Exploring the copper binding ability of Mets7 hCtr-1 protein domain and His7 derivative: An insight in Michael addition catalysis

Article abstract of DOI:10.1002/psc.3289

Mets7 is a methionine-rich motif present in hCtr-1 transporter that is involved in copper cellular trafficking. Its ability to bind Cu(I) was recently exploited to develop metallopeptide catalysts for Henry condensation. Here, the catalytic activity of Me

Full text of DOI:10.1002/psc.3289

Received: 31 July 2020
DOI: 10.1002/psc.3289
Revised: 18 September 2020
Accepted: 21 September 2020
R E S E A R C H A R T I C L E
Exploring the copper binding ability of Mets7 hCtr-1 protein
domain and His7 derivative: An insight in Michael addition
catalysis
Isabella Rimoldi1 | Raffaella Bucci1 | Lucia Feni1 | Laura Santagostini2
Giorgio Facchetti1 | Sara Pellegrino1
|
1Dipartimento di Scienze Farmaceutiche,
Università degli Studi di Milano, Milan, Italy
Mets7 is a methionine-rich motif present in hCtr-1 transporter that is involved in
2Dipartimento di Chimica, Università degli
Studi di Milano, Milan, Italy
copper cellular trafficking. Its ability to bind Cu(I) was recently exploited to develop
metallopeptide catalysts for Henry condensation. Here, the catalytic activity of
Mets7-Cu(I) complex in Michael addition reactions has been evaluated. Furthermore,
His7 peptide, in which Met residues have been substituted with His ones, was also
prepared. This substitution allowed His7 to coordinate Cu (II), with the obtainment of
a stable turn conformation as evicted by CD experiments. His7-Cu (II) proved also to
be a better catalyst than Mets7-Cu(I) in the addition reaction. In particular, when the
substrate was the (E)-1-phenyl-3-(pyridin-2-yl)prop-2-en-1-one, a conversion of 71%
and a significative 58% of e.e. was observed.
Correspondence
Giorgio Facchetti and Sara Pellegrino,
Dipartimento di Scienze Farmaceutiche,
Università degli Studi di Milano, Via Venezian
21, Milan 20133, Italy.
Email: giorgio.facchetti@unimi.it; sara.
pellegrino@unimi.it
K E Y W O R D S
chalcone, copper, hybrid catalyst, metallopeptide, Michael reaction
1
|
INTRODUCTION
levels of efficiency and of chemoselectivity, regioselectivity and
stereoselectivity,17–20 giving thus an inspiration for the development
Metal coordination to proteins is often a necessary requirement to
provide functional structures as well as enzymatic catalytic activi-
ties.1 Commonly, amino acids bind metal ions through the formation
of stable five-membered chelates, but the presence of different side
chains, comprising more than one set of donor atoms, greatly
increases coordination geometries variability and thermodynamic
stability.2–4 Enzymes active sites possess unique architectures deter-
mined by the nature and the position of the amino acid residues
involved in metal ion coordination.5 This intricate three-dimensional
network of interactions creates an asymmetric environment around
the metal centre allowing an efficient substrate recognition and ori-
entation. It is thus not surprising that chemists have been exploring
the advantageous properties of peptides as ligands for metal ions for
asymmetric catalysis.6–9 Indeed, despite the achievements obtained
using transition metal catalysts based on chiral diphosphines and
N-chelating ligands, many transformations are still hampered by
several issues concerning reactivity and selectivity.10–16 Conversely,
enzymes often outperform transition metal catalysts with higher
of enzyme-like transition metal hybrid catalysts.19–23 Combining the
rational design of the peptide ligand and the choice of an appropri-
ate metal, it is possible to merge the high reactivity of transition
metals with the structural and functional groups versatility of
peptides, achieving levels of reactivity otherwise unrealisable. A
common approach in this regard takes advantage of natural peptide
sequences known for their ability to coordinate to metal ions.24
Indeed peptides can be used as templates for inserting non-natural
amino acids or synthetic scaffolds stabilizing a specific secondary
structure,25–27 and/or allowing the coordination to a specific transi-
tion metal.28 In this context, Roelfes' research group modified bovine
pancreatic polypeptide, a 36-mer member of pancreatic hormones,29
by truncating it to
a 31-mer derivative and introducing non-
proteinogenic amino acids in order to develop a functional ligand for
Cu2+ catalysis. These hybrid systems, applied to the asymmetric
Diels-Alder and Michael addition reactions on α,β-unsaturated
carbonyl compounds, afforded the desired products in moderate-
to-good enantioselective levels. Recently, starting from ATCUN
J Pep Sci. 2020;e3289.
wileyonlinelibrary.com/journal/psc
© 2020 European Peptide Society and John Wiley & Sons, Ltd.
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https://doi.org/10.1002/psc.3289

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RIMOLDI ET AL.
motif,30,31 a Cu (II)- and Ni (II)-binding sequence found at the
N-terminus of many naturally occurring proteins, ultrashort peptides
were developed as iridium binding catalytic systems for asymmetric
transfer hydrogenation of substituted ketones in water.32,33 The
same scaffolds were successfully employed in the preparation of
cobalt-based hybrid electrocatalysts, for the selective six-electron
reduction of nitrite and hydroxylamine intermediates to ammonium
in aqueous buffer.34,35
spectrometer at 300 and 75 MHz, respectively. Chemical shifts are
given as δ values in ppm relative to residual solvent peaks as the inter-
nal reference. 13C NMR spectra are 1H-decoupled, and the determina-
tion of the multiplicities was achieved by the APT pulse sequence.
2.2 | General procedure for the synthesis of peptide
In our research group, we recently reported the catalytic ability of
the complex between Mets7 and Cu(I) in Henry condensation reac-
Peptide His7 and Mets7 were synthetized on Rink-amide resin
(250 mg, 0.69 loading) using microwave-assisted solid phase pep-
tide synthesis.41 A five-fold molar excess of Fmoc-protected amino
acids (0.2M in DMF), and Oxima/DIC (5:5 eq) as activators were
used. Coupling reactions were performed for 5 min at 40 W with a
maximum temperature of 75ꢀC. His coupling was performed for
2 min at room temperature (rt) at 0 W followed by 4 min at 35 W
with a maximum temperature of 50ꢀC. Deprotection was performed
twice using 20% piperidine in DMF (5 and 10 min each). Acetyla-
tion was performed manually on resin using Ac2O (10 eq.) and
DIPEA (10 eq.), two couplings of 1 h each. The final cleavage cock-
tail was made by trifluoroacetic acid (TFA, 8 ml), triisopropylsilane
(400 μl), thioanisole (400 μl), water (200 μl) and phenol (0.6 g) for
4 h. After the cleavage, the peptides were precipitated in cold
diethyl ether and centrifuged three times (3 min, 6000 rpm, 4ꢀC).
tion.28 Mets7 is
a methionine-rich motif naturally present in
hCtr1copper transporters, devoted to the trafficking of cellular copper
and recognized for being involved in inducing resistance to therapeu-
tically approved platinum drugs by their sequestration.36 In our
previous work, we developed a series of hybrid catalysts obtaining
significative e.e. and yield in the reaction between substituted benzal-
dehydes with nitromethane.
Here, we investigated the catalytic ability of Mets7-Cu(I) complex
in asymmetric Michael addition of nitromethane to a series of
homocycle and heterocycle chalcones. This reaction was selected as it
affords valuable intermediates in the synthetic pathway for GABAB
receptor agonists.37 Furthermore, we developed a new chiral ligand
called His7, in which Met residues have been replaced with His. This
replacement allowed Cu (II) binding,38,39 with the obtainment of a
stable turn conformation as evicted by CD experiments. In Michael
addition,40 His7-Cu (II) proved to be a better catalyst than Mets7-Cu
(I). In particular, when the substrate was the (E)-1-phenyl-3-(pyridin-
2-yl)prop-2-en-1-one, a conversion of 71% and a significant 58% of
e.e. were obtained.
Mets7 was purified by RP-HPLC using
a gradient elution of
5%–70% solvent (solvent A: water/acetonitrile/TFA, 95:5:0.1;
B
solvent B: water/acetonitrile/TFA, 5:95:0.1) over 20 min at a flow
rate of 20 ml/min. His7 was purified by fractioned precipitation
from tBuMe.ether/hexane 1:1 mixture. The peptides were analysed
by analytical RP-HPLC, showing a purity higher than 90%, and then
freeze-dried and stored at 0ꢀC.
2
| MATERIALS AND METHODS
Calcd
MW
Found
MW
rta
Yield
(mg)
Peptide
Mets7
His7
Sequence
(min)
2.1 | General
M T G M K G M S
H T G H K G H S
882.36
900.94
883.61
901.72
17.5
11.8
50
80
All reactions were carried out in oven-dried glassware and dry sol-
vents under nitrogen atmosphere. Fmoc Rinkamide resin, Fmoc-
protected (L)-amino acids, HBTU, HOBt and DIEA were purchased
from Iris Biotech Gmbh (Germany). Solvents, piperidine and other
reagents were purchased from Sigma-Aldrich (Germany). His7 and
Mets7 were synthesized using a CEM Liberty Blue peptide synthe-
sizer and purified using RP-HPLC with a Jasco BS-997-01 instrument
a
Gradient elution of 2%–60% solvent B (solvent A: water/+0.1% TFA;
solvent B: acetonitrile/+0.1% TFA) over 20 min at a flowrate of
0.8 ml/min.
2.3 | Synthesis of Cu(I) complex
and
a DENALI C-18 column from GRACE VYDAC (10 μm,
250 × 22 mm). MS analyses were performed with a Thermo Finnigan
(MA, USA) LCQ Advantage system MS spectrometer with an
electrospray ionization source and an ‘Ion Trap’ mass analyser. The
MS spectra were obtained by direct infusion of a sample solution in
MeOH under ionization, ESI positive. Circular dichroism (CD) spectra
were recorded on a Jasco-810 spectropolarimeter. Catalytic reactions
were monitored by HPLC analysis with Merck-Hitachi L-7100
equipped with Detector UV6000LP and chiral column (AD, OJ-H Chi-
ralcel, Lux Cellulose-4, Lux Cellulose-2 or Lux Amylose-2). 1H NMR
and 13C NMR spectra were recorded with Varian Oxford 300 MHz
In a Schlenk tube containing 8 mg of Mets7 or His7 in 2 ml of MeOH,
4.6 mg of solid Cu (OAc)2 (1 eq.) was added. Ascorbic acid (2 eq.) of
8.1 mg was introduced for in situ reduction of Cu (II) to Cu(I). The
solution was stirred overnight at rt, and a formation of precipitate was
observed. The precipitate was filtered and dried in vacuum. The solid
was washed three times with Et2O and analysed by ESI-MS.
Mets7-Cu(I) (pale yellow powder, 4.5 mg) yield 49%. ESI-MS for
[C34H62N10O11S3Cu]+ (calcd 945.31) found 945.3. His7-Cu(I) (pale
brown powder, 5.2 mg) yield 57%. ESI-MS for [C37H56N16O11Cu]+
(calcd 962.36) found 963.38 [M + H]+.

RIMOLDI ET AL.
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2.4 | Synthesis of Cu (II) complex
The conversion and the enantiomeric excess were determined by
HPLC analysis (see Table T2 in the Supporting Information).
In a Schlenk tube containing 8 mg of His7 in 2 ml of MeOH, 4.6 mg of
solid Cu (OAc)2 (1 eq.) was added. The solution was stirred overnight
at rt, and a formation of precipitate was observed. The precipitate
was filtered and dried in vacuum. The solid was washed three times
with Et2O and analysed by ESI-MS. His7-Cu (II) (blue-green powder,
7.3 mg) yield 80%. ESI-MS for [C37H56N16O11Cu]2+ (calcd 962.36)
found 963.44 [M + H]+.
3
| RESULTS AND DISCUSSION
Mets7 (MTGMKGMS) is a 8-mer peptide derived from hCtr1 trans-
port protein able to chelate Cu(I).42,43 In 2007, Arnesano and
coworkers demonstrated that the sulphur atoms of Mets7 are able to
chelate both Cu(I) and its Pt (II) bioisoster. This coordination allows
the peptide conformational change from an unordered to two consec-
utive β-turns structure. This structuring process is responsible of the
copper trafficking inside the cell and of cis-platinum transport through
the membranes.42 In our previous work,28 we demonstrated the cata-
lytic ability of Mets7-Cu(I) complex in Henry condensation reaction.
In particular, the presence of a stable β-turn conformation was found
crucial for enhancing both the reaction rate and the enantioselection.
Here, Mets7-Cu(I) catalyst was investigated in asymmetric Michael
addition of nitromethane to a series of homocycle and heterocycle
chalcones. Furthermore, we developed His7 peptide (HTGHKGHS), in
which the Met residues have been replaced by His ones. The presence
of the imidazole ring of His could indeed allow the binding of both
Cu(I) and Cu (II), without interfering with the peptide structural
rearrangement because of the metal complexation. Furthermore, the
HSAB principles establish border-line Cu (II) ion preference for the
imidazole histidyl N-donor as the best match allowing thus a substan-
tial change in the geometry of the related complex achieving a
square-planar rather than tetrahedral complex. The obtainment of
metal complexes with Cu (II) could give the possibility to extend the
scope of reactions thanks to different coordination geometries and to
improve stability towards oxidation.
2.5 | Circular dichroism
Solutions of peptides Mets7 and His7 and complexes Mets7-Cu(I),
His7-Cu(I) and His7-Cu (II) were prepared in H2O (50 μM, 1.5 ml).
Spectra were obtained from 195 to 250 nm with a 0.1 nm step and
1 s collection time per step, measuring three averages. The spectrum
of the solvent was subtracted to eliminate interference from cell,
solvent and optical equipment. The CD spectra were plotted as mean
residue ellipticity θ (degree × cm2 × dmol−1) versus wave length λ
(nm). Noise reduction was obtained using a Fourier-transform filter
program from Jasco.
2.5.1 | General procedure for asymmetric Michael
reaction
A mixture of powdered flame-dried CsF (2 eq.), peptide (0.022 eq.),
Cu (OAc)2 (0.02 eq.) and chalcone (1 eq.) in toluene (0.15 ml) was
treated with nitromethane (10 eq.). The mixture was stirred at rt for
18 h and then diluted with 400 μl of Et2O and 400 μl of H2O. The
organic phase was separated, dried over anhydrous Na2SO4 and
evaporated.
Both His7 and Mets7 peptides were synthetized by Fmoc solid
phase peptide synthesis, using Rink-amide resin (0.69 mmol/g)
following standard procedures.41,44 The complexes were prepared by
mixing equimolar quantities of Mets7 or His7 with Cu (OAc)2 in
MeOH and adding two equivalent of ascorbic acid for inducing the Cu
In the case of Mets7-Cu(I) and His7-Cu(I), the reaction was
performed applying the same procedure by using the preformed
complex as described above.
FIGURE 1 CD spectra (50 μM in water) of (left) Mets7 and Mets7/Cu(I) complex and (right) His7, His7/Cu(I) and His7/Cu (II) complexes

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RIMOLDI ET AL.
(II) reduction to Cu(I). The formation of the corresponding Cu(I)-
complexes was evinced from ESI-MS spectra. For His7, using the
same copper precursor, the Cu (II) complex was synthetized, and also
in this case, its formation was proved by ESI-MS. The coordination of
His7 to copper metal centre was then confirmed by the bathochromic
shift of the metal-to-ligand charge transfer transition band45 in the
UV spectra at 407 nm, more intense for His7-Cu (II) complex than in
the case of the His7-Cu(I) one (see Supporting Information).
The conformational behaviour of peptides and their complexes
were investigated in water by CD experiments (Figure 1). CD is indeed
a powerful tool to detect conformational changes also in short pep-
tides.41,46 As expected, the CD spectra of His7 and Mets7 alone were
characterized by the presence of
a negative band at around
195–198 nm, indicating the absence of a preferred conformation. CD
spectra of Cu(I) complexes suggested for both peptides a conforma-
tional rearrangement. For Mets7-Cu(I), the metal coordination
TABLE 1 Enantioselective Michael addition
Entry
1
Catalyst
Substrate
Conversion %a
e.e. %a
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
His7-Cu (II)
Mets7-Cu(I)
Mets7-Cu(I)
Mets7-Cu(I)
His7-Cu(I)
1a
1b
1c
1d
1e
1f
1g
1h
1i
1l
1m
2
85
99
99
99
99
99
99
99
99
99
99
14
15
71
99
39
83
35
25
43
-
2
32 (1 ent)
3
-
4
-
5
-
6
5 (2 ent)
7
-
8
-
9
8 (2 ent)
10
11
12
13
14
15
16
17
18
19
20
10 (1 ent)
-
7 (1 ent)
3
11 (2 ent)
4
58 (2 ent)
1a
3
-
-
4
13 (2 ent)
1a
3
-
His7-Cu(I)
5
-
His7-Cu(I)
4
Note: Reaction were conducted with peptide (0.022 eq.), Cu (OAc)2 (0.02 eq.), CsF (2 eq.) and CH3NO2 (10 eq.) in toluene, final substrate concentration of
350 mM at room temperature for 18 h.
aConversion and enantiomeric excess were determined by HPLC (see Table T2 in the Supporting Information).

RIMOLDI ET AL.
5 of 7
FIGURE 2 Proposed catalytic cycle for asymmetric Michael addiction with His7-Cu (II)
between the three sulfur atoms present on the three Met led to an
important structural change in the peptide, as previously reported.28
In the CD spectrum, indeed, the blue shift of the band at 195 nm and
the positive band at around 208 nm indicated the presence of a turn
or a bended conformation. In the case of His7-Cu(I) a change of pep-
tide conformation was also suggested by the presence of a negative
peak at 195 nm and two shoulders at 208 and 235 nm. Anyway, the
conformational induction upon complexation was more evident in the
case of His7-Cu (II) complex, with the obtainment of a β-turn-like
spectrum characterized by a strong negative value at 190 nm, a
positive Cotton effect at 205 nm and a negative minimum at 225 nm.
From these findings we can thus confirm that the His/Met substitu-
tion does not interfere with the peptide rearrangement upon
complexation.
the (E)-1-phenyl-3-(pyridin-2-yl)prop-2-en-1-one (Table 1, entry 14).
In the case of substrate 4, a modest 13% e.e. was also obtained when
Mets7-Cu(I) was used as catalyst, probably because of an additional
coordination in the transition state of the pyridinic N to the metal.48
The obtained data agreed with the proposed reaction mechanism
depicted in Figure 2. First, nitromethane displaces acetate in the
His7-Cu (II) complex, with the obtainment of complex A. Then a
nucleophilic addition to the chalcone occurred with the formation of
O-enolate. The pivotal role of the peptide turn conformation around
the copper centre makes the catalytic moiety assessable only by
one of the two pro-chiral faces of the substrate dictating the
enantioselectivity of the product. The elimination of the product
regenerates the precatalyst, able to react with another nitromethane.
Furthermore, both His7-Cu (II) and Mets7-Cu(I) complexes led to
the same enantiomers, suggesting a similar coordination mode of the
two peptides around the metal centre as highlighted by CD data. On
the other hand, the drastic decrease of the enantioselectivity for
Mets7-Cu(I) and His7-Cu(I) complexes could be ascribed to a thermo-
dynamically more stable conformation of His7 when coordinated to
Cu (II).
The catalytic performance of peptide–copper complexes was
then investigated in the asymmetric Michael condensation of different
substituted chalcones47 with nitromethane. This reaction represents
an important synthetic tool to form new C–C bonds, and it is com-
monly used for the obtainment of valuable synthetic intermediates.
Three different benzoyl group-activated olefins such as the 2- or
4-imidazolyl enones (2 and 3 respectively) and the 2-pyridinyl enone
4 were synthesized and evaluated as substrates for the synthesis of
the corresponding chiral products using the obtained Mets7 and His7
complexes.13 A preliminary screening (See Supporting Information) in
which different reaction conditions were tested, changing the solvent,
substrate/catalyst ratio, basic additive and temperature, was first
performed. As a result, toluene was confirmed as the solvent of choice
and CsF as the best performing base. A substrate/catalyst ratio of
50/1 was used for determining conversion in 18 h, and the reaction
was conducted at rt to increase the reaction rate without harming the
enantioselectivity.
4
| CONCLUSION
In this work, we investigated the catalytic ability of peptides derived
from hCtr1 copper transporter domain in asymmetric Michael addition
of nitromethane to chalcones. The introduction of His residues
favoured the formation of a stable Cu (II) complex characterized by a
β-turn conformation. In particular, the reaction proceeded with good
enantioselectivity when compound 4 ((E)-1-phenyl-3-(pyridin-2-yl)
prop-2-en-1-one) was used as the substrate, reaching a valuable
58% e.e.
His7-Cu (II) revealed to be the best catalyst, both in terms of con-
version and enantioselection, except in the case of substrates 2 and
3 carrying the imidazole moiety (Table 1, entries 12, 13, 16 and 19). In
particular, significative e.e. were obtained for compounds 1b, the (E)-
3-(3-nitrophenyl)-1-phenylprop-2-en-1-one (Table 1, entry 2,), and 4,
ORCID
Sara Pellegrino
https://orcid.org/0000-0002-2325-3583

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RIMOLDI ET AL.
21. Santi N, Morrill LC, Luk LY. Streptavidin-hosted organocatalytic aldol
addition. Molecules. 2457;2020:25.
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