The synthesis of an EDTA-like chelating peptidomimetic building block suitable for solid-phase peptide synthesis

Article abstract of DOI:10.1039/c6cc10203d

A novo trifunctional EDTA-like peptidomimetic amino acid is described. This unique building block, which is prepared in a straightforward manner from commercialized starting materials, contains three moieties: a hexadentate chelating unit similar to that

Full text of DOI:10.1039/c6cc10203d

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
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3
N@C2n (2n
=
68 and 80). Synthesis of the
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DOI: 10.1039/C6CC10203D
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Synthesis of an EDTA-like Chelating Peptidomimetic Building Block
Suitable for Solid-Phase Peptide Synthesis
Received 00th January 20xx,
Accepted 00th January 20xx
a,b,c*
Jan Spengler,
a,d
a
a
a,e
Michael Barker, Constanze Schelhorn, Jesús García, Maria J. Macias and
a,b,f,g*
Fernando Albericio
DOI: 10.1039/x0xx00000x
www.rsc.org/
A novo trifunctional EDTA-like peptidomimetic amino acid is modified to contain an additional amino and an additional
described. This unique building block, which prepared in a carboxylic groups crafted to the pendant arms of the EDTA-like
straighforward manner from commercialized starting materials, moiety.
O
CO
NHBoc
Br HO
2
H
contains three moieties: a hexadentate chelating unit similar to
that present in EDTA, and also, the amino and carboxylic groups,
which faciliate its introduction into the backbone of peptides
using conventional SPPS. As a proof of concept, this building block
is introduced into a cyclic peptide inspired in the family of Gratisin
analogues. The designed peptide model contains the amino acid
FmocHN
CO t
1
2
Bu
O
O
FmocHN
O
N
t
Br
CO2 Bu
N
tBuO
2
C
Ca2+
N
CO t
2
Bu
tBuO
C
2
Br NH
O
2
N
tBuO
2
C
2
HO C
O
O
CO
2
H
2
CO Bzl
2
+
Fmoc-NH-EDTA(tBu)4-OH, 10
4
O
analogue in one of the turns, and chelates Ca with nanomolar
affinity at physiological pH.
Figure 1. EDTA and Fmoc-NH-EDTA(tBu) -COOH 10
4
Bifunctional chelating agents (BFCAs) derived from polyamino
polycarboxylic ligands are efficient and widely used for the
Here we report the synthesis of Fmoc-NH-EDTA(tBu)
4
-COOH
1
coordination of metal ions. In addition to a chelating moiety,
1
0
, its introduction into a cyclic model peptide, and the
2
+
BCFAs have only one functional group, which allows for
covalent attachment to biomolecules—mostly peptides. The
resulting conjugates attract interest in biomedicine as metal-
based pharmaceuticals, with applications such as metal-
labeled drugs for diagnostic purposes or radioactively labeled
characterization of the peptide´s Ca
chelation at
physiological pH by CD, NMR and ITC. Compound 10 has the
EDTA substructure running through its backbone, which is, in
turn, stable to proteolytic degradation (Figure 1). The
functional groups are adequately protected for standard
coupling conditions used in solid-phase peptide synthesis
2
drugs for therapy. In these cases, the metal ion coordination
with BCFAs is mostly restricted to the peptide N or C termini.
To expand the potential use of these ligands, we sought to
design a chelating agent (CA) that can be introduced into the
(
SPPS).
The building block 10 is derived from diamino propionic acid
Dap) and aspartic acid (Asp) derivatives, connected by an
(
1
4
3
backbone of peptides using conventional SPPS.
ethylene bridge, and decorated with additional carboxymethyl
substituents (Figure 1). We attempted several synthetic
strategies to prepare 10 from commercially available
compounds. However, only one rendered 10 efficiently
As the building block, we envisioned an EDTA-like molecule,
which provides all six coordination sites of EDTA and is
(Scheme 1). The intermediate bromo-product 8 is described in
the literature and can be obtained in 25-30% yield starting
4
from H-Asp(Bzl)-OH after three steps. It represents the Asp-
part of the target molecule carrying the terminal bromo-
substituted ethylene bridge to be connected to the Dap-part
For the synthesis of enantiomerically pure , we used the
HClO -catalyzed transesterification of tert-butylacetate and
3.
2
4
1
to satisfy the requirement of epimerization-free conditions for
5
tert-butyl ester formation.
The reaction of
2 with tert-butylbromoacetate to render 3 by
stirring in ACN-phosphate buffer mixture (a heterogenic
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system, “Rapoport-conditions”) proceeded slowly and had to exclude unspecific interactions between the NH group of the
2
be stopped when N-disubstitution started at the expense of N- Orn residues with the carboxylic groups present in the EDTA
monosubstituted product (monitored by HPLC). The mildly amino acid analogue. Therefore, the model cyclic peptide (cP)
6
basic conditions do not affect the Fmoc-group.
sequence is composed by 8 aliphatic amino acids connected to
the pendant arm functionalities of 10, with the substructure of
The reaction between 3 and 8 to prepare
9
using Rapoport- the EDTA analogue running through the peptide backbone.
conditions is a slow but continuous process that requires The sequence of the model peptide ensures that at
several days. Added excesses of can be partially recovered by physiological pH, any interaction with metal ions will take
column chromatography when isolating . Under these place exclusively at the analogue.
conditions, parts of undergo intramolecular cyclization to
render morpholine 9b, which cannot be separated from
8
9
8
7
.
9
The linear precursor of cP was synthesized by stepwise
standard Fmoc-SPPS on 2-chlorotrityl (2-CTT) resin (Scheme 2).
Among the conditions tested for the removal of the Bzl-group As cP is a cyclic peptide, its synthesis can start at any of the
from by catalytic hydrogenation the best results were amino acids of the sequence. Starting by loading 10 on the 2-
obtained with Pd-C at atmospheric pressure in EtOH and CTT resin has several advantages. First, only 1 equiv. of 10 is
monitoring the conversion by HPLC-UV Otherwise, required for the loading step (introduction in a later position
9
,
.
hydrogenolysis of the Fmoc-group occurs. After flash would require excess), and second, the EDTA amino acid does
chromatography, 10 was obtained with 31-43% yield in 4 steps not undergo epimerization when activated for cyclization. The
and starting from
1
and
8.
elongation of the peptide chain took place smoothly using DIC-
9
OxymaPure as coupling cocktail, and after removal of the
Once the building block was obtained, we characterized its Fmoc of the last residue (Val), the protected peptide was
incorporation into a cyclic peptide inspired in the antibiotic cleaved from the resin with 2% TFA in DCM. Cyclization was
Gratisin family, well-studied in the literature, and with a high carried out using PyBOP and HOBt following the usual
8
10
tendency to populate beta-hairpin structures. The sequence conditions developed in our laboratory. Fully protected
of this antibiotic is (D-Phe-Pro-Val-Orn-Leu) and is known to intermediate cP was purified by semi-preparative HPLC.
2
populate an amphiphilic beta-sheet conformation in solution, Removal of tert-butyl protecting groups from the EDTA moiety
with two hairpins whose formation is favored by the presence was slow (15 hr). After lyophilization, HPLC pure cP was
of a D-Phe followed by Pro residue in the sequence. We obtained in 19% overall yield from the crude linear precursor.
decided to introduce the new amino acid analogue in this
2
+
scaffold, in the second hairpin, substituting one of the D-Phe- To detect and quantify whether cP binds Ca at pH7, we used
Pro motifs. The logic behind this choice is to introduce an Isothermal Titration Calorimetry (ITC) experiments. An
unambiguously measurable effect resulting from the metal aqueous solution of cP in Tris-HCl buffer at 25ºC and 37ºC was
2
+
binding such as an increase in the flexibility of the peptide titrated with increasing amounts of Ca , displaying
2
+
directly related to Ca coordination. We have also substituted dissociation constants of 40 and 70 nM, respectively (SI).
both Orn by Ala residues in positions 2 and 7, in order to
2
| J. Name., 2012, 00, 1-3
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The CD spectrum of cP showed a minimum at 200 nm and a behavior in the NMR time scale. Consistent with the CD
2
+
weak negative band around 220 nm at pH 7.2 (Scheme 2, results, the NMR analysis also suggests that Ca -binding
bottom) in agreement with the presence of –turn distorts the -turn-like structure of cP, as deduced from C
conformations in solution. Interestingly, the addition of Ca , chemical shifts and JαNH coupling constants corroborating that
or of other metal salts), the band at 220 nm disappeared and Ca is bound by the BFCA analogue, even under the restraints
the CD curve observed resembles that of random coil samples. imposed by its location in the cyclic peptide.
This process was reversible, and the presence of –turn In summary, we describe here a method for preparing the
conformations are detected when the solution was either EDTA amino acid building block Fmoc-NH-EDTA(tBu) -COOH 10
from commercial sources efficiently. This building block can be
used in standard SPPS, as we have demonstrated with the
β
β
α
2
+
3
(
β
4
acidified or in the presence of excess of EDTA.
2
+
cyclopeptide cP. We also showed the effectiveness of Ca
chelation of this analogue, even under severe steric
constraints, as those imposed by its location at one of the
hairpins of the peptide model used as an example. We believe
that the use of 10 as a BFCA will open new possibilities for the
preparation of peptide analogs modified to efficiently interact
with metal ions along the backbone.
Figure 2. CD and NMR titration of cP in the presence or
2
+
absence of Ca . On the left: Light green: cP (0.1 mM) in 10
mM phosphate buffer pH 7.2. Dark green: cP + 1 equiv.
Ca(tfms) . After addition of Ca(tfms) , the solution was
Notes and references
2
2
‡
Acknowledgements: We thank the NMR facility of the
acidified with HClO to pH1 (blue), or EDTA was added (red).
4
Scientific and Technological Centre of the University of
Barcelona (CCiT UB) and to Dr. Thomas Bruckdorfer, IRIS
Biotech, Innovationsgutschein der Bayrischen Regierung, for
technical support. M.B. acknowledges Erasmus+ funding for 9
months provided by the University of Glasgow.
This work was partially funded by MEC (CTQ2015-67870-P), the
Generalitat de Catalunya (2014 SGR 137), the IRB Barcelona, and
the National Research Foundation (NRF, South Africa). IRB
On the right 13C-HSQC expansion of cP. Changes are indicated
with arrows.
2
+
The influence of bound Ca on the conformational behavior of
cP was further studied by bidimensional NMR experiments
1
13
2+
C HSQC). In the Ca -free
(TOCSY, COSY, ROESY, and
H
sample, NMR parameters such as H and C chemical shifts, Barcelona is a recipient of a Severo Ochoa Award of Excellence
α
α
3
JαNH coupling constants, NH amide temperature coefficients, from MINECO (Government of Spain). M.J.M. is an ICREA
Programme Investigator.
and NOE patterns, suggest the presence of β–like structures in
the Leu3-Ala7 segment as well as backbone flexibility in and
around the second hairpin where the aa analogue 10 is
introduced, (Supplementary Information).
1
L. Lattuada, A. Barge, G. Cravotto, G.B. Giovenzana, L. Tei,
Chem. Soc. Rev., 2011, 40, 3019-3049,and references cited
therein.
2
3
K.L. Haas, K.J. Franz, Chem. Rev., 2009, 109, 4921-60.
Chelating linkers for holding two peptides have been
proposed by Achilefu group: a) S.I. Achilefu, PCT Int. Appl.
(2001), WO 2001052900 A2 20010726; b) S.I. Achilefu, PCT
Int. Appl. (2001), WO 2001052899 A1 20010726; c) S.I.
Achilefu, A. Srinivasan, PCT Int. Appl. (2001), WO
2001052898 A1 20010726.
cP
Cl
Val6
COOH
N
(
10) Fmoc-NH-EDTA(tBu)
4
-OH, 1 equiv.;
Pro5
O
O
H
CO
2
H
H
DIEA in DCM
N
N
N
N
H
N
H
O
O
O
CO
2
H
Fmoc-NH-EDTA(tBu)
4
-O
H
N
O
N
O
H
N
H
N
N
Fmoc-SPPS (Oxyma-DIC activation, 3 equiv.;
double coupling), 20% piperidine in DMF
H
COOH
10)
DPhe4
O
O
4
5
S. Aime, M. Galli, L. Lattuada, P. Morosini, F. Uggeri, R.
Kondareddiar, WO 2006/002873 A2.
(
Leu3
V1A2L3-DPhe4-P5V6A7L8-NH-EDTA(tBu)
4
-O
Val6
Esterification
epimerization (> 10%) of the Dap subunit as detected by
with
tert-BuOH/DCC/DMAP
caused
2
% TFA in DCM
Pro5
H-Val-Ala-Leu-DPhe-Pro-Val-Ala-Leu-NH-EDTA(tBu)
4
-OH
HPLC-UV when
mass.
M.A. Williams, H. Rapoport, J. Org. Chem., 1993, 58, 1151-
9 is separated in two peaks with the same
PyBop/HOBt/DIEA in DCM 1 mg/mL;
HPLC purification,
then 95% TFA/2.5% H2O/ 2.5% TIPS
overnight
6
7
1158.
9
methods. 9b was detected by its HPLC-MS peak ([M-56+H] =
and 9b could not be separated by chromatographic
cP
DPhe4
Leu3
+
1
08) and by the higher integration values of signals in the H
3
NMR. The amount can be estimated by integration of the
signals of the benzyl-methylene signals at 5.0 (
ppm, the only signals those are well separated.
M. Tamaki, M. Kokuno, I. Sasaki, Y. Suzuki, M. Iwama, K.
Saegusa, Y. Kikuchi, M. Shindo, M. Kimura, Y. Uchida. Bioorg
Med. Chem Lett. 2009, 19, 2856-2859.
9) vs 5.1 (9b)
Scheme 2. SPPS of the peptide cP, sequence and hairpins.
The DPhe-Pro configuration is trans (highlighted in the 3D
representation shown below the cP sequence)
8
2
+
Addition of Ca induced major changes in the NMR spectra of
9
1
R. Subirós-Funosas, R. Prohens, R. Barbas, A. El-Faham, F.
Albericio. Chem. Eur. J., 2009, 15, 9394-9403.
0 M. Pelay-Gimeno, F. Albericio, J. Tulla-Puche. Nature
Protocols, 2016, 11, 1924-1947.
cP. In agreement with the nM affinity obtained by ITC
2
experiments, Ca -binding to cP showed slow exchange
+
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