50 Years of Chemistry in Opole
Errors in the studies of enzymatic reactions resulting
from unforeseen oxidation-reduction reactions
Beata GĄSOWSKA-BAJGER, Hubert WOJTASEK* – Faculty of Chemistry, Opole University,
Opole, Poland
Please cite as: CHEMIK 2014, 68, 4, 341–346
This article is dedicated to Professor Paweł Kafarski
on the occasion of his 65th birthday
The Division of Biochemistry, Faculty of Chemistry, Opole
University for more than 10 years has been involved in analysis
of reactions catalysed by tyrosinase (monophenol oxidoreductase,
L-dopa:O2, EC 1.14.18.1). This is a key enzyme in melanization
process that catalyses its two first reactions: o-hydroxylation
of tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and oxidation
of dopa to dopaquinone (Fig. 1) [1].
is found. For phosphonic analogs of aromatic amino acids it was
found that amino-(3,4-dihydroxyphenyl)methylphosphonic acid
reduced dopaquinone by oxidating itself to respective quinone,
that subsequently decomposed to 3,4-dihydroxybenzaldehyde
(Fig. 2) [3]. Obviously, the monophenol derivatives (amino-(3hydroxyphenyl)methylphosphonic acid and amino-(4-hydroxyphenyl)
methylphosphonic acid) did not undergo such reactions and thus
inhibition was not observed.
Fig. 1. Initial reactions of melanization pathway [1]
The research started from the analysis of inhibition mechanism
of aromatic aminophosphonic acids against this enzyme. In 1987,
the information was published that amino-(3,4-dihydroxyphenyl)
methylphosphonic acid is a strong inhibitor of tyrosinase, while
monophenol derivatives (amino-(3-hydroxyphenyl)methylphosphonic
acid and amino-(4-hydroxyphenyl)methylphosphonic acid) practically
shown no such an activity [2]. These data were interesting for
the Authors, as the mechanism of tyrosinase action [1] did not
supported in any way such differences. The detailed analysis of
the oxidation of natural tyrosinase substrates (tyrosine and dopa)
in the presence of these compounds using spectrophotometric
and electrochemical methods has shown that strong inhibition of
enzyme by amino-(3,4-dihydroxyphenyl)methylphosphonic acid)
is only apparent. The inhibition constants determined previously
were calculated based on spectrophotometric measurements. In
the analysis of tyrosinase-catalysed reactions, spectrophotometric
measurements of dopachrome concentration are used as a routine
procedure. Dopachrome is not a product of enzymatic reaction, but
is produced from direct product of this reaction (dopaquinone) as
a result of non-enzymatic cyclization (to leukodopachrome) and
oxidation (Fig.1). Both reactions are sensitive to nucleophiles and
reducers. Strong nucleophiles (e.g. thiol groups or amines) might
compete with dopaquinone amine group in its attack at o-quinone
system. Reducers in turn might reduce dopaquinone back to dopa, in
this way preventing its cyclization or oxidation of leucodopachrome.
All these reactions prevent production of dopachrome, and thus if
progress of enzymatic reaction is monitored by the measurement
of dopachrome absorbance – an apparent strong inhibition effect
Corresponding author:
Hubert Wojtasek – Sc.D, e-mail: [email protected]
344 •
Fig. 2. Reactions occuring in the mixture containing tyrosinase, dopa
and amino-(3,4-dihydroxyphenyl)methylphosphonic acid [3]
False results in analysis of enzymatic reaction kinetics due
to unexpected oxidation-reduction reactions are well exemplified
by results obtained from studies on effect of compounds
containing hydrazine group on activity of mammalian peroxidases.
2,2’-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium
salt (ABTS) is used commonly for monitoring of lactoperoxidase
activity. While studying the activity of this enzyme against this
substrate in the presence of carbidopa (drug used in Parkinson’s
disease) containing catechol and hydrazine groups, the delayed
appearance of ABTS oxidation product (ABTS+•, Fig. 3) was
observed. This might have indicated that carbidope is preferentially
oxidised by this enzyme till its depletion or reduces ABTS+•.
Discoloration of ABTS+• solution after addition of carbidopa has
shown that the second reaction is at least partially responsible for
lag-phase observed for enzymatic reactions . The literature review
has confirmed that such effect was found previously for other
reducers [4, 5], and ABTS+• is commonly used for determination
of antioxidative activity [6]. The inhibition parameters (Ki, IC50)
nr 4/2014 • tom 68
by indirect hydrazide group oxidation by tyrosinase, using phenols or
catechols as electron carriers (Fig.4). Such reactions were carried out
i.a. with 4-tert-butylcatechol, which is oxidased by tyrosinase to stable
4-tert-butyl-o-benzoquinone, resistant to attacks by nucleophiles,
e.g. water. In the presence of traces of this catechol the deprotection
reaction of amino acid phenylhydrazides was many times faster when
significantly smaller amounts of enzyme were used [14].
Fig. 3. Absorbance changes at λ = 415 nm during the oxidation of ABTS
(25 µM) by bovine lactoperoxidase (0.1 µM) without carbidopa (1) and in
its presence: 2 – 2 µM, 3 – 4 µM, 4 – 6 µM, 5 – 8 µM, 6 – 10 µM
Similar cases, where unexpected redox reactions have led
to the erroneous interpretation of results, have been described
multiple times in the literature. Such situations occur usually when
enzymatic reactions (usually with oxyreductases, but not only)
are monitored spectrophotometrically at one wavelength using
routine methodology. Whereas, many of such mistakes could have
been avoided even by measurement of UV-VIS spectra of analysed
reaction mixtures or by extending analysis by other methods, e.g.
electrochemical or chromatographic ones. The further part of
article presents few cases based on literature and own experience
of the Authors in this regard.
In 2004 one of the Authors (H.W.) has found an article describing
effect of tetrahydropterins on tyrosinase activity [7], which was
explicit criticism of research published by other authors [8]. The
British research group was proving that tetrahydropteridines
allosterically inhibit tyrosinase activity [7], while the group from South
Korea has shown that the inhibition is apparent and the observed
effect is a result of dopaquinone reduction by tetrahydropteridines
[8]. However, the publication of British group contained number
of errors and shortcomings – in the research i.a. measurements
of oxygen consumptions were not used, basing the results solely
on point spectrophotometric measurements. This led the Author
to prepare polemical article presenting these shortcomings and lack
of addressing many facts described previously in the literature [9].
Unfortunately, this critique did not lead to correction of mistakes. On
the contrary – in the response, the British group repeated previous
mistakes and added few new ones [10]. Interestingly, the stimulation
of monophenolase activity of tyrosinase by tetrahydropteridines in
similar way as other reducers, such as ascorbic acid have been found
almost four decades earlier [11]. The phenomenon is consistent with
the model proposed by the Korean group, while contrary to the
report of the British group.
The Authors of this publication decided to use in their research
oxidation-reduction reactions where part would be played by products
of phenols and catechols oxidised by tyrosinase. Soon after the
explanation of tyrosinase “inhibition” mechanism by amino-(3,4dihydroxyphenyl)methylphosphonic acid, the Authors have found
articles describing use of this enzyme for oxidation of amino acid
hydrazides [12, 13]. The reaction was used in peptide synthesis, but its
yield was very low. They concluded that the process might be improved
nr 4/2014 • tom 68
Fig. 4. The reaction occuring between generated enzymatically 4-tertbutyl-o-benzoquinone and amino acid phenylhydrazide [14]
In the literature, the hydrazine derivatives are often mentioned in
relation to reactions catalyzed by tyrosinase. This enzyme has been
applied among others in electrochemical biosensor for detection of
hydrazine derivatives, as it was observed that production of o-quinones
by this enzyme is inhibited in the presence of these compounds [15].
The natural amino acid hydrazide, agaritine (5-[2-[4-(Hydroxymethyl)
phenyl]-L-glutamohydrazide]), that commonly occurs in mushrooms
Agaricus, has been also descirbed as inhibitor of polyphenol oxidase and
[16] melanization process [17]. In both cases, the observed inhibition
effects have been likely apparent, resulting from dopaquinone
reduction by hydrazine derivatives and not from direct effect of these
compounds on enzyme activity. In both cases, the mistakes in the result
interpretation have been caused again by insufficient knowledge of the
literature as the reduction of dopaquinone by hydrazine derivatives has
been described two decades earlier [18]. Unfortunately, as it was once
said by Prof. Mirosław Soroka, “Careful reading of scientific literature
becomes very rare” (see aforementioned case of tetrahydropteridines).
The researchers prefers to write than read, because it just pays off.
Amino acid phenylhydrazides decomposition after their indirect
oxidation by tyrosinase inspired the Authors to use similar reaction
to release anti-cancer compounds from prodrugs in melanoma
treatment. As tyrosinase is enzyme present only in melanocytes, its use
for activiation of such compounds has been considered for a long time
[19]. The prodrugs proposed by the Authors were to be composed of
activator (phenol or catechol group oxidased by tyrosinase), effector
in form anti-cancer drug (e.g. nitrogen mustard or 5-fluorouracil)
and hydrazine linker. Before starting to synthesise such compounds,
they decided to carry out trail reactions on commercially available
model compound with phenol or catechol and hydrazine group.
They have chosen carbidopa, whose effect on lactoperoxidase they
have described earlier. As a result of tyrosinase action on this alkyl
hydrazine derivative they obtained mixture of products resulting from
oxidation and subsequent elimination of hydrazine group (amino-(3,4dihydroxyphenyl)methylphosphonic acid), as well as nucleophilic attack
of this group on generated enzymatically o-quinone (5,6-dihydroxy-3methylquinoline, product of cyclization reaction ) [20]. To prevent the
cyclization reaction that would compete with the release of the effector
from the prodrug, they decided to prepare only acylated hydrazine
• 345
50 Years of Chemistry in Opole
are calculated routinely based on such measurements (see
aforementioned case of phosphonic amino acids). At the same time,
Figure 3 shows explicitly that values of these parameters will vary
greatly depending on chosen measurement time and concentration
range, and all will be incorrect. This method is just not suitable for
their determination.
50 Years of Chemistry in Opole
derivatives. They synthesised nitrogen mustard derivative – N-{4-[bis(2-chloroethyl)amino]benzoyl}-N’-(4-hydroxybenzyl)hydrazine – and
carried out oxidation of this compound by tyrosinase and identified
reaction products. Surprisingly, the main product was 5,6-dihydroxy1H-indazo-1-yl 4-[bis-(2chloroethyl)amino]beonzoate formed as
a result of nucleophilic attack of acylated nitrogen atom of hydrazine
linker on generated enzymatically o-quinone. Transiently, also N-{4[bis-(2-chloroethyl)amino]benzoyl}-N’-(4-hydroxybenzylidene)
hydrazine was formed – probably as a result of intermolecular redox
reaction between o-quinone and hydrazine group of substrate molecule,
and subsequent tautomerization of produced diazene to hydrazone
(Fig. 5) [21]. However, none of these compounds released anti-cancer
effector (nitrogen mustard). The occurring reactions turned out
to be completely different from expected by the Authors and basically
the project failed. They rather could not have foreseen cyclization
reaction – all literature data indicated that it should not have occurred.
However, results suggesting that tautomerization of benzyldiazene
to hydrazone might occur, and even faster than hydrolysis, have been
previously reported [22]. Unfortunately, the Authors have found them
by accident, when project was basically completed, as the article had
no direct relation with their research subject.
Fig. 5. Reactions occuring during the oxidation of potential anti-melanoma prodrug, N-{4-[bis-(2-chloroethyl)amine]benzoyl}-N’-(4-hydroxybenzyl)hydrazine, by tyrosinase [21]
The scientific literature is growing to the size that makes it
sometimes difficult to cope with. The scientists, publication authors,
reviewers and journal editors alike, have less and less time. It results
in research carried out and described in greater and greater rush. If
the situation concerning research worker evaluation systems does
not change soon, the mistakes will be more widespread and the
science less credible.
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346 •
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Beata Gąsowska-Bajger – Ph.D., graduated from the Institute
of Chemistry, Opole University. She received her Ph.D. degree from
the Faculty of Chemistry, Opole University under the supervision of
Dr. Hubert Wojtasek. Scientific fields: oxidoreductase mechanism of action,
search for inhibitors of these enzymes and their application in activation
of anti-cancer prodrugs. She is the author of 8 publications in national and
international journals.
e-mail: [email protected]; phone: +48 77 452 71 20
* Hubert Wojtasek – Sc.D., graduated from the Institute of Chemistry,
State Higher Pedagogical College currently Opole University). He received
his Ph.D. degree from the Department of Chemistry, State University of New
York at Stony Brook, USA, under supervision of Prof. Glenn D. Prestwich.
He received fellowship from Japanese Science and Technology Agency at
National Institute of Agrobiological Sciences in Tsukuba in the laboratory of
Dr Walter S. Leal and fellowship from Japan Society for the Promotion of
Science at the School of Medicine, Mie University in laboratory of Prof. Yasuo
Chinzei. Scientific fields: mechanisms of actions of enzymes invovlved
in melanization and sclerotization processes, molecular mechanisms of insects’
development and olfactory perception. He is the author of 1 monograph,
1 international patent and 28 publications in international journals.
e-mail: [email protected];
phone: +48 77 452 71 22; Fax: +48 77 452 71 01
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