Synthesis and antioxidant activity of DOPA peptidomimetics by a novel IBX mediated aromatic oxidative functionalization

Article abstract of DOI:10.1039/c5ra09464j

DOPA peptidomimetics with stable O-C and N-C covalent bonds between amino acid residues have been prepared by aromatic oxidative functionalization of tyrosine with 2-iodoxybenzoic acid (IBX). The reaction involves the Michael-like nucleophilic addition of

Full text of DOI:10.1039/c5ra09464j

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Synthesis and antioxidant activity of DOPA
peptidomimetics by a novel IBX mediated aromatic
Cite this: RSC Adv., 2015, 5, 60354
oxidative functionalization†
Bruno Mattia Bizzarri,a Cristina Pieri,a Giorgia Botta,a Lili Arabuli,c Pasquale Mosesso,a
b
a
a
*
Serena Cinelli, Angelo Schinoppi and Raffele Saladino
DOPA peptidomimetics with stable O–C and N–C covalent bonds between amino acid residues have been
prepared by aromatic oxidative functionalization of tyrosine with 2-iodoxybenzoic acid (IBX). The reaction
involves the Michael-like nucleophilic addition of different oxygen and nitrogen protected amino acids on a
reactive DOPA quinone intermediate. Similar results were obtained in heterogeneous conditions using
supported IBX-amide for more runs. Among the novel derivatives, compounds containing glycine
residues showed a more pronounced antioxidant activity in the 2,2-diphenyl picrylhydrazyl (DPPH)
radical scavenging cell free assay. Instead, valine derivatives showed the highest biological effect in
L5178Y mouse lymphoma cells, by assessing the ability to reduce H2O2 induced DNA breakage in the
alkaline comet assay.
Received 20th May 2015
Accepted 7th July 2015
DOI: 10.1039/c5ra09464j
www.rsc.org/advances
absorbed more efficiently than L-DOPA via the peptide trans-
porter in Caco-2 cells, which are considered to be a good model
for in vivo intestinal absorption in humans.8 In a similar way, D-
1. Introduction
Peptidomimetics are small molecules which are designed to
mimic a naturally occurring peptide. They are typically obtained
by modication of parent peptides or by total synthesis1 in order
to optimize pharmacological properties, such as bioavailability
and biological activity.2 The modications can involve N-alkyl-
ation, Ca-substitution, cyclization, N-replacement, carbonyl
replacement, heterocyclic generation, Ca-replacement, and
backbone or side-chain transformations, as well as the incor-
poration of unnatural amino acids.3 Among peptidomimetics,
DOPA derivatives play a crucial role in the therapy of Parkinson
disease (PD). PD is one of the most important neurodegenera-
tive disorder, characterized by dopamine (DA) depletion in
dopaminergic neurons of the striatum of the brain, inducing
rigidity, tremor, and postural instability as some of the most
important symptoms.4 DOPA peptides are able to increase the
capacity of DOPA in penetration of the blood brain barrier
(BBB)5 by specic peptide-mediated carrier transport systems
(PMCTS), thus restoring adequate DA concentration and
inhibiting oxidative cell damage.6 They also act as pro-drugs,
preserving DOPA from fast metabolic decarboxylation and
avoiding the peripheral DA-related side effects.7 L-DOPA-L-Phe is
Phe-L-DOPA showed 31-fold higher oral bioavailability and anti-
Parkinson activity than L-DOPA in rats.9 DOPA peptides and
peptidomimetics are usually synthesized by solution or solid
phase procedures, which show a different degree of complexity
depending on the method used for the activation/protection of
amino acids.10,11 Irrespective to experimental conditions, these
syntheses requires tedious and long time protecting/
deprotecting steps and have, in principle, an intrinsic low
selectivity. This study is focused on the design of a novel
synthetic procedure for the preparation of DOPA peptidomi-
metics by oxidative side chain modication of amino acid
residues.12–17 In this context, DOPA-peptides have been previ-
ously synthesized with complete stereochemical integrity by
oxidation of Tyr residues with tyrosinase from Agaricus bisporus
in organic solvent.18 1-hydroxy-1-oxo-1H-1l5-benz[d][1,2]
iodoxol-3-one (2-iodoxybenzoic acid, IBX) was also used in
similar trensformations.19,20 IBX performs the ortho-hydroxyl-
ation of phenol to catechols, with a selectivity similar to natural
polyphenol oxidases.21–25 The regioselectivity of the oxidation is
a consequence of the concerted intramolecular oxygen transfer,
from iodine (V) in l5-iodanyl intermediate (I), to ortho-position
of the phenol moiety, with concomitant reduction to l3-iodanyl
orthoquinol monoketal (II) (Scheme 1).26
aDepartment of Ecology and Biology, University of Tuscia, Via S. Camillo de Lellis,
01100 Viterbo, Italy. E-mail: saladino@unitus.it
bResearch Toxicology Center Menarini, Via Tito Speri 12/14, 00040 Pomezia (Roma),
In this reaction, the chirality of L-DOPA residues is not
affected, the L-enantiomer being the only stereoisomer
obtained.27 The replacement of the natural amide bond with
more stable covalent linkages In DOPA peptides can signi-
cantly improve the bioavailability and activity.28,29 It is well
Italy
cDepartment of Chemistry, Javakhishvili Tbilisi State University, Georgia
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra09464j
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aromatic oxidative functionalization of Tyr with different
oxygen and nitrogen protected a-amino acids. The procedure is
able to oxidize the phenol moiety to catechol group with
concomitant introduction of the a-amino acid residues on the
aromatic ring, exploiting the reactivity of the DOPA-quinone
intermediate.37 The general synthetic pathway is described in
Scheme 2.
2. Results and discussions
2.1. Synthesis of O–C bonded L-DOPA peptidomimetics
Following the general procedure in Scheme 1, we analyzed the
reactivity of N-Boc-Tyr-OMe (1) with a panel of N-Boc protected
a-amino acids, including glycine (N-Boc-Gly, 2), alanine (N-Boc-
Ala, 3), valine (N-Boc-Val, 4), leucine (N-Boc-Leu, 5), phenylala-
nine (N-Boc-Phe, 6), proline (N-Boc-Pro, 7), tryptophan (N-Boc-
Trp, 8) and methionine (N-Boc-Met, 9). The formation of the
L-DOPA quinone intermediate was obtained applying the
experimental conditions previously reported for the oxidation
of Tyr to L-DOPA with IBX.19 Briey, compound 1 (0.1 mmol) was
dissolved in THF (1.5 mL) in the presence of an excess of (2) (1.0
mmol, as selected nucleophile) and treated with IBX (0.3 mmol)
Scheme 1 Mechanism of oxidation of phenol by IBX.
ꢀ
at 25 C for 3.0 h. The color turned into brown-orange, that is
characteristic for the formation of quinone species. Aer work-
up and purication procedures, the desired N-Boc-Gly-N-Boc-
DOPA-OMe (10) was obtained in low yield, besides to unreac-
ted substrate and N-Boc-DOPA-OMe (11) (Scheme 2, Table 1,
entry 1). In the 1H-NMR of 10, the presence of two aromatic
hydrogens (singlets at 7.24 and 7.42 ppm), associated to that of
all expected side-chain absorption signals (i.g. two OMe moie-
ties at 3.74 and 4.11, and the Gly CH2 group at 3.93 ppm)
conrmed the mono-substitution pattern. Better results were
obtained increasing the temperature (45 ꢀC) and the reaction
time (72 h) to afford 10 in higher conversion and product yield
(Table 1, entry 2). On the basis of these data, the reaction was
extended to a-amino acids 3–9 to obtain the corresponding L-
DOPA peptidomimetics N-Boc-Ala-N-Boc-DOPA-OMe (12), N-
Boc-Val-N-Boc-DOPA-OMe (13), N-Boc-Leu-N-Boc-DOPA-OMe
(14), N-Boc-Phe-OMe (1) with N-protected a-amino acids.
Scheme 2 General reactivity scheme of Tyr with IBX in the presence
of protected a-amino acids. Step A: oxidation of Tyr to DOPA-
quinone. Step B: in situ Michael-like 1-4-addition of protected a-
amino acids on DOPA-quinone intermediate, followed by a reduction
step.
known that Tyr residues cross-link to protein receptors under
laccase and tyrosinase oxidation, by nucleophilic addition of
sulydryl groups.30–34 As a general trend, very complex protein
agglomerates are obtained, oen used as glues.35 Although few
informations are available for the structure of these products, it
is expected that the reaction proceeds through nucleophilic
addition on reactive DOPA ortho-quinone intermediate
following the Michael-like 1-4-regiochemistry.36 With the aim to
synthesize new L-DOPA-peptidomimetics characterized by
stable O–C and N–C bonds, we report here the IBX mediated
Table 1 Synthesis of O–C bonded L-DOPA peptidomimetics
Entry
Amino acid
R
R1
Products
Conversion (%)
Yieldsa (%)
1
2
3
4
5
6
7
2
2
3
4
5
6
7
H
H
CH3
CH(CH3)2
CH2CH(CH3)2
CH2C6H5
CH2CH2CH2
H
H
H
H
H
H
10(11)
10(11)
12(11)
13(11)
14(11)
15(11)
16(11)
70
21(19)b
70(10)
65(15)
58(8)
56(10)
50(16)
52(12)
$98
$98
$98
$98
$98
$98
8
8
9
H
H
17(11)
18(11)
$98
$98
51(12)
57(3)
9
CH2CH2SCH3
a
Reaction conditions: compound 1 (0.1 mmol) was dissolved in THF (1.5 mL) in the presence of the appropriate protected a-amino acids 2–9 (1.0
b
mmol) and treated with IBX (0.3 mmol) at 45 ꢀC for 72 h. Reaction performed at 25 ꢀC for 3 h.
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In the case of aliphatic a-amino acids, the yield increased by conversion of substrate (Table 2, entries 2, 4, 6 and 8 versus
decreasing of the steric hindrance of the side chain (Table 1, entries 1, 3, 5 and 7).
entries 2 and 3 versus entries 4 and 5). Note that, possible side-
In this latter case, we were not able to recognize any specic
products due to IBX side-chain oxidation of others low redox relationship between the steric hindrance of the side-chain and
potential residues (e.g. tryptophan), were not detected in the the yield of desired product. A different reaction pathway was
reaction mixture. This result is in accordance with the high observed with a-amino acids 24–26, in which case THF was the
selectivity of IBX towards the oxidation of phenolic aromatic best reaction solvent (Table 2, entries 9–16). In some cases (e.g.
moieties.38
Table 2, entries 3, 5, 14 and 16), the relatively low yield of
desired product with respect to the high value of conversion of
substrate can be due to formation of undesired side-products
difficult to be detected and recovered from the reaction
mixture. Note that in the case of the reactions performed with
25 and 26 in THF, a dimeric DOPA derivative (compound 35,
Scheme 3) was detected in low amount as a side-product (7%
and 9%, respectively). This compound showed all signals (and
2.2. Synthesis of N–C bonded L-DOPA peptidomimetics
The procedure was generalized by the use of a-amino acid
methyl ester derivatives Gly-OMe (19), Ala-OMe (20), Val-OMe
(21), Leu-OMe (22), Phe-OMe (23), Pro-OMe (24), Trp-OMe
(25), Met-OMe (26) to synthesize N–C bonded L-DOPA peptido-
mimetics. The oxidation of compound 1 (0.1 mmol) with IBX
(0.3 mmol) in THF (1.5 mL) in the presence of 19 (1.0 mmol)
afforded Gly-N-Boc-DOPA-OMe (27) in low yield, besides to
traces of N-Boc-DOPA-OMe (11) (Scheme 4, Table 2, entry 1). The
reactivity of IBX can be tuned by the use of organic solvents with
different properties, the efficacy of the oxidation depending on
the nature of the substrate.39 For this reason, we repeated the
reaction in the presence of MeOH (1.5 mL) as an alternative
solvent, under previously described experimental conditions. As
reported in Table 2 (entry 2 versus entry 1), the reaction in
MeOH afforded compound 27 in highest yield and conversion
of substrate. Further oxidations were then performed with both
THF and MeOH solvents. Better results were obtained for
aliphatic a-amino acids 20–22 in MeOH, to afford Ala-N-Boc-
DOPA-OMe (28), Val-N-Boc-DOPA-OMe (29), and Leu-N-Boc-
DOPA-OMe (30) in appreciable yield and quantitative
1
multiplicity) in H-NMR analysis (e.g. 7.46 ppm and 5.70 ppm
singlets for symmetric couples of aromatic protons) expected
for a dimeric structure, and was probably obtained by oxidative
coupling of radical intermediates. This hypothesis is in accor-
dance with previously reported results during IBX oxidation of
2-methoxy- and 2-methyl-substituted phenols in THF.40 The
C(6)–C(6) regiochemistry in 35 was assigned by comparison of
the 1H NMR multiplicity of aromatic protons with similar dimer
species characterized in the polymerization of 3-(3,4-dihydrox-
yphenyl) propionic acid (DHPA) with Fe3+ ions.41
Finally, in view of possible large scale applications, we eval-
uated the use of heterogeneous conditions by applying polymer
supported IBX-amide, that is an easily recoverable and reusable
oxidant.42 The oxidation of Boc-Tyr-OMe (1) in the presence of
Gly-OMe (19) in MeOH was performed under previously opti-
mized experimental conditions. Comparable results in terms of
Table 2 Synthesis of N–C bonded L-DOPA peptidomimeticsa
Entry
Amino acid
Solvent
R
R1
Product
Conversion (%)
Yield (%)
1
2
3
4
5
6
7
8
19
19
20
20
21
21
22
22
23
23
24
24
THF
MeOH
THF
MeOH
THF
MeOH
THF
MeOH
THF
MeOH
THF
H
H
CH3
CH3
CH(CH3)2
CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH2C6H5
CH2C6H5
CH2CH2CH2
CH2CH2CH2
H
H
H
H
H
H
H
H
H
H
27(11)
27(11)
28(11)
28(11)
29(11)
29(11)
30(11)
30(11)
31(11)
31(11)
32(11)
32(11)
85
27(5)
$98
$98
$98
70
95
87
96
$98
81
87
85
65(10)
48(10)
80(5)
40(16)
60(13)
43(21)
65(11)
90(8)
9
10
11
12
45(25)
53(13)
11(36)
MeOH
13
25
THF
H
H
33(11) (35)
$98
$98
80(4)(7)
14
25
MeOH
33(11)
32(20)
15
16
26
26
THF
MeOH
CH2CH2SCH3
CH2CH2SCH3
H
H
34(11)(35)
34(11)
$98
96
72(5)(9)
40(20)
a
Reaction conditions: compound 1 (0.1 mmol) was dissolved in the appropriate solvent (1.5 mL) in the presence of protected a-amino acids 19–26
(1.0 mmol) and treated with IBX (0.3 mmol) at 45 ꢀC for 72 h.
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Table 3 Reusability of heterogeneous IBX-amide in oxidative func-
tionalization of N-Boc-Tyr-OMe (1) with Gly-OMea (19)
Run
Amino acid
Product
Conversion (%)
Yield (%)
1
2
3
4
5
19
19
19
19
19
27(11)
27(11)
27(11)
27(11)
27(11)
$98
$98
$98
$98
$98
65(8)
67(9)
63(10)
65(11)
66(7)
a
Reaction conditions: compound 1 (0.1 mmol) was dissolved in MeOH
(1.5 mL) in the presence of protected a-amino acids 19 (1.0 mmol) and
treated with IBX-amide at 45 ꢀC for 72 h.
Scheme 3 IBX mediated oxidative functionalization of N-Boc-Tyr, N-
Boc-DOPA-OMe (15), N-Boc-Pro-N-Boc-DOPA-OMe (16), N-Boc-
Trp-N-Boc-DOPA-OMe (17), N-Boc-Met-N-Boc-DOPA-OMe (18),
from acceptable to high yield (Scheme 2, Table 1, entries 3–9).
3. Antioxidant activity
Radical oxygen centered species (ROS) are formed in the cell
under normal metabolic and physiologic processes.44 In PD,
hydroxyl and superoxide radicals, which are produced by
oxidative phosphorylation,45 can damage mitochondrial DNA
(mtDNA), causing modulation in the electron transport chain
(ETC).46 The catechol pharmacophore in DOPA plays a key role
in scavenging ROS by formation of stable phenoxyl radical
species,47 as evaluated by standard and modied comet assays
in mammalian cells.48
Furthermore, the administration of L-DOPA reduces hypoxia
conditions and induces the over-expression of ORP150 (oxygen
regulated protein 150 kDa) with concomitant cytoprotective
effects, and possible activation of endogenous antioxidant
mechanisms.49 On the basis of these data, we started to evaluate
the antioxidant activity of novel synthesized DOPA peptidomi-
metic derivatives. The in vitro antioxidant activity of catecol
compounds is usually determined by spectrophotometric
analyses. We evaluated the 2,2-diphenyil picrylhydrazyl (DPPH)
radical scavenging properties of compounds 10, 12–18 and 27–
34, using 11 as reference. Briey, the appropriate compound
was added to freshly prepared DPPH solution (6 ꢁ 10ꢂ5 M in
EtOH) and the decrease in absorbance (475 nm) was deter-
mined at different times until the reaction reached a plateau.
The kinetic of the process was analyzed for each concentration
tested, and the rate of DPPH remaining at the steady state was
Scheme 4 IBX mediated oxidative functionalization of N-Boc-Tyr-
OMe (1) with O-protected a-amino acids.
Scheme 5 Supported IBX-amide mediated oxidative functionalization
of N-Boc-Tyr-OMe (1) with Gly-OMe (19).
conversion of substrate and yield of 27 were obtained with
respect to IBX. Recycling experiments proceeded with success
(Scheme 5). Aer the disappearance of compound (1), the IBX-
amide was recovered by ltration, regenerated with Oxone® as
reported43 and reused in further oxidations. As shown, IBX-
amide was used for at least ve cycles to give 27 without
appreciable loss of efficiency (Table 3, runs 1–5).
Fig. 1 2,2-Diphenyil picrylhydrazyl (DPPH) radical scavenging prop-
erties of compounds 10, 12–18 and 27–34. (a) IC50 value of O–C
bonded L-DOPA peptidomimetics 10, and 12–18 (b) IC50 value of N–C
bonded L-DOPA peptidomimetics 27–34. IC50 is the drug concen-
tration causing 50% inhibition of the desired activity. Each experiment
was conducted in triplicate.
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 60354–60364 | 60357