Ultrastructure of metallopeptide-based soft spherical morphologies

Article abstract of DOI:10.1039/c4ra10532j

Peptides and proteins offer interesting starting points for triggering self-assembly processes owing to the chemical diversity of side-chains, ease of chemical modifications and the possibility of exploiting several non-covalent and metal-assisted interac

Full text of DOI:10.1039/c4ra10532j

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Ultrastructure of metallopeptide-based soft
spherical morphologies†
Cite this: RSC Adv., 2014, 4, 64457
a
b
b
a
*
*
Gagandeep Kaur, Lihi A. Abramovich, Ehud Gazit and Sandeep Verma
Peptides and proteins offer interesting starting points for triggering self-assembly processes owing to the
chemical diversity of side-chains, ease of chemical modifications and the possibility of exploiting several
non-covalent and metal-assisted interactions, to stabilize higher order ensembles. Consequently, a
variety of nanoscale morphologies such as fibers, vesicles, nanotubes are observed for modified amino
acids and short peptides and these biocompatible soft materials have been used for diverse biological,
medical and material applications. Herein, we report metal-mediated modification of spherical soft
assemblies, by introducing a coordinating linker for the Phe–Phe dipeptide, which results in the
coalescence of soft structures. The possibility of copper ion coordination, with the metal-binding
peptide conjugate, was confirmed by single crystal analysis. Based on these observations, a model
depicting possible interactions leading to soft structure formation and metal-aided coalescence is also
presented. The coalescence could be reversed in the case of Au-mediated soft structures with the help
of thiol interference. Such an approach, exploiting interfacial metal ion interactions, is expected to
provide an entry into novel metallopeptide materials.
Received 16th September 2014
Accepted 18th November 2014
DOI: 10.1039/c4ra10532j
www.rsc.org/advances
Three different approaches could be envisaged for con-
structing MPFs: (i) introducing amino acids side chains capable
of metal binding11 (ii) use of exogenous ligands supporting MOF
Introduction
Self-assembling peptides offer a versatile platform towards
formation of ordered nanoscale systems, with various applica-
tions in catalysis, delivery, and tissue engineering, to name a
few.1–8 Control through different physicochemical properties of
constituent amino acids, possibility of predictable design based
on secondary structural signatures, and high compatibility
interaction with other biological systems, are the hallmarks of
peptide-based nanoscale materials. Consequently, it is quite
advantageous to employ short self-assembling peptides, to
obtain diverse hierarchical structures with desired functions.
Recent advances along these lines concern interaction of metal
ions with self-assembling peptides to afford bio-inspired metal–
peptide frameworks (MPF),9,10 which may offer combined
investigated properties of metal–organic frameworks (MOF)
with biocompatibility.
architecture12 (iii) use of covalently linked chiral/achiral
connectors. These approaches represent facile route for the
design of novel, abiotic oligomers for supramolecular studies.
As a specic example, Lehn, Huc and other groups have
reported family of oligoamides based on achiral connectivity of
2,6-pyridinedicarboxylic acid, where the resultant oligomers not
only self-organized into single helices, but also support forma-
tion of double helices under specic conditions.13,14 Notably,
short oligomers with 2,6-pyridinedicarboxylic acid and related
connectors afford planar ‘crescent-like’ conformation, while
longer oligomers exhibit more pronounced propensity to form
helical structures.15 In particular, pyridine diacid oligomers
offer hydrogen bonding at the inner rim of helices resulting in
ꢀ4.5 monomer per turn of the ensuing helix.
Diphenylalanine (FF) dipeptide motif exhibits spontaneous
self-assembly to generate peptide nanotubes in solution.16 FF
nanotubes offer interesting applications as scaffolds for the
synthesis of metal nanowires, possess improved mechanical
properties, semi-conductivity and serve as potential drug
delivery agents.17–23 Side chain aromaticity in phenylalanine is
implicated for self-assembly properties and key structural
features in the solid state.24 In current study, given our
continued interest in hierarchical self-assembled peptides
systems and our ongoing efforts to prepare covalently linked
conjugates of FF dipeptide,18,25,26 we decided to synthesize a
symmetric FF conjugate using pyridinedicarboxylic acid as an
aDepartment of Chemistry, DST Thematic Unit of Excellence on So Nanofabrication,
Center for Environmental Sciences and Engineering, Indian Institute of Technology
Kanpur, Kanpur-208016, India
bDepartment of Molecular Biology and Biotechnology, Department of Materials Science
and Engineering, Tel Aviv University, Tel Aviv-69978, Israel. E-mail: sverma@iitk.ac.
in; ehudg@post.tau.ac.il
† Electronic supplementary information (ESI) available: Crystallographic data,
details structures showing H-bonding and CH/p interactions in Cu242
complex. HR-SEM images of
4 + metal ions on copper grid and spectral
characterisation. CCDC 1005702. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c4ra10532j
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achiral linker. The ability of this new peptide conjugate to self-
assemble and its binding to various metals including copper,
silver and gold leading to formation of metallopeptide so
structures is described.
Results and discussions
Molecular design of peptide conjugate, 4
Fig. 1 (a) Molecular structure of 4. (b) Predicted curvature of 4.
We designed and synthesized 4, which is a hybrid of two diphe-
nylalanine moieties conjugated using 2,6-pyridinedicarboxylic
acid as a linker (Scheme 1). We envisioned the following advan-
tages for the proposed conjugate: [i] possibility to engender a
structure containing the ability of ‘crescent-like’ conformation in
an aggregating dipeptide (Fig. 1a); [ii] introduction of metal ion
co-ordination sites to further interrogate peptide self-assembly
process.
2,6-Pyridyl disubstituted linkers have been utilized to
engender a crescent conformation or a curvature in many fol-
dameric systems.15,27 In addition, pyridyl ring also offers addi-
tional advantage of metal ion interaction, which may further
lead to useful coordination architectures and varied geometries.
It can thus be surmised that such curved conformation (Fig. 1),
upon solution phase self-assembly, may eventually culminate
into spherical structures.
and diffraction quality crystals were grown by slow evaporation
method. Crystal analysis revealed M2L2 coordination, via
carboxylate groups, to afford a Cu(II)-supported paddle-wheel
like structure.28–31 The dimeric units were further stabilized by
hydrogen bonding between –N–H and –C]O of adjacent
˚
dimeric units in the range of 2.11–2.12 A (Fig. S1a†) as well as
through C–H/p interactions (2.47 A)32–35 (Fig. S1b†), to form a
˚
3D lattice structure (Fig. 2b; Table S2†). Notably, Cu(II) ions in
this case did not interact with pyridyl nitrogen.
Formation of M2L2-type of discreet dinuclear copper cluster
evinces interest not only due to interesting coordination
geometry, but also due to the fact that coordination-driven self-
assembled structures offer facile entry to the construction of
novel metallosupramolecular ensembles such as two-
dimensional polygons and three-dimensional spherical cages,
prisms, and polyhedral.36–39 It is envisaged that these designed
superstructures, which combine multiple weak interactions
including p–p stacking, CH–p interactions, hydrogen bonds
and metal-coordination, could extend application in domains
ranging from optoelectronic materials to drug delivery to
storage and catalysis. In case of 4, it could be imagined from
crystal structure that metal–carboxylate interactions offer a
benecial cooperative effect, along with other stabilizing non-
covalent interactions, to form interesting superstructures.
Thus, we started with the determination of solution-phase
morphology of 4, followed by investigating the effect of metal
ions, in the solution phase, to generate metallopeptide
ensembles.
Crystal structure of 4 with Cu ions
Presence of pyridyl group as a linker gave us an impetus to focus
our attention to metal ion coordination. As envisaged, we
surmised that such interaction may offer an entry metal-
mediated peptide so matter, perhaps with tunable size and
cavities. To start, we decided to assess metal ion interaction by
growing appropriate crystal to ascertain the possible site of
metal binding. 4 was interacted with Cu(II) ions and studied
through single-crystal diffraction analysis (Fig. 2a and Table
S1†). Methanolic solution of 4 was treated with CuSO4$5H2O
Solution phase self-assembly of 4
The propensity of peptide conjugate 4 to self-assemble was
studied. It was dissolved in 50% methanol–water at a concen-
tration of 1 mM and unlike nanotube forming FF dipeptide,16
4 leads to instantaneous formation of spherical structures with
a diameter of 350–650 nm, as conrmed by atomic force
microscopy (AFM) (Fig. 3a and b) and dynamic light scattering
(DLS) analysis (Fig. 3c). Spherical nature of these assemblies
could be attributed to the crescent-shaped structural design of
this conjugate. These spherical morphologies were further
analysed using scanning electron microscopy (SEM) and
transmission electron microscopy (TEM) (Fig. 3d and e).
Stability of these so structures was probed by subjecting
them to high-energy ion beam manipulation (Fig. 3f). Conse-
quently, a focused ion beam (FIB) system, using a gallium ion
beam, was employed to site-selectively machine a spherical
particle, at an operating voltage of 15.00 kV. This experiment
Scheme 1 Synthetic scheme of peptide conjugate 4: (i) 75% TFA-
DCM, 4 h, N2 atm. (ii) HOBt, DCC, 0 ꢁC, TEA, N2 atm. (iii) THF, 1 N
NaOH, 6 h, N2 atm.
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Fig. 2 Crystal structure of Cu2L2 metallopeptide (L ¼ 4): (a) dinuclear paddle wheel-type of structure (b) lattice structure showing peripheral
interactions.
Fig. 4 Microscopic images of 4 + Cu(II) complex. (a and b) SEM images
on silicon wafer after 12 h and 24 h respectively. (c) TEM image (inset:
magnified view of a metal-bound spherical structure). (d) EDX spec-
trum after 24 h.
T-shaped geometries. Such instances lead to formation of
hydrophobic microenvironments by excluding water. Moreover,
it is also known that aromatic–aromatic interactions further
help short peptides to efficiently access energetically favored
conformations.41–44 It should be emphasized here that mixed
Fig. 3 Microscopy analysis of 4 (a and b) AFM micrographs. (c) DLS
spectrum showing the spherical structure size distribution. (d) SEM
image. (e) TEM image. (f) FIB-SEM image of the sphere after ion beam
damage revealing the inner surface (1 mM, 50% aqueous methanol) on
silicon wafer.
solvent used in this case, due to solubility constraints, may also
exert some effect in stabilizing FF dipeptide side-chain inter-
actions. Notably, stacked arrangements are more stable in
water, while amphiphilicity of methanol interferes with both
stacked and T-shaped geometries, by attenuating aromatic–
aromatic interactions.45
revealed a solid inner core in the so structure, suggesting
formation of highly compact structures that are able to with-
stand ion beams without any measurable loss of morphological
integrity.40
Stabilization of these structures could be attributed to
known aromatic amino acid side-chain interactions during
peptide aggregation process to form parallel-stacked or
Interaction of 4 with copper ions
Cu(II) ions form stable four-, ve- and six-coordinate complexes.
These complexes show their potential use as anti-inammatory,46
antimicrobial,47 antitumor agents,48–51 enzyme inhibitors, and
chemical nucleases, generally bind through nitrogen of the
ligands. Conjugate 4 offers pyridyl ring nitrogen as well as free
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carboxylate groups as probable metal binding sites. Samples
were prepared by co-incubating 4 (1 mM, 0.5 mL, MeOH) with
CuCl2$2H2O solution (1 mM, 0.5 mL, H2O), followed by micros-
copy evaluation. Minor morphological changes appeared within
2 h of mixing, which became prominent during prolonged incu-
bation as conrmed by SEM and TEM microscopy (Fig. 4). These
images clearly showed vesicle coalescence in presence of metal
ions, leading to the formation of larger-sized vesicles in 24 h
(Fig. 4b). The monitoring was further continued for 120 h through
DLS measurements. The size of spherical structures increased up
to ꢀ3.4 mm in 120 h upon copper interaction perhaps due to
fusion of these vesicles. Notably, the presence of copper ions in
vesicles was conrmed by energy-dispersive X-ray spectroscopy
(EDX) measurements (Fig. 4d).
One of the hallmarks of Cu(II) interaction was the coalescence
of self-assembled structures. As Phe–Phe interactions are primarily
guided by stacking interactions, phenyl rings prefer to be buried
inside the spherical structures. Thus, it is likely that free carboxylic
groups and pyridyl ring nitrogens in 4 would possibly be exposed
at the surface of spherical structures for metal ion binding.52
Fig. 6 Microscopic images of 4 + Au(III) complex. (a and b) SEM images
on Si wafer after 12 h and 24 h respectively. (c) TEM image, (inset:
magnified view of a spherical structure). (d) EDX spectrum after 24 h.
Interaction of 4 with silver ions
Ag(I) ions generally interact through nitrogen centers in the
ligands53 and many such complexes are known to exhibit biolog-
ical activities.54,55 They are also known to trigger conformational
changes in self-assembled peptides.56 As pyridyl nitrogen of
4 could be an interesting metal binding site for Ag(I), samples were
prepared in presence of AgNO3 as described before. We were able
to observe changes in morphology 12 h aer the addition of metal
ions (Fig. 5a). Interestingly, coalescence of spherical vesicles was
observed again, where so structures of 4 exhibited formation of
fused structures, as evidenced by SEM and TEM micrographs
(Fig. 5b and c). As this morphology change was slightly different
compared to copper-mediated morphology, it is possible that Ag(I)
coordination in this case is distinct to Cu(II) interactions.57
Fig. 7 (a) SEM image of 4. (b) SEM image of 4 + Au(III) complex after 12
h. (C) Magnified view of 4 + Au(III) complex. (d) SEM image of 4 + Au(III)
+ MPA after 5 h incubation.
Unfortunately, our efforts to grow single crystals with Ag ions were
not successful.
Interaction of 4 with Au(III) ions
Chloroauricꢂacid (HAuCl4) protonates pyridyl ring with counter
anion AuCl4 ion balancing overall charge.58–61 Latter possesses
square-planar geometry, relatively diffuse charge distribution
and a weak interaction with solvent (water) molecules, with
nearest solvent molecules occupying two orthogonal quasi-
elliptical surfaces.62 Dissolving 4 along with HAuCl4 also
revealed morphological changes, where spherical structures
were found to afford a network-like morphology within 12 h
incubation (Fig. 6). Given the anionic nature of AuCl4ꢂ, it is
likely that carboxylate anions will not have any role in coordi-
nation. Thus, observed coalescence could be attributed to pyr-
idyl nitrogen coordination of Au(III). Indeed, several groups have
reported formation of Au(III) coordination complexes using
Fig. 5 Microscopic images of 4 + Ag(I) complex. (a and b) SEM images
on silicon wafer after 12 h and 24 h respectively. (c) TEM image (inset:
magnified view of a Ag(I)-mediated soft spherical structures). (d) EDX
spectrum after 24 h.
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experiments was also probed by doing this procedure on copper
grids (Fig. S2†). Similar results were obtained in all three cases
irrespective of the surface used.
Thiol displacement studies
Au(III) ion binds very effectively with 4 to form network-like
metal–peptide framework. As Au(III) has thiophilic binding
preference over nitrogen or oxygen centers,64,65 we decided to
treat 4 + Au(III) complex with 1-mercaptopropanoic acid (MPA)
to achieve its displacement from the framework. Samples were
prepared by mixing 4 (0.5 mL, 1 mM, MeOH) with HAuCl4
solution (0.5 mL, 1 mM, H2O), incubated for 12 h. This was
followed by addition of MPA solution (0.5 mL, 10 mM, 50%
methanol–water) and further incubation of 5 h (Fig. 7). Notably,
network-like structure disappeared conrming crucial role of
Au(III) ions in causing metalized vesicles to stick together.
Fig. 8 DLS spectra: (a) size distribution of 4 (50% MeOH–H2O). (b, d
and f) Size distributions after 24 h. (c, e and f) Size distributions after
120 h.
Dynamic light scattering analysis
Effect of metal ions on vesicle coalescence was conrmed by
DLS analysis using similar metal–peptide solutions used for
microscopy studies.66 The samples were prepared by mixing 4
(1 mL, 10 mm, MeOH) with metal ion solutions (1 mL, 10 mm,
H2O). It was observed that the peptide vesicles alone have a
hydrodynamic diameter of ꢀ350–650 nm (Fig. 8a), which upon
Cu(II) ions addition changes to ꢀ1.2 mm (Fig. 8b) aer 24 h
incubation and it further matured to an average diameter of
ꢀ3.4 mm aer 120 h (Fig. 8c). For Ag(I) ions, average size of
metalated vesicles changed to ꢀ1 mm aer 24 h (Fig. 8d) and
matured to an average diameter of 2.2 mm aer 120 h (Fig. 8e).
Similar trend was also observed for Au(III) interactions (Fig. 8f
and g). Such a change in size may be ascribed to metal-mediated
vesicle fusion process.
pyridyl and phenanthronyl ligands.63 Vesicles linked in a
network-like fashion were conrmed by various microscopy
methods and EDX analysis data.
Thus, different possibilities could be envisaged for metal–
peptide interactions: in case of Cu(II) ions, free carboxyl group
side-chains in 4 could interact as suggested by the solid state
structure; but, this does not preclude solution interaction of
pyridyl nitrogens with copper ions. In case of Ag(I) and Au(III)
ions, interaction could possibly be supported by pyridyl
nitrogen. Despite repeated attempts, we were unable to grow
crystals with Ag(I) and Au(III) ions to arrive at precise sites of Ag
or Au interaction with 4. Finally, role of surface in these
Fig. 9 Proposed model of metal–peptide interactions.
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Peptide conjugate 4 afforded formation of spherical struc-
tures supported by H-bonding and p–p stacking interactions.
Moreover, possible crescent-shape of monomers, engendered
by pyridyl linker, further assisted self-organization of 4 to
maximize non-covalent interactions.
Based on the occurrence of these forces, a model could be
proposed to depict formation of spherical vesicles from
crescent-shaped conjugate (Fig. 9). Observation of coalescence
upon metal ion addition was attributed to metal-coordination,
which could possibly bring these so peptide-based structures
in close proximity. As seen in Au experiment, exogenous addi-
tion of MPA could revert coalescence perhaps by complexing
metal ions thus releasing spherical structures.
Synthesis of peptide conjugate
4 was prepared by solution phase peptide synthesis.
N-(tert-Butyloxycarbonyl)-L-phenylalanine-L-phenylalanine
methyl ester (1)
N-(Boc)-L-phenylalanine (5 g, 18.8 mmol), and HOBt (2.55 g,
18.8 mmol) were dissolved in dry DMF (25 mL) under nitrogen
atmosphere and the reaction mixture was cooled to 0 C in an
ꢁ
ice bath. A solution of DCC in DCM (4.66 g, 22.6 mmol) was then
added drop wise to the reaction mixture. The reaction mixture
ꢁ
was stirred at 0 C for 1 h, aer which, L-phenylalanine methyl
ester hydrochloride (4.05 g, 22.6 mmol) was added to it followed
by triethylamine (13.1 mL, 94.2 mmol). The reaction mixture
was monitored and stirred for 24 h at room temperature under
nitrogen atmosphere. Reaction mixture was concentrated in
vacuo, redissolved in ethyl acetate and ltered to remove DCU.
The organic layer was then washed with 1 N HCl (3 ꢃ 30 mL),
10% NaHCO3 (3 ꢃ 30 mL) and brine (30 mL). The organic layer
was dried over anhydrous sodium sulfate and concentrated in
vacuo. The crude compound was puried through silica gel
column chromatography by using hexane and ethyl acetate
(80 : 20) solvent system to isolate pure 1 (6.17 g, 76.76% yield).
m.p. 102–105 ꢁC, Rf [30% ethyl acetate in hexane] ¼ 0.5, 1H
NMR (500 MHz, CDCl3, TMS, d ppm): 1.38 (s, 9H); 2.99–3.08 (m,
4H); 3.65 (s, 3H); 4.32 (m, 1H); 4.77–4.93 (m, 1H); 6.96–6.97 (m,
2H); 7.17–7.29 (m, 10H, overlapped aromatic signal and CDCl3
peak); 13C NMR (125 MHz; CDCl3, d ppm): 28.3, 38.0, 38.3, 52.3,
53.3, 55.7, 77.3, 80.4, 127.2, 128.6, 128.7, 129.3, 129.4, 170.8,
171.4; HRMS (M + H)+ for C24H30N2O5: 427.2233 (calcd),
427.2236 (anal.).
Conclusions
Conjugate 4 was designed having a crescent-type of structure,
synthesized and studied for its interaction with metal ions in
the solid state and in solution. Crystal analysis of copper
complex of 4 revealed M2L2 coordination, via carboxylate
groups, to afford a Cu(II)-supported paddle-wheel like structure.
Lattice structure offered insight into further interactions of
these dinuclear units via noncovalent interactions. Solution
phase self-assembly peptide conjugate revealed formation of
spherical structures, which possessed metal ion binding prop-
erties leading to coalescence, as conrmed by microscopy.
Cu(II), Ag(I) and Au(III) to form large sized vesicles: while Cu(II)
ions bound free acid side chains of 4 as conrmed by solid state
structure, Ag(I) and Au(III) ions could also invoke pyridyl
nitrogen as additional binding sites. Reversal of coalescence
was achieved in the case of Au(III) ions supported metal–peptide
structures, when treated with 1-mercaptopropanoic acid. Thus,
it can be stated that metal coordination plays an important role
in connecting these spherical structures and exploit an inter-
esting interfacial interaction to provide expeditious entry into
novel metallopeptide materials.
L-Phenylalanine-L-phenylalanine methyl ester (2)
1 (5 g, 9.5 mmol) was dissolved in 75% TFA-DCM and stirred for
1 h under nitrogen atmosphere. Aer completion of the reac-
tion, the solvent was evaporated in vacuo and was subsequently
washed with diethylether resulting in a white solid. The white
solid then dissolved in methanol and passed through activated
anion exchange resin and evaporated under reduced pressure to
obtain pure 2 (3.27 g, 85.4% yield). Rf [50% ethyl acetate in
hexane] ¼ 0.5; 1H NMR (500 MHz, DMSO-d6, TMS, d ppm): 2.89–
3.09 (m, 4H), 3.57 (s, 3H); 4.02 (m, 1H); 4.51–4.55 (m, 1H); 7.18–
7.30 (m, 10H), 8.16 (s, 3H); 13C NMR (125 MHz; DMSO-d6, d
ppm): 39.8, 40.0, 40.1, 40.3, 52.2, 54.1, 54.3, 128.4, 128.7, 129.5,
162.8, 172.1; HRMS (M + H)+ for C19H22N2O3: 327.1709 (calcd),
327.1700 (anal).
Materials and methods
General procedures
N,N0-Dicyclohexylcarbodiimide (DCC), N-hydroxybenzotriazole
(HOBt), di-tert-butyl dicarbonate (Boc), triuoroacetic acid
(TFA), and L-phenylalanine were purchased from SRL, Mumbai,
India and used without further purication. Dichloromethane
(DCM), N,N-dimethylformamide (DMF), methanol (MeOH) and
triethylamine (TEA) were distilled according to standard
procedures prior to use. 1H and 13C NMR spectra were recorded
on JEOL-DELTA2 500 model operating at 500 and 125 MHz,
respectively. HRMS (ESI+ and ESIꢂ) was recorded at IIT Kanpur,
Pyridyl-bis-L-phenylalanine-L-phenylalanine methyl ester (3)
India, on WATERS, Q-Tof Premier Micromass HAB 213 mass 2,6-Pyridinedicarboxylic acid (0.25 g, 1.49 mmol) was dissolved
spectrometer using capillary voltage 2.6–3.2 kV. IR spectra were in DMF and cooled to 0 ꢁC in ice bath under nitrogen atmo-
recorded as KBr pellets on a Perkin-Elmer Model 1320 spec- sphere. To this solution, HOBt (0.404 mg, 2.99 mmol) and a
trophotometer operating from 400 to 4000 cmꢂ1. For thin layer solution of DCC (0.617 g, 2.99 mmol) in DCM was added and
chromatography (TLC), Merck pre-coated TLC plates (MEARK stirred for 1 h under nitrogen atmosphere at 0 ꢁC. Subsequently
60 F254) were used, and compounds were visualized with a UV 2 (1.07 g, 3.29 mmol) was added into the reaction mixture fol-
light at 254 nm. Chromatographic separations were performed lowed by triethylamine (0.76 mL, 9.48 mmol). The reaction
on S. D. Fine-Chem 100–200 mesh silica gel.
mixture was monitored and stirred for 24 h at room
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temperature under nitrogen atmosphere. Reaction mixture was
concentrated in vacuo, redissolved in ethyl acetate and ltered
to remove DCU. The organic layer was then washed with 1 N HCl
(3 ꢃ 30 mL), 10% NaHCO3 (3 ꢃ 30 mL) and brine (30 mL). The
organic layer was dried over anhydrous sodium sulfate and
concentrated in vacuo. Crude compound was then puried
through silica gel column chromatography by using methanol–
DCM (4 : 96) resulting in pure 3. (0.45 g, 38.5% yield). m.p. ¼
142–144 ꢁC, Rf [5% methanol in DCM] ¼ 0.5; 1H NMR (500 MHz,
DMSO-d6, TMS, d ppm) ¼ 2.46–3.18 (m, 8H), 3.54 (s, 6H), 4.48–
Focused ion beam-scanning electron microscopy (FIB-SEM)
10 mL aliquots of the samples (1 mM in 50% methanol–water)
were deposited on a silicon wafer (100) and allowed to dry at
room temperature. Subsequently the samples were dried in
vacuo for 30 min prior to imaging. FIB-SEM images were
acquired on FEI make Nova 600 Nanolab workstation equipped
with a eld emission Ga ion source, operating at 15 kV.
Transmission electron microscopy (TEM)
4.53 (m, 2H), 4.70–4.77 (m, 2H), 7.08–7.29 (m, 20H) 8.07 (m, 10 mL aliquots of the sample (1 mM in 50% methanol–water)
2H), 8.59–8.60 (m, 1H), 8.92–8.93 (2s, 4H); 13C NMR (125 MHz; were placed on a 400-mesh copper grid. Aer 1 min any excess
DMSO-d6, d ppm): 37.1, 37.8, 39.5, 40.0, 40.4, 40.5, 52.3, 54.3, uids were removed. Due to the present of metal samples were
54.7, 125.2, 126.8, 127.0, 128.7, 137.5, 139.9, 149.0, 163.5, 170.3, imaged without any further staining. Samples were viewed
172.1; HRMS (M + Cl)ꢂ for C45H45ClN5O8: 818.2957 (calcd), using a JEOL 1200EX electron microscope operating at 80 kV.
818.2957 (anal.).
Dynamic light scattering (DLS)
Pyridyl-bis-L-phenylalanine-L-phenylalanine conjugate (4)
Particle size distribution of 4, 4 + Cu(II), 4 + Ag(I) and 4 + Au(III)
complexes were measured using dynamic light scattering
analyzer at a wavelength 657 nm, with Delsa Nano C Particle
analyzer (Beckman Coulter). Samples were measured at 25 C.
3 (0.1 g, 0.127 mmol) was dissolved in tetrahydrofuran to form a
clear solution. To this 1 N NaOH (0.03 g, 0.765 mmol) was added
and stirred for 6 h at room temperature. The solution obtained
was passed over activated cation exchange resin. The ltrate was
evaporated under reduced pressure to obtain pure 4. (0.07 g,
ꢁ
1
72.9% yield); m.p. 120–122 ꢁC; H NMR (500 MHz, DMSO-d6,
Acknowledgements
TMS, d ppm): 2.46–3.34 (m, 8H), 4.46–4.73 (m, 2H), 4.74–4.83
(m, 2H), 7.06–7.34 (m, 20H), 8.12–8.13 (m, 1H), 8.41–8.46, (m,
2H), 8.85–8.95 (m, 4H), 9.23–9.31 (m, 2H); 13C NMR (125 MHz;
DMSO-d6, d ppm): 37.1, 37.4, 39.5, 40.0, 40.2, 40.3, 54.3, 55.5,
125.3, 126.7, 126.9, 128.6, 129.6, 139.0, 148.9, 157.1, 163.9,
171.8, 173.4; FTIR (KBr, cmꢂ1): 1220, 1528, 1655 (C]O, amide I,
II and III), 1736 (C]O, acid), 2931 (CH-aliph.), 3029 (CH-Ar),
3310 (NH, str); HRMS (M ꢂ H)ꢂ for C43H41N5O8: 754.2877
(calcd), 754.2877 (anal).
We thank Ion Beam Centre, Nuclear Physics unit, IIT Kanpur,
for FIB-SEM and DST Thematic Unit of Excellence in So
Nanofabrication, IIT Kanpur, for FE-SEM and DLS. We thank Z.
Barkay for help with the SEM analysis and Y. Delarea for help
with TEM experiments. We thank member of the Gazit group
for helpful discussions. G.K. thanks IIT Kanpur and UGC, India
for nancial support. This work is supported by an Outstanding
Investigator Award to S.V. from DAE-SRC, Department of
Atomic Energy, India, J. C. Bose National Fellowship, DST,
India, and by MHRD supported Centre for Excellence in
Chemical Biology, IIT Kanpur.
Field emission scanning electron microscopy (FE-SEM)
10 mL aliquots of the samples (1 mM in 50% methanol–water)
were deposited on a silicon wafer (100) and allowed to dry at
room temperature. Subsequently the samples were dried in
vacuo for 30 min prior to imaging. The samples were imaged
with and without (for EDX) gold coating. SEM images were
acquired on FEI Quanta 200 microscope, equipped with a
tungsten lament gun, operating at WD 3 mm and 10 kV.
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