Solid phase synthesis of functionalised SAM-forming alkanethiol- oligoethyleneglycols

Article abstract of DOI:10.1039/c4tb00573b

We present an efficient solid phase synthesis methodology that provides easy access to a range of functionalised long-chain alkanethiol- oligoethyleneglycols that form well-defined self-assembled monolayers on gold and are compatible with pre- or post-ass

Full text of DOI:10.1039/c4tb00573b

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Materials Chemistry B
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Solid phase synthesis of functionalised SAM-
forming alkanethiol–oligoethyleneglycols†
Cite this: J. Mater. Chem. B, 2014, 2,
741
3
ab
bc
bd
e
James Murray, Dominika Nowak, Laurynas Pukenas, Rizuan Azhar,
a
c
bd
e
Mathieu Guillorit, Christoph Walti, Kevin Critchley, Steven Johnson
¨
Received 10th April 2014
Accepted 22nd April 2014
ab
and Robin S. Bon*
DOI: 10.1039/c4tb00573b
www.rsc.org/MaterialsB
We present an efficient solid phase synthesis methodology that However, commercial availability of functionalised LCAT–OEGs
provides easy access to a range of functionalised long-chain alka- is severely limited and synthetic routes oen laborious. In this
nethiol–oligoethyleneglycols that form well-defined self-assembled study, we present a versatile solid phase synthesis route that
monolayers on gold and are compatible with pre- or post-assembly provides easy access to a range of functionalised LCAT–OEGs.
conjugation of (bio)molecules. We demonstrate the versatility of our Using a range of surface characterisation techniques including
synthetic route by synthesising LCAT–OEGs with a range of functional surface plasmon resonance (SPR) and electrochemical spec-
moieties, including peptides, electro-active redox groups, chemical troscopy, we demonstrate that the resulting LCAT–OEGs form
handles for post-assembly conjugation of (bio)molecules, and dense, well-dened mixed SAMs of high quality, which are
demonstrate the application of our LCAT–OEG monolayers in compatible with on-surface bioorthogonal ligation reactions.
immunosensing, where they show good biocompatibility with minimal
Customised SAMs presenting ligands and/or biomolecules
can be prepared according to two general strategies (Fig. 1): (i)
conjugation of (bio)molecules to LCAT–OEGs aer SAM
formation (post-assembly conjugation); (ii) total synthesis of
functionalised LCAT–OEGs followed by SAM formation (pre-
assembly conjugation). Post-assembly conjugation requires
robust, chemoselective, and mild chemistry to ensure complete
biofouling.
Self-assembled monolayers (SAMs) of functionalised long-chain
1
alkanethiolates (LCATs) on metals provide an excellent and
versatile platform for the chemoselective immobilisation of
(
bio)molecules on planar and nanoparticle surfaces. Their well-
2,5
surface functionalisation without side reactions. For pre-
2
dened thickness, structure, and dielectric properties make
them ideally suited for applications where a high degree of
control of the physical and chemical properties of surfaces is
required. Functionalised LCATs also enable the creation of
assembly conjugation approaches, total synthesis of LCAT–
6
6c,7
OEGs terminated with, amongst others, biotin, peptides,
8
9
9
10
ferrocene, azides, aminooxy groups, and sugars has been
demonstrated. Such functionalised LCAT–OEGs are usually
synthesised in solution, which requires multiple chromato-
graphic steps and suffers from poor overall yields. Solid phase
chemistry presents an attractive alternative for synthesising
3
patterned surfaces critical for protein and DNA microarrays.
SAMs used for protein biosensing applications usually require
LCATs incorporating oligoethylene glycol (OEG) linkers to help
4
reduce non-specic protein adsorption and functional groups
that allow the further tuning of surface chemistry providing
control over the immobilisation, density and orientation of
capture molecules. Thus, easy access to a variety of functional-
ised LCAT–OEGs is essential for optimisation of, for example,
immobilisation chemistry and OEG and alkanethiolate length.
a
School of Chemistry, University of Leeds, LS2 9JT, UK. E-mail: r.bon@leeds.ac.uk
b
Astbury Centre for Structural Molecular Biology, University of Leeds, UK
c
School of Electronic and Electrical Engineering, University of Leeds, UK
d
School of Physics and Astronomy, University of Leeds, UK
e
Department of Electronics, University of York, Heslington York, YO10 5DD, UK
Fig. 1 SAM functionalisation strategies. (a) SAM formation (in the
example: a mixed SAM, with a hydroxy-terminated diluent, on a gold
surface); (b) conjugation of LCAT 1 with reagent 2 (in which functional
†
Electronic supplementary information (ESI) available: Synthesis and
characterisation of compounds, XPS, EIS, AFM, contact angle measurements,
CV, colorimetry and SPR. See DOI: 10.1039/c4tb00573b
0
0
groups X and Y react to form linker X Y ).
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LCAT–OEGs. The groups of Whitesides and Mrksich have coupled to 2-chlorotrityl resin-bound 11-mercaptoundecanoic
demonstrated the attachment of a thiol-protected LCAT–OEG to acid (11-MUA) 9 (Scheme 1B). The terminal alcohol of 10c was
7
b,11
resin-bound peptides,
while Albericio et al. have successfully tosylated and treated with sodium azide to afford resin-bound
6b
used 2-chlorotrityl chloride resin to construct biotinylated and azide 12. Reduction of 12 under Staudinger conditions proved
peptide-containing LCAT–OEGs. However, for applications sluggish, but proceeded rapidly with tin thiophenolate to
that require a quick, iterative ne-tuning of SAM properties/ afford resin-bound amine 14.
7a
15
functionalities, currently available syntheses are oen not
Using Fmoc-solid phase peptide synthesis, we demonstrated
sufficient. To address this need, we developed a exible solid that 14 is an excellent starting point for the synthesis of LCAT–
phase synthesis that gives access to a range of LCAT–OEGs OEG–peptide conjugates such as 17 (for pre-assembly func-
through late-stage functionalisation, while minimising inter- tionalisation strategies) or LCAT–OEG 18 incorporating the
mediate purication.
redox probe methylene blue (MB; Scheme 1C). MB was chosen
OEGs are oen linked to LCATs through an ether bond. for electrochemical analysis of SAMs (see below) because of its
6b,7a
16
However, in analogy to Albericio's approach,
we chose to use superior aqueous stability compared to ferrocene. MB-func-
an amide linker, which has been shown to improve SAM tionalised LCAT–OEG 18 was synthesised by acylating resin-
stability through the formation of networks of hydrogen bound amine 14 with carboxylate 16, which was prepared by
12
bonds. We rst synthesised amino-OEGs 6 through the sil- adapting a procedure described by Pheeney et al. (Scheme
17
ver(I) oxide-mediated mono-tosylation of oligoethyleneglycols 4 S1.1†). Reactions were allowed to run until all resin-bound
ref. 13) followed by tosyl displacement with sodium azide and starting material was consumed, according to LC-MS analysis
(
14
Staudinger reduction (Scheme 1A). Next, 6a, 6b and 7 were aer micro-cleavage. The immobilised molecules can be
Scheme 1 Synthesis of LCAT–OEGs: (A) preparation of amino-OEGs 6a and 6b. (B) Solid phase synthesis of functionalised LCAT–OEGs on 2-
chlorotrityl chloride resin 8. (C) On-resin synthesis of peptide-labelled LCAT–OEGs 17 and methylene blue-labelled LCAT–OEG 18. Yields of 11a,
1
1
1b, 13, 15, and 16–18 were calculated after chromatography. Insets: Structures of 2-(2-aminoethoxy)ethanol 7 and methylene blue derivative
6. Abbreviations: Ac ¼ acetyl; DCM ¼ dichloromethane; DIC ¼ diisopropylcarbodiimide; DIPEA ¼ N,N-diisopropylethylamine; DMAP ¼ 4-
dimethylaminopyridine; DMF ¼ N,N-dimethylformamide; Fmoc ¼ 9-fluorenylmethyloxycarbonyl; HOBt ¼ 1-hydroxybenzotriazole; 11-MUA ¼
1
1-mercaptoundecanic acid; SPPS ¼ solid phase peptide synthesis; TES ¼ triethylsilane; TFA ¼ trifluoroacetic acid; Ts ¼ 4-toluenesulfonyl.
3742 | J. Mater. Chem. B, 2014, 2, 3741–3744
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cleaved from the solid support at any point using triuoroacetic (AFM) of a SAM of 11a : 17a (1 : 1) revealed uniform surfaces
acid and triethylsilane (at least one equivalent to scavenge resin- absent of macroscopic islands resulting from phase separation
bound trityl cations). Aer purication by chromatography (Fig. S5.1†). Cyclic voltammetry (CV) measurements of mixed
(
either SiO or C18), the pure LCAT–OEGs 11a, 11b, 13, 15, 17 SAMs formed from different ratios of 11b : 15 (Fig. S6.1–S6.3†)
2
ꢀ
6
ꢀ1
and 18 were obtained in good total yields, based on the loading revealed SAM capacitance values of 4.4 ꢂ 10 F cm , which
of 2-chlorotrityl chloride resin 8.‡ corresponds well with the capacitance of simple LCAT mono-
Next, we analysed the quality of SAMs assembled from our layers. Furthermore, the absence of redox peaks due to electron
functionalised LCAT–OEGs on sputtered gold surfaces. Using X- transfer to solution phase redox probe conrms the absence of
ray photoelectron spectroscopy (XPS), we found S 2p peaks of pin holes within the LCAT–OEG monolayers.
19
SAMs of 11a, 11b, and 13 to have binding energies consistent
To demonstrate the potential suitability of MB-functional-
with the formation of thiolate bonds (Fig. S2.1†). The C 1s region ised LCAT–OEGs for molecular electronics applications, we
consisted of three peaks for all SAMs, corresponding to the alkyl assessed the redox behaviour of surface-bound MB by CV. CV
chain, the OEG chain, and carbonyls (Fig. 2A). The analysis of the measurements on SAMs of 18 showed clear oxidation and
N 1s region for SAMs of 13 was complicated due to rapid degra- reduction peaks associated with the MB moiety around ꢀ130
dation of the azide group under XPS conditions (see the ESI†). mV vs. Ag/AgCl (Fig. 2C). No redox peaks were observed in
The azide-terminated SAM 13 resulted in a surface that was of equivalent CV measurements performed on non-redox active
slightly lower surface energy than SAMs of 11a or 11b. This LCAT–OEG SAMs (compounds 15 and 17a, Fig. S6.1–S6.3†). The
enabled the degree of mixing in the SAMs between diluents 11a peaks are reasonably symmetric with only a small peak sepa-
ꢀ1
or 11b and azide 13 to be estimated by contact angle analysis by ration (9 mV at a scan rate of 200 mV s ), typical of the elec-
18
applying the Cassie equation (Fig. 2B). The insulating proper- trochemical behaviour of
a fully reversible redox-active
ties of SAMs containing different ratios of 11a : 15 or 11a : 17a monolayer. Furthermore, the peak anodic and cathodic current
were assessed by electrochemical impedance spectroscopy was found to increase linearly with scan rate (see inset of
ꢁ
ꢁ
(
Fig. S3.1 and S3.2†) The minimum phase angles of ꢀ88 to ꢀ83
Fig. 2C), again characteristic of a surface-tethered redox-active
ꢁ
ꢁ
for SAMs of 11a : 15 and ꢀ88 to ꢀ87 for SAMs of 11a : 17a, group. From the gradient of the straight line t to peak current
1
3
measured at 0.1 Hz, correspond to the formation of well-packed, vs. scan rate we calculated a surface coverage, G ¼ 8.8 ꢂ 10
2
insulating monolayers that are almost free of pinholes and molecules per cm . This is slightly lower than the theoretical
collapsed sites effects. Furthermore, atomic force microscopy density predicted for a perfect SAM formed from LCATs only (4
1
4
2
ꢂ 10 molecules per cm ) and is likely due to the steric
hindrance of the bulky OEG and MB groups that inhibit denser
monolayer assembly. Finally, the FWHM of the oxidation and
ꢀ
1
reduction peaks was found to be 37 mV (at 200 mV s ). While
this is lower than the theoretical ideal (45.3 mV for a 2 electron
process) we note that deviations in the FWHM are not
uncommon in densely packed redox-active monolayers due to
electrostatic interactions between adjacent charged species.
Condent that our molecules formed well-dened SAMs, we
demonstrated that they also allow (bio)molecule immobilisa-
tion by common post-assembly conjugation techniques:
Copper-catalysed azide-alkyne cycloaddition (CuAAC) and bis-
(
sulfosuccinimidyl) suberate (BS3)-mediated amide coupling.
First, we ‘clicked’ propargyl biotin onto a SAM of 11a : 13
1 : 1; see ESI†) and tested for the presence of biotin attached
(
covalently to the surface using a colorimetric assay (Fig. 3A).
Briey, we introduced streptavidin-alkaline phosphatase fusion
protein to the surface and used western blue as a stain. Only
spots where biotin had been successfully linked to the surface
Fig. 2 Characterisation of LCAT–OEG SAMs. (A) XPS data of C 1s region were stained purple (Fig. 3A, S7.1 and S7.2†). Finally, we tested
for reference SAMs of 11a (dotted), 11b (dashed) and 13 (solid) normalised
to the alkyl chain peak at 284.9 eV. Spectra reflect similar carbonyl (288.3
eV), but varying amount of OEG (286.9 eV) carbon. (B) A linear change in
cosine of the contact angle with molar ratio of azide 13 in mixed SAMs
was observed. Triangles facing down represent 11a : 13; facing up –
the suitability of SAMs containing amine 15 for immobilisation
of the clinically relevant human chorionic gonadotropin anti-
20
body (anti-hCG). A SAM of 11a : 15 (1 : 1) was formed and
activated using the bis-succinimide crosslinker BS3 on a Bia-
11b : 13 SAMs; advancing and receding angles are filled and open core SPR chip (Scheme S8.1†). Injection of anti-hCG gave a
symbols, respectively. Dotted and solid lines represent the line of best fit
for the Cassie equation for contact angles for 11a : 13 and 11b : 13 SAMs,
respectively. (C) Typical cyclic voltammograms (left) for the 2-electron
oxidation/reduction of SAM-bound methylene blue (right) at scan rates
of 200 (black), 400 (red), 600 (blue) and 800 mV s (green). The inset
shows the linear relationships between current and scan rate.
much stronger signal in the channel pre-treated with BS3 than
in the control channel (no BS3), indicating successful covalent
immobilisation of anti-hCG (Fig. 3B). The lack of binding
observed on the control channel suggests the LCAT–OEG
monolayer is efficient at minimising non-specic adsorption.
ꢀ1
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blue, thereby signicantly expanding the range of LCAT–OEGs
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All LCAT–OEGs could be
8
9
isolated in good yields as pure materials, and were used to form
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phase methodology also allows the use of bioorthogonal post-
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Centre of the University of Leeds (RSB and SJ), a Henry Ellison
PhD Studentship (JM), EPSRC Grants EP/J010731/1 (RSB, DN and
SJ), EP/J01513X/1 (KC), and EP/I014039/1 (LP), the Centre for
Chronic Diseases and Disorders (C2D2; RA), the University of
York (SJ), and WELMEC, a Centre of Excellence in Medical
Engineering funded by the Wellcome Trust and EPSRC, under
grant number WT 088908/Z/09/Z (DN and CW). We thank Dr.
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‡
We routinely perform the synthesis of 11a, 11b, 13, 15, 17 and 18 on a 0.1–0.6
mmol scale per SPE tube, which provides plenty material for optimisation studies
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3744 | J. Mater. Chem. B, 2014, 2, 3741–3744
This journal is © The Royal Society of Chemistry 2014