Chiral Gold Nanoparticles Decorated with Pseudopeptides

Article abstract of DOI:10.1002/ejoc.201500549

Peptide-functionalized gold nanoparticles are supramolecular systems that can mimic natural proteins or DNA. In this work, we describe the preparation, the analysis, and the biological evaluation of gold nanoparticles linked to pseudopeptide foldamers containing one to eight L-Ala-D-Oxd (Ala = alanine; Oxd = 4-carboxy-5-methyloxazolidin-2-one) residues. The nanoparticles become increasingly organized as the number of such moieties increases. Moreover, from the analysis of chiroptical signals we find that the chirality of the gold surface becomes more evident with decreasing foldamer length. Finally, these systems display no cytotoxicity towards HeLa cells and are good candidates to promote drug delivery once equipped with biologically active moieties.

Full text of DOI:10.1002/ejoc.201500549

FULL PAPER
DOI: 10.1002/ejoc.201500549
Chiral Gold Nanoparticles Decorated with Pseudopeptides
Rossana Fanelli,[a] Lorenzo Milli,[b] Andrea Cornia,[c] Alessandro Moretto,[d]
Nicola Castellucci,[b] Nicola Zanna,[b] Giulia Malachin,[d] Regina Tavano,[e] and
Claudia Tomasini*[b]
Keywords: Nanoparticles / Gold / Peptidomimetics / Foldamers / Circular dichroism / Conformation analysis
Peptide-functionalized gold nanoparticles are supramolec- the number of such moieties increases. Moreover, from the
ular systems that can mimic natural proteins or DNA. In this
work, we describe the preparation, the analysis, and the bio-
logical evaluation of gold nanoparticles linked to pseudo-
peptide foldamers containing one to eight L-Ala-D-Oxd (Ala
= alanine; Oxd = 4-carboxy-5-methyloxazolidin-2-one) resi-
dues. The nanoparticles become increasingly organized as
analysis of chiroptical signals we find that the chirality of the
gold surface becomes more evident with decreasing fold-
amer length. Finally, these systems display no cytotoxicity
towards HeLa cells and are good candidates to promote drug
delivery once equipped with biologically active moieties.
copies of a peptide is a nanosystem that resembles a protein
in size (2–20 nm), shape (globular), and, possibly, function.
A variety of synthetic methodologies for the preparation
of AuNPs within a narrow size distribution are available.[8]
Such systems may be assembled by “wet chemistry” pro-
cedures, whereby a surface-capping ligand binds to the
metal, controlling its growth and avoiding agglomeration or
coalescence into the bulk material.[9]
The design strategy for the synthesis of a peptide-based
capping ligand must address some key requirements. For
instance, it is important to avoid attractive interparticle in-
teractions promoted by ammonium and carboxylate groups.
Moreover, peptides need to have a strong affinity for gold
and must be able to self-assemble into a dense layer that
excludes water, while a hydrophilic terminus is required to
achieve solubility and stability in water.[10]
In this work, we describe the preparation, the analysis,
and the biological evaluation of AuNPs capped by pseudo-
peptide foldamers containing the l-Ala-d-Oxd (Ala = alan-
ine; Oxd = 4-carboxy-5-methyloxazolidin-2-one) moiety
and functionalized with a trityl mercaptopropanoic linker
(Figure 1). In our previous work we fully demonstrated that
oligomers containing several l-Ala-d-Oxd residues tend to
become organized into β-bend ribbon spirals.[11]
Introduction
Colloidal solutions have been known and widely used
since antiquity.[1] However, colloid science had development
at a slower pace compared with other branches of chemistry
because of several difficulties encountered in the prepara-
tion, reproduction, and characterization of colloidal solu-
tions.
More recently, nanoparticles (NPs) functionalized with
various molecular and biomolecular units were assembled
into complex hybrid systems.[2] Composite layered or aggre-
gated structures of (macro)molecule-cross-linked NPs on
surfaces have been prepared,[3] and specific sensing of sub-
strates, tunable electroluminescence, and enhanced photo-
electrochemistry[4] have been observed. Several reviews have
addressed the recent advances in the synthesis and proper-
ties of NPs[5] and the progress made in the integration of
composite NP systems with surfaces.[6]
Peptide-functionalized gold nanoparticles (AuNPs)[7] are
intriguing supramolecular systems that can mimic natural
proteins or DNA. A cluster of gold atoms surrounded by
[a] School of Chemistry Food & Pharmacy, University of Reading,
Whiteknights, P. O. Box 217, Reading, Berkshire, RG6 6AH,
United Kingdom
[b] Departimento di Chimica “G. Ciamician”, Università di
Bologna,
2, Via Selmi, 40126 Bologna, Italy
E-mail: claudia.tomasini@unibo.it
[c] Dipartimento di Scienze Chimiche e Geologiche, University of
Modena and Reggio Emilia,
183, Via G. Campi, 41125 Modena, Italy
[d] Dipartimento di Scienze Chimiche, Università di Padova,
1, Via Marzolo, 35131 Padova, Italy
[e] Dipartimento di Scienze Biomediche, Università di Padova,
3, Viale G. Colombo, 35121 Padova, Italy
Figure 1. General structure of the pseudopeptide foldamers described
in this work.
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500549.
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Results and Discussion
Synthesis
Compounds 1–4 were prepared by cleavage of the N-ter-
minal Boc moieties with anhydrous TFA in dichlorometh-
ane followed by coupling with trityl mercaptopropionic
acid in the presence of HBTU and TEA in acetonitrile at
room temperature under a nitrogen atmosphere (Scheme 2).
The reaction was complete after one hour and the desired
compounds were obtained with a yield ranging from 52 to
63%.
We prepared a series of oligomers Boc-(L-Ala-d-Oxd)n-
OBn (n = 1, 2, 4, 8) (Boc = tert-butyloxycarbonyl)
(Scheme 1) by conventional methods in solution, starting
from l-Ala and d-Thr (Thr = threonine). Boc-l-Ala-d-
Oxd-OBn (5) was obtained in high yield by addition of Boc-
l-Ala-OH to d-Oxd-OBn in the presence of O-(benzotri-
azol-1-yl)-N,N,NЈ,NЈ-tetramethyluronium hexafluorophos-
phate (HBTU) and triethylamine (TEA) in anhydrous
acetonitrile,[12] whereas d-Oxd-OBn can be easily synthe-
sized in multigram scale starting from d-Thr.
Conformational Analysis of 1–4
Before linking compounds 1–4 to AuNPs, the possible
adoption of a well-defined secondary structure in these
compounds, as in the previously studied N-Boc deriva-
tives,[13] was investigated by IR and NMR spectroscopy and
by single-crystal X-ray diffraction. The N–H stretching re-
gion enables intramolecular C=O···H–N hydrogen bonds
formed in solution to be detected; free (non-hydrogen-
bonded) amide NH groups exhibit a stretching signal above
3400 cm–1, whereas hydrogen-bonded amides[14] produce a
stretching band below 3400 cm–1. The FTIR spectra were
recorded in CH2Cl2 at a compound concentration of 3 mm,
so as to avoid compound self-aggregation (Table S1, for the
IR spectra see the Supporting Information).
The spectrum of 1 clearly shows the presence of only
one band at 3419 cm–1 attributed to free amide groups. By
contrast, two bands at 3436 and 3347 cm–1 are seen in the
spectrum of 2, indicating that both free and hydrogen-
bonded amide NH groups are present in solution. Signifi-
cantly, the stretching band above 3400 cm–1 disappears in
3 and 4, suggesting that these compounds adopt a folded
conformation, as could be anticipated from the analysis of
their N-Boc protected counterparts.[13] A similar trend was
observed for the C=O stretching band of the amide groups,
which shift from 1679 cm–1 in 1 to 1663 cm–1 in 4.
Scheme 1. Reagents and conditions: (i) HBTU (1.1 equiv.), TEA
(2 equiv.), anhydrous CH3CN, 1 h, r.t.; (ii) H2, Pd/C (10%), MeOH,
16 h, r.t.; (iii) TFA (18 equiv.), anhydrous CH2Cl2, 4 h, r.t.; (iv) HBTU
(1.1 equiv.), TEA (3 equiv.), anhydrous CH3CN, 1 h, r.t.
1H NMR analysis supports the foregoing interpretation
of IR spectroscopy results. The NH group of 1 resonates at
The other oligomers 7, 9, and 11 were synthesized in δ = 6.08 ppm, whereas the NH groups of 2 resonate at δ =
solution by selective deprotection of the C-terminal benzyl 5.94 and 7.35 ppm and the NH groups of 4 are very de-
esters with H2 in methanol in the presence of Pd/C (10%) shielded, ranging between 7.9 and 8.5 ppm; these pro-
and by cleavage of the N-terminal Boc moieties with an- nounced downfield shifts are usually indicative of hydrogen-
hydrous trifluoroacetic acid (TFA) in dichloromethane. The bonded NH groups.[14] The occurrence of intramolecular
two deprotected moieties were then coupled by using C=O···H–N hydrogen bonds in oligomers 2 and 4 was fur-
HBTU and TEA in anhydrous acetonitrile under an inert ther confirmed by following the dependence of NH proton
atmosphere to give the longer oligomers in satisfactory chemical shifts upon addition of [D6]dimethyl sulfoxide
yield. All the deprotection steps were performed with excel- ([D6]DMSO). This solvent is a strong hydrogen-bond ac-
lent yields, whereas the yields of the coupling step ranged ceptor and, when bound to a NH proton, it is expected to
from 72 to 88% (Scheme 1).
have a dramatic deshielding effect.[15] The results of [D6]-
Scheme 2. Reagents and conditions: (i) TFA (18 equiv.), anhydrous CH2Cl2, 4 h, r.t.; (ii) HBTU (1.1 equiv.), TEA (2 equiv.), anhydrous CH3CN,
1 h, r.t.
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Chiral Gold Nanoparticles Decorated with Pseudopeptides
DMSO/CDCl3 titrations of the NH protons in pseudopept- turn (310-helical structure) is not formed. Instead, the
ides 2 and 4 are presented in Figure 2.
strongest intramolecular H-bonding interaction involves
amide nitrogen N2 and the d-Oxd2 carbonyl oxygen O4
[d(HN2···O4) = 2.141(18) Å]. Notably, the amide group
N4–HN4 of l-Ala1 is not engaged in intramolecular
hydrogen bonds in the solid state, and the resulting picture
1
matches remarkably well the results of IR and H NMR
investigations in solution. Molecules related by the 21 axis
are hydrogen bonded into chains through amide group N4–
HN4 and carbonyl oxygen O4 [d(HN4···O4i)
=
2.103(11) Å], but crystal packing also relies on a number
of other short C–H···O contacts (Table S3). The backbone
conformational parameters gathered in Table S4 show that
the torsion angles (φ and ψ) at the l-Ala1 residue are sim-
ilar to those of poly-l-proline II helix, as in related folda-
mers.[16] However, the l-Ala2 residue has quite different
conformational parameters that fall in the right-handed α-
helix region of the Ramachandran plot.
Figure 2. Variation of NH proton chemical shifts (ppm) of 2 (a), and
4 (b) as a function of increasing percentages of [D6]DMSO added to
the CDCl3 solution (v/v) (concentration: 3 mm).
Of the two NH protons in 2, one is totally free (Δδ =
1.40 ppm over the explored range of DMSO concentration),
whereas the second is quite H-bonded (Δδ = 0.34 ppm). The
former is most likely the group closer to the S terminus,
because its chemical shift in CDCl3 solution is virtually the
same as that found in compound 1. By contrast, all NH
protons in 4 are DMSO-insensitive, confirming that 4 is
fully organized in a secondary structure supported by
C=O···H–N interactions.
Figure 3. Partially labeled ORTEP-3[18] plot of 2 with displacement
ellipsoids at 50% probability level and H atoms drawn as spheres
with arbitrary radius. Dashed lines indicate intramolecular hydrogen-
bonding interactions discussed in the text. For simplicity, only H
atoms engaged in intra- or intermolecular hydrogen bonds have been
labeled. Atom labeling follows the system used previously.[16]
X-ray quality crystals of 2 were grown as long colorless
needles by slow evaporation of a toluene solution. Unfortu-
nately we were not able to grow crystals of oligomers 1, 3,
or 4. Crystal data and refinement parameters for 2 are given
in Table S2; the geometry of hydrogen-bond interactions are
detailed in Table S3 and conformational data are gathered
in Table S4. The crystals are monoclinic, space group P21,
and the unit cell comprises two molecules related by the
twofold screw axis (Figure 3). The molecule exhibits a turn-
like structure. As found in related foldamers,[16] the l-Ala1
α-carbon atom C22 has a H-contact with the endocyclic
C=O of the neighboring d-Oxd1 residue [d(H22···O8) =
2.300(13) Å]. However, no significant H-bonding interac-
tion is established between the carbonyl oxygen O10 and
the amide group N2–HN2 of l-Ala2 [d(HN2···O10) =
The ECD spectra of compounds 1–4 were not recorded
because the spectral features related to the secondary struc-
ture are obscured around 200 nm by the very strong B tran-
sitions of trityl groups, that are removed upon formation of
the AuNPs. Strong support for the formation of a robust
secondary structure for the longer oligomers of the (l-Ala-
d-Oxd)n series comes from analysis of the Boc-(L-Ala-d-
Oxd)n-OH oligomers, that we reported previously.[11] We
could demonstrate that a stable 310 helix is formed when n
Ն 5.
Preparation and Analysis of Gold Nanoparticles
Pseudopeptide-functionalized gold nanoparticles AuNPs1,
2.808(16) Å], so that a ten-membered hydrogen-bonded AuNPs2, AuNPs3, AuNPs4 were obtained by reduction of
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HAuCl4 with NaBH4 in the presence of compounds 1–4,
Electronic circular dichroism (ECD) analysis of AuNPs1–4
respectively.
was performed in water solution (Figure 5), both in the far-
Structural information on the NPs was obtained by using UV region (190–250 nm) and in the UV/Vis region (300–
several techniques (Table 1). The average number of Au atoms 700 nm), where chiroptical signals are expected to arise from
per NP was calculated by assuming spherical particles and the chirality of the gold surface (chiral staples).
using the diameter of the metallic core observed in trans-
mission electron microscopy (TEM) images and the density of
bulk metal (55 atoms/nm3) (Figure S3).
Table 1. Chemical structure data for AuNPs1–4.
AuNP Core diameter No. of Au atoms No. of foldamers Footprint
[nm][a]
per NP[b]
per NP[c]
[nm2][d]
AuNPs1
AuNPs2
AuNPs3
AuNPs4
2.1Ϯ0.3
2.3Ϯ0.3
2.3Ϯ0.4
2.5Ϯ0.3
289
376
376
508
167
156
135
97
0.10
0.11
0.15
0.56
[a] Calculated by averaging the size of at least 200 NPs. [b] Calcu-
lated by assuming a spherical model. [c] Determined by combining
TEM and TGA analysis, as described in the Supporting Infor-
mation. [d] Defined as the area occupied by the projection of the
foldamer ligand onto the gold cluster surface.
The number of foldamers conjugated to the inorganic core
was calculated by using the weight loss observed in thermo-
gravimetric analysis (TGA),[17] assumed to arise from the loss
of the organic coating monolayer, and the molecular weight
of the appropriate oligomer (Figure S4).
Finally the foldamer footprints were calculated by relating
the number of oligomers linked to the gold surface with the
dimensional information obtained by TEM analysis (Fig-
ure S5).
The UV/Vis absorption spectra (Figure 4) are consistent
with the results gathered in Table 1, because a plasmonic band
(ca. 520 nm) indicative of a NP diameter Ն 2.5 nm was ob-
served only for AuNPs4.[19]
Figure 5. ECD spectra of AuNPs1–4 recorded in water at 20 °C:
(a) far-UV ECD spectra indicating the foldamer chirality; (b) UV/Vis
ECD spectra indicating the chirality of the gold surface.
The ECD spectra in the far-UV region are reported for
AuNPs1–4 in Figure 5 (a). The spectra of AuNPs1 and
AuNPs3 show only a deep minimum at 195 nm, which is usu-
ally associated with a random coil conformation,[21] whereas
AuNPs2 shows a deep minimum at 191 nm and a weak maxi-
mum at 215 nm. All these spectra resemble closely those of
unordered peptides,[21] the ECD response of which entails a
strong negative band near 197 nm and a weak band at ap-
proximately 220 nm, that may be a negative shoulder on the
short-wavelength band. In contrast, the spectrum of AuNPs4
outlines the typical conformation of a 310 helix, displaying a
maximum at 195 nm, a deep minimum at 208 nm, and a
shoulder at 227 nm.[22] The four separate ECD spectra of
AuNPs1–4 are reported in Figure S6.
The chiroptical signals associated with the gold surface are
reported in Figure 5 (b).[23] The signals associated with
AuNPs1–3 show a variation of the molar ellipticity, with a
maximum ranging between 360 and 430 nm, whereas AuNPs4
Figure 4. UV/Vis spectra of AuNPs1–4 recorded in H2O at 20 °C in
the 350–650 nm region.
Another important feature of AuNPs is chirality, which shows a steadier trend. This outcome suggests that only an
may derive from several factors:[20] (1) A chiral arrangement unordered conformation may influence the chirality of the
of metal atoms in the core; (2) the binding of thiolates on the whole AuNP, whereas the more robust secondary structure 4
gold surface to form chirally arranged staples; (3) the chirality does not communicate with the AuNPs. This is a remarkable
of the monolayer of passivating organic molecules. Pseudo- effect that confirms previous observations on AuNPs passiv-
peptide oligomers, binding to the gold core, may influence the ated with short oligopeptides based on Aib-l-Ala repeating
whole NP to assume a diverse organization.
units (Aib = aminoisobutyric acid).[24] These NPs presented
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Chiral Gold Nanoparticles Decorated with Pseudopeptides
relevant ECD signals in the 300–650 nm wavelength region IR and NMR spectroscopy and X-ray crystallography. In par-
only for short peptides. These signals vanished when the se- ticular, both in solution and in the solid state, only one NH
quences were long enough to assume a folded conformation.
group of the (l-Ala-d-Oxd)2 moiety is engaged in intramolec-
ular H-bonding interactions. By contrast, all NH groups of
the longer-chain derivatives are involved in intramolecular
hydrogen bonds.
Effect of AuNPs on Cell Viability
According to the ECD spectra, such a scenario is largely
retained when pseudopeptide foldamers are linked to AuNPs,
with the (l-Ala-d-Oxd)8 moiety forming a well-organized 310
helix.
Moreover, chiroptical signals in the UV/Vis region suggest
that the chirality of the gold surface becomes more pro-
nounced with decreasing foldamer length. This important out-
come demonstrates that oligopeptide chirality may be commu-
nicated to the whole NPs with better efficiency through an
unordered oligopeptide conformation than through a well-
organized secondary structure.
Finally, regardless of their conformation, none of the
capped AuNPs displayed acute cytotoxicity towards HeLa
cells. However, detectable amounts of ROS were induced, with
various efficacies, by all the studied AuNPs. Thus, these sys-
tems can be regarded as promising candidates for drug deliv-
ery once properly equipped with biologically active moieties.
The cytotoxic acute effect of the different AuNPs developed
in this study was evaluated in HeLa cells, which is a human
cell line from cervical cancer. After incubation at different
nanoparticle concentrations, cell viability was assessed by the
MTS assay. In this test, the MTS compound is reduced by
living cells into a colored formazan product that is soluble
in tissue culture medium. So, the quantity of formazan, the
absorbance of which was measured at 490 nm, is directly pro-
portional to the number of living cells in the culture. As shown
in Figure 6, none of the four types of AuNPs used are toxic
for human HeLa cells up the high dose of 200 μg/mL.
Experimental Section
General: Routine NMR spectra were recorded with spectrometers
working at 400 or 200 MHz (1H NMR) and at 100, 75 or 50 MHz
(13C NMR). Chemical shifts are reported in δ values relative to the
solvent peak of CHCl3 (δ = 7.27 ppm), CH3OH (δ = 3.31 ppm), and
DMSO (δ = 2.54 ppm). Proton signals were assigned based on
gCOSY spectra. IR spectra were recorded with an FTIR spectrome-
ter. Melting points were determined in open capillaries and are uncor-
rected. High-quality infrared spectra (64 scans) were obtained at
2 cm–1 resolution by using a 1 mm NaCl solution cell and a Bruker
Laser class FT-infrared spectrometer. Spectra were recorded on 3 mm
solutions in CH2Cl2 at 297 K. All compounds were dried in vacuo,
and all sample preparations were performed in a nitrogen atmosphere.
The ECD spectra were obtained with a Jasco J-810 spectropolari-
meter. Cylindrical fused quartz cells of 0.02 cm and 1 cm path length
were used. The values are expressed in terms of [θ]T, the total molar
ellipticity (degcm2 g–1). UV/Vis absorption spectra were measured in
water with a Perkin–Elmer Lambda 45 UV/Vis spectrophotometer
with 1 cm path length quartz cuvettes. TEM micrographs were regis-
tered with a Jeol 300PX instrument. Samples were prepared by 100-
fold dilution of a 2 mg/mL Milli-Q water solution of the appropriate
AuNPs1–4. A glow discharged carbon coated grid was floated on a
Figure 6. Cell viability evaluated in HeLa cells by the MTS assay,
after incubation for 24 h with different concentrations of NPs. The
percentage of living cells was calculated with respect to control cells
(no NPs). Values are mean ϮSE (N = 2).
We also determined the ability of AuNPs to induce the pro-
duction of reacting oxygen species (ROS). To this end, HeLa
cells were incubated with AuNPs1–4 for three hours and the
produced ROS were measured using the Carboxy-H2DCFDA
(6-carboxy-2Ј,7Ј-dichlorodihydrofluorescein diacetate) probe.
This nonfluorescent molecule is converted into a green-
fluorescent form by ROS, as they remove the acetate groups.
As shown in Figure S7, in spite of their lack of cytotoxicity,
all AuNPs can induce ROS production. AuNPs1 was the least small drop of solution and excess was removed by #50 hardened
Whatman filter paper. TGA was run on 5–8 mg nanoparticle samples
with a SDT-2960 model TA instrument from 25 to 1200 °C under a
continuous N2 flow.
active, whereas AuNPs3 induced the strongest ROS pro-
duction.
Detailed information for the synthesis and characterization of oligo-
mers are reported in the Supporting Information.
Conclusions
Synthesis of AuNPs4: Compound 4 (53 mg, 0.026 mmol) was dis-
solved in a mixture of Milli-Q water, MeOH, and CH2Cl2. A solution
of HAuCl4 (0.040 g, 0.11 mmol) in water was added and the mixture
was stirred for 20 min to permit the polymerization. A solution of
NaBH4 (0.020 g, 0.53 mmol) dissolved in water was then added to the
mixture very rapidly. A brown solution was obtained and, after 2 h,
Gold nanoparticles capped with pseudopeptide foldamers
based on the l-Ala-d-Oxd repeating unit and featuring a ter-
minal mercaptopropanoic linker were prepared and analyzed.
With increasing number of l-Ala-d-Oxd units (n = 1, 2, 4, and
8), the pseudopeptides become increasingly more organized in
their secondary structure, as shown by the combined use of the reaction reached completion. The reaction was quenched by ad-
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pressure and the dark residue was dissolved in Milli-Q water (4 mL).
Subsequently, the so-obtained solution was filtered through a 45 µm
microfilter, the nanoparticles precipitated by adding a large amount
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X-ray Crystallography: Single-crystal X-ray structure determination
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parameters. Aromatic and methyl hydrogen atoms were added in ide-
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U(H) = 1.2Ueq(C) and U(H) = 1.5Ueq(C), respectively (torsion angle
was refined for methyl groups using AFIX 137 instruction). The re-
maining hydrogen atoms were refined by forcing ID parameters to be
identical within each methylene group, by restraining C–H distances
to be the same (within 0.015 Å) for all tertiary and all methylene
hydrogen atoms, and by fixing N–H distances to 0.880(15) Å. The
expected absolute configuration at C9, C10, C14, C17, C18, and C22
was confirmed by anomalous dispersion effects; Flack parameter:
0.01(4).
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CCDC-1060459 contains the supplementary crystallographic data for
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Cell Culture and MTS Assay: HeLa cells were maintained in DMEM
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biotics (penicillin and streptomycin, Invitrogen) at 37 °C in a humidi-
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Acknowledgments
L. M., N. Z., R. T., G. M., and C. T. thank the Italian Ministero
dell’Istruzione, dell’Università e della Ricerca (MIUR) (program
PRIN 2010NRREPL_009) and the Consorzio Spinner Regione Emi-
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Received: April 29, 2015
Published Online: August 12, 2015
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