Enhanced biocatalytic performance of bacterial laccase from Streptomyces sviceus: Application in the Michael addition sequence towards 3-arylated 4-oxochromanes

Article abstract of DOI:10.1002/cctc.201500142

A fast and efficient laccase-catalysed oxidation/Michael addition sequence is performed using the bacterial laccase Ssl 1 from Streptomyces sviceus under basic conditions to provide a new class of 3-arylated 4-oxochromanes. This approach has advantages compared to previous biocatalytic arylation protocols that use fungal laccases under slightly acidic conditions to allow a significant decrease in reaction time with improved yields and maintained regio- and diastereoselectivity. Furthermore, a successful diastereoselective, consecutive one-pot approach with the use of a hydrogenation flow system combined with the laccase-catalysed arylation was performed. Finally, the general utility of this enzyme as a superior biocatalyst for Michael additions using several nucleophiles was demonstrated. The corresponding starting material was obtained in a straightforward esterification/hydrogenation process with the latter accomplished by using the flow system.

Full text of DOI:10.1002/cctc.201500142

DOI: 10.1002/cctc.201500142
Full Papers
Enhanced Biocatalytic Performance of Bacterial Laccase
from Streptomyces sviceus: Application in the Michael
Addition Sequence Towards 3-Arylated 4-Oxochromanes
[a]
[a]
[b]
[b]
´
Sanel Suljic, Frederik B. Mortzfeld, Matthias Gunne, Vlada B. Urlacher, and
Jçrg Pietruszka*[a, c]
A fast and efficient laccase-catalysed oxidation/Michael addi-
tion sequence is performed using the bacterial laccase Ssl 1
from Streptomyces sviceus under basic conditions to provide
a new class of 3-arylated 4-oxochromanes. This approach has
advantages compared to previous biocatalytic arylation proto-
cols that use fungal laccases under slightly acidic conditions to
allow a significant decrease in reaction time with improved
yields and maintained regio- and diastereoselectivity. Further-
more, a successful diastereoselective, consecutive one-pot ap-
proach with the use of a hydrogenation flow system combined
with the laccase-catalysed arylation was performed. Finally, the
general utility of this enzyme as a superior biocatalyst for Mi-
chael additions using several nucleophiles was demonstrated.
The corresponding starting material was obtained in a straight-
forward esterification/hydrogenation process with the latter ac-
complished by using the flow system.
Introduction
Laccases (benzenediol: oxygen oxidoreductase EC 1.10.3.2.)
belong to the family of multi-copper oxidases that contain
four copper ions. They catalyse the one-electron oxidation of
the substrate (through the T1 copper ion) and transfer the ab-
stracted electrons to molecular oxygen, which is reduced to
water (at the trinuclear cluster of one T2 copper and two T3
copper ions).[1] Suitable substrates such as phenols, catechols,
hydroquinones or aromatic amines are oxidised readily by lac-
cases in the presence of atmospheric oxygen.[2] As a result of
their compatibility towards organic solvents, commercial avail-
ability and mild reaction conditions, fungal laccases represent
a green alternative as an oxidative catalyst for arylation reac-
tions in organic synthesis.[3] Despite their high activities caused
by redox potentials between 0.5–0.8 mV versus the normal hy-
drogen electrode (NHE),[4] the use of fungal laccases requires
neutral or acidic conditions (depending on the substrate
used).[5a] In basic media, fungal laccases usually suffer from
a strong inhibition of the electron transfer between the copper
centres essential for its biocatalytic activity caused by the bind-
ing of hydroxide ions at the trinuclear cluster.[5b] However, alka-
lophilic fungal laccases are highly desirable biocatalysts in, for
example, the pulp and paper industry and organic synthesis,[5c]
and examples of laccases adapted successfully to high pH
values by directed evolution are well documented.[5d,e]
In our previous protocols, laccase-catalysed oxidation/aryla-
tion sequences were performed successfully under acidic con-
ditions. However, one question that needs to be asked is
whether we could significantly reduce general reaction times
for these conversions that vary from 18–72 h[3c,6] while main-
taining yields and selectivities. The circumstance that Michael
additions require a base or basic reaction conditions to acti-
vate the reaction donor to proceed well and the fact that bio-
catalytic performances under basic reaction conditions are still
elusive leaves a gap for the optimisation of this type of reac-
tion.[7]
Recently, we cloned and characterised a laccase from Strep-
tomyces sviceus (Ssl 1) with an activity profile towards the oxi-
dation of several substrates at different pH values. Ssl 1
showed an outstanding activity for the oxidation of 2,6-dime-
thoxyphenol (1) at pH 9, guaiacol (2) at pH 8 and 2,2’-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid) (3) at pH 4. Al-
though it originates from a mesophilic organism, Ssl 1 demon-
strates high stability at elevated temperatures (t1/2,608C =88 min)
and in a wide pH range (pH 5.0–11.0). A number of detergents
and organic co-solvents do not affect Ssl 1 stability.[8] As
a result of the electronic and steric similarity of catechol (4) to
1 and 2, a basic pH value seems to be promising for its oxida-
tion and possible subsequent 1,4-addition using the new lac-
case. As o-quinone, the corresponding oxidised analogue of 4,
is not commercially available because of its high reactivity, the
laccase-catalysed in situ formation allows its utilisation as an
attractive arylating agent (Figure 1).
´
[a] S. Suljic, F. B. Mortzfeld, Prof. Dr. J. Pietruszka
Institut fꢀr Bioorganische Chemie
Heinrich-Heine-Universitꢁt Dꢀsseldorf im Forschungszentrum Jꢀlich
Stetternicher Forst, Geb. 15.8, 52426 Jꢀlich (Germany)
Fax: (+49)2461-616196
E-mail: j.pietruszka@fz-juelich.de
[b] Dr. M. Gunne, Prof. Dr. V. B. Urlacher
Institut fꢀr Biochemie, Heinrich-Heine-Universitꢁt Dꢀsseldorf
Universitꢁtsstrasse 1, Geb. 26.42, 40225 Dꢀsseldorf (Germany)
[c] Prof. Dr. J. Pietruszka
Institut fꢀr Bio- und Geowissenschaften (IBG-1: Biotechnologie)
Forschungszentrum Jꢀlich
52425 Jꢀlich (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cctc.201500142.
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Results and Discussion
To demonstrate the utility of the laccase Ssl 1 for oxidative CÀ
C couplings in organic synthesis, we started our investigation
by performing an arylation sequence that employed the chro-
manone ester 7 prepared previously by esterification[16] and
flow hydrogenation[17] from the corresponding chromone (Sup-
porting Information). As the initial model reaction, this ester 7
and 3-methylcatechol (8) as the arylating agent were chosen
as substrates. The oxidation of catechol 8 with the new laccase
was expected to furnish the Michael acceptor, and the chroma-
none 7 acted as the nucleophile (Table 1). Compared to a previ-
Figure 1. Suitable substrates for the Ssl 1-catalysed oxidation (1–4) as well as
pharmacologically promising chromone scaffolds (5 and 6).
Table 1. Optimisation of the laccase-catalysed synthesis of chromanone
9.[a]
As we are also interested in the development of active scaf-
folds for medicinal purposes by establishing biocatalytic meth-
ods for the synthesis of new CÀC bonds, the chromones[9]
caught our attention. Chromones 5 (4H-chromen-4-one, 4H-1-
benzopyran-4-one) are heterocyclic planar compounds that are
highly distributed in nature, especially in the plant kingdom
(Figure 1).[10] They regularly possess interesting biological activ-
ities and were used, for example, as antidiabetics and cardio-
vascular agents,[11] calcium antagonists,[12] diuretics[13] and anti-
oxidants.[14] One basic scaffold of this class is 4-oxo-4H-chro-
mene-3-carboxylic acid (6), which acts as an irreversible selec-
tive human monoamine oxidase-B (hMAO-B) inhibitor for the
treatment of Parkinson’s disease. In general, structure–activity
relationship (SAR) studies performed by Borges and co-work-
ers[15] showed that chromone derivatives with substituents in
the 3-position act as MAO-B inhibitors, with IC50 values down
to the nanomolar range (Figure 1).
Entry Laccase
source
Buffer/solvent
(ratio)
Unit Reaction time Yield
[U]
[h]
[%][e]
1
2
3
4
5
6
7
8
A. bisporus[b] buffer/MeCN (2:1) 15[c] 48
62
31
67
86
72
39
21
70
89
71
52
A. bisporus
Ssl 1
Ssl 1
buffer/MeCN (2:1) 15[c] 48
buffer/MeCN (4:1) 16[d]
3
4
buffer/MeCN (4:1)
buffer/MeOH (4:1)
buffer/MeCN (4:1)
buffer
8
8
4
8
8
8
8
8
Ssl 1
4
Ssl 1
3
Ssl 1
5
Ssl 1
Ssl 1
Ssl 1
Ssl 1
buffer/MeCN (1:1)
buffer/MeCN (2:1)
buffer/MeOH (2:1)
buffer/THF (2:1)
24
3
3
9
10
11
Although examples of alkalophilic bacterial laccases and “en-
gineered” fungal laccases are known,[5d–h] to the best of our
knowledge, biocatalytic Michael-type reactions that use alkalo-
philic laccases under basic conditions have not been investi-
gated yet.
3
[a] Reaction conditions: 0.25 mmol 7, 1.2 equiv. 8, 3 mL solvent, 228C,
buffer: 50 mm Gly/NaOH, pH 9.0. [b] Reaction performed in a KPi-buffer of
pH 6.0. [c] Activity as provided by the supplier with catechol 4 as sub-
strate at pH 6 (6.8 UmgÀ1). Activity determined with
3 at pH 5.5
(0.9 UmgÀ1). [d] Activity determined with 3 at pH 4 (2.5 UmgÀ1). [e] Yields
Herein, we report the biocatalytic performance of the lac-
case Ssl 1 towards the 3-arylation of 3-substituted 4-oxochro-
manes (A), which leads to potentially pharmacologically inter-
esting entities (B) that bear an all-carbon quaternary stereo-
genic centre. As the key step, the laccase-catalysed oxidation
of commercially available catechols (C) to form the highly reac-
tive o-quinone analogue (D) was performed at pH 9 followed
by a regio- and diastereoselective 1,4-addition with the activat-
ed donor (E) as a soft nucleophile (Scheme 1).
of isolated products.
ous approach that used Pb-mediated arylations of chromanone
motifs,[18] we present an environmentally benign alternative. In
a first attempt to prove the general suitability of the chroma-
none ester 7, the optimised reaction conditions from a previous
protocol were considered in which the commercially available
fungal laccase from Agaricus bisporus was used under slightly
acidic conditions.[6b] Under acidic conditions, we could instantly
isolate the desired product as a single regioisomer as deter-
1
mined by H NMR spectroscopy (62%, Table 1, entry 1). Howev-
er, full conversion was not achieved even after 48 h of reaction
time. As anticipated, if the fungal laccase is used under basic
conditions, its activity towards the oxidation of 8 decreased
significantly, which was indicated by a poor yield (31%,
entry 2). Even with an increased nucleophilicity of the nucleo-
phile under basic conditions, the limiting factor seemed to be
the low efficiency of catechol oxidation. We started the investi-
gation with the bacterial laccase Ssl 1 from Streptomyceus svi-
Scheme 1. Laccase-catalysed 3-arylation of 3-substituted 4-oxochromanes A.
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ceus[8] and used a basic pH of 9 in all cases for its optimal activ-
ity towards catechol oxidation. Acetonitrile was chosen as
a co-solvent. The Ssl 1 was stored in 50 mm KPi-buffer (potassi-
um phosphate buffer: KH2PO4/K2HPO4) of pH 7.5. The deter-
mined volumetric activity of the laccase solution was 40 U/mL
towards the oxidation of 3 at pH 4. For the first attempt, 16 U
of Ssl 1 was used, and the desired product 7 was formed in
a good yield (67%, entry 3). Again, as expected, only one re-
gioisomer was isolated because of the favoured coupling at
the 1’-position. Remarkably, the reaction time could be re-
duced significantly to 3 h for full conversion. In contrast to the
first approaches that used the fungal laccase, both
proach satisfactorily under acidic conditions. Moreover, Ssl 1
laccase showed an outstanding tolerance towards organic co-
solvents as well as short reaction times and complete regiose-
lectivity. As a result of solubility issues, the use of organic co-
solvents was mandatory for the successful outcome of these
approaches.
Next, we investigated the scope of the reaction. For this pur-
pose, commercially available catechols substituted in the 3’-
and 4’-position as well as chromanone esters prepared previ-
ously (Supporting Information) substituted in the 2’- and 6’-po-
sition were considered (Table 2). First, we used 7 and 3’-substi-
the oxidation of the catechol as well as the increase
Table 2. Laccase-catalysed oxidation/Michael addition sequence.[a]
of nucleophilicity of the chromanone were addressed
in the second approach that employed the bacterial
laccase, which thus accelerated the desired reaction.
If the amount of Ssl 1 was decreased to 8 U, the yield
could be further increased and the reaction time was
extended slightly to 4 h (86%, entry 4). We suggest
that this outcome could be explained by a possible
shift of the pH value in the reaction mixture. As we
applied this laccase directly as a solution in 50 mm
KPi-buffer with a pH of 7.5 and diluted it afterwards
with the Gly/NaOH-buffer (pH 9) to the desired ratio,
a higher amount of laccase could lead to more acidic
reaction conditions and consequently to a decrease
of the rate. If we used methanol as a co-solvent
under the same conditions, the outcome was similar
to that of the previous approach. With the same ratio
of buffer to co-solvent, full conversion was achieved
Entry
R1
R2
R3
Ester
Catalyst
Product
Reaction time
[h]
Yield
[%][b]
1
2
3
4
5
H
H
H
Me
F
MeO
H
H
H
Me
H
H
Me
H
H
7
7
7
15
17
10
4
13
4
11
12
14
16
18
4
8
7
8
3
64
70
66
85
74
8
[a] Reaction conditions: 0.25 mmol ester, 1.2 equiv. catechol, 8 U (40 UmLÀ1) laccase
from Streptomyces sviceus, 3 mL solvent with MeCN/buffer 1:2, 228C, buffer: 50 mm
Gly/NaOH, pH 9.0. [b] Yields of isolated products.
after 4 h with a good yield (72%, entry 5). A further decrease
of the amount of enzyme to 4 U did not have a positive effect;
the reaction was incomplete after 3 h and the yield decreased
significantly (39%, entry 6). As anticipated, if we used only
buffer as the reaction medium, the amount of isolated product
was very low (21%, entry 7). The solubility of 7 was very poor
in water only. If we increased the amount of co-solvent to an
equal ratio with the buffer, a good yield was achieved (70%,
entry 8), but the reaction time until full conversion was in-
creased to 24 h. However, the laccase showed a high tolerance
to organic co-solvents even at high ratios. Fortunately, if we
used a ratio of 2:1 buffer to acetonitrile we obtained full con-
version after 3 h and a very good yield (89%, entry 9). If the
ratio was shifted to 4:1 towards buffer, the outcome of the re-
action was nearly the same in terms of yield (86%, entry 4),
but the reaction time was increased slightly (4 h). Finally, with
this optimised ratio of buffer to co-solvent in hand, we re-eval-
uated the organic solvent and used methanol and THF as alter-
natives. The result for methanol did not change compared to
the previous approach (71%, entry 10 vs. 72%, entry 5), but
the shift towards a higher ratio of buffer resulted in an in-
creased reaction time of 4 h. With THF as a co-solvent, the
yield of isolated product decreased further (52%, entry 11). In
summary, the bacterial laccase Ssl 1 (8 U) and a ratio of 2:1
buffer/acetonitrile at a pH of 9 were proven to be the best re-
action conditions for this arylation (89%, entry 9). The commer-
cially available fungal laccase could not perform in this ap-
tuted catechols such as 3-methoxycatechol (10). The desired
product 11 was obtained in a good yield after 4 h (64%,
Table 2, entry 1). As expected, coupling only occurred in the 5’-
position of the catechol because of the electron-donating
group in the 3’-position that makes the ortho position unat-
tractive for nucleophilic attack. If the unsubstituted catechol 4
was used, a slightly better yield was obtained after 8 h reaction
time for full conversion (70%, entry 2). The use of 4-methylca-
techol (13) gave the expected single regioisomer 14 after 7 h
in a good yield (66%, entry 3). The 4’-position of the catechol
13 was clearly not available for the coupling. Next, the chro-
manone scaffold was varied, and 6’-methylchromanone ester
15 was used first. A very good yield was obtained after 8 h re-
action time if unsubstituted catechol 4 was used as the accept-
or (85%, entry 4). Last, 6’-fluorochromanone ester 17 was re-
acted selectively with 8, and within 3 h regioisomer 18 was
formed in good yield (74%, entry 5).
We were intrigued to investigate the simple diastereoselec-
tivity of the coupling reaction and hence introduced the 2-
methyl-substituted chromanone 19 (Supporting Information)
to the reaction conditions. To our delight, if racemic ester 19
and 8 were used, complete regio- and diastereoselectivity of
>99:1 (determined by chiral HPLC) was achieved with a very
good yield of 91% (Scheme 2). For both racemic diastereoiso-
mers 19 the arylation occurred in the anti position with re-
spect to the methyl group in the 2’-position, which is in full
agreement with a related report.[3c]
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Scheme 2. Diastereoselective arylation of racemic 2’-methylchromanone 19.
As both the reduction of 2-methylchromene 21 under flow
conditions (95%, Table S2, entry 4) and the laccase-catalysed
arylation of chromanone ester 7 (52%, Table 1, entry 11) could
be performed using THF as a solvent, an additional consecu-
tive one-pot approach to combine these two reaction steps
was anticipated. Again 2-methylchromene 21 was reduced in
under flow conditions and the formed ester 19 was introduced
directly into a flask that contained Ssl 1 solution in buffer to
which catechol 8 was added by using a syringe pump
(Scheme 3), a set-up that was successfully established pre-
Scheme 4. Laccase-catalysed sequence using 3-halocatechols 22 and 23.
synthesis, the scope was broadened by using alternative nucle-
ophiles from our protocols developed previously in which
commercially available fungal laccases were applied. As a first
comparison, the b-ketoester 26 was employed as a suitable
nucleophile again using 8 as the arylating agent. Previously,
the reaction was performed successfully, and the desired prod-
uct 27 was obtained in a very good yield using the fungal lac-
case from Pleurotus ostreatus (86% after 72 h). Almost com-
plete regioselectivity was achieved with a ratio of 96:4 toward
the arylated b-ketoester 27.[3c] The use of the optimised reac-
tion conditions and Ssl 1 gave a comparable yield and the se-
lectivity was increased to provide the 5’-coupling product ex-
clusively (86%; Scheme 5A). However, the most pleasing
Scheme 3. Diastereoselective synthesis of ester 20 in a consecutive one-pot
approach.
viously.[6b] However, as the outcome of the arylation in THF
was not satisfactory in the first place (vide supra), the amount
of enzyme was doubled (16 U instead of 8 U) and the flow
rates were adjusted. We were pleased to find that despite the
higher dilution (recommended concentration for the reduction
in flow conditions: 0.5 m) for the enzyme-catalysed transforma-
tion (a factor of 6), the results matched the batch reactions.
The regio- and diastereoselectivity was excellent as was the
yield (96%, diastereomeric ratio (dr)>99:1, 4 h; Scheme 3).
Finally, the substituents of the catechol moiety were altered.
For this approach, 3-fluorocatechol (22) and 3-bromocatechol
(23) were utilised as arylation agents for chromanone 7. After
7 h reaction time, the desired products were isolated in good
to very good yields of 79% for the fluorine derivative 24 and
63% for the bromine derivative 25 (Scheme 4). As expected,
both possible regioisomers were obtained. In the case of the
fluorine-containing scaffold 24, the ratio of regioisomers was
determined to be 73:27 in favour of 24A (arylation in the 5’-
position). In contrast, if we used bromocatechol 23, the ratio
decreased to 52:48 albeit slightly in favour of 25A. Neverthe-
less, the outcome was surprising as laccase-catalysed arylations
with 23 were described previously to provide coupling exclu-
sively in the 4’-position.[19] Clearly, the choice of nucleophile
also influences the outcome of the regioselectivity significantly.
To demonstrate the general superiority of the laccase Ssl 1
as a new biocatalyst for oxidative CÀC couplings in organic
Scheme 5. Improved laccase-catalysed arylation protocols.
aspect was that the reaction time could be decreased drastical-
ly from 72 to 3 h. Scale-up was unproblematic, and to our de-
light, we were able to isolate the desired arylated b-ketoester
27 as major regioisomer on a 5.0 mmol scale with comparable
yield and selectivity (85%; see Supporting Information). Similar
results were obtained if the protected oxindole 28 was used as
the coupling partner for 4 (Scheme 5B). With the acidophilic
fungal laccase, product 29 was synthesised in two steps in
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3J2a,3 =8.2 Hz, 2a-H), 4.63 (dd, 1H, J2b,2a =11.6 Hz, J2b,3 =4.4 Hz, 2b-
H), 3.83 (s, 3H, CO2CH3)*, 3.79 (s, 3H, CO2CH3), 3.75 ppm (dd, 1H,
3J3,2a =8.2 Hz, 3J3,2b =4.4 Hz, 3-H); 13C NMR (151 MHz, CDCl3): d=
186.87 (C-4), 167.66 (CO2CH3), 162.82 (CO2CH3)*, 161.36 (C-8a),
157.66 (C-4)*, 136.44 (C-7), 133.07 (C-7)*, 127.64 (C-5), 124.56 (C-5)*,
124.55 (C-8a)*, 121.86 (C-6), 121.48 (C-6)*, 120.43 (C-4a), 118.24 (C-
4a)*, 117.92 (C-8), 116.45 (C-8)*, 91.73 (C-3)*, 68.15 (C-2), 63.76 (C-
2)*, 52.79 (C-3), 52.46 (CO2CH3), 51.69 ppm (CO2CH3)* (*signals from
the corresponding enol form): IR (ATR): n˜ =2955, 2921, 1737, 1725,
1682, 1457, 1329, 761 cmÀ1; HRMS (ESI): m/z: calcd for C11H10O4Na+
229.04713 [M+Na+]; found 229.04710.
2
3
69% yield; the oxidative laccase-catalysed coupling took
16 h.[6a] With the use of Ssl 1, not only was the reaction time re-
duced significantly to 4 h but also the yield could be increased
to 78% over two steps towards oxindole 29 (Scheme 5B).
Again, because of purification issues, the methylation step was
necessary for this substrate. As in the last example, we consid-
ered the structural motif of coumarins as a suitable coupling
partner and in particular the acetyl-substituted 3,4-dihydrocou-
marin 30 because the lowest yield for this scaffold was ob-
served previously (63% after 18 h).[6b] The use of Ssl 1 to syn-
thesise derivative 31 again improved the yield and decreased
the reaction time (79% after 3 h; Scheme 5C).
Laccase-catalysed arylation: Methyl 3-(3,4-dihydroxyphenyl)-
4-oxochromane-3-carboxylate (12)
Conclusions
Chromanone 7 (51.6 mg, 0.25 mmol) and catechol 4 (33.1 mg,
0.30 mmol) were dissolved in acetonitrile (1.00 mL) and buffer
(1.80 mL; 50 mmol Gly/NaOH, pH 9). The laccase Ssl 1 (0.20 mL,
8 U) was added dropwise to the vigorously stirred mixture, and
stirring was continued for 8 h at 228C under air. The reaction was
quenched by the addition of 1m HCl (1–2 drops), EtOAc (5.00 mL)
and water (5.00 mL). The layers were separated and the aqueous
phase was extracted with EtOAc three times. The combined organ-
ic layer was washed with brine, dried with MgSO4 and filtered, and
the solvent was evaporated under reduced pressure. The crude
product was purified using flash column chromatography (PE/
EtOAc 70:30) to provide product 12 as a yellow-brown viscid solid
The synthesis of a new compound class of 3-arylated 4-oxo-
chromanes was established successfully by an efficient three-
step sequence, which gave excellent yields and regioselectivi-
ties. The key to success was the use of the bacterial Ssl 1 lac-
case from S. sviceus for the environmentally benign oxidation
with aerial oxygen. It showed an outstanding performance in
terms of reaction time, yield and tolerance towards organic
solvents, which thus proved its superiority compared to fungal
laccases. Moreover, complete simple diastereoselectivity was
already induced by an additional methyl group. High yields for
the single-step transformations also rendered a convenient
consecutive one-pot approach possible, which also furnished
the pharmacologically promising scaffold in excellent yield and
diastereoselectivity. These findings consolidate the Ssl 1 laccase
as a valuable biocatalyst for Michael additions. Its catalytic
properties enabled us to perform these oxidative CÀC cou-
plings in a highly efficient manner under basic conditions for
the first time.
(70%, 0.17 mmol, 54.7 mg). Mp 1288C; Rf =0.11 (PE/EtOAc 70:30);
4
1H NMR (600 MHz, CDCl3): d=7.95 (dd, 1H, 3J5,6 =8.0 Hz, J5,7
=
3
3
4
1.8 Hz, 5-H), 7.48 (ddd, 1H, J7,8 =8.6 Hz, J7,6 =7.1 Hz, J7,5 =1.8 Hz,
7-H), 7.03 (ddd, 1H, 3J6,5 =8.1 Hz, 3J6,7 =7.1 Hz, 4J6,8 =1.1 Hz, 6-H),
6.94 (dd, 1H, 3J8,7 =8.4 Hz, 4J8,6 =1.1 Hz, 8-H), 6.81 (d, 1H, J2’,6’
2.3 Hz, 2’-H), 6.79 (d, 1H, J5’,6’ =8.4 Hz, 5’-H), 6.72 (dd, 1H, J6’,5’
4
=
=
3
3
4
8.4 Hz, J6’,2’ =2.3 Hz, 6’-H), 5.83 (s, 1H, OH), 5.64 (s, 1H, OH), 5.04
(d, 1H, 2J2a,2b =12.0 Hz, 2a-H), 4.87 (d, 1H, 2J2b,2a =12.0 Hz, 2b-H),
3.76 ppm (s, 3H, CO2CH3); 13C NMR (151 MHz, CDCl3): d=189.20 (C-
4), 169.35 (CO2CH3), 160.86 (C-8a), 144.11 (C-3’), 143.73 (C-4’),
136.45 (C-7), 128.19 (C-5), 124.91 (C-1’), 121.91 (C-6), 120.18 (C-6’),
120.08 (C-4a), 117.83 (C-8), 115.46 (C-2’), 115.28 (C-5’), 71.83 (C-2),
61.64 (C-3), 53.32 ppm (CO2CH3); IR (ATR): n˜ =3396, 2955, 1727,
1679, 1604, 1232, 1209, 1037, 757 cmÀ1; HRMS (ESI): m/z: calcd for
Experimental Section
Representative procedures
+
C17H15O6 [M+H+] 315.08631; found 315.08629.
Reductions under flow conditions: Methyl 4-oxochromane-3-
carboxylate (7)
Consecutive one-pot approach: Methyl 3-(3,4-dihydroxy-5-
methylphenyl)-2-methyl-4-oxochromane-3-carboxylate (20)
The H-Cube Pro flow system[17] by ThalesNano was equipped with
a 20% Pd(OH)2/C (30 mm) catalyst cartridge and absolute THF and
the conditions (7 atm, 228C, 40% H2, 0.3 mLminÀ1) were entered.
Methyl 4-oxo-4H-chromene-3-carboxylate (1.20 g, 5.89 mmol) was
dissolved in absolute THF (120 mL). After the flow system had sta-
bilised the desired conditions, the reduction was started, and the
dead volumes of the system (2.59 mL) and of the catalyst cartridge
(0.19 mL) were taken into consideration. The crude product solu-
tion was evaporated and purified using flash column chromatogra-
phy (PE/EtOAc 75:25). Compound 7 was obtained as a pale-rose
solid (86%, 5.04 mmol, 1.04 g). Mp 588C; Rf =0.60 (PE/EtOAc
60:40); 1H NMR (600 MHz, CDCl3): d=11.95 (br s, 1H, OH)*, 7.93
The H-Cube Pro flow system[17] by ThalesNano was equipped with
a 20% Pd(OH)2/C (30 mm) catalyst cartridge and absolute THF. The
reduction conditions (7 atm, 248C, 60% H2, 0.3 mLminÀ1) were en-
tered. Methyl 2-methyl-4-oxo-4H-chromene-3-carboxylate (21)
(54.5 mg, 0.25 mmol) was dissolved in absolute THF (5.00 mL). 3-
Methylcatechol (8) (38.1 mg, 0.30 mmol) was dissolved in absolute
THF (1.00 mL) and taken up into a syringe (to a volume of
3.00 mL). A syringe pump was prepared and equipped with the sy-
ringe. Its flow rate was adjusted to that of the flow system
(0.07 mLminÀ1). The laccase Ssl 1 (0.40 mL, 16 U) was dissolved in
buffer (12.0 mL; 50 mmol Gly/NaOH, pH 9) in a round-bottomed
flask (100 mL) equipped with a stirring bar. After the flow system
had stabilised the desired conditions, the reduction was started.
The dead volumes of the system (2.59 mL) and of the catalyst car-
tridge (0.19 mL) were considered. Simultaneously, the syringe
pump was started, and catechol 8 and the crude reagent solution
from the flow system were introduced into the flask that contained
3
4
3
(dd, 1H, J5,6 =7.9 Hz, J5,7 =1.7 Hz, 5-H), 7.66 (dd, 1H, J5,6 =7.7 Hz,
4J5,7 =1.7 Hz, 5-H)*, 7.51 (ddd, 1H, J7,8 =8.7 Hz, J7,6 =7.2 Hz, J7,5
=
3
3
4
3
3
4
1.8 Hz, 7-H), 7.32 (ddd, 1H, J7,8 =8.2 Hz, J7,6 =7.3 Hz, J7,5 =1.7 Hz,
7-H)*, 7.06 (ddd, 1H, 3J6,5 =7.9 Hz, 3J6,7 =7.2 Hz, 4J6,8 =1.1 Hz, 6-H),
7.00 (ddd, 1H, 3J6,5 =7.7 Hz, 3J6,7 =7.3 Hz, 4J6,8 =1.1 Hz, 6-H)*, 6.99
3
4
3
(dd, 1H, J8,7 =8.7 Hz, J8,6 =1.1 Hz, 8-H), 6.87 (dd, 1H, J8,7 =8.2 Hz,
4J8,6 =1.1 Hz, 8-H)*, 4.96 (s, 2H, 2-H)*, 4.81 (dd, 1H, J2a,2b =11.6 Hz,
2
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Full Papers
f) S. Riva, Trends Biotechnol. 2006, 24, 219–226; g) A. Kunamneni, S. Ca-
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the laccase. The reaction was stirred vigorously for 4 h at 228C
under air. The mixture was acidified with 1m HCl (1–2 drops) and
quenched with EtOAc (10.0 mL) and water (10.0 mL). The layers
were separated, and the aqueous phase was extracted with EtOAc
three times. The combined organic layer was washed with brine
and dried with MgSO4. After filtration, the solvent was evaporated
under reduced pressure, and the crude product was purified using
flash column chromatography (PE/EtOAc 80:20). An orange viscid
solid (96%, 0.24 mmol, 78.8 mg) was obtained for the anti diaste-
reoisomer 20.[3c] Rf =0.10 (PE/EtOAc 80:20); Rf =0.22 (PE/EtOAc
ˇ
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70:30); 1H NMR (600 MHz, CDCl3): d=7.95 (dd, 1H, 3J5,6 =8.0 Hz,
4
4J5,7 =1.7 Hz, 5-H), 7.49 (ddd, 1H, 3J7,8 =8.7 Hz, 3J7,6 =7.1 Hz, J7,5
=
3
3
4
1.8 Hz, 7-H), 7.04 (ddd, 1H, J6,5 =8.1 Hz, J6,7 =7.2 Hz, J6,8 =1.1 Hz,
6-H), 6.98 (dd, 1H, 3J8,7 =8.5 Hz, 4J8,6 =1.1 Hz, 8-H), 6.53 (d, 1H,
4J2’,6’ =2.3 Hz, 2’-H), 6.50 (d, 1H, 4J6’,2’ =2.4 Hz, 6’-H), 5.94 (s, 1H,
3
OH), 5.44 (s, 1H, OH), 4.90 (q, 1H, J2,2ÀCH =6.5 Hz, 2-H), 3.69 (s, 3H,
3
CO2CH3), 2.21 (s, 3H, 5’-CH3), 1.54 ppm (d, 3H, 3J2ÀCH ,2 =6.5 Hz, 2-
3
CH3); 13C NMR (151 MHz, CDCl3): d=190.73 (C-4), 168.43 (CO2CH3),
160.66 (C-8a), 143.20 (C-3’), 142.38 (C-4’), 136.12 (C-7), 128.29 (C-5),
125.77 (C-5’), 124.70 (C-1’), 122.41 (C-6’), 121.73 (C-6), 120.64 (C-4a),
117.83 (C-8), 113.16 (C-2’), 80.43 (C-2), 65.74 (C-3), 52.72 (CO2CH3),
16.24 (2-CH3), 15.80 ppm (5’-CH3); IR (ATR): n˜ =3419, 2924, 1727,
1688, 1606, 1462, 1295, 1222, 762 cmÀ1; HRMS (ESI): m/z: calcd for
´
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C19H19O6 [M+H+] 343.11761; found 343.11760.
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Acknowledgements
We gratefully acknowledge the Ministry of Innovation, Science
and Research of the German Federal State of North Rhine-West-
phalia (NRW) and the Heinrich-Heine-Universitꢁt Dꢀsseldorf
(HHU), the Deutsche Forschungsgemeinschaft, and the For-
schungszentrum Jꢀlich GmbH for the support of our projects. We
additionally thank Birgit Henßen for carrying out the HPLC ana-
lytics and Melanie Wachtmeister for preparing the laccase.
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Keywords: enzyme catalysis
addition · multicomponent reactions · oxidation
·
hydrogenation
·
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Received: February 12, 2015
Published online on && &&, 0000
&
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FULL PAPERS
´
It’s all in the laccase: A laccase-cata-
lyzed oxidative regio- and diastereo-
selective Michael addition sequence
towards a new compound class of 3-
arylated 4-oxochromanes, with the crea-
tion of a quaternary carbon stereogenic
center, is described. Reactions are per-
formed in basic media using an alkalo-
philic laccase from Streptomyces sviceus.
Its general utility as a biocatalyst with
several different nucleophiles is demon-
strated. Bn=Benzyl; Boc=tert-butoxy-
carbonyl.
S. Suljic, F. B. Mortzfeld, M. Gunne,
V. B. Urlacher, J. Pietruszka*
&& – &&
Enhanced Biocatalytic Performance of
Bacterial Laccase from Streptomyces
sviceus: Application in the Michael
Addition Sequence Towards 3-
Arylated 4-Oxochromanes
ChemCatChem 0000, 00, 0 – 0
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ꢀ 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ