T. K. Mandal et al.
6.88–6.85 (d, J=8.3 Hz, 2H; Tyr(1)/Tyr(2)/Tyr(3) ring hydrogen), 6.84–
6.81 (d, J=8.4 Hz, 2H; Tyr(1)/Tyr(2)/Tyr(3) ring hydrogen), 6.74–6.71 (d,
J=8.4 Hz, 2H; Tyr(1)/Tyr(2)/Tyr(3) ring hydrogen), 6.71–6.68 (d, J=
8.4 Hz, 2H; Tyr(1)/Tyr(2)/Tyr(3) ring hydrogen), 6.50–6.48 (d, J=6.2 Hz,
Acknowledgements
S.S. thanks the CSIR, Government of India for providing a Senior Re-
search Fellowship. This research was supported by grants [Grant No. BT/
PR3648/BRB/10/301/2003] from the Department of Biotechnology, New
Delhi. Thanks are also due to the partial support from the Nanoscience
and Nanotechnology Initiatives, DST, New Delhi. We also thank the
SAIF, Bose Institute, Kolkata for recording the EPR spectra.
ꢀ
1H; Tyr(1)/Tyr(2)/Tyr(3) N H), 6.41–6.39 (d, J=7.5 Hz, 1H; Tyr(1)/
ꢀ
Tyr(2)/Tyr(3) N H), 5.09–5.06 (d, J=7.62 Hz, 1H; Tyr(1)/Tyr(2)/Tyr(3)
a
ꢀ
N H), 4.68–4.61 (m, 1H; Tyr(1)/Tyr(2)/Tyr(3) C H), 4.50–4.43 (m, 1H;
Tyr(1)/Tyr(2)/Tyr(3) CaH), 4.24–4.22 (m, 1H; Tyr(1)/Tyr(2)/Tyr(3) CaH),
3.68 (s, 3H; OCH3), 3.02–2.78 (m, 6H; Tyr(1)+Tyr(2)+Tyr(3) CbH),
1.39 ppm (s, 9H; Boc-CH3); elemental analysis calcd (%) for C33H39N3O9
(621): C 63.77, H 6.28, N 6.76; found: C 63.21, H 6.29, N 6.13; MS (ESI)
(35 eV): m/z (%): 644 (100) [M++Na].
[1] U. Kreibig, M. Vollmer in Optical Properties of Metal Clusters,
Springer Series in Materials Science, No. 25, Springer, Berlin, 1995.
[2] J. R. Krenn, B. Lamprecht, H. Ditlbacher, G. Schider, M. Salerno,
A. Leitner, F. R. Aussenegg, Europhys. Lett. 2002, 60, 663–669.
[3] J. J. Storhoff, R. Elghanian, R. C. Mucic, C. A. Mirkin, R. L. Letsing-
er, J. Am. Chem. Soc. 1998, 120, 1959–1964.
[4] A. Gole, C. Dash, V. Ramakrishnan, S. R. Sainkar, A. B. Mandale,
M. Rao, M. Sastry, Langmuir 2001, 17, 1674–1679.
[5] A. P. Alivastos, K. P. Johnson, X. Peng, T. E. Wilson, C. J. Loweth,
M. P. Bruchez, P. G. Schultz, Nature 1996, 382, 609–611.
[6] S. Mann, W. Shenton, M. Li, S. Connolly, D. Fitzmaurice, Adv.
Mater. 2000, 12, 147–150.
[7] C. A. Mirkin, R. L. Letsinger, R. C. Mucic, J. J. Storhoff, Nature
1996, 382, 607–609.
[8] W. Shenton, S. A. Davis, S. Mann, Adv. Mater. 1999, 11, 449–452.
[9] Y. Zhou, W. Chen, H. Itoh, K. Naka, Q. Ni, H. Yamane, Y. Chujo,
Chem. Commun. 2001, 2518–2519.
[10] P. R. Selvakannan, A. Swami, D. Srisathiyanarayanan, P. S. Shirude,
R. Pasricha, A. B. Mandale, M. Sastry, Langmuir 2004, 20, 7825–
7836.
[11] R. R. Bhattacharjee, A. K. Das, D. Haldar, S. Si, A. Banerjee, T. K.
Mandal, J. Nanosci. Nanotechnol. 2005, 5, 1141–1147.
[12] B. A. Barry, M. K. El-Deeb, P. O. Sandusky, G. T. Babcock, J. Biol.
Chem. 1990, 265, 20139–20143.
Synthesis of peptide-3, (NH2-Tyr(1)-Tyr(2)-Tyr(3)-OMe): We added
formic acid (10 mL, 98%) to 5 mm of Boc-Tyr(1)-Tyr(2)-Tyr(3)-OMe and
the removal of the Boc-group was monitored by TLC. After, 8 h, the
formic acid was removed under vacuum. The residue was taken up in
water (20 mL), and was washed with diethyl ether (220 mL). The pH of
the aqueous solution was then adjusted to 8 with sodium bicarbonate and
the solution was then extracted with ethyl acetate (330 mL). The organ-
ic extract was washed with saturated brine, dried over sodium sulfate,
and was concentrated to a viscous liquid that gave a positive ninhydrin
test. 1H NMR (300 MHz, CDCl3 + 5% DMSO, TMS): d=8.41 (br, 2H;
ꢀ
Tyr(1) NH2), 7.7–7.68 (d, J=8.4 Hz, 1H; Tyr(2)/Tyr(3) N H), 7.00–6.65
(m, 12H; Tyr(1)+Tyr(2)+Tyr(3) ring hydrogen), 4.73–4.66 (m, 1H;
Tyr(1)/Tyr(2)/Tyr(3) CaH), 4.59–4.52 (m, 1H; Tyr(1)/Tyr(2)/Tyr(3) CaH),
4.24–4.21 (m, 1H; Tyr(1)/Tyr(2)/Tyr(3) CaH), 3.68 (s, 3H; OCH3), 3.02–
2.85 ppm (m, 6H; Tyr(1)+Tyr(2)+Tyr(3) CbH); elemental analysis calcd
(%) for C28H31N3O7 (521): C 64.49, H 5.95, N 8.06; found: C 64.13, H
6.43, N 7.89; MS (ESI) (35 eV): m/z (%): 544 (100) [M++Na].
Synthesis of metal–peptide nanoconjugates: The detailed synthesis of
gold-peptide-1 nanoconjugates has been described in our earlier
report.[11] Similarly, 40 mm of a solution of peptide-2 and peptide-3 (in
methanol) was added to an aqueous solution of HAuCl4 (10 mm), such
that the final concentration of respective peptide and HAuCl4 was 5 mm
and 2.5 mm respectively. In both the reactions the desired pH values
were maintained using standard NaOH solution.
[13] S. Kim, J. Liang, B. A. Barry, Proc. Natl. Acad. Sci. USA 1997, 94,
14406–14411.
[14] M. Sjodin, S. Styring, B. ꢁkermark, L. Sun, L. Hammarstrom, J.
Am. Chem. Soc. 2000, 122, 3932–3936.
[15] W. Hofbauer, A. Zouni, R. Bittl, J. Kern, P. Orth, F. Lendzian, P.
Fromme, H. T. Witt, W. Lubitz, Proc. Natl. Acad. Sci. USA 2001, 98,
6623–6628.
[16] R. Ghanem, Y. Xu, J. Pan, T. Hoffmann, J. Andersson, T. Polivka, T.
Pascher, S. Styring, L. Sun, V. Sundstrom, Inorg. Chem. 2002, 41,
6258–6266.
[17] C. Carra, N. Iordanova, S. Hammes-Schiffer, J. Am. Chem. Soc.
2003, 125, 10429–10436.
[18] L. W. Smith, T. E. Eling, R. J. Kulmacz, L. J. Marnett, A.-L. Tsai,
Biochemistry 1992, 31, 3–7.
[19] M. M. Whittaker, J. W. Whittaker, J. Biol. Chem. 1990, 265, 9610–
9613.
[20] A. Larsson, B.-M. Sjoberg, EMBO J. 1986, 5, 2037–2040.
[21] J. A. Creighton, D. G. Eadon, J. Chem. Soc. Faraday Trans. 1991, 87,
3881–3891.
[22] R. Levy, N. T. K. Thanh, R. C. Doty, I. Hussain, R. J. Nichols, D. J.
Schiffrin, M. Brust, D. G. Fernig, J. Am. Chem. Soc. 2004, 126,
10076–10084.
Characterization
NMR experiments: All NMR studies were carried out on a Bruker DPX
300 MHz spectrometer. Peptide concentrations were in the range 1–
10 mm in CDCl3 and DMSO.
GNP formation by UV/Vis spectrophotometry: UV/Vis absorption spectra
of all the nanoconjugate suspensions were measured in a Hewlett–Pack-
ard 8453 spectrophotometer. For the kinetic study, the UV/Vis spectra
were recorded from time-to-time during the reaction with samples in a
0.1 cm quartz cuvette. The rate of formation of GNPs was monitored by
the time-dependent evaluation of the SP band of the gold nanoparticles.
Fluorescence spectroscopy: Emission spectra of peptides/nanoconjugates
were recorded on a Perkin–Elmer LS55 fluorimeter. The emission spec-
tra of gold-peptide-1 nanoconjugates were also recorded occasionally
during their formation by conducting the reaction in a 1 cm quartz cell
and by using an excitation wavelength of 325 nm.
EPR study: Formation of intermediate tyrosyl radicals during the reac-
tion of an alkaline solution of the peptide-1 and HAuCl4 or Cu(NO3)2
was monitored by recording the instantaneous EPR spectra of the above-
mixture using a Varian E-112 spectrometer.
[23] J. Odo, K. Matsumoto, E. Shinmoto, Y. Hatae, A. I. Shiozaki, Anal.
Sci. 2004, 20, 707–710.
[24] T. G. Huggins, M. C. Wells-Knecht, N. A. Detorie, J. W. Baynes,
S. R. Thorpe, J. Biol. Chem. 1993, 268, 12341–12347.
[25] E. Assemand, M. Lacroix, M. A. Mateescu, Biotechnol. Appl. Bio-
chem. 2003, 38, 151–156.
Mass spectral study: First, an alkaline solution of the peptide-1 was treat-
ed with Cu(NO3)2. The product of this reaction, that is supposed to be a
mixture of unreacted peptide-1 and the dityrosine form of the peptide,
was then extracted back from the reaction solution and used for ESI
mass analysis in a quadrupole time-of-flight (Qtof) Micro YA263 mass
spectrometer.
[26] K. J. A. Davies, J. Biol. Chem. 1987, 262, 9895–9901.
[27] J. S. Jacob, D. P. Cistola, F. F. Hsu, S. Muzaffar, D. M. Mueller, S. L.
Hazen, J. W. Heinecke, J. Biol. Chem. 1996, 271, 19950–19956.
[28] D. A. Malencik, J. F. Sprouse, C. A. Swanson, S. R. Anderson, Anal.
Biochem. 1996, 242, 202–213.
[29] I. Pockrand, A. Brillante, D. Mobius, Chem. Phys. Lett. 1980, 69,
499–504.
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