Broadening the chemical scope of laccases: Selective deprotection of N-benzyl groups

Article abstract of DOI:10.1039/c5gc00525f

Laccase from Trametes versicolor together with TEMPO has been found to be a very efficient system to deprotect N-benzylated primary amines, differing from previously described methods since it uses oxygen as a mild oxidant in aqueous medium. Chemoselective removal of the benzyl group was achieved with excellent yields when secondary amines and alcohol moieties were also present.

Full text of DOI:10.1039/c5gc00525f

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Broadening the chemical scope of laccases:
selective deprotection of N-benzyl groups†
Cite this: DOI: 10.1039/c5gc00525f
Received 9th March 2015,
Accepted 30th March 2015
Lía Martínez-Montero, Alba Díaz-Rodríguez,‡ Vicente Gotor,
Vicente Gotor-Fernández* and Iván Lavandera*
DOI: 10.1039/c5gc00525f
www.rsc.org/greenchem
Laccase from Trametes versicolor together with TEMPO has been alkali metal bromide formed by mixing KBr and Oxone®,11
found to be a very efficient system to deprotect N-benzylated among others. Finally, Lewis acids such as boron reagents
primary amines, differing from previously described methods since have also been applied to this process.12
it uses oxygen as a mild oxidant in aqueous medium. Chemoselec-
Unfortunately, these methodologies make use of organic
tive removal of the benzyl group was achieved with excellent solvents and, in some cases, high temperatures and stoichio-
yields when secondary amines and alcohol moieties were also metric amounts of poisoning reagents. Therefore, biocatalytic
present.
methods can provide clear advantages in the development of
a synthetic route employing mild reaction conditions in
aqueous media. In fact, one of the most appreciated advan-
tages ligated to their use is the ability to achieve selective pro-
tection/deprotection steps avoiding multi-step protocols.13
Moreover, enzymes have proven their effectiveness in the selec-
tive modification of a wide range of functionalities, mainly
using hydrolytic or redox enzymes. For instance, acylases,
esterases, lipases and proteases are recognized as efficient
enzymes for the deprotection of alkyl esters including benzylic
ones,14–18 amides19 and carbamates.20 Also, oxidoreductases
have shown the potential to carry out N-deprotection reactions.
Cytochromes P450 have been described as catalysts to perform
debenzylations,21 and laccases to cleave the p-methoxyphenyl
(PMP) group.22
Herein we wish to report our latest results in the develop-
ment of an efficient N-benzyl deprotection reaction taking
advantage of the use of laccases (Scheme 1),23–27 which are
multi-copper enzymes found in a myriad of bacteria, fungi and
plants. These oxidases reduce O2 into H2O at the expense of
the oxidation of the corresponding substrate. In the case of
some specific substrates such as primary amines or primary
and secondary alcohols, it is compulsory to use a chemical
The development of efficient deprotection protocols is crucial
in organic synthesis.1 The presence of multiple functionalities
with similar reactivities in the target molecule, and harsh con-
ditions often employed in deprotection reactions, hamper the
implementation of these methodologies. Therefore, there is a
need for alternative, more sustainable and chemoselective pro-
cesses to modify a specific position.
The reactivity of free amines can be a challenge when
preparing increasingly complex compounds, thus requiring
protection during a chemical transformation. Due to the abun-
dance of amines in organic molecules,2 the employment of
amino protecting groups is commonplace in many synthetic
schemes, such as carbamates, amides and sulfonamides. In
particular, the benzyl group is commonly used as a protecting
group in different synthetic areas. Its simple introduction with
benzyl halides and inherent stability under basic conditions
provide a myriad of possibilities to perform further transfor-
mations. N-Benzyl groups are typically cleaved using reductive
methods, with palladium-catalyzed hydrogenolysis often
employed.1,3–7 Also oxidative or photolytic debenzylations can
be carried out with a variety of reagents such as cerium
ammonium nitrate (CAN),8 diisopropyl azodicarboxylate
(DIAD),9 titanium(III) chloride in combination with iodine,10 or
Department of Organic and Inorganic Chemistry, Biotechnology Institute of Asturias,
University of Oviedo, Avenida Julián Clavería 8, 33006 Oviedo, Spain.
E-mail: vicgotfer@uniovi.es, lavanderaivan@uniovi.es
†Electronic supplementary information (ESI) available: Experimental pro-
cedures, substrate characterization, E-factor calculations, analytical data, and
NMR spectra. See DOI: 10.1039/c5gc00525f
‡Present address: Medicines Research Centre, GlaxoSmithKline R&D Ltd,
Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK.
Scheme 1 Laccase-catalyzed debenzylation protocol vs. typical chemi-
cal methods.
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mediator that acts as the true oxidant species, giving access to
Despite this unexpected result, we found the first debenzy-
the so-called laccase mediator systems (LMSs). In particular, lation step promising, as this method utilized a substoichio-
2,2,6,6-tetramethylpiperidin-1-yl)oxy radical (TEMPO) has been metric amount of the oxidant and proceeded under aerobic
recurrently applied as a compatible oxidant,28–32 but also other conditions in aqueous medium. In fact, this methodology
mediators such as 2,2′-azino-bis(3-ethylbenzothiazoline-6-sul- could turn into an orthogonal deprotection strategy. While
phonic acid) (ABTS) and 2-azaadamantane N-oxyl (AZADO) not suitable for amines over primary positions due to the
have been successfully used.33 Recently, this oxoammonium formation of the corresponding aldehydes, this method could
salt has been employed in combination with laccases34 or perfectly work over N-benzylated amines at secondary posi-
metal catalyst systems35 to achieve mild and selective oxi- tions. Thus, to study the scope of this reaction, a series of
dations of alcohols and amino alcohols.
primary amines was benzylated to obtain derivatives 2a–8a
The application of our laccase/TEMPO system using oxygen (Table 1), and the laccase-mediated deprotection reaction was
as the final oxidant to a selected panel of N-benzylated performed under the previously described conditions. Among
primary amines will be disclosed, demonstrating its regio- and the different substrates tested, racemic and enantiomerically
chemoselectivity in the presence of protected secondary pure amines were considered, also including amino alcohols
amines and alcohols, also comparing its eco-efficiency with and amino esters.
other traditional chemical deprotection strategies.
While the blank reactions without laccase and/or TEMPO
As a continuation of our previous study related to the syn- did not provide any conversion (see the ESI†), this method-
thesis of interesting lactones and lactams using the laccase/ ology could be successfully applied over different aliphatic,
TEMPO system,30,36 5-(benzylamino)pentan-1-ol (1a) was syn- cyclic and aromatic amines affording deprotected derivatives
thesized and exposed to the previously optimized conditions. 2b–8b smoothly and in a very clean fashion, with no undesired
After reaction optimization (see the ESI†), laccase from by-products due to, e.g., oxidative transformations (conv.
Trametes versicolor and TEMPO (33% mol) were employed in >97%). Moreover, when using enantiopure derivatives 3a and
Tris·HCl 200 mM buffer (pH 5) at 30 °C under orbital stirring, 8a (entries 2 and 7), the optical purity of the corresponding
expecting the formation of the corresponding N-benzylated amines remained untouched. Also, no loss of diastereo-
lactam 1c.36 Surprisingly,
a mixture of hemiacetal 1d selectivity was observed for substrate ( )-trans-5a (entry 4).
and lactone 1e was attained in quantitative conversion Hence, interesting derivatives such as amphetamine 7a
(Scheme 2a). This can be explained as the oxidation takes (entry 6) or phenylalanine methyl ester 8a (entry 7) could be
place preferentially at the benzylic position (leaving the
primary alcohol intact), affording amino alcohol 1b after
hydrolysis of the imine intermediate. This compound would
Table 1 Debenzylation of N-protected amino derivatives 2a–8a using
the laccase/TEMPO systema
lead to derivatives 1d and 1e after chemoselective reoxidation
of the amino group (Scheme 2b).36
Entry
Substrate
2b–8bb (%)
1
2
3
4
5
6
7c
( )-2a
(S)-3a
4a
( )-trans-5a
( )-6a
( )-7a
L-8a
97
97
99
99
97
>99
99
a Reaction conditions: 2a–8a (0.08 mmol), T. versicolor laccase (41 U)
and TEMPO (33% mol) in an oxygen-saturated Tris·HCl 200 mM buffer
(pH 5) at 30 °C for 16 h at 250 rpm. b Conversion values measured by
GC. c Reaction time: 8 h.
Scheme 2 (a) Formation of hemiacetal 1d and lactone 1e starting from
N-benzylated amino alcohol 1a. (b) Sequence pathway involving an
N-debenzylation step.
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Table 2 Reactivity of N-benzylated amino alcohols 1a, 5a and 11a
using laccase/TEMPO, laccase/AZADO and CuCl/AZADO systemsa
Fig. 1 Substrates showing low or depleted conversion using this
method.
Oxidation
Entry system
N-oxidationb O-oxidationb
Substrate
(%)
(%)
1
2
3
4
5
6
7
8
9
Laccase/TEMPO 1a
>97c
80d
<3
99
55
<3
<1
<3
<3
<3
<3
Laccase/AZADO
CuCl/AZADO
1a
1a
18e
<1
Laccase/TEMPO ( )-trans-5a
Laccase/AZADO
CuCl/AZADO
( )-trans-5a
( )-trans-5a
<3
33
<1
<3
Laccase/TEMPO 11a
Laccase/AZADO
CuCl/AZADO
11a
11a
>97
a For reaction conditions, see the ESI. b Conversion values measured by
GC or NMR. c Mixture of 1d : 1e (1.5 : 1). d Mixture of 1d : 1e (7.7 : 1).
e Formation of enamine 1f.
amines in very high isolated yields (>80%, see the ESI† for
more information).
Scheme 3 Chemoselective N-debenzylation of primary amines vs. sec-
ondary amines or alcohols using the laccase/TEMPO system.
Encouraged by these promising results, we decided to
compare the reactivity of our methodology with both laccase/
AZADO and CuCl/AZADO pairs, described as selective oxidative
methods.34,35 For this study, N-benzylated amino alcohols 1a,
( )-trans-5a and 11a were used as model substrates (Table 2).
The laccase-dependent system showed similar behaviour, oxi-
dizing the N-position rather than the oxygen moiety (entries 1,
2, 4, 5, 7 and 8), although at a lower extent. CuCl/AZADO
favoured the formation of the carbonylic compounds via
alcohol oxidation (entries 3, 6 and 9). These complementary
methods can overcome a challenge in synthetic organic chem-
istry such as the chemoselective oxidation of alcohols in the
presence of amino functional groups and vice versa.42,43
Remarkably, in the case of substrate 1a, the laccase/AZADO
system, after N-benzyl deprotection and subsequent amine
reoxidation led to a mixture of hemiacetal 1d and lactone 1e
(Scheme 2) in a 7.7 : 1 ratio. In contrast, for the CuCl/AZADO
pair, after alcohol oxidation into the corresponding aldehyde
and intramolecular cyclization, the hemiaminal intermediate
dehydrated giving access to enamine 1f (18% conv., see
Scheme S3 in the ESI†).
To demonstrate the excellent chemoselectivity of the
laccase/TEMPO system for the deprotection of N-benzylated
amines, different chemical methods were assessed and
applied to N,O-dibenzylated compound ( )-trans-13a. Among
the previous methodologies that were described as mild and
selective to achieve similar reactions, we chose Pd/C in the
presence of H2 or 1,4-cyclohexadiene,39 CAN,8,40 and DIAD.9
The application of Pd/C and 1,4-cyclohexadiene did not lead to
any measurable conversion and CAN preferentially deprotected
the ether moiety affording ( )-trans-5a. Only the employment
obtained in excellent conversions. It must be mentioned that
in the last example, the reaction was stopped after 8 h since
longer times favoured the ester moiety hydrolysis.
Although showing a broad scope, some limitations were
found for this methodology (Fig. 1). For instance, this system
did not work for N,N-dibenzylated amine ( )-9a, O-benzylated
amino alcohol ( )-trans-10a, and N-protected piperidin-4-ol
11a. In the first case just minor quantities of the mono- and
deprotected compounds were detected, and for the other two
derivatives no conversion was attained. This is in line with pre-
vious reports based on Cu-nitroxyl oxidative systems, which
show the oxidation of benzylic amines into the corresponding
imines when they are mono- or di- (but not tri-)
substituted.37,38
Based on these results, we decided to test the behaviour of
diprotected substrates 12a and ( )-trans-13a (Scheme 3).
Primary amines were selectively deprotected irrespective of
whether a secondary amine or an alcohol moiety appeared also
benzylated in the molecule. These selective transformations
are remarkable as there are just few examples described in the
literature.8,9,39–41 These results open the door for the develop-
ment of regio- and chemoselective processes that can hardly
be performed by means of more conventional chemical
methods.
Furthermore, some of these deprotection reactions were
done on a 100 mg scale and the amount of TEMPO could be
reduced to 20% mol without any conversion decrease obtain-
ing, after a simple extraction protocol, the corresponding
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of hydrogen with Pd/C and DIAD allowed the synthesis of the
desired derivative ( )-trans-10a in 27% and 80% yield, respecti-
vely, after column chromatography isolation.
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