Unveiling the Biocatalytic Aromatizing Activity of Monoamine Oxidases MAO-N and 6-HDNO: Development of Chemoenzymatic Cascades for the Synthesis of Pyrroles

Article abstract of DOI:10.1021/acscatal.6b03081

A chemoenzymatic cascade process for the sustainable production of pyrroles has been developed. Pyrroles were synthesized by exploiting the previously unexplored aromatizing activity of monoamine oxidase enzymes (MAO-N and 6-HDNO). MAO-N/6-HDNO whole cell biocatalysts are able to convert 3-pyrrolines into pyrroles under mild conditions and in high yields. Moreover, MAO-N can work in combination with the ruthenium Grubbs catalyst, leading to the synthesis of pyrroles from diallylamines/-anilines in a one-pot cascade metathesis-aromatization sequence.

Full text of DOI:10.1021/acscatal.6b03081

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Article
Unveiling the biocatalytic aromatizing activity of
monoamino oxidases MAO-N and 6-HDNO: development
of chemoenzymatic cascades for the synthesis of pyrroles
Nicolo Scalacci, Gary W. Black, Giulio Mattedi, Nicola L. Brown, Nicholas J Turner, and Daniele Castagnolo
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b03081 • Publication Date (Web): 27 Dec 2016
Downloaded from http://pubs.acs.org on December 27, 2016
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Unveiling the biocatalytic aromatizing activity of
monoamino oxidases MAOꢀN and 6ꢀHDNO:
development of chemoenzymatic cascades for the
synthesis of pyrroles.
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Nicolό Scalacci,‡,† Gary W. Black,† Giulio Mattedi,‡ Nicola L. Brown,† Nicholas J. Turner,±
Daniele Castagnolo*,‡
‡Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street, SE1 9NH,
†
London, United Kingdom. Department of Applied Sciences, Northumbria University, Ellison
±
Place, NE1 8ST, Newcastle upon Tyne, United Kingdom. School of Chemistry, Manchester
Institute of Biotechnology, University of Manchester, 131 Princess Street, M1 7DN, Manchester,
United Kingdom.
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ABSTRACT: A chemoenzymatic cascade process for the sustainable production of pyrroles has
been developed. Pyrroles were synthesised exploiting the previously unexplored aromatizing
activity of monoamino oxidase enzymes (MAOꢀN and 6ꢀHDNO). MAOꢀN/6ꢀHDNO whole cell
biocatalysts are able to convert 3ꢀpyrrolines into pyrroles under mild conditions and high yields.
Moreover, MAOꢀN can work in combination with ruthenium Grubbs' catalyst leading to the
synthesis of pyrroles from diallylꢀamines/anilines in a oneꢀpot cascade metathesisꢀaromatization
sequence.
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KEYWORDS: MAOꢀN, biocatalysis, chemoenzymatic cascade, metathesis, pyrrole
INTRODUCTION
The occurrence of aromatic nitrogen heterocycles in many natural and synthetic biologically
active compounds represents an incentive toward the development of new synthetic
methodologies toward these important chemicals.1 It is estimated that more of 50% of the
bestselling drugs contain a nitrogen heterocyclic nucleus, thus fueling a demand for broadly
applicable synthetic methods that deliver aromatic heterocycles in high yield. We recently
discovered a new class of pyrroles endowed with potent activity against drugꢀresistant
tuberculosis and thus we became interested in finding new sustainable approaches for the
production of this class of compounds.2 Typical approaches for the synthesis of pyrroles include
the Paal−Knorr and Clauson−Kass reactions,3 the azaꢀWittig reaction4 or other multicomponent
approaches.5 Approaches to pyrroles under relatively mild conditions via iron or palladium
catalysed PaalꢀKnorr reactions on water have been also described.6 However, the structural
features of the reagents required in the latter methods or the side products formed may represent
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a limitation in the scope of these reactions. Recently, olefin ringꢀclosing metathesis (RCM)
emerged as a powerful and effective reaction for the two steps synthesis of many functionalized
pyrroles from acyclic precursors. Donohoe7 and Rutjies8 first developed a two steps sequence to
pyrroles from diallylamines 1 via ringꢀclosing metathesis (RCM) followed by aromatization
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t
mediated by RuCl3, Pd/C, FeCl3 or BuOOH (Figure 1). This latter step is supposed to occur
through the oxidation of the amine 2 into the corresponding iminium intermediate 3 leading in
turn to pyrrole 4 by tautomerization. The direct oxidation of amines into imines or iminium ions
still represents a challenge in organic synthesis and only few approaches have been described so
far. These methods rely on the use of metal catalysts such Fe, Pd, Ru in the presence of oxygen
or TEMPO and often require harsh reaction conditions.9 However, in Nature, the conversion of
an amine group into the corresponding imine is a common biochemical transformation that is
catalysed by monoamino oxidase (MAO) enzymes.10 MAO enzymes catalyse the oxygenꢀ
dependent oxidation of amines into imines which are in turn hydrolysed into aldehydes under
aqueous conditions. Due to their unique properties, the monoamino oxidase variants from
Aspergillus niger (MAOꢀN) have found a broad application also as biocatalysts for
stereocontrolled syntheses11 and have been extensively used in a variety of synthetic
transformations. In particular, MAOꢀN have been widely studied as biocatalysts for the
production of enantiomerically pure amines through the selective oxidation and deracemization
of a range of chiral aliphatic substrates.11 Herein, we describe a new sustainable approach for the
synthesis of pyrroles unveiling for the first time the ability and versatility of MAOꢀN enzymes in
catalysing the oxidationꢀaromatization of 3ꢀpyrrolines 2 into pyrroles 4. In addition, a oneꢀpot
chemoenzymatic cascade reaction for the synthesis of pyrroles through the inꢀsitu combination of
RCM reactions with the MAOꢀN aromatizing biocatalysts is described.
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RESULTS AND DISCUSSION
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The MAOꢀN aromatization of 3ꢀpyrrolines substrates 7 and 10, synthesised via RCM as
reported in Scheme 1,12 was first investigated. Freezeꢀdried whole cells containing MAOꢀN
variants D5, D9 and D11 were selected on the basis on their known activity and selectivity
toward structurally related pyrrolidines.11 In addition, the oxidizing/aromatizing properties of the
recently developed nicotine oxidase biocatalyst 6ꢀHDNO E350L/E352D13 were also explored.
All the enzymatic biotransformations were initially carried out at 37 oC in a buffer solution (pH =
7.8) using DMF as coꢀsolvent where appropriate, according to standard protocols.11a The data are
summarised in Table 1 and Table 2. The arylꢀpyrrolines 7 were converted into pyrroles 11 with
moderate to excellent yields depending on the substituent on the aryl group. The phenylꢀ
pyrroline 7a was converted into pyrrole 11a by MAOꢀD5 and 6ꢀHDNO in good amount (61%),
whilst the variant D9 led to 11a in only 32% conversion. Full conversion (>99%) of pyrrolines
7d and the 2ꢀmethylꢀsubstituted 7i was observed with MAOꢀD5, whilst D9 and 6ꢀHDNO proved
to be less active. On the other hand, no conversion of the pyrrolines 7j-k into pyrroles bearing a
phenyl substituent at C2 and C3 respectively was observed. Steric factors prevented 7j-k to enter
into the catalytic site of MAOꢀD5 as shown by docking simulation in Figure 2.14Docking
simulations were carried out in order to show the interaction of pyrrolines 7a and 7k within the
catalytic site of MAOꢀD5 and explain the experimental data. Whilst 7a fits within the catalytic
site of MAOꢀD5, the phenyl substituent (in yellow) of the bulkier 7k lays in a region of the
catalytic site where an amino acid residue is located, thus preventing 7k from entering the
enzyme's active site and being aromatized. Again, no conversion was observed when 7j-k were
treated with the variant MAOꢀD11.The substrates 7g and 7h bearing an electron withdrawing
substituent on the aromatic ring (ꢀNO2 and ꢀCN respectively) were not converted into
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corresponding pyrroles. Despite the exact mechanism of action of MAO has not fully
elucidated,15 one of the proposed model suggests that the oxidation of amines proceeds through a
nucleophilic mechanism where the amine attacks the FAD by nucleophilic addition and it is in
turn oxidised to imine leading to the formation of the reduced FADH2.16 Thus, it is likely that the
electron withdrawing substituents on the phenyl ring of 7g-h reduce the electronic density and
consequently the nucleophilicity of the pyrroline nitrogen, thus preventing the attack of 7g-h to
FAD and their following oxidation into pyrroles.17 As further confirmation of this assumption,
the more nucleophilic alkyl pyrrolines 10a-f were fully converted (>99%) by MAOꢀD5. Table 2.
Lower conversion (51ꢀ61%) values were observed when the same biotransformations were
carried out with MAOꢀD9. The nicotine oxidase 6ꢀHDNO was able to fully oxidise the benzylꢀ
pyrroline 10a (95%) while lower conversion (65%) was observed for the bulkier cyclohexyl
derivative 10f. Pyrroline 10g bearing a bulky substituent on the nitrogen was oxidised in low
yields as well as the benzyl pyrrolines 10h-i bearing two methyl substituents on the heterocyclic
nucleus (48ꢀ51% of 12h-12i). Finally, the aromatization of 10j-k bearing a phenyl substituent on
the pyrroline nucleus was investigated. Interestingly, the secondary pyrroline 9c was poorly
converted into the pyrrole 12l by all the set of amino oxidase catalysts, whilst excellent
conversion values were observed for the tertiary pyrroline 10j. Lower conversion for 10k was
observed due to steric factors.
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One of the most intriguing challenge for chemists and biologists is represented by the
possibility to combine chemoꢀ and enzymatic catalysis in a concurrent fashion, due to the
compatibility issues of the catalysts and the different reaction conditions in which these generally
operate.18 The combination of chemoꢀ and bioꢀcatalysis offers opportunities to outperform
sequential transformations and the development of oneꢀpot multistepꢀcascade reactions
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containing both transitionꢀmetal catalysts and enzymes is highly appealing in terms of both
selectivity and synthetic efficiency. Moreover, cascade reactions offer additional benefits other
than the immediate succession of the individual transformations, such as the almost
instantaneous consumption of toxic or unstable intermediates without the requirement of
intermediate isolation or functional group protection strategies, leading to safer processes and to
the reduction of undesired side products. Thus, the combination of RCM reactions with MAO
biocatalysts in the same reaction medium was investigated with the aim to develop a
chemoenzymatic cascade synthesis of pyrroles from allylꢀamines/anilines as shown in Table 3.
The diallylaniline 6a was first dissolved in a 1:60 DMF/Buffer pH=7.8 solution and treated
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simultaneously with 5mol% Grubbs' catalyst GII and MAOꢀD5 at 37 C. Aniline 6a was
recovered from the reaction mixture after 24h and only 10% of 3ꢀpyrroline 7a was obtained,
whilst no traces of the pyrrole 11a were detected (entry 1). On the opposite, 90% of 7a was
obtained when the same biotransformation was performed in 1:60 acetone/buffer mixture. Again
no traces of 11a were observed (entry 2). It is known that acetone is a more suitable solvent for
RCM reaction than DMF, thus accounting for the higher amount of 7a detected in the second
case.19 Increasing the coꢀsolvent/buffer ratio to 1:4 and using different waterꢀmiscible coꢀ
solvents (THF and DMSO) (entries 4ꢀ7) did not affect the outcome of the biotransformation. In
all cases, variable amounts of the RCM product 7a were detected but no traces of the desired
pyrrole 11a were obtained. Previous works demonstrated that MAO biocatalysts suffer the coꢀ
presence of chemocatalysts in the same reaction medium.11c Recently, Hartwig and Zhao showed
that isoꢀoctane can work as excellent coꢀsolvent in chemoenzymatic biotransformations, due to
its ability to form a biphasic system with low mass transfer together with the buffer solution.20,18a
When the chemoenzymatic reaction was carried out in an isoꢀoctane/buffer 1:4 mixture (entry 8),
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the pyrrole 11a was obtained as main product in high yield together with negligible amount of
7a. The use of the nonꢀwater miscible coꢀsolvent isoꢀoctane proved to be crucial to prevent the
interaction between the Ruꢀcatalyst and the MAOꢀD5 and avoid the deactivation of the enzyme
probably due to the Ru binding. In fact, in an immiscible isoꢀoctaneꢀbuffer mixture, the
homogeneous Ruꢀcatalyst is partioned in the organic phase, while the biocatalyst is suspended in
water. The diene 6a is converted by GII into the 3ꢀpyrroline 7a in the isoꢀoctane phase, and
then7a is oxidized by MAOꢀD5 leading to the desired pyrrole 11a. The biphasic reaction
medium acts mimicking the compartmentalization of cellular processes allowing thus the
cascade reactions to take place in an efficient manner. The scope of the chemoenzymatic
cascade was then investigated as shown in Table 4. Pyrrole 11a was obtained in 88% conversion
and 78% isolated yield (method A). With the aim to improve the yield of the reaction, a two
steps method was also set up. Allylaniline 6a was mixed with GII and MAOꢀD5 and stirred at 37
oC for 6 h, after which time an additional amount of MAOꢀD5 was added (method B). Higher
conversion (95%) was observed with the oneꢀpot two steps protocol, but 11a was isolated in
lower yield (65%) (entry 2). It is noteworthy that the treatment of 7a with MAOꢀD5 led to 11a
with 61% conversion, whilst the chemoenzymatic cascade led to 11a from 6a in 95%
conversion.It is plausible that in the chemoenzymatic cascade the aniline 6a is converted into the
pyrroline 7a slowly. As soon as 7a if formed, it is immediately oxidised by MAOꢀD5 affording
11a. In this way only a low amount of 7a is oxidised by MAOꢀD5 time by time allowing a more
rapid enzyme turnover. Similarly, pyrroles 11b and 11l (entries 5ꢀ6, 11ꢀ12) were isolated in high
yields when the oneꢀpot oneꢀstep method A was used whilst higher conversion values were
observed with the two steps oneꢀpot protocol B. As general trend, excellent conversion and high
yields were observed for pyrroles bearing chloroꢀ (11l), methylꢀ(11m) and alkoxyꢀsubstituents
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(11n, 11o) (entries 11ꢀ17). Medium to good conversion (50ꢀ75%) was observed for pyrroles
11d-e (entries 6ꢀ8) probably due to the lower reactivity of electronꢀrich diallylanilines toward the
RCM reaction. In fact, several factors must be taken into account in the chemoenzymatic
cascade, such as the reactivity of anilines in the metathesis reaction, where electron donating
substituents on the phenyl ring disfavour the ring closure, as well as the biocatalytic oxidation,
where electron withdrawing substituents prevent the pyrroline oxidation.
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The chemoenzymatic cascade of alkylꢀdiallylamines was finally investigated. Not surprisingly,
the benzylpyrrole 12a was obtained in low yield (entry 18) due to the poor reactivity of alkylꢀ
diallylamines toward RCM reactions.21 It is well documented that tertiary amines inactivate GII
forming stable complexes and thus preventing the RCM cyclization. On the other hand, the
inactivation of GII by amines can be overcome if branched diallylamines are used as substrates.
In fact, branched pyrroles 12e and 12g were obtained from the corresponding allylamines in
good yields (entries 19ꢀ20).
The attempt to obtain the pyrrole 12l via the chemoenzymatic cascade from the corresponding
secondary amine was also unsuccessful (entry 21). An alternative route was thus developed
(Scheme 2) leading to 12l in oneꢀpot from the chloroethylꢀcarbamate 8. The carbamate
protecting group allows the RCM reaction of 8 to take place almost instantaneously (around 20
min) leading to the intermediate 13. The chloroethylꢀcarbamate protecting group is labile under
the reaction conditions and it is slowly cleaved leading to pyrroline 9c. The latter is in turn
oxidised by MAOꢀD5 affording the desired pyrrole 12l in 42% overall yield. Finally, the present
methodology has been applied to the synthesis of a series of pyrrole analogues of the
antitubercular agents recently described by us.2 As an example, the pyrroline 7b was converted
in a single step into the pyrrole 14 by treatment with MAOꢀD5 followed by in situ Mannich
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reaction with phenylpiperazine and formaldehyde. (Scheme 3). In addition, the pyrrole 12m,22
precursor of the natural alkaloid myrmicarin217, was synthesised from the diene 6t via the
chemoenzymatic method A in 20% isolated yield.23 (Scheme 3).
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CONCLUSIONS
In conclusion, the aromatizing properties of MAOꢀN biocatalysts have been disclosed for the
first time. MAOꢀN, and in particular the variant D5, catalyse the aromatization of a wide range
of Nꢀarylꢀ and Nꢀalkylꢀ3ꢀpyrrolines into the corresponding pyrroles. The ability of MAOꢀN
biocatalysts to work in a concurrent way together with Grubbs’ catalyst has been also
investigated leading to the development of a chemoenzymatic cascade reaction for the one pot
synthesis of pyrroles from dilallylꢀanilines and diallylꢀamines. This work represents the first
example of a chemoenzymatic cascade combining in the same reaction medium MAOꢀN with a
metalꢀcatalyst, other than boron reducing agents. The new methodology represents a robust and
sustainable alternative to standard catalytic methods for the synthesis of pyrroles, which
generally require the use of group specific or poorly available reagents, higher temperatures to
promote the aromatization step and lead to the formation of several side products. Finally, the
chemoenzymatic methodology is currently used in our laboratory for the synthesis of novel
antitubercular pyrrole derivatives.
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FIGURES
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Figure 1. Classical RCM approaches for the synthesis of pyrroles and MAOꢀN
aromatization of 3ꢀpyrrolines.
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Figure 2.Docking of pyrroline 7a into the MAOꢀD5 catalytic site and superposition of 7k on the
predicted conformation of 7a. The additional phenyl ring at C3 (in yellow) lay outside the
catalytic site.
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SCHEMES
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Scheme 1. Synthesis of 3ꢀpyrroline substrates
7 and 10.
Scheme 2.Chemoenzymatic synthesis of pyrrole 12l
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Scheme 3. a) MAOꢀMannich synthesis of pyrrole 14. b) Synthesis of 12m, precursor of
myrmicarin217
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TABLES.
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Table 1. MAOꢀN and 6ꢀHDNO aromatization of pyrrolines 7a-k
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38
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40
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53
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60
3-Pyr.
7a
Ar
R1
R2
R3
Biocat.a
Pyrrole Conv (%)b
MAOꢀD5
MAOꢀD9
6ꢀHDNO
MAOꢀD5
MAOꢀD5
MAOꢀD5
MAOꢀD9
6ꢀHDNO
MAOꢀD5
MAOꢀD9
6ꢀHDNO
MAOꢀD5
MAOꢀD9
6ꢀHDNO
MAOꢀD5
MAOꢀD5
MAOꢀD5
61
Ph
H
H
H
32
60
60
40
>99
50
27
82
54
21
45
34
30
2
11a
4ꢀFꢀPh
H
H
H
H
H
H
7b
7c
11b
11c
4ꢀBrꢀPh
4ꢀiPrꢀPh
4ꢀMeOꢀPh
2,5ꢀMeꢀPh
H
H
H
H
H
H
H
H
H
7d
7e
7f
11d
11e
11f
4ꢀNO2ꢀPh
4ꢀCNꢀPh
H
H
H
H
H
H
7g
7h
11g
11h
2
>99
51
55
0
Ph
H
H
Me MAOꢀD9
6ꢀHDNO
7i
11i
MAOꢀD5
MAOꢀD9
6ꢀHDNO
Ph
Ph
Ph
H
H
H
H
11jc
0
7j
0
Ph MAOꢀD5
11kc
0
7k
MAOꢀD9
0
aFreezeꢀdried E.coli whole cells were used. Conversion values were measured by
GCꢀMS spectroscopy and/or HꢀNMR. Pyrrolines 7j-k were also treated with
MAOꢀD11 variant, but no conversion was observed.
b
1
c
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Table 2. MAOꢀN and 6ꢀHDNO catalysed aromatization of pyrrolines 10a-h and 9
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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
Conv
3-Pyr.
Alk
R1
R2
R3
Biocat.a
Pyrrole
(%)b
MAOꢀD5
MAOꢀD9
6ꢀHDNO
>99
61
95
Bn
H
H
H
H
H
H
10a
10b
12a
12b
4ꢀClꢀBn
MAOꢀD5
MAOꢀD5
MAOꢀD5
88
H
H
H
H
H
H
H
H
H
>99
>99
10c
10d
10e
12c
12d
12e
Isovaleryl
Ph
MAOꢀD5
MAOꢀD5
MAOꢀD9
6ꢀHDNO
MAOꢀD5
>99
>99
51
H
H
H
H
H
H
10f
12f
65
56
10g
12g
Me Me
Me Me
H
H
MAOꢀD5
48
51
10h
10i
12h
12i
4ꢀClꢀBn
Me
MAOꢀD5
MAOꢀD5
92
78
87
H
H
H
H
H
H
Ph MAOꢀD9
6ꢀHDNO
10j
10k
9c
12j
12k
12l
iPr
Ph MAOꢀD5
31
MAOꢀD5
33
57
10
H
Ph MAOꢀD9
6ꢀHDNO
aFreezeꢀdried E.coli whole cells were used. Conversion values were
b
measured by GCꢀMS spectroscopy and/or 1HꢀNMR.
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Table 3. Optimization of the chemoenzymatic cascade
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9
10
11
12
13
14
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21
22
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30
31
32
33
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42
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59
60
Ratio 6a/7a/11a
Entry
Co-Solvent Buffer/Cosolvent
(%)a
1
2
3
4
5
6
7
8
DMF
Acetone
ꢀ
60:1
60:1
100:0
4:1
90/10/0
10/90/0
5/95/0
DMSO
DCM
73/27/0
75/25/0
77/23/0
92/8/0
4:1
DMF
4:1
THF
4:1
Isoꢀoctane
4:1
0/12/88
aConversion values were measured both by GCꢀMS
spectroscopy and/or 1HꢀNMR.
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Table 4. Chemoenzymatic cascade of diallyl anilines ꢀ substrate scope
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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
Entry
Pyrrole
11a
11a
R
Ph
Ph
4ꢀFꢀPh
4ꢀFꢀPh
4ꢀBrꢀPh
4ꢀiPrꢀPh
4ꢀiPrꢀPh
4ꢀMeOꢀPh
2,5ꢀMeꢀPh
Ph
R1
H
Conv. (%)a
Yield (%)b
Method
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
88
78
A
B
A
B
B
A
B
B
A
B
A
B
A
B
A
B
B
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
95
86
99
15
35
75
50
25
87
73
90
99
90
94
81
84
9
65
63
59
10
22
70
42
20
45
65
56
50
45
84
72
57
5
11b
11b
11c
11d
11d
11e
11f
11i
11l
11l
11m
11m
11n
11n
11o
12a
4ꢀClꢀPh
4ꢀClꢀPh
4ꢀMeꢀPh
4ꢀMeꢀPh
2ꢀMeOꢀPh
2ꢀMeOꢀPh
3,4ꢀ(OCH2O)ꢀPh
Bn
A & B
19
H
63
41
B
12e
Ph
20
21
H
37
0
21
0
B
B
12g
12l
H
Ph
aConversion values were measured both by GCꢀMS spectroscopy as well as 1HꢀNMR. bIsolated yields
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AUTHOR INFORMATION
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Corresponding Author
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*Daniele Castagnolo, Institute of Pharmaceutical Science, King’s College London, 150 Stamford
Street, SE1 9NH, London, United Kingdom. Eꢀmail: daniele.castagnolo@kcl.ac.uk
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures and full characterization for new compounds
and intermediates are reported in the Supporting Information. Additional unsuccessful
1
experiments are described. Copies of HꢀNMR and 13CꢀNMR spectra for new compounds are
reported.
ACKNOWLEDGMENT
We gratefully acknowledge the EPSRC UK National Mass Spectrometry Facility for providing
the mass spectrometry data. DC thanks the Royal Society of Chemistry (Research Fund 2015)
and EPSRCꢀKCLꢀStrategic Fund for financial support. GM thanks University of Padova for
Erasmus leaving.
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SYNOPSIS
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