Gold nanoclusters protected by conformationally constrained peptides

Article abstract of DOI:10.1021/ja0560581

The preparation and properties of a series of gold nanoclusters protected by thiolated peptides based on the α-aminoisobutyric acid (Aib) unit are described. The peptides were devised to form 0-3 C=O...H-N intramolecular hydrogen bonds, as required by their 3₁₀-helical structure. The monolayer-protected clusters (MPCs) were prepared, using a modified version of the two-phase Brust-Schiffrin preparation, and fully characterized with ¹H NMR spectrometry, IR and UV-vis absorption spectroscopies, transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and X-ray photoelectron spectroscopy (XPS). The MPCs were obtained with core diameters in the range of 1.1-2.3 nm, depending on the reaction conditions. Structured peptides formed smaller clusters. The smallest MPC obtained is in agreement with the average formula Au₃₈PeP₁₈. The results showed that the chemical integrity of the peptide is maintained upon monolayer formation and that the average number of peptide ligands per gold cluster is typically 75-85% the value calculated for alkanethiolate MPCs of similar sizes. The IR and NMR spectra indicated that in the monolayer the peptides are involved in both intra- and interligand C=O-H-N hydrogen bonds.

Full text of DOI:10.1021/ja0560581

Published on Web 12/14/2005
Gold Nanoclusters Protected by Conformationally
Constrained Peptides
Laura Fabris,‡ Sabrina Antonello,‡ Lidia Armelao,§ Robert L. Donkers,‡
Federico Polo,‡ Claudio Toniolo,‡ and Flavio Maran*,‡
Contribution from the Department of Chemistry, UniVersity of PadoVa, Via Marzolo 1, 35131
PadoVa, Italy, and Department of Chemistry, ISTM-CNR, Via Marzolo, 1, 35131 PadoVa, Italy
Received September 10, 2005; E-mail: f.maran@chfi.unipd.it
Abstract: The preparation and properties of a series of gold nanoclusters protected by thiolated peptides
based on the R-aminoisobutyric acid (Aib) unit are described. The peptides were devised to form 0-3
CdO‚‚‚H-N intramolecular hydrogen bonds, as required by their 310-helical structure. The monolayer-
protected clusters (MPCs) were prepared, using a modified version of the two-phase Brust-Schiffrin
preparation, and fully characterized with 1H NMR spectrometry, IR and UV-vis absorption spectroscopies,
transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and X-ray photoelectron
spectroscopy (XPS). The MPCs were obtained with core diameters in the range of 1.1-2.3 nm, depending
on the reaction conditions. Structured peptides formed smaller clusters. The smallest MPC obtained is in
agreement with the average formula Au38Pep18. The results showed that the chemical integrity of the peptide
is maintained upon monolayer formation and that the average number of peptide ligands per gold cluster
is typically 75-85% the value calculated for alkanethiolate MPCs of similar sizes. The IR and NMR spectra
indicated that in the monolayer the peptides are involved in both intra- and interligand CdO‚‚‚H-N hydrogen
bonds.
Introduction
adsorbates interact mainly through intermolecular van der Waals
forces. Additional interactions, however, may enhance the
Understanding and controlling the physical and chemical
properties of monolayer-protected gold clusters (MPCs) is an
active area of current research.1,2 The MPCs are rather easily
fabricated and possess high stability.2,3 The protecting ligands
provide a way to decorate the outer MPC shell with molecular
groups to carry out specific functions, which is an issue of
paramount relevance for the use of these nanosystems as devices
and sensors.1-4 Exploiting MPCs for applied purposes thus
requires a proper control of the properties of the ligands
employed, which are usually thiolate adsorbates. Monolayer
formation on gold substrates is driven by the chemical bonding
between the sulfur and gold atoms and by interactions between
neighboring adsorbate molecules. Such a driving force controls
the properties and stability of monolayers protecting small metal
clusters (3D architectures) as well as those of self-assembled
monolayers (SAMs) on extended (2D) metal surfaces. Al-
kanethiolates are often employed to make SAMs, and thus the
stability of SAMs, such as intermolecular CdO‚‚‚H-N hydro-
gen bonding between embedded amide groups, as found with
various 2D SAMs.5 Peptides are thus expected to be particularly
convenient molecules to use, also in view of their intrinsic
relevance for biological applications. A variety of strategies to
prepare peptide 2D SAMs has been reported, where oligopep-
tides are usually modified by introducing thiol groups at either
the N- or C-terminus.6 Generally, peptide monolayers are well-
packed, and their stability may be strengthened by CdO‚‚‚H-N
(5) (a) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990,
112, 558-569. (b) Lenk T. J.; Hallmark, V. M.; Hoffmann, C. L.; Rabolt,
J. F.; Castner, D. G.; Erdelen, C.; Ringsdorf, H. Langmuir 1994, 10, 4610-
4617. (c) Wagner, P.; Hegner, M.; Guntherodt, H.-J.; Semenza, G. Langmuir
1995, 11, 3867-3875. (d) Tam-Chang, S. W.; Biebuyck, H. A.; Whitesides,
G. M.; Jeon, N.; Nuzzo, R. G. Langmuir 1995, 11, 4371-4382. (e) Clegg,
R. S.; Hutchison, J. E. Langmuir 1996, 12, 5239-5243. (f) Clegg, R. S.,
Reed, S. M.; Hutchison, J. E. J. Am. Chem. Soc. 1998, 120, 2486-2487.
(g) Sabapathy, R. C.; Bhattacharyya, S.; Leavy, M. C.; Cleland, W. E., Jr.;
Hussey, C. L. Langmuir 1998, 14, 124-136. (h) Zhang, J.; Zhang, H. L.;
Chen, M.; Zhao, J.; Liu, Z. F.; Li, H. L. Ber. Bunsen-Ges. Phys. Chem.
1998, 102, 701-703. (i) Clegg, R. S.; Hutchison, J. E. J. Am. Chem. Soc.
1999, 121, 5319-5327. (j) Valiokas, R.; Svedhem, S.; Svensson, S. C. T.;
Liedberg, B. Langmuir 1999, 15, 3390-3394. (k) Clegg, R. S.; Reed, S.
M.; Smith, R. K.; Barron, B. L.; Rear, J. A.; Hutchison, J. E. Langmuir
1999, 15, 8876-8883. (l) Smith, R. K.; Reed, S. M.; Lewis, P. A.; Monnell,
J. D.; Clegg, R. S.; Kelly, K. F.; Bumm, L. A.; Hutchison, J. E.; Weiss, P.
S. J. Phys. Chem. B 2001, 105, 1119-1122. (m) Valiokas, R.; Svedhem,
S.; O¨ stblom, M.; Svensson, S. C. T.; Liedberg, B. J. Phys. Chem. B 2001,
105, 5459-5469. (n) Lewis, P. A.; Smith, R. K.; Kelly, K. F.; Bumm, L.
A.; Reed, S. M.; Clegg, R. S.; Gunderson, J. D.; Hutchison, J. E.; Weiss,
P. S. J. Phys. Chem. B 2001, 105, 10630-10636. (o) Valiokas, R.; O¨ stblom,
M.; Svedhem, S.; Svensson, S. C. T.; Liedberg, B. J. Phys. Chem. B 2002,
106, 10401-10409. (p) Petoral, R. M.; Uvdal, K. J. Phys. Chem. B 2003,
107, 13396-13402. (q) Inman, C. E.; Reed, S. M.; Hutchison, J. E.
Langmuir 2004, 20, 9144-9150.
‡ Department of Chemistry, University of Padova.
§ Department of Chemistry, ISTM-CNR.
(1) (a) Zhang, J. Z.; Wang, Z.; Liu, J.; Chen, S.; Liu, G. Self-Assembled
Nanostructures; Kluwer Academic/Plenum: New York, 2003. (b) Schmid,
G. Nanoparticles; Wiley-VCH: Weinheim, Germany, 2004. (c) El-Sayed,
M. A. Acc. Chem. Res. 2001, 34, 257-264.
(2) (a) Templeton, A. C.; Wuelfing, W. P.; Murray, R. W. Acc. Chem. Res.
2000, 33, 27-36. (b) Schmid, G.; Corain, B. Eur. J. Inorg. 2003, 3081-
3098. (c) Daniel, M.-C.; Astruc, D. Chem ReV. 2004, 104, 293-346.
(3) Brust, M.; Kiely, C. J. Colloids Surf., A 2002, 202, 175-186.
(4) (a) Jahn, W. J. Struct. Biol. 1999, 127, 106-112. (b) Shipway, A. N.; Katz,
E.; Willner, I. ChemPhysChem 2000, 1, 18-52. (c) Shenhar, R.; Rotello,
V. Acc. Chem. Res. 2003, 36, 549-561. (d) Nanobiotechnology: Concepts,
Applications and PerspectiVe; Niemeyer, C. M., Mirkin, C. A., Eds.; Wiley-
VCH: Weinheim, Germany, 2004. (e) Katz, E.; Willner, I. Angew. Chem.,
Int. Ed. 2004, 43, 6042-6108.
(6) Wen, X.; Linton, R. W.; Formaggio, F.; Toniolo, C.; Samulski, E. T. J.
Phys. Chem. A 2004, 108, 9673-9681.
9
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J. AM. CHEM. SOC. 2006, 128, 326-336
10.1021/ja0560581 CCC: $33.50 © 2006 American Chemical Society

Gold Nanoclusters Protected by Select Peptides
A R T I C L E S
interchain hydrogen bonds. Similar issues are expected to affect
the stability of SAMs formed on small gold clusters, although
for them the radial distribution of the ligands makes the
interchain distance a subtle function of the distance from the
core as well as of the core size and geometry.7
increase the possibility that exogenous species, such as solvent
or electrolytes, could penetrate the capping monolayer, which
for some applications might be quite unsought. In this paper,
we describe the results of a study aimed at preparing gold
clusters protected by peptides displaying a definite conforma-
tional preference and thus remarkable stiffness. We used a series
of structurally well-defined peptide systems based on the
R-aminoisobutyric acid (Aib) residue. The Aib homooligomers
are characterized by marked steric hindrance at the R-carbon,
resulting in restricted torsional freedom.19,20 Consequently, they
are significantly rigid even when they are short,21 which is a
general feature shared by oligopeptides based on CR-tetrasub-
stituted R-amino acids.19,20 It is worth stressing that this 3D-
structural propensity is not encountered with peptide systems
based on coded R-amino acids, which start to form helices only
for rather long oligomers.22 Instead, the tendency of Aib
homopeptides to form â-turns and 310-helices starts from the
tripeptide.20 In the 310-helix, each intramolecular CdO‚‚‚H-N
hydrogen bond involves residues i and i + 3 and a single helical
turn requires 3.24 amino acid residues,21b while the R-helix is
characterized by a pitch of 3.63 residues/turn. Formation of the
310-helix is accompanied by the onset of a strong oriented dipole
moment that develops along the peptide backbone.23 As opposed
to simple alkanethiols, for which the increase in the number of
methylene units increases the flexibility of the ligand, the key
motif of the selected peptides is that the increase of the number
of units is accompanied by a concomitant increase of the number
of intramolecular hydrogen bonds and thus of the peptide
stiffness. This feature and, particularly, the unique relative
rigidity of short Aib homopeptides are being successfully
exploited as spacers or templates in electrochemical and
spectroscopic investigations.24-26 Since 2D or 3D SAM forma-
tion already causes the conformational freedom of alkanethiols
to decrease dramatically, we expected that for the same reason
our peptides would have behaved as even more rigid ligands.
Several studies have been carried out on gold nanoparticles
protected by peptides, particularly in connection to possible
biomedical applications.4e,8 The core diameters employed are
generally larger than 10 nm, and thus the properties of the
particles resemble those of the bulk material; few investigations
concern MPCs in the size region marking the transition to the
molecular-like behavior (1-2 nm). Murray and co-workers
described the synthesis of tiopronin MPCs (down to 1.8 nm
size) and their modification through ligand place exchange to
prepare nanoparticles displaying redox and fluorophoric proper-
ties.9 Peptide backbones have been added onto preformed MPCs
by ligand place exchange10 or multistep synthesis.11 In addition
to place exchange, mixed monolayers based on tiopronin or
glutathione ligands were shown to directly form under controlled
conditions.12 It has also been reported that large gold nanopar-
ticles can be prepared by using the amino acid itself as the
reductant.13 Glutathione MPCs proved to be a particularly
efficient ligand to passivate gold nucleation to very limited
amounts of atoms, yielding molecule-like particles displaying
interesting optical properties.14 Metal nanoparticles protected
by single amino acids or linear peptides showed a subtle
dependence of size and stability on the metal/ligand combina-
tion.15 Interesting studies for applications in the area of nuclear
targeting are also worth mentioning, in which gold nanoclusters
are capped by suitable peptides for crossing cellular mem-
branes.16 Amino acids and peptides are also useful to create
complex assemblies of nanoparticles17 and to form stable
functionalized gold nanoparticles for molecular recognition.8,18
As opposed to the SAMs formed on extended surfaces, the
radial orientation of the thiolate ligands in small MPCs make
their outer terminals prone to lateral motion. These fluctuations
In this paper, we describe the synthesis, characterization, and
properties of peptide MPCs having average core diameters
ranging from 1.1 to 2.3 nm. We used the thiolated peptides 0-3,
where the number of amino acids is such to allow forming
structures characterized by zero, one, two, or three intramo-
lecular CdO‚‚‚H-N hydrogen bonds, respectively.
(7) (a) Hostetler, M. J.; Stokes, J. J.; Murray, R. W. Langmuir 1996, 12, 3604-
3612. (b) Boal, A.; Rotello, V. M. Langmuir 2000, 16, 9527-9532.
(8) Rosi, N. L.; Mirkin, C. A. Chem. ReV. 2005, 105, 1547-1562.
(9) (a) Templeton, A. C.; Chen, S.; Gross, S. M.; Murray, R. W. Langmuir
1999, 15, 66-76. (b) Templeton, A. C.; Cliffel, D. E.; Murray, R. W. J.
Am. Chem. Soc. 1999, 121, 7081-7089. (c) Huang, T.; Murray, R. W.
Langmuir 2002, 18, 7077-7081.
(10) (a) Pengo, P.; Broxterman, Q. B.; Kaptein, B.; Pasquato, L.; Scrimin, P.
Langmuir 2003, 19, 2521-2524. (b) Le´vy, R.; Thanh, N. T. K.; Doty, C.;
Hussain, I.; Nichols, R. J.; Schiffrin, D. J.; Brust, M.; Fernig, D. G. J. Am.
Chem. Soc. 2004, 126, 10076-10084.
(11) (a) Templeton, A. C.; Hostetler, M. J.; Warmoth, E. K.; Chen, S.; Hartshorn,
C. M.; Krishnamurthy, V. M.; Forbes, M. D. E.; Murray, R. W. J. Am.
Chem. Soc. 1998, 120, 4845-4849. (b) Fan, J.; Chen, S.; Gao, Y. Colloid
Surf., B 2003, 28, 199-207. (c) Sung, K.-M.; Mosley, D. W.; Peelle, B.
R.; Zhang, S.; Jacobson, J. M. J. Am. Chem. Soc. 2004, 126, 5064-5065.
(12) Zheng, M.; Huang, X. J. Am. Chem. Soc. 2004, 126, 12047-12054.
(13) (a) Selvakannan, P. R.; Mandal, S.; Phadtare, S.; Gole, A.; Pasricha, R.;
Sastry, M. Langmuir 2003, 19, 3545-3549. (b) Bhargava, S. K.; Booth, J.
M.; Agrawal, S.; Coloe, P.; Kar, G. Langmuir 2005, 21, 5949-5956.
(14) (a) Schaaff, T. G.; Knight, G.; Shafigullin, M. N.; Borkman, R. F.; Whetten,
R. L. J. Phys. Chem. B 1998, 102, 10643-10646. (b) Schaaff, T. G.;
Whetten, R. L. J. Phys. Chem. B 2000, 104, 2630-2641. (c) Link, S.;
El-Sayed, M. A.; Schaaff, T. G.; Whetten, R. L. Chem. Phys. Lett. 2002,
356, 240-246. (d) Link, S.; Beeby, A.; FitzGerald, S.; El-Sayed, M. A.;
Schaaff, T. G.; Whetten, R. L. J. Phys. Chem. B 2002, 106, 3410-3415.
(e) Negishi, Y.; Takasugi, Y.; Sato, S.; Yao, H.; Kimura, K.; Tsukuda, T.
J. Am. Chem. Soc. 2005, 126, 6518-6519. (f) Negishi, Y.; Nobusada, K.;
Tsukuda, T. J. Am. Chem. Soc. 2005, 127, 5261-5270.
(15) Slocik, J. M.; Wright, D. W. Biomacromolecules 2003, 4, 1135-1141.
(16) (a) Tkachenko, A. G.; Xie, H.; Coleman, D.; Glomm, W.; Ryan, J.;
Anderson, M. F.; Franzen, S.; Feldheim, D. L. J. Am. Chem. Soc. 2003,
125, 4700-4701. (b) Xie, H.; Tkachenko, A. G.; Glomm, W. R.; Ryan, J.;
Brennaman, M. K.; Papanikolas, J. M.; Franzen, S.; Feldheim, D. L. Anal.
Chem. 2003, 75, 5797-5805. (c) Tkachenko, A. G.; Xie, H.; Liu, Y.;
Coleman, D.; Ryan, J.; Glomm, W. R.; Shipton, M. K.; Franzen, S.;
Feldheim, D. L. Bioconjugate Chem. 2004, 15, 482-490.
(17) (a) Wong, M. S.; Cha, J. N.; Choi, K.-S.; Deming, T. J.; Stucky, G. D.
Nano Lett. 2002, 2, 583-587. (b) Chakrabarti, R.; Klibanov, A. M. J. Am.
Chem. Soc. 2003, 125, 12531-12540. (c) Stevens, M. M.; Flynn, N. T.;
Wang, C.; Tirrell, D. A.; Langer, R. AdV. Mater. 2004, 16, 915-918. (d)
Zhong, Z.; Subramanian, A. S.; Highfield, J.; Carpenter, K.; Gedanken, A.
Chem. Eur. J. 2005, 11, 1473-1478.
(18) Wang, Z.; Le´vy, R.; Fernig, D. G.; Brust, M. Bioconjugate Chem. 2005,
16, 497-500.
(19) Karle, I.; Balaram, P. Biochemistry 1990, 29, 6747-6756.
(20) Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C. Biopolymers (Pept.
Sci.) 2001, 60, 396-419.
(21) (a) Toniolo, C.; Bonora, G. M.; Barone, V.; Bavoso, A.; Benedetti, E.; Di
Blasio, B.; Grimaldi, P.; Lelj, F.; Pavone, V.; Pedone, C. Macromolecules
1985, 18, 895-902. (b) Toniolo, C.; Benedetti, E. Trends Biochem. Sci.
1991, 16, 350-353.
(22) Goodman, M.; Toniolo, C.; Pallai, P. In Forum Peptides; Castro, B.,
Martinez, J., Eds.; Dhor: Nancy, France, 1985; pp 146-174.
(23) Shin, Y.-G.; Newton, M. D.; Isied, S. S. J. Am. Chem. Soc. 2003, 125,
3722-3732.
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A R T I C L E S
Fabris et al.
ice, were added to the polymeric AuSR solution under vigorous stirring.
A black color, indicative of MPC formation, was immediately produced
in the solution mixture and a gas evolved. The reaction temperature
was maintained at 0 °C in the ice bath and the mixture vigorously stirred
for 2 h, after which the flask content was poured into 40 mL of water
and then extracted after addition of 20 mL of methylene chloride. The
organic extracts were dried over Na2SO4, filtered, and evaporated, pro-
viding a brown solid. The MPC was separated from most unreacted
peptide thiol and peptide disulfide by dissolving the MPC in 5 mL of
acetone, leaving a white precipitate behind, and filtering on a fine fritted
glass filter. Residual peptide impurities were removed by collecting
the MPC in the filtrate, evaporating the acetone, and repeating the ace-
tone wash (2 mL). Purification of each MPC was different, depending
on the specific peptide, and is described in the Supporting Information.
Infrared Absorption Spectroscopy. Both the solid-state (KBr disk
technique) and solution (CH2Cl2, using cells having 1 mm optical path
and CaF2 windows) IR absorption spectra were recorded with a Perkin-
Elmer model 1720X FT-IR spectrophotometer, nitrogen-flushed, equipped
with a sample-shuttle device, at 2 cm-1 nominal resolution, and
averaging 100 scans.
UV-Vis Absorption Spectroscopy. UV-vis spectra were taken
in methanol and CH2Cl2 solutions with a Varian Cary 5 UV-vis
spectrophotometer. The MPC concentrations were chosen to provide
an optical absorbance A near 400 nm of ∼1.0. The experiments were
carried out at room temperature, the absorption spectra were recorded
from 300 to 1500 nm, and the contribution of the background was
accounted for.
1H Nuclear Magnetic Resonance Spectroscopy. 1H NMR spectra
were obtained by using a Bruker model AC 200 spectrometer, a Bruker
model Avance 300 spectrometer, a Bruker model AM 400 spectrometer,
or a Bruker model Avance 600 instrument. Deuteriochloroform
(99.96%, d; Acros Organics), deuterated acetonitrile (99.95%, d3; Acros
Organics), deuterated methanol (99.8%, d4; Acros Organics), and
deuterated dimethyl sulfoxide (99%, d6; Aldrich) were used as the
solvents. Chemical shifts (δ) are reported as parts per million (ppm)
downfield from tetramethylsilane. Two-dimensional spectra were
obtained from ROESY experiments carried out in deuteriochloroform
and CD3CN.
The MPC synthesis follows a modified version of the two-
phase preparation described by Brust et al.27 and commonly used
for the production of aryl and alkanethiolate coated MPCs. The
1
clusters were characterized by H NMR spectrometry, IR and
UV-vis absorption spectroscopies, transmission electron mi-
croscopy (TEM), thermogravimetric analysis (TGA), and X-ray
photoelectron spectroscopy (XPS). Our results indicate that a
rigid peptide structure is maintained in the monolayer as well
as in the free ligand. By altering the ratio of the Au(III) salt
and capping thiol, the core diameter of the cluster could be
varied down to 1.1 nm, in agreement with the formation of Au38
clusters, which were recently prepared using phenylethanethi-
olate and hexanethiolate as the ligands.28 Spectral data indicated
that the peptides are involved in both intra- and interligand
CdO‚‚‚H-N hydrogen bonds, resulting in densely packed
monolayers.
Experimental Section
Chemicals. All chemicals were commercially available and used
as received. The syntheses of peptide precursors and ligands are
described in detail in the Supporting Information. All peptides were
stable and could be stored at low temperature for months. The synthesis
of additional peptides, having general formula CH3CO-(Aib)n-NHtBu,
was carried out as previously reported.25c
Synthesis of Peptide-Coated MPCs. A general procedure for the
preparation of the crude reaction product of the 3-protected gold
nanocluster using a 3:1 thiol-to-Au ratio at 0 °C is described. After
adding 0.102 g (0.26 mmol) of HAuCl4‚(H2O)3 in 10 mL of water to
a solution of 0.403 g (0.74 mmol) of tetra-n-octylammonium bromide
(Oct4NBr) in 25 mL of toluene in a 250 mL flask, the mixture was
stirred for 15 min until the water phase changed from yellow to colorless
and the toluene layer from colorless to a dark red. The aqueous layer
was removed, the organic phase was dehydrated with Na2SO4, and 0.375
g (0.75 mmol) of 3 was added in 5 mL of methanol with stirring. After
5 min, the solution became colorless (if the synthesis is carried out
using a 0.5:1 thiol-to-Au ratio, the color does not disappear completely),
signaling reaction of the gold salt with the thiol to form a soluble
polymeric AuSR compound. The reaction was carried out for 30 min,
and then the flask was cooled to 0 °C in an ice water bath. Then, 0.100
g (2.7 mmol) of NaBH4, dissolved in 5 mL of methanol and cooled on
Thermogravimetric Analysis. TGA was performed with a TA
Instruments SDT 2960 system on accurately weighed, carefully dried,
1-8 mg cluster samples, under N2 and in standard Al pans. In the
temperature ramp (25-600 °C at 15 °C/min.), the organic volatilization
and ensuing mass loss occurred for all samples at temperatures above
200 °C, leaving a gold metal residue in the pan.
(24) Toniolo, C.; Crisma, M.; Formaggio, F.; Peggion, C.; Broxterman, Q. B.;
Kaptein, B. Biopolymers (Pept. Sci.) 2004, 76, 162-176.
(25) (a) Antonello, S.; Formaggio, F.; Moretto, A.; Toniolo, C.; Maran, F. J.
Am. Chem. Soc. 2003, 125, 2874-2875. (b) Improta, R.; Antonello, S.;
Formaggio, F.; Maran, F.; Rega, N.; Barone, V. J. Phys. Chem. B 2005,
109, 1023-1033. (c) Polo, F.; Antonello, S.; Formaggio, F.; Toniolo, C.;
Maran, F. J. Am. Chem. Soc. 2005, 127, 492-493.
Transmission Electron Microscopy. TEM phase contrast images
were obtained with a JEOL JEM 2010 microscope operating at 200
keV. The MPC samples were prepared by spreading a droplet of 1
mg/mL MPC in methylene chloride, methanol, or ethanol on standard
carbon-coated (20-30 nm) Formvar films on copper grids (400 mesh).
To analyze a statistically significant number of particles (usually 100-
130), three regions were typically imaged for each sample in the 100-
250 K magnification range. The core sizes were quantified on the
digitized photographic images by using the Photoshop Elements
software; values from obvious twins or aggregates of particles were
discarded. The histograms were obtained by using Scion Image Beta
Release 2. The dispersity data will refer to the standard deviations (σ);
the average deviations are ∼20% smaller than the σ values.
X-ray Photoelectron Spectroscopy. XPS analyses were performed
with a Perkin-Elmer Φ 5600-ci spectrometer using non-monochroma-
tized (15 kV, 400 W) Mg KR radiation (1253.6 eV). The appropriate
gold cluster solution, in ethanol or dichloromethane, was drop-cast onto
a platinum substrate. The carefully dried sample was mounted on a
steel sample holder and introduced directly, by a fast-entry lock system,
into the XPS analytical chamber. The sample analysis area was 800
µm in diameter, and the working pressure was <5 × 10-8 Pa. The
spectrometer was calibrated assuming the binding energy (BE) of the
(26) (a) Hanson, P.; Millhauser, G.; Formaggio, F.; Crisma, M.; Toniolo, C. J.
Am. Chem. Soc. 1996, 118, 7618-7625. (b) Polese, A.; Mondini, S.; Bianco,
A.; Toniolo, C.; Scorrano, G.; Guldi, D. M.; Maggini, M. J. Am. Chem.
Soc. 1999, 121, 3446-3452. (c) Pispisa, B.; Stella, L.; Venanzi, M.;
Palleschi, A.; Viappiani, C.; Polese, A.; Formaggio, F.; Toniolo, C.
Macromolecules 2000, 33, 906-915. (d) Pispisa, B.; Stella, L.; Venanzi,
M.; Palleschi, A.; Polese, A.; Formaggio, F.; Toniolo, C. J. Pept. Res. 2000,
56, 298-306. (e) Pispisa, B.; Mazzuca, C.; Palleschi, A.; Stella, L.; Venanzi,
M.; Wakselman, M.; Mazaleyrat, J.-P.; Rainaldi, M.; Formaggio, F.;
Toniolo, C. Chem. Eur. J. 2003, 9, 4084-4093. (f) Sartori, E.; Toffoletti,
A.; Rastrelli, F.; Corvaja, C.; Bettio, A.; Formaggio, F.; Oancea, S.; Toniolo,
C. J. Phys. Chem. A 2003, 107, 6905-6912. (g) Sartori, E.; Toffoletti, A.;
Corvaja, C.; Moroder, L.; Formaggio, F.; Toniolo, C. Chem. Phys. Lett.
2004, 385, 362-367. (h) Venanzi, M.; Valeri, A.; Palleschi, A.; Stella, L.;
Moroder, L.; Formaggio, F.; Toniolo, C.; Pispisa, B. Biopolymers (Bio-
spectroscopy) 2004, 75, 128-139.
(27) Brust, M.; Walker, M.; Bethell, D.; Schiffrin, D. J.; Whyman, R. J. Chem.
Soc., Chem. Commun. 1994, 801-802.
(28) (a) Lee, D.; Donkers, R. L.; DeSimone, J. M.; Murray, R. W. J. Am. Chem.
Soc. 2003, 125, 1182-1183. (b) Lee, D.; Donkers, R. L.; Wang, G.; Harper,
A. S.; Murray, R. W. J. Am. Chem. Soc. 2004, 126, 6193-6199. (c)
Donkers, R. L.; Lee, D.; Murray, R. W. Langmuir 2004, 20, 1945-1952.
(d) Jimenez, V. L.; Georganopoulou, D. G.; White, R. J.; Harper, A. S.;
Mills, A. J.; Lee, D.; Murray, R. W. Langmuir 2004, 20, 6864-6870. (e)
Wang, G.; Huang, T.; Murray, R. W.; Menard, L.; Nuzzo, R. G. J. Am.
Chem. Soc. 2005, 127, 812-813.
9
328 J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006

Gold Nanoclusters Protected by Select Peptides
A R T I C L E S
Au4f7/2 line at 83.9 eV with respect to Fermi level. The standard
deviation for the BE values was 0.2 eV. A low-energy flood gun was
used to minimize charge when necessary. The residual BE shifts (on
the order of 0.4-0.6 eV) were corrected by assigning to the C1s peak
associated with the methyl groups a value of 285.0 eV. Survey scans
were run in the 0-1100 eV range (pass energy, 187.85 eV), while
detailed scans were recorded for the Au4f, C1s, O1s, N1s, and S2p
regions. The analyses involved Shirley-type background subtraction29
and, whenever necessary, spectral deconvolution, which was carried
out by nonlinear least-squares curve fitting, adopting a Gaussian-
Lorentzian sum function. The atomic composition of the drop-cast MPC
samples was calculated by peak integration, using the sensitivity factors
supplied by the spectrometer manufacturer and taking into account the
geometric configuration of the apparatus.
conformations. This conclusion is confirmed by the number and
position of the high-resolution enhanced bands exhibited by Aib
homooligomers with 5 NH groups.33
Bidimensional 1H NMR spectra were obtained from ROESY
experiments carried out in CDCl3 and CD3CN (Supporting
Information, Figure S1). Through-space connectivities of the
CâH(i-1)fNH(i) type (between the methyl protons of an Aib
and the amide proton of the following residue) and NH(i)fNH-
(i+1) type were used for the assignment of the proton
resonances. The rather intense NH(i)fNH(i+1) cross-peak
signals obtained for 1a suggest that even this short peptide
adopts a prevailing folded structure (â-turn).34 The same
outcome is observed with 3a. Noteworthy, the same conforma-
tion is kept unaltered in both CDCl3 and CD3CN, the latter
solvent being an appreciable H-bond acceptor. To test the
participation of the amide protons in intramolecular hydrogen
bonds, the 1H NMR spectra of 1a and 3a were obtained in the
3D structure supporting solvent CDCl3 and their evolution was
studied as a function of the addition of the good H-bond acceptor
Me2SO-d6.21a The N(1)H proton of both peptides was found to
be very sensitive to Me2SO-d6 addition, the peaks shifting to
lower fields. A similar but less pronounced behavior was
observed for the N(2)H proton, while the other amide protons
were essentially insensitive. A similar outcome was observed
with the corresponding CH3CO-(Aib)n-NHtBu peptides, which
rules out a possible involvement of the sulfur atom in the
secondary structure. This trend is in line with the normal
behavior of Aib homopeptides for which the sensitivity of the
N(1)H protons is notably higher than that of the N(2)H
protons.21a The absence of effects brought about on the other
NH protons points to the failure of Me2SO-d6 to disrupt the
intramolecular H-bonds involving them. Since in the 310-helix
the formation of stable intramolecular H-bonds starts from the
N(3)H proton, we infer that these peptides adopt such an ordered
secondary structure.
Results and Discussion
Synthesis, Characterization, and Properties of the Peptide
Ligands. The syntheses of peptide ligands 0-3 were carried
out as follows. The tert-butyl amides of the Z-protected peptides,
(Z = benzyloxycarbonyl) Z-(Aib)n-NHtBu, were obtained by
condensation of Z-Aib-OH30 with the appropriate H-(Aib)n-1
-
NHtBu segments. Carboxyl activation was achieved by use of
the EDC/HOAt method.31 The NR-deprotected peptides, obtained
by catalytic hydrogenation of the Z-(Aib)n-NHtBu oligomers,
were converted to their Ph3C-S-(CH2)2-CO-(Aib)n-NHtBu
analogues, compounds 0a-3a, by reaction with Ph3C-S-
(CH2)2-COOH. Finally, the triphenylmethyl (trityl) sulfides
were treated with trifluoroacetic acid and triisopropylsilane to
yield the corresponding thiols.
FT-IR absorption spectra of 0a-3a and 0-3 were obtained
in CH2Cl2, which is a solvent of low polarity having no
propensity to behave as a hydrogen-bond acceptor, and the
analysis focused on the N-H and CdO stretching modes. The
N-H stretching region (amide A, 3450-3250 cm-1) is com-
posed by a high-energy region corresponding to the NH groups
that are not involved in hydrogen bonds and a low-energy region
pertaining to NH groups involved in hydrogen bonds.21a As one
passes from 0 to 3 (or, similarly, from 0a to 3a), the low-energy
N-H stretching band appears starting from 1 and 1a, which
are the shortest peptides for which formation of an incipient
310-helix is possible.32 As the peptide chain length increases,
the frequency of the N-H stretching band of intramolecularly
H-bonded peptides decreases, which is a marker of the increase
of peptide stiffness.21a In a previous IR absorption study, we
could show that Aib homooligomers with 3-5 NH groups (such
as peptides 1-3 and 1a-3a in this work) are characterized by
41, 69, and 80% of intramolecular H-bonds of the 310-helix type,
respectively.21a These data highlight the great efficiency of the
Aib unit in stabilizing folded and helical species even in very
short peptides, but also explain the broadness of the IR bands
(the spectra will be illustrated later, in Figure 7, in comparison
with those obtained for the corresponding peptide MPCs), which
result from a combination of contributions from multiple
Preparation of Peptide MPCs. Peptides 0-3 are soluble in
many organic solvents, and thus the MPCs could be prepared
by using a procedure similar to that reported by Brust et al. at
0 °C.27 We focused on two thiol concentrations, at 3:1 and 0.5:1
ratios to the gold salt. Details are provided in the Experimental
Section and in the Supporting Information. The MPC syntheses
generally led to materials with a narrow range of core diameters,
in line with the best results obtained in MPC syntheses based
on alkanethiolate ligands.9a,35-38 This tendency and the fact that
separation from unreacted peptide is easily performed by taking
advantage of solubility differences are useful features that make
the MPC purification procedure rather straightforward.
The TEM analysis was employed to determine the average
MPC size and to test the sensitivity of the preparation procedure
on experimental parameters. Similarly to previous reports of
alkanethiolate MPCs,35 we found that decreasing the temperature
(33) Kennedy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Biochemistry 1991,
30, 6541-6548.
(29) Shirley, D. A. Phys. ReV. B 1972, 55, 4709-4714.
(34) Wu¨thrich, K. In NMR of Proteins and Nucleic Acids; Wiley: New York,
1986; pp 192-196.
(30) (a) McGahren W. J.; Goodman, M. Tetrahedron 1967, 23, 2017-2030.
(b) Valle, G.; Formaggio, F.; Crisma, M.; Bonora, G. M.; Toniolo, C.;
Bavoso, A.; Benedetti, E.; Di Blasio, B.; Pavone, V.; Pedone, C. J. Chem.
Soc., Perkin Trans. 2 1986, 1371-1376. (c) Leplawy, M. T.; Jones, D. S.;
Kenner, G. W.; Sheppard, A. C. Tetrahedron 1960, 11, 29-51.
(31) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398. EDC = 1-(3-
dimethyl-aminopropyl)-3-ethylcarbodiimide; HOAt = 1-hydroxy-7-aza-
1,2,3-benzotriazole.
(35) Hostetler, M. J.; Wingate, J. E.; Zhong, C.-J.; Harris, J. E.; Vachet, R. W.;
Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.;
Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Langmuir 1998,
14, 17-30.
(36) Shimmin, R. G.; Schoch, A. B.; Braun, P. V. Langmuir 2004, 20, 5613-
5620.
(37) (a) Chen, S.; Murray, R. W. Langmuir 1999, 15, 682-689. (b) Shon, Y.-
S.; Gross, S. M.; Dawson, B.; Porter, M.; Murray, R. W. Langmuir 2000,
16, 6555-6561.
(32) We also found that the investigated peptides do not display any appreciable
tendency to self-aggregate in solution, e.g., by head-to-tail intermolecular
hydrogen bonding, even at concentrations as large as 10 mM.
(38) Zheng, M.; Li, Z.; Huang, X. Langmuir 2004, 20, 4226-4235.
9
J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006 329

A R T I C L E S
Fabris et al.
although some steric control of the growing-passivation com-
petition during the reduction step can be inferred from literature
data pertaining to poly(ethylene glycols)(PEG)36 and alkanethi-
olates,39 there is no perceptible similar trend in the 1-3 MPC
series. The case of 2-MPC obtained at a S:Au ratio of 3:1 is
particularly interesting as the core diameter is only ∼1.1 nm,
which is a value corresponding to nominal Au38 particles.40 Such
clusters were previously obtained by using alkanethiol or phenyl-
ethanethiol ligands.28,41 The outcome observed with peptide 2
would thus provide an alternative way to obtain Au38 clusters.
Because of the intrinsic relevance of this aspect, this and the
other small peptide MPCs will be the specific subject of a
forthcoming study.42
1H NMR Spectrometry. The peptide MPCs were studied
by 1H NMR spectrometry. The data confirmed that the chemical
integrity of the peptides is preserved upon MPC formation and
that the signals are slightly sensitive to the distance of the
protons from the gold cluster and the size of the latter, as already
described for alkanethiolate MPCs.35,43,44
ROESY experiments were carried out for 2-MPC in CDCl3.
As described for the free peptides, assignment of the proton
resonances was based on the analysis of the CâH(i-1)fNH(i)
type and NH(i)fNH(i+1) type (Figure S6) through-space
connectivities. The analysis indicates that also when assembled
in the monolayer, the peptides adopt a prevailing folded structure
in keeping with the pattern expected for the 310-helix. Note-
worthy, we found that the signal of the amide N(1)H proton,
8.58 ppm, is downfield by 3 ppm with respect to the signal of
the free peptide 2a, 5.60 ppm (5 mM concentration). A smaller
but still significant downfield shift was also observed for the
N(2)H proton, the values being 7.14 and 6.35 ppm for 2-MPC
and 2a, respectively. To further check whether the peptide
behaves differently in the monolayer or in solution, we
stepwisely added Me2SO-d6 to a 3-MPC solution in CDCl3.
Compared to the spectrum of 3a in CDCl3, one of the NH
protons shifts to a much higher value, 8.79 ppm (Figure 2). In
analogy with the outcome of the ROESY experiments performed
on 2-MPC, this peak is attributed to the amide N(1)H and thus
the shift also is on the order of 3 ppm (3.24 or 3.04 ppm,
compared to the signals of 1 or 5 mM 3a, respectively). A
comparable shift (2.5 ppm) was reported for MPCs formed by
amide-containing thiolate ligands in which the NH group is at
similar distances from the gold core.7b Noteworthy, none of the
peptide-MPC NH-proton chemical shifts is appreciably affected
by Me2SO-d6 addition. Only the chemical shift of the N(1)H
proton slightly decreases as the concentration of Me2SO-d6
increases above ∼7%; this shift, however, is opposite with
respect to the trend already verified for the free peptide 3a. First,
we can conclude that Me2SO-d6 again fails to disrupt the
Figure 1. TEM images of 2-MPC obtained from preparations at (a) 0.5:1
S:Au ratio and (b) 3:1 S:Au ratio. Insets show histograms of the
corresponding distribution of core sizes.
Table 1. MPC Core Diameters Determined from TEM Images or
Extrapolated from Analysis of the Derivative of the UV-Vis
Absorption Spectra
peptide
S:Aua
dTEMb (nm)
d
UV(2.10eV)c (nm)
d
UV(2.65eV)c (nm)
0
1
2
3
0
1
2
3
0.5:1
2.3(0.6)
1.8(0.4)
2.0(0.3)
1.9(0.4)
1.8(0.2)
1.3(0.3)
1.1(0.2)
1.3(0.3)
2.2
1.8
2.2
2.1
1.7
1.2
1.2
1.2
2.3
1.7
2.1
1.9
1.8
1.3
1.1
1.3
3:1
a T ) 0 °C. b The number in parentheses is the standard deviation
(dispersity). c From a correlation between the derivative of absorbance and
the TEM diameters (see text).
reduces the average core diameter (d). For example, the 0.5:1
preparations of 3-MPC at room temperature and 0 °C yield
average d values of 2.1 and 1.9 nm, respectively. In the search
for suitable conditions to prepare small peptide MPCs, all
syntheses described below were thus carried out at 0 °C. Figure
1 pertains to 2-MPC and illustrates the effect of increasing the
S:Au ratio from 0.5:1 to 3:1, the average core diameter
decreasing from 2.0 to 1.1 nm. The same two S:Au feed ratios
were used in the syntheses of the other MPCs, and a summary
of the core diameters is provided in Table 1. A similar
relationship between the S:Au feed ratio and core diameter was
previously pointed out by Hostetler et al. for alkane-coated
MPCs.35 The peptide MPCs appear to display smaller core
dispersity and core size than are usually observed for alkanethio-
late MPCs prepared under otherwise identical conditions.
Overall, the TEM data indicate that for both sets of
experimental conditions d decreases for peptides capable of
forming a helical secondary structure, i.e., with 1-3. However,
(39) Hicks, J. F.; Templeton, A. C.; Chen, S.; Sheran, K. M.; Jasti, R.; Murray,
R. W.; Debord, J.; Schaaff, T. G.; Whetten, R. L. Anal. Chem. 1999, 71,
3703-3711.
(40) Ha¨kkinen, H.; Barnett, R. N.; Landman, U. Phys. ReV. Lett. 1999, 82, 3264-
3267.
(41) (a) Alvarez, M. M.; Khoury, J. T.; Schaaff, T. G.; Shafigullin, M. N.;
Vezmar, I.; Whetten, R. L. Chem. Phys. Lett. 1997, 266, 91-98. (b) Schaaff,
T. G.; Shafigullin, M. N.; Khoury, J. T.; Vezmar, I.; Whetten, R. L.; Cullen,
W. G.; First, P. N.; Gutie´rrez-Wing, C.; Ascensio, J.; Jose-Yacama´n, M. J.
J. Phys. Chem. B 1997, 101, 7885-7891. (c) Chen, S.; Ingram, R. S.;
Hostetler, M. J.; Pietron, J. J.; Murray, R. W.; Schaaff, T. G.; Khoury, J.
T.; Alvarez, M. M.; Whetten, R. L. Science 1998, 280, 2098-2101.
(42) Fabris, L.; Maran, F. Work in progress.
(43) Badia, A.; Lennox, R. B.; Reven, L. Acc. Chem. Res. 2000, 33, 475-481.
(44) Badia, A.; Gao, W.; Singh, S.; Demers, L.; Guccia, L.; Reven, L. Langmuir
1996, 12, 1262-1269.
9
330 J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006

Gold Nanoclusters Protected by Select Peptides
A R T I C L E S
Figure 3. UV-vis absorption spectra of the peptide MPCs. The spectra
were normalized to unity absorbance at 4 eV and displaced vertically for
better viewing. The dashed lines correspond (top to bottom) to the 0.5:1
preparations and peptides 0, 2, 3, and 1; the solid lines (top to bottom) to
the 3:1 preparations and peptides 0, 3, 1, and 2.
UV-Vis Spectroscopy. Optical absorption spectra provide
information on the electronic structure of MPCs.41b,45,46 Whetten
and co-workers45 described the evolution of the absorption
spectra as a function of the core size of alkanethiolate MPCs.
For sufficiently small particles (d < 1.7 nm) the absorption
pattern is sensitive to the nature of monolayer, while it is largely
independent of it for larger particles. As shown in Figure 3,
only for the largest peptide MPCs there is a shoulder at 520
nm (2.38 eV), where the surface plasmon absorption of gold is
expected to emerge as a true band exhibiting a maximum only
for particles having diameters larger than ∼2.4 nm.35,45 Inde-
pendently of the ligand size, the shoulder fades away and
eventually disappears for d < 1.6 nm.
The plots of the derivative of the absorbance (A) with respect
to the photon energy near the plasmon band (Figure S5) allow
one to better appreciate the dependence of the spectrum on the
MPC size. In particular, we observed that at photon energies
of 2.10 and 2.65 eV the spectra are particularly sensitive to the
particle size. The values of the derivatives of the optical
absorption with respect to the photon energy are plotted in
Figure 4 as a function of the core diameters obtained from the
TEM analysis. In the plot, we included one additional 1-MPC
obtained by carrying out the synthesis at an S:Au ratio slightly
smaller than 0.5:1. The two solid lines mark quite satisfactorily
the trends (r2 ) 0.968 and 0.926 for 2.65 and 2.10 eV,
respectively), particularly if one takes into account the uncer-
tainty associated with the TEM determinations. The d values
calculated by using the two linear regressions are gathered in
Table 1. Figure 4 would thus indicate that also for (related)
peptide MPCs the optical size estimate may provide a useful
Figure 2. 1H NMR titration in CDCl3: Variation of the chemical shifts of
the five amide protons of 1 mM 3a (upper graph) and 3-MPC (bottom graph)
as a function of the addition of Me2SO-d6.
intrachain H-bonding network and thus the 310-helix secondary
structure. The new aspect is the absence of the expected shift
for the N(1)H proton: this may indicate that Me2SO-d6 is
incapable of penetrating the inner part of the peptide monolayer
or, if it does, it is incapable of interacting significantly with the
otherwise free NH groups. This finding implies that the latter
NH protons are involved in H-bonds with CdO groups of
neighboring peptide chains, as similarly inferred for MPCs based
on amide-containing thiolate ligands.7b The chemical shift of
the amide N(2)H revealed by the ROESY experiments would
suggest that this group also is involved in interligand interac-
tions.
We should also mention that in CD3OD solution the 1H NMR
spectrum of 3-MPC is very similar, within a few tenths of ppm,
to the pattern described for the CDCl3/ Me2SO-d6 mixture; in
particular, the N(1)H proton peak is at 8.52 ppm. This result
also indicates that the H-bonding network involving the peptide
NH groups in the monolayer is essentially unaffected by
otherwise strongly interacting solvents. Finally, we observed
that the chemical shift of the peak attributed to the N(1)H proton
significantly increases with the peptide length: for the structured
peptides, we measured (CDCl3) 8.02 (1), 8.58 (2), and 8.79 ppm
(3). Since a downfield shift is associated with an increase of
hydrogen bonding, we may conclude that an increase of the
peptide (intrachain) stiffness also optimizes the interligand
network. As we will describe later, the presence of such complex
H-bonding peptide network is fully supported by our IR
absorption results.
(45) Alvarez, M. M.; Khoury, J. T.; Schaaff, T. G.; Shafigullin, M. N.; Vezmar,
I.; Whetten, R. L. J. Phys. Chem. B 1997, 101, 3706-3712.
(46) (a) Mie, G. Ann. Phys. 1908, 25, 377-445. (b) Kreibig, U.; Genzel, L.
Surf. Sci. 1985, 156, 678-700. (c) Quinten, M.; Kreibig, U. Surf. Sci. 1986,
172, 557-577. (d) Barnett, R. N.; Cleveland, C. L.; Ha¨kkinen, H.; Luedtke,
W. D.; Yannouleas, C.; Landman, U. Eur. Phys. J. D 1999, 9, 95-104.
(e) Kelly, K. L.; Coronado, E.; Zhao, L. L.; Schatz, G. C. J. Phys. Chem.
B 2003, 107, 668-677.
9
J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006 331

A R T I C L E S
Fabris et al.
preparation procedure. This behavior is unusual because nor-
mally Tdec increases as the ligand molecular mass51 increases.9a,35,50
This is illustrated in Figure 5 for PEGs,38 alkanethiols,50 and
arenethiols,37a also showing that ligands belonging to different
families afford similar slopes. With all due caution associated
with the complexity of TGA decomposition processes,49 the
pattern displayed by the peptide MPCs might be related to the
peptide molecular dipole moment pointing its positive end
toward the metal core and thus diminishing the charge density
at sulfur (normally,35,52 ca. -0.2). The observed TGA pattern
would thus reflect a compensation between the increasing
strength of the molecular dipole with the peptide length and
the “normal” effect caused by increasing the molecular mass.
The importance of the peptide dipole moment in affecting the
MPC properties is supported by our recent data on its effect on
the redox properties of small MPCs.42
Figure 4. Plots of the derivatives of the UV-vis spectra calculated at
photon energies of 2.10 (blue series) and 2.65 eV (red series) as a function
of the cluster core diameter determined by TEM analysis. The circles pertain
to the 3:1 series and the squares to the 0.5:1 series. The two solid lines are
the corresponding linear fit to the data.
Table 2 shows the weight losses measured by TGA for the
investigated MPCs. By combining these data with the TEM
diameters, the number of peptide ligands on each cluster can
be calculated along with the ensuing surface coverage (θ ) ratio
of ligands to surface Au atoms) and peptide footprint. Concern-
ing the number of Au atoms (NAu), we may model the cluster
as a sphere and calculate NAu from the core diameters and the
density of bulk fcc-Au (59 atoms/nm3). Thus, by using the
sphere model50 and the TGA results, θ can be estimated. A
different strategy is based on Au clusters whose sizes reflect a
discrete sequence of fcc structures of truncated octahedral
shape.53 Each d value is associated with one of the “magic
numbers” of Au atoms,46d,53 allowing calculation of the Au mass
pertaining to each cluster. By using the TGA weight loss, the
number of ligands (NL) is then estimated. The values of θ and
of the peptide footprint are eventually obtained from the number
of gold surface atoms.35
Analysis of our data shows that the polyhedron (Table 3)
and sphere models (Supporting Information) yield comparable
θ and NL values; in the following, we make reference to the
polyhedron model. For alkanethiolate ligands a θ value of ∼0.6
was calculated for ∼2 nm size clusters;35,50 this value almost
doubles the θ value of corresponding SAMs formed on extended
gold surfaces, namely, 0.33.54 In fact, the curvature of nano-
clusters increases the monolayer packing with respect to flat
gold surfaces, particularly for small particles and thanks to the
presence of edges and vertexes.55 Consequently, the alkanethi-
olate footprint has been reported to decrease from 0.22 to 0.15
nm2 on going from 2D SAMs to MPCs of ca. 2.3 nm.56 Because
of the steric hindrance of the helical structure, a larger footprint
is associated with the peptide ligands. In particular, we find
that the 0.5:1 series, which also is characterized by MPCs of
∼2 nm, yields an average θ value of 0.43. For the smallest
Table 2. TGA Data: Decomposition Temperature (Corresponding
to a 5% Weight Loss) and Organic Weight Loss (wl%)
peptide
Tdec (0.5:1)
Tdec (3:1)
wl% (0.5:1)
wl% (3:1)
0
1
2
3
239.1
251.0
247.2
240.6
236.8
250.7
251.6
250.6
22.7
31.1
39.0
37.1
35.3
49.6
46.7
55.7
(and convenient) investigation tool, further stressing the im-
portance of the optical absorption spectra to characterize small
MPCs.47 We should finally notice that for the smallest particle
obtained some fine structure starts to be perceptible in the optical
absorbance curve (Figure 3), but much less clearly as that
displayed by Au38 clusters capped by alkanethiolate ligands.28
This observation also would support the notion that the fine
structure of the optical absorption spectra displayed by
molecular-like MPCs may be affected by the capping ligand.48
Thermogravimetric Analysis and Monolayer Coverage.
TGA49 allows one to estimate the composition of the MPC.
Previous TGA of gold nanoparticles protected by various types
of thiolate ligand showed that the Au-S bond is broken at
relatively high temperature (150-300 °C)37,38,50,51 and the
decomposition yields a metallic gold residue and volatile organic
compounds, namely, the corresponding disulfides.35 We verified
the same outcome by IR absorption spectroscopy analysis of
the organic vapors of 3-MPC. The TGA was carried out on
both series of peptide MPCs, and the results are summarized in
Table 2. The decomposition temperature (Tdec), taken as the
temperature corresponding to 5% weight loss, does not exhibit
any significant dependency on the peptide length or the
(52) Bourg, M. C.; Badia, A.; Lennox, R. B. J. Phys. Chem. B 2000, 104, 6562-
6567.
(53) Whetten, R. L.; Khoury, J. T.; Alvarez, M. M.; Murthy, S.; Vezmar, L.;
Wang, Z. L.; Stephens, P. W.; Cleveland, C. L.; Luedtke, W. D.; Landman,
U. AdV. Mater. 1996, 8, 428-432.
(47) See also: Scaffardi, L. B.; Pellegri, N.; de Sanctis, O.; Tocho, J. O.
Nanotechnology 2005, 16, 158-163.
(48) A similar quite featureless optical absorption behavior was observed by
Chen and Kimura for gold MPCs having diameters in the range of 1.0-
1.1 nm: Chen, S.; Kimura, K. Langmuir 1999, 15, 1075-1082.
(49) Wendlandt, W. W. M. Thermal Analysis, 3rd ed.; Wiley: New York, 1986.
(50) Terrill, R. H.; Postlethwaite, T. A.; Chen, C.-H.; Poon, C.-D.; Terzis, A.;
Chen, A.; Hutchison, J. E.; Clark, M. R.; Wignall, G.; Londono, J. D.;
Superfine, R.; Falvo, M.; Johnson, C. S., Jr.; Samulski, E. T.; Murray, R.
W. J. Am. Chem. Soc. 1995, 117, 12537-12548.
(54) (a) Widrig, C. A.; Alves, C. A.; Porter, M. D. J. Am. Chem. Soc. 1991,
113, 2805-2810. (b) Kim, Y.-T.; McCarley, R. L.; Bard, A. J. J. Phys.
Chem. 1992, 96, 7416-7421.
(55) (a) Luedtke, W. D.; Landman, U. J. Phys. Chem. 1996, 100, 13323-13329.
(b) Badia, A.; Cuccia, L.; Demers, L.; Morin, F.; Lennox, R. B. J. Am.
Chem. Soc. 1997, 119, 2682-2692.
(56) (a) Camillone, N., III; Chidsey, C. E. D.; Lui, G.-Y.; Scoles, G. J. Chem.
Phys. 1993, 98, 4234-4245. (b) Sellers, H.; Ulman, A.; Schnidman, Y.;
Eilers, J. E. J. Am. Chem. Soc. 1993, 115, 9389-9401. (c) Badia, A.; Singh,
S.; Demers, L.; Cuccia, L.; Brown, G. R.; Lennox, R. B. Chem. Eur. J.
1996, 2, 359-363.
(51) A similar pattern can be obtained from other literature MPC data: Foos,
E. E.; Snow, A. W.; Twigg, M. E.; Ancona, M. G. Chem. Mater. 2002,
14, 2401-2408.
9
332 J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006

Gold Nanoclusters Protected by Select Peptides
A R T I C L E S
Figure 5. Plots of the MPC decomposition temperature versus the ligand
molecular weight for the 0.5:1 (1) and 3:1 (4) peptide MPCs, PEGs38 ([),
alkanethiols50 (b), and arenethiols37a (9).
Figure 6. XPS spectra in the Au4f, C1s, S2p, and N1s regions for 3-MPC
(0.5:1 preparation). The C1s signal has been deconvoluted into the three
components of the carbon types of the peptide ligands.
Table 3. Surface Coverage, Number of Peptide Ligands, and
Peptide Footprints (MPC Surface Area over the Number of
Ligands) from TEM and TGA Data and Using the Polyhedron
Model
Table 4. Binding Energy (eV) and Number of Peptide Ligands per
Cluster Au Atoms
footprint (nm2)
MPC
Au4f7
O1s
N1s
S2p
S:Au
S:Aua
/
2
peptide
S:Au
dTEM (nm)
NAu
θ
NL
3, 0.5:1
2, 0.5:1
0, 0.5:1
2, 3:1
84.0
84.0
84.0
84.2
531.7
532.4
532.6
532.0
400.0
399.9
400.0
399.8
162.6
162.8
162.6
163.0
0.32
0.33
0.28
0.44
0.23
0.30
0.24
0.42
0
1
2
3
0
1
2
3
0.5:1
2.3
1.8
2.0
1.9
1.8
1.3
1.1
1.3
309
201
225
225
201
55
0.45
0.42
0.49
0.37
0.69
0.77
0.47
0.65
73
54
68
52
88
32
16
27
0.27
0.28
0.22
0.29
0.17
0.20
0.29
0.24
3:1
a From TGA data, using the TEM diameters and the polyhedron model.
38
55
X-ray Photoelectron Spectroscopy. We carried out the XPS
analysis on four MPC samples: three of them were taken from
the 0.5:1 series, chosen to test the ligand effect on particles
(the MPCs of 0, 2, and 3) having comparable sizes, and the
last sample served to test the effect of decreasing the particle
size (from 2.0 to 1.1 nm), while keeping the peptide ligand, 2,
constant. Figure 6 illustrates spectral regions pertaining to the
XPS analysis of 3-MPC. For all samples, we obtained signals
from gold, carbon, oxygen, nitrogen, and sulfur; no other
elements were detected. The Au4f level is characterized by a
sharp doublet having a peak-to-peak separation of 3.6 eV and
a full width at half-maximum (fwhm) of 1.6 eV.
Within the 0.5:1 MPC series, the binding energy (BE) of the
Au4f7/2 peak (Table 4) is constant to 84.0 eV, which is the
typical value expected when thiolate monolayers self-assemble
onto gold nanoclusters or extended gold surfaces.35,52,57 Only
for the small 2-MPC a shift to a higher BE is perceptible, as
expected for molecular-like MPCs.35,58 The C1s band is broad
due to the presence of three components: methyl and methylene
carbon, C-N type carbon, and carbonyl carbon. Deconvolution
of this peak (Figure 6) allows determination of the corresponding
BEs. For 3-MPC we obtained, e.g., 285.0 eV (fwhm, 1.5 eV),
286.5 eV (fwhm, 1.4 eV), and 288.1 eV (fwhm, 1.5 eV),
respectively. Such values are in very good agreement with the
XPS analysis of methylene-chain carbons,59 amide-containing
thiolates,5k,60 and 2D SAMs composed by similar Aib-based
particles, obtained in the 3:1 preparations, a larger average θ is
calculated; namely, θ ∼ 0.65. A similar relative variation is
observed for alkanethiolate and phenyethanethiolate MPCs for
which θ increases to 0.75 when the core diameter is reduced to
1.1 nm.28
If the peptide helix is approximated, using molecular models,
as a cylinder, a footprint of 0.64 nm2 can be calculated. This
estimate pertains to the theoretical footprint on a flat surface.
On the other hand, from the NL and core-size values of Table
3, we calculated the peptide-helix footprints to be in the range
of 0.17-0.29 nm2. The smaller experimental footprint values
clearly indicate that these peptides are extremely well-packed
in the monolayer. The reasons for this behavior can be related
to the radial distribution of the ligands on the small gold clusters
and the fact that our peptides are rather thin near the gold surface
because of the -S-CH2-CH2- groups. That the core size matters
is also supported by a comparison with very recent literature
data pertaining to much larger gold nanoparticles (d ) 12.3
nm) capped by peptides of similar sizes and for which an
experimental footprint of 0.55 nm2 could be estimated.10b
Finally, the results pertaining to both the 3:1 and 0.5:1 series
were compared with the results reported for alkanethiolate
MPCs. We calculated that the peptide surface coverage is, on
the average, smaller by 15-25% relative to the thinner
alkanethiolate ligand monolayers (polyhedron model). For
example, if we focus on the largest and the smallest clusters
prepared, the experimental MPC formulas (average of the two
models) are Au38Pep18 and Au309Pep80 as opposed to (filled
(57) (a) van der Putten, D.; Zanoni, R.; Coluzza, C.; Schmid, G. J. Chem. Soc.,
Dalton Trans. 1996, 1721-1725. (b) Heister, K.; Zharnikov, M.; Grunze,
M.; Johansson, L. S. O. J. Phys. Chem. B 2001, 105, 4058-4061. (c)
Joseph, Y.; Besnard, I.; Rosenberger, M.; Guse, B.; Nothofer, H.-G.;
Wessels, J. M.; Wild, U.; Knop-Gericke, A.; Su, D.; Schlo¨gl, R.; Yasuda,
A.; Vossmeyer, T. J. Phys. Chem. B 2003, 107, 7406-7413.
28
alkanethiolate shell) Au38L24 and Au309L98,37a respectively.
(58) Zhang, P.; Sham, T. K. Phys. ReV. Lett. 2003, 90, 245502-1-245502-4.
9
J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006 333

A R T I C L E S
Fabris et al.
thiolated peptides.6 The BEs pertaining to the O1s peaks agree
well with previous observations for peptide or amide CdO
oxygen signals, which are in the range 531-533 eV.5b,d,e,k,p,61
The N1s peak also has the BE value expected for amide groups,
within experimental uncertainty. The intensity of the S2p region
is very low because of the tiny amount of sulfur in the samples.
Nonetheless, we could observe that the S2p BE values are
somewhat larger than the value of ∼162 eV typical of thiols
strongly interacting with gold in 2D and 3D SAMs,5k,p,35,52,57c,60c,62
but still significantly smaller than those found for free thiols
and elemental sulfur (163.5-164.2 eV).52,59a,62b,63 In addition,
the BE appears to increase as the MPC size decreases. By using
the approach described by Lennox and co-workers,52 based on
a correlation between the S2p3/2 binding energy and the charge
density on the S atom,64 we estimate a lower and a maximum
charge of -0.17 and -0.09, which should be compared with
the charge generally estimated for alkanethiolates, -0.2.52 The
difference would be in keeping with the effect brought about
by the peptide dipole moment.
The final issue stemming from the XPS data concerns the
elemental analysis. Reference was made to nitrogen, which is
almost unaffected by adventitious environmental contamination.
For 3-MPC (the longer the peptide, the more precise is the
calculation), we found the following theoretical and experimental
ratios (C,N,O,S): 4.6, 1, 1, 0.2 and 5.1, 1, 0.95, 0.15. In addition,
whereas the relative amounts of the different carbon species
determined by deconvolution of the XPS signal are 60% (methyl
and methylene carbon), 21% (C-N type-carbon), and 19%
(carbonyl carbon), the corresponding theoretical values are 57,
22, and 22%. The good agreement thus ensures that the integrity
of the peptide ligand is preserved in the MPC. Similar results
are found for the other MPCs. The second analysis is about NL
per cluster gold atoms, which is easily calculated from the S:Au
ratios (sixth column of Table 4). These ratios can be compared
with the corresponding data (last column) stemming from
TGA: overall, the agreement between the two sets of data is
satisfactory, the average uncertainty being ∼15%. The very good
agreement between the S:Au data of the last entry is particularly
worth noting, and this further supports the notion that the 2-MPC
obtained in the 3:1 preparation is particularly small.
Figure 7. FT-IR absorption spectra for the N-H stretch region of peptides
0a-3a (graph a), 0.5:1 MPCs (graph b), and 3:1 MPCs (graph c). The
spectra correspond (bottom to top) to 0 (black), 1 (red), 2 (green), and 3
(blue). The spectra were acquired in CH2Cl2 solutions, and the absorbance
was corrected for the peptide concentration.
IR Absorption Spectroscopy. Vibrational spectroscopy has
been used occasionally to study MPCs but proved to be a
powerful tool to characterize the structure and orientation of
3D SAMs.7,35,65 We already discussed the IR absorption
behavior of the free ligands and particularly how the N-H
stretch region (amide A) allows one to distinguish the amide
protons that are involved in CdO‚‚‚H-N hydrogen bonds from
those that are not. The spectra obtained in the N-H stretch
region with the two series of MPCs are shown in Figure 7
(graphs b and c) together with those recorded for the trityl
precursors 0a-3a (graph a) in the same solvent (CH2Cl2). The
latter series clearly shows that the onset of intramolecular
CdO‚‚‚H-N hydrogen bonding is significantly evident starting
from 1a. It also shows that while the frequency of the band
corresponding to the two free N-H groups does not change
(3421 ( 1 cm-1), the frequency of the N-H band of intramo-
lecularly H-bonded peptides decreases as the peptide length
(59) (a) Bain, C. D.; Blebuyck, H. A.; Whitesides, G. M. Langmuir 1989, 5,
723-727. (b) Laibinis, P. E.; Whitesides, G. M.; Allara, D. L.; Tao, Y.-
T.; Parikh, A. N.; Nuzzo, R. G. J. Am. Chem. Soc. 1991, 113, 7152-7167.
(60) (a) Beamson, G.; Briggs, D. High-Resolution XPS of Organic Polymers:
Wiley: Chichester, U.K., 1992. (b) Uvdal, K.; Bodo¨, P.; Liedberg, B. J.
Colloid Interface Sci. 1992, 149, 162-173. (c) Cavalleri, O.; Oliveri, L.;
Dacca`, A.; Parodi, R.; Rolandi, R. Appl. Surf. Sci. 2001, 175-176, 357-
362.
(61) (a) Clark, D. T.; Peeling, J.; Colling, L. Biochim. Biophys. Acta 1976, 453,
533-545. (b) Bomben, K. D.; Dev, S. B. Anal. Chem. 1988, 60, 1393-
1397. (c) Petoral, R. M., Jr.; Uvdal, K. J. Electron Spectrosc. Relat. Phenom.
2003, 128, 159-164.
(62) (a) Ishida, T.; Choi, N.; Mizutani, W.; Tokumoto, H.; Kojima, I.; Azehara,
H.; Hokari, H.; Akiba, U.; Fujihira, M. Langmuir 1999, 15, 6799-6806.
(b) Castner, D. G.; Hinds, K.; Grainger, D. W. Langmuir 1996, 12, 5083-
5086. (c) Maye, M. M.; Luo, J.; Lin, Y.; Engelhard, M. H.; Hepel, M.;
Zhong, C.-J. Langmuir 2003, 19, 125-131.
(63) (a) Riga, J.; Verbist, J. J. J. Chem. Soc., Perkin Trans. 2 1983, 1545-
1551. (b) Nuzzo, R. G.; Zegarski, B. R.; Dubois, L. H. J. Am. Chem. Soc.
1987, 109, 733-740. (c) Moulder, J. F.; Stickle, W. F.; Sobol, P. W.;
Bomben, K. D. Handbook of X-ray Photoelectron Spectroscopy; Perkin-
Elmer: Eden Prairie, MN, 1992.
(65) (a) Templeton, A. C.; Hostetler, M. J.; Kraft, C. T.; Murray, R. W. J. Am.
Chem. Soc. 1998, 120, 1906-1911. (b) Paulini, R.; Frankamp, B. L.;
Rotello, V. M. Langmuir 2002, 18, 2368-2373.
(64) Lindberg, B. J.; Hamrin, K.; Johansson, G.; Gelius, U.; Fahlman, A.;
Nordling, C.; Siegbahn, K. Phys. Scr. 1970, 1, 286-298.
9
334 J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006

Gold Nanoclusters Protected by Select Peptides
A R T I C L E S
increases. This decrease is related to the peptide stiffness and
thus to the extent of intramolecular H-bond formation.
To better appreciate the relative similarities and differences,
also in terms of absorbance maxima, we normalized the MPC
spectra of Figure 7 by taking into account the actual number of
peptides per particle as obtained from TGA. The absorption
values of Figure 7 thus reflect our best estimate of the peptide
concentration, whether corresponding to an actual molar con-
centration (1 mM) or to the same concentration calculated as if
the peptides forming the monolayer were free diffusing species.
Graphs b and c of Figure 7, which correspond to the 0.5:1 and
3:1 series, respectively, are qualitatively very similar but also
significantly differ from those of the free ligands. First, the band
intensity of the free N-H groups of the MPCs of 0-3 (at 3433
( 1 cm-1) is only about one-fourth of that recorded for 1a-
3a. Second, starting from the shortest ligand an absorption band
appears at low frequencies; this band is roughly constant (from
derivative analysis of the absorption spectrasnot shown) in
terms of both position (3287 ( 5 cm-1) and intensity. Third,
the intensity of the band at intermediate frequencies is an
increasing function of the peptide length. As the length increases,
the frequency undergoes the same red shift observed for the
free peptide, although the values are on the average lower by 8
cm-1. The frequency shift and the intensity increase eventually
causes this band to overlap the above-mentioned low-energy
band, thus making the resulting band quite broad. Whereas the
vibrational mode observed at intermediate frequencies is as-
signed to the intrachain CdO‚‚‚H-N hydrogen bonds, the low-
frequency component is attributed to the N-H groups that are
hydrogen-bonded to neighbor peptides. In fact, the observed
frequencies are close to the values expected for such interactions,
for example as found with â-sheets66 or for MPCs with
embedded amide groups.5d,e,7b Close inspection of the graphs b
and c shows that the intensity of the main N-H band of the
0.5:1 MPC preparation series is, overall, slightly larger than
that of the 3:1 series. The reason for such a trend may be related
to the size of the 0.5:1 MPCs, which are substantially larger
than the corresponding 3:1 MPCs and are thus characterized
by the presence of flatter surfaces. Thus, the band intensity may
be related to the surface selection rules, according to which the
absorbance of bands with a transition moment essentially
perpendicular to the surface are expected to appear with
enhanced intensities.67 The phenomenon is expected to be more
evident when the monolayer forms on a flatter surface. In this
framework, the above observation would thus support the notion
that the peptides are assembled by directing their main axis (and
thus, according to the 310-helical structure, the intrachain
hydrogen-bonded CdO and N-H groups) essentially perpen-
dicular to the surface.
Figure 8. FT-IR absorption spectra for the amide I and amide II regions
of peptides 0a-3a (graph a), 0.5:1 MPCs (graph b), and 3:1 MPCs (graph
c). The spectra correspond (bottom to top) to 0 (black), 1 (red), 2 (green),
and 3 (blue). The spectra were acquired in CH2Cl2 solutions, and the
absorbance was corrected for the peptide concentration.
∼8 cm-1). The spectral region pertaining to the amide II band
(for fully formed 310-helices the amide II band is expected at
1531-1533 cm-1 68
is complex because the peptides that we
)
investigated are short oligomers. We can note, however, the
tendency to develop a well-defined amide II band as we move
from 0a to 3a. Interestingly, the corresponding MPCs display
this tendency even more promptly, which suggests that the
system now is stiffer (more stable 310-helices for peptides buried
in the monolayer). We note also that, compared to the free
peptides, the directions of the shifts of the amide I and the amide
II bands are different: for example, the values shown by peptide
3 are 1674 and 1666 cm-1 (amide I) and 1526 and 1530 cm-1
(amide II). These shifts are associated with chemisorption to
Information on peptide conformation is also found in the
amide I and amide II regions. Although the fine structure of
these bands is quite complex,33 we now will focus only on
relative comparisons. For amide I, which corresponds to the
CdO stretch and for fully stable 310-helices occurs at 1662-
1666 cm-1, the main difference between precursors 0a-3a and
corresponding MPCs (Figure 8: once again, the absorption
values were normalized for the actual peptide concentration) is
that the latter systems exhibit lower frequencies (typically, by
(66) Toniolo, C.; Palumbo, M. Biopolymers 1977, 16, 219-224.
(67) Greenler, R. G. J. Chem. Phys. 1966, 44, 310-315.
(68) A similar enhancement could be observed for a 2D-SAM formed by an
Aib-containing hexapeptide.6
9
J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006 335

A R T I C L E S
Fabris et al.
Conclusions
the gold surface and thus to the interactions between the peptides
in the adsorbed layer, as also noticed for peptide 2D SAMs.5b
The relative absorption intensities of the amide I and the amide
II bands is particularly worth noting: the intensity ratio
(amide I to amide II absorbance) increases from 1.82 to
2.08 to 2.37 (peptide 3) as we go from the free ligand to the
3:1 MPCs and finally to the 0.5:1 MPCs, respectively. This
comparison also supports the view that the peptides (and thus
the intrachain CdO‚‚‚H-N hydrogen bonds) are oriented
(mostly) perpendicular to the surface, thereby experiencing a
surface enhancement for the amide I (and amide A) vibrational
mode.68
As final experimental notes, we mention that the IR absorp-
tion behavior of the free ligand in solution is rather different
from that obtained in a KBr pellet, where the peptides are packed
favoring the formation of interligand CdO‚‚‚H-N hydrogen
bonds. In particular, whereas in KBr the amide I band shifts to
smaller energies, the amide II experiences the opposite trend.
On the other hand, almost no difference is observed (Figure
S2) for the MPCs, in keeping with the notion that when the
peptides are buried in the monolayer they feel no difference
whether the MPC is in solution. We also carried out IR
absorption experiments (with 3-MPC and 3a: see Supporting
Information, Figures S3 and S4) in acetonitrile, which is a
solvent prone to interact by hydrogen bonding with free NH
groups. Remarkably, we did not notice any significant difference
(both in the amide A and the amide I and II regions) with respect
to the results obtained in CH2Cl2, while the effect is evident
with the free ligand. These results also support the view that
the properties of the peptide monolayer are not affected by
exogenous species, as already discussed for the NMR results
(Me2SO-d6 titrations).
We have prepared a series of gold nanoclusters in which the
capping monolayer is composed by thiolated peptide ligands.
The peptides are oligomers based on the Aib unit and were
chosen for their propensity to promptly form 310-helical struc-
tures. The corresponding MPCs were prepared under different
experimental conditions and characterized with various physi-
cochemical tools. The core diameters of the MPCs are smaller
when more structured peptides are used (i.e., 1-3), which would
suggest that sterically hindered and conformationally constrained
peptide ligands are better suited to passivate the cluster growth
at an earlier stage of its formation. The clusters prepared by
using a 3:1 S:Au ratio and structured peptides (1-3) are
particularly small, being in the 1.1-1.3 nm range.
The structural integrity of the peptides in the monolayer was
assessed. The data indicate that the peptides are remarkably well-
packed in the monolayer and that the surface coverage is
typically 75-85% the value calculated for alkanethiolate MPCs
of similar sizes. Spectral analyses indicate that the secondary
structure does not change when the peptide is in the monolayer
and that H-bond acceptors do not alter the H-bond interactions.
The results also provide evidence for the presence of interligand
CdO‚‚‚H-N hydrogen bonds in the MPCs and suggest that
the peptides are mostly oriented perpendicular to the gold
surface. The peptide secondary structure and the interligand
H-bonding network are envisaged as responsible for the
successful preparation of this new class of small MPCs. For
future applications, the rigidity of these peptide monolayers
could be employed to control the relative distance and thus the
physicochemical behavior of electro- or photoactive groups
attached on the MPC periphery.
The above IR absorption data and comparisons indicate that
the peptides are both intraligand (according to the 310-helical
structure) and interligand hydrogen-bonded. The latter interac-
tion should involve the first two N-H groups, which are not
involved in intrachain interactions. This conclusion also implies
that neighbor peptide adsorbates should provide CdO groups
that are already involved in intrachain CdO‚‚‚H-N hydrogen
bonds. These three-center hydrogen bonds allow formation of
a strong interchain bonding network and, since they are
embedded inside the monolayer, they are largely unaffected by
an increase of the peptide length.
Acknowledgment. This work was financially supported by
the Ministero dell’Istruzione, dell’Universita` e della Ricerca
(MIUR) and the National Consortium of Materials Science and
Technology (INSTM). S.A. and L.F. also acknowledge a grant
from the Universita` di Padova (Progetto Giovani Ricercatori).
We thank Dr. G. Pace for the TGA.
Supporting Information Available: Additional preparation
and characterization data of peptides and MPCs. This material
is available free of charge via the Internet at http://pubs.acs.org.
JA0560581
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336 J. AM. CHEM. SOC. VOL. 128, NO. 1, 2006