Synthesis of phosphorylcholine-oligoethylene glycol-alkane thiols and their suppressive effect on non-specific adsorption of proteins

Article abstract of DOI:10.1016/j.tetlet.2009.04.091

A series of phosphorylcholine-oligoethylene glycol-alkane thiols were synthesized, and their suppressive effect on the non-specific adsorption of proteins was evaluated by comparison with corresponding oligoethylene glycol-alkane thiols. It was found that

Full text of DOI:10.1016/j.tetlet.2009.04.091

Tetrahedron Letters 50 (2009) 4092–4095
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Tetrahedron Letters
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Synthesis of phosphorylcholine–oligoethylene glycol–alkane thiols
and their suppressive effect on non-specific adsorption of proteins
*
Mutsuo Tanaka , Takahiro Sawaguchi, Yukari Sato, Kyoko Yoshioka, Osamu Niwa
National Institute of Advanced Industrial Science and Technology (AIST), Institute for Biological Resources and Functions, Central 5, 1-1-1, Higashi, Tsukuba, Ibaraki 305-8565, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of phosphorylcholine–oligoethylene glycol–alkane thiols were synthesized, and their suppressive
effect on the non-specific adsorption of proteins was evaluated by comparison with corresponding oligo-
ethylene glycol–alkane thiols. It was found that phosphorylcholine–oligoethylene glycol–alkane thiols
had a greater suppressive effect on the non-specific adsorption of proteins.
Received 13 April 2009
Accepted 24 April 2009
Available online 3 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
The complete deciphering of the human genome has made it
important to analyze various biomolecules to reveal the functions
of genes. The non-specific adsorption of proteins that occurs during
biomolecule detection often has a serious detrimental effect on
selectivity and the detection limit. Therefore, research on surface
modification materials has attracted great interest with a view to
avoiding the interference caused by the non-specific adsorption
of proteins. The properties of polyethylene glycol (PEG) such as
flexibility, chemical stability, water solubility, and low cytotoxicity
make it a versatile surface modification material.1 The most attrac-
tive property of PEG is its suppressive effect on the non-specific
adsorption of proteins. While polymers consisting of the ethylene
glycol moiety have been studied extensively, it has also been re-
ported that self-assembled monolayers (SAMs) of oligoethylene
glycol–alkane thiols suppress the non-specific adsorption of pro-
teins in a similar way to surfaces modified with PEG.2 This finding
led to the examination of various SAMs of various alkane thiol
derivatives for use in surface modification to suppress the non-spe-
cific adsorption of proteins with the goal of developing bioanalyt-
ical devices.3 PEG and oligoethylene glycols are recognized as
versatile components for surface modification. In our previous
work,4 we showed that SAMs of oligoethylene glycol–alkane thiols
effectively suppressed the non-specific adsorption of proteins
allowing us to realize the high performance detection of proteins.
On the other hand, phosphorylcholine derivatives could be
other candidates for suppressing the non-specific adsorption of
proteins because the phosphorylcholine group is one of the pri-
mary lipid components of plasma membranes. After polymers con-
sisting of the phosphorylcholine moiety were reported to reduce
the non-specific adsorption of proteins,5 various polymers bearing
the phosphorylcholine moiety have been synthesized and their
properties examined.6 Although SAMs of phosphorylcholine-al-
kane thiols were also reported to suppress the non-specific adsorp-
tion of proteins3f, there have been relatively few studies related to
SAMs of phosphorylcholine-alkane thiols compared with those on
SAMs of oligoethylene glycol–alkane thiols. Therefore, we are
interested in the properties of phosphorylcholine SAMs, especially,
SAMs of phosphorylcholine-alkane thiols bearing the oligoethylene
glycol moiety, namely phosphorylcholine–oligoethylene glycol–al-
kane thiol SAMs, with the expectation of achieving a superior sup-
pressive effect on the non-specific adsorption of proteins. In this
Letter, we report preliminary results on the synthesis of phospho-
rylcholine–oligoethylene glycol–alkane thiols and the nature of
those phosphorylcholine SAMs by comparison with SAMs of corre-
sponding oligoethylene glycol–alkane thiols.
The synthesis route is summarized in Scheme 1. Similar to our
previous work, the synthesis of oligoethylene glycols7, the starting
materials and the procedures were chosen to make the synthesis as
straightforward as possible. Therefore, Bittman’s procedure8 was
adopted for the introduction of phosphorylcholine group in order
to avoid using trimethyl amine, which is problematic in terms of
handling because it vaporizes causing a serious odor. On the other
hand, the trityl group was chosen as a protecting group for the
mercapto group, as it is reported that deprotection of the trityl
group was carried out without the hydrolysis of the phosphoryl
group.9
The mono-introduction of the oligoethylene glycol moiety into
1,12-dibromododecane was achieved by using potassium t-butox-
ide as a base in THF. Oligoethylene glycol mono-12-bromododecyl
ether 11b–e was purified with silica-gel column chromatography.
12-Bromo-1-dodecanol 11a was commercially available. Bromide
was converted to thiol with a one-pot procedure, the reaction of
bromide with thiourea and subsequent hydrolysis using ethylene
diamine. The obtained crude thiols 12a–e were used for a subse-
quent reaction without purification. To protect the mercapto
group, trityl chloride was reacted with 12a–e. The selective reac-
tion of trityl chloride with the mercapto group was accomplished
by using an equimolar amount of trityl chloride in the presence
of potassium carbonate in acetonitrile. The products 13a–e were
* Corresponding author. Tel.: +81 29 861 6233.
E-mail address: mutsuo-tanaka@aist.go.jp (M. Tanaka).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.04.091

M. Tanaka et al. / Tetrahedron Letters 50 (2009) 4092–4095
t-BuOK
4093
Br
Br
O
+
Br
HO
OH
THF
m
m = 0~3
H2NCSNH2
EtOH
H2N(CH2)2NH2
EtOH
OH
OH
O
n
n=0~4:11a~e
TrCl, K2CO3
CH3CN
HS
O
n
n=0~4:12a~e
(12a = 6)
POCl3,NEt3
CHCl3
H2O
cholinetosylate
C5H5N
TrS
OH
n
O
C5H5N
n=0~4:13a~e
O
TrS
O
n
O
+
O
P
NMe3
O-
n=0~4:14a~e
CH2Cl2-CF3COOH
SiHEt3
CH2Cl2-CF3COOH
SiHEt3
1~5
7~10
HS
O
O
O
+
P
NMe3
1
2
O-
O
HS
O
O
+
O
O
P
NMe3
O-
O
O
O
O
+
HS
O
O
P
NMe3
3
O-
O
HS
O
O
+
O
O
P
NMe3
4
O-
O
O
O
O
+
HS
O
O
P
NMe3
5
O-
HS
OH
O
6
7
8
9
O
O
HS
OH
O
HS
O
O
OH
O
O
HS
OH
HS
O
O
OH
10
Scheme 1. Synthesis of phosphorylcholine–oligoethylene glycol–alkane thiols and corresponding oligoethylene glycol–alkane thiols.
purified with HPLC, gel permeation chromatography. The introduc-
tion of the phosphorylcholine moiety is the key step in this synthe-
sis. The adopted Bittman’s procedure is a one-pot introduction of
the phosphorylcholine group which consists of three steps, namely
the reaction of alcohols with phosphoryl chloride, the introduction
of the choline moiety, and hydrolysis.8 Based on the results of con-
trol experiments, pyridine was used as a solvent instead of chloro-
form, and the second step of the reaction was carried out at 0 °C.
Purification was conducted with silica-gel column chromatogra-
phy. In the final step, deprotection of the trityl group was achieved
using triethyl silane in a mixture of dichloromethane and trifluoro-
acetic acid.9 The products 1–5 were purified with silica-gel column
chromatography. Oligoethylene glycol–alkane thiols 7–10 were
obtained by deprotection of the trityl group for 13b–e because
the purification of 12 afforded only 6 with sufficient purity.
Gold surfaces were modified with 1–10, and the nature of those
surfaces was explored. First, a single crystal gold surface was mod-
ified with a water–ethanol solution of 1–10, and then the molecu-
lar concentrations on the surfaces were determined with the
electrochemical method.10 The results are given in Table 1. As
shown in Table 1, the molecular concentration of the surface mod-
ified with 6 was 7.2 Â 10À10 mol/cm2. As the theoretically calcu-
lated molecular concentration for alkane thiol SAMs is known to
be 7.7 Â 10À10 mol/cm2, terminally hydroxylated alkane thiol 6
seems to form SAMs with a high density in a similar way. The
introduction of oligoethylene glycol moieties at the terminals of al-
kane thiols reduced the molecular concentrations from 7.2 Â 10-10
mol/cm2 to 6.4 Â 10À10 mol/cm2 in the increase of the oligoethyl-
ene glycol chain length for 6–10. The additional introduction of
the phosphorylcholine moiety to oligoethylene glycol–alkane thi-

4094
M. Tanaka et al. / Tetrahedron Letters 50 (2009) 4092–4095
Table 1
Properties of SAMs of phosphorylcholine–oligoethylene glycol–alkane thiols 1–5 and oligoethylene glycol–alkane thiols 6–10a
1
2
3
4
5
6
7
8
9
10
Bare
gold
Surface concentration
Fibrinogen adsorption
Concanavalin A
5.2 0.2
5.3 0.4
5.3 0.3
5.2 0.4
5.1 0.5
7.2 0.2
7.1 0.3
7.2 0.4
7.0 0.4
6.4 0.8
—
0.91 0.20 1.09 0.12 0.82 0.32 1.12 0.53 0.97 0.38 3.51 0.39 2.09 0.30 1.68 0.06 2.40 0.24 2.60 0.45 20.6
0.60 0.06 0.85 0.03 0.25 0.21 0.37 0.07 0.12 0.05 2.48 0.31 0.92 0.20 1.05 0.32 0.78 0.52 0.86 0.09 26.8
adsorption
a
The units for surface concentrations and protein adsorption amounts are Â10À10 mol/cm2 and ng/cm2, respectively. The adsorption measurements were conducted in the
presence of a surfactant, Tween 20.
ols resulted in a more significant reduction in the molecular con-
centrations being 5.1–5.3 Â 10À10 mol/cm2 for 1–5. However, the
molecular concentrations were almost the same regardless of the
length of the oligoethylene glycol chain. These results suggest that
alkane thiols 1–10 form SAMs on gold surfaces and the molecular
concentrations of SAMs reflect the bulkiness of the moieties intro-
duced into the terminals of the alkane thiols, where the phospho-
rylcholine moiety is bulkier than the oligoethylene glycol moiety.
There have been several reports on SAMs of a phosphorylcho-
line-alkane thiol, 11-mercaptoundecyl-phosphorylcholine.11,12
According to the literature providing a detailed characterization
of SAMs,11 it has been concluded that with respect to the structure
of phosphorylcholine-alkane thiols in SAMs the orientation of the
phosphorylcholine head groups is nearly parallel to the gold sur-
face. Therefore, the reported SAM thickness is only 13.7 Å, and
the molecular concentration is thought to be less than half of that
of 11-mercaptoundecanol. However, our results for the molecular
concentrations of 12-mercaptododecylphosphorylcholine 1 and
12-mercapto-dodecanol 6 were 5.2 Â 10-10 mol/cm2 and 7.2 Â 10-10
mol/cm2, respectively, and the observed difference between their
molecular concentrations was far smaller than that suggested in
the literature. At this stage, it is not clear why the difference of only
one methylene-chain-length in alkyl groups induces such a differ-
ence in the molecular concentration of SAMs as regards phospho-
rylcholine-alkane thiols.
To evaluate the suppressive function of the modified surfaces for
thenon-specific adsorption of proteins, weexamined theamountsof
fibrinogen and concanavalin A adsorbed on the modified surfaces
using the BIACOREÒ system (T100, GE healthcare). Fibrinogen is a
well-known blood coagulation factor and is used to evaluate anti-
thrombogenic surfaces. The modified surfaces in this work consist
of the phosphorylcholine moiety, which is reported to show anti-
thrombogenicity.13 In addition, concanavalin A is a commonly used
protein in various studies. Therefore, these two proteins were used
to evaluate the suppressive function of the modified surfaces for
the non-specific adsorption of proteins. The adsorption amounts of
both proteins are summarized in Table 1 together with the molecu-
lar concentrations. Compared with a bare gold surface, the adsorp-
tion amounts of both proteins were greatly reduced for all
modified surfaces, even for a surface modified with terminally
hydroxylated alkane thiol 6. While the introduction of oligoethylene
glycolmoietiesinto alkanethiolsdidnotenhancethesuppressiveef-
fect on the adsorption amounts of fibrinogen, the adsorption
amounts of concanavalin A were reduced by half, from 2.48 ng/
cm2 for 6 to 0.78–1.05 ng/cm2 for 7–10 by the incorporation of the
oligoethylene glycol moieties. In this case, the oligoethylene glycol
chains exhibited no length dependence although chain-length
dependence was reported for the SAMs of some oligoethylene glycol
derivatives.14 Surfaces modified with phosphorylcholine-alkane
thiol 1 and phosphorylcholine–oligoethylene glycol–alkane thiols
2–5 suppressed fibrinogen adsorption more effectively, where the
adsorptionamounts were abouthalf of those for 6–10. This tendency
supports the view that modification with SAMs of phosphorylcho-
line derivatives produces antithrombogenic surfaces possessing
the potential for medical applications. Furthermore, similar to the
cases of 6–10, the incorporation of oligoethylene glycol moieties in
phosphorylcholine-alkane thiol 1 did not enhance the suppressive
effect on the adsorption of fibrinogen. In contrast, the adsorption
amounts of concanavalin A were reduced from 0.60–0.85 ng/cm2
for 1 and 2 to 0.12–0.37 ng/cm2 for 3–5 again by the incorporation
of oligoethylene glycol moieties. These results indicate that surfaces
modified with SAMs consisting of phosphorylcholine moiety exhibit
a more suppressive effect on fibrinogen adsorption than those with
SAMs of oligoethylene glycol–alkane thiols, and the incorporation of
oligoethylene glycol moieties in phosphorylcholine-alkane thiols
does not enhance the suppressive effect on fibrinogen adsorption.
By contrast, the incorporation of oligoethylene glycol moieties in al-
kane thiols enhances the suppressive effect on concanavalin A
adsorption even in the presence of the phosphorylcholine moiety.
In other words, it is implied that fibrinogen interacts with the phos-
phorylcholine moiety, while concanavalin A tends to interact with
both the phosphorylcholine and oligoethylene glycol moieties.
The adsorption amounts of fibrinogen were reported to be 3–
400 ng/cm2 for SAMs of 11-mercaptoundecyl-phosphorylcho-
line.11,12 These amounts are relatively high compared with our re-
sult of 0.91 ng/cm2 in Table 1. This difference might be caused by
the presence of a surfactant Tween 20Ò in the BIACOREÒ system,
as the adsorption amount of fibrinogen on bare gold in our exper-
iments was also very low at about 20 ng/cm2 compared with the
reported values of about 1000 ng/cm2.12 Surfaces modified with
co-polymers consisting of phosphorylcholine and oligoethylene
glycol moieties have also been examined.15,16 It was reported that
the co-polymers effectively reduced the adhesion of platelets to
the modified surface15, but the adsorption amounts of fibrinogen
unexpectedly increased from about 50 ng/cm2 to 250 ng/cm2 as
the oligoethylene glycol chain length increased from 0 to 9-
mer.16 In our results, oligoethylene glycol moieties had no influ-
ence on fibrinogen adsorption, however some effect derived from
oligoethylene glycol moieties was observed with concanavalin A
adsorption. These results imply that the influence of oligoethylene
glycol moieties on the non-specific adsorption of proteins depends
on not only the length of the oligoethylene glycol chains but also
the nature of proteins, and might be reduced under packed condi-
tions caused by SAM formation.
As a typical SAM surface feature, a surface modified with 3 was
observed with an electrochemical scanning tunneling microscope
(EC-STM) 17 as shown in Figure 1. In Figure 1a, the surface roughness
was less than 0.3 nm indicating that the molecules were closely
packed with some dark spots indicating dents. As shown in Figure
1b, in some areas a double-stripe shaped structure was also ob-
served, which resulted from the molecular arrangements of the
SAMs. It has been reported that these dark spots are dents on the
SAMs derived from 2.4 Å deep etching pits on the gold surface and
the formation of such etching pits during SAMs fabrication is known
to be a typical phenomenon that occurs during gold surface modifi-
cation with alkane thiols.18 Furthermore, these etching pits are
reportedly covered with SAMs similar to other areas.18 It has also
been suggested that such dents on the surface of SAMs could signif-
icantly influence the suppressive function for the non-specific
adsorption of proteins.19 However, no significant difference could

M. Tanaka et al. / Tetrahedron Letters 50 (2009) 4092–4095
4095
References and notes
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be found in the numbers or shapes of the dents in our experiments. A
detailed analysis of the relationship between the surface structure
and the function suppressing the non-specific adsorption of proteins
is now in progress.
In conclusion, it was found that phosphorylcholine–oligoethyl-
ene glycol–alkane thiols formed SAMs on gold surfaces and sup-
pressed fibrinogen adsorption more effectively than the
corresponding oligoethylene glycol–alkane thiols. The incorpora-
tion of oligoethylene glycol moieties in phosphorylcholine-alkane
thiols enhanced the suppressive effect on concanavalin A adsorp-
tion but not on fibrinogen adsorption.
Acknowledgments
We thank Mrs. Takashima for experimental assistance. This
work was supported by Grant-In-Aid for Scientific Research (C)
(No. 17550140) from the Ministry of Education, Culture, Sports,
Science, and Technology, Japan.
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Supplementary data
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Supplementary data (synthesis procedures and evaluation for
SAMs) associated with this article can be found, in the online ver-
sion, at doi:10.1016/j.tetlet.2009.04.091.