Enzymatic oxidative cyclisation reactions leading to dibenzoazocanes

Article abstract of DOI:10.1055/s-0030-1258486

From simple N-isovanillyltyramine derivatives double oxidative biotransformations can be achieved using tyrosinase leading to the corresponding hydroxylated dibenzoazocanes. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0030-1258486

LETTER
1919
Enzymatic Oxidative Cyclisation Reactions Leading to Dibenzoazocanes
Cyclisation
R
e
r
actions
a
L
eadingto
n
Dibenzoazoc
c
anes esco Tozzi, Steven V. Ley, Matthew O. Kitching, Ian R. Baxendale
Innovative Technology Centre, Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK
Fax +44(1223)336442; E-mail: theitc@ch.cam.ac.uk
Received 5 April 2010
ducted, rather the enzymes were used as supplied without
further purification although an SDS PAGE analysis of
the commercial extracts gave us some idea of their purity
(Figure 1).
Abstract: From simple N-isovanillyltyramine derivatives double
oxidative biotransformations can be achieved using tyrosinase lead-
ing to the corresponding hydroxylated dibenzoazocanes.
Key words: enzymes, oxidation, biotransformation, heterocycles
The development of new oxidation methods for phenolic
compounds remains an enduring area of research interest.
Not only are these methods desirable because of their po-
tential similarity to biological processes,1 the iterative ap-
plication of such oxidations can rapidly lead to ever more
complex structures. Unfortunately, current chemical ap-
proaches to these individual oxidation steps usually re-
quire stoichiometric reagents and can suffer from
problems of chemoselectivity,2 harsh conditions, high re-
agent costs, and the generation of toxic byproducts. By
comparison enzymatic methods address many of these
concerns but can often only be applied to very specific or
simple substrates.3 Having developed some expertise with
enzymatic oxidation methods for organic synthesis4 we
decided to investigate the potential of enzyme-mediated
oxidative cyclisation reactions of biaryl compounds con-
taining phenolic groups, in the hope they would lead to
new natural-product-like scaffolds.
Figure 1 SDS-PAGE analysis of extracts containing tyrosinase
(Tyr) and horseradish peroxidase (HRP)
Unfortunately, despite extensive screening of the reaction
conditions no identifiable products could be isolated from
the incubation of substrate 1 with these enzymes. In all
cases, only unreacted starting material 1 or highly insolu-
ble polymeric byproducts were observed. We therefore
decided to protect the amine functionality due to its sus-
pected role in inhibiting the desired oxidative cyclisation
reaction. Formylation of 1 was achieved under microwave
irradiation in neat ethylformamide with a catalytic amount
of formic acid which gave 87% yield of the crystalline
product 4 on standing.8 Similarly, we also prepared the tri-
fluoroacetyl derivative 510 by standard methods in 78%
isolated yield following column chromatography
(Scheme 1).
Previous work using enzymes to effect inter- and intramo-
lecular hetero-bond formation is known although carbon–
carbon bond-forming events are rare.5 Our aim therefore
was to devise suitable substrates and investigate their se-
lective oxidative biotransformations to architecturally
more elaborate products. For the initial phase of these
studies reported here, we prepared the norbelladine deriv-
ative, N-isovanillyltyramine (1) as a model substrate since
the products could be expected to mimic natural products
from the amaryllidaceae family6 and are of particular in-
terest to our group.7
The secondary amine 1 was readily obtained via a reduc-
tive amination procedure using isovanillin (2) and
tyramine (3) in methanol to afford 18 in 86% isolated yield
following crystallisation from methanol (Scheme 1). The
oxidase enzymes selected for screening were horseradish
peroxidase (HRP, EC 1.11.1.7) and tyrosinase (Tyr, EC
1.14.18.1) as these two had shown good substrate scope in
other studies.9 Since our focus was on the reactivity of the
substrate no specific enzymatic activity study was con-
OH
OH
MeO
OH
MeO
O
R
a
2
3
N
H
H2N
1 R = H
b
4 R = CHO
c
5 R = COCF3
OH
Scheme 1 Synthetic route to the starting materials. Reagents and
conditions: (a) MeOH, r.t., 3 h, then NaBH4, 0 °C to r.t., 3 h, 86%; (b)
HCOOEt, HCO2H (cat.), MW 120 °C, 30 min, 87%; (c) TFAA (1.5
equiv), pyridine, 0 °C to r.t., 2 h, 78%.
SYNLETT 2010, No. 13, pp 1919–1922
Advanced online publication: 09.07.2010
DOI: 10.1055/s-0030-1258486; Art ID: D08510ST
© Georg Thieme Verlag Stuttgart · New York
x
x
.x
x
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0
1
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1920
F. Tozzi et al.
LETTER
The N-formyl-protected amine 4 was then treated with boat-chair (TBC) conformation of the eight-membered
HRP but afforded only decomposition products, however, ring existing as a racemic mixture of atropoisomers.15
more promising results were obtained using the tyrosinase This result was confirmed by optical rotation measure-
1
enzyme system. A basic set of test conditions using a 4:1 ments as well as H NMR studies which supported the
mixture of buffer and acetone (to solubilise the substrate) same observation in solution. Moreover, all the NMR sig-
under an oxygen atmosphere at ambient temperature was nals were duplicated in a general ratio of between 7:3 and
employed. Conversion to a single product was observed 8:2 depending on the exact deuterated solvent used. Even
which, following chromatographic purification, could be using VT-NMR analysis a complete coalescence of the
isolated in 17% yield and its structure determined to be peaks could not be achieved (DMSO, 140 °C). Separation
that of compound 6.11 Further optimisation experiments of the species via preparative supercritical chromatogra-
using lower enzyme loadings proceeded well without del- phy (CO2–i-PrOH) and NMR analysis of the major frac-
eteriously impacting on the rate of conversion while high- tion afforded again the same ratio, thus strongly
er dilution led to faster reaction times and increased yields suggesting the presence of a rotameric equilibrium of the
of around 50% (Scheme 2). Moreover, we discovered it formamide group. A favourable p-stacking interaction
was possible to use air as the co-oxidant in these experi- can be observed between the carbonyl and aryl ring lead-
ments, although necessitating extended reaction times, ing to the high stability of these structural forms at least in
pleasingly gave no reduction in the final yields.
the crystalline state.
This result suggests that the enzyme initially induces a To further investigate the mechanism of the reaction we
very specific chemo- and regioselective ortho oxidation found that the catechol intermediate 8 could be isolated in
of the mono phenolic ring. This is then followed by a sec- 68% yield if the reaction was conducted in the presence of
ond oxidation step presumably via an unstable ortho- excess reductant such as L-sodium ascorbate (Scheme 2,
quinone which itself then undergoes rapid intramolecular conditions b). Having this intermediate available allowed
nucleophilic attack by the second electron-rich aryl ring us to intercept the catalytic cycle and further investigate
eventually yielding the substituted dibenzoazocane 6 the documented lag time period that has been observed in
(Scheme 2). Under optimised conditions the reaction pro- many tyrosinase-mediated reactions.16
ceeded to full conversion in 1.5 hours using 1% bulk
It has been established that the active site of tyrosinase is
weight of enzyme (14 KU/mmol). The product was readi-
present in three forms: deoxy (Tydeoxy), oxy (Tyoxy), and
ly isolated by direct quenching of the reaction mixture
met (Tymet, Figure 3). The first of these is a free form that
with 1 M hydrochloric acid, followed by extraction of the
is able to bind molecular oxygen producing the oxy form,
solution with EtOAc, and finally purification of the resi-
which is active in both the cresolase and the catecholase
due by column chromatography (Scheme 2).12
cycle.17 The met form on the other hand is only able to ca-
talyse oxidation to the dione18 but is the predominant form
HO
found in the crude enzyme (Figure 3). The observed in-
duction period (lag time) is due to an autocatalytic mech-
MeO
anism where the small amount of oxy form present
facilitates a chemical oxidation producing an ortho-dihy-
R
a
N
4 or 5
droxy benzene. Once present, this intermediate can relay
all of the met form to the corresponding deoxy state. The
deoxy form is then capable of binding oxygen to generat-
HO
6 R = CHO
7 R = COCF3
HO
ing the oxy form allowing the catalytic cycle to reach
b
steady-state operation.16
OH
c
This was exemplified by adding 5 mol% of the intermedi-
ate 8 to the standard reaction mixture. This yielded the de-
sired product 6 in under 1 hour instead of the typical 1.5
hours. As a control, doping the reaction mixture with the
product 6 or catechol as the exogenous bis-ortho-hydrox-
ylated activator, respectively, had no influence at all, or in
the case of catechol, produced secondary unwanted
byproducts and deactivated the enzyme (Table 1).
MeO
R
N
OH
OH
8 R = CHO
9 R = COCF3
Scheme 2 Oxidative enzymatic reactions. Reagents and conditions:
(a) 4, Tyr (14 KU/mmol), O2, buffer–acetone (4:1), 1.5 h, 50%; 5, Tyr
(28 KU/mmol), O2, buffer–acetone (3:1), 2 h, 53%; (b) 4, Tyr (28 KU/
mmol), O2, buffer–acetone (4:1), sodium ascorbate (10 equiv), 4 h,
68%; 5, Tyr (56 KU/mmol), O2, buffer–acetone (3:1), sodium ascor-
bate (10 equiv), r.t., 6 h, 68%; (c) 8, Tyr (14 KU/mmol), O2, buffer–
acetone (4:1), 1 h, 58%; 9, Tyr (28 KU/mmol), O2, buffer–acetone
(3:1), 1 h, 56%.
Modification of the protecting group with a more labile
trifluoroacetoxy moiety proved equally viable, and re-
quired only minor modifications (acetone/buffer 1:3, 28
KU/mmol) to the enzymatic reaction conditions
(Scheme 2). The alternative product 7 could be isolated in
almost identical yield (58%) after two hours. As in the
case of compound 6, product 7 was obtained as a racemic
atropoisomeric mixture and in a TBC conformation of the
Crystal structure analysis by X-ray methods of this new
dibenzoazocane derivative 613 (which is related to the
apogalanthamine alkaloid family)14 revealed a twisted
Synlett 2010, No. 13, 1919–1922 © Thieme Stuttgart · New York

LETTER
Cyclisation Reactions Leading to Dibenzoazocanes
1921
Figure 2 Crystal structures of 6 and 7, respectively
dibenzoazocane ring as confirmed by its X-ray analysis In conclusion, we have demonstrated the power of en-
(Figure 2). However, no rotameric forms were observed zyme oxidases to bring about selective late tailoring oxi-
in the 1H NMR at ambient conditions.
dation processes on less functionalised substrates to
afford new natural-product-like structures.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
Acknowledgment
We gratefully acknowledge financial support from the Foundation
Toso-Montanari of the University of Bologna (to F.T.), the EPSRC
(M.O.K.), the Royal Society (to I.R.B.) and the BP endowment (to
S.V.L.). We also wish to thank Dr. J. E. Davies for determining the
crystal structures of compounds 6 and 7 and the EPSRC for a finan-
cial contribution towards the purchase of the diffractometer. In ad-
dition we would like to acknowledge and thank Pawel Sledz for
performing the SDS-PAGE analysis.
References and Notes
(1) (a) Stasiuk, M.; Kozubek, A. Cell. Mol. Life Sci. 2010, 67,
841. (b) Li, L.; Zhang, Y.; Shang, H.-L.; Xie, J.-Q. Huaxue
Gongcheng (Chem. Eng.) 2008, 36, 36. (c) Kotali, A.
ARKIVOC 2009, (i), 81. (d) Dohi, T.; Kita, Y. Chem.
Commun. 2009, 2073. (e) Maki-Arvela, P.; Holmbom, B.;
Salmi, T.; Murzin, D. Y. Catal. Rev. Sci. Eng. 2007, 49,
197. (f) Itoh, S.; Fukuzumi, S. Acc. Chem. Res. 2007, 40,
592. (g) Shetty, K.; McCue, P. Food Biotechnol. 2003, 17,
67. (h) Moonen, M. J. H.; Fraaije, M. W.; Rietjens,
I. M. C. M.; Laane, C.; van Berkel, W. J. H. Adv. Synth.
Catal. 2002, 344, 1023. (i) Eickhoff, H.; Jung, G.; Rieker,
A. Tetrahedron 2001, 57, 353.
(2) For example, see: Oxidation, In Comprehensive Organic
Synthesis, Vol. 7; Trost, B. M.; Fleming, I., Eds.; Pergamon
Press: Oxford, 1991.
(3) (a) Waldmann, H.; Drauz, K. In Enzyme Catalysis in
Organic Synthesis: A Comprehensive Handbook, Vols. 1
and 2; Wiley-VCH: Weinheim, 1995. (b) Liese, A.;
Wandrey, C.; Seelbach, K. In Industrial
Figure 3 Catalytic active forms of tyrosinase and associated cataly-
tic cycle
Table 1 Investigation of Lag Time
Entry
Activatora
Reaction time (h) Conversion (%)b
1
2
3
4
none
1.5
1
99
99
99
80c
8
6
1.5
3.5
catechol
Biotransformations: A Comprehensive Handbook; Wiley-
VCH: Weinheim, 2000. (c) Vulfson, E. N. In Enzymes in
Nonaqueous Solvents: Methods and Protocols (Methods in
Biotechnology); Humana Press: Totowa, NJ, 2001.
a Conditions: 5 mmol%.
b As determined by LC-MS analysis.
c Additional products with no additional turnover after 1.5 h.
Synlett 2010, No. 13, 1919–1922 © Thieme Stuttgart · New York

1922
F. Tozzi et al.
LETTER
(4) (a) Hollinshead, D. M.; Howell, S. C.; Ley, S. V.; Mahon,
M.; Ratcliffe, N. M.; Worthington, P. A. J. Chem. Soc.,
Perkin Trans. 1 1983, 1579. (b) Ley, S. V.; Sternfeld, F.;
Taylor, S. Tetrahedron Lett. 1987, 28, 225. (c) Ley, S. V.;
Sternfeld, F. Tetrahedron Lett. 1988, 29, 5305.
(9) Jin, Z. Nat. Prod. Rep. 2005, 22, 111.
(10) Schwartz, M. A.; Holton, R. A. J. Am. Chem. Soc. 1970, 92,
1090.
(11) Preparation data X-ray structure file reference: CCDC
770222. See Supporting Information for full experimental
details and characterisation.
(12) Alumina (neutral and basic) and Florisil were also used as
well as several basic ion exchange resins with a catch and
release procedure although these showed no benefits in
terms of isolated yield or purities.
(d) Baxendale, I. R.; Ernst, M.; Krahnert, W.-R.; Ley, S. V.
Synlett 2002, 1641. (e) Baxendale, I. R.; Griffith-Jones,
C. M.; Ley, S. V.; Tranmer, G. K. Synlett 2006, 427.
(5) (a) Pandey, G.; Muralikrishna, C.; Bhalerao, U. T.
Tetrahedron 1989, 45, 6867. (b) Sugumaran, M.; Dali, H.;
Kundzicz, H.; Semensi, V. Bioorg. Chem. 1989, 17, 443.
(c) Bhalerao, U. T.; Murali Krishna, C.; Pandey, G. J. Chem.
Soc., Chem. Commun. 1992, 1176. (d) Land, J. E.;
Ramsden, C. A.; Riley, P. A.; Yoganathan, G. Org. Biomol.
Chem. 2003, 1, 3120. (e) Gademann, K.; Bethuel, Y.;
Locher, H. H.; Hubschwerlen, C. J. Org. Chem. 2007, 72,
8361. (f) Leutbecher, H.; Hajdok, S.; Braunberger, C.;
Neumann, M.; Mika, S.; Conrad, J.; Beifuss, U. Green
Chem. 2009, 11, 676.
(6) For reviews and recent prospects, see: (a) Burton, S. G.
Catal. Today 1994, 22, 459. (b) Solomon, E. I.; Chen, P.;
Metz, M.; Lee, S.; Palmer, A. E. Angew. Chem. Int. Ed.
2001, 40, 4570. (c) Veitch, N. C. Phytochemistry 2004, 65,
249.
(7) (a) Baxendale, I. R.; Deeley, J.; Griffiths-Jones, C. M.; Ley,
S. V.; Saaby, S.; Tranmer, G. K. Chem. Commun. 2006,
2566. (b) Baxendale, I. R.; Ley, S. V. Curr. Org. Chem.
2005, 9, 1521. (c) Baxendale, I. R.; Ley, S. V. Ind. Eng.
Chem. Res. 2005, 44, 8588. (d) Baxendale, I. R.; Ley, S. V.
Curr. Org. Chem. 2005, 9, 1521. (e) Baxendale, I. R.; Ley,
S. V.; Nesi, M.; Piutti, C. Tetrahedron 2002, 58, 6285.
(f) Baxendale, I. R.; Ley, S. V.; Piutti, C. Angew. Chem. Int.
Ed. 2002, 41, 2194. (g) Ley, S. V.; Baxendale, I. R.;
Brusotti, G.; Caldarelli, M.; Massi, A.; Nesi, M. Farmaco
2002, 57, 321.
(13) Preparation data X-ray structure file reference: CCDC
770223. See Supporting Information for full experimental
details and characterisation.
(14) Appukkuttan, P.; Van der Eycken, E. Eur. J. Org. Chem.
2008, 5867.
(15) In one of the rotamers H-6a and the H-6b are, respectively,
located at d = 4.80 and 2.99 ppm are in an AB system with a
coupling constant of 13.5 Hz. The high shift of H-6a is due
to its position in the plane of the carbonyl. In the minor
rotamer they are in the same system with a coupling constant
of 14.5 Hz but at d = 4.40 and 3.40 ppm, respectively.
Moreover, COSY experiment showed that the protons H-9a
and H-8b, and the H-9b and H-8a do not, respectively,
couple because of their ca. 90° dihedral angle, thus
confirming the TBC conformation. See also: Landais, Y.;
Robin, J. Tetrahedron 1992, 48, 7185.
(16) Evidence of the indirect formation of the catecholic
intermediate substrate responsible for the autoactivation
kinetics of tyrosinase. See: Cooksey, C. J.; Garratt, P. J.;
Land, E. J.; Pavel, S.; Ramsden, C. A.; Riley, P. A.; Smit, N.
P. M. J. Biol. Chem. 1997, 272, 26226.
(17) Cresolase activity is the hydroxylation of phenols while
catecholase is reserved for the oxidation of catechols.
(18) Sànchez-Ferrer, A.; Rodriguez-López, J. N.; Garcia-
Cànovas, F.; Garcia-Carmona, F. Biochim. Biophys. Acta
1995, 1247, 1.
(8) Szewczyk, J. J. Heterocycl. Chem. 1988, 25, 1809.
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