Ecofriendly syntheses of phenothiazones and related structures facilitated by laccase – a comparative study

Article abstract of DOI:10.1016/j.tetlet.2016.07.016

The biocatalytic synthesis of phenothiazones and related compounds has been achieved in an aqueous system under mild conditions facilitated by laccase oxidation. It was found that by coupling 2-aminothiophenol directly with 1,4-quinones, the product yield

Full text of DOI:10.1016/j.tetlet.2016.07.016

Accepted Manuscript
Ecofriendly syntheses of phenothiazones and related structures facilitated by
laccase – A comparative study
Mark D. Cannatelli, Arthur J. Ragauskas
PII:
S0040-4039(16)30838-3
DOI:
http://dx.doi.org/10.1016/j.tetlet.2016.07.016
TETL 47878
Reference:
Tetrahedron Letters
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25 June 2016
5 July 2016
Please cite this article as: Cannatelli, M.D., Ragauskas, A.J., Ecofriendly syntheses of phenothiazones and related
structures facilitated by laccase – A comparative study, Tetrahedron Letters (2016), doi: http://dx.doi.org/10.1016/
j.tetlet.2016.07.016
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Ecofriendly syntheses of phenothiazones and
related structures facilitated by laccase – A
comparative study
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Mark D. Cannatelli
Arthur J. Ragauskas

1
Tetrahedron Letters
Ecofriendly syntheses of phenothiazones and related structures facilitated by
laccase – A comparative study
Mark D. Cannatellia and Arthur J. Ragauskas a,b,c 
a Renewable Bioproducts Institute, School of Chemistry & Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA
b Joint Institute for Biological Sciences, Biosciences Division, Oak Ridge National Laboratory, TN 37831, USA
c Department of Chemical & Biomolecular Engineering, Department of Forestry, Wildlife & Fisheries, University of Tennessee, Knoxville, TN 37996, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The biocatalytic synthesis of phenothiazones and related compounds has been achieved in an
aqueous system under mild conditions facilitated by laccase oxidation. It was found that by
coupling 2-aminothiophenol directly with 1,4-quinones, the product yields could be significantly
increased compared to generating the 1,4-quinones in situ from the corresponding
hydroquinones via laccase oxidation. However, laccase still proved to be pivotal for achieving
highest product yields by catalyzing the final oxidation step. Furthermore, a difference in
reactivity of aromatic and aliphatic amines towards 1,4-naphthoquinone is observed. This study
provides a sustainable approach to the synthesis of a biologically important class of compounds.
Available online
Keywords:
Biocatalysis
Green chemistry
Laccase
2009 Elsevier Ltd. All rights reserved.
Phenothiazones
Thiol-amine
———
 Corresponding author. Tel.: +1-865-974-2042; fax: +1-865-974-7076; e-mail: aragausk@utk.edu

2
Tetrahedron Letters
Enzyme assisted processes in chemical synthesis have
MnO2, or ceric ammonium nitrate to oxidize phenothiazines to
the corresponding phenothiazones at elevated temperatures in
organic solvents.21, 24 More contemporary syntheses involve the
proved to be a viable alternative to traditional chemical catalysis
in the past few decades.1 Their nontoxic nature, renewability and
biodegradability, high activity and stability in aqueous systems,
high selectivity, cost effectiveness, and general ease of use will
ensure their continuing application as sustainable solutions to
pollution prevention and toxic waste control within many areas
of the chemical industry. Laccases (benzenediol:oxygen
oxidoreductases, EC 1.10.3.2) are a class of copper-containing
enzymes of biotechnological importance that have received
growing amounts of attention in previous years.2 These multi-
copper oxidases catalyze the mono-electron oxidation of four
substrate molecules (typically phenolic compounds) coupled with
the reduction of O2 to 2H2O.3 Their existence in nature is
widespread, ranging from fungi,4 bacteria,5 and insects,6 to
plants7 and algae.8 Owing to their environmentally benign
character and catalytic properties, they find significant
application in a variety of fields, such as delignification and
biobleaching of pulps,9 lignocellulosic fiber modification,10
bioremediation of industrial wastes,11 biosensors,12 as well as
organic synthesis.13
condensation of 2-aminothiophenol with 1,4-quinones;21,
25
however, these reactions are all conducted in organic solvents.
Thus, there lacks a method that is conducted both in an aqueous
solvent system and free of stoichiometric, transition-metal
oxidants. Herein, we provide a green, biocatalytic approach to the
synthesis of phenothiazones and related structures.
To determine if the reaction would proceed to give the
desired product, an initial experiment was conducted that reacted
2-aminothiophenol (1) with naphthohydroquinone (2a) in the
presence of laccase (Method A), shown in Scheme 1. The
product, 5H-benzo[a]phenothiazine-5-one 3a, which has shown
antiproliferative activity towards human tumor cells,26 was
achieved, albeit in very low yield (11%). The low yield can be
rationalized by the formation of a S-S dimer of 1, which was also
observed in a previous study.19b Studying the kcat values for
laccase oxidation of phenols and their thiol analogs, it can be
seen that benzenethiols are oxidized at a significantly greater rate
than phenols (e.g. kcat catechol = 3300 min-1, kcat 1,2-
benzenedithiol = 45000 min-1).27 In fact, reacting 1 with laccase
alone can yield the S-S dimer product 4 (Scheme 2), which
possesses antimicrobial properties and was historically used to
treat syphilis,28 in very high conversion (83%). Thus, simply
reacting 1 with hydroquinones and laccase in a one-step process
is not a feasible method for the synthesis of phenothiazones.
Laccases have attracted increasing use as green tools for the
synthesis of fine chemicals, as demonstrated by a growing
number of related publications.14 Continuing our research on
laccase-catalyzed synthesis of heterocyclic compounds,15 we
focused our attention towards coupling hydroquinones with
compounds containing both the thiol and amine functional
groups. While laccase-catalyzed coupling reactions involving
nucleophiles derived from carbon,16 nitrogen,17 or sulfur15, 18 have
been widely studied, the use of compounds containing two
nucleophilic centers capable of forming multiple bonds to yield
cyclic products is much less explored.19 The laccase-catalyzed
coupling of 2-aminothiophenol with hydroquinone for the
synthesis of 3H-phenothiazin-3-one has been previously
achieved;19b however, the process suffers from low product yield
(21%). We present an alternative methodology that significantly
increases product yields of several synthesized phenothiazones.
Scheme 1. Laccase-catalyzed coupling of 2-aminothiophenol (1) with
naphthohydroquinone (2a). Reaction conditions: 1 eq. (0.50 mmol) 1, 1.25
eq. (0.625 mmol) 2a, 50 U laccase, 8.5 mL 0.10 M sodium acetate buffer pH
5.0 : 1.5 mL MeOH, rt, 6 h.
The phenothiazones are an important class of compounds that
possess a variety of biological activities and practical use. Early
studies demonstrated that these compounds exhibit lethal effects
on liver fluke as well as paralytic effects on the human parasitic
worm Ascaris lumbricoides.20 More recently, derivatives and
analogs, particularly 4-bromo-2,7-dimethoxy-3H-phenothiazin-3-
one (Figure 1), have shown inhibitory effects on 5-lipoxygenase
Scheme 2. Laccase-catalyzed dimerization of 2-aminothiophenol (1).
Reaction conditions: 0.50 mmol 1, 50 U laccase, 6 mL 0.10 M sodium acetate
buffer pH 5.0, rt, 4 h.
Figure 1. Representative structures of phenothiazones.
Table 1
and mammalian leukotriene biosynthesis, thus, they find
therapeutic use in treating allergies, inflammation, asthma, and
cardiovascular disorders.21 They have also displayed
tuberculostatic, antibacterial, and analgesic properties and have
been used to treat oxidative stress disorders.22 Furthermore, they
offer protection to mild steel from acidic corrosion, and find use
in organic semiconductors and dyes (e.g. methylene violet, Fig.
1).
Laccase-catalyzed coupling of 2-aminothiophenol (1) with hydroquinones
(2)a
The first reported synthesis of a phenothiazone compound
involved the oxidation of 3-hydroxyphenothiazine by FeCl3.23
Most early syntheses relied upon the use of stoichiometric,
transition-metal-containing oxidants, such as FeCl3, K2Cr2O7,
Entry
2
Product Yield
1
a
3a 20%

3
2
3
4
b
c
R1, R2, R3 = H
3b 24%
3c 53%
regioselectivity of addition for the reaction of 1 with 5c (Table
2, entry 3) is similar to that observed by Terdic, who conducted
R1 = H, R2, R3 = OCH3
R1, R3 = H, R2 = CH3
the coupling reaction in ethanol.25c
d
3d
9% R1 = CH3, R2, R3 = H
Analysis of the gas chromatograms for the reaction of 1 with
5b using both Methods C and D (Fig. 2) provides a qualitative
picture of the reaction systems and reveals the role laccase has in
improving product yields. In Fig. 2a (Method C), the peak with
m/z 213, corresponding to product 3b, is relatively small,
indicating a low product yield. In comparison, the same peak in
Fig. 2b (Method D) is the predominant peak in the
chromatogram, corresponding to a high product yield. Further
analysis of the chromatogram in Fig. 2a shows a sizeable peak
with m/z 215, which is negligible in Fig. 2b. We believe this
compound to be the reduced form of product 3b. Furthermore,
allowing 1 to react with 5b in the absence of laccase for 72 h
does not significantly improve the yield of 3b. Thus, laccase
appears to be crucial for completely oxidizing the phenothiazine
form to the phenothiazone form and providing greatest product
yields. Scheme 3 shows the proposed reaction mechanism. Initial
addition of the aromatic amino group of 1 to a carbonyl group of
1,4-quinone 5 yields the imine 6, which is followed by addition
of sulfur to an adjacent alkene carbon and subsequent
tautomerization to produce the phenothiazine intermediate 7. A
final oxidation of the phenothiazine affords the phenothiazone 3.
3e 12% R1, R3 = H, R2 = CH3
a Reaction conditions: 1 eq. (0.50 mmol) 1, 1.25 eq. (0.625 mmol) 2, 50 U
laccase added at t = 2 h, 0.10 M sodium acetate buffer pH 5.0 with 10-15%
MeOH (v:v), rt, 6 h.
Based on the aforementioned findings, we then experimented
with a two-step process in which 1 is added to the reaction
mixture 2 hours after the hydroquinone and laccase (Method B),
similar to what has been done previously.19b We hypothesized
that this would allow for a substantial formation of laccase-
generated 1,4-quinone capable of rapidly reacting with 1 before it
is oxidized by laccase. The results are shown in Table 1. As can
be seen, the product yields are still quite low, with the exception
of 3c (Table 1, entry 3). These results indicate that this is a viable
procedure for the synthesis of 2,4-disubstituted phenothiazones,
which are produced via the highly stable laccase-generated 2,6-
disubstituted-1,4-quinone intermediate. However, for the
remaining hydroquinones examined, this is still not a practical
synthetic method.
There seemed to be two factors contributing to low product
yields: 1) oxidation of 1 by laccase, and 2) poor conversion of the
hydroquinone to the corresponding 1,4-quinone by laccase. Thus,
to overcome these problems, we reacted the 1,4-quinones 5
directly with 1 both without (Method C) and with (Method D)
a)
Table 2
Coupling of 2-aminothiophenol (1) with 1,4-quinones (5) in the absence
(Method C) and presence (Method D) of laccase
Entry
5
Product Yield
Product Yield
b)
Method C a
Method D b
1
3a 44%
3a 52%
a
2
3
b
c
R1, R2 = H
R1 = H, R2 = CH3 3d
3b
9%
9% R1 = CH3, R2 = H
3e 12% R1 = H, R2 = CH3
3b 61%
3d 24%
3e 29%
a Method C reaction conditions: 1 eq. (0.50 mmol) 1, 1.25 eq. (0.625 mmol)
5, 0.10 M sodium acetate buffer pH 5.0 with 10-15% MeOH (v:v), rt, 6 h.
b Method D reaction conditions: 1 eq. (0.50 mmol) 1, 1.25 eq. (0.625 mmol)
5, 50 U laccase added at t = 2 h, 0.10 M sodium acetate buffer pH 5.0 with
10-15% MeOH (v:v), rt, 6 h.
Figure 2. Qualitative gas chromatograms with m/z values of peaks for the
reaction of 1 with 5b using a) Method C and b) Method D.
laccase. The results are displayed in Table 2. First of all, it can be
seen that by using Method D, the product yields can be increased
compared to employing Method B (Table 1). Thus, it seems as
though the aforestated problems can be reduced or eliminated by
using this methodology. Furthermore, when comparing the
results of Methods C and D, it is noticed that the product yields
can be substantially increased when laccase is utilized.
Comparing the data in Tables 1 and 2 for the synthesis of
phenothiazones using different methods, we can see that when
employing Method D, the product yields can be increased on
average by 2.5 fold compared to using Method B, and up to 6.8
fold compared to when Method C is used. For comparison, the

4
Tetrahedron Letters
Scheme 3. Reaction mechanism for the laccase-facilitated synthesis of
phenothiazones.
form. The increased nucleophilicity of the amino group of 1 is
With the chemistry for the coupling of an aromatic thiol-
what allows it to rapidly react with the carbonyl carbon of a 1,4-
naphthoquinone, whereas for 8, the sulfur adds first to an alkene
carbon.
amine with 1,4-quinones developed, we then attempted to apply
the principles using a simple aliphatic thiol-amine, cysteamine
(8). Since we observed that laccase does not directly oxidize 8, it
was possible to react this compound in one-pot with the
hydroquinone 2 and laccase in one-step. Using a variety of
hydroquinones and catechols,29 the results of these reactions were
generally unsuccessful, providing a vast mixture of products
(based on GC-MS and TLC analyses). One reaction was
successful, however; the laccase-catalyzed coupling of 8 with 2a
yielded product 9 (Scheme 4), a compound that possesses
tuberculostatic potential and is also an important structural
moiety present in compounds that exhibit potent antibacterial and
antifungal activities.30 A possible reason for the success in
achieving a desired product when compound 2a was used may be
the increased stability of the 1,4-naphthoquinone intermediate
compared to less substituted 1,4-quinones.
The presented synthetic protocol for the preparation of
phenothiazones offers an ecofriendly alternative to the synthesis
of these important compounds, further progressing sustainability
within the field of chemical synthesis. The process addresses
many of the principles of green chemistry,32 such as waste
prevention, atom economy, catalysis (laccase), benign solvents
(aqueous solvent system), renewable feedstocks (1,4-quinones
and hydroquinones are biomass derived), and a safe and energy
efficient synthetic procedure. Using the reaction of 1 with 5b
(Method D) for the synthesis of 3b as an example, green
chemistry can be quantified. The E factor, which measures kg of
waste produced per kg of desired product, was calculated to be
17.7, which falls toward the lower limit expected for fine
chemical syntheses.33 The ratio of the incorporation of all
reactant atoms into the desired product (i.e. the atom economy)34
is also very good, calculated as 86%, as is the space time yield,
calculated to be 0.005 mol x L-1 x h-1.
A proposed reaction mechanism for the laccase-catalyzed
coupling of 8 with 2a is provided in Scheme 5. First, laccase
oxidizes 2a to the corresponding 1,4-quinone 5a, which is
followed by addition of sulfur to an alkene carbon. Following
subsequent tautomerization and oxidation, the nitrogen then adds
to the neighboring alkene carbon, forming a six-membered
heterocyclic ring. After another tautomerization and final
oxidation, product 9 is reached. From this result, we observe a
difference in reactivity of the aliphatic amino group of 8 and the
aromatic amino group of 1 towards nucleophilic addition to 1,4-
naphthoquinone. This difference in reactivity may be rationalized
by the differences in basicity of the amino groups. The pKb of the
aromatic amino group of 1 is 9.49, whereas the pKb of the
aliphatic amino group of 8 is 3.19.31 Thus, in the aqueous
reaction medium, the amino group of 8 is predominantly in its
cationic form, rendering it less nucleophilic and less reactive than
the aromatic amino group of 1, which is mostly in its neutral
In summary, we have developed an environmentally friendly
approach for the synthesis of phenothiazones and related
structures facilitated by laccase. By coupling 1,4-quinones with
2-aminothiophenol in an aqueous reaction medium in the
presence of laccase, the yields of phenothiazones can be
substantially increased compared to when laccase is not present
or the 1,4-quinones are generated in situ via laccase-catalyzed
oxidation of the corresponding hydroquinone. Furthermore, a
difference in reactivity between aromatic and aliphatic amines
towards nucleophilic addition to 1,4-naphthoquinone was also
observed. This study adds to the ever-growing toolkit of enzyme
assisted processes in chemical synthesis, aiding in the increasing
global effort of promoting sustainability within the field.
Acknowledgment
The authors are thankful for a student fellowship supported
by the Renewable Bioproducts Institute at Georgia Institute of
Technology.
Supplementary data
Scheme 4. Laccase-catalyzed coupling of cysteamine (8) with
naphthohydroquinone (2a). Reaction conditions: 5 eq. (2.50 mmol) 8, 1 eq.
(0.50 mmol) 2a, 50 U laccase, 8.5 mL 0.10 M sodium acetate buffer pH 5.0 :
1.5 mL MeOH, rt, 12 h.
Supplementary data associated with this article can be found,
in the online version, at
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dihydroxyacetophenone,
ethyl
2,5-dihydroxybenzoate,
3-
methylcatechol, 4-methylcatechol, 3-methoxycatechol, and 1,2-
dihydroxynaphthalene.
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6
Tetrahedron
Ecofriendly syntheses of
phenothiazones and related
structures facilitated by laccase – A
comparative study
Highlights
Mark D. Cannatelli and Arthur J. Ragauskas





Biocatalytic synthesis of phenothiazones in one-pot under
mild conditions.
Laccase-catalyzed coupling of 2-aminothiophenol with
hydroquinones.
Increased yields achieved when 1,4-quinones are used
compared to hydroquinones.
Difference in reactivity of aromatic and aliphatic amine
towards nucleophilic addition.
Favorable green chemistry metrics, such as E factor and
atom economy.