Dithio-phospholipids for oriented immobilization of proteins to gold surfaces

Article abstract of DOI:10.1016/S0040-4039(01)00261-1

We report the syntheses of phospholipids containing a dithio-group on their hydrophobic end and a choline or a biotin-group on their hydrophilic end. As demonstrated by surface plasmon resonance, a monolayer of these dithiolipids on gold affords specific binding of streptavidin and of biotinylated molecules in further steps.

Full text of DOI:10.1016/S0040-4039(01)00261-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 2677–2680
Dithio-phospholipids for oriented immobilization of proteins to
gold surfaces
Gerald Kada, Christian K. Riener and Hermann J. Gruber*
Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstr. 69, A-4040 Linz, Austria
Received 14 February 2001; accepted 15 February 2001
Abstract—We report the syntheses of phospholipids containing a dithio-group on their hydrophobic end and a choline or a
biotin-group on their hydrophilic end. As demonstrated by surface plasmon resonance, a monolayer of these dithiolipids on gold
affords specific binding of streptavidin and of biotinylated molecules in further steps. © 2001 Elsevier Science Ltd. All rights
reserved.
1
0–13
Lipid monolayers and bilayers supported on solid sub1-
strates have attracted great interest in the past decade.
A variety of applications have been developed, such as
interest.
Subsequently, the 2D-crystal can be trans-
4
ferred to an electron microscopy (EM)-grid or to
highly oriented pyrolytic graphite (HOPG, for AFM
2
3
13
design of biosensors, immobilization of DNA, and
studies ), due to the strong hydrophobic interaction
4
2
D-crystallization of proteins. The choice of support is
between lipid tails and solid substrate.
dominated by the measuring technique applied. Noble
metal surfaces are suitable for surface plasmon reso-
nance (SPR) and atomic force microscopy (AFM). This
choice determines the type of chemistry required for
surface modification which is needed for optimal condi-
tions of self assembly and attachment of further
In order to apply the same strategy to gold substrates,
we designed phospholipids with a disulfide element at
their hydrophobic end. These dithio-phospholipids can
be spread at the air–water interface and the resulting
monolayer can be transferred to a gold substrate. There
it covalently binds to the surface, which is of particular
advantage for AFM experiments where pulling forces
are applied. The detergent resistance of this monolayer
is essential when it comes to binding of integral mem-
brane proteins from detergent solutions. In all experi-
ments, the predominant lipid (‘host’) was the
dithio-derivative of phosphatidylcholine (5). For the
5
biomolecules to the support. Metal surfaces can easily
be modified by sulfur-bearing components. Long-chain
alkylthiols, derivatized with different 8 functional
6
,7
groups, or phospholipids and cholesterol with thiol
headgroups have been adsorbed from solution onto
gold surfaces, yielding densely packed self assembled
monolayers (SAMs). However, the composition of a
SAM generated by coadsorption of more than one
component is usually different from that in solution,
due to the influence of molecular structure on the
biospecific capture of streptavidin (or of His -tagged
6
proteins), a small fraction (‘guest’) of the analogous
phosphatidylethanolamine with a biotin-group (8) (or
with a nitrilotriacetic acid function, NTA, currently
under study) was included. The surface density of the
‘guest’ lipids was easily adjusted during spreading at the
air–water interface.
9
kinetics of chemisorption.
One way to solve this problem is spreading surface-
active molecules at the air–water interface, which is
only possible with hydrophobic molecules, such as
phospholipids. In addition, the resulting phospholipid
monolayers are an ideal matrix for 2D-crystallization of
proteins when 10–50% of the phospholipid headgroups
contain a specific anchoring site for the protein of
The synthetic steps are shown in Scheme 1. v-Hydroxy-
hexadecanoic acid (1) was converted into v-bromohex-
6
adecanoic acid with hydrobromic acid. v-Mercapto-
hexadecanoic acid (2) was formed by converting v-
bromohexadecanoic acid into its thiouronium hydro-
bromide, followed by cleavage of the complex with
NaOH and precipitating compound 2 with sulfuric
Keywords: phospholipid; disulfide; gold; streptavidin; atomic force
microscopy; surface plasmon resonance.
14
acid. Reaction with propylmethanethiosulfonate gave
*
Corresponding author. E-mail: hermann.gruber@jk.uni-linz.ac.at
propyldithiohexadecanoic acid (3) in good yield (about
0
040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00261-1

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678
G. Kada et al. / Tetrahedron Letters 42 (2001) 2677–2680
Scheme 1. Reagents and conditions: (i) 1:1 mixture of 48% HBr and 98% acetic acid; (ii) thiourea in 98% ethanol followed by
NaOH and sulfuric acid treatment; (iii) propylmethanethiosulfonate/diisopropylethylamine in methanol; (iv) DCC/DMAP in
CH Cl followed by silica gel chromatography; (v) biotin-cap-NHS/diisopropylethylamine in CHCl /methanol; (vi) DCC/DMAP
2
2
3
in CH Cl followed by silica gel chromatography.
2
2
6
try.
0%). The purity was confirmed by NMR spectrome-
For synthesis of the ‘guest’-lipid 8, 1-myristoyl-2-
hydroxy-sn-glycero-3-phosphoethanolamine (6) (Avanti
Polar Lipids) was reacted with N-hydroxysuccinimidyl-
biotinamidocaproate (biotin-cap-NHS) to give the
biotin–lysolipid 7. Finally, esterification of propyldithio-
hexadecanoic acid (3) with the biotin–lysolipid gave
molecule 8, which was purified by silica gel column chro-
1
5
The ‘host’-lipid 5 was obtained by esterification of
propyldithiohexadecanoic acid (3) with 1-myristoyl-
2
-hydroxy-sn-glycero-3-phosphocholine (4) (Avanti
Polar Lipids Inc., Alabaster, AL) using equimolar
16–18
20
DCC and N,N-dimethylaminopyridine (DMAP).
matography and characterized by NMR spectrometry.
Lipid 5 was purified by silica gel column chromatogra-
phy (35% yield) and characterized by NMR spectrome-
For AFM experiments, 1 mg/mL ‘host’ lipid 5 in
chloroform/hexane 1:1 (v/v) was spread on a small round
1
9
try.
Figure 1. (A) MAC-mode™ AFM image of a monolayer of dithiolipid 5 (1 mm in square, height of the lipid film between 2.5 and
®
3
nm) scanned in PBS buffer solution; (B) BIACORE sensorgram showing the specific binding of streptavidin to a dithiolipid
monolayer (containing biotin-dithiolipid 8 as a scavenger) adsorbed on an untreated gold chip. Enhanced signal response during
incubation is due to different protein buffer and transient weak adsorption. Only the solid lines after the washing step reflect
specifically bound protein.

G. Kada et al. / Tetrahedron Letters 42 (2001) 2677–2680
2679
Teflon trough (4 mm diameter) filled with a drop of
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sion of proper anchor lipids, such as 8, as demonstrated
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1
9. H NMR of compound 5 (500 MHz, CDCl ) l (ppm):
3
5
.20 (m, 1H; CH); 4.39 (m, 1H; 1-myristoyl-COO-
+
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-myristoyl-COO-CH (II)); 3.94 (m, 2H; CH-CH -O-P);
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.67 (m, 4H; -CH -S-S-CH -); 2.28 (m, 4H; -CH -COO);
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1.54-1.75 (m, 8H; -CH -CH -S-S-CH -CH -, CH -CH -
2
2
2
2
2
2
COO); 1.25 (s, 42H; -CH -); 0.99 (t, 3H, J=7.4 Hz; CH -
2
3
of propyldithio); 0.88 (t, 3H, J=6.8 Hz; CH - of
3
Acknowledgements
myristoyl).
1
2
0. H NMR of compound 8 (200 MHz, CDCl ) l (ppm):
3
5
.19 (m, 1H; b); 4.50 (m, 2H; c(I) and 6a); 4.30 (m, 1H;
The authors would like to thank Dr. Steffi Bachem
from Biacore AB for help with BIACORE measure-
ments. Helpful advice from Prof. K. Grubmayr and Dr.
T. G. Dax is gratefully acknowledged. This work was
supported by the Austrian Science Foundation project
P12801-MED.
3a); 4.1–3.8 (m, 5H; c(II), a, m); 3.44 (m, 1H; 4); 3.1–3.3
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g%, d); 1.25 (s, 42H; -CH -); 0.99 (t, 3H; i ); 0.88 (t, 3H,
f ).
2

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