Synthesis and redox behavior of flavin mononucleotide-functionalized single-walled carbon nanotubes

Article abstract of DOI:10.1021/ja076598t

In this contribution, we describe the synthesis and covalent attachment of an analogue of the flavin mononucleotide (FMN) cofactor onto carboxylic functionalities of single-walled carbon nanotubes (SWNTs). The synthesis of FMN derivative (12) was possible by coupling flavin H-phosphonate (9) with an aliphatic alcohol, using a previously unreported N-(3-dimethylaminopropyl)- N′-ethylcarbodiimide hydrochloride coupling. We found that the flavin moiety of 12-SWNT shows a strong π-π interaction with the nanotube side-walls. This leads to a collapsed FMN configuration that quenches flavin photoluminescence (PL). The treatment of 12-SWNT with sodium dodecyl sulfate (SDS) overcomes this strong nanotube/isoalloxazine interaction and restores the FMN into extended conformation that recovers its luminescence. In addition, redox cycling as well as extended sonication were proven capable to temporally restore PL as well. Cyclic voltammetry of FMN onto SWNT forests indicated profound differences for the extended and collapsed FMN configurations in relation to oxygenated nanotube functionalities that act as mediators. These findings provide a fundamental understanding for flavin-related SWNT nanostructures that could ultimately find a number of usages in nanotube-mediated biosensing devices.

Full text of DOI:10.1021/ja076598t

Published on Web 12/15/2007
Synthesis and Redox Behavior of Flavin
Mononucleotide-Functionalized Single-Walled
Carbon Nanotubes
Sang-Yong Ju and Fotios Papadimitrakopoulos*
Nanomaterials Optoelectronics Laboratory (NOEL), Polymer Program, Department of
Chemistry, Institute of Materials Science, UniVersity of Connecticut, Storrs,
Connecticut 06269-3136
Received August 31, 2007; E-mail: papadim@mail.ims.uconn.edu
Abstract: In this contribution, we describe the synthesis and covalent attachment of an analogue of the
flavin mononucleotide (FMN) cofactor onto carboxylic functionalities of single-walled carbon nanotubes
(SWNTs). The synthesis of FMN derivative (12) was possible by coupling flavin H-phosphonate (9) with an
aliphatic alcohol, using a previously unreported N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydro-
chloride coupling. We found that the flavin moiety of 12-SWNT shows a strong π-π interaction with the
nanotube side-walls. This leads to a collapsed FMN configuration that quenches flavin photoluminescence
(PL). The treatment of 12-SWNT with sodium dodecyl sulfate (SDS) overcomes this strong nanotube/
isoalloxazine interaction and restores the FMN into extended conformation that recovers its luminescence.
In addition, redox cycling as well as extended sonication were proven capable to temporally restore PL as
well. Cyclic voltammetry of FMN onto SWNT forests indicated profound differences for the extended and
collapsed FMN configurations in relation to oxygenated nanotube functionalities that act as mediators.
These findings provide a fundamental understanding for flavin-related SWNT nanostructures that could
ultimately find a number of usages in nanotube-mediated biosensing devices.
Scheme 1. Chemical Structure of Isoalloxazine (1), Riboflavin (2),
and Flavin Mononucleotide (FMN) (3)
Introduction
Flavin is an important redox moiety for one- and two-electron-
transfer cycles in cell membranes.1,2 Scheme 1 illustrates a
number of flavoprotein cofactors vital to cellular redox chem-
istry, such as riboflavin (Vitamin B2, 2) and flavin mononucle-
otide (FMN, 3).3 A number of applications have surfaced
involving these cofactors in the presence or absence of apo-
proteins in the area of biosensors,4,5 biocatalysis,6 biolumines-
cence,7,8 and biofuel cells.9 Out of these flavin derivatives, FMN
(3) is an important luciferin (i.e., bioluminescent cofactor) when
reconstituted with apo-bacterial luciferase from photobacterium
fischeri.7,10,11 Upon introduction of an n-alkyl aldehyde and
molecular oxygen substrates, the reduced holo-bacterial lu-
ciferase (containing FMNH2) emits visible light at around 495
nm.7
The unique electronic and electrochemical properties of
single-walled carbon nanotubes (SWNTs) have inspired a
number of biosensing methodologies for proteins,12-14 DNA,15
and aptamers,16 using electrochemical- and transistor-based
configurations. Flavin adenine dinucleotide (FAD) was shown
to spontaneously adsorb onto nanotubes and facilitate quasi-
reversible electron transfer with SWNT electrodes.17 A recent
theoretical study indicates that the flavin moiety of FAD forms
strong π-π interactions with both metallic and semiconducting
(1) Massey, V. Biochem. Soc. Trans. 2000, 28, 283-296.
(2) Stryer, L. Biochemistry, 4th ed.; W. H. Freeman & Company: New York,
1995.
(3) Walsh, C. Acc. Chem. Res. 1980, 13, 148-155.
(4) Willner, B.; Katz, E.; Willner, I. Curr. Opin. Biotechnol. 2006, 17, 589-
596.
(5) Wang, J. Electroanalysis 2001, 13, 983-988.
(6) Niazov, T.; Baron, R.; Katz, E.; Lioubashevski, O.; Willner, I. Proc. Natl.
Acad. Sci. U.S.A. 2006, 103, 17160-17163.
(12) Wang, J.; Liu, G.; Jan, M. R. J. Am. Chem. Soc. 2004, 126, 3010-3011.
(13) Yu, X.; Munge, B.; Patel, V.; Jensen, G.; Bhirde, A.; Gong, J. D.; Kim, S.
N.; Gillespie, J.; Gutkind, J. S.; Papadimitrakopoulos, F.; Rusling, J. F. J.
Am. Chem. Soc. 2006, 128, 11199-11205.
(7) Hastings, J. W. Ann. ReV. Biochem. 1968, 37, 597-630.
(8) Palomba, S.; Berovic, N.; Palmer, R. E. Langmuir 2006, 22, 5451-5454.
(9) Katz, E., Shipway, A. N., Willner, I., Eds. Biochemical fuel cells. In
Handbook of Fuel Cells: Fundamentals, Technology and Applications;
Wiley: 2003; Vol. 1, 1-27.
(14) Barisci, J. N.; Wallace, G. G.; Chattopadhyay, D.; Papadimitrakopoulos,
F.; Baughman, R. H. J. Electrochem. Soc. 2003, 150, E409-E415.
(15) He, P.; Dai, L. Chem. Commun. 2004, 348-349.
(16) So, H.-M.; Won, K.; Kim, Y. H.; Kim, B.-K.; Ryu, B. H.; Na, P. S.; Kim,
H.; Lee, J.-O. J. Am. Chem. Soc. 2005, 127, 11906-11907.
(17) Guiseppi-Elie, A.; Lei, C.; Baughman, R. H. Nanotechnology 2002, 13,
559-564.
(10) Wilson, T.; Hastings, J. W. Annu. ReV. Cell. DeV. Biol. 1998, 14, 197-
230.
(11) Meighen, E. A.; MacKenzie, R. E. Biochemistry 1973, 12, 1482-1491.
9
10.1021/ja076598t CCC: $40.75 © 2008 American Chemical Society
J. AM. CHEM. SOC. 2008, 130, 655-664
655

A R T I C L E S
Ju and Papadimitrakopoulos
SWNTs.18 In addition, the flavin adsorption energy onto SWNTs
is an order of magnitude higher in strength (ca. 2 eV) than that
of an equicyclic anthracene.19
obtained from Carbon Nanotechnologies, Inc. Fourier Transform
Infrared (FTIR) spectra were obtained from a Nicolet Magna FTIR
spectrometer using a HgGeTe detector (cooled or not cooled) at a
spectral resolution of 4 cm-1. KBr disks were used as supports and an
incident angle of ca. 45° was used to minimize interference noise.26
Room-temperature UV-vis-near IR absorption and photoluminescence
(PL) spectra were obtained using a Perkin-Elmer LAMDA-900 UV-
NIR spectrometer and a Perkin-Elmer LS50B Fluorometer, respectively.
Atomic force microscopic (AFM) characterization was performed on
a Topometrix Explorer and Asylum Research MFP-3D in contact mode
and tapping mode, respectively. Cyclic voltammetry (CV) was measured
using a CH Instruments 840B electrochemical workstation. Phosphate-
buffer saline solution (PBS, pH 7.4, 0.1 M) was used as an electrolyte.
The solution was rigorously purged with nitrogen prior to use, and all
electrochemical data were recorded under a nitrogen atmosphere. A
three electrode configuration was used with working electrodes made
from either pyrolytic graphite (PG) or SWNT forests assembled on
PG, a calomel reference electrode, and a platinum wire as a counter
electrode.
The covalent attachment of redox cofactors onto a variety of
conductive substrates (i.e., Au, nanotubes, etc.) has sparked
considerable interest in mediatorless electrochemistry.20,21 Pa-
tolsky et al. have realized the highest turnover rate to glucose
oxidase (GOx) by the covalent attachment of FAD cofactor to
the tips of SWNTs forests.21 Such holoenzyme reconstruction
could prove challenging for a side-wall grafted, flavin-containing
cofactor, owing to the aforementioned strong π-π attraction
with the nanotube.
In this contribution we report the synthesis and covalent
functionalization of a synthetic FMN cofactor (12) to carboxylic
functionalized SWNTs. The synthetic design for this cofactor
was based on a theoretical and experimental study indicating
greater activity of bacterial luciferase than natural FMN.11,22
To provide adequate distance between SWNT and FMN
cofactor, an n-hexyl-spacer (toward CNT) and an n-pentyl spacer
(toward flavin) on either sides of the phosphate group were
introduced. A novel phosphite-assisted coupling methodology
is also described based on N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDC). As expected, the flavin
moiety of (12) strongly adheres to the side-walls of SWNTs,
which quenches its luminescence. The detachment of the flavin
cofactor from the side-walls of SWNTs was realized by treating
with a surfactant (sodium dodecyl sulfate (SDS)) or by reducing
the attached FMN to FMNH2. These findings pave the way to
ultimately link SWNTs with bacterial luciferase toward nano-
structured bioluminescence devices, wherein the redox cycling
of the flavin cofactor is electrically provided through the
nanotube as opposed to the use of mediators.23
Synthesis. N-(5′-Hydroxypentyl)-3,4-dimethylaniline (5). A mix-
ture of 9.1 g (75 mmol) of 3,4-dimethylaniline (4) and 3.05 g (24.88
mmol) of 5-chloropentanol in 15 mL of triethylamine (TEA) was stirred
at 110 °C for 6 h. After the mixture was cooled and methylenechloride
(200 mL) was added, the organic solution was washed with aqueous
Na2CO3 solution (10%, 40 mL). The aqueous layer was extracted twice
with methylene chloride (2 × 100 mL). The combined organic extracts
were dried over MgSO4 and rotary evaporated to dryness. The thin-
layer chromatography retention factor (Rf) of the target compound 5
was 0.1 with methylene chloride (MC)-methanol (MeOH) (95:5) and
was confirmed by ninhydrin test. Compound 5 was purified by flash
chromatography on silica gel in MC:MeOH (95:5) and crystallized
from n-hexane to produce 4.1 g of white crystals (80% yield): mp
39-40 °C. 1H NMR (300 MHz, CDCl3, Figure S1a of Supporting
Information (SI)): δ 6.93 (1H, d, J) 8 Hz), 6.44 (1H, d, J < 2 Hz),
6.38 (1H, dd, J) 8, J < 2 Hz), 3.66 (2H, t), 3.10 (2H, t), 2.19 (3H, s),
2.15 (3H, s), 1.63 (4H, m), 1.47 (2H, m). 13C NMR (75 MHz, CDCl3):
δ 146.7, 137.5, 130.4, 125.5, 115, 110.6, 62.9, 44.5, 32.6, 29.5, 23.53,
20.2, 18.9. Anal. Calcd for C13H21NO (MW ) 207.16): C, 75.32; H,
10.21; N, 6.76. Found: C, 74.92; H, 10.39; N, 6.65.
Experimental
Materials and Instrumentation. All reagents were purchased from
Sigma-Aldrich unless otherwise mentioned. All solvents were reagent
grade. 5-Chloropentanol and D2O (99.5%) were purchased from TCI
and Acros, respectively. Precautions were taken to avoid prolonged
exposure of isoalloxazine derivatives to direct sunlight and the blue/
UV part of the visible spectra by performing the experiments in a
laboratory lit with yellow fluorescent bulbs. Silica gel with 230-400
mesh was used for flash chromatography. Reverse phase C-18 columns
(40 and 80 g purification capacity) was performed in a Companion
flash chromatography apparatus (Teledyne Isco Inc.), equipped with a
single-wavelength UV detector. Millipore quality deionized water with
6-[N-(5′-Hydroxypentyl)-3,4-xylidino] Uracil (6). To a solution of
compound 5 (2.9 g, 14 mmol) in water-dioxane (1:1; 30 mL) refluxed
under argon, 0.672 g of 6-chlorouracil (4.59 mmol) was added with
stirring. After 15 h of reflux, the solvent was removed, the residue
was redissolved in water (40 mL), and the pH was increased to 11 by
the addition of aqueous NaOH solution (10 w/v %). The resulting
solution was extracted thrice with methylene chloride (3 × 70 mL) to
remove unreacted starting compound 5, which can be recycled. Aqueous
HCl solution (38 wt. %) was added to reduce the pH of the aqueous
extract down to pH 3, which induced precipitation of 6. The resulting
precipitate was collected by filtration and washed with water. Additional
compound 6 was obtained from the filtrate after water evaporation,
followed by dissolution in methanol, removal of undissolved moieties,
and rotary evaporation of the methanol filtrate. Both fractions of 6 were
combined and further purified by flash chromatography on a silica gel
with MC/MeOH ) 8:2 to recover 0.99 g (68% yield) of a yellow solid
with bright blue luminescence in MeOH solution, mp 228 °C. 1H NMR
(300 MHz, DMSO-d6, Figure S1b of SI): δ 10.34 (1H, br s), 10.10
(1H, br s), 7.23 (1H, d, J ) 4 Hz), 7.04 (1H, d, J < 2 Hz), 6.97 (1H,
dd, J ) 8, <2 Hz), 4.08 (1H, s), 3.57 (2H, t), 3.34 (2H, t), 2.24 (6H,
s), 1.46 (2H, m), 1.38 (2H, m), 1.26 (2H, m). 13C NMR (75 MHz,
DMSO-d6): 164.1, 154.8, 151.7, 140.2, 138.5, 136.2, 131.2, 129.0,
125.4, 77.4, 61.0, 51.3, 32.6, 27.6, 22.8, 19.9, 19.44. Anal. Calcd for
1
resistivity greater than 18 MΩ was used for all experiments. H, 13C,
and 35P NMR spectra were acquired on an Avance spectrometer
operating at a Larmor frequency of 300, 75, and 121.6 MHz,
respectively, unless otherwise mentioned. All spectra were recorded
in 5 mm NMR tubes containing 0.65 mL of DMSO-d6 or D2O at 295
K, unless otherwise mentioned. SWNTs, prepared by the high-pressure
carbon monoxide (HiPco) chemical vapor deposition process,24,25 were
(18) Lin, C. S.; Zhang, R. Q.; Niehaus, T. A.; Frauenheim, T. J. Phys. Chem.
C 2007, 111, 4069-4073.
(19) Lu, J.; Nagase, S.; Zhang, X.; Wang, D.; Ni, M.; Maeda, Y.; Wakahara,
T.; Nakahodo, T.; Tsuchiya, T.; Akasaka, T.; Gao, Z.; Yu, D.; Ye, H.;
Mei, W. N.; Zhou, Y. J. Am. Chem. Soc. 2006, 128, 5114-5118.
(20) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003,
299, 1877-1881.
(21) Patolsky, F.; Weizmann, Y.; Willner, I. Angew. Chem., Int. Ed. 2004, 43,
2113-2117.
(22) Lin, L. Y.-C.; Sulea, T.; Szittner, R.; Vassilyev, V.; Purisima, E. O.;
Meighen, E. A. Protein Sci. 2001, 10, 1563-1571.
(25) Bronikowski, M. J.; Willis, P. A.; Colbert, D. T.; Smith, K. A.; Smalley,
R. E. J. Vac. Sci. Technol. A 2001, 19, 1800-1805.
(26) Papadimitrakopoulos, F.; Konstadinidis, K.; Miller, T. M.; Opila, R.;
Chandross, E. A.; Galvin, M. E. Chem. Mater. 1994, 6, 1563-1568.
(23) Karatani, H.; Konaka, T. Bioelectrochem. Bioenerg. 1998, 46, 227-235.
(24) Nikolaev, P.; Bronikowski, M. J.; Bradley, R. K.; Rohmund, F.; Colbert,
D. T.; Smith, K. A.; Smalley, R. E. Chem. Phys. Lett. 1999, 313, 91-97.
9
656 J. AM. CHEM. SOC. VOL. 130, NO. 2, 2008

Synthesis of FMN-Functionalized SWNTs
A R T I C L E S
C17H23N3O3‚0.6H2O (MW ) 328.20): C, 62.21; H, 7.43; N, 12.80.
Found: C, 62.14; H, 7.33; N, 12.69.
from a gradient of methylene chloride/methanol with a gradual increase
of methanol to produce 9.6 g of HHCar (89% yield), a waxy solid,
mp 35-36 °C, followed with subsequent decomposition. 1H NMR (300
MHz, DMSO-d6, Figure S1i of SI): δ 6.75 (1H, br t), 4.33 (1H, t),
3.37 (2H, q), 2.88 (2H, q), 1.37 (13H, m), 1.26 (4H, m). 13C NMR (75
MHz, DMSO-d6): 156.0, 77.7, 61.1 33.0, 30.0, 28.7, 26.7, 25.7. Anal.
Calcd for C11H23NO3 (MW ) 217.17): C, 60.80; H, 10.67; N, 6.45.
Found: C, 60.99; H, 10.90; N, 6.88.
Isoalloxazine 5-Oxide (7). Sodium nitrite (0.35 g, 5.14 mmol) was
added to a solution of compound 6 (0.35 g, 1.1 mmol) in acetic acid
(5 mL) in a dark environment. The mixture was stirred at room
temperature for 3 h, and 2 mL of water was subsequently added. The
suspension was stirred for 3 h prior to rotary evaporation out of all
solvents. Compound 7 was obtained by filtration after the addition of
5 mL of ethanol and brief sonication. This was recrystallized from acetic
acid or ethanol to produce 0.3 g of 7 (80% yield), mp 228 °C, dec. 1H
NMR (500 MHz, DMSO-d6, Figure S1c of SI): δ 11.02 (1H, s), 8.09
(1H, s), 7.79 (1H, s), 4.52 (2H, t), 4.40 (1H, t), 3.44 (2H, q), 2.48 (3H,
s), 2.39 (3H, s), 1.72 (2H, m), 1.50 (4H, m). 13C NMR (125 MHz,
DMSO-d6): 157.2, 155.1, 153.1, 147.1, 136.1, 133.0, 132.5, 125.4,
120.4, 117.5, 61.1, 44.5, 32.7, 27.0, 23.2, 20.6, 19.4. Anal. Calcd for
C17H20N4O4‚0.5H2O (MW ) 353.38): C, 57.78; H, 5.99; N, 15.85.
Found: C, 57.41; H, 5.83; N, 15.55.
(6-([Flavin-pentyloxy]-H-phosphoryl)-hexyl)-carbamic Acid tert-
Butyl Ester (10). Compound 9 (0.284 g, 0.72 mmol) was dissolved in
dry pyridine (100 mL) at 100 °C under argon in dark environment and
cooled to 30 °C prior to adding HHCar (0.315 mg, 1.45 mmol) and
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC,
0.6 g, 2.9 mmol). The mixture was stirred for 24 h and the resulting
organic solvents were removed via rotary evaporation. TLC indicated
a new spot (i.e., compound 10) with Rf ) 0.45 using methylenechloride/
methanol (9:1). Compound 10 was purified by chromatography on
silica gel in methylene chloride-methanol (95:5) (0.263 g, 78%): mp
131 °C, followed with subsequent decomposition. 1H NMR (300 MHz,
DMSO-d6, Figure S1f of SI): 11.3 (1H, br s), 7.91 (1H, s), 7.81 (1H,
s), 6.82 (1H, d, JP-H ) 693 Hz), 6.76 (1H, br t), 4.59 (2H, br t), 4.0
(4H, m), 2.89 (2H, q), 2.51 (3H, s), 2.41 (3H, s), 1.71 (4H, m), 1.56
(4H, m), 1.36 (9H, s), 1.26 (6H, m). 13C NMR (75 MHz, DMSO-d6):
160.4, 156.1, 156.0, 150.5, 147.1, 137.5, 136.2, 134.2, 131.5, 131.1,
116.5, 77.7, 65.5,65.4, 65.3, 65.2, 49.1, 44.4, 30.3, 30.2, 30.0, 29.8,
28.7, 26.4, 26.2, 25.1, 22.7, 21.0, 19.2. 31P NMR (121.5 MHz, 85%
N-(5′-Hydroxypentyl) Isoalloxazine (8). An aqueous solution of
dithiothreitol (DTT, 1.4 g, 3.18 mmol, 7 mL of water) was added to a
suspension of 7 (0.25 g, 0.66 mmol) in ethanol (200 mL). The mixture
was refluxed under argon for 4 h. After the solution became clear, the
organic solvent was evaporated, and the residue was soaked with 5
mL of ethanol and filtered. The yellowish-orange solid was recrystal-
lized from ethanol to provide 0.23 g of 8 (96% yield), mp 293 °C,
followed with subsequent decomposition. 1H NMR (500 MHz, DMSO-
d6, Figure S1d of SI): δ 11.28 (1H, br s), 7.90 (1H, s), 7.79 (1H, s),
4.58 (2H, br t, J ) 7 Hz), 4.40 (1H, t, J ) 5 Hz), 3.43 (2H, q), 2.52
(3H, s), 2.41 (3H, s), 1.73 (2H, m), 1.50 (4H, m); 13C NMR (125 MHz,
DMSO-d6): 160.4, 156.1, 150.5, 147.0, 137.6, 136.2, 134.3, 131.5,
131.2, 116.5, 61.024, 44.67, 32.65, 26.87, 23.32, 21.08, 19.3. Anal.
Calcd for C17H20N4O3‚0.3H2O (MW ) 333.78): C, 61.17; H, 6.22; N,
16.79. Found: C, 61.46; H, 6.23; N, 16.46.
H3PO4 as a external reference, 1H-decoupled, D2O): 16.58, 10.87 (J1
P-H
) 660 Hz, d, J3P-H ) 2.4 Hz, quintet). Anal. Calcd for C28H42N5O7P‚CH3-
OH (MW ) 623.67): C, 55.85; H, 7.43; N, 11.23; P, 4.97. Found: C,
56.63; H, 7.48; N, 11.27; P, 4.75.
(6-([Flavin-pentyloxy]-phosphoryloxy)-hexyl)-carbamic Acid tert-
Butyl Ester (11). Compound 10 (84 mg, 0.142 mmol) was dissolved
in 0.2 M iodine in pyridine-THF (1:1, 10 mL) in a dark environment
and triethylamine (1 mL) was added. The mixture was stirred for 5 h,
and then water was added. TLC showed a new spot with Rf ) 0 (i.e.,
compound 11) using MC/MeOH (9:1), whereas the rest of starting
reagents have higher Rf. The resulting organic solution was evaporated,
and the resulting residue was purified with a reverse phase C-18 column
(80 g pack, Teledyne ISCO) using water-MeOH gradient (starting from
100:0 and finishing at 50:50) to produce 40 mg of 11 (46% yield), mp
120 °C, followed with subsequent decomposition. 1H NMR (300 MHz,
DMSO-d6, Figure S1 g of SI): 11.31 (1H, br s), 7.87 (1H, s), 7.81
(1H, s), 6.76 (1H, br t), 3.62 (4H, m), 2.85 (2H, m), 2.51 (3H, s), 2.38
(3H, s), 1.72 (2H, m), 1.40-1.65 (6H, m), 1.34 (9H, s), 1.23 (6H, m).
13C NMR (75 MHz, DMSO-d6): 160.4, 156.2, 156.0, 150.4, 147.2,
137.1, 136.2, 134.2, 131.5, 131.2, 116.54, 77.7, 64.3, 56.5, 31.0, 30.9,
30.7, 30.0, 26.8, 25.7, 23.3, 21.0, 19.3. 31P NMR (121.5 MHz, 85%
H3PO4 as a external reference, D2O): -1.10 (1H, s).
Flavin Pentyl H-Phosphonates (9). Compound 8 (52.54 mg, 0.16
mmol) was dissolved in dry pyridine (10 mL) at 100 °C under argon
in a dark environment. The solution was subsequently cooled to
30 °C, and phosphorous acid (54 mg, 0.66 mmol) was added prior to
the addition of 97 mg (0.32 mmol) of 2,4,6-triisopropylbenzenesulfonyl
chloride. The mixture was stirred at room temperature for 15 h, and
the solvent was evaporated. The residue was purified by flash
chromatography either on a reverse C-18 column (40 g cartridge,
Teledyne ISCO) in water or on a silica gel in MC-MeOH gradient
(which takes significantly longer than the reverse C-18 column) to yield
compound 9. In the case of using reverse phase C-18 column, 9 comes
out together with pyridine and water, both of which need to be removed
by washing with ethanol. The final yellow solid was obtained with a
yield of 80% (mixture of acid and its pyridinium salt (1:2 ratio, as
1
inferred by the H NMR of Figure S1e)), mp 175 °C, followed with
subsequent decomposition. 1H NMR (300 MHz, DMSO-d6, Figure S1e
of SI): δ 11.31 (1H, br s), 8.71 (1.5H, pyridine, d, J ) 4 Hz), 8.1
(0.75H, pyridine, t, J ) 7 Hz), 7.84 (1H, s), 7.77 (1H, s), 6.72 (1H, d,
JP-H ) 665 Hz), 4.55 (2H, br t), 3.91 (2H, br q), 2.50 (3H, s), 2.38
(3H, s), 1.69 (4H, m), 1.52 (2H, m). 13C NMR (75 MHz, DMSO-d6):
160.4, 156.2, 150.4, 147.2, 140.4, 137.5, 136.3, 134.2, 131.4, 131.1,
125.6, 116.5, 64.23, 44.5, 30.1, 26.44, 22.85, 21.0, 19.3. 31P NMR
(121.5 MHz, 85% H3PO4 as a external reference, D2O): 9.66 (1H, d,
JHP ) 660 Hz). Anal. Calcd for C17H21N4O5P‚1.6H2O (MW )
421.18): C, 48.48; H, 5.79; N, 13.30; P, 7.35. Found: C, 48.27; H,
5.59; N, 12.94; P, 7.11.
6-([Flavin-pentyloxy]-phosphoryloxy)-hexyl-ammonium-tri-
fluroro Acetic Acid Salt (12). Trifluoroacetic acid (1 mL) was added
to a solution of compound 11 (36 mg, 0.0593 mmol) in methylene
chloride (10 mL). The mixture was rapidly stirred at room temperature
for 30 min, and the organic solvent was completely evaporated. The
resulting residue was purified using reverse C-18 column (80 g pack,
Teledyne Isco Inc.) using water-MeOH gradient (starting from 100:0
and ending to 0:100) as eluent to produce 35 mg of 12 (95% yield),
1
mp 120 °C. H NMR (300 MHz, DMSO-d6, Figure S1h of SI): 7.58
(1H, s), 7.54 (1H, s), 4.52 (2H, br t), 3.76 (2H, q), 3.69 (2H, q), 2.87
(2H, t), 2.43 (3H, s), 2.30 (3H, s), 1.75 (2H, m), 1.62 (2H, m), 1.35-
1.6 (6H, m), 1.23 (4H, m). 13C NMR (75 MHz, DMSO-d6): 160.5,
158.6, 156.2, 147.2, 137.6, 136.3, 134.3, 131.5, 131.2, 116.6, 66.1,
44.2, 44.5, 30.1, 30.0, 27.3, 26.6, 25.7, 25.0, 22.8, 21.1, 19.3. 31P NMR
(121.5 MHz, 85% H3PO4 as a external reference, D2O): -1.49
(1H, s).
(6-Hydroxy-hexyl)-carbamic Acid tert-Butyl Ester (HHCar). To
a mixture of di-tert-butyl dicarbonate (11 g, 50 mmol) in methanol
(100 mL), 6-amino hexyl alcohol (5.8 g, 49.5 mmol) dissolved in
methanol (200 mL) was added dropwise. The solution was stirred
overnight at room temperature, and the methanol was removed away
by rotary evaporation. The resulting solid was extracted with methylene
chloride against water. The organic layer was evaporated, and the
residue was purified using flash chromatography on a silica gel column
Acid Functionalization of SWNTs (a-SWNTs). A 100 mg portion
of HiPco SWNTs (Lot. CM26-0036AB-1) was shortened and carboxylic
9
J. AM. CHEM. SOC. VOL. 130, NO. 2, 2008 657

A R T I C L E S
Ju and Papadimitrakopoulos
Scheme 2. Synthetic Route for Realizing Amine-Terminated Analogues of FMNa
a Conditions: (a) HO(CH2)5Cl/TEA, reflux; (b) 6-chlorouracil/H2O/dioxane, reflux; (c) NaNO2/AcOH, room temp; (d) DTT/EtOH, reflux; (e) H3PO3/
TPSO2Cl/Py, room temp; (f) HO(CH2)6NH-t-BOC/EDC/Py, room temp; (g) I2/THF/Py, room temp followed by H2O addition; (h) triflic acid/CH2Cl2, room
temp.
functionalized by sonication-assisted oxidation in 40 mL of a 3/1 v/v
mixture of H2SO4 (96.4%) and HNO3 (98%), on the basis of a
previously established method.27,28 The resulting black solution was
mildly sonicated for 4 h prior to filtering it from a 0.45 µm Teflon
filter. The precipitate was washed with copious amount of water until
the pH of the filtrate reached 6, and then it was vacuum-dried at 60 °C
for 1 day to provide a-SWNTs.
Coupling 12 with a-SWNTs and Purification (12-SWNT). A 5
mg portion of a-SWNT was dispersed in DMF (20 mL) using mild
sonication (80 W) for 1 h. To this 5 mg (8 µmol) of 12 and 20 mg
(100 µmol) of EDC were added, and the mixture was stirred overnight.
After adding 50 mL of acetone to the mixture to facilitate precipitation
of the 12-SWNT adduct, the flocculate was filtered and washed with
copious amount of water and then dried in vacuum. A 3 mg portion of
12-SWNT was redispersed in 10 mL of deionized (DI) water and mildly
sonicated (80 W, Cole Parmer 8891) for 10 min to generate a greenish-
gray homogeneous suspension of 12-SWNT and physisorbed 12.
Subjecting this suspension to a 10 min, 5000g centrifugation resulted
in precipitating out the 12-SWNT adduct while leaving behind a bright
greenish supernatant solution of 12 that was easily decanted. This
sonication-centrifugation-decantation cycle was repeated four times
to remove the noncovalently coupled 12 from the SWNTs.
Covalent Attachment of 12 onto SWNT Forests. SWNT forests
on pyrolytic graphite (PG) electrode were prepared according to
literature.30 In brief, polished PG electrodes were immersed in 90%
v/v methanolic solution of Nafion (5 wt. %, Sigma-Aldrich) for 15
min, followed by washing with water. Subsequently, these electrodes
were immersed in aqueous iron(III) chloride solution (5 mg/mL) for
15 min, followed by washing the excess of FeCl3 in DMF before
immersing it in a DMF dispersion of a-SWNT (ca. 0.1 mg/mL) for 30
min, to facilitate the vertical nanotube forest assembly.13,30,31 These
nanotube forest (NTF)-modified electrodes were rinsed with copious
amounts of methanol, dried and covalently functionalized by reacting
12 (2 mM) with carboxyl moiety of the NTF in the presence of EDC
(20 mM), and dissolved in 50 µL of water for 3 h.13 These electrodes
were then rinsed with copious amounts of water and dried in air.
Results and Discussion
The multiplicity of hydroxyl groups in natural flavin mono-
nucleotide (FMN) (3), renders challenging the controllable
covalent attachment of 3 to carboxyl-functionalized SWNTs (a-
SWNTs). This directed us to investigate hydroxyl-lacking FMN
derivatives that possess similar cofactor activity with lu-
ciferase.11,22 As explained in the Introduction section, the
phosphate derivative of 8 (see Scheme 2) does meet this
requirement, and its synthesis has been previously reported by
Frier et al.32 The introduction of a n-pentyl, as opposed to
n-hexyl spacer between the isoalloxazine ring (1) and phosphate
group was found to provide a much higher binding affinity
toward apo-luciferase.11 Steps a-e in Scheme 2 illustrate the
previously established methodology for the synthesis of the
H-phosphite derivative of 8, which upon I2-assisted oxidation
produces the phosphate derivative of 8. Further functionalization
of the n-hexyl H-phosphite derivative has been also reported
using adamantine carboxylic acid chloride activation to attach
this to the 3′-end of DNA oligomers. As will become apparent
later on in the discussion, the adamantine carboxylic acid
chloride activation is unsuitable for realizing a phosphodiester
derivative of 9. In this contribution, a versatile coupling
methodology has been developed using EDC to link an
N-protected aminoalcohol with the H-phosphite group (Scheme
Dispersion of 12-SWNT Using Sodium Dodecyl Sulfate (SDS).
A 0.5 mg portion of 12-SWNT (or a-SWNT) was mildly sonicated
(80 W) in 4 mL of DI water containing 1 wt. % of SDS, according to
a previously described methodology.29
Redox Cycling of 12-SWNT. A mixture of 1 mg of 12-SWNT in
4 mL of D2O was suspensioned for 20 min using a cup-horn sonication
(Cole Parmer ultrasonicprocessor) at 600 W intensity. The resulting
suspension was subjected to two sonication-centrifugation-decantation
cycles described above, prior to taking the supernatant. The suspension
was then purged with Argon (99.998%, oxygen level around few ppm)
in a capped cuvette and purged for 20 min prior to collecting both
UV-vis-NIR and PL spectra. Following the addition of an adequate
amount of a 1 M of sodium dithionite (Na2S2O4) D2O solution, the
12-SWNT D2O suspension (with or without SDS) was purged with Ar
for an additional 10 min prior to collecting both UV-vis-NIR and
PL spectra. Similar redox cycling was performed with 8 as the model
compound.
(27) Liu, J.; Rinzler, A. G.; Dai, H.; Hafner, J. H.; Bradley, R. K.; Boul, P. J.;
Lu, A.; Iverson, T.; Shelimov, K.; Huffman, C. B.; Rodriguez-Macias, F.;
Shon, Y.-S.; Lee, T. R.; Colbert, D. T.; Smalley, R. E. Science 1998, 280,
1253-1256.
(28) Chattopadhyay, D.; Galeska, I.; Papadimitrakopoulos, F. J. Am. Chem. Soc.
2003, 125, 3370-3375.
(30) Yu, X.; Kim, S. N.; Papadimitrakopoulos, F.; Rusling, J. F. Mol. BioSyst.
2005, 1, 70-78.
(29) O’Connell, M. J.; Bachilo, S. M.; Huffman, C. B.; Moore, V. C.; Strano,
M. S.; Haroz, E. H.; Rialon, K. L.; Boul, P. J.; Noon, W. H.; Kittrell, C.;
Ma, J.; Hauge, R. H.; Weisman, R. B.; Smalley, R. E. Science 2002, 297,
593-596.
(31) Wei, H.; Kim, S. N.; Marcus, H. L.; Papadimitrakopoulos, F. Chem. Mater.
2006, 18, 1100-1106.
(32) Frier, C.; Decout, J.-L.; Fontecave, M. J. Org. Chem. 1997, 62, 3520-
3528.
9
658 J. AM. CHEM. SOC. VOL. 130, NO. 2, 2008

Synthesis of FMN-Functionalized SWNTs
A R T I C L E S
2f). Upon I2 oxidation and amine deprotection, the n-pentyl-
FMN cofactor 12 was covalently coupled to the carboxyl
functionalities of a-SWNTs.
The synthetic steps that lead to the synthesis of 9 are listed
in Scheme 2 and were reproduced from a previous report.32 To
provide adequate distance between a carbon nanotube and the
phosphate group of the FMN cofactor, while ensuring electron
tunneling from the nanotube to isoalloxazine moiety, HO-
(CH2)6-NH2 was chosen as an FMN-SWNT spacer. For this,
the N-t-Boc protected aminohexylalcohol (HHCar) was first
synthesized, as described in the Experimental section. The
coupling of HHCar to 9 was proven unsuccessful using
adamantine carboxylic acid chloride activation of the H-
phosphite group of 9. While the production of adamantine
carboxylic ester of 9 was confirmed by TLC, this compound
was proven quite stable against subsequent displacement by
HHCar.
An alternative methodology to link HHCar to 8 and produce
compound 11 was also attempted using the N,N-diisopropyl-
chloro phosphoramidite and proved unsuccessful on the basis
of the extreme O2 and light susceptibility of the intermediate
that quickly returned to the phosphate derivative of 8.32 For
this, a new coupling methodology has been developed using
carbodiimide chemistry, such as EDC.
Figure 1. 31P NMR of compounds 9-12 in D2O. H3PO3 was used as
internal standard for only compound 9 and is indicated with a star. The
indicated 31P NMR shifts were against H3PO4 as an external standard.
EDC is well-known coupling agent for amide formation
between carboxylic acids and amine groups33 or phosphoramide
formation between phosphate and amine groups.34 To the best
of our knowledge, EDC has never been used for the coupling
of a hydroxyl group on H-phosphite with primary alcohols.
Compound 9 was first dissolved in pyridine at 100 °C, and the
clear solution was cooled to room temperature to avoid possible
deprotection of the t-Boc group of HHCar. Subsequently,
HHCar and excess of EDC ( 2 equiv of limiting reagent) were
added and stirred for 24 h to afford a yield of 78% (Scheme
2f). Here, it is worth mentioning that the pyridine used in this
coupling was reagent grade and not dried, unlike the two
aforementioned coupling methodologies.32,35 Compound 9 can
be regenerated if EDC is not used in excess to remove the
residual water in pyridine, thereby lowering the yield. As
around 9.66 and 6.18 ppm,36 respectively, against H3PO4 as an
external standard. When the proton decoupler was off (1H
coupled), the phosphite proton of 9 was split into two triplets
centered around 12.39 and 6.95 ppm, respectively, with short-
range coupling of phosphite proton to 31P at J1
) 660 Hz.
) 8.92 Hz) originates from the
P-H
Such triplet splitting (J3
P-H
neighboring -O-CH2- group, while the acidic nature of the
P-OH proton renders it highly exchangeable with deuterium
from the D2O solvent and therefore does not participate in this
splitting. Similar short-range coupling was observed for the H3-
PO3 (H-PO-(OH)2) of the internal standard, with no further
long-range coupling. For 10, the presence of two neighboring
-O-CH2- groups produce shift the 31P peak to 13.7 ppm (with
the proton decoupler on). When the proton decoupler was turned
off, two quintets were produced with a short- and long-range
coupling from the phosphite and methylene protons, respec-
1
presented in the Experimental section, H, 13C, and 31P liquid
NMR, as well as elemental analysis has been used to confirm
the formation of 10. The 31P NMR of 9, 10, 11, and 12 will be
further discussed below. The more acidic nature of hydroxyl
group of phosphite group, as compared to carboxylic acid, is
currently believed to be responsible for the efficient coupling
of H-phosphite with the primary hydroxyl group.
Following the successful formation of 10, the H-phosphite
diester was oxidized using I2 in the presence of pyridine to afford
compound 11 with a yield of 46%. This was followed by amine
group deprotection using trifluoroacetic acid, while the phosphor-
diester linkage remains intact. Figure 1 illustrates the 31P NMR
of compounds 9-12. Spectra were obtained with and without
proton decoupling to confirm the local environment of the
phosphorus atom.36 With the proton decoupler on, the 31P NMR
of compound 9 and H3PO3 (internal standard) produced a singlet
tively, centered at 16.6 and 10.9 ppm, (J1
) 693 Hz and
P-H
J3
) 7.43 Hz). In the case of compound 11, the lack of
P-H
phosphite H resulted in single 31P peak (ca. -1.10 ppm) for
both the proton decoupler on and off. The poor signal-to-noise
ratio along with the two remote -O-CH2- groups renders
1
1
unresolvable the H long-range coupling when the decoupler
was off. Compound 12 showed a similar 31P position (ca. -1.49
ppm) with 11, while this time the higher signal-to-noise ratio
revealed a poorly resolved quintet with significant line broaden-
ing, when the proton decoupler was off.
Following the successful synthesis and characterization of
12, its amine moiety was EDC-coupled with the carboxylic acid
groups on SWNTs (12-SWNT). This was realized by dispersing
a-SWNTs in DMF, subsequently adding 12 in the presence of
EDC, and allowing the mixture to stir overnight (Scheme 3).
Since the flavin moiety was shown to strongly adsorb onto
SWNTs,17,18 extensive sonication-centrifugation-decantation
(SCD) cycles (at least four times using 10 min bath sonication
at 80 W intensity) were required to completely remove phys-
isorbed 12 moieties on SWNTs, while leaving behind the
(33) Sheehan, J. C.; Ledis, S. L. J. Am. Chem. Soc. 1973, 95, 875-879.
(34) Adessi, C.; Matton, G.; Ayala, G.; Turcatti, G.; Mermod, J. J.; Mayer, P.;
Kawashima, E. Nucleic Acids Res. 2000, 28, E87.
(35) Usman, N.; Ogilvie, K. K.; Jiang, M. Y.; Cedergren, R. J. J. Am. Chem.
Soc. 1987, 109, 7845-7854.
(36) Gorenstein, D. G. Phosphorus-31 NMR: Principles and Applications, 1st
ed.; Academic Press: Orlando, FL, 1984.
9
J. AM. CHEM. SOC. VOL. 130, NO. 2, 2008 659

A R T I C L E S
Ju and Papadimitrakopoulos
Figure 2. FTIR spectra of 12-SWNT (top), and 12 (bottom).
Figure 3. UV-vis-NIR spectra of SDS-dispersion of a-SWNT (i, dashed
line), 12-SWNT (ii, solid line), and 12 (iii, dotted line). Left inset shows
the spectral subtraction of ii - i as compared to iii, while the two right
insets depict AFM traces and line profiles of 12-SWNT on mica.
Scheme 3. Covalent Attachment of 12 to Acid-Treated SWNTs
(a,b) and Flavin-Tethered SWNTs and Its Conformation in the
Absence (c) and Presence (d) of Sodium Dodecylsulfate (SDS)
be enhanced because of the strong flavin adsorption onto the
side-walls of the SWNTs.37 Such strong absorption of the flavin
moiety on the SWNTs is expected to also lead to a conforma-
tional collapse of the FMN onto the side-wall of the nanotube
(see Scheme 3c). The asymmetric and symmetric C-H stretch-
ing bands at 2917 and 2856 cm-1, respectively, provide an initial
indication for such collapsed conformation. Unlike the broad
and weak 2958 and 2858 cm-1 C-H stretching in 12, the sharp
2918 and 2850 cm-1 C-H stretching in 12-SWNT indicates
an entirely different -(CH2)- conformation in either case.38,39
On comparison with linear paraffin CnH2n+2 evaporated on gold,
one can conclude that the alkyl groups of 12 are packed
irregularly in its salt form.38,39 Once 12 is coupled on the
SWNTs, the pronounced increase in C-H stretching intensity
suggests a substantial ordering of the alkyl spacers at the vicinity
of the nanotubes. This might originate from favorable hydro-
phobic interactions with the highly ordered graphene side-walls
of SWNTs.38,39 This is schematically illustrated at the bottom
right part of Scheme 3, which is synergistic with the facile
adsorption of the flavin moiety onto SWNTs. Such adsorption
will also force the hydrophilic phosphate diester in the vicinity
of the nanotube. As will become evident later on in the
discussion, such a configuration renders 12-SWNT dispersible
in aqueous media, yet quenches the strong photoluminescence
efficiency of the isoalloxazine ring (1).
Figure 3 illustrates the UV-vis spectra of aqueous dispersions
of a-SWNTs (i) and 12-SWNTs (ii), in comparison to an
aqueous solution of 12 (iii). In excess of four successive
sonication-centrifugation decantation cycles have been applied
to 12-SWNT to ensure that only covalently linked FMN groups
remain onto the SWNTs. The lack of sharp E22 and E11
absorptions, at 600-1000 and 1000-1600 nm ranges, respec-
tively, are indicative of the substantial side-wall oxidation and/
or the presence of small bundles of SWNTs. The two pro-
nounced peaks of iii at 371 and 444 nm are characteristic of
the isoalloxazine ring. The top-left inset of Figure 3 illustrates
the spectral subtraction of i from ii, in comparison to iii. The
close spectral resemblance of this subtracted peak (ii - i) to iii
provides an additional confirmation for the successful attachment
of 12 to SWNTs. Upon closer inspection, however, a 4 nm red-
covalently grafted adduct of 12. The complete removal of
residual physisorbed 12 was confirmed using H NMR with
1
98% deuterated SDS-d25 surfactant (see Figure S2 in Supporting
Information). In the case of physisorbed 12 on SWNT, treatment
with SDS-d25 was intended to disperse SWNTs in D2O and to
liberate any residual 12 that can be easily detected from its
aromatic region of 1H that persisted in the four SCD cycles. As
proven by Figure S2, no residual 12 was detected, indicating
that the four SCD cycles were sufficient to completely remove
the physisorbed 12 from 12-SWNT adduct.
Figure 2 illustrates the FTIR of 12-SWNT and 12. The
increase in the broad 3450 cm-1 N-H stretching vibration from
12 to 12-SWNT is in accordance with the formation of an amide
group on top of the uracil N-H of the isoalloxazine ring.37 This
is in agreement with the simultaneous decrease of the broad
N-H stretching of the RNH3+ salt of 12 (ranging from 3300-
2600 cm-1) in the 12-SWNT spectrum during the formation of
the amide linkage. While the amide-I band overlaps with the
already existing amide linkages from the isoalloxazine ring, an
increase is witnessed for the 1578 cm-1 (amide-II) mode as
compared to the 1540 cm-1 CdN out-of-plane stretching mode
of flavin moiety or the 1209 and 1182 cm-1 absorptions of the
phosphate diester group.37 The pronounced increase of the 1385
cm-1 absorption in 12-SWNT as compared to 12 is presently
unknown. However, this peak might originate from the N1-
C2-N3 antisymmetric stretching accompanied by the deforma-
tion of the uracil moiety of the isoalloxazine group and might
(38) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem.
Soc. 1987, 109, 3559-3568.
(39) Nuzzo, R. G.; Fusco, F. A.; Allara, D. L. J. Am. Chem. Soc. 1987, 109,
2358-2368.
(37) Abe, M.; Kyogoku, Y. Spectrochim. Acta 1987, 43A, 1027-1037.
9
660 J. AM. CHEM. SOC. VOL. 130, NO. 2, 2008

Synthesis of FMN-Functionalized SWNTs
A R T I C L E S
the Supporting Information). On the other hand, the as-
synthesized 12-SWNT showed almost no flavin-originated PL,
with an exception of a shoulder at ca. 520 nm, situated on top
of a broad emission feature (370-600 nm) of a-SWNTs. This
broad a-SWNT PL emission is believed to originate from a
variety of carbonaceous fragments during acid-purification
procedure of SWNTs.44 The π-π stacking between the isoal-
loxazine ring and the side-walls of SWNTs17,18 appears to be
responsible for such quenching, as shown in Scheme 3c. Similar
PL quenching has been reported for pyrene,45-47 and metal-
loporphyrin derivative,48 onto the sidewalls of SWNTs. The
sonication-assisted dispersion of 12-SWNT with sodium dodecyl
sulfate (SDS) (0.1 w/v %) is capable of recovering the flavin
luminescence, as shown in Figure 4. Such PL recovery originates
from the effective micellerization of SWNTs by SDS that
organize tightly along the nanotube side-walls and prevent
readsorption of the flavin moiety to SWNT. This is schemati-
cally illustrated in Scheme 3d.
Figure 4. Solutions PL spectra of various constructs. Inset illustrates a
close up at 520 nm. Excitation wavelength was 350 nm.
shift is observed for the λmax of (ii - i)) (448 nm) as opposed
to the λmax of iii (444 nm). This slight red-shift might originate
from the strong physisorbtion of flavin onto SWNTs (as
indicated by both experimental17 and theoretical studies18). A
recent report on DNA-wrapped nanotubes also indicates similar
red-shifts due to π-π stacking of both purine and pyrimidine
bases.40 While π-π stacking is known to produce slight
hypochromism,40 one can utilize the flavin extinction coefficient
at the 444 nm absorption peak (12600 L/mol‚cm)32 to make a
rough estimate of the % of covalent functionalization of FMN
on SWNTs. Since not all SWNTs get suspended, and a
significant fraction remains as a precipitate, the amount of
suspended nanotubes was estimated by the extinction coefficient
reported for acid-treated HiPco SWNTs at 9750 cm-1 (1025
nm) in terms of carbon equivalency (260 ( 20 L/mol‚cm).41
On the basis of these two values, the covalent functionalization
of 12-SWNT was estimated to one FMN residue for every 43-
50 carbons. This value is in good agreement with the percent
carboxylic acid functionalization on the SWNT, as introduced
by the specific acid treatment.42
The upper right inset of Figure 3 illustrates a representative
tapping-mode height AFM-image of 12-SWNT deposited on
mica substrate. The line-scans along positions 1 and 2 are shown
below. The approximate height of SWNTs produced by the
HiPco synthesis methodology is on the order of 1 nm,43 which
is in good agreement with the indicated height of line-scan 1.
A number of protruding features can be seen distributed
unevenly along this nanotube with height varying from 2 to 3
nm, believed to originate from the attached FMN groups.
Photoluminescence Behavior. Figure 4 illustrates the 350
nm excited photoluminescence (PL) spectra of 12, SDS-
dispersed 12 (12/SDS), and SDS-dispersed 12-SWNT (12-
SWNT/SDS), normalized with respect to their maximum
intensity at ca. 517 nm. In addition, the PL spectra of 12-SWNT
and a-SWNT are also plotted and normalized at the 460 nm
with respect to the absorption of 12-SWNT/SDS sample. 12
emits brightly at green, with an emission maxima of 519 in
dilute (10-4 M and below) water solutions, which is the
characteristic emission of the flavin moiety (see Figure S3 of
Upon closer inspection of the PL emission of 12, 12/SDS,
and 12-SWNT/SDS one can witness a progressive broadening
of their PL full-width-half-maximum (FWHM) from 42 to 48
and 63 nm as well as a gradual blue-shift of the PL peak
emission from 519 to 517 and 516 nm, respectively. Such blue-
shift is believed to originate by lowering of the dielectric
constant of the surrounding media from water to the aliphatic
SDS tail group.49 Similarly, the progressive broadening of fwhm
demonstrates the inhomogeneity that the flavin moiety experi-
ences in the proximity of SDS and SDS/SWNT.
Chemical Redox Behavior. The redox properties of the
flavin group are of profound importance to biocatalysis.6 Figure
5 illustrates the UV-vis and PL behavior of 8 as a function of
its redox state. Initially, the aqueous solution of 8 was purged
with argon for 20 min to completely remove any dissolved O2.
Subsequently, two aliquots of aqueous solution of dithionite
(Na2S2O4) (3.3 × 10-4 M each) was added sequentially to 9 ×
10-6 M of 8 to reduce the oxidized form of flavin to the reduced
form,1,11 as shown in Scheme 4, and its UV-vis and PL (350
nm excitation) were collected prior to the addition of the next
dithionite aliquot. Next, 100 mL of air (9.3 × 10-4 M of O2)
was slowly bubbled to the solution of dithionite and 8, to return
back the flavin to its original oxidation state.1 Such reduction
is associated with luminescence quenching and a profound
conformational change for the isoalloxazine ring away from
planarity, as can be witnessed from Scheme 4.50,51 This redox
cycle is also accompanied by major changes in the UV-vis
spectra of 8, as shown in Figure 5. The oxidized and reduced
forms of 8 have their first absorption maximums at 444 and
316 nm, indicative of the presence and lack of extended
(44) Xu, X.; Ray, R.; Gu, Y.; Ploehn Harry, J.; Gearheart, L.; Raker, K.; Scrivens,
W. A. J. Am. Chem. Soc. 2004, 126, 12736-12737.
(45) Alvaro, M.; Atienzar, P.; Bourdelande, J. L.; Garcia, H. Chem. Phys. Lett.
2004, 384, 119-123.
(46) Liu, L.; Wang, T.; Li, J.; Guo, Z.-X.; Dai, L.; Zhang, D.; Zhu, D. Chem.
Phys. Lett. 2002, 367, 747-752.
(47) Qu, L.; Martin, R. B.; Huang, W.; Fu, K.; Zweifel, D.; Lin, Y.; Sun, Y.-P.;
Bunker, C. E.; Harruff, B. A.; Gord, J. R.; Allard, L. F. J. Chem. Phys.
2002, 117, 8089-8094.
(40) Hughes, M. E.; Brandin, E.; Golovchenko, J. A. Nano Lett. 2007, 7, 1191-
1194.
(48) Murakami, H.; Nomura, T.; Nakashima, N. Chem. Phys. Lett. 2003, 378,
481-485.
(41) Zhao, B.; Itkis, M. E.; Niyogi, S.; Hu, H.; Zhang, J.; Haddon, R. C. J.
Phys. Chem. B 2004, 108, 8136-8141.
(49) Greaves, M. D.; Deans, R.; Galow, T. H.; Rotello, V. M. Chem. Commun.
1999, 785-786.
(42) Marshall, M. W.; Popa-Nita, S.; Shapter, J. G. Carbon 2006, 44, 1137-
(50) Zhang, W.; Zhang, M.; Zhu, W.; Zhou, Y.; Wanduragala, S.; Rewinkel,
D.; Tanner, J. J.; Becker, D. F. Biochemistry 2007, 46, 483-491.
(51) Dixon, D. A.; Lindner, D. L.; Branchaud, B.; Lipscomb, W. N. Biochemistry
1979, 18, 5770-5775.
1141.
(43) Fantini, C.; Jorio, A.; Souza, M.; Strano, M. S.; Dresselhaus, M. S.; Pimenta,
M. A. Phys. ReV. Lett. 2004, 93, 147406.
9
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A R T I C L E S
Ju and Papadimitrakopoulos
Figure 6. Tracewise changes of PL (left) and UV-vis absorption (right)
at 520 and 444 nm, respectively of 12-SWNT, upon sequential addition of
Na2S2O4 (reduction) and air (O2) (oxidation), in the absence (A) and presence
(B) of SDS. Lines were added to guide the eye. Excitation wavelength was
350 nm.
Figure 5. UV-vis (left) and PL (right) spectra of 8 (9 × 10-6 M) (trace
1) upon sequential sodium dithionite reduction (traces 2 and 3, for 3.3 ×
10-4 M Na2S2O4 added each) and air (O2) reoxidation (trace 4, 9.3 × 10-4
M O2 added), respectively. Inset shows tracewise changes of PL (left) and
UV-vis absorption (right) at 518 and 444 nm, respectively. Lines were
added to guide the eye. Excitation wavelength was 350 nm.
while the UV-vis absorption of 12-SWNT at 444 nm was
completely restored, after the second air addition, the PL
emission recovered only by ca. 30%. Subsequent addition of
air did not increase the PL emission from the 30% level. On
the other hand, in the presence of SDS, the PL emission
recovered completely (trace no. 3 and no. 4 of Figure 6B). This
provides an additional indication that in the absence of SDS
nanotube coating, the strong π-π interactions of the reoxidized
FMN promote a collapsed configuration of the flavin on the
nanotube side-walls as shown in Scheme 3c.
Scheme 4. Chemical Structure and Side View of Molecular
Conformation of Flavin in Its Oxidized (left) and Reduced (right)
State, along with Its Luminescence Characteristics
Electrochemical Studies of Flavin Derivatives on SWNT
Forests Arrays. The electrochemical response of redox reagents
greatly depends on their ability to rapidly diffuse to and from
the working electrode. While this is the case for small molecules,
the large hydrodynamic volume of 12-SWNT profoundly
impedes the aforementioned diffusion. To overcome this
problem, the redox behavior of covalently and noncovalently
attached flavin moieties was studied on top of SWNT forest
arrays.17,21 These vertically aligned arrays of nanotubes provide
good electrical contacts with the underlying electrode, as well
as the necessary carboxylic functionalities for covalent attach-
ments of 12. A large number of studies have shown that such
forest configuration provides approximately an order of mag-
nitude enhancement in signal-to-noise ratio that has been
instrumental for development of ultrasensitive electrochemical
sensors.13,30,52 While the exact nature of such enhancement is
not completely elucidated, it is strongly believed that is related
to the presence of oxygenated sites (i.e., phenols and corre-
sponding quinones) at the tips of these nanotube forests.53 These
oxygenated sites provide effective mediation in the vicinity of
these tips, which are responsible for obtaining an ideal separation
between oxidation and reduction peaks at 25 °C (i.e., 59/n mV,
where n is the number of electrons per molecule).53 The only
drawback of these nanotube forest (NTF) electrodes is their
susceptibility to sonication, which disassembles such forest
assembly. This prevents the use of sonication for desorbing the
flavin moiety from nanotubes, thereby limiting us only to redox-
assisted desorption (described above).
π-conjugation in the isoalloxazine ring, respectively. The
dithionite reduction and O2-assisted reoxidation is traced in the
inset of Figure 5 by tracking both the 444 and 519 nm UV-vis
absorption and PL emission, respectively. The addition of two
aliquots of dithionite was found capable to completely convert
8 to its reduced form (trace #3).50,51 The fully reversible redox
of 8 was demonstrated by the addition of an excess amount of
molecular oxygen that returns the flavin moiety back to its
oxidized form (trace #4). Compounds 8-12 exhibited similar
reversible redox behavior in terms of their absorption and PL
characteristics that helped us establish an important control
toward the investigation of the redox properties of 12-SWNT.
Figure 6 panels A and B illustrate the redox-dependent
changes of the 444 nm UV-vis absorption and 520 nm PL
emission of 12-SWNT in the absence and presence of SDS,
respectively (as obtained from Figure S4 of the Supporting
Information). Since the as-synthesized 12-SWNT exhibits little
or no PL emission at 516-520 nm because of the strong π-π
interactions of flavin with SWNTs, intense sonication treatment
was required to temporarily detach the tethered flavin moiety
from the nanotube side-walls. Upon addition of one aliquot of
dithionite, all covalently attached flavin moieties were trans-
formed to their reduced form (in the presence or absence of
SDS), since the flavin concentration onto 12-SWNT (5.8 × 10-7
M) was substantially lower that that of dithionite (9 × 10-6
M). Subsequently, two additions of 3 and 15 mL of air (2.8 ×
10-5 and 1.4 × 10-4 M of O2) were added, respectively, shown
in trace no. 3 and no. 4. In the absence of SDS (Figure 6A),
Figure 7 illustrates the cyclic voltamograms (CVs) of two
flavin derivatives 8 and 12 in the presence and absence of SDS.
(52) Yu, X.; Chattopadhyay, D.; Galeska, I.; Papadimitrakopoulos, F.; Rusling,
J. F. Electrochem. Commun. 2003, 5, 408-411.
(53) Gooding, J. J. Electrochim. Acta 2005, 50, 3049-3060.
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662 J. AM. CHEM. SOC. VOL. 130, NO. 2, 2008

Synthesis of FMN-Functionalized SWNTs
A R T I C L E S
physisorbed flavins, whose strong interaction with the nanotube
side-walls has been demonstrated by Guiseppi-Elie et al.17 Such
a strong π-π interaction might split the combined two-electron
reduction of FMN to FMNH2 into two separate waves, for
reasons that are not currently known. The equal spacing (ca.
160 mV) between anodic and cathodic peaks for both waves
appear to support such hypothesis. While the second redox wave
(marked with +) of Figure S5 coincides with that of Figure 7d,
the first redox wave (marked with /) is shifted by 170 mV
toward a higher negative potential. Such a shift might originate
from the fact that the physisorbed flavin moieties are expected
to physisorb stronger to the stems, as opposed to the oxygenated
tips of the SWNT forests, where stable π-π interaction with
the pristine graphene side-walls of SWNT can prevent their
desorption from multiple PBS washing. Correspondingly, the
separation of such stem-physisorbed flavin moieties from the
mediation-prone oxygenated tips of SWNT forests could account
for the aforementioned -170 mV shift of the first redox wave.
Figure 7e illustrates the comparable CV of Figure 7d, only
this time SDS was added along with 8. While all three waves
maintained their redox-potential positions, a generic reduction
in current intensity was observed for all waves. Since SDS is
known to physisorb on nanotubes,55 such current reduction
appears to originate from the insulating properties of SDS.
Figure 7 panels f and g illustrate the CVs of 12 covalently
attached on the tips of SWNT forests in the absence and
presence of SDS, respectively. Considerable washing and redox
cycling was performed to ensure that no unreacted 12 remains
in the vicinity of forests. The most striking feature of Figure
7f,g as compared to Figure 7d,e is that covalent functionalization
resulted in the narrowing of peak separation for both star and
cross waves (from 160 to ca. 40-50 mV). This enabled the
reappearance of the cathodic peak of the wave marked with the
cross, which led further credence to the assignment of the
marked waves to physisorbed FMN. Such dramatic narrowing
is believed to originate from the effective mediation that the
oxygenated forest tips provide to the attached 12 on these tips.
The relative magnitude of redox activity (amperometric current)
from Figure 7d to 7f illustrates the profound electrochemical
activity of covalently attached FMN onto SWNTs. However,
the relative magnitude of the star and cross-peaks (corresponding
to physisorbed FMN) in Figure 7f are twice as large as that of
Figure 7d. This is in direct agreement with the aforementioned
tendency of 12 to adopt a collapsed configuration onto SWNTs.
While the insulative nature of SDS lowers the magnitude of all
three redox waves, the star and cross waves (attributed to
collapsed 12) appeared to be impacted at greater extent, which
is in accordance with the chemical redox behavior of 12-SWNT,
observed spectroscopically.
Figure 7. Cyclic voltamograms obtained from a different electrode in PBS
(pH 7.4, 0.1 M). Each CV was offset by 2 × 10-6 A. Scan rate was 50
mV/s.
In addition, the CVs of PG and NTF on PG working electrodes
are provided as controls (Figure 7a,c). These CVs were recorded
in a phosphate buffer saline (PBS) solution in the absence of
oxygen to prevent unwanted oxygen reduction of ca. -0.6 V.
Both PG and NTF working electrodes show featureless CVs
from 0.1 to -0.9 V versus calomel reference electrode. Upon
closer inspection (5× magnification), the bare NTF electrode
shows a minute anodic hump around -0.3 V which is believed
to originate from the oxygenated functionalities (i.e., phenolic/
quinoid moieties) at the tips of the severed, acid-purified carbon
nanotubes.52
Figure 7 panels b and d illustrate the CVs of the dilute (5 ×
10-5 M) solution of 8 in PBS, in the absence and presence of
the nanotube forest (NTF). In the absence of NTF, 8 shows a
reversible redox wave centered ca. -0.49 V, separated by 50
mV.21 The anodic wave peaking at -0.52 V has been assigned
to the sequential two-electron flavin reduction, which along with
two-proton addition, transforms FMN to FMNH2.54 In the
presence of NTF, two additional reduction peaks (-0.37 and
-0.67 V) are witnessed in the anodic wave, marked with a star
and a cross, respectively. In the cathodic wave, an additional
oxidation peak is witnessed at ca. -0.15 V, marked with a star.
The peak separation between the oxidation and reduction peaks
marked with stars is ca. 220 mV. On the basis of such redox
peak separation, the corresponding oxidation peak of the cross-
marked anodic wave at -0.67 V should overlap with the main
cathodic wave of 8 (-0.47 V, see Figure 7b,d). To investigate
the nature of these two additional redox waves, extensive
washing with PBS of the 8+NTF electrode was performed, and
its CV was obtained in PBS in the absence of 8. (see Figure S5
of the Supporting Information). Much to our surprise, a
significant signal was observed, although the majority of 8
should have been washed away. Two broadened waves are
visible (cathodic/anodic at -0.51/-0.35 V (marked with stars)
and -0.64 and -0.47 V (marked with crosses)), both separated
by ca. 160 mV. These two waves are believed to originate from
Conclusions
In this contribution, the covalent functionalization of SWNTs
with flavin mononucleotide (FMN) is reported. This function-
alization was realized via a novel EDC-assisted coupling of an
H-phosphite derivative of FMN (9) to an alcohol-terminated
spacer, containing an amine-protected moiety on its other end.
Upon oxidation of the phosphite group to phosphate and
subsequent amine-deprotection, the FMN derivative of (12) was
(54) Cooke, G.; Duclairoir, F. M. A.; John, P.; Polwart, N.; Rotello, V. M. Chem.
Commun. 2003, 2468-2469.
(55) Richard, C.; Balavoine, F.; Schultz, P.; Ebbesen, T. W.; Mioskowski, C.
Science 2003, 300, 775-778.
9
J. AM. CHEM. SOC. VOL. 130, NO. 2, 2008 663

A R T I C L E S
Ju and Papadimitrakopoulos
covalently attached to acid-functionalized SWNTs via EDC
coupling (12-SWNT). The strong π-π interaction of aromatic
flavin moiety causes a collapsed FMN configuration onto the
side-walls of SWNTs that quenches its fluorescence. The
detachment of flavin moiety can be temporally achieved by
either sonication or chemical reduction of FMN to FMNH2 and
reoxidation back to the fluorescent FMN state. The miceller-
ization of the SWNT with sodium dodecyl sulfate (SDS)
surfactant was proven to be an effective way to prevent the long-
term collapse of FMN onto the SWNTs. Fluorescence and UV-
vis absorption spectroscopy, in conjunction with cyclic volta-
mmetry (CV) were utilized to characterize the redox behavior
of 12-SWNT, in comparison to a nanotube-unbound FMN
derivative (8). The dithionite- and O2-assisted reduction and
reoxidation to FMNH2 and FMN, respectively, was quantita-
tively achieved as long as the flavin moieties did not exhibit a
collapsed conformation onto the nanotube side-walls. The CV
of 12 attached to the tips of SWNT forests showed that the
redox behavior of extended and collapsed FMN is significantly
impacted by the presence of oxygenated moieties, which act as
mediators. These findings establish a basic understanding in
terms of synthesis and redox-assisted conformational behavior
of covalently bound SWNT FMN derivatives toward the
ultimate goal of incorporating them into functional bioelectronic
devices.
Acknowledgment. The authors wish to thank Dr. Martha
Morton and Dr. Minhua Zhao for their assistance in 31P NMR
and AFM imaging, respectively. Helpful discussions with Prof.
K. Noll are also acknowledged. Financial support from Grants
ARO-DAAD-19-02-1-10381, AFOSR FA9550-06-1-0030, NSF-
NIRT DMI-0422724, NIH-ES013557, and US Army Medical
Research W81XWH-05-1-0539 is greatly appreciated.
Supporting Information Available: 1H NMR spectra for
compounds 5-12, HHCar, and 12-SWNT (encapsulated in
perdeuterated SDS micelles); UV-vis and PL spectra of
compounds 8-12 and 12-SWNT in the presence and absence
of SDS while exposed to dithionite and air (O2), respectively;
the CV of physisorbed 8 onto SWNT forests is. This material
is available free of charge via the Internet at http://pubs.acs.org.
JA076598T
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664 J. AM. CHEM. SOC. VOL. 130, NO. 2, 2008