S1

Supporting Information for

Anodic photocurrent generation by porphyrin-terminated helical peptide monolayers on gold

Hirotaka Uji, Yuji Yatsunami, and Shunsaku Kimura*

Department of Material Chemistry, Graduate School of Engineering, Kyoto University,

Kyoto-Daigaku-Katsura, Nishikyo-ku, Kyoto 615-8510, Japan

Tel: +81-75-383-2400 Fax: +81-75-383-2401 E-mail: shun@scl.kyoto-u.ac.jp

S2

Materials

All chemicals were purchased from commercial suppliers and used without further purification. SA16M were

synthesized according to the literature.1-4

All the intermediates were identified by 1H NMR spectroscopy (Bruker

DPX-400) and the final products were further confirmed by MALDI mass spectrometry (Bruker ultraflexIII-KE).

The purity of the intermediates was checked by thin-layer chromatography and the purity of the final compounds was

checked by HPLC (TOSOH System 8020).

General procedures for the liquid-phase peptide coupling reactions. A carboxyl acid component and an amine

component were added to a round bottom flask under argon atmosphere and dissolved with dimethyl formamide

(DMF). The flask was cooled to 0 °C. A concentrated DMF solution of HATU and HOAt was added into the mixture.

N,N-diisopropylethylamine (DIEA) was then added to the mixture. The mixture was stirred for 12 hours under argon

atmosphere. The mixture was concentrated under reduced atmosphere and purified by one or combination of the

following three methods: (1) taking up with chloroform (CHCl3) and successive washes with 4 wt% aq. NaHCO3 (3

times), brine, 4 wt% aq. KHSO4 (3 times), and brine, followed by drying of the organic layer over with Na2SO4; (2)

purification by silica gel column chromatography; or (3) purification by Sephadex LH-20 chromatography.

TPP-CONH-Et-NHBoc

N-tert-butoxycarbonyl-ethylene diamine (NH2-Et-NHBoc, 73.0 mg, 455 mol, 3 eq.) was coupled to

5-(4-carboxyphenyl)-10,15,20-triphenyl porphyrin (TPP-COOH, 100 mg, 152 mol, 1 eq.) with addition of DIEA

(132 L, 759 mol, 5 eq.), HATU (86.6 mg, 228 mol, 1.5 eq.) and HOAt (31.0 mg, 228 mol, 1.5 eq.) for 1 h at 0

oC under argon atmosphere and additionally for 12 h at room temperature. Then the solvent was removed under

S3

reduced pressure and the residue was purified by column chromatography (silica gel, eluent: CHCl3/MeOH = 50/1

v/v) and then the product was washed with methanol to afford TPP-CONH-Et-NHBoc (120 mg, 150 mol, 98%

yield) as a solid.

1H NMR (400 MHz, CDCl3) (ppm): -2.80 (br s, 2H, ring NH), 1.47 (s, 9H, (CH3)3C), 3.55 (t, 2H, CH2CH2), 3.73

(t, 2H, CH2CH2), 5.07 (br s, 1H, urethane NH), 7.54 (br s, 1H, amide NH), 7.75 (m, 9H, phenyl Ho,p), 8.21 (m, 8H,

phenyl Hm), 8.26 (m, 2H, phenyl Ho), 8.78 (m, 2H, pyrrole Ha), 8.83 (m, 6H, pyrrole Hb).

FAB-MS m/z: [M+H]+ calcd for C52H45N6O3, 801.35; found, 801.4.

Boc-(Ala-Aib)4-CONH-Et-NHCO-TPP (BA8TPP)

The Boc group of TPP-CONH-Et-NHBoc (30.0 mg, 37.5 mol) was deprotected by treating with 4 N

HCl/dioxane (10 mL) after diluted in chloroform (0.5 mL). After an additional 1 h of stirring at room temperature,

the reaction mixture was concentrated under reduced pressure to obtain the deprotected product

HCl.H2N-Et-NHCO-TPP, and this product was not further purification. HCl

.H2N-Et-NHCO-TPP (25.0 mg, 33.9

mol, 1.5 eq.) was coupled to BA8OH (Boc-(Ala-Aib)4-OH, 16.8 mg, 22.6 mol, 1 eq.) with addition of DIEA

(29.5 L, 170 mol, 7.5 eq.), COMU (14.5 mg, 61.0 mol) and Oxyma Pure (4.82 mg, 33.9 mol, 1.5 eq.) for 1 h

at 0 oC under argon atmosphere and thereafter for 12 h at room temperature. Then the solvent was removed under

reduced pressure and the product was purified by column chromatography (silica gel, eluent: CHCl3/MeOH = 20/1

v/v). The product was recrystallized from diisopropyl ether to afford BA8TPP (24.2 mg, 16.5 mol, 73% yield) as a

purple solid.

1H NMR (400 MHz, CDCl3) (ppm): -2.80 (br s, 2H, porphyrin ring NH), 1.3-1.7 (45H, (CH3)3C, AlaC

H3,

AibCH3), 3.58 (t, 2H, CH2CH2), 3.7-4.1 (5H, CH2CH2, AlaC

H), 4.30 (1H, AlaC

H), 5.13 (br s, 1H, urethane NH),

S4

6.58 (br s, 1H, amide NH), 7.5 (m, 6H, amide NH), 7.76 (m, 9H, phenyl Ho,p), 8.20 (m, 8H, phenyl Hm), 8.36 (m,

2H, phenyl Ho), 8.83 (m, 8H, pyrrole H).

MALDI-MS m/z: [M+H]+ calcd for C80H93N14O11, 1425.71; found, 1426.1.

Lipoic acid-(Ala-Aib)8-CONH-Et-NHCO-TPP (SA16TPP)

The Boc group of BA8TPP (24.2 mg, 16.5 mol) was deprotected by treating with 4 N HCl/dioxane (20 mL)

after diluted in chloroform (0.5 mL). After an additional 1 h of stirring at room temperature, the reaction mixture

was concentrated under reduced pressure to obtain the deprotected product HCl.HA8TPP, and this product was not

further purification. HCl.HA8TPP (20.0 mg, 14.7 mol, 1 eq.) was coupled to SA8OH (Lipoic acid-(Ala-Aib)4-OH,

24.4 mg, 29.4 mol, 2 eq.) with addition of DIEA (20.5 L, 117 mol, 8 eq.), COMU (18.9 mg, 26.4 mol, 3 eq.),

Oxyma Pure (6.26 mg, 26.4 mol, 3 eq.) and BHT (9.69 mg, 26.4 mol, 3 eq.) for 1 h at 0 oC under argon

atmosphere and thereafter for 12 h at room temperature in chloroform. Then the mixture was purified by HPLC

(Shodex K-2002, eluent: CHCl3 containing BHT 200 mg/L). The product was recrystallized from hexane and

diisopropyl ether containing triethylamine to afford SA16TPP (12.9 mg, 6.02 mol, 41% yield) as a purple solid.

1H NMR (400 MHz, CDCl3) (ppm): -2.80 (br s, 2H, porphyrin ring NH), 1.3-1.7 (78H, lipoic acid

CH2CH2CH2CH2CO, AlaCH3, AibC

H3), 1.76, 2.58 (m, 2H, lipoic acid SSCH2CH2CH), 2.42 (m, 2H, lipoic acid

CH2CH2CH2CH2CO), 3.3- 3.6 (m, 4H, lipoic acid SSCH2CH2CH, NCH2CH2N), 3.8-4.1 (9H, NCH2CH2N,

AlaCH), 4.23 (1H, AlaC

H), 6.7-8.1 (m, 10H, amide NH), 7.77 (m, 9H, phenyl Ho,p), 8.20, 8.28 (m, 8H, phenyl

Hm), 8.42 (m, 2H, phenyl Ho), 8.82 (m, 8H, pyrrole H).

MALDI-MS m/z: [M+Na]+ calcd for C111H144N22O18S2Na, 2161.039; found, 2161.1.

S5

Circular dichroism spectroscopy (CD)

Figure S1. CD spectra of SA16TPP and SA16M in methanol.

CD spectra of the peptides were measured on a CD spectropolarimeter (J-1500, JASCO, Tokyo). The

measurements were carried out with an optical cell of 0.1 cm optical path length. The residue concentration in

methanol was set to be 0.999 × 10-3

M for SA16M, and 0.263 × 10-3

M for SA16TPP.

The CD spectra show a negative double-minimum pattern with comparable negative peaks at around 208 nm and

222 nm indicating that these peptides take -helical conformation.5 The helix contents of the peptides were calculated

by a fomula in the literature,5

30300

)2340]([ 222

Hf

where fH and [θ]222 represent the helix content and molar ellipticity in residue concentration at 222 nm. The helix

contents in methanol were calculated as 41% and 54% for SA16M and SA16TPP, respectively.

S6

Characterization of SA16M SAMs

Figure S2. (A) IRRAS spectra of SA16M SAMs. (B) Cyclic voltammograms of SA16M SAMs, TPPM10,

TPPM11, TPPM12 and bare Au substrate in a 1 mM K4[Fe(CN)6] and 1 M KCl aqueous solution at a scan rate of

0.1 V/s.

SA16M was immobilized on gold to compare SAM properties. Gold glass substrates were incubated in 0.1 mM

SA16M ethanol solution. The SAMs were analyzed by IRRAS showing amide I absorption at 1680 cm-1

with

stronger intensity than amide II absorption at 1540 cm-1

(Figure S2A). The tilt angles of the helix axis from the

surface normal were calculated, based on the absorbance ratios of amide I and amide II, to be 26.0 ± 3.1°. The

structural defects in these peptide SAMs were evaluated by cyclic voltammetry in the presence of K4[Fe(CN)6] in

aqueous solution (Figure S2B). Figure S2B shows SA16M SAM could not block the access of ferrocyanide ions to

the electrode, even though these peptides tilt angles were smaller than the other SAMs. The reason is attributed to

the small methyl ester group at the C terminal of SA16M facing the aqueous phase about the SAM, resulting in less

blocking ability than the bulky porphyrin group. The porphyrin group therefore has two opposing effects on the

blocking experiment, where one is blocking due to the bulky hydrophobic moiety on the surface and the other is

S7

leaky due to the disordered SAM structure due to the bulky moiety. Accordingly, the 1:1 composition of SA16TPP

and SA16M, TPPM11, showed the better blocking properties.

S8

Absorption spectra of peptide SAMs

Figure S3. UV-Vis absorption spectra of TPPM10, TPPM11 and TPPM12.

The absorption spectra of the peptide SAMs were carried out by transition mode. Peptides were immobilized on

gold quartz substrates whose gold thickness was 20 Å.

Figure S3 shows the UV-Vis absorption spectra in the region of the porphyrin soret band of the peptide SAMs. The

SAM compositions on gold substrates were evaluated by the ratio of absorption area from 380 nm to 480 nm. The

concentrations of SA16TPP/SA16M were analyzed to be 57/43 and 36/64 for TPPM11 and TPPM12, respectively.

S9

Figure S4. UV-Vis absorption spectra of SA16M, TPPM10, TPPM11 and TPPM12. (A) The spectra were

measured by the integral sphere method. (B) Differential spectra of TPPM10, TPPM11 and TPPM12. (C) The

original spectra of Figure S3.

We have studied on the Q-bands by UV-Vis absorption spectroscopy. As shown in Figure S4A and S4C,

unfortunately there was typical absorption of gold substrate in the range from 500 nm to 700 nm obscuring the

Q-bands on gold even with using the integral sphere method to remove the scattering effect. Figure S4B shows the

differential spectra of the SAMs and the reference SA16M spectrum.

S10

Fluorescence spectra of peptide SAMs

Figure S5. (A) Emission spectra of TPPM10, TPPM11 and TPPM12. (B) Excitation spectra of TPPM10,

TPPM11 and TPPM12.

The fluorescence (emission and excitation) spectra of the peptide SAMs were recorded on a Jasco FP-6600

fluorometer. The excitation wavelengths for emission spectra were using peak top value of absorption spectra in soret

band, and the emission wavelengths for excitation spectra were using peak top value of emission spectra around 650

nm. The excitation spectra were consistent with the absorption spectra (Figure 3A).

S11

Cyclic voltammetry

Figure S6. Cyclic voltammograms of tetraphenyl porphyrin (TPP), SA16TPP and bare Pt electrode in a 0.1 M

TBAPF6 dichloromethane solution at a scan rate of 0.5 V/s.

Cyclic voltammograms were obtained with using a BAS model 604 voltammetric analyzer. A standard

three-electrode setup was used under non-aqueous conditions with a platinum electrode as the working electrode,

Ag/Ag+ in 0.1 M tetrabutylammonium hexafluorophosphate (TBAPF6) acetonitrile solution as the reference

electrode, and a platinum wire as the counter electrode in a glass vessel capped with a silicon rubber. The solution

was a 0.1 M TBAPF6 in dichloromethane solution, and it was deaerated with argon gas for 15 min prior to the

experiments. After every sample measured, the reference electrode was checked with Fc compound. The applied

potentials of the working electrode reported here are with respect to SCE using the literature value of Fc/Fc+ in

TBAPF6 dichloromethane to be 0.41 V vs SCE.

S12

Characterization of SA16TPP SAMs

Figure S7. (A) IRRAS spectra of SA16TPP SAMs. (B) Cyclic voltammograms of SA16TPP SAMs and bare Au

substrate in a 1 mM K4[Fe(CN)6] and 1 M KCl aqueous solution at a scan rate of 0.1 V/s.

The SA16TPP was dissolved in ethanol and chloroform mixed solution (v/v, 9/1, ca. 0.1 mM). The gold

substrate was then immersed in the solution for 120 h. After that, the substrate was rigorously rinsed with ethanol

and ethanol/chloroform (1/1 v/v) in this order, and dried with a nitrogen stream and in vacuum for 15 min. Three

samples were measured by IRRAS, ellipsometry, CV and photocurrent generation.

Figure S7A shows IRRAS spectra of obtained SA16TPP SAMs. The tilt angle was calculated to be 28.8 ± 2.3°.

This value was smaller than TPPM10. In Figure S7B the molecular packing of SA16TPP SAMs were more

densely packed than TPPM10 (Figure 2B). This behavior is consistent the smaller value of the peptide tilt angle.

The molecular thickness was obtained as 31.1 ± 1.9 Å by ellipsometry.

S13

Action spectra with Q-bands

Figure S8. Action spectra in a 0.05 M TEOA and 0.05 M MV and 0.1 M Na2SO4 aqueous solution of SA16TPP

SAM. (A) Raw photocurrent action spectra of SA16TPP SAM. (B) Normalized action spectra of SA16TPP SAM

and normalized UV-Vis absorption spectra of SA16TPP in chloroform solution. Vertical axis of inset graph

modified for convenient comparison.

In Figure S8A action spectra including porphyrin Q-bands were measured. Measured wavelength was in the

range from 400 nm to 700 nm with measuring point intervals of 10 nm. Figure S8B shows that photoirradiation at

porphyrin Q-bands also generated photocurrents.

S14

Reference.

1. Kai, M.; Takeda, K.; Morita, T.; Kimura, S., Distance dependence of long-range electron transfer through

helical peptides. J Pept Sci 2008, 14 (2), 192-202.

2. Kitagawa, K.; Morita, T.; Kimura, S., Observation of single helical peptide molecule incorporated into

alkanethiol self-assembled monolayer on gold by scanning tunneling microscopy. J Phys Chem B 2004, 108 (39),

15090-15095.

3. Morita, T.; Kimura, S., Long-range electron transfer over 4 nm governed by an inelastic hopping mechanism in

self-assembled monolayers of helical peptides. J Am Chem Soc 2003, 125 (29), 8732-8733.

4. Watanabe, J.; Morita, T.; Kimura, S., Effects of dipole moment, linkers, and chromophores at side chains on

long-range electron transfer through helical peptides. J Phys Chem B 2005, 109 (30), 14416-14425.

5. Chen, Y. H.; Yang, J. T.; Martinez, H. M., Determination of Secondary Structures of Proteins by

Circular-Dichroism and Optical Rotatory Dispersion. Biochemistry-Us 1972, 11 (22), 4120-4131.