Development of tyrosinase labile protecting groups for amines

Article abstract of DOI:10.1021/ol040042i

(Chemical Equation Presented) The development of two novel protecting groups for amines is described. Thus, a range of amines have been converted to ureas, and the deprotection of these upon exposure to mushroom tyrosinase (E.C. 1.14.18.1) has been demonstrated.

Full text of DOI:10.1021/ol040042i

ORGANIC
LETTERS
2004
Vol. 6, No. 18
3111-3113
Development of Tyrosinase Labile
Protecting Groups for Amines
Helen M. I. Osborn* and Nana Aba O. Williams
School of Chemistry, UniVersity of Reading, Whiteknights, Reading, RG6 6AD, UK
h.m.i.osborn@rdg.ac.uk
Received June 16, 2004
ABSTRACT
The development of two novel protecting groups for amines is described. Thus, a range of amines have been converted to ureas, and the
deprotection of these upon exposure to mushroom tyrosinase (E.C. 1.14.18.1) has been demonstrated.
The need for efficient protection/deprotection protocols
within synthetic strategies is well recognized, and the
selection of protecting groups that will be compatible with
specific synthetic strategies plays a fundamental part in the
design of synthetic pathways. Although a large number of
protecting groups for amines are already known and ex-
ploited,1 relatively few amine protecting groups that can be
removed using enzymes are known.2 Enzyme labile protect-
ing groups are particularly attractive due to the highly chemo-
and stereoselective reactions and mild reaction conditions
required for their removal. We have recently demonstrated
that mushroom tyrosinase (E.C. 1.14.18.1) can promote
release of cytotoxic agents from prodrugs by an oxidation/
cyclization pathway analogous to that documented for the
synthesis of melanin from L-tyrosine.3 We now report a
development of this strategy that has led to the identification
of two classes of enzyme labile protecting groups for amines.
The mushroom tyrosinase enzyme is a particularly attractive
reagent for deprotection protocols since it is commercially
available or can be readily isolated from the regular com-
mercially available mushrooms, Agaricus bisporus.4 More-
over, its use for the efficient cleavage of amino acid and
peptide phenyl hydrazides has recently been demonstrated.5
The first stage of this study necessitated the selection of
tyrosinase substrates for attachment to the amines, to serve
as both protecting groups for the amines and triggers for
tyrosinase-mediated deprotection. From the knowledge that
the tyrosinase enzyme exhibits phenolase and catecholase
activity,6 and our previous oximetry investigations,3 4-amino-
phenol and 3-hydroxytyramine were selected as the tyrosi-
nase substrates. It was decided to attach these to the amines
via a urea linkage, which would serve to form urea-based
protecting groups for the amines. Upon exposure to tyrosi-
nase, it was hypothesised that these ureas would produce
intermediates that were unstable to aqueous conditions,
regenerating the deprotected amines. The mechanisms by
which these deprotections are postulated to occur are
illustrated in Schemes 1 and 2.
* Fax: +44 (0) 118 931 6331.
(1) (a) Kocienski, P. J. Protecting Groups, 3rd ed.; Georg Thieme
Verlag: 2003. (b) Greene, T. W.; Wuts, P. G. M. ProtectiVe Groups in
Organic Synthesis, 3rd ed.; Wiley: New York, 1999. (c) Robertson, J.
Protecting Group Chemistry; O.U.P., 2000.
A range of amines was selected for protection, and these
encompassed aromatic and aliphatic amines, and the methyl
(2) For recent reviews see (a) Kadereit, D. K.; Waldmann, H. Chem.
ReV. 2001, 101, 3367-3396. (b) Kadereit, D.; Waldmann, H. Monatsh.
Chem. 2000, 131, 571-584.
(3) (a) Jordan, A. M.; Khan, T. H.; Osborn, H. M. I.; Photiou, A.; Riley,
P. A. Bioorg. Med. Chem. 1999, 7, 1775-1780. (b) Jordan, A. M.; Khan,
T. H.; Malkin, H.; Osborn, H. M. I.; Photiou, A.; Riley, P. A. Bioorg. Med.
Chem. 2001, 9, 1549-1558. (c) Jordan, A. M.; Khan, T. H.; Malkin, H.;
Osborn, H. M. I. Bioorg. Med. Chem. 2002, 10, 2625-2633.
(4) Mu¨ller, G. H.; Lang, A.; Seithel, D. R.; Waldmann, H. Chem. Eur.
J. 1998, 4, 2513-2522.
(5) (a) Mu¨ller, G. H.; Waldmann, H. Tetrahedron Lett. 1999, 40, 3549-
3552. (b) Vo¨lkert, M.; Koul, S.; Mu¨ller, G. H.; Lehnig, M.; Waldmann, H.
J. Org. Chem. 2002, 67, 6902-6910.
(6) For a recent review, see: Land, E. J.; Ramsden, C. A.; Riley, P. A.
Acc. Chem. Res. 2003, 36, 300-308.
10.1021/ol040042i CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/10/2004

Scheme 1
Scheme 2
ester of an amino acid. Amines 1-4 were easily converted
Before the tyrosinase-mediated deprotections were inves-
tigated, the stabilities of the two classes of ureas to some
reaction conditions commonly used for the removal of other
alcohol and amine protecting groups were investigated. Thus,
ureas 12 and 16 derived from cyclohexylamine were treated,
in turn, with neat TFA, NaOH (1 M), and K2CO3/MeOH
for 8 h at room temperature. TLC and 1H NMR spectroscopic
analysis of the materials obtained after this time illustrated
that no deprotection of the ureas occurred, with starting
material being recovered in quantitative yields in all cases.
Our studies next probed the utility of tyrosinase for
effecting deprotection of the ureas, to re-form the amines.
Ureas 9 and 13 were dissolved in a mixture of acetonitrile
and phosphate buffer (1:5, 100 mg of urea per 72 mL of
solvent), and after complete dissolution, tyrosinase in
phosphate buffer was added. Deprotections were performed
into the urea derivatives by initial reaction with triphosgene,
in anhydrous dichloromethane, to afford isocyanate deriva-
tives 5-8 in quantitative yield. These could either be isolated
and purified by column chromatography on silica gel or used
directly in coupling reactions with either 4-aminophenol or
3-hydroxytyramine to afford the two classes of protected
amines. Couplings were performed in anhydrous pyridine
under an inert atmosphere for 2-6 h, and the progress of
the reaction could be conveniently monitored by IR analysis
of the reaction mixture, for complete disappearance of the
NCO stretch. Urea products 9-16 were purified by column
chromatography on silica gel and obtained in synthetically
useful yields (Scheme 3, Table 1).7
Scheme 3
Table 1.
tyrosinase
substrate
urea,
%
amine
aniline (1)
benzylamine (2)
L-phenyl alanine methyl ester (3) 4-aminophenol
cyclohexylamine (4)
aniline (1)
benzylamine (2)
4-aminophenol
4-aminophenol
9, 92
10, 83
11, 86
12, 99
4-aminophenol
3-hydroxytyramine 13, 78
3-hydroxytyramine 14, 75
L-phenyl alanine methyl ester (3) 3-hydroxytyramine 15, 72
cyclohexylamine (4)
3-hydroxytyramine 16, 78
3112
Org. Lett., Vol. 6, No. 18, 2004

Scheme 4
Scheme 5
with and without the bubbling of oxygen through the
solutions and with two different quantities of the tyrosinase
enzyme (0.005 equiv by mass and 0.05 equiv by mass). The
reaction mixtures were analyzed by TLC for complete
disappearance of starting material, and then the tyrosinase
was removed by filtration. Extraction of the aqueous solution
with dichloromethane, and purification of the crude reaction
products by filtration through silica, allowed isolation of the
deprotected aniline in pure form in excellent synthetic yields
(Scheme 4, Table 2).8
utilized and when oxygen was continuously bubbled through
the solution. Moreover, catechol-derived urea (13) was
deprotected at a faster rate than phenol-derived urea (9),
which correlates with a lag period for the oxidation of
phenols to catcehols by tyrosinase.6 Control experiments
illustrated that bubbling oxygen through the solutions without
adding tyrosinase did not effect deprotection of the ureas.
The optimized deprotection conditions (0.05 equiv by mass
of tyrosinase, and bubbling of oxygen through the reaction
mixtures) were then utilized to effect deprotection of ureas
9-16, to allow formation of the amines 1-4 in excellent
yields (Scheme 5, Table 3). As expected, spectroscopic data
for the products were identical to those for the starting
material substrates 1-4.
Table 2.
mass
equiv of
oxygen bubbled time (h) for complete
through
solution
disappearance
of urea
urea tyrosinase
1, %
9
9
9
0.005
0.005
0.05
no
yes
no
yes
no
yes
no
96
72
60
24
84
48
24
12
78
75
75
82
74
82
71
91
Table 3.
urea
deprotected amine, yield %
9
0.05
9
10
11
12
13
14
15
16
aniline, 82
13
13
13
13
0.005
0.005
0.05
benzylamine, 73
phenyl alanine, 87
cyclohexylamine, 80
aniline, 91
benzylamine, 76
phenylalanine, 93
cyclohexylamine, 74
0.05
yes
These studies illustrated that optimum results were ob-
tained when larger mass equivalents of tyrosinase were
(7) Typical Experimental Procedure for Formation of Protected Urea
Derivatives: Step 1. The amine (1 equiv) and NEt3 (2 equiv) were dissolved
in anhydrous DCM and cooled in an ice bath at 0-5 °C. Triphosgene (2
equiv) was added, and the reaction was gradually brought to reflux under
N2 (g). The reaction was monitored by IR analysis until the formation of a
strong NCO stretch (2-6 h) was observed. The reaction mixture was cooled
and then filtered through a short pad of silica, and the filtrate was
concentrated in vacuo to yield the pure isocyanate product (g90%). Step
2. Either 3-hydroxytyramine (1 equiv) or 4-aminophenol (1 equiv) was
dissolved in pyridine, and this was added to the isocyanate (1 equiv) in
pyridine. The reaction mixture was then stirred under N2 (g) for 6 h. The
solvent was then removed in vacuo, and products were purified using
chromatography on silica gel.
(8) Typical Experimental Procedure for Deprotection of Ureas to Generate
the Amines. The protected amine was dissolved in MeCN and phosphate
buffer (1:5, v:v) and sonicated for 20 min at rt. After complete dissolution,
tyrosinase (0.05 equiv by mass) in phosphate buffer was added. After
addition of the enzyme, oxygen was bubbled through the solution until the
disappearance of starting material was evidenced by TLC analysis. The
reaction was filtered through Celite and the MeCN removed by evaporation.
The remaining solution was extracted with DCM, and the organic layers
were combined and dried over MgSO4; the solvents were removed in vacuo,
and the residue was purified by chromatography on silica gel to afford the
free amines.
Thus, the deprotection strategy proved to be equally
effective for the two classes of ureas (9-12 and 13-16).
This study has therefore illustrated the efficient synthesis of
two classes of protected amines and has illustrated that both
classes can be deprotected to regenerate the amines upon
exposure to tyrosinase. In addition, preliminary studies have
illustrated that the protected amines are stable to reaction
conditions that are often used to remove other widely used
alcohol and amine protecting groups. This protocol may also
be applicable for the protection of alcohols as tyrosinase
labile carbamates, and we are currently exploring the scope
of this within our laboratory.
Acknowledgment. We are grateful for financial support
from the University of Reading’s Research Endowment Trust
Fund and the University of Reading’s School of Chemistry.
OL040042I
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