introduced as a new AFM mode of operation in which the AFM
tips are chemically modified to have a specific functionality by
the covalent attachment of a molecular monolayer.6,7,11-13 An
extension of this technique, the “force titration”, in which the tip-
sample interaction is monitored as a function of pH,7,13-20 can be
used to determine and map surface pKa with nanometer resolution.
Critical to the development of the technique is the availability of
well-characterized model surfaces, and therefore, the design and
fabrication of thin molecular films that simulate the chemistry of
biological surfaces is extremely important and is the subject of
this present report.
Scheme 1. Synthesis of
11-Thioundecyl-1-phosphonic Acida
aConditions: (i) BnSH (85%); (ii) POCl3, Et3N THF; (iii) NaHCO3
(aq) (65%); (iv) Na, NH3 (l) (62%).
the alkyl chain is long enough to form regular SAMs on the gold
surface. 11-Bromoundecanol was reacted with benzyl mercaptan
to give the corresponding benzyl thioether 2 . Phosphorylation
was then accomplished with phosphorus oxychloride,22 to give,
after partial hydrolysis, phosphonic acid 3 . The thiol was then
synthesized by deprotection of the benzyl group using sodium in
liquid ammonia, giving 1 in good yield, and its structure was
A particularly important molecular group in biology is the
phosphate group. Phosphates occur at the surface of phosphory-
lated proteins, in nucleic acids, and in cell membranes. Often
phosphorylation or dephosphorylation of proteins is a controlling
factor in the determination of protein conformation. More specif-
ically in sketetal tissue, interactions between extracellular matrixes
and the mineral phase, calcium hydroxyapatite, are thought to
be mediated by phosphate groups.8 These interactions are
considered to be important in both intiating and modulating
skeletal crystal growth.
To investigate the behavior of surface-immobilized phosphate
groups, we have synthesized and prepared self-assembled mono-
layers (SAMs) with a phosphate headgroup. Here we report on
the synthesis and characterization of this novel ω-functionalized
alkanethiol and present CFM force-titration curves for probes
and substrates both modified with the phosphate SAM. In this
way, the surface ionization states of the phosphate headgroup and
the ionic strength dependence of the surface pKa have been
determined.
1
confirmed with FAB-MS, H NMR and FT-IR.23
Benzyl mercaptan and 11-bromoundecanol were purchased
from Aldrich Chemical Co. Solvents and reagents were used as
received. Flash column chromatography was performed on Merck
silica gel (No. 109385), and TLC was carried out on precoated
plates (silica gel 60 F254, Merck No. 5715). The products were
visualized using UV light or potassium permanganate dip as
appropriate. 1H NMR spectra were acquired using a Bruker 250-
MHz instrument, and chemical shifts are relative to tetramethyl-
silane. Mass spectra were recorded on a VG instrument and
microanalyses were carried out by Warwick Analytical Services.
(i) P reparation of 1 1 -Benzylthioundecanol. Sodium (1.01
g, 43.8 mmol, 1.1 equiv) was added to dry ethanol (40 mL) at 0
°C. Benzyl mercaptan (4.7 mL, 4.94 g, 39.81 mmol, 1 equiv) was
added via syringe, followed by a solution of 11-bromo-1-undecanol
(10 g, 39.81 mmol) in dry ethanol (20 mL), and the reaction
mixture refluxed overnight. The mixture was cooled, poured into
saturated ammonium chloride solution (100 mL), and then
extracted with chloroform (3 × 50 mL). The combined organic
phases were dried with magnesium sulfate and filtered and the
solvent evaporated under reduced pressure to give a yellow oil
which solidified after 1 h at room temperature. The crude product
was dissolved in ether (200 mL) and the white precipitate removed
by filtration. The organic phase was concentrated under reduced
pressure to give the 11-benzylthioundecanol as a yellow solid (9.95
g, 85%).
EXPERIMENTAL SECTION
Solvents used in this study were reagent grade or better, and
in particular, the solvents used in substrate and probe function-
alization were HPLC grade to reduce particulate matter. Ultrapure
water with a resistivity of above 18 MΩ‚cm was used throughout
the experiments. Constant ionic strength phosphate buffer solu-
tions at different pH values were prepared according to methods
reported in the literature.7,21
Synthesis of 11-Thioundecyl-1-phosphonic Acid. We chose
phosphonic acid 1 (Scheme 1) as a target for synthesis because
(10) (a) Marti, O.; Ribi, H. O.; Drake, B.; Albrecht, T. R.; Duate, C. F.; Hansma,
P. K. Science 1 9 8 8 , 239, 50-54. (b) Formmer, J. Angew. Chem., Int. Ed.
Engl. 1992, 31, 1298-1304. (c) Mate, C. M.; McClelland, G. M.; Erlandsson,
R.; Chiang, S. Phys. Rev. Lett. 1 9 8 7 , 59, 1942-1945.
(11) Frisbie, C. D.; Rozsnyai, L. F.; Noy, A.; Wrighton, M. S.; Lieber, C. M. Science
1 9 9 4 , 265, 2071-2076.
(ii, iii) P reparation of 11-Benzylthioundecyl-1-phosphonic
Acid. To a stirred solution of phosphorus oxychloride (0.815 mL,
1.34 g, 8.74 mmol, 1.3 equiv) in dry THF (30 mL) at 0 °C was
added triethylamine (1.42 mL, 1.03 g, 10.19 mmol, 1.5 equiv)
dropwise via syringe, and the resultant mixture was stirred for
10 min until a white precipitate appeared. After a further 10 min
stirring, a solution of 11-benzylthioundecanol (1.98 g, 6.72 mmol)
was added to the reaction via dropping funnel over a period of 20
(12) Green, J.-B. D.; McDermott, M. T.; Porter, M. D.; Siperko, L. M. J. Phys.
Chem. 1 9 9 5 , 99, 10960-10965.
(13) Takano, H.; Kenseth, J. R.; Wong, S.-S.; O’Brien, J. C.; Porter, M. D. Chem.
Rev. 1 9 9 9 , 99, 2845-2890.
(14) He, H. X.; Li, C. Z.; Song, J. Q.; Mu, T.; Wang, L.; Zhang, H. L.; Liu, Z. F.
Mol. Cryst. Liq. Cryst. 1 9 9 7 , A294-295, 99-102.
(15) Van der Vegte, E. W.; Hadziioannou, G. Langmuir 1 9 9 7 , 13, 4357-4362.
(16) Zhang, H.; He, H. X.; Mu, T.; Liu, Z. F. Appl. Phys. 1 9 9 8 , A66, S269.
(17) Hu, K.; Bard, A. J. Langmuir 1 9 9 7 , 13, 5114-5119.
(18) Ito, T.; Namba, M.; Buhlmann, P.; Umezawa, Y. Langmuir, 1 9 9 7 , 13, 4323-
4332.
(22) Phosphorous oxychloride: toxic by inhalation and contact with skin, irritant,
reacts violently with water. Liquid ammonia: toxic by inhalation and contact
with skin, irritant, flammable. Sodium metal: reacts violently with water;
releases extremely flammable gas upon contact with water.
(19) Zhang, H.; He, H. X.; Mu, T.; Liu, Z. F. Thin Solid Films 1 9 9 8 , 778, 327-
329.
(20) Zhang, H.; Zhang, H. L.; He, H. X.; Zhu, T.; Liu, Z. F. Mater. Sci. Eng. C,
(23) Spectral data of 1 : 1HNMR (250 MHz, CDCl3), δ 4.04 (2H, q, CH2O), 3.56
(2H, br, m, P(OH)2), 2.50 (2H, t, SCH2CH2), 1.80-1.10 (18H, m, CH2); FT-
IR, νmax (solid) 3120, 2923, 2850 (C-H), 2539 (S-H), 1465, 1140 (PdO),
1034 (P-O-C) cm-1; MS (FAB) M + 1, 285).
1 9 9 9 , 8-9, 191-194.
(21) CRC Handbook of Chemistry and Physics, 72nd ed.; Lide, D. R., Ed.; CRC
Press: Boca Raton, FL, 1991.
1974 Analytical Chemistry, Vol. 72, No. 9, May 1, 2000