Efficient solid-phase synthesis of peptide-substituted alkanethiols for the preparation of substrates that support the adhesion of cells

Full text of DOI:10.1021/jo981113s

7552
J . Org. Chem. 1998, 63, 7552-7555
Sch em e 1
Efficien t Solid -P h a se Syn th esis of
P ep tid e-Su bstitu ted Alk a n eth iols for th e
P r ep a r a tion of Su bstr a tes Th a t Su p p or t
th e Ad h esion of Cells
Benjamin T. Houseman and Milan Mrksich*
Department of Chemistry, The University of Chicago,
Chicago, Illinois 60637
Received J une 10, 1998
Self-assembled monolayers that present short peptide
ligands are an important class of model substrates for
studies of the interactions of mammalian cells with
surfaces.1 Substrates that present the Arg-Gly-Asp trip-
eptide, for example, have been important in understand-
ing the adhesion and spreading of cells.2-6 Although self-
assembled monolayers of alkanethiolates on gold are a
versatile class of model substrates for these studies, the
effort required to synthesize peptide-terminated alkaneth-
iols remains a disadvantage of this methodology.2 In this
note, we describe a rapid and efficient method, based on
solid-phase peptide synthesis, for preparing alkanethiols
terminated with peptide ligands. We utilize this meth-
odology to synthesize a Gly-Arg-Gly-Asp-Ser alkanethiol
conjugate and demonstrate that monolayers prepared
from this compound support the adhesion and spreading
of fibroblast cells.
Our strategy required a suitably protected alkanethiol
that could be coupled to a peptide on solid support.
Simultaneous deprotection and cleavage of the adduct
from the resin would afford the final product. Scheme 1
shows the preparation of compound 1 from undec-1-en-
11-ylhexa(ethylene glycol) (2).7 Alkylation of 2 with ethyl
diazoacetate in the presence of 10 mol % BF3‚Et2O
afforded ester 3. Photochemical addition of thiolacetic
acid to the terminal olefin of 3 gave thioacetate 4 in 93%
yield. Acidic hydrolysis of 4, followed by protection of
the resultant thiol as a trityl thioether, provided 5. This
intermediate was saponified with aqueous lithium hy-
droxide in THF/MeOH to afford carboxylic acid 1 (35%
yield over five steps).
and cleavage of the peptide from the resin can be
performed simultaneously using aqueous trifluoroacetic
acid. The product of cleavage contains an amide termi-
nus that resembles the amide bond following the Arg-
Gly-Asp sequence in extracellular matrix proteins.8 We
prepared the Gly-Arg-Gly-Asp-Ser pentapeptide moiety
of 6 on this support using routine protocols.10 Removal
of the terminal glycinyl Fmoc-carbamate with 20% pip-
eridine/DMF, followed by coupling with acid 1 in the
presence of DCC and 1-hydroxybenzotriazole (HOBT),
provided the fully protected conjugate. Cleavage of the
peptide from the resin, followed by precipitation with
ether and purification by gel permeation chromatogra-
phy, afforded 6 in 51% overall yield based on initial
loading of the resin. The chemical shift of the methylene
protons adjacent to the sulfur (t, 2.4 ppm, -CH2S)
demonstrated the presence of a sulfhydryl group; no
disulfide (t, 2.7 ppm) was observed. A thin layer chro-
matogram of the product (65:30:5 CHCl3/MeOH/H2O v/v)
turned bright yellow upon application of Ellman’s re-
agent11 (1 mg/mL in ethanol), confirming the presence
of the sulfhydryl group. We have synthesized several
other peptide-terminated alkanethiols with similar re-
sults.
Scheme 2 illustrates the preparation of peptide-
substituted alkanethiol 6, which contains the Gly-Arg-
Gly-Asp-Ser sequence known to bind integrin receptors
on the surface of mammalian fibroblast cells.8 For this
work, we chose Fmoc-Rink amide MHBA polystyrene
as the solid support.9 This resin was attractive for
several reasons. The initial amino acid coupling step is
accomplished in near quantitative yield. Deprotection
* To whom correspondence should be addressed.
(1) Mrksich, M. Cell Mol. Life Sci. 1998, 54, 653. Mrksich, M.;
Whitesides, G. M. Annu. Rev. Biophys. Biomol. Struct. 1996, 25, 55.
(2) Roberts, C.; Chen, C. S.; Mrksich, M.; Martichonok, V.; Ingber,
D. E.; Whitesides, G. M. J . Am. Chem. Soc. 1998, 120, 6548.
(3) Massia, S. P.; Hubbell, J . A. Anal. Biochem. 1990, 187, 292; J .
Cell Biol. 1991, 114, 1089.
(4) Brandley, B. K.; Schnaar, R. L. Anal. Biochem. 1988, 172, 270.
(5) Danilov, Y. N.; J uliano, R. L. Exp. Cell Res. 1989, 182, 186.
(6) Herbert, C. B.; McLernon, T. L.; Hypolite, C. L.; Adams, D. N.;
Pikus, L.; Huang, C. C.; Fields, G. B.; Letourneau, P. C.; Distefano,
M. D.; Hu, W. Chem. Biol. 1997, 4, 731.
We next examined the ability of self-assembled mono-
layers prepared from 6 to support cell adhesion. Glass
coverslips coated with an optically transparent (12 nm)
layer of gold were immersed for 5 h in an ethanolic
solution containing (1-mercaptoundec-11-yl)tri(ethylene
glycol)7 and alkanethiol 6 (1 mM total thiol; 10 µM
compound 6). The tri(ethylene glycol) groups at the
surface of the monolayer resist both nonspecific adsorp-
tion of protein and nonspecific attachment of cells.12 The
(7) Pale-Grosdemange, C.; Simon, E. S.; Prime, K. L.; Whitesides,
G. M. J . Am. Chem. Soc. 1991, 113, 12.
(8) Ruoslahti, E. Annu. Rev. Cell Dev. Biol. 1996, 12, 697 and
references cited therein.
(9) Rink, H. Tetrahedron Lett. 1987, 28, 3787.
(10) Solid-Phase Peptide Synthesis: A Practical Approach; Atherton,
E., Sheppard, R. C., Eds.; IRL Press: New York, 1989.
(11) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.
S0022-3263(98)01113-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 09/22/1998

Notes
J . Org. Chem., Vol. 63, No. 21, 1998 7553
Sch em e 2
cycles were repeated until assembly of the Fmoc-protected Gly-
Arg-Gly-Asp-Ser peptide was complete.
monolayers were rinsed with ethanol and dried under a
stream of nitrogen. 3T3 Swiss Albino fibroblast cells
suspended in serum-free culture medium were allowed
to attach to these substrates for 5 h at 37 °C. The
substrates with attached cells were removed from the
culture medium, fixed with a solution of 4% paraform-
aldehyde in phosphate-buffered saline, and photographed
at 20× magnification (Figure 1). Adherent cells exhibited
morphology similar to those attached to fibronectin or
treated tissue culture dishes. Cells did not attach to
monolayers that presented tri(ethylene glycol) groups
alone.
We have demonstrated an efficient methodology for the
preparation of peptide-terminated alkanethiols. These
molecules are important precursors for the preparation
of monolayers of alkanethiolates on gold that support the
adhesion of cells. We believe that this methodology will
be valuable for the preparation of a wide range of
functionalized alkanethiols for studies of the adhesion,
migration, and differentiation of cells.
P r ep a r a tion of Self-Assem bled Mon ola yer s. Substrates
were prepared as described previously.2,14 Briefly, titanium (1
nm) and then gold (12 nm) were evaporated onto glass slides.
The slides were cut into pieces approximately 1 cm2 in size and
immersed in 0.3 mL of an ethanolic solution containing (1-
mercaptoundec-11-yl)tri(ethylene glycol) and 6 (1 mM total thiol;
10 µM compound 6). After 5 h, the substrates were removed
from the solutions, rinsed with absolute ethanol, and dried under
a stream of nitrogen.
Cell Cu ltu r e a n d Micr oscop y. Swiss Albino 3T3 cells
(ATCC, Rockville, MD) were grown in Dulbecco’s modified Eagle
medium containing 10% fetal bovine serum (Gibco BRL, Gaith-
ersburg, MD). Gentamicin (Gibco BRL) was included in the
culture medium to prevent bacterial growth. All cultures were
maintained at 37 °C in a 10% CO2 atmosphere. Near confluent
monolayers of cells were detached by incubation in a solution of
0.05% trypsin/0.53 mM Na4EDTA (Gibco BRL) for 5 min. The
trypsin was neutralized by the addition of culture medium
containing 10% fetal bovine serum, and the cells were centrifu-
gated and resuspended in serum-free culture medium. A fixed
number of cells (15 000 cells in 1 cm3 culture medium) were
allowed to attach onto self-assembled monolayers presenting
peptide and tri(ethylene glycol) groups. After 5 h at 37 °C, the
substrates were gently rinsed with phosphate-buffered saline
(pH 7.4, Gibco BRL) and fixed for 30 min in a solution of 4%
paraformaldehyde in phosphate-buffered saline. Photographs
were taken on Ilford PanF film (Malelo Camera, Chicago, IL)
using a Zeiss Axiovert 135 microscope at 20× magnification.
Exp er im en ta l Section
Gen er a l P r oced u r es. 1H NMR and 13C NMR spectra were
recorded on a Bruker 500 MHz spectrometer with chemical shifts
reported in parts per million relative to tetramethylsilane. All
reagents for solution phase chemical synthesis were purchased
from Aldrich Chemical Co. (Milwaukee, WI). Amino acids and
the Fmoc-Rink amide MBHA resin were purchased from
Anaspec, Inc. (La J olla, CA). THF was distilled from sodium/
benzyl ketyl, and dichloromethane was distilled from calcium
hydride. Anhydrous DMF was purchased from Aldrich and used
without further purification. The BCA protein assay reagent
was purchased from Pierce Chemical Co. (Rockford, IL). Flash
chromatography was carried out using EM Science Kiselgel 60
(230-400 mesh).
Solid -P h a se P ep tid e Syn th esis. Fmoc-Rink amide MBHA
resin (312 mg, 0.15 mmol, substitution 0.48 mmol/g) was placed
in a 10 mL polypropylene reaction vessel, washed with DMF (2
× 5 mL), and swollen for 30 min in DMF. The resin was rinsed
with an additional portion of DMF before a solution of 20%
piperidine in DMF (5 mL, 2 × 15 min) was added. The resin
was washed with DMF (2 × 5 mL) before a solution of the Fmoc-
amino acid (0.45 mmol), HOBT (68 mg, 0.5 mmol), and DCC
(95 mg, 0.46 mmol) in DMF (5 mL) was added to the vessel.
The mixture was agitated for 2 h, at which point the Kaiser
ninhydrin test13 was negative. The deprotection and coupling
Hexa (eth ylen e glycol) Eth yl Ester 3. To a solution of
undec-1-en-11-ylhexas(ethylene glycol) (2)7 (4 g, 9.22 mmol) in
dry dichloromethane (20 mL) at 0 °C was added ethyl diazoac-
etate (1.4 mL, 11.06 mmol) and BF3‚Et2O (97 µL, 0.92 mmol).
After the mixture was stirred for 30 min at 0 °C, saturated
aqueous ammonium chloride (10 mL) was added and the reaction
mixture was placed in a separatory funnel. The organic phase
was collected, and the aqueous phase was extracted with
dichloromethane (5 × 50 mL). The combined organic phases
were dried over magnesium sulfate and concentrated to a yellow
oil. Silica gel chromatography using gradient elution (1:1 ethyl
acetate/hexanes f ethyl acetate) afforded ester 3 (2.97 g, 5.69
mmol, 62%) as a clear oil: 1H NMR (500 MHz, CDCl3) δ 1.26-
1.36 (m, 16H), 1.53-1.58 (m, 2H), 2.00-2.04 (dd, 2H, J ) 7.02,
6.77 Hz), 3.43 (t, 2H, J ) 6.79 Hz), 3.49-3.76 (m, 24H), 4.13 (s,
2H), 4.20 (q, 2H, J ) 7.14 Hz), 4.88-4.96 (m, 2H), 5.74-5.84
(m, 1H); 13C NMR (CDCl3) δ 14.19, 26.06, 28.90, 29.09, 29.40,
29.44, 29.51, 29.61, 33.78, 60.76, 68.71, 70.04, 70.57, 70.61, 70.87,
71.52, 114.08, 139.20, 170.44; IR (thin film) 2922, 2855, 1744
cm-1; HRMS (FAB) calculated for C27H53O9 (MH+) m/ e 521.3690,
found m/ e 521.3678.
Th ioa ceta te 4. A solution of olefin 3 (1.64 g, 3.14 mmol) in
dry THF (32 mL) containing thiolacetic acid (0.55 mL, 7.85
(12) Mrksich, M.; Whitesides, G. M. In Chemistry and Biological
Applications of Polyethylene Glycol; ACS Symposium Series; American
Chemical Society: Washington, DC, 1997; Vol. 680,
references therein.
p 361 and
(13) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook P. I. Anal.
Biochem. 1970, 71, 59.
(14) Mrksich, M.; Dike, L. E.; Tien, J . Y.; Ingber, D. E.; Whitesides,
G. M. Exp. Cell Res. 1997, 235, 305.

7554 J . Org. Chem., Vol. 63, No. 21, 1998
Notes
methanolic ammonium hyroxide. The solution was concentrated
in vacuo to a volume of 10 mL and extracted with dichlo-
romethane (6 × 50 mL). The combined organic phases were
dried over magnesium sulfate and concentrated to afford the free
thiol as a pale yellow oil. To a solution of this compound in dry
THF (8 mL) under nitrogen was added trityl chloride (1.23 g,
4.4 mmol). The reaction mixture was allowed to stir at room
temperature for 24 h and reduced in vacuo to a yellow oil.
Purification by silica gel chromatography (eluent 30:1 f 20:1
CH2Cl2/MeOH) provided 5 as a clear oil (1.67 g, 2.09 mmol, 71%
over two steps): 1H NMR (500 MHz, CDCl3) δ 1.13-1.23 (m,
18H), 1.25-1.36 (m, 2H), 1.55-1.69 (m, 2H), 2.12 (t, 2H, J )
7.36 Hz), 3.43 (t, 2H, J ) 6.83 Hz), 3.56-3.74 (m, 24H), 4.14 (s,
2H), 4.20 (q, 2H, J ) 7.12 Hz), 7.20 (t, 3H, J ) 7.31 Hz), 7.27 (t,
6H, J ) 7.36 Hz), 7.40 (d, 6H, J ) 7.45 Hz); 13C NMR (CDCl3)
δ 14.20, 26.07, 28.57, 29.00, 29.16, 29.38, 29.46, 29.47, 29.54,
29.62, 32.01, 60.78, 66.33, 68.71, 70.04, 70.57, 70.52, 70.87, 71.53,
126.47, 127.76, 129.59, 145.07, 170.45; IR (thin film) 2921, 2855,
1750 cm-1; HRMS (FAB) calculated for C46H67O9S (MH+) m/ e
795.4506, found m/ e 795.4501.
Acid 1. To a solution of 5 (500 mg, 0.63 mmol) in 1:1 THF/
MeOH (8 mL) was added 1 M aqueous lithium hydroxide (2 mL).
The reaction mixture was stirred at room temperature for 3 h
and cooled to 0 °C. The solution was acidified to a pH of 2 with
1 N HCl and extracted with ethyl acetate (3 × 20 mL). The
combined organic phases were washed with brine (10 mL), dried
over magnesium sulfate, and concentrated to afford 1 (412 mg,
0.54 mmol, 85%) as a clear oil: 1H NMR (500 MHz, CDCl3) δ
1.13-1.30 (m, 15H), 1.35-1.41 (m, 2H), 1.53-1.59 (m, 2H), 2.13
(t, 2H, J ) 7.36 Hz), 3.44 (t, 2H, J ) 6.84 Hz), 3.56-3.77 (m,
24H), 4.16 (s, 2H), 7.20 (t, 3H, J ) 7.31 Hz), 7.27 (t, 6H, J )
7.36 Hz), 7.41 (d, 6H, J ) 7.45 Hz); 13C NMR (CDCl3) δ 26.02,
28.56, 28.97, 29.13, 29.35, 29.42, 29.45, 29.50, 29.55, 31.99, 66.32,
68.92, 69.98, 70.34, 70.42, 70.45, 70.49, 70.52, 70.54, 70.61, 70.65,
71.32, 71.50, 126.44, 127.74, 129.57, 145.05, 171.95; IR (thin film)
3057, 2916, 2852, 1735 cm-1; HRMS (FAB) calcd for C44H64O9-
SK (M + K+) m/ e 807.3908, found m/ e 807.3909.
Gly-Ar g-Gly-Asp -Ser Alk a n eth iol 6. To the resin contain-
ing the Fmoc-protected Gly-Arg-Gly-Asp-Ser peptide (0.15 mmol)
was added a solution of 20% piperidine in DMF (5 mL, 2 × 15
min). The solid support was washed thoroughly with DMF to
remove excess piperidine, and a solution of compound 1 (340
mg, 0.45 mmol), HOBT (68 mg, 0.5 mmol), and DCC (95 mg,
0.46 mmol) in THF (5 mL) was added. The mixture was agitated
for 4 h before the resin was washed thoroughly with DMF and
dichloromethane. The resin was placed in a 25 mL round-bottom
flask, and a solution of 5 mL TFA containing 0.25 mL of H2O,
0.375 g of phenol, 0.25 mL of ethanedithiol, and 0.25 mL of
thioanisole was added. The yellow mixture was stirred at room
temperature for 2 h and filtered to remove the polystyrene
support. The beads were washed with TFA (2 × 2 mL), and the
combined filtrates were concentrated in vacuo. Repeated pre-
cipitation from cold ether (4 × 40 mL) afforded a clear oil that
was purified using gel permeation chromatography (Sephadex
G-10, 20% aqueous MeOH/0.1% TFA). Fractions that stained
positive with the BCA protein reagent were combined and
lyophilized to afford 6 as a fluffy, white powder (75 mg, 51%
based on loading of resin): 1H NMR (500 MHz, CD3OD) δ 1.13-
1.32 (m, 15H), 1.46-1.69 (m, 7H), 1.84-1.92 (m, 1H), 2.41 (t,
2H, J ) 7.13 Hz), 3.13 (t, 2H, J ) 6.93 Hz), 3.39 (t, 2H, J ) 6.63
Hz), 3.49-3.66 (m, 24H), 3.73-3.84 (m, 4H), 3.91 (s, 2H), 4.00
(s, 2H), 4.28-4.33 (m, 2H), 4.66 (t, 1H, J ) 6.52 Hz); 13C NMR
(CD3OD) δ 24.98, 26.09, 27.21, 29.41 29.66, 30.08, 30.22, 30.58,
30.65, 30.72, 31.36, 32.90, 35.23, 36.27, 41.91, 43.19, 43.98, 51.61,
54.46, 56.99, 57.08, 62.96, 71.12, 71.17, 71.32, 71.40, 71.44, 71.49,
72.05, 72.36, 158.56, 171.96, 172.01, 172.93, 173.85, 174.15,
174.81; HRMS (FAB) calculated for C41H80O16N9S (MH+) m/ e
998.5444, found m/ e 998.5447.
F igu r e 1. (A) Representation of the structure of a mixed self-
assembled monolayer presenting Gly-Arg-Gly-Asp-Ser peptides
and tri(ethylene glycol) groups. (B) Optical micrograph of 3T3
fibroblast cells attached to a self-assembled monolayer shown
in (A) wherein 1% of the alkanethiolates present the peptide
ligand. Cells were plated onto the substrate in serum-free
culture medium and allowed to attach at 37 °C. This photo-
graph was taken at 20× magnification after 5 h in culture.
mmol) and AIBN (51 mg, 0.31 mmol) was irradiated in a
photochemical reactor (Rayonet reactor lamp, Southern New
England Ultraviolet Co., model no. RPR-100) for 5 h under an
atmosphere of nitrogen. Concentration of the reaction mixture,
followed by flash chromatography (eluent 30:1 CH2Cl2/MeOH)
gave compound 4 as a clear oil (1.75 g, 2.93 mmol, 93%): 1H
NMR (500 MHz, CDCl3) δ 1.25-1.34 (m, 18H), 1.52-1.59 (m,
4H), 2.31 (s, 3H), 2.85 (t, 2H, J ) 7.35 Hz), 3.43 (t, 2H, J ) 6.81
Hz), 3.56-3.74 (m, 24H), 4.14 (s, 2H), 4.21 (q, 2H, J ) 7.14 Hz);
13C NMR (CDCl3) δ 14.16, 26.02, 28.74, 29.03, 29.07, 29.38,
29.40, 29.43, 29.48, 29.57, 30.58, 60.71, 68.66, 69.99, 70.53, 70.57,
70.82, 71.46, 170.39, 195.96; IR (thin film) 2921, 2854, 1750, 1691
Ack n ow led gm en t. We are grateful for support
provided by The University of Chicago, the NIH (Grant
GM54621), the Searle Scholars Program/The Chicago
Community Trust, and the Camille & Henry Dreyfus
Foundation (New Faculty Award). B.T.H. is supported
by MD/PhD Training Grant HD-09007 and a Searle
Fellowship from the University of Chicago.
cm-1; HRMS (FAB) calculated for C29H57O10
S
(MH+) m/ e
597.3672, found m/ e 597.3670.
Tr ityl th ioeth er 5. A solution of 4 (1.75 g, 2.94 mmol) in
absolute ethanol (35 mL) containing concentrated HCl (1.5 mL)
was heated to reflux for 12 h. The reaction mixture was cooled
to room temperature and adjusted to a pH of 7 with 5%

Notes
Su p p or tin g In for m a tion Ava ila ble: 1H NMR spectra of
compounds 1 and 3-6 (5 pages). This material is contained
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