Nanoscale biomolecular structures on self-assembled monolayers generated from modular pegylated disulfides

Article abstract of DOI:10.1002/chem.200902439

A solid-phase synthetic strategy was developed that uses modular building blocks to prepare symmetric oligo(ethylene glycol)-terminated disulfides with a variety of lengths and terminal functionalities. The modular disulfides, composed of alkyl amino groups linked by an amide group to oligoethylene chains were used to generate self-assembled monolayers (SAMs), which were characterised to determine their applicability for biomolecular applications. X-ray photoelectron spectroscopy (XPS) of the SAMs obtained from these molecules demonstrated improved stability towards displacement by 16-hexadecanethiol, while surface plasmon resonance (SPR) analyses of SAMs prepared with the hydroxy-terminated oligoethylene disulfide showed equal resistance to non-specific protein adsorption in comparison to 11-mercaptoundecyl tri(ethylene glycol). SAMs made from these adsorbates were amenable to nanoscale patterning by scanning near-field photolithography (SNP), facilitating the fabrication of nanopatterned, protein-functionalised surfaces. Such SAMs may be further developed for bionanotechnology applications such as the fabrication of nanoscale biological arrays and sensor devices. Play it again SAM! A synthetically expedient method for the assembly of functionalised pegylated alkyldisulfides employing an alternative solid-phase synthetic strategy was successfully demonstrated. Self-assembled monolayers (SAMs) of the synthesised disulfides were characterised and shown to possess a number of desirable properties that were relevant for biological applications and amenable to near-field photolithography.

Full text of DOI:10.1002/chem.200902439

DOI: 10.1002/chem.200902439
Nanoscale Biomolecular Structures on Self-Assembled Monolayers
Generated from Modular Pegylated Disulfides
AHCTUNGTRENNUNG
Lu Shin Wong,[a] Stefan J. Janusz,[b] Shuqing Sun,[b, c] Graham J. Leggett,[b] and
Jason Micklefield*[a]
Abstract: A solid-phase synthetic strat-
egy was developed that uses modular
building blocks to prepare symmetric
oligo(ethylene glycol)-terminated disul-
fides with a variety of lengths and ter-
minal functionalities. The modular di-
sulfides, composed of alkyl amino
groups linked by an amide group to
electron spectroscopy (XPS) of the
SAMs obtained from these molecules
demonstrated improved stability to-
wards displacement by 16-hexadecane-
thiol, while surface plasmon resonance
(SPR) analyses of SAMs prepared with
the hydroxy-terminated oligoethylene
disulfide showed equal resistance to
non-specific protein adsorption in com-
parison to 11-mercaptoundecyl tri
ACHTUNGTRENNUNG(eth-
AHCTUNGTRENNUNG
adsorbates were amenable to nanoscale
patterning by scanning near-field pho-
tolithography (SNP), facilitating the
fabrication of nanopatterned, protein-
functionalised surfaces. Such SAMs
may be further developed for bionano-
technology applications such as the
fabrication of nanoscale biological
arrays and sensor devices.
oligo
ACHTUNGTRENNUNG
Keywords: bionanotechnology
monolayers photolithography
·
·
erate
·
(SAMs), which were characterised to
determine their applicability for bio-
molecular applications. X-ray photo-
protein immobilization · self-assem-
bly · solid-phase synthesis
Introduction
phy,[15,16] dip-pen nanolithography[11,17,18] and scanning near-
field photolithography (SNP),[14,19,20] patterning of SAMs
can be achieved with nanoscale resolution.
SAMs have been widely used as platforms for the study
of biological interactions and assembly of biomolecular
structures down to the nanoscopic scale. For example,
SAMs have been utilised for studies involving enzymatic
processing of DNA,[21,22] DNA computing,[23] protein
assays[24,25] and studies of cellular responses towards sur-
Nanofabrication has been exploited in a diverse range of ap-
plications from high-sensitivity biomedical diagnostic tools,
sensor devices, surface coatings, nanoelectronics, through to
single-molecule enzymology and the development of molec-
ular motors.[1–5] Self-assembled monolayers (SAMs) of al-
kylthiolates on metals have proved to be particularly impor-
tant platforms for nanotechnology development. Here,
SAMs offer a straightforward route to the control of chemis-
try over macroscopic areas.[6,7] When combined with a litho-
graphic technique,[3,8–14] such as electron beam lithogra-
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
to develop protein and oligosaccharide functionalised chips
for proteomics- and glycomics-based research.[29–31] Also,
with the ever increasing drive towards smaller sample sizes
and higher throughput for whole-organism analysis, has
come the need to develop nanometer-scale, biomolecular,
patterned surfaces.[3,32,33] However, in order to further ex-
ploit SAM-based systems in this context, a number of issues
remain to be fully addressed. Firstly, convenient strategies
for the synthesis of bespoke thiols (or their equivalents) that
are tailored towards specific applications are required.[34]
This is especially the case in applications involving patterned
surfaces, since several thiol species, one to cover the bulk
surface and one or several others at the patterned areas, are
required. An additional requirement in biomolecular appli-
[a] Dr. L. S. Wong, Prof. J. Micklefield
School of Chemistry & Manchester Interdisciplinary Biocentre
University of Manchester, 131 Princess Street
Manchester M1 7DN (UK)
Fax : (+44)161 306 5199
E-mail: j.micklefield@manchester.ac.uk
[b] Dr. S. J. Janusz, Dr. S. Sun, Prof. G. J. Leggett
Department of Chemistry, University of Sheffield
Brook Hill, Sheffield S3 7HF (UK)
[c] Dr. S. Sun
National Centerfor Nanoscience and Technology,
Beijing 100190 (China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200902439.
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FULL PAPER
cations is the need to control non-specific adsorption of bio-
molecules, particularly for proteins for which adsorption can
rapidly become irreversible. In this respect, thiols bearing
polyethylene glycol (PEG) moieties have emerged as the
most widely used materials for the fabrication of such bio-
ACHTUNGTRENNUNG
resistant SAMs.[7,24,25,35–37] However, these thiols are usually
prepared by time-consuming, linear, multistep syntheses,
which are inconvenient for the generation of a range of
chain lengths and the introduction of further chemical diver-
sity.
In this report, the use of a solid-phase synthesis for the
preparation of “pegylated” disulfides, containing oligomeric
ethylene glycol units, is explored. The SAMs generated
from the modular disulfides were characterised with a suite
of surface analyses techniques and scanning near-field pho-
tolithography (SNP) was used to generate nanoscale pat-
terns on their surfaces, thus demonstrating the suitability of
these materials for nanofabrication.
Results and Discussion
Solid-phase synthesis: It was envisaged that a strategy remi-
niscent of solid-phase peptide synthesis could be adopted
generate a variety of functionalised thiols for SAMs.[38–41]
For the purposes of this synthesis, a solid-phase strategy is
more efficient than solution-phase synthesis, because it
allows excess of reagents to force high yields and simple
washing steps obviates the need for purification of inter-
mediates. Accordingly, two pegylated alkyldisulfides 1 and
2, were chosen as synthetic targets, which are more stable to
oxidation and reactions with electrophiles than the corre-
sponding thiols, but are still able to form SAMs (Scheme 1).
The SAMs generated from 1 would be able to provide a
bulk bioresistant surface,[42,43] while in the featured areas, 2
would allow further chemical elaboration at the amino
group. The latter disulfide also incorporated an additional
PEG spacer to allow greater accessibility to this terminal
moiety above the bulk surface of the SAM. Thus, a modular
strategy was envisaged in which the more simple building
blocks 3–5 were synthesised and then combined on the solid
support 6. It was also envisaged that the internal amide
bonds of 1 and 2, apart from acting as the link between the
building blocks, would also impart improved SAM stability
by virtue of lateral hydrogen bonding between the SAM
Scheme 1. Retrosynthetic outline for the solid-phase synthesis of pegylat-
ed alkanedisulfides 1 and 2. The structure of 11-mercaptoundecyl tri-
AHCTUNGTREG(NNUN ethylene) glycol 7 is also included for comparison.
units[44,45] in contrast to 11-mercaptoundecyl tri
ACTHUNRTGNE(GNU ethylene)
glycol (7), the material commonly used in the preparation of
bioresistant SAM surfaces[20,24,26] which lacks the internal
amide bond.
Scheme 2. Reagents and conditions: a) tert-Butyl acrylate, Na (cat.),
THF, 18 h, 75%; b) Ac2O, Et3N, CH2Cl2, 16 h, 99%; c) 50% TFA,
CH2Cl2, 2 h, 86%; d) mesyl chloride, Et3N, CH2Cl2, 2 h; e) NaN3, DMF,
7 d, 95% over 2 steps; f) 4m HCl, 1,4-dioxane, 2 h, 80%.
Synthesis of the pegylated building blocks 3 and 4
(Scheme 2) from triethylene glycol was acheived without
chromatography,[46] via common intermediate 8. Acetylation
of 8 gave 9 followed by trifluoroacetic acid (TFA) acidolysis
to generate building block 3. Mesylation of 8 and displace-
ment with sodium azide gave 10, which on acidolysis re-
vealed the azido–PEG acid 4 with the azido group function-
ing as a masked amine for solid-phase synthesis. The al-
kylthiol
5
was prepared from 11-bromoundecanol
(Scheme 3), which on treatment with potassium phthalimide
gave 11. Activation of the alcohol of 11 and substitution
with potassium thioacetate resulted in 12, which on treat-
ment with sodium methanethiolate[47] gave the free thiol 5.
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J. Micklefield et al.
pled to 14, but this step was found to be slow. Even when
the highly active coupling agent N,N’-tetramethylchloro-
AHCTUNGTREGfNNUN ormamidium hexafluorophosphate (TCFH) was used, 2.5
days was required for complete acylation (as determined by
Kaiser tests). Deacetylation with hydrazine gave the hy-
droxy-functionalised 16. Cleavage from the resin was affect-
ed with 5% TFA and the crude material subjected to air ox-
idation to form the disulfide. Finally, purification by HPLC
yielded the pure 1 with a 75% overall yield based on the
Fmoc quantification of the loaded aminoalkylthiol 14 (94%
average conversion per step).
Scheme 3. Reagents and conditions: a) PhtNK, DMF, 608C, 4 h, 98%;
b) mesyl chloride, Et3N, CH2Cl2, 2 h; c) AcSH, K2CO3, DMF, 15 h, 97%
over 2 steps; d) NaSMe, MeOH, 2 h, 94%.
For the synthesis of amino-functionalised disulfide 2, the
azido building block 4 was first coupled with the amine of
14, which was also slow requiring 2.5 days for completion. A
solid-phase version of the Staudinger reduction[50,51] gave the
free amine to which the next pegylated unit could be at-
tached and the azide reduced in a stepwise manner to give
17. Cleavage and oxidation under similar conditions for 1
followed by HPLC purification gave 2 in 65% yield based
on the Fmoc quantification (95% per step). It should be
noted that although both 1 and 2 were isolated here as the
disulfides for convenience, if desired, omission of the final
air oxidation step would yield the free thiols.[48]
For the solid-supported synthesis (Scheme 4), the acid-
labile 4-methoxytrityl polystyrene resin 6 commonly used
for the immobilisation of thiols was employed.[48] The hy-
droxy resin was activated using thionyl chloride[49] and thiol
component 5 attached to give 13. Similar to a previously re-
ported solid-phase strategy,[41] attachment through its S
atom served to protect the thiol from reaction during the
solid-phase assembly. On-resin hydrazinolysis of the phthali-
mide revealed the amine 14. A portion of the amine resin
14 was treated with fluorenylmethyloxycarbonyl chloride
(FmocCl) to give 15 and UV/Vis quantification of the subse-
quently cleaved piperidine–fulvene adduct, revealed a 53%
resin loading of the thiol relative to the original amount of
hydroxytrityl groups on the resin, in agreement with previ-
ous reports with resin 6.[48] To complete the synthesis of the
target compound 1, the acetoxy building block 3 was cou-
Stability of SAMs: As noted above, it was intended that in-
corporation of the amide bond would improve the stability
of the SAMs of 1 and 2 compared to the widely used 11-
mercaptoundecyl tri
ACHTUNGTRENN(UNG ethylene) glycol (7). In order to assess
this, the displacement of pegylated adsorbates by 16-hexade-
Scheme 4. Reagents and conditions: a) SOCl2, CH2Cl2, 1 h; b) 5, DIPEA, CH2Cl2, 2 h; c) 15% N2H4, DMF/MeOH (8:2), 16 h; d) FmocCl, DIPEA,
CH2Cl2/DMF (1:1), 2 h; e) 3, TCFH, DIPEA, CH2Cl2/DMF (1:1), 65 h; f) 4, TCFH, DIPEA, CH2Cl2/DMF (1:1), 65 h; g) Me3P, H2O, 1,4-dioxane/THF,
18 h; h) 5% TFA, CH2Cl2, 3 minꢁ6 cycles; i) air, aq. (NH4)HCO3 pH 8–10, 16 h.
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Nanoscale Biomolecular Structures
FULL PAPER
Figure 1. Typical XPS C1s spectra of films of a) 1, b) 2 and c) 7 following immersion in the 16-hexadecanethiol for a range of durations. Arrows indicate
the position of the CCO peak on each spectrum of the freshly prepared SAMs. Peak table of C1s binding energies derived from the fitting of XPS spec-
tra can be found in supporting information (Table S1 in the Supporting Information).
canethiol was studied. Our hypothesis was that if the incor-
poration of amide groups into the adsorbate would confer
greater stability on the resulting monolayer, then the rate of
displacement should be slowed. The proportion of the SAM
thiolates that were displaced by immersion in a 10 mm solu-
tion of 16-hexadecanethiol in ethanol over varying periods
of time was readily quantified by XPS, following analysis of
the C1s spectra (Figure 1 and Table S1 in the Supporting In-
formation).
the peak corresponding to the amide functionality together
with the decrease in CCO is also observed and is consistent
with this hypothesis. In the spectra for films of 2, the CCO
peak was initially greater than in 1 due to the increased size
of the ether component in this molecule. This peak also un-
derwent a similar decrease over time.
To quantify the changes observed in these data, the area
of the CCO component relative to the area of the AuACHTUNGTRENNUNG(4f 7/
2) peak was calculated and normalised to the initial (0 h)
value. The resulting data are plotted as a function of the im-
mersion time (Figure 2). SAMs of the hydroxy-terminated
monolayer of 1 yielded a larger ratio than that measured for
a monolayer of 7 at all immersion times, suggesting en-
hanced stability, although when allowance is made for the
error bars, it may be concluded that the improvement in sta-
bility is modest. This enhancement in stability is attributed
Spectra were first acquired for freshly prepared samples
(0 h). In agreement with previously reported data,[52] the
C1s spectrum of SAMs prepared from 7 exhibits two major
components, one at a binding energy of ꢀ286.8 eV due to
the ether unit (henceforth CCO), and one due to the carbon
atoms in the alkyl chain (CCC), always adjusted to 285 eV.
In comparison, spectra of films from 1 contain three peaks,
attributable to CCO, CCC and NC=O (the latter at
ꢀ288.3 eV). Films of 2 also include these three major peaks.
Consideration of the molecular structure of 2 suggests that a
peak may additionally be expected at 286.65 eV, due to the
ammonium functional group. However, because of the close
proximity of this peak to the main C1s peak, we did not at-
tempt to resolve it during fitting. In comparison, C1s spectra
of n-alkylthiols such as 16-hexadecanethiol consist of a
single CCC component at 285 eV. C1s spectra were then ac-
quired from films of 1, 2 and 7 following immersion in 16-
hexadecanethiol for a range of durations (Figure 1). If im-
mersion of the pegylated SAMs in solutions of 16-hexadeca-
nethiol leads to displacement of the pegylated adsorbate, it
will thus be accompanied by an increase in the size of the
CCC component in the C1s spectrum and a concomitant re-
duction in the size of the CCO component. In line with this,
the spectra for films made from 1 showed a marked de-
crease in the area of this peak over time, while the CCC
peaks (to the right of the CCO peak at 285 eV) show an in-
crease, indicating that molecules in the film are gradually
displaced by 16-hexadecanethiol over time. A decrease in
Figure 2. Graph of XPS CCO peak intensity ratio for the SAMs prepared
~
&
^
from 1 ( ), 2 ( ) and 7 ( ) against immersion time in a hexadecanethiol
solution.
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J. Micklefield et al.
to the formation of lateral (intra-monolayer) hydrogen
bonds between amide groups, which causes an increase in
the desorption energy and hence reduces the rate-constant
for desorption of thiolates from the gold surface in monolay-
ers of 1 relative to that for 7. At all immersion times, SAMs
of 2 yielded a clearly larger CCO/Au4f ratio than was mea-
sured for monolayers of either 1 or 7, indicating that they
had a much greater resistance to displacement by hexadeca-
nethiol. This was thought to be due to the capacity of 7 to
form two lateral, intra-monolayer amide bonds, which is ex-
pected to yield a significant increase in the desorption en-
thalpy, reducing the rate of desorption from the surface. It is
also possible that lateral hydrogen-bonding interactions be-
tween amine terminal groups may further contribute to the
stabilisation of the monolayers. SAMs of both of the amide-
bearing 1 and 2 were thus more stable than those formed
from thiol 7, and after 180 h, 49% and 75% of the thiolates
from 1 and 2, respectively, remained at the surface, com-
pared to less than 35% in the SAM of 7.
Resistance to protein adsorption: The SAMs of 1, 2 and 7
were also assessed for their resistance to the non-specific ad-
sorption of proteins by SPR (surface plasmon resonance)
spectroscopy. Here, the change in SPR was measured as sol-
utions of individual proteins were flowed over the SAMs
followed by buffer solutions to desorb any loosely bound
protein. The differences in the SPR response level after the
buffer wash was used as an indicator of the amount of resid-
ual protein adsorbed to the surfaces, which was compared
between the different SAMs.[42] Three test proteins were ex-
amined; bovine serum albumin (BSA), the acyl carrier pro-
tein BtrI from Bacillus circulans and phosphopantethienyl
transferase Sfp from Bacillus subtilis,[53,54] which represent a
range of protein types. The hydroxy-terminated SAMs from
1 that possessed the internal amide bond were found to pos-
sess marginally superior resistance to adsorption compared
to hydroxy-terminated SAMs produced from 7 for all the
proteins tested, confirming that these disulfides formed bio-
resistant monolayers (Figure 3). This difference in bioresist-
ance may be due to the internal hydrogen-bonding resulting
in a tighter packing of the hydrophobic alkyl chains and a
reduction of their exposure to the proteins. As expected, the
proteins adhered to SAMs from the amino-functionalised di-
sulfide 2, probably through electrostatic interactions since
the termini of the SAMs present charged ammonium species
under these physiological conditions. Indeed, such interac-
tions are commonly used as a means of non-covalent protein
immobilisation.[31,55] The SPR curve for 2 appears to differ
slightly from those of 1 and 7 in the association region. The
reason for this is not certain, although the most likely ex-
planation would be that it results from the charged nature
of the terminal group for adsorbate 2 (an ammonium ion,
compared to the hydroxyl termini of the other two systems
studied).
Figure 3. SPR sensograms of SAMs composed from 1, 2 and 7 with expo-
sure to the proteins: a) BSA, b) BtrI and c) Sfp.
surfaces employing these disulfides, hydroxy-terminated
SAMs composed of 1 were subjected to SNP. Light with a
wavelength of 244 nm was coupled to the optical fiber probe
and the sample exposed to the optical near field associated
with the aperture at its apex. This enabled the photooxida-
tion of the thiolates in the SAM to the corresponding sulfo-
nates, which could be displaced subsequently with a second
thiol.[14] Firstly, an arrangement of dots was fabricated by
holding the probe stationary at each location in a 6ꢁ6
matrix of points. The sample was then immersed in a solu-
tion of 16-hexadecanethiol. After rinsing with ethanol and
drying, the friction force microscopy (FFM) image (Fig-
ure 4a) revealed dark contrast on the exposed regions com-
pared to the rest of the surface, as expected, due to the re-
duced coefficient of friction in these areas. Figure 4b shows
an FFM image of a pattern formed by writing six parallel
lines into a SAM of 1, and subsequently immersing the
sample in a solution of 3-mercaptopropanoic acid. The ex-
posed regions now exhibited brighter contrast than the sur-
rounding material; again consistent with existing literature
that shows that the coefficients of friction of hydroxy-termi-
nated SAMs are 50% those of carboxylic acid terminated
monolayers. Finally, the pattern formed by writing into a
SAM of 1 and incorporation of 2 into the exposed regions
Scanning near-field photolithography (SNP): In respect to
the further aim of developing functionalised nano-patterned
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Figure 4. Friction force microscopy images of SAMs of 1 with features
generated by SNP followed by displacement of the oxidised thiol with
a) 16-hexadecanethiol, b) 3-mercaptopropionic acid and c) 2.
was imaged using FFM (Figure 4c). In this case the dots ex-
hibit brighter contrast, which may result from the electro-
static interaction between the negatively charged probe and
positive charges on the ammonium terminated regions (the
outer surface of a silicon nitride probe is composed of a thin
layer of silicon dioxide, which is expected to carry a net neg-
ative charge). In all cases submicron features with a mean
full width at half maximum height (fwhm) of ꢀ190 nm were
generated. These data indicate that oxidation down to the S
atom was successful and the entire molecule was displaced
by the incoming thiol, giving rise to the complimentary lat-
eral force images after exposure to 16-hexadecanethiol or 3-
mercaptopropionic acid (Figure 4a and b).
SAM 18, presenting a “dashed” pattern of the ammoni-
um-terminated features from disulfide 2 on a bulk surface of
the hydroxy-pegylated 1, was then used to demonstrate the
immobilisation of proteins by both covalent and non-co-
ACHTUNGTRENNUNGvalent means (Scheme 5). Here, the electrostatic interactions
mentioned above were exploited to immobilise immunoglo-
bulin G (IgG) as a model protein. For covalent binding, 18
was treated with the cross-linking agent glutaraldehyde to
generate SAMs with aldehyde-functionalised features 19.
IgG was then covalently immobilised by imine formation.
The surfaces with the immobilised IgG were then imaged by
AFM in tapping mode for more accurate height measure-
ments. The protein nanofeatures that were generated had a
mean fwhm of 180 nm for the electrostatically immobilised
protein and 178 nm for the covalent immobilisation
(Figure 5). Also, the electrostatically bound IgG features
had a height of 3.0 nm while the glutaraldehyde-bound pro-
tein features had a height of 3.5 nm compared to the bulk
Scheme 5. Reagents and conditions: a) SNP at 244 nm, then 2, EtOH,
18 h; b) 25% aq. glutaraldehyde, 30 min; c) IgG, PBS pH 7.4, 2.5 h.
surface, consistent with previous results.[56] In comparison,
the features prior to protein immobilisation gave a small
height decrease of ꢀ0.2 nm, possibly due to the looser pack-
ing of the incoming pegylated units. Thus, these experiments
demonstrate that patterns of nanoscale protein features
could be generated on these materials.
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J. Micklefield et al.
since these promise to be effective methods for the fabrica-
tion of protein nano-features.[20,56–59] It is expected that
under optimum conditions, truly nanoscopic feature sizes as
small as 9 nm may be fabricated.[20]
The work shown in this report will be useful in the future
development of biomolecular “nanoarrays” for high
throughput screening and wider applications in biomedical
diagnostics, biosensors, organic material electronics and the
assembly of more complex three-dimensional structures.[60]
Nevertheless, the nanopatterned SAMs described can poten-
tially find more immediate use in the investigation of cell
biology mediated by cell surface interactions. These surfaces
may also be a route to the isolation of single molecules in
the study of single molecule dynamics and enzymology.
Experimental Section
Figure 5. Tapping mode AFM images of SAMs with IgG immobilised
through a) electrostatic interactions to 18 with b) a topographic plot of a
representative feature; and c) imine formation to 19 with d) a topograph-
ic plot of a representative feature.
Materials and equipment: 4-Methoxytrityl hydroxide polystyrene resin
with an initial loading of 1.5 mmolgÀ1 was purchased from Iris Biotech
(Marktredwitz, Germany) All DMF used was of peptide synthesis grade
from Rathburn Chemicals (Walkerburn, UK). Substrates used were boro-
silicate glass slides (Chance number 2 thickness, 64 mmꢁ22 mm). Pegy-
lated thiol 7 was synthesised according to a published procedure.[52] All
EtOH used was of HPLC grade and degassed prior to use. Hexadecane-
thiol and mercaptopropionic acid were purchased from Sigma–Aldrich
(Gillingham, UK). BSA and IgG from human serum was purchased from
Sigma–Aldrich and reconstituted in 100 mm phosphate buffered saline
(PBS). BtrI and Sfp were expressed and purified as previously de-
scribed[53] with BtrI in 50 mm HEPES buffer (pH 7.0), 50 mm NaCl, 2 mm
dithiothreitol, 0.5 mm EDTA and Sfp in 50 mm HEPES buffer (pH 7.6),
200 mm NaCl. These proteins were donated by Dr. J. L. Thirlway (Man-
chester Interdisciplinary Biocentre, University of Manchester). All the
proteins were used at a concentration of 100 mgmlÀ1 for the SPR experi-
ments.
Conclusion
In summary, a synthetically expedient method for the assem-
bly of functionalised pegylated alkyldisulfides employing an
alternative solid-phase synthetic strategy was successfully
demonstrated, providing the disulfides in high yields and re-
quiring only one chromatographic purification during the
final isolation. Up until now researchers in this field have
primarily relied on a limited number of alkyl thiols that are
commercially available. However, it is envisaged that by
using the simple building blocks and solid-phase methodolo-
gy presented here, and together with other strategies, even
laboratories with little synthetic experience will be able to
prepare bespoke pegylated thiols in a similar way to which
peptides are synthesised. Indeed, by harnessing the full spec-
trum of available solid-phase and combinatorial synthetic
methodologies, it is expected that a plethora of SAM mate-
rials with functional groups not previously studied will now
become accessible.
Further, SAMs of both the synthesised disulfides 1 and 2
were characterised and shown to possess a number of desir-
able properties which were relevant for biological applica-
tions. These SAMs, which incorporated internal amide link-
ages, had improved stability towards displacement by hexa-
decanethiol when compared to the more commonly used
comparison material 7 which lacked the amide moiety. The
SAMs prepared from the hydroxy-functionalised disulfide 1
demonstrated at least equal biocompatibility in comparison
to 7 as determined by SPR for a range of proteins. Addition-
ally, these SAMs are amenable to SNP that allowed the gen-
eration of features of less than 190 nm. The fabrication of a
SAM of 1 with amino-presenting features of 2 allowed the
generation of protein patterns through both covalent and
non-covalent immobilisation methods, a significant result
Equipment: Reversed-phase HPLC was conducted on a Varian ProStar
320 with a Phenomenex Gemini C18 5 mm 250ꢁ10 mm column using
water with 0.1% formic acid and MeCN with 0.1% formic acid as elu-
ents. A gradient from 5 to 100% MeCN/HCO2H from 0–20 min followed
by an isocratic elution of 100% MeCN/HCO2H from 20–37 min was used
in all cases. Biocompatibility studies were carried out using a Biacore
3000 SPR Instrument (GE Healthcare UK, Little Chalfont, UK) with an
automatic sample changer maintained at 258C. XPS was carried out
using a Kratos Axis Ultra DLD (Kratos Analytical, Manchester, UK)
with monochromated AlKa X-ray source. SAM samples were analysed by
contact mode lateral force microscopy (LFM) using a ThermoMicro-
scopes Explorer and in tapping mode with a Nanoscope IIIa atomic force
microscope (Veeco, Cambridge, UK). SNP was performed using Thermo-
Microscopes Aurora III NSOM systems (Veeco).
tert-Butyl 12-hydroxy-4,7,10-trioxadodecanoate (8): This compound was
synthesised based on
a
previous procedure.[46] Sodium (200 mg,
8.7 mmol) was dissolved in anhydrous triethylene glycol (126 mL,
940 mmol) under N2 and anhydrous THF (500 mL) was added. tert-Butyl
acrylate (49 mL, 334.5 mmol) was dissolved in anhydrous THF (500 mL)
under N2 and added dropwise over 5 h to the glycol solution. The entire
mixture was stirred under N2 until all the acrylate was consumed (
ꢀ12 h). The mixture was adjusted to pH 7–8 by dropwise addition of 1m
aq. HCl (ꢀ8 mL) and the THF evaporated under reduced pressure. The
residual oil was redissolved in EtOAc (1 L) and extracted with brine (1 L
once then 0.4 L once). The organic layer was dried with Na2SO4, evapo-
rated under reduced pressure and the residual oil dried in vacuo to yield
a pale yellow oil (69.47 g, 249.6 mmol, 75% with less than 1% diester by
NMR). Rf =0.33 (EtOAc); IR (BaF2): n˜ =3438 (OH), 2872 (alkyl), 1727
(COO), 1368 (CH2O), 1161, 1119 cmÀ1 (COO or CH2O); 1H NMR
(400 MHz, CDCl3): d=1.41 (s, 9H; CH3), 2.48 (t, J=7 Hz, 2H; 2-CH2),
12240
www.chemeurj.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 12234 – 12243

Nanoscale Biomolecular Structures
FULL PAPER
2.67 (brs, 1H; OH), 3.65–3.73 ppm (m, 14H; 3,5,6,8,9,11,12-CH2O); 13C
NMR (100 MHz, CDCl3): d=28.2 (CCH3), 36.3 (2-CH2CO), 61.8 (12-
CH2OH), 67.0 (3-CH2O), 70.4, 70.6, 70.7 (5,6,8,9-CH2O), 72.6 (11-CH2O),
80.6 (CCH3), 171.0 ppm (COO); MS (CI): m/z (%): 223 (20)
[MÀtBu+H]+, 240 (100) [MÀtBu+NH4]+, 296 (40) [M+NH4]+.
tert-Butyl 12-acetoxy-4,7,10-trioxadocecanoate (9): The pegylated ester 8
(5567 mg, 20 mmol) was dissolved in CH2Cl2 (distilled, 30 mL) and Et3N
(4181 mL, 30 mmol) was added followed by Ac2O (2363 mL, 25 mmol). A
silica drying tube was attached and the mixture stirred for 16 h after
which water (50 mL) was added and the mixture was extracted Et2O
(50 mLꢁ3). The organic layer dried with Na2SO4, evaporated under re-
duced pressure and dried in vacuo to yield the desired acetate as a col-
ourless oil (6331 mg, 19.76 mmol, 99%). Rf =0.66 (hexane:EtOAc, 1:3);
IR (BaF2): n˜ =2882 (alkyl), 1732 (COOR), 1367 (Me), 1244 (COOR),
1123 cmÀ1 (ROR); 1H NMR (400 MHz, CDCl3): d=1.44 (s, 9H; C-
was washed with CH2Cl2 (10 mLꢁ3). The organic phases were combined
and evaporated under reduced pressure and the residue dried in vacuo to
yield 4 as a brown oil (1304 mg, 5.28 mmol, 80%). Rf =0.15 (hexane:
EtOAc, 1:2); IR (BaF2): n˜ =3343 (COOH), 2871 (alkyl), 2109 (N3), 1727
(COOH), 1182 (COOH), 1120 cmÀ1 (ROR); 1H NMR (400 MHz,
CDCl3): d=2.66 (t, J=6 Hz, 2H; 2-CH2CO), 3.40 (t, J=5 Hz, 2H; 12-
CH2N3), 3.56–3.83 (m, 12H; 3,5,6,8,9,11-CH2O), 9.06 ppm (brs, 1H;
COO13C NMR (100 MHz, CDCl3): d=CH2O), 70.1, 70.5, 70.7 (5,6,8,9,11-
CH2O), 176.2 ppm (COOH); MS (ES+): m/z (%):270 (100) [M+Na]+,
286 (60) [M+K]+; HRMS: m/z calcd for [M+Na]+: 270.1060; found
270.1058 (d=0.9 ppm).
11-(N-Phthalimido)undecan-1-ol (11): 11-Bromoundecanol (5008 mg,
20 mmol) and potassium phthalimide (4078 mg, 22 mmol) were mixed in
DMF (100 mL) and refluxed at 608C for 4 h. The mixture was evaporat-
ed to a small volume under reduced pressure, resuspended in CHCl3
(25 mL) and filtered through a celite bed. The filtrate was evaporated
under reduced pressure to yield the desired product as a white solid
(6215 mg, 19.58 mmol, 98%). Rf =0.37 (hexane:EtOAc, 2:1); m.p. 858C
(lit.[61] 85–868C); IR (BaF2): n˜ =3344 (OH), 2926 (alkyl), 2848 (alkyl),
1717 (imide), 1399 (alkyl), 1057 (OH), 725 cmÀ1 (aryl); 1H NMR
(300 MHz, CDCl3): d=1.10–1.40 (m, 14H; 3–9-CH2), 1.42–1.75 (m, 4H;
2,10-CH2), 3.56–3.75 (m, 4H; 1,11-C ppm; 13C NMR (75 MHz, CDCl3):
d=25.8, 26.9, 28.7, 29.3, 29.5 (d), 29.6 (3–10-CH2), 32.9 (2-CH2), 38.2 (11-
CH2N), 63.2 (1-CH2OH), 123.3 (2,5-Phth), 132.3 (1,6-Phth), 134.0 (3,4-
Phth), 168.6 ppm (CON); MS (ES+): m/z (%): 381 (100)
[M+Na+MeCN]+, 657 (50) [2M+Na]+.
ACHTUNGTRENNUNG(CH3)3), 2.07 (s, 3H; CH3CO), 2.50 (t, J=7 Hz, 2H; 2-CH2CO), 3.55–
3.75 (m, 12H; 3,5,6,8,9,11-CH2O), 4.21 ppm (t, J=7 Hz, 2H; 12-
CO2CH2); 13C NMR (75 MHz, CDCl3): d=21.4 (CH3CO), 28.5 (C-
ACHTUNGTRENNUNG(CH3)3), 36.6 (2-CH2CO), 64.0 (3-CH2O), 67.3 (12-CO2CH2), 69.5 (11-
CH2O), 70.8, 71.0 (5,6,8,9-CH2O), 80.9 (CACHTUNGRTNEUNG(CH3)3), 171.3 (1-CO2),
171.4 ppm (CH3CO); MS (CI): m/z (%): 338 (100) [M+NH4]+; HRMS:
m/z calcd for [M+NH4]+: 338.2173; found 338.2172 (d=0.4 ppm).
12-Acetoxy-4,7,10-trioxadocecanoic acid (3): The acetoxy ester
9
(6326 mg, 19.75 mmol) was dissolved in CH2Cl2 (50 mL), TFA (50 mL)
was added and the mixture was stirred for 2 h. Subsequently, the solution
was evaporated under reduced pressure and the residual oil was redis-
solved in CH2Cl2 (100 mL) and stirred with Amberlyst A-21 for 1 h. The
resin was filtered and the filtrate evaporated under reduced pressure to
give 3 as a pale yellow oil (4486 mg, 16.98 mmol, 86%). Rf =0.05 (hexa-
ne:EtOAc, 1:3); IR (BaF2) n˜ =3483 (COOH), 2878 (alkyl or COOH),
1737 (COOR or COOH), 1248 (COOR or COOH), 1111 cmÀ1 (ROR);
1H NMR (400 MHz, CDCl3): d=2.06 (s, 3H; CH3CO), 2.62 (t, J=6 Hz,
2H; 2-CH2CO), 3.55–3.71 (m, 10H; 5,6,8,9,11-CH2O), 3.75 (t, J=6 Hz,
2H; 3-CH2O), 4.21 ppm (t, J=5 Hz, 2H; 12-CO2CH2); 13C NMR
(100 MHz, CDCl3): d=21.1 (CH3CO), 34.9 (2-CH2CO), 63.7 (12-
CO2CH2), 66.4 (3-CH2O), 69.2 (11-CH2O), 70.5, 70.6, 70.7 (5,6,8,9-
CH2O), 171.4 (CH3CO), 176.3 ppm (1-CO2H); MS (ES+): m/z (%): 287
(100) [M+Na]+; HRMS: m/z calcd for [M+Na]+: 287.1101; found
287.1094 (d=2.5 ppm).
1-[11-(N-Phthalimido)]undecyl thioacetate (12): Compound 11 (2365 mg,
7.45 mmol) and Et3N (1740 mL, 12.50 mmol) were dissolved in CH2Cl2
(25 mL). The solution was cooled in an ice bath, mesyl chloride (774 mL,
10 mmol) was added dropwise while stirring and the mixture was stirred
at RT for a further 2 h. CH2Cl2 (50 mL) and Et2O (25 mL) were added to
the mixture and the resulting solution was extracted with 0.5m aq. HCl
(50 mLꢁ2), water (50 mL) and 5% w/v NaHCO3 aq. solution (100 mL).
The organic layer was dried with MgSO4 and evaporated under reduced
pressure to give the desired mesylate as a yellow oil which solidified on
standing to a pale yellow solid. This was added to a vigorously stirring
mixture of AcSH (620 mL, 8.70 mmol) and K2CO3 (1500 mg, 10.85 mmol)
in DMF (50 mL) and stirred for 15 h. The suspension was reduced to a
small volume under reduced pressure and the residue was resuspended
with CHCl3 (50 mL). This was extracted with water (150 mLꢁ2), 0.5m
aq. HCl (100 mLꢁ2) and 5% w/v NaHCO3 aq. solution (150 mLꢁ3),
dried with MgSO4 and evaporated under reduced pressure to yield the
thioacetate as a buff semi-crystalline solid (2728 mg, 7.26 mmol, 97%).
Rf =0.47 (hexane:EtOAc, 6:1); m.p. 788C; IR (BaF2): d=2918 (alkyl),
2851 (alkyl), 1698 (CONCO and COS), 1467 (aryl), 1407 cmÀ1 (aryl); 1H
NMR (400 MHz, CDCl3): d=1.15–1.45 (m, 14H; 3–9-CH2), 1.54 (tt, J=
7, 7 Hz, 2H; 10-CH2), 1.66 (tt, J=7, 7 Hz, 2H; 2-CH2), 2.31 (s, 3H;
SCOCH3), 2.85 (t, J=7 Hz, 2H; 1-CH2COS), 3.67 (t, J=7 Hz 2H;
NCH2), 7.70 (dd, J=3, 5 Hz, 2H; ppm Hz,1H NMR : d=2,5-Phth), 132.3
(1,6-Phth), 134.0 (3,4-Phth), 168.6 (CON), 196.3 ppm (COS); MS (ES+):
m/z calcd: 376 (85) [M+H]+, 393 (25) [M+NH4]+, 768 (100) [2M+NH4]+
tert-Butyl 12-azido-4,7,10-trioxadocecanoate (10): A solution of hydroxy–
PEG ester 8 (9116 mg, 32.75 mmol) and Et3N (9220 mL, 66.15 mmol) in
CH2Cl2 (distilled, 50 mL) was cooled in an ice bath with a CaCl2 drying
tube. Mesyl chloride (3800 mL, 49.10 mmol) was added dropwise while
stirring and the mixture was stirred at RT for a further 2.5 h. The mixture
was reduced to a small volume under reduced pressure, water (350 mL)
was added and the suspension was extracted with EtOAc (250 mLꢁ3).
The organic layers were combined and dried with Na2SO4 then evaporat-
ed under reduced pressure to yield the mesylate as a yellow oil. This oil
was redissolved in DMF (100 mL), NaN3 (6387 mg, 98.25 mmol) and the
suspension was stirred for 7 d. The mixture was reduced to a small
volume under reduced pressure, water (200 mL) was added and extracted
with Et2O (150 mLꢁ2). The organic layers were combined, dried with
Na2SO4, evaporated under reduced pressure and dried in vacuo to yield
the desired compound as a pale yellow oil (9460 mg, 31.19 mmol, 95%).
Rf =0.59 (EtOAc); IR (BaF2): n˜ =2865 (alkyl), 2103 (N3), 1722 (COOR),
1158 (COOR), 1119 cmÀ1 (ROR); 1H NMR (400 MHz, CDCl3): d=1.44
(s, 9H; CH3), 2.50 (t, J=7 Hz, 2H; 2-CH2), 3.39 (t, J=5 Hz, 2H; 12-
CH2), 3.57–3.80 ppm (m, 12H; 3,5,6,8,9,11-CH2); 13C NMR (100 MHz,
CDCl3): d=28.2 (CCH3), 36.4 (2-CH2CO), 50.8 (12-CH2N3), 67.0 (3-
CH2O), 70.2, 70.5, 70.8 (t; 5,6,8,9,11-CH2O), 80.7 (CCH3), 171.1 ppm
(COO); MS (CI): m/z (%): 265 (100) [MÀtBu+NH4]+, 321 (80)
[M+NH4]+; HRMS: m/z calcd for [M+NH4]+: 321.2132; found 321.2125
(d=2.3 ppm).
;
HRMS: m/z calcd for [M+NH4]+: 393.2206; found 393.2205 (d=
0.4 ppm).
11-(N-Phthalimido)undecane-1-thiol (5): The thioacetate 12 (2306 mg,
6.45 mmol) was suspended in MeOH (anhydrous, degassed, 60 mL)
under an inert atmosphere. Separately, NaSMe (456 mg, 6.50 mmol) was
dissolved in MeOH (anhydrous, degassed, 6.5 mL) under an inert atmos-
phere and added to the thioacetate suspension. The mixture was stirred
for 2 h during which the solids were observed to gradually dissolve and
the reaction was quenched with 0.1m aq. HCl (140 mL) and extracted
twice with Et2O (degassed, 75 mL). The organic layer was dried with
MgSO4, evaporated under reduced pressure and dried in vacuo to give 5
an oil, which solidified into a buff solid which was pure by NMR spec-
troscopy (2031 mg, 6.09 mmol, 94%). Rf =0.57 (hexane:EtOAc, 4:1); IR
(BaF2): n˜ =2925, 2851 (alkyl), 1713 (CONCO), 1393, 1365 cmÀ1 (SH or
alkyl); 1H NMR (400 MHz, CDCl3): d=1.15–1.45 (m, 14H; 3–9-CH2),
1.54–1.73 (m, 4H; 2,10-CH2), 2.51 (td, J=7, 7 Hz, 2H; 1-CH2S), 3.67 (t,
J=7 Hz, 2H; NCH2), 7.70 (dd, J=3, 5 Hz, 2H; ppm Hz13C NMR : d=
12-Azido-4,7,10-trioxadocecanoic acid (4): The azido–PEG ester 10
(2000 mg, 6.59 mmol) was stirred with 4m HCl in 1,4-dioxane (10 mL) for
12 h and the mixture was then evaporated under reduced pressure. The
residual oil was redissolved in CH2Cl2 (10 mL) and stirred with Amber-
lyst A-15 resin (2 g) for 15 min. The solution was eluted and the resin
Chem. Eur. J. 2010, 16, 12234 – 12243
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12241

J. Micklefield et al.
2,5-Phth), 132.3 (1,6-Phth), 134.0 (3,4-Phth), 168.6 ppm (CON); MS
(ES+): m/z (%): 334 (100) [M+H]+; HRMS: m/z calcd for [M+H]+:
334.1835; found 334.18.40 (d=1.4 ppm).
CH2Cl2, DMF, CH2Cl2, Et2O (3 mLꢁ3 each solvent in order) and dried
in vacuo to give a resin that returned a negative ninhydrin test result.
This dried resin was resuspended in 1,4-dioxane/water (4:1, 2 mL), 1m
Me3P in THF (960 mL, 960 mmol) was added and the mixture was gently
swirled for 18 h. The solution was eluted and the resin rinsed with 1,4-di-
oxane, CH2Cl2, 1,4-dioxane, CH2Cl2 (3 mLꢁ3 each solvent in order). This
resin was dried in vacuo and gave a strongly positive result to the ninhy-
drin test.
11-(N-Phthalimido)undecyl 1-thio-(4-methoxytrityl) polystyrene resin
(13): Resin 6 (2 g, 3.0 mmol) was placed in a sealed sintered glass column
and purged with N2. SOCl2 (330 mL, 4.53 mmol) was dissolved in CH2Cl2
(distilled, 9 mL) and added to the resin. The mixture was shaken for 1 h,
the resin drained by applying N2 pressure to the top of the column and
washed with CH2Cl2 (distilled, 10 mLꢁ3). Thiol 5 (1250 mg, 3.75 mmol)
and N,N-diisopropylethylamine (DIPEA; 2080 mL, 12.02 mmol) were dis-
solved in CH2Cl2 (distilled, 9 mL) then added to the resin and shaken for
2 h. The resin was drained, washed with CH2Cl2/MeOH/DIPEA (17:2:1,
10 mLꢁ3), CH2Cl2, MeOH, CH2Cl2, MeOH and Et2O (10 mLꢁ3 each
solvent in sequence). The resin was dried in vacuo and gave a negative
result to the ninhydrin test.[62]
11-[12-(12-amino-4,7,10-trioxadocecamido)-4,7,10-trioxadocecamido]-
AHCTUNGTERGuNNUN ndecyl-1-thio-(4-methoxytrityl) polystyrene resin (17): The coupling
above was repeated (negative ninhydrin test), followed by the reduction
step above (positive ninhydrin test) on the resin (ꢀ160 mmol).
Di-1-{[12-(12-amino-4,7,10-trioxadocecamido)-4,7,10-trioxadocecami-
do]undecyl}disulfide diformate salt (2): The resin above (ꢀ160 mmol)
was treated with TFA and the residue oxidised using the same procedure
as for 1 to yield a pale yellow oil. This oil was purified by RP-HPLC to
give a white hygroscopic solid (116 mg, 60% based on the Fmoc quantifi-
cation). HPLC (256 nm) 13.5 min; 1H NMR (400 MHz, CD3OD): d=
1.30–1.50 (m, 32H; 3–10-CH2), 1.72 (tt, J=7, 7 Hz, 4H; 2-CH2), 2.45–
2.55 (m, 8H; 2’,2’’-CH2CO), 2.72 (t, J=7 Hz, 4H; 1-CH2S), 3.02 (brs,
2H; CONH), 3.14 (t, J=5 Hz, 4H; 12’-CH2N), 3.21 (t, J=7 Hz, 4H; 11-
CH2N), 3.41 (t, J=6 Hz, 4H; 12’’-CH2NH3+), 3.58 (t, J=6 Hz, 4H; 11’’-
CH2O), 3.61–3.85 ppm (m, 44H; 3’,5’,6’,8’,9’11’,3’’,5’’,6’’,8’’,9’’-CH2O); 13C
NMR (100 MHz, CD3OD): d=28.9 (9-CH2), 30.3 (3-CH2), 31.1, 31.2,
31.3, 31.4, 31.5, 31.6 (d) (2,4–8,10-CH2), 38.3 (br), 38.5 (1,2’,2’’-CH2), 40.6
(12’’-CH2NH3+), 41.3 (11-CH2N), 41.6 (12’-CH2N), 69.1, 69.2 (3’,3’’-
CH2O), 71.4 (11’’-CH2O), 72.1 (d), 72.2, 72.3, 72.4 (d)
(5’,6’,8’,9’,10’,11’,5’’,6’’,8’’,9’’,10’’-CH2O), 171.2 (HCO2À), 174.6 (1’-CONH),
174.9 ppm (1’’-CONH); MS (ES+): m/z (%): 609 (100) [M+2H]2+, 1218
(50) [M+H]+, 1240 (10) [M+Na]+; HRMS: m/z calcd for [M+H]+:
1217.7968; found: 1217.7940 (d=2.3 ppm).
11-Aminoundecyl-1-thio-(4-methoxytrityl) polystyrene resin (14): The
phthalimido-resin (200 mg) was shaken with 15% v/v N2H4·H2O in DMF/
CH2Cl2 (8:2, 1.5 mL) for 16 h. The solution removed and the resin
washed with DMF, CH2Cl2, DMF, CH2Cl2 (3 mLꢁ3 each solvent in
order) and dried in vacuo.
N-Fmoc-11-aminoundecyl-1-thio-(4-methoxytrityl) polystyrene resin (15):
The aminoundecyl resin 7 (5 mg) was mixed for 2 h with CH2Cl2 (100 mL)
containing DIPEA (5.2 m, 30 mmol) and FmocCl (3.9 mg, 15 mmol). The
resin was rinsed with DMF, CH2Cl2, DMF, CH2Cl2 (3 mLꢁ3 each solvent
in order) and dried in vacuo to give a resin which yielded a negative nin-
hydrin test result and an Fmoc loading[63] of 0.80 mmolgÀ1 (53% over 3
steps relative or hydroxy-resin loading).
11-(12-Hydroxy-4,7,10-trioxadocecamido)undecyl-1-thio-(4-methoxytri-
tyl) polystyrene resin (16): The acetoxy acid 3 (134 mg, 507 mmol) was
dissolved in CH2Cl2/DMF (1:1, 800 mL), DIPEA was added (262 mL,
1514 mmol) and the mixture was cooled in an ice bath. TCFH (142 mg,
500 mmol) was added to the cooled solution, gently swirled for 2 min and
the entire solution was added to the aminoundecyl resin 7 (316 mg,
ꢀ250 mmol). The reaction was shaken for 65 h at RT, and the resin was
drained, washed with DMF, CH2Cl2, DMF, CH2Cl2, Et2O (3 mLꢁ3 each
solvent in order) and dried in vacuo to give a resin which returned a neg-
ative ninhydrin test result. This resin was shaken with 15% v/v N2H4·H2O
in DMF/CH2Cl2 (8:2, 2 mL) for 16 h. The solution removed and the resin
washed with DMF, CH2Cl2, DMF, CH2Cl2 (3 mLꢁ3 each solvent in
order) and dried in vacuo.
Preparation of SAMs of 1, 2 and 7: Gold (50 nm) was evaporated on to
chromium-primed (2 nm) slides at a rate of 0.1–0.2 nmsÀ1 at 10À7 bar.
Slides and other glassware were cleaned using piranha solution, a 3:7 mix
of 30% H2O2 and conc. H2SO4, before washing with copious amounts (6
rinses) of deionised water (18.2 MW) and drying in an oven overnight.
Caution: the preparation of piranha solution is highly exothermic and po-
tentially violent in the presence of organic materials. Extreme care should
be taken. SAMs were formed by the incubation of gold-coated slides for
a minimum of 18 h in 0.25 mm solutions of the appropriate disulfide in
ethanol for 1, or deionised water for 2; or in 0.5 mm ethanolic solutions
of 7. The equilibrated SAMs were removed from solution, washed with
HPLC grade ethanol, dried under a jet of N2 and cut to size on a clean
surface using a diamond scribe as needed.
Di-1-[11-(12-hydroxy-4,7,10-trioxadocecamido)undecyl]disulfide (1): The
hydroxy-pegylated resin 16 above (~250 mmol) was mixed with 5% TFA
v/v in CH2Cl2 (1 mL) for 3 min and the solution was eluted. This was re-
peated for a total of six cycles; the eluents were combined and evaporat-
ed under reduced pressure. The residual oil was resuspended in 0.1m aq.
(NH4)HCO3 (20 mL) and air was bubbled through the mixture until di-
sulfide formation was complete (ꢀ16 h). The mixture was lyophilised
and purified by RP-HPLC, the desired fractions were combined and
lyophilised to yield the desired product as a white solid (132 mg, 65%
overall yield based on original Fmoc-resin loading). HPLC (256 nm)
24.5 min; 1H NMR (400 MHz, CD3OD): d=1.25–1.57 (m, 32H; 3–10-
CH2), 1.68 (tt, J=7, 7 Hz, 4H; 2-CH2), 2.43 (t, J=7 Hz, 4H; 2’-CH2CO),
2.68 (t, J=7 Hz, 4H; 1-CH2S), 3.17 (t, J=7 Hz, 4H; 11-CH2N), 3.56 (t,
J=5 Hz, 4H; 11’-CH2O), 3.59–3.69 (m, 20H; 5’,6’,8’,9’,12’-CH2O),
3.72 ppm (t, 4H; J=5, 3’-CH2O); 13C NMR (100 MHz, CD3OD): d=28.0
(9-CH2), 29.5 (3-CH2), 30.2, 30.3, 30.4, 30.5, 30.6, 30.7 (d) (2,4–8,10-CH2),
37.64 (2’-CH2CO), 39.8 (1-CH2S), 40.4 (11-CH2N), 62.2 (12’-CH2OH),
68.3 (3’-CH2O), 71.2, 71.3, 71.4, 71.6 (5’,6’,8’,9’-CH2O), 73.6 (11’-CH2O),
173.8 ppm (CONH); MS (ES+): m/z (%): 407 (65) [M+2H]2+, 418 (15)
[M+H+Na]2+, 814 (100) [M+H]+, 836 (40) [M+Na]+; HRMS: m/z calcd
for [M+H]+: 813.5327; found: 813.5328 (d=0.1 ppm).
SAM thiolate displacement by hexadecanethiol and XPS analysis: SAMs
of 1, 2, and 7 were immersed in 10 mm solutions of hexadecanethiol in
EtOH for a range of durations (4.5, 9, 18, 36, 48, 72 and 180 h). Samples
were washed in HPLC grade ethanol (1 and 7) or nanopure water and
then ethanol (2) then blow dried under an N2 stream. Scans were collect-
ed at 20 keV and the C1s spectra for the different immersion durations
were compared to virgin SAMs to determine rate of loss of ether car-
bons. Scans for each time point were taken for three different SAMs
made on three different days. The ratio of the area of the CCO peak to
the Au(4f) peak was therefore calculated for each sample.
SAM protein adsorption measurements by SPR analysis: Prior to each
experiment, SAM chips were primed by flowing sample-matched aque-
ous biological buffer over them at 5 mLsÀ1 for a period of 30 min. For
each type of protein and SAM chip, 5 mgmlÀ1 protein solutions were in-
jected into appropriately matched buffer carrier streams at a flow rate of
10 mLsÀ1 for a duration of 5 min. These were then followed with a buffer-
only elution for 3 min, a 5 s injection of 0.1% w/v SDS and then buffer
flow for a further minute.
11-(12-amino-4,7,10-trioxadocecamido)undecyl-1-thio-(4-methoxytrityl)
polystyrene resin: The azido acid 4 (79 mg, 319 mmol) was dissolved in
CH2Cl2/DMF (1:1, 500 mL), DIPEA (166 mL, 959 mmol) was added and
the resulting solution was cooled in an ice bath. TCFH (90 mg, 302 mmol)
was added and the mixture gently swirled for 2 min before addition to
the aminoundecyl resin 14 (200 mg, ꢀ160 mmol). This resin suspension
was shaken for 65 h at RT, and the resin was filtered, washed with DMF,
Scanning near-field photolithography and protein array fabrication: Light
from the frequency doubled argon ion laser was coupled to the end of
the fiber probe through an optical coupler. Laser power of 4 mW was
coupled to the NSOM fiber probe with an aperture size of 100 nm and a
scan speed of 5 s per dot was used to generate the dot arrays and
0.2 mmsÀ1 was used to create patterned lines. After patterning, the sam-
12242
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Chem. Eur. J. 2010, 16, 12234 – 12243

Nanoscale Biomolecular Structures
FULL PAPER
ples were dipped in a thiol/disulfide solution with contrasting terminal
group (1 mm 16-hexadecanethiol, 1 mm 3-mercaptopropionic acid in
EtOH) for at least 30 min to create the compositional chemical patterns.
For patterns of 2, a solution of 0.1 mm was used and the patterned chips
were immersed for 18 h to give 18. For the non-covalent immobilisation
of proteins on patterned SAMs, the SNP-patterned SAM 18 was im-
mersed in the IgG solution for 2.5 h, washed with PBS (5 times), water
(once) and dried under an N2 jet. For covalent immobilisation, SAM 18
was immersed in a 25% aq. solution of glutaraldehyde for 30 min,
washed with water (5 times) and dried under an N2 stream to give 19.
These SAMs were then exposed to IgG under the same conditions as
above.
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The authors wish to thank the EPSRC for funding through the “Snomi-
pede” Basic Technology grant (EP/C523857/1) and a Life Science Inter-
face fellowship (EP/F042590/1) for L.S.W. The donation of protein sam-
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Revised: May 21, 2010
Published online: September 14, 2010
Chem. Eur. J. 2010, 16, 12234 – 12243
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12243