Laccase-mediated deprotection of para-methoxyphenyl (PMP)-protected amines

Article abstract of DOI:10.1002/adsc.200700060

A novel enzymatic procedure for the oxidative deprotection of p-methoxyphenyl (PMP)protected amines is described. By using laceases (E.C. 1.10.3.2) under mildly acidic conditions, a variety of PMP-protected amines were successfully deprotected. The rate of deprotection was solvent-and pH-dependent, and the reaction scope could be increased by using so-called mediators

Full text of DOI:10.1002/adsc.200700060

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DOI: 10.1002/adsc.200700060
Laccase-Mediated Deprotection of para-Methoxyphenyl (PMP)-
Protected Amines
Jorge M. M. Verkade,a Lieke J. C. van Hemert,a Peter J. L. M. Quaedflieg,b
Hans E. Schoemaker,b Martin Schürmann,b Floris L. van Delft,a
and Floris P. J. T. Rutjesa,*
a
Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen,
The Netherlands
Fax : (+31)-24–365–3393; e-mail: F.Rutjes@science.ru.nl
DSM Pharmaceutical Products – Advanced Synthesis, Catalysis & Development, P.O. Box 18, 6160 MD Geleen,
b
The Netherlands
Received: January 24, 2007
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: A novel enzymatic procedure for the oxi-
dative deprotection of p-methoxyphenyl (PMP)-
protected amines is described. By using laccases
(E.C. 1.10.3.2) under mildly acidic conditions, a va-
riety of PMP-protected amines were successfully
deprotected. The rate of deprotection was solvent-
and pH-dependent, and the reaction scope could be
increased by using so-called mediators.
oxidative enzymatic method for the effective removal
of the PMP group from nitrogen atoms.
Having observed from our recent work[9] that a
rather large variety of oxidative reagents can be em-
ployed for PMP removal, we reasoned that this trans-
formation might also be executed with oxidative en-
zymes. In view of the many applications of laccases
(E.C. 1.10.3.2) in oxidative transformations,[10] we en-
visioned that they are potential candidates for selec-
tive PMP removal as well. Laccases are multi-copper
oxidases found in several trees and fungi, catalyzing
the oxidation of various types of substances with con-
comitant reduction of oxygen to water, avoiding the
formation of the hazardous hydrogen peroxide.
Keywords: biocatalysis; enzymatic deprotection;
green chemistry; laccase; para-methoxyphenyl pro-
tecting group; organocatalysis
Our investigation into the applicability of laccases
for PMP group removal commenced with developing
The para-methoxyphenyl (PMP) group is being in- an HPLC assay to screen laccases by monitoring the
creasingly used as a nitrogen protecting group for conversion of starting material, that is, the N-PMP-
amines[1–3] and imines.[4] In particular, in proline-cata- protected amine into the desired free amine. As a
lyzed asymmetric Mannich reactions, the PMP pro- benchmark substrate, we chose the protected 1,3-
tecting group appeared to be a crucial element for amino alcohol 1 (Scheme 1).[11] In a first attempt, ad-
reaching high enantioselectivities.[5] Until recently, in- dition of 4 mg of laccase T (from Trametes versicolor)
dustrial application of the PMP protecting group has to 1 mL of a solution of 1 (1.0 mg) in an acetonitrile/
been thwarted by a lack of cheap and practical depro- aqueous buffer (pH 5.0) mixture immediately altered
tection methods. In most cases, ceric ammonium ni- the color of the reaction mixture from colorless to
trate (CAN) has been used for deprotection, which is purple, indicating enzymatic activity. Subjection of the
expensive and highly toxic.[6] Phenyl iodoacetate[7] crude reaction mixture to the HPLC assay clearly
and also electrochemical deprotection[8] have been demonstrated the formation of the free amine 3 and
proposed instead, but both approaches do not provide benzoquinone (4, Scheme 1). After this promising
viable alternatives for industrial application. Recently, first result, a more thorough screen of reaction condi-
however, we demonstrated that the cheap oxidants tions was carried out in which co-solvent and pH
trichloroisocyanuric acid (TCCA) and periodic acid were varied. Two commercially available laccases
both can be efficiently applied for PMP deprotection were employed without modification in lyophilized
of amines.[9] In conjunction with this latter research form, namely laccase T and laccase AB (from Agari-
and the growing need for green and sustainable cata- cus bisporus).
lytic deprotection strategies, we herewith report an
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Deprotection of para-Methoxyphenyl (PMP)-Protected Amines
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Scheme 1. Deprotection of the PMP group.
Because of the generally low solubility of organic vealed that laccases are often deactivated by organic
substrates in aqueous solution, we decided to investi- solvents.[12] Thus, a series of experiments was carried
gate the use of co-solvents from the start. We consid- out in a phosphate buffer (50 mM, pH 5)/co-solvent
ered this especially important, since earlier studies re- mixture, thereby varying the co-solvent. The conver-
Table 1. Screening of the reaction parameters.[a]
Entry Laccase Solvent Vol%
buffer
pH t
Conversion to Entry Laccase Solvent Vol%
buffer
pH t
Conversion to
[h] 3 [%][b]
[h] 3 (%)[b]
1
2
3
4
5
6
7
8
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
AB
AB
AB
AB
AB
AB
AB
AB
T
T
T
T
T
MeCN 60
MeCN 70
MeCN 80
MeCN 90
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
1
2
3
4
5
6
7
24 32
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
MeCN 80
7
18 23
24 46
24 46
24 73
24 27
24 47
24 79
24 51
MeCN 80
MeCN 60
MeCN 70
MeCN 80
MeCN 90
MeCN 60
MeCN 70
MeCN 80
MeCN 90
8.5 18
0
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
42
68
77
79
THF
THF
THF
THF
60
70
80
90
20 53
20 76
20 80
20 85
9
EtOAc 60
EtOAc 70
EtOAc 80
EtOAc 90
toluene 60
toluene 70
toluene 80
toluene 90
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
MeCN 80
24 0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
24 12
24 21
24 58
24 11
24 23
24 44
24 44
THF
THF
THF
THF
THF
THF
THF
THF
60
70
80
90
60
70
80
90
4
4
4
4
19
48
58
70
20 31
20 72
20 75
20 82
18 84
18 85
18 89
18 91
18 75
18 78
18 82
18 87
18 47
18 64
18 72
18 87
18
18
18
5
2
4
DMSO 60
DMSO 70
DMSO 80
DMSO 90
MeOH 60
MeOH 70
MeOH 80
MeOH 90
MTBE 60
MTBE 70
MTBE 80
MTBE 90
18 24
18 25
18 20
18 15
8.5 18 13
1
2
3
4
5
6
18
18
0
8
18 82
18 74
18 61
18 37
T
[a]
Conditions: laccase T or AB (4 mg) was added to a solution of 1 (1 mg) in the buffer/solvent mixture (1 mL), room tem-
perature.
Conversions were determined using HPLC (Inertsil ODS-3 column) on crude samples taken from the reaction mixture.
[b]
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Jorge M. M. Verkade et al.
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sions of substrate 1 into the free amine 3, as deter-
The best result was obtained with 2,2’-azinobis(3-
mined by HPLC, are depicted in Table 1 (entries 1– ethylbenzothiazoline-6-sulfonic acid) (ABTS, 5) as
16). These results led us to conclude that the laccase- the mediator [84% conversion to 3 (instead of 25%
mediated PMP deprotection proceeds faster in homo- in entry 21), 18 h, r.t., 1 equiv. of ABTS]. For each
geneous systems (e.g., MeCN, THF, MeOH, DMSO), mediator, a blank reaction without enzyme was also
but that the use of a biphasic system (e.g., EtOAc, conducted. In addition to increasing the conversion, it
toluene, MTBE) is also applicable. Additionally, it was also found that the substrate scope could be ex-
must be noted that higher amounts of co-solvent, al- tended by using mediators. For instance, N-PMP pro-
though rendering the substrate more soluble, signifi- tected 4-phenylbutan-2-amine (10) could not be enzy-
cantly decrease the overall reaction rate through matically deprotected as such with laccases T and AB,
enzyme deactivation. Considering these initial results, which could be due to the fact that this is not a ben-
we continued with evaluation of the pH dependence zylic amine. Gratifyingly, introduction of any of the
of the reaction in a buffer mixture/MeCN (4:1) using mediators 5–9 led to high conversions to the corre-
both laccases T and AB (entries 17–32). Clearly, the sponding amine 11 (Table 2).
conversions increased with lower pH values, albeit
that too acidic conditions (pH<3) led to enzyme de-
activation. Having concluded that 3 was the optimum
pH value and laccase T the more suited enzyme, a
series of industrially viable solvents was evaluated in
varying ratios with the buffer solution (entries 33–60).
No real solvent limitations were identified in these ex-
periments, although the use of acetonitrile and
DMSO gave slightly better results. In addition, there
was no clear difference between mono- and biphasic
systems both giving high conversions to the desired
product.
Subjection of protected amino alcohol 1 to laccase
AB gave a maximum conversion of 25% (phosphate
buffer pH 5/MeCN 4:1, entry 21). Since the scope of
laccases is known to be enlarged by invoking so-
called mediators,[13] we investigated the influence of
mediators (Figure 1) on the efficiency of this particu-
lar reaction. Mechanistically the mediator, after being
oxidized by the laccase, is responsible for the conver-
sion of the substrate into the oxidized form. Since the
Table 2. Influence of the mediators.
Entry Mediator Conversion (18 h) Conversion (48 h)
1
2
3
4
5
6
-
0
-
5
6
7
8
9
43
50
53
30
71
87
88
80
75
83
To validate the screening results, we performed a
reduced mediator can be reoxidized by the enzyme, series of preparative experiments using the initial sub-
both laccase and mediator can be used in a catalytic strate 1 as well as substrates 10, 13-16 with laccases T
fashion using oxygen as the stoichiometric oxidant and AB (Table 3). Surprisingly, subjection of PMP-
thereby producing water as the by-product.
protected benzylamine 12 to these enzymes did not
produce benzylamine (17) itself. Instead, formation of
benzaldehyde was observed, suggesting oxidation at
the benzylic position, followed by hydrolysis of the re-
sulting imine. In all other cases, however, the corre-
sponding free amines 11, 18–21 were isolated after
work-up in reasonable to good yields and excellent
purity. The isolated yields are generally lower than
the HPLC conversions, which reflects the difficulty to
separate the emulsions formed in the extraction step.
Moreover, the high dilution of the reaction mixtures
(1 mg/mL) might contribute to the low yield of the
products. Finally, realizing that higher concentrations
are required to render processes industrially viable,
we repeated one of the deprotection experiments in a
10 mg/mL suspension to obtain comparable results
(63% yield vs. 67% in entry 5). Further concentration
of the reaction mixture (100 mg/mL) led to a consid-
erably lower reaction rate, although a clean conver-
Figure 1. Mediators.
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Deprotection of para-Methoxyphenyl (PMP)-Protected Amines
Table 3. Preparative deprotections using laccase T and AB.[a]
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addition of the substrate, are currently under investi-
gation.
In conclusion, the increasing use of the p-methoxy-
phenyl (PMP) group in organic synthesis requires
cost-efficient, environmentally friendly and scalable
deprotection procedures to render these processes in-
dustrially viable. We have developed a novel enzy-
matic oxidative deprotection procedure for N-PMP
protected benzylic amines involving laccases, which is
effective at a pH below 4. Additionally, we have
found that the use of mediators leads to an extension
of the substrate scope and an increase in reaction
rate. Studies to elucidate the exact mechanism and
further exploration of the scope and limitations of lac-
case-mediated oxidative deprotection reactions are
currently ongoing.
Experimental Section
Commercially available laccase T from Trametes versicolor
was purchased from Jülich Fine Chemicals in two batches as
a light brown lyophilized powder. The activity of the first
batch (used in the experiments as shown in Table 1, en-
tries 1–32) was 24 U/mg (using syringaldazin). The activity
of the second batch (used in the experiments as shown in
Table 1, entries 33–60, Table 2 and Table 3) was 1.2 U/mg
(using syringaldazin). Laccase AB from Agaricus bisporus
was commercially available from Jülich Fine Chemicals as a
brown lyophilized powder [activity 7.9 U/mg (using cate-
chol)].
Representative Procedure for the Laccase-Mediated
PMP Deprotection
To a solution of 1 (250 mg, 0.92 mmol) in THF (40 mL) and
phosphate buffer (160 mL, pH 3, 50 mM) was added laccase
T (120 mg) and if required ABTS (10 mol%). The resulting
suspension was stirred for 20 h at room temperature, acidi-
fied to pH 1 with 5N HCl and filtrated over Celite. The fil-
trate was washed with CH2Cl2 (3”100 mL). The resulting
aqueous phase was subsequently brought to pH 10.5 via ad-
dition of 5N NaOH and extracted with EtOAc (4”75 mL).
The combined organic fractions were brought to pH 1 via
addition of HCl/EtOAc, dried (Na2SO4) and concentrated
under reduced pressure to afford 2·HCl as a white solid;
[a]
Conditions: laccase T or AB (4 mg) was added to a solu-
tion of 1 (1 mg) in the buffer/solvent mixture (1 mL),
room temperature.
Conversions were determined using HPLC (Inertsil
ODS-3 column) on crude samples taken from the reac-
tion mixture.[a] Conditions: PMP-protected amine
(0.92 mmol), laccase (120 mg), MeCN/buffer mixture
(1:4, pH 3, 200 mL), room temperature.
Reaction conducted in the presence of ABTS (10
mol%).
[b]
1
yield: 124 mg (67%); [a]2D0: À24.3 (c 0.18, MeOH); H NMR
(400 MHz, D2O): d=1.12 (d, J=6.9 Hz, 3H), 2.36 (m, 1H),
3.37 (dd, J=5.4, 11.4 Hz, 1H), 3.42 (dd, J=5.4, 11.4 Hz,
1H), 4.33 (d, J=8.2 Hz, 1H), 7.50 (m, 5H); 13C NMR
(75 MHz, D2O): d=12.3, 37.9, 57.8, 62.6, 126.9, 128.7, 128.7,
134.6; IR: n=3330, 3112, 2975, 2871, 1489, 1473, 1026, 706,
[b]
[c]
Products were isolated as HCl salts.
558 cmÀ1
;
HRMS (ESI+): m/z=166.1248, calcd. for
C10H16NO: 166.1232; anal. calcd. for C10H16ClNO: C 59.55,
H 8.00, N 6.94; found: C 58.05, H 7.87, N 6.84.
Spectroscopic data for new compounds are available in
the Supporting Information.
sion to the desired product was still observed. Alter-
native ways to attain rapid deprotection reactions in a
more concentrated fashion, such as the use of elevat-
ed temperatures or other organic co-solvents to in-
crease the solubility of the substrates or portionwise
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Jorge M. M. Verkade et al.
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Acknowledgements
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This work forms part of the Ultimate Chiral Technology pro-
ject supported in part with funds provided by SNN (Coopera-
tion Northern Netherlands) and EFRO (European Fund for
Regional Development).
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