Conformational modulation of in vitro activity of cyclic RGD peptides via aziridine aldehyde-driven macrocyclization chemistry

Article abstract of DOI:10.1021/bc300239a

Here, we demonstrate a conjugation strategy whereby cyclic RGD-containing macrocycles are prepared using aziridine aldehydes, isocyanides, and linear peptides, followed by conjugation to a cysteamine linker. Our method involves site-selective aziridine ring-opening with the nucleophilic sulfhydryl group of cysteamine. Fluorescein was then efficiently conjugated to the primary amine of cysteamine by NHS-chemistry. This strategy may be expanded to provide easy access to a wide variety of fluorescent dyes or radiometal chelators. Modeling studies showed that aziridine aldehyde cyclization chemistry stabilized the RGD motif into the required bioactive conformation and that this cyclization chemistry modulated the geometry of macrocycles of different residue lengths. In vitro studies showed that cPRGDA and cPRGDAA both selectively bound to α_Vβ₃-overexpressing U87 glioblastoma cells, and that cPRGDA had a better binding affinity compared to cPRGDAA. The improved binding affinity of cPRGDA was attributed to the fixed Pro-C^α- Asp-C^α distance surrounding the stabilized RGD motif in cPRGDA.

Full text of DOI:10.1021/bc300239a

Communication
pubs.acs.org/bc
Conformational Modulation of in Vitro Activity of Cyclic RGD
Peptides via Aziridine Aldehyde-Driven Macrocyclization Chemistry
§
,†
†
‡
‡
,‡
́
Aron Roxin, Juan Chen, Conor C. G. Scully, Benjamin H. Rotstein, Andrei K. Yudin,*
,§,†
and Gang Zheng*
§
‡
Department of Pharmaceutical Sciences and Department of Chemistry, University of Toronto, Toronto, Ontario, Canada
†Ontario Cancer Institute and Princess Margaret Hospital, University Health Network, Toronto, Ontario, Canada
*
S Supporting Information
ABSTRACT: Here, we demonstrate a conjugation strategy
whereby cyclic RGD-containing macrocycles are prepared using
aziridine aldehydes, isocyanides, and linear peptides, followed
by conjugation to a cysteamine linker. Our method involves
site-selective aziridine ring-opening with the nucleophilic
sulfhydryl group of cysteamine. Fluorescein was then efficiently
conjugated to the primary amine of cysteamine by NHS-
chemistry. This strategy may be expanded to provide easy
access to a wide variety of fluorescent dyes or radiometal
chelators. Modeling studies showed that aziridine aldehyde
cyclization chemistry stabilized the RGD motif into the required
bioactive conformation and that this cyclization chemistry
modulated the geometry of macrocycles of different residue
lengths. In vitro studies showed that cPRGDA and cPRGDAA
both selectively bound to α β -overexpressing U87 glioblastoma cells, and that cPRGDA had a better binding affinity compared
V
3
α
α
to cPRGDAA. The improved binding affinity of cPRGDA was attributed to the fixed Pro-C -Asp-C distance surrounding the
stabilized RGD motif in cPRGDA.
INTRODUCTION
these additional residues may hinder binding of the biologically
4
■
−6
relevant peptide regions
and often require cumbersome
Research into peptides targeting biomarkers associated with
cancer actively pursues the creation of cyclic peptides that
display specific biological activity and that can be functionalized
for targeted imaging or therapy. A recent cyclization method
developed in our laboratories employs amphoteric aziridine
aldehydes and isocyanides to cyclize linear peptides through a
7
−10
protection/deprotections protocols.
A method that enables
one to constrain a linear epitope of interest into a macrocycle,
while offering an exocyclic handle that can be modified at a late
stage of synthesis without perturbing the desired conformation
11
of the molecule, is highly desirable. Here, we show a novel
modification strategy wherein the thiol of cysteamine
nucleophilically attacks aziridine aldehyde-cyclized macrocycles,
resulting in aziridine ring-opening. Fluorescein was then
conjugated to the macrocycles through the primary amine of
cysteamine using N-hydroxysuccinimide (NHS)-chemistry
1
rerouted Ugi reaction. Aziridine aldehyde-driven cyclization
proceeds rapidly, giving high yields at higher concentrations
than is possible for conventional peptide cyclizations. This
original procedure reported the conjugation of a thiolated
coumarin fluorophore after cyclization and has since been
refined to include several functional inputs. For instance, in
addition to postcyclization fluorophore labeling, macrocycles
have been labeled with a solvatochromic naphthalimide
(
Scheme 1). This strategy removes the necessity of
incorporating superfluous residues and allows the conjugation
of a variety of biological entities containing carboxylic acid,
isothiocyanate (ITC), or NHS functionalities by well-
established amide coupling.
2
isocyanide during cyclization. Most recently, novel thioester
isocyanide reagents were developed with the goal of subsequent
Arg-Gly-Asp (RGD)-containing peptides were chosen as a
model for our investigations. The RGD motif is known to bind
to the α β integrin receptor, which is overexpressed by several
macrocycle−peptide ligation to access cycle-tail peptide
3
scaffolds. We sought to expand these recent conjugation
strategies by developing a versatile modification chemistry
whereby the conjugation of a wide variety of fluorophores,
radiometal chelators, and other biological entities could be
achieved.
V 3
12−15
types of cancers and by tumor neovasculature.
To obtain
biologically active RGD motifs, computer models of RGD-
Conventional amide-cyclized peptides often require addi-
tional lysine, cysteine, or glutamate residues to support side
chain-selective conjugations. However, conjugations through
Received: May 1, 2012
Revised: June 7, 2012
Published: June 18, 2012
©
2012 American Chemical Society
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Scheme 1. General Strategy for Synthesizing the Fluorescein-Labeled Aziridine Aldehyde-Cyclized RGD Macrocycles
Chart 1. Novel Fluorescein-Labeled Macrocycles cPRGDA (13), cPRGDAA (14), and cPRDGA (15)
based aziridine aldehyde-cyclized pentapeptide and hexapeptide
macrocycles were constructed in an attempt to recapitulate the
observed backbone geometry from solution phase and
crystallographic studies of potent integrin binding peptides.
Docked conformations of promising RGD macrocycles and the
α β integrin receptor were examined to investigate if these
EXPERIMENTAL PROCEDURES
■
Chemicals. Organic solvents were purchased from Fisher
Scientific and Sigma Aldrich. Amino acids and resins were
purchased from Nova Biochem. Cysteamine, zinc chloride, L-
serine, triphenyl phosphene (PPh ), diisopropyl azodicarbox-
3
V
3
ylate (DIAD), diisobutylaluminum hydride (DIBAL), tert-butyl
isocyanide, and crystal violet were purchased from Sigma
Aldrich. Fluorescein-NHS was purchased from Thermo
Scientific. The chelator 1,4,7,10-tetraazacyclododecane-1,4,7-
tris(t-butyl acetate)-10-acetate mono-succinimide ester
DOTA-NHS) was purchased from Macrocyclics. Pyropheo-
phorbide-succinimide ester (Pyro-NHS) was synthesized
following a previously published procedure.
RGD macrocycles could attain binding geometries that were
similar to the known high-affinity ligand cRGDf(N-Me)V
1
6−18
(
Cilengitide).
We proposed that if our RGD macrocycles
could stabilize the RGD motif in a bioactive conformation
similar to that of cRGDf(N-Me)V, then these peptides would
be suitable for in vitro α β integrin-binding. Linear RGD-
(
V
3
containing peptides were cyclized and labeled with fluorescein
to experimentally validate the biological activity of the proposed
functional macrocycles (Chart 1). Cell adhesion assays
compared the binding affinity of the pentapeptide cPRGDA
19
General Methods. Proton NMR was performed using a
Bruker Ultrashield 400 Plus 400 MHz NMR. Mass
spectrometry was performed using an Agilent Technologies
(
13) and the hexapeptide cPRGDAA (14) to investigate how
6
130 Mass Spectrometer. The identity and purity of all peptide
the geometry of these peptides correlated to their binding
derivatives were assessed using a Waters 2695 HPLC with a
Waters Delta Pak C18, 5 μm 3.9 × 150 mm column under
reverse-phase conditions (0−100% acetonitrile in 0.1% TFA
over 15 min at 0.8 mL/minute) while monitoring with a Waters
2996 photodiode array detector and a Waters Micromass ZQ
mass spectrometer. Macrocycles were purified using a Waters
Prep LC System with an Agilent 300SB-C3, 5 μm 9.4 × 250
mm column under reverse-phase conditions (0−100%
acetonitrile in 0.1% TFA over 40 min at 3.0 mL/minute)
affinity for the α β integrin receptor of U87 glioblastoma cells.
V
3
Confocal microscopy was then used to further investigate if
these fluorescein-labeled macrocycles could specifically target
the α β integrin receptor of U87 cells for in vitro cancer
V
3
imaging. Our study provides a guide to experimentally verify
the biological activity and specificity of our constrained RGD
macrocycles proposed by computational geometry-based
modeling that can be extended for screening other biologically
active receptor-targeting motifs.
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while monitoring with a Waters 2487 Dual λ absorbance
detector set to 254, 300, 411, or 441 nm. Peptide derivatives
were quantified spectrophotometrically by measuring the
absorbance of Fmoc ((1:4) piperidine: DMF) or fluorescein
stituted aziridine-aldehyde dimer (18) and 3.5 equiv of tert-
butyl isocyanide in TFE. The progress of cyclization reactions
were assessed using HPLC-MS monitoring by integrating UV−
vis spectra scanned at 254 nm. After 17−19 h, these cyclization
reactions produced the desired macrocycles containing aziridine
rings with the sequences PR(Pbf)GD(OtBu)A (4), PR(Pbf)-
GD(OtBu)AA (5), and PR(Pbf)D(OtBu)GA (6) where yields
were estimated at ∼47%, ∼38%, and ∼43%, respectively. Mass
spectrometry showed the production of 4 (ESI-MS: 960 (M+1)
m/z), 5 (ESI-MS: 1031 (M+1) m/z), and 6 (ESI-MS: 960 (M
(
0.1 M NaOH) using a Varian 50 Bio UV−visible
spectrophotometer. Binding affinity assays measured absorb-
ance values using a Molecular Devices Spectra Max M5 plate
reader. Confocal experiments were analyzed with an Olympus
FV1000 laser confocal scanning microscope.
Synthesis of Unsubstituted Aziridine-Aldehyde (18).
The unsubstituted aziridine-aldehyde (18) was synthesized as
previously reported in our lab. Briefly, 10 g (95 mmol) of L-
serine was esterified to a serine propyl ester HCl salt (16) using
+1) m/z). The reaction mixtures were precipitated using Et O.
2
1,20
The crude mixtures were dried with a speed-vac and were
stored at −20 °C.
Aziridine Ring-Opening with Cysteamine. The reaction
crudes from the previous aziridine aldehyde-driven cyclization
2
00 mL 1-propanol and 7.7 mL (106 mmol) SOCl . This
2
reaction yielded 16.77 g (91.2 mmol) of precursor (96% yield).
1
1
The identity was confirmed with H NMR. H NMR (CDCl ,
were dissolved in CHCl . Cysteamine was dissolved in DMSO
3
3
4
4
4
00 MHz): δ 0.90 (t, J = 7.4 Hz, 3H), 1.62 (m, 2H), 3.51 (t, J =
.1 Hz, 1H), 3.68 (dd, J = 10.9, 5.5 Hz, 1H), 3.76 (dd, J = 10.7,
.0 Hz, 1H), 4.05 (t, J = 6.6 Hz, 2H).
to make a 3 mg/μL stock solution, while zinc chloride was
dissolved in (95:5) CHCl /DMF to make a 0.1 mg/μL stock
3
solution. Cysteamine and zinc chloride was added to the CHCl3
solution containing the peptide crude. The molar ratio of
peptide/cysteamine/zinc chloride was 1:3:1. The progress of
this aziridine ring-opening reaction was assessed using HPLC-
MS monitoring as described. Once the starting material was
consumed, the reaction mixture was dried using a speed-vac.
The crude was dissolved in a minimal volume of DMSO and
was purified using a prep-HPLC. This gave the cysteamine-
modified macrocycles with the sequences PR(Pbf)GD(OtBu)A
(7), PR(Pbf)GD(OtBu)AA (8), and PR(Pbf)D(OtBu)GA (9)
with yields of ∼23%, ∼33%, and ∼5%, respectively. The
identity and purity of each product was confirmed using HPLC-
MS (Figure S1, S2, and S3). Mass spectrometry showed the
isolation of 7 (ESI-MS: 1037 (M+1) m/z), 8 (ESI-MS: 1108
(M+1) m/z), and 9 (ESI-MS: 1037 (M+1) m/z).
Amide Cyclization. The linear PR(Pbf)GD(OtBu)K(Boc)
peptide (19) was cleaved from the 2-chlorotrityl chloride resin
and the Fmoc-protecting group was removed as described. The
NH-PR(Pbf)GD(OtBu)K(Boc)-COOH peptide (15 μmol)
was cyclized using HBTU (1 equiv) and HOBt (1 equiv)
while dissolved in 25 mL of DMF (1.76 mM) with 2.3% DIPEA
(v/v). Reaction progress was monitored with HPLC-MS as
described. The starting material was consumed within 20 h.
The solution was aliquoted to 1.5 mL Eppendorf tubes and
dried with a speed-vac. The dried product (21) was precipitated
The HCl salt (16) was converted to the free base (13.37 g or
9
0.9 mmol) using NH OH. A Mitsonobu reaction was
4
performed using 23.85 g (90.9 mmol) of PPh and 17.96 mL
3
(
90.9 mmol) of DIAD in anhydrous DCM at −10 °C for 3 h
and at room temperature for 18 h. The workup required Et O
2
precipitation to remove the white PPh O byproduct and a
Kugelrohr apparatus (heated to 86 °C) to distill the aziridine
propyl ester (17). This gave a 4.44 g (34.4 mmol) sample of
this precursor (38% yield). The identity was confirmed with H
NMR. H NMR (CDCl , 400 MHz): δ 0.92 (t, J = 7.4 Hz, 3H),
3
1
1
3
1
1
.22 (m, 3H), 1.65 (m, 2H), 1.82 (d, J = 4.6 Hz, 1H), 1.96 (s,
H), 2.49 (s, 1H), 4.08 (m, 2H).
The aziridine propyl ester (17) (2.0 g or 15.5 mmol) was
reduced with 38.8 mL (38.8 mmol) of DIBAL (1.0 M in
toluene) at −78 °C for 4.5 h. The reaction was quenched using
MeOH after 2 h. Gelation was initiated with sat. Na SO
after 1 h and 45 min. The gel was filtered and washed with
MeOH to give 532 mg (7.44 mmol) of the unsubstituted
aziridine aldehyde dimer (18) (48% yield). Mass spectrometry
was used to identify the product (ESI-MS: 143.1 (M+1) m/z).
TLC was performed using acetonitrile/water (4:1) and showed
2
4 (aq)
1
,20
an R = 0.4 (lit. R = 0.32).
any byproducts.
KMnO staining did not reveal
f
f
4
Manual Linear Peptide Synthesis. The linear peptides
PRGDA (1), PRGDAA (2), PRDGA (3), PRGDK (19), and
RGD (20) were synthesized on the solid-phase with 2-
chlorotrityl chloride resin (loading ∼1.5 mmol/g) using
standard Fmoc-chemistry. The amino acids Arg, Asp, and Lys
had side chains protected by Pbf, OtBu, and Boc, respectively.
Amide coupling of Fmoc-protected amino acids (3 equiv) was
achieved after 2 h using HBTU (3 equiv) in (1:1) DCM/DMF
containing 5% DIPEA (v/v). Resin capping was performed
using 1-acetylimidazole (3 equiv) in a solution of (1:1) DCM/
DMF containing 5% (v/v) DIPEA. Peptides were cleaved from
the resin using (1:2:97) TFA/TIS/DCM after 2 h. Peptides
were synthesized on a 250−550 μmol scale.
with DMF and Et O as described and dried with a speed-vac.
2
The identity and purity of the product was confirmed using
HPLC-MS (Figure S4). Mass spectrometry showed the product
21 (ESI-MS: 963 (M+1) m/z).
Fluorescein Conjugation. Fluorescein-NHS (1.5 equiv)
was conjugated to the cysteamine-modified macrocycles in
DMF containing 4% DIPEA (v/v) within 30 min. This reaction
gave the side-chain protected fluorescein labeled macrocycles
with the sequences PR(Pbf)GD(OtBu)A (10), PR(Pbf)GD-
(OtBu)AA (11), and PR(Pbf)D(OtBu)GA (12). ESI-MS(+)
showed the production of 10 (ESI-MS: 1395 (M+1) m/z), 11
(ESI-MS: 1466 (M+1) m/z), and 12 (ESI-MS: 1395 (M+1)
m/z). The Pbf and OtBu protecting groups were removed
using TFA for 2 h. This side-chain deprotection step gave the
final fluorescein-labeled macrocycles cPRGDA (13),
cPRGDAA (14), and cPRDGA (15). These solutions were
Aziridine-Driven Cyclization. The peptides PR(Pbf)GD-
(
(
OtBu)A (1), PR(Pbf)GD(OtBu)AA (2), and PR(Pbf)D-
OtBu)GA (3) were cleaved from the 2-chlorotrityl chloride
resin as described and the N-terminal Fmoc was removed using
standard piperidine cleavage. The peptides were precipitated
precipitated with Et O and purified using a prep-HPLC using
2
from a minimal volume of DMF using cold Et O seven times.
the described method while detecting products with the 254
and 441 nm absorbance channels. HPLC-MS was used to
determine the purity (>95% pure) and to confirm the identity
2
These peptides (10 μmol) were cyclized by the aziridine
1
,20
aldehyde-driven reaction
using 2.5 equiv of the unsub-
1
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of each product (Figure S5, S6, and S7). Mass spectrometry
showed the isolation of 13 (ESI-MS: 1087 (M+1) m/z), 14
studies were obtained from the conformational search detailed
above.
(
ESI-MS: 1158 (M+1) m/z), and 15 (ESI-MS: 1087 (M+1)
Binding Affinity. U87 glioblastoma cells were cultured in
Corning 75 cm flasks in MEM containing 10% FBS. Cells were
2
m/z). The peptides were then quantified spectrophotometri-
cally.
allowed to reach ∼70% confluence and were treated with
4
The Pbf, OtBu, and Boc protecting groups were removed
from amide-cyclized PR(Pbf)GD(OtBu)K(Boc) peptide (21)
using (95:5) TFA/DCM after 2 h. The production of the side-
chain deprotected amide-cyclized PRGDK (22) was confirmed
by mass spectrometry (ESI-MS: found 550 m/z). Fluorescein-
NHS (1.3 equiv) was conjugated to the ε-NH of Lys residue of
the macrocycle (22) (7.5 μmol) in DMF containing 2.5%
DIPEA (v/v) within 30 min. This solution was precipitated
with Et O and purified using a prep-HPLC using the described
method while detecting product with the 254 and 441 nm
absorbance channels. HPLC-MS was used to determine the
purity (>95% pure) and to confirm the identity of this
fluorescein-labeled cyclic peptide amide-cPRGDK (23) (Figure
S8). Mass spectrometry showed the isolation of amide-
cPRGDK (23) (ESI-MS: 913 (M+1) m/z). The peptide was
then quantified spectrophotometrically.
DOTA Conjugation. DOTA-NHS (3.3 equiv) was
conjugated to the side-chain protected cysteamine-modified
PR(Pbf)GD(OtBu)A macrocycle (7) (0.2 μmol or 1 equiv) in
trypsin containing EDTA for 15 min. The U87 cells (5 × 10
cells/well) were coincubated with various concentrations of the
fluorescein-labeled cyclic peptides cPRGDA (13), cPRGDAA
(14), cPRDGA (15), and amide-cPRGDK (23) (0−20 μM) on
human vitronectin-coated 96-well plates (R&D Systems,
Minneapolis, MN, USA) for 1 h at 37 °C. After washing,
U87 cells were stained with 0.5% (v/v) crystal violet. The wells
were washed and the absorbencies were measured at 627 nm in
2
27
2
0.1 M HCl using a plate reader. IC50 values were calculated
using Graph-Pad PRISM by a log(inhibitor) vs response three
parameter nonlinear regression from x−y scatter plots (bottom
constraint at 18) of all normalized values from 3 independent
experiments.
Confocal Microscopy. The U87 glioblastoma cells served
as a positive cell line for the α β integrin receptor, while HT-
V
3
29 colon cancer cells served as a negative cell line since these
2
8
express low levels of the human α β integrin receptor. U87
V 3
2
cells and HT-29 cells were cultured in Corning 75 cm flasks in
MEM and DMEM media, respectively, containing 10% FBS.
Cells were allowed to reach ∼70% confluence and were treated
5
0 μL of DMF containing 6% DIPEA (v/v) within 1 h. The
4
crude was purified by prep-HPLC as described while detecting
the product using a 254 nm absorbance channel. HPLC-MS
was used to determine the purity (>90% pure) and to confirm
the identity of the product (Figure S9). Mass spectrometry
showed the production of cPRGDA-DOTA (24) (ESI-MS:
1
with trypsin containing EDTA for 15 min. Cells (3 × 10 cells/
well) were transferred to 8-well Lab-Tek 155411 #1.0
borosilicate plates and were incubated with 10 μM solutions
of fluorescein-labeled cPRGDA (13) and cPRGDAA (14)
0.5% v/v DMSO in media) for 2 h at 37 °C. The fluorescein-
labeled cPRDGA scramble peptide (15) was used as a negative
control, while the fluorescein-labeled amide-cPRGDK peptide
(
+
423 (M ) m/z).
Pyropheophorbide a Conjugation. Pyro-NHS (2
1
9
equiv) was conjugated to the side chain-protected cyste-
amine-modified cPR(Pbf)GD(OtBu)A (7) (0.4 μmol or 1
equiv) in 70 μL of DMF containing 4% DIPEA (v/v) within 2
h. The crude was purified by prep-HPLC as described while
detecting the product using 254 and 411 nm absorbance
channels. HPLC-MS was used to determine the purity (>95%
pure) and to confirm the identity of the product (Figure S10).
(
23) served as a positive control. The targeting specificity of
cPRGDA and cPRGDAA was investigated by blocking the αVβ3
integrin receptor of U87 cells with a 50-fold molar excess of the
linear RGD peptide (20). Laser confocal scanning microscopy
was then performed with excitation wavelength of 488 nm
using 5% laser power, PMT voltage of 760 V, and 120 μM
pinhole diameter.
Mass spectrometry showed the production of cPRGDA-Pyro
+
(25) (ESI-MS: 1553 (M ) m/z).
RESULTS
■
Computer Modeling. The cPRGDA (13) and cPRGDAA
(14) peptides, as well as the amide-cPRGDK (23) peptide,
Aziridine Ring-Opening and Fluorophore/Chelator
Conjugation. The general synthetic strategy included four
distinct steps: (1) aziridine aldehyde-driven cyclization of linear
peptides, (2) aziridine ring-opening by a heterobifunctional
linker, (3) dye conjugation, and (4) full deprotection of amino
acid side chains. The linear peptides with the sequences
PR(Pbf)GD(OtBu)A, PR(Pbf)GD(OtBu)AA, and PR(Pbf)D-
(OtBu)GA were successfully cyclized by our recently reported
aziridine aldehyde-driven cyclization chemistry. Cysteamine
was attached to these macrocycles by zinc chloride-catalyzed
aziridine ring-opening. The cysteamine-modified macrocycles
(7, 8, and 9) were then conjugated to fluorescein-NHS using
DIPEA in DMF in 30 min (10, 11, and 12). The side-chain
protecting groups were then removed using TFA to produce
cPRGDA (13), cPRGDAA (14), and cPRDGA (15). We also
extended the exocylic conjugation of the cysteamine-modified
macrocycles to other reporters. The radiometal chelator DOTA
was efficiently conjugated to the free amine of cysteamine on
the macrocycle PR(Pbf)GD(OtBu)A (7) using NHS-chemistry
(24). The multimodal fluorescent dye, photosensitizer, and
radiometal chelator pyropheophorbide a was similarly con-
were modeled using the Monte Carlo Multiple Minima
(
the OPLS2005 force field and a Generalized-Born/Surface-
Area (GB/SA) water model. During this simulation, torsion
angles of the macrocycles were varied randomly using a Monte
21
MCMM) algorithms implemented in Macromodel using
22
23
2
4
Carlo procedure and the conformational states were selected
2
5
based on the Metropolis algorithm according to the
Boltzmann probabilities for each state based on its internal
energy. We performed 1 000 000 steps of Monte Carlo
sampling using 100 steps per each rotatable bond with
retention of unique conformers. A 21 kJ/mol energy window
was applied to discard high-energy conformers and we used a
0
.5 Å maximum atom deviation cutoff to discard redundant
conformers. An average structure was calculated for each
peptide from their respective conformer families using
Macromodel. Docking of cPRGDA, the N -lysine-acetylated
amide-cPRGDK and the highly studied cRGDf(N-Me)V
peptide with the extracellular α β integrin protein (PDB
ε
V
3
2
6
entry 1L5G) was performed using Glide 5.7 (Schrodinger
Inc.). The average ligand conformations used in the docking
1
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α
α
Figure 1. 3D models of (A) cPRGDA (orange) and (B) cPRGDAA (purple) separately showing Pro-C −Asp-C vectors and (C) overlaid with
amide-cPRGDK (yellow). The cPRGDA peptide (orange) was overlaid with cRGDf(N-Me)V (green) in a docked configuration with the α β
V
3
protein to illustrate structural similarity at the RGD-motif.
jugated to the free amine of cysteamine on the macrocycle
PR(Pbf)GD(OtBu)A (7) using NHS-chemistry (25).
of the RGD-motif of cPRGDA to the α β integrin binding site
V 3
compared to the larger cPRGDAA.
Ligand docking to the extracellular α β integrin protein
Computer Modeling. Macromodel was used to generate
conformational ensembles of cPRGDA (13), cPRGDAA (14),
and amide-cPRGDK (23). The average conformations of
cPRGDA (Figure 1A), cPRGDAA (Figure 1B), and the amide-
cPRGDK were analyzed to investigate suitability for binding to
the α β integrin receptor. A preliminary study indicated that
V
3
ε
showed that cPRGDA and the N -acetyl-lysine analogue of
amide-cPRGDK possessed similar stabilizing binding inter-
actions to cRGDf(N-Me)V from the crystal structure. The Lys
ε
residue of amide-cPRGDK (22) was N -acetylated in its
docking study to mimic the presence of fluorescein in amide-
cPRGDK (23). It was found that cPRGDA and cRGDf(N-
Me)V docked to the α β integrin protein through their
V
3
the pentapeptides, cPRGDA and amide-cPRGDK, preferred a
backbone conformation that overlaid well with the RGD motif
of cRGDf(N-Me)V (bound to the extracellular α β integrin
V
3
respective Arg residues to Asp150 and Asp218 of α β and
V
3
V 3
26
ε
protein (PDB: 1L5G) ) (Figure 1D), while the hexapeptide
through their Asp residues to Mn1 (Figure 2A). The N -acetyl-
cPRGDAA showed significant backbone deviation in this area
(
Figure 1C). Conformational searches generated between 233
and 368 conformers for each of these macrocycles from which
α
α
the mean Pro-C −Asp-C distances within cyclic peptides
cPRGDA, cPRGDAA, and amide-cPRGDK (Table 1) were
α
α
Table 1. Calculated Pro-C −Asp-C Distances of
Macrocycles Using Averaged Conformer Geometries
α
α
a
peptide
Pro-C −Asp-C distance (Å)
N
amide-cPRGDK
cPRGDA
5.24 ± 0.15
6.11 ± 0.17
8.37 ± 0.24
368
264
233
Figure 2. 3D modeling showing the comparative docking interactions
of (A) cPRGDA (yellow) and cRGDf(N-Me)V (blue) and (B) amide-
cPRGDK (orange) and cRGDf(N-Me)V (blue) with the extracellular
binding regions of the α β integrin receptor.
cPRGDAA
a
Number of generated conformers.
V
3
calculated. Both cPRGDA and cPRGDAA contain a γ-turn-like
backbone conformation similar to cRGDf(N-Me)V at the RGD
lysine of the amide-cPRGDK analogue formed contacts to the
same residues and also to Asp126 through the amide proton of
the N -acetyl-lysine residue (Figure 2B). Heavy atom distances
α
α
motif (Arg-C −Asp-C < 7 Å). However, the cycle topology is
ε
significantly different when the residue preceding the RGD
α
motif is included in the backbone comparison. The Val-C -Asp-
between the respective docking regions of the α β integrin
V
3
α
C distance was measured at 6.9 Å in the crystal structure of
protein to cPRGDA, amide-cPRGDK, and cRGDf(N-Me)V
were calculated (Table 2). These measurements showed that,
while cPRGDA and cRGDf(N-Me)V used all three nitrogen
atoms of their respective Arg residues for optimal binding to
Asp150 and Asp218, the binding mode of the Arg of amide-
cPRGDK is inferior. This observed binding mode uses two of
cRGDf(N-Me)V which is similar to the distance calculated for
α
α
α
α
Pro-C −Asp-C in cPRGDA (6.1 Å). The Pro-C −Asp-C
distance in cPRGDK is shorter (5.2 Å) and is much longer in
cPRGDAA (8.7 Å). This suggests that differing peptide
backbone topology could account for the enhanced binding
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Table 2. Calculated Heavy Atom Distances between
Macrocycles and the Extracellular Binding Regions of the
α β Integrin Receptor
Though cRGDf(N-Me)V was not tested in this study, a review
̈
by Ruegg et al. reported that Cilengitide displays a similar αVβ3
integrin binding affinity (IC ∼1 μM) to displace α β
V
3
50
V 3
29
integrin-expressing cells from adhering to vitronectin. The
peptide
ligand atom receptor atom heavy atom distance (Å)
IC of cPRGDA (4.2 μM) was significantly lower compared to
50
cPRGDA
Asp-OD
Arg-NE
Mn1
2.1
3.1
3.1
3.2
2.1
3.1
2.9
3.0
3.6
2.7
2.9
2.8
3.3
cPRGDAA (11.1 μM, p < 0.05) (Table 3). These differential
D218-OD1
D218-OD2
D150-OD
Mn1
IC values were attributed to modulated geometries as a result
50
Arg-NZ1
Arg-NZ2
Asp-OD
Arg-NZ1
Arg-NZ1
Arg-NZ2
Ac-Lys-NZ
Asp-OD
Arg-NE
of peptide length and the presence of the constrained RGD
motif in cPRGDA. It was also found that there was no
significant difference in the IC50 of cPRGDA compared to
amide-cPRGDK.
amide-cPRGDK
D218-OD1
D218-OD2
D150-OD
D126-OD
Mn1
Confocal Microscopy. Confocal microscopy investigated
the specific binding of the fluorescein-labeled cPRGDA (13)
and cPRGDAA (14) peptides to the human α β integrin
V
3
cRGDf(N-Me)V
receptor of U87 glioblastoma cells. These studies showed that
cPRGDA, cPRGDAA, and the amide-cPRGDK (23) each
bound to U87 cells (Figure 3A,C,D) but none bound to HT-29
cells (Figure 3G,I,J). The cPRDGA scramble control (15)
showed nonspecific binding to U87 cells (Figure 3B) but not to
HT-29 cells (Figure 3H). The targeting specificity of cPRGDA
and cPRGDAA was investigated by blocking the α β integrin
D218-OD1
D218-OD2
D150-OD
Arg-NZ1
Arg-NZ2
its three available nitrogen atoms to bind to the dual aspartate
motif of the integrin.
V
3
Binding Affinity. With computational insights in hand, the
binding affinity of each fluorescein-labeled RGD-macrocycle for
the α β integrin receptor was investigated experimentally
receptor of U87 cells with a 50-fold molar excess of the linear
RGD peptide (20). This blocking reduced specific αVβ3
integrin binding for both RGD-containing macrocycles, but
the remaining fluorescence signals suggested additional non-
specific binding (Figure 3E,F).
V
3
using competitive cell adhesion assays. The peptide concen-
tration-dependent displacement of U87 cell adherence from
vitronectin-coated wells was used to calculate the respective
IC50 values for fluorescein-labeled macrocycles cPRGDA (13),
cPRGDAA (14), and amide-cPRGDK (23). The IC50 value of
these RGD-containing macrocycles was on the same order (low
μM) as previously reported amide-cyclized RGD sequences
DISCUSSION
■
The aziridine aldehyde-driven cyclization method has been
successfully developed to synthesize functional α β integrin-
V
3
27
targeting fluorescein-labeled macrocycles. We were able to
show that increasing the peptide length (from five to six
residues) and switching the Gly and Asp residues in the middle
of the sequence did not affect the aziridine aldehyde-driven
cyclization reaction. We used a simple zinc chloride salt to
catalyze the sulfhydryl-driven aziridine ring-opening of the
exocyclic unsubstituted aziridine handle of our macrocycles by
the heterobifunctional linker cysteamine. The versatility of this
synthetic strategy was illustrated by conjugating the common
radiometal chelator DOTA and the multimodal fluorescent
dye/radiometal chelator/photosensitizer pyropheophorbide a
using NHS-chemistry. This conjugation could be easily adapted
to a wide variety of other fluorescent dyes, radiometal chelators,
or extended linkers using conventional amide-coupling, the
described NHS-chemistry, or by analogous ITC-chemistry.
Aziridine aldehyde-driven macrocyclization reactions showed
clear advantages over conventional end-to-end amide cycliza-
tion. The former reactions were performed at a much higher
analyzed by this competitive cell adhesion method (Table 3).
Table 3. Cell Adhesion Competition Assay Summary of IC50
Values of Fluorescein-Labeled Macrocycles
a
b
c
peptide
log IC50 (M)
SE (log M)
IC50 (μM)
amide-cPRGDK
cPRGDA
−5.114
−5.381
−4.955
±0.2
±0.2
±0.06
7.7
4.2*
11.11*
-
cPRGDAA
cPRDGA
d
d
n.d.
n.d.
a
Calculated in Graph-Pad PRISM using a log(inhibitor) vs response,
three parameter nonlinear regression from an x−y scatter plot (bottom
constraint at 18) of all normalized values from 3 independent
b
experiments. Standard error calculated for log IC presented as log
50
c
d
of molar concentrations. Transformed from log IC . n.d., Not
50
*
determined. Significantly different using a two-tailed unpaired t test
p < 0.05).
(
Figure 3. Confocal microscopy fluorescence images merged with DIC showing the differential binding of fluorescein-labeled macrocycles to U87
α β +) (A−F) and HT-29 (α β −) cells (G−J).
(
V
3
V 3
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concentration (50 mM in TFE) wherein no noticeable
polymerization was observed. The amide cyclization required
high dilution (0.6 mM in DMF) to ensure that amide coupling
was predominantly due to intramolecular bonding of the free
N- and C-termini within each monomeric linear PRGDK
The amide-cPRGDK also fulfills the α carbon requirement
for a γ-turn-like structure, having a distance of 4.6 Å between
the Arg and Asp α carbons. However, the β carbons are much
closer than in either PRGDA or cRGDf(N-Me)V where a
distance of 7.2 Å separates them. This means that the backbone
dihedral angles are significantly different for amide-cPRGDK,
which could contribute to a reduction in affinity compared to
cRGDf(N-Me)V despite its similar ring size.
3
0
peptide. In addition, the conventional amide cyclization
reactions require an additional amino acid to provide a thiol,
carboxylic acid, or primary amine for subsequent functional
group conjugations. These amino acids should be specially
protected and selectively deprotected for functional group
Computational docking studies identified the binding mode
ε
of cPRGDA and the N -acetyl-lysine analogue of amide-
7
−10
cPRGDK with the extracellular α β integrin receptor. The Lys
conjugation.
On the contrary; the aziridine aldehyde-driven
V 3
of amide-cPRGDK was acetylated to mimic the hindrance
imposed by fluorescein at the Lys residue in amide-cPRGDK
macrocyclization offers a free amine for conjugation once
cysteamine has become bonded to the macrocycle by aziridine
ring-opening. We are thereby able to avoid the challenges of
protection/deprotection often required after amide cyclization.
Finally, after the aziridine ring-opening of the aziridine
aldehyde-cyclized macrocycles, the space between the binding
motif of the macrocycle and the conjugation site may have
allowed the desired receptor binding to proceed without
imposing significant steric hindrance.
(
23). We found that both macrocycles interacted with the
Asp150 and Asp218 residues of the protein through their
respective Arg residues, much the same as cRGDf(N-Me)V.
Interestingly, amide-cPRGDK only used two of its three
available Arg nitrogen atoms to bind to Asp150 and Asp218,
while cPRGDA and cRGDf(N-Me)V used all three nitrogen
atoms of their respective Arg residues for optimal integrin
ε
binding. In addition, the N -acetyl-lysine residue of amide-
Analysis of the X-ray crystal structure of integrin-bound
cPRGDK interacted with Asp126. Therefore, it was not obvious
which binding interaction would have the greatest contribution
to the binding affinity of cPRGDA and amide-cPRGDK.
Competitive cell adhesion assays experimentally showed that
the IC50 of cPRGDA for U87 cells was not significantly
different compared to amide-cPRGDK. This suggests that the
aforementioned difference observed in the described docking
studies compensated to create geometries that allowed similar
in vitro binding to the α β integrin receptor on U87 cells.
cRGDf(N-Me)V shows a significant difference between it and
26
the solution-phase structure proposed by Kessler et al. Cyclic
pentapeptides with (LDLDL) relative stereochemistry often
adopt a βII′/γ turn architecture in solution. In the case of
cRGDf(N-Me)V, Gly occupies the i+1 position of the γ turn
while D-Phe occupies the i+1 position of the βII′ turn. The X-
ray crystal structure of integrin-bound cRGDf(N-Me)V shows
that the γ turn is maintained in the bound state, with a 6.4 Å
separation between the Arg and Asp α carbons and a 8.7 Å
distance separating the Arg and Asp β carbons. cPRGDA
docked to α β and shows that it adopts the same γ turn
V
3
The chemical structures of the cPRGDA, cPRGDAA, and
cPRDGA macrocycles are inherently different from analogous
peptide macrocycles generated through backbone amide
coupling. The aziridine aldehyde-driven cyclization and the
subsequent aziridine ring-opening reaction result in the
V
3
conformation as the docked cRGDf(N-Me)V, which is critical
for the polar side chains of Arg and Asp to engage in receptor
α
β
binding. The Arg-Asp intercarbon (C and C ) distances of
cPRGDA were similar to those of cRGDf(N-Me)V where the
3
formation of an sp -hybridized carbon atom at the non-amino
acid portion of the macrocycles. The described geometric and
binding affinity comparisons between cPRGDA and amide-
cPRGDK are similar to the investigations published by Geyer et
α
α
β
β
Arg-Asp (C -C ) distance is 6.0 Å and the Arg-Asp (C -C )
distance is 8.7 Å. Kessler et al. have shown that the identity of
the remaining residues in the peptide are important for
structural rigidity, but due to the fact that residues not in the
RGD motif do not make contact with the integrin surface,
ample room exists to splice different residues and functional
32
al. in 1994. They developed the cyclic peptide cRGDfψ-
[
CH −NH]V that contained a reduced amide bond that
2
imposed geometric differences when compared to the parent
cRGDfV, whereas with cRGDfψ[CH −NH]V, they found that
31
2
handles into the macrocycle sequence, hence the observed
binding affinity for cPRGDA.
a β-turn forms between the Val and Asp residues (a γ turn lies
between Arg and Asp in potent RGD cyclic peptides). They
also found that the structural perturbation caused by the
introduction of a reduced amide bond into a cyclic RGD
peptide caused a significant drop in activity. This suggests that
RGD macrocycles containing only amide bonds would stabilize
geometries that result in superior αVβ3 integrin binding
affinities compared to analogues that do not contain a fully
amide-bonded backbone. Interestingly, the binding affinity of
cPRGDA to the α β integrin receptor was not significantly
Computer modeling data showed that cPRGDA contained a
similar backbone conformation at the RGD motif to cRGDf(N-
Me)V, while cPRGDAA differs significantly. This suggested
that the stabilized RGD motif within cPRGDA would
contribute to a better binding affinity for the α β integrin
V
3
receptor compared to cPRGDAA. Competitive cell adhesion
assays experimentally showed that the IC50 of cPRGDA for
α β integrin-overexpressing U87 glioblastoma cells was
V
3
V
3
significantly lower compared to cPRGDAA. This may be
explained by the backbone shape and macrocycle ring size
differences between the two peptides. It is possible that other
RGD-containing pentapeptide sequences cyclized by the
aziridine aldehyde-driven reaction may also contain this desired
turn at the RGD motif. Nevertheless, confocal analysis showed
that both the fluorescein-labeled cPRGDA and cPRGDAA
macrocycles could successfully bind to U87 cells for in vitro
fluorescence cancer imaging while avoiding binding to HT-29
cells that express low levels of the α β integrin receptor.
different compared to amide-cPRGDK. Though the amino acid
residues of cPRGDA and amide-cPRGDK were not identical, it
3
could be inferred that the sp carbon-containing macrocycles
generated through aziridine aldehyde-driven cyclization and
subsequent aziridine ring-opening may not hinder the binding
affinity of cyclic RGD sequences to the same extent as the
aforementioned study when compared to analogues that
contain only amide bonds.
We have shown that the aziridine aldehyde-driven cyclization
modulated the geometry of similar five- and six-membered
V
3
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RGD macrocycles and was responsible for tuning the binding
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V 5
AUTHOR INFORMATION
■
Corresponding Author
*
Prof. Andrei K. Yudin: Tel. 416-946-5042; E-mail: ayudin@
chem.utoronto.ca. Prof. Gang Zheng: Tel. 416-581-7666, Fax
16-581-7667, E-mail: gang.zheng@uhnres.utoronto.ca.
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ACKNOWLEDGMENTS
■
Synthesis of peptide macrocycles using unprotected amino aldehydes.
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