bound KYN can react with ascorbate (an antioxidant present in
signaling cascades regulating physiological functions. However, they the lens), leading to a variety of compounds known to generate ad
can also lead to mitochondria dysfunction followed by cell death, em vanced glycation end products (AGE) on Lys and Arg residues of pro
phasizing the importance of the regulation of oxidation reactions (e.g. teins. These AGE adducts are believed to contribute to the pigmentation
via the presence of antioxidants). Interestingly, the site specific oxida of the human lens and thus contribute to the development of cataracts
tion of Trp residues to NFK in ATP synthase required binding of iron to [252]. While many studies have focused on KYN protein adducts, 3-
a metal binding site in close proximity to the AA residue, probably hydroxykynurenine adducts may also increase the potential for protein
facilitating Fenton-mediated oxidation reactions [238] and illustrating crosslinks via formation of quinonimines that can undergo successive
the crucial role of the environment in the regulation of those reactions. Michael addition reactions with Lys, His and Cys residues in the pre
Due to the irreversibility of tryptophan oxidation, these covalent sence of metals such as copper [253].
modifications may constitute a marker for the mitochondrial quality Alternatively, Trp residues of the lens protein may themselves be
control system and thus might contribute to the removal of damaged oxidized to NFK or KYN and since these forms are better photo
proteins [238]. sensitizers than Trp, they may, upon UV light exposure, lead to the
Whereas exposure to high level of superoxide radical anions (e.g. in generation of ROS, which exacerbate the UV-induced protein damage.
the mitochondria) was demonstrated to yield mainly hydroperoxides The specificity of the Trp oxidation was shown by Finley et al. to de
that are decomposed to NFK, KYN or alcohol/diol products [239], in pend on metal binding sites in the protein, supporting metal-catalyzed
anaerobic conditions, following exposure to peroxyl radicals or under oxidation [254]. Altogether, these data indicate that the oxidation of
conditions where the superoxide radical anion is absent or very tryptophan residues in proteins and the occurrence of Trp mediated
minimal, Trp residues can also form highly reactive tryptophanyl ra protein crosslinking contribute to the development of multiple patho
dicals that can react with other radicals leading to protein dimers or logical conditions.
higher molecular weight aggregates formed by crosslinking [240]. In
vitro models using Trp-containing peptides and pulse radiolysis have 5. Current stabilization methods for Trp
been used to study these radicals. Results have shown that tryptophanyl
radicals formed using UV laser pulses can oxidize other peptide residues As shown previously, oxidative degradation of tryptophan, either as
containing an aromatic side chain via hydrogen atom transfer, yielding a free AA in solution or as a protein residue, is often the result of en
Trp-Trp dimers (proposed structures for Trp-Trp dimers are the N1–C3 vironmental factors, and therefore may be minimized by careful control
linked dimer 124 and the C3–C3 linked dimer 125) [226,241] and Tyr- of the environment, i.e. minimizing oxygen exposure, light exposure,
Trp adducts [242,243] (Fig. 12). Trp-Trp crosslinks have been involved and high temperatures. This is already common practice in the food and
in the structure of protein via formation of hyperstable peptide β- drink industry, where packaging is carefully chosen to minimize the
hairpin and α-helix scaffolds [244]. In addition, tryptophanyl radicals degradative effects of oxygen and light (e.g. choice of colored glass over
may be oxidized further to KYN residues that can also form crosslinks. clear glass [255]). Minimization of UV-light induced degradation of
Both Trp-Trp and KYN-KYN crosslinks have been monitored in en food and beverages may also be achieved by the inclusion of a physical
dogenous proteins such as the copper/zinc superoxide dismutase quencher, as has been demonstrated for flavonoids [256]. Two other
(hSOD1), where they have been linked to protein misfolding and methods to protect against degradation of Trp in complex solutions will
711
be explored here in detail: the addition of antioxidants to formulations/ ascorbic acid was able to inhibit this process by quenching the triplet
systems containing Trp, and the utilization of functional derivatives of excited state of the photosensitizer [266]. Ascorbic acid reduced oxi
Trp in place of Trp. dation induced by the photosensitizer riboflavin in the same manner,
wherein the Trp photooxidation rate was decreased approximately 4-
5.1. Antioxidants to inhibit Trp degradation fold in the presence of the antioxidant [267].
When free Trp in solution or Trp residues in proteins have been
When seeking an antioxidant to inhibit oxidative degradation of a oxidized to a radical form, in which the free electron is delocalized
species of interest, some studies aim to identify the type ROS causing around the indole ring, it is possible for another compound (e.g. an
the oxidation, and to choose an antioxidant which has a high reactivity antioxidant) to reduce the radical back to the neutral state, effectively
with that exact ROS. However, reaction of the antioxidant with one “repairing” the Trp molecule. For this spontaneous redox reaction to
type of ROS may in fact generate a different type of ROS. Some ex occur, the reduction potential of the antioxidant needs to be lower than
amples include hexyl glycosides [258] and ascorbic acid [259] produ that of the radical which it is intended to repair. However, the pH of the
cing hydrogen peroxide from reaction with singlet oxygen, 5-hydro solution affects the reduction potential of Trp. At acidic pHs (< 4), the
xytryptophan or tyrosol and its derivatives reacting with molecular Trp radical is always the protonated cationic species, and the reduction
oxygen under radiation to form singlet oxygen [260,261], and ascorbic potential is independent of pH, whereas at higher pH values, there is
acid and phenols generating superoxide radicals in the presence of the possibility for the neutral Trp radical to also exist, and now the
molecular oxygen [262,263]. In these situations, the antioxidant is, reduction requires the protonation of the radical along with the re
somewhat confusingly, simultaneously also a prooxidant, i.e. a com duction to return to the neutral Trp species. Harriman et al. used cyclic
pound which promotes the formation of a particular ROS. voltammetry to determine the reduction potential of Trp vs. NHE
More complications in antioxidant investigations arise when the (normal hydrogen electrode) at varying pHs and found that it had a
system under investigation enables the conversion of one ROS into linear relationship to pH, varying from 0.65 at pH 13 up to 1.15 at pH 4
another. This is common in systems that contain photosensitizers or and below.
transition metals and is exceptionally problematic in systems where the Nonetheless, repair of Trp radicals has been demonstrated for many
target of oxidation itself or even the degradation products of the target organic compounds and kinetics and efficiency of reactions have been
species can cause ROS interconversion. Additionally, it is common that reviewed recently [268]. Both uric acid (E○ = 0.59 V vs NHE) and
multiple or even all ROS in a given system are capable of oxidizing the Trolox C (calculated 1e− reduction potential = 0.48 V at pH 7) [269]
species under investigation, but perhaps at different rates, and with were shown to repair tryptophan radicals (1e− reduction poten
different resultant oxidized forms. tial = 1.0 V at pH 7 vs NHE) [270–272]. Carotenoids such as can
There are commonly two metrics for measuring the strength of an thaxanthin and L-carotene were shown to repair Trp radical cations in
antioxidant - its ability to react with a particular ROS, and its ability to aqueous micellar solutions in the pH range 2–5.5 with the reaction rate
reduce the oxidation of the original ROS target. Due to the complexities increasing with decreasing pH [273], and flavonoids also enhanced the
mentioned above, the connection between the two is not always direct decay of neutral Trp radicals [274]. Glutathione was shown to inhibit
and is not always easy to establish in a multicomponent system, and all photosensitized oxidation in anaerobic solutions of α-crystallins in lens
of these aspects need to be considered when interpreting literature on proteins, and this was proposed to be the result of glutathione under
the topic. going 1e− reduction by radicals (including Trp radicals), and subse
Two compounds that were studied for their ability to inhibit mAb quently dimerizing to stop the radical chain reaction [266].
oxidation were hexyl glucoside and hexyl maltoside. Both glycosides Generally speaking, phenolic compounds seem to be the most
were found to reduce the light-induced oxidation of Trp residues in a popular antioxidants to inhibit Trp oxidation, being functional against a
monoclonal antibody, but their application for protecting the whole wide variety of ROS, and generally more reactive to these ROS than Trp
protein was limited, as they conversely increased the oxidation of Met itself.
residues [258]. Singlet oxygen-induced degradation of free Trp was
reduced in varying degrees by ascorbic acid, trolox and 2- and 3-hy 5.2. Trp derivatives
droxylphenylethyl alcohol, but curiously not by 4-hydroxylphenylethyl
alcohol [260]. Grewal et al. acknowledged that tryptophan itself may When reviewing the literature on the reactivity of tryptophan-like
be used to protect against oxidation of proteins, but as light-induced compounds, it is important to note that the vast majority of the scien
oxidation of Trp is prooxidative (generating hydrogen peroxide and tific literature in this area focuses on the potential role of these struc
singlet oxygen [80,203]), they conducted studies to find derivatives of tures as antioxidants, i.e. these works aim to identify structures which
Trp which would potentially be more suitable candidates to inhibit are more reactive than tryptophan. In addition, the antioxidant-focused
protein oxidation in a recombinant mAb [235]. Hydroxyindole deri literature rarely investigates the mechanism of oxidation or the typical
vatives (5-hydroxytryptophan, 5-hydroxyindole, 7-hydroxyindole, ser degradation pathway of the antioxidant, meaning that drawing links
otonin) produced less hydrogen peroxide than Trp and were found to between structural features of these compounds and the mechanism of
protect protein Trp residues against oxidation by alkyl peroxides, their reactivity, and thus their stability, is challenging. To determine
singlet oxygen, superoxide radical anion, and hydrogen peroxide [235]. which structural changes would likely offer a stabilization effect on
This effect may be linked to the overall higher reactivity of hydro tryptophan, the literature data needs to be carefully scrutinized and
xyindole compounds compared to their non-hydroxylated counterparts. often reinterpreted.
This is demonstrated in the quenching of singlet oxygen by 5-hydro The main approaches that could decrease reactivity are: (a) altering
xytryptophan versus Trp, where the hydroxylated compound achieved a the key reactive functional groups on the Trp structure to inhibit re
quenching rate 3-fold faster than the non-hydroxylated compound actions of these functionalities with external compounds; (b) altering
[261]. Some other phenols, such as ellagic acid, flavanols, and hydro the compound to increase its reduction potential, so as to reduce its
xycinnamic acids, have also been found to have protective effects on susceptibility to radical-induced oxidation or other non-specific oxida
Trp, inhibiting both H2O2 [264] and Fe2+/hexanal-induced oxidation tion.
[265]. As mentioned earlier, the main organic compounds which lead to
Photooxidation of Trp can also be inhibited by the addition of an the degradation of Trp, are carbonyls. The initial reaction is a con
tioxidants that react preferentially with the excited photosensitizer. In densation with the primary amine of Trp, forming a Schiff base. This
anaerobic conditions, kynurenic acid was found to sensitize the type I initial reaction was able to be inhibited by attaching a substituent to the
photooxidation of tryptophan in α-crystallin proteins in eye lenses, and amine (e.g. alkyl, carbonyl), which in turn inhibited all of the
712
subsequent degradation pathways (e.g. Maillard reactions) and forma [275,282], though a thorough study into the substituent effects of
tion of degradation products (e.g. βCs) that occur following the Schiff melatonin showed that the effect of the N-acyl functionalization on
base formation [78]. However, Saito et al. noted that aldehydes may compound reactivity can be highly dependent on the oxidant in ques
still react with Trp slowly, but now only with the nitrogen in the indole tion and its combination with other functionalities in the structure
core [78]. [277].
Most research investigating the ease of oxidation of tryptophan-like While chemical modifications can reduce the reactivity of trypto
compounds focused on the ability of these Trp-like compounds to sca phan, if the derivative is intended to be used as a more stable re
venge typical oxidants, including ROS such as the hydroxyl radical, placement for tryptophan, for example as a pharmaceutical, in cell
peroxyl radicals and singlet oxygen, or RNS, such as the peroxynitrite culture, or in animal feeds, then the derivatization needs to be one
anion. The superoxide radical was the only ROS shown to react very which the organism/cell culture is capable of reverting to the functional
negligibly with all Trp-related compounds [275–278]. Melatonin has L-Trp molecule. While structural changes to the amine and carboxylic
been a key scaffold for investigating substituent effects on the anti acid group have a high potential for being reverted by enzymes such as
oxidative action of indole derivatives, and comprehensive reviews and hydrolases or esterases in cells and organisms, changes to the indole
investigations of this topic already exist [277,279]. ring are not likely to be reversed in the same manner, and as such may
The indole scaffold is noted for the ease with which it reacts with not be as applicable as approaches to stabilize tryptophan for cell cul
radicals, undergoing oxidation. This is often attributed to its high ture, animal feed, or pharmaceutical application.
electron density, largely due to the nitrogen, and the ability of the
unpaired electron in the radical product to delocalize over two aromatic 6. Conclusion
rings. Consequently, one would assume that alterations of this nitrogen
to decrease its electron-donating effect may lower this reactivity. This The amino acid tryptophan is crucial for any organism both as a
could theoretically be achieved by replacing the ring nitrogen with precursor of key metabolites and as a common protein residue. It is
another atom or attaching another functional group to it. Indeed, both therefore a key requirement in animal nutrition and in cell culture in
replacement of the nitrogen in melatonin with an oxygen [277] and biomanufacturing processes.
substitution at the nitrogen of the indole ring with alkyl groups [275] In these applications, degradation of Trp can lead to significant
were shown to decrease the reactivity of indole-based structures with problems. In nutrition, either the loss of Trp leads to detrimental effects
oxidants. due to inadequate supply for the organism, or the byproducts them
As high electron density seems to promote the oxidation of indole- selves can be toxic. This was most prominently demonstrated by the
containing compounds, it is reasonable to assume that the addition of outbreak of EMS, caused by the byproducts generated in the microbial
electron-donating groups (EDG) to the indole core would increase the manufacture of commercial Trp [63–65]. In biomanufacturing, condi
oxidation potential (and conversely lower the reduction potential) of tions that degrade Trp in the CCM or in the protein product can lead to
these structures, while the incorporation of electron-withdrawing poor cell growth or suboptimal therapeutic product. For all of these
groups (EWG) would do the reverse, decreasing the potential for oxi reasons, understanding the conditions that lead to Trp degradation can
dative decomposition. This theory is supported by the trend in the re enable avoidance or inhibition of these detrimental consequences.
duction potentials (as convention is to measure reduction potentials In this review, we have summarized the key conditions that lead to
rather than oxidation potentials) for 5-substituted Trp-related com Trp degradation. The major causes are exposure to light, reactive
pounds. EDG-substituted compounds (hydroxy, methoxy, amino) had oxygen species, photosensitizers, and carbonyl-containing organic mo
lower reduction potentials than the unsubstituted indole species, while lecules and the minor causes are pH changes, increased temperature
the EWG-substituted derivative (fluoro) had a higher reduction poten and exposure to metal cations. For all degradation processes, de
tial [235]. This means that EDG-substituted indoles will be more prone gradation pathways and products were summarized. The major de
to oxidation than EWG-substituted indoles. In addition, the location of gradation products were often similar across the range of conditions
the substituent on the indole ring was proposed to have an effect on the investigated: NFK, KYN, diOia, Oia, pyrolloindole species and the series
reactivity, however the literature is not in agreement as to which di of hydroxytryptophan regioisomers. Common minor degradation pro
rection. Computational analysis suggested that compounds substituted ducts include decarboxylated or deaminated Trp species, dimers, and
at the 5- and 7- positions would react better with oxidants than the oxidized forms of these. One particularly specific group of degradation
corresponding 6-substituted species [280], whereas Mor et al. showed products were the β-carbolines, which arose from condensation of
higher antioxidative ability for the 6-substituted melatonin analogue carbonyl-containing organic compounds with Trp. Methods of pre
over the parent 5-methoxy melatonin [281]. However, as stated above, venting such degradation have also been covered, including some an
antioxidative strength is not necessarily correlated with compound tioxidants, and some Trp structural modifications, but to date, there are
stability. very few well-studied ideas or viable applications for the prevention of
Similar predictions of reactivity by measuring reduction potentials Trp degradation in aqueous, complex solutions.
have been used to investigate the effects on reactivity of a free primary While each individual study tends to focus on isolated conditions,
amine versus a protected amine, but the literature is inconclusive. demonstrating the extent and type of Trp degradation, in reality, the
Grewal et al. reported a slightly lower reduction potential for the pro conditions that Trp is exposed to are usually a complex combination of
tected amines [235], while Jovanovich measured a higher reduction all of these scenarios. In nutrition, cooking food can lead to massive Trp
potential for protected amines versus primary amines [282]. losses; the combination of high temperatures and the presence of at
More concrete explorations have been undertaken to determine the mospheric oxygen, other compounds in the food that can behave as
effects of altering or substituting the primary amine and carboxylic acid photosensitizers, and reactive carbonyl-containing compounds will
groups, with a view to minimizing potential oxidation reactions. For generate vast losses and many byproducts, which could themselves be
example, incorporation into a di- or polypeptide at the C- or N-terminus harmful. Storage and transport of aqueous solutions of Trp is most
was found to offer no protection to tryptophan against oxidation by detrimental with light exposure. In the presence of atmospheric oxygen,
singlet oxygen [283–285], but Phe-Trp exhibited less Trp degradation Trp behaves as a photosensitizer, inducing its own decomposition. This
than Ala-Trp or lone Trp upon oxidation with hydrogen peroxide [286]. initiates the production of a plethora of damaging species, not only the
Therefore, linking Trp to another AA may afford some stabilization highly destructive ROS, but also the direct Trp degradation products
against certain types of ROS. that are also photosensitizers. This is exacerbated in complex solutions
In the same vein, Trp-like structures containing a primary amine are such as drink products or CCM, where additional compounds, such as
more prone to oxidation than those with a functionalized amine transition metals, carbonyls and other photosensitizers also enhance the
713
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Tryptophan Degradation

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Free Radical Biology and Medicine 160 (2020) 696–718

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Free Radical Biology and Medicine

Reactivity and degradation products of tryptophan in solution and proteins T

ARTICLE INFO ABSTRACT

Abbreviations GSH glutathione

Fig. 3. Trp degradation products formed in presence of singlet oxygen.

2.5. Interaction of Trp with carbonyls or aldehydes

One of the most common reactions of tryptophan with organic

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