Peptide Films on Surfaces: Preparation and Electron Transfer

A Stokes ellipsometer LSE (Gaertner Scientific. Corporation, Skokie, IL, fixed angle (70°), fixed wavelength (632.8 nm)) was used, and the data were ...
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Chapter 27

Peptide Films on Surfaces: Preparation and Electron Transfer

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G. A. Orlowski and H. B. Kraatz Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, Saskatchewan S7N 5C9, Canada

A new method for the electrodeposition of peptide disulfides onto gold surfaces is described together with a comparison of these surfaces to those prepared by conventional methods. For this purpose we are using acylic peptides of the general formula [Fc-Xxx-CSA] (Fc = ferrocenoyl, X x x = amino acid, C S A = cysteamine) and cyclic compounds Fc[Xxx-CSA] . These two classes of peptides show significant differences in their electron transfer behavior, which is most likely due to the ability of the cyclic systems to engage in two Au-S linkages. 2

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© 2006 American Chemical Society

Schubert et al.; Metal-Containing and Metallosupramolecular Polymers and Materials ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Introduction Understanding electron-transfer (ET) processes in proteins is of fundamental importance. * In a series of photophysical studies of well-behaved peptide model systems, it has become evident that the ET through the peptide spacer is greatly influenced by the separation between the acceptor (A) and the donor (D), the nature of the peptide backbone, the amino acid sequence, and the resulting flexibility. ' In particular, it was suggested in the literature that the presence of Η-bonding will increase the rate of E T , and there is experimental evidence (mostly in proteins) to suggest that Η-bonding indeed increases the rate of E T . ' We have been involved in electrochemical measurements of the E T process in redox-labeled peptide thiol films. The formation of the peptide film is achieved via a Au-thiolate linkage. E T has to proceed from the redox probe through the peptide spacer to the A u surface. Electrochemical measurements allow us to evaluate the forward and backward E T processes in these systems. 1

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We have demonstrated before that short Fc-peptides form poorly ordered films and leave about 20% of the surface accessible to solvent and supporting electrolyte. Using hexanethiol it was possible to fill these holes and to evaluate the E T kinetics in great detail. The question arose i f the poor packing is a result of the self-assembly procedure, which usually entails soaking of a clean gold substrate for several days. Thus we embarked on a study aimed at developing a method that allows us to form densely packed and well-ordered peptide films from disulfides. Here, we investigate the role of the attachment of the peptide on a gold surface and describe the results of an electrochemical study of ferrocenoyl (Fc)labeled peptides. We made use of two classes of Fc-peptides: acylic ferrocenoyl (Fc)-peptide disulfides [Fc-CSA] (la), [Fc-Gly~CSA] (2a), [Fc-Ala-CSA] (3a), [ F c - V a l - C S A ] (4a) and [Fc-Leu-CSA] (5a) and cyclo-l,l'-Fc-peptide disulfides Fc[CSA] (lc), F c [ G l y - C S A ] (2c), F c [ A l a - C S A ] (3c), F c [ V a l C S A ] (4c) and Fc[Leu-CSA] (5c) (see Scheme 1). These compounds were prepared as described before. " In addition, we describe an electro-deposition method for the preparation of cyclic and acyclic Fc-peptide disulfides leading to tightly-packed films. 18

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Experimental Ellipsometry. A u on Si(100) (Platypus Technologies, Inc) wafers were incubated in a 1 mMFc-peptide ethanolic solution for 5 days and finally rinsed with EtOH and H 0 . A Stokes ellipsometer L S E (Gaertner Scientific Corporation, Skokie, I L , fixed angle (70°), fixed wavelength (632.8 nm)) was used, and the data were collected and analyzed using L G E M P (Gaertner Ellipsometer Measurement Software) o n a P C . Ellipsometry constants were as 2

Schubert et al.; Metal-Containing and Metallosupramolecular Polymers and Materials ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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Scheme 1. Chemical drawings of allferrocene-peptide disulfide conjugates.

Schubert et al.; Metal-Containing and Metallosupramolecular Polymers and Materials ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

395 follows: n = 0.00 and K$ = 1.44 was used as the refractive index of the monolayer. s

Electrochemistry was performed on two types of potentiostats: home made and CHI660B, C H I instruments Inc. A l l potentials are given vs A g / A g C l which was used as a reference electrode. Supporting electrolyte in all cases was 2 M NaC10 . Calculation of electron transfer rate is described elsevere. Experiments were performed on gold ultramicroelectrode with a diameter of 25 μπι. 18

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Electrodeposition. 10 m M solutions of Fc-peptide disulfide in pure EtOH were prepared and a potential of -1.3 V (vs Ag/AgCl) was applied for 3 0 minutes. Lengthening of the electrodeposition time does not significantly change the film.

Results and Discussion Film of cyclic Fc-peptides lc-5c and of the acylic systems la-5a were prepared as schematically shown in Figure 1. The electrochemical properties are evaluated by cyclic voltammetry (CV) and chronoamperometry (CA). A l l films exhibit reversible one electron redox waves (Figure 2).

Figure I. Schematic representation of the two methods used to prepare Fc-peptide films.

Schubert et al.; Metal-Containing and Metallosupramolecular Polymers and Materials ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

396 Integration of the Faradaic current provides the Fc surface concentration, from which a specific area per molecule can be calculated. Surface concentration of the monolayer ranges from 2.3 - 4.6 * 10' mol/cm' . The theoretical area (calculated from crystal structure data of the acylic Fc-peptides and Ι,Γ-cyclo-Fc-peptides are -30 and -40 A molecule" , respectively. The thickness of the films prepared by electrodeposition and by soaking was determined by ellipsometry and gave values of 9(3) Â, which is in good agreement with data obtained from x-ray crystallography and by molecular modeling (Spartan). The signal of the cylcopeptides lc-5c is shifted to higher potential as expected for disubstituted ferrocene derivatives. The monolayers prepared by the electrodeposition exhibited remarkable stability and showed about a 5% of monolayer loss after more than 100 cycles (100 m V to 1000 m V vs. Ag/AgCl) (Figure 3). 18

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e/mv

V / V e

«

Figure 2. (left) Electrochemical response of immobilized films for acyclic [Fc-Gly-CSA] (2a) and Fc[Gly-CSA] (2c). Inset shows linear dependency between scan rate and peak current indicating successful immobilization of cyclic and acyclic compounds on gold surface; (right): Comparison of two methods: electrodeposition and incubation (soak). Graph presenting successful immobilization (linear dependency of scan rate and peak current) of cyclic Fc[Ala-CSA] (3c) by application of both methods. 2

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A l l C V s showed reversible Fc to Fc* redox reactions as measured by peak current ratios. A l l of the cyclic compounds showed a higher redox potential compared to the non-cyclic analog, which is typical when comparing mono- to Ι,Γ-di-substituted Fc. The electron withdrawing capability of the amides makes the disubstituted Fc more difficult to oxidize. A l l electrochemical parameters are included in Table 1 and Table 2.

Schubert et al.; Metal-Containing and Metallosupramolecular Polymers and Materials ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

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397

Ε/ν

Figure 3. Multiple CVs of Fc[AlaCSA] (3c) taken every 0.05 seconds for 60 seconds with a 12.5 μηχ radius Au-modified electrode, 2 MNaCl0 supporting electrolyte and a Ag/AgCl/(3.5 MKCl) reference electrode. 2

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Table 1· Summary of electrochemical parameters analyzed by C V and C A . Values in parentheses are the standard deviations from 5 electrode measurements Incubation

Electrodeposition Comp.

2

1-c

670(7)

9.5

Area/A molecule' 45(7)

1-a

465(9)

8.0

40(7)

473(6)

7.0

Ef'/mV

s'*

3

2

660(9)

kerxlO / Area/A s~' * molecule' 8.0 120(9)

Ef'/mV

1

50(3)

2-c

688(6)

14.0

47(8)

682(8)

13.0

150(20)

2-a

464(6)

13.5

50(8)

445(9)

12.0

78(8)

3-c

635(6)

12.0

68(9)

624(8)

11.0

141(20)

3-a

490(7)

6.0

36(5)

468(8)

6.9

53(9)

4-c

670(7)

12.0

60(9)

665(9)

11.0

130(25)

4-a

488(7)

9.5

65(8)

484(7)

10.0

70(10)

5-c

686(8)

17.0

60(8)

680(7)

14.0

220(10)

5-a

484(7)

11.0

72(8)

476(9)

11.0

101(9)

* Error for k

calculations was 1.5 χ 10 s" 3

ET

Schubert et al.; Metal-Containing and Metallosupramolecular Polymers and Materials ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

398 Electron transfer kinetics was assessed by C V and C A and both electrochemical methods yielded similar values (Table 1). The E T rate, £ , for all compounds are between 17(1.5) and 6(1.5) χ 10 s". In general, the cyclic Fc-peptide systems exhibit higher k s compared to the corresponding acyclic systems. The enhanced E T for the cyclic peptides is attributed to the double junction - d ouble ρ eptide w ire ο f t he 1,1 '-Fc ρ eptide s ystem w ith b oth s ulfur atom linked to the A u surface, compared to die single connection o f acyclic systems. Intramolecular Η-bonding in the cyclopeptide, as confirmed by X-ray structure analysis, may contribute to the increased electron transfer rate for cyclic compounds. ET

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Table 2. Electrochemical parameters calculated from C V experiments using the electrochemical deposition and incubation methods. Electrodeposition

Incubation ΔΕ / mV Ρ

ΔΕ / mV

Efr/hm/

90(9)

190(10)

0.92(5)

220(10)

1.00(5)

170(8)

1.00(5)

ρ

mV

mV

lc

90(8)

200(15)

1.00(9)

la

120(6)

240(15)

1.00(9)

2c

60(8)

180(10)

0.90(9)

120 (10) 60(7)

2a

65(5)

195(10)

1.00(9)

140(10)

175(8)

0.92(5)

3c

85(7)

190(15)

0.90(9)

55(7)

160(8)

0.94(5)

3a

90(5)

200(15)

1.00(9)

111(10)

190(10)

0.90(5)

4c

80(7)

210(8)

0.90(5)

55(7)

170(8)

0.98(5)

4a

85(5)

230(15)

0.90(5)

120(10)

190(10)

1.00(5)

5c

70(5)

200(10)

0.90(5)

62(7)

157(10)

0.90(3)

5a

95(7)

210(10)

0.90(5)

110(10)

190(10)

0.90(3)

In summary, we have presented an electrochemical method to form Fcpeptide films from Fc-peptide disulfides, giving raise to well-packed films on gold. This method should find wide-spread applications for die formation of films from disulfides. Our studies allowed a direct comparison of the E T kinetics of cyclic and acyclic Fc-peptide disulfide systems. Our results show faster E T kinetics for the cyclic systems compared to the acyclic systems, which may be

Schubert et al.; Metal-Containing and Metallosupramolecular Polymers and Materials ACS Symposium Series; American Chemical Society: Washington, DC, 2006.

399 result of the enhanced rigidity of the molecules or simple increase number o f "conductive peptide wires" (one versus two) to the surface. We are now investigating this phenomenon in more detail.

Acknowledgment

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The authors thank N S E R C for funding. H.B.K. is the Canada Research Chair in Biomaterials.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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18. Bediako-Amoa, I.; Sutherland, T.C.; Li, C.-Z.; Silerova, R.; Kraatz, H.B. J. Phys. Chem. B. 2004, 108, 704-714 19. Bediako-Amoa, I.; Silerova, R.; Kraatz, H . B . Chem. Commun. 2002, 24302431. 20. Chowdhury, S.; Schatte, G.; Kraatz, H . B . J. Chem. Soc., Dalton Trans. 2004, 1726-1730.

Schubert et al.; Metal-Containing and Metallosupramolecular Polymers and Materials ACS Symposium Series; American Chemical Society: Washington, DC, 2006.