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-assembly conjugation of (bio)molecules. We demonstrate the versatility of our synthetic route by synthesising LCAT-OEGs with a range of functional moieties, including peptides, electro-active redox groups, chemical handles for post-assembly conjugation of (bio)molecules, and demonstrate the application of our LCAT-OEG monolayers in immunosensing, where they show good biocompatibility with minimal biofouling

Bartra

Bertin

Booth

Bouzide

Cassie

Cole

Derda

Góes

Houseman

Jonkheijm

Kang

Kleinert

Laurent

Love

Pheeney

Prats-Alfonso

Prime

Ryan

Samanta

Svedhem

Tam-Chang

Tolstyka

Zaccari

Zhou

PubMed

We present an efficient solid phase synthesis methodology that provides easy access to a range of functionalised longchain alkanethiol–oligoethyleneglycols that form well-defined self-assembled monolayers on gold and are compatible with pre- or post-assembly conjugation of (bio)molecules.</p

James Murray

Dominika Nowak

Laurynas Pukenas

Rizuan Azhar

Mathieu Guillorit

Christoph Wälti

Kevin Critchley

Steven Johnson

Robin S. Bon

Crossref

Journal of Materials Chemistry B

Solid phase synthesis of functionalised SAM-forming alkanethiol–oligoethyleneglycols

Murray, J

Nowak, D

Pukenas, L

Azhar, R

Guillorit, M

Wälti, C

Critchley, K

Johnson, S

Bon, RS

White Rose Research Online

Journal ofMaterials Chemistry BCOMMUNICATIONOpen Access Article. Published on 23 April 2014. Downloaded on 03/07/2014 12:43:36.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View IssueaSchool of Chemistry, University of Leeds, LbAstbury Centre for Structural Molecular BiocSchool of Electronic and Electrical EngineerdSchool of Physics and Astronomy, UniversiteDepartment of Electronics, University of Yo† Electronic supplementary informatiocharacterisation of compounds, XPS, EICV, colorimetry and SPR. See DOI: 10.103Cite this: J. Mater. Chem. B, 2014, 2,3741Received 10th April 2014Accepted 22nd April 2014DOI: 10.1039/c4tb00573bwww.rsc.org/MaterialsBThis journal is © The Royal Society of CSolid phase synthesis of functionalised SAM-forming alkanethiol–oligoethyleneglycols†James Murray,ab Dominika Nowak,bc Laurynas Pukenas,bd Rizuan Azhar,eMathieu Guillorit,a Christoph Wälti,c Kevin Critchley,bd Steven Johnsoneand Robin S. Bon*abWe present an efficient solid phase synthesis methodology thatprovides easy access to a range of functionalised long-chain alka-nethiol–oligoethyleneglycols that form well-defined self-assembledmonolayers on gold and are compatible with pre- or post-assemblyconjugation of (bio)molecules. We demonstrate the versatility of oursynthetic route by synthesising LCAT–OEGs with a range of functionalmoieties, including peptides, electro-active redox groups, chemicalhandles for post-assembly conjugation of (bio)molecules, anddemonstrate the application of our LCAT–OEG monolayers inimmunosensing, where they showgood biocompatibility withminimalbiofouling.Self-assembled monolayers (SAMs) of functionalised long-chainalkanethiolates (LCATs) on metals1 provide an excellent andversatile platform for the chemoselective immobilisation of(bio)molecules on planar and nanoparticle surfaces. Their well-dened thickness, structure, and dielectric properties2 makethem ideally suited for applications where a high degree ofcontrol of the physical and chemical properties of surfaces isrequired. Functionalised LCATs also enable the creation ofpatterned surfaces3 critical for protein and DNA microarrays.SAMs used for protein biosensing applications usually requireLCATs incorporating oligoethylene glycol (OEG) linkers to helpreduce non-specic protein adsorption4 and functional groupsthat allow the further tuning of surface chemistry providingcontrol over the immobilisation, density and orientation ofcapture 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.S2 9JT, UK. E-mail: r.bon@leeds.ac.uklogy, University of Leeds, UKing, University of Leeds, UKy of Leeds, UKrk, Heslington York, YO10 5DD, UKn (ESI) available: Synthesis andS, AFM, contact angle measurements,9/c4tb00573bhemistry 2014However, commercial availability of functionalised LCAT–OEGsis severely limited and synthetic routes oen laborious. In thisstudy, we present a versatile solid phase synthesis route thatprovides easy access to a range of functionalised LCAT–OEGs.Using a range of surface characterisation techniques includingsurface plasmon resonance (SPR) and electrochemical spec-troscopy, we demonstrate that the resulting LCAT–OEGs formdense, well-dened mixed SAMs of high quality, which arecompatible with on-surface bioorthogonal ligation reactions.Customised SAMs presenting ligands and/or biomoleculescan be prepared according to two general strategies (Fig. 1): (i)conjugation of (bio)molecules to LCAT–OEGs aer SAMformation (post-assembly conjugation); (ii) total synthesis offunctionalised LCAT–OEGs followed by SAM formation (pre-assembly conjugation). Post-assembly conjugation requiresrobust, chemoselective, and mild chemistry to ensure completesurface functionalisation without side reactions.2,5 For pre-assembly conjugation approaches, total synthesis of LCAT–OEGs terminated with, amongst others, biotin,6 peptides,6c,7ferrocene,8 azides,9 aminooxy groups,9 and sugars10 has beendemonstrated. Such functionalised LCAT–OEGs are usuallysynthesised in solution, which requires multiple chromato-graphic steps and suffers from poor overall yields. Solid phasechemistry presents an attractive alternative for synthesisingFig. 1 SAM functionalisation strategies. (a) SAM formation (in theexample: a mixed SAM, with a hydroxy-terminated diluent, on a goldsurface); (b) conjugation of LCAT 1 with reagent 2 (in which functionalgroups X and Y react to form linker X0Y0).J. Mater. Chem. B, 2014, 2, 3741–3744 | 3741Journal of Materials Chemistry B CommunicationOpen Access Article. Published on 23 April 2014. Downloaded on 03/07/2014 12:43:36.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineLCAT–OEGs. The groups of Whitesides and Mrksich havedemonstrated the attachment of a thiol-protected LCAT–OEG toresin-bound peptides,7b,11 while Albericio et al. have successfullyused 2-chlorotrityl chloride resin to construct biotinylated6b andpeptide-containing7a LCAT–OEGs. However, for applicationsthat require a quick, iterative ne-tuning of SAM properties/functionalities, currently available syntheses are oen notsufficient. To address this need, we developed a exible solidphase synthesis that gives access to a range of LCAT–OEGsthrough late-stage functionalisation, while minimising inter-mediate purication.OEGs are oen linked to LCATs through an ether bond.However, in analogy to Albericio's approach,6b,7a we chose to usean amide linker, which has been shown to improve SAMstability through the formation of networks of hydrogenbonds.12 We rst synthesised amino-OEGs 6 through the sil-ver(I) oxide-mediated mono-tosylation of oligoethyleneglycols 4(ref. 13) followed by tosyl displacement with sodium azide andStaudinger reduction (Scheme 1A).14 Next, 6a, 6b and 7 wereScheme 1 Synthesis of LCAT–OEGs: (A) preparation of amino-OEGs 6achlorotrityl chloride resin 8. (C) On-resin synthesis of peptide-labelled LC11b, 13, 15, and 16–18 were calculated after chromatography. Insets: Str16. Abbreviations: Ac ¼ acetyl; DCM ¼ dichloromethane; DIC ¼ diisopdimethylaminopyridine; DMF ¼ N,N-dimethylformamide; Fmoc ¼ 9-fluo11-mercaptoundecanic acid; SPPS ¼ solid phase peptide synthesis; TES3742 | J. Mater. Chem. B, 2014, 2, 3741–3744coupled to 2-chlorotrityl resin-bound 11-mercaptoundecanoicacid (11-MUA) 9 (Scheme 1B). The terminal alcohol of 10c wastosylated and treated with sodium azide to afford resin-boundazide 12. Reduction of 12 under Staudinger conditions provedsluggish, but proceeded rapidly with tin thiophenolate15 toafford resin-bound amine 14.Using Fmoc-solid phase peptide synthesis, we demonstratedthat 14 is an excellent starting point for the synthesis of LCAT–OEG–peptide conjugates such as 17 (for pre-assembly func-tionalisation strategies) or LCAT–OEG 18 incorporating theredox probe methylene blue (MB; Scheme 1C). MB was chosenfor electrochemical analysis of SAMs (see below) because of itssuperior aqueous stability compared to ferrocene.16 MB-func-tionalised LCAT–OEG 18 was synthesised by acylating resin-bound amine 14 with carboxylate 16, which was prepared byadapting a procedure described by Pheeney et al. (SchemeS1.1†).17 Reactions were allowed to run until all resin-boundstarting material was consumed, according to LC-MS analysisaer micro-cleavage. The immobilised molecules can beand 6b. (B) Solid phase synthesis of functionalised LCAT–OEGs on 2-AT–OEGs 17 andmethylene blue-labelled LCAT–OEG 18. Yields of 11a,uctures of 2-(2-aminoethoxy)ethanol 7 and methylene blue derivativeropylcarbodiimide; DIPEA ¼ N,N-diisopropylethylamine; DMAP ¼ 4-renylmethyloxycarbonyl; HOBt ¼ 1-hydroxybenzotriazole; 11-MUA ¼¼ triethylsilane; TFA ¼ trifluoroacetic acid; Ts ¼ 4-toluenesulfonyl.This journal is © The Royal Society of Chemistry 2014Communication Journal of Materials Chemistry BOpen Access Article. Published on 23 April 2014. Downloaded on 03/07/2014 12:43:36.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Onlinecleaved from the solid support at any point using triuoroaceticacid and triethylsilane (at least one equivalent to scavenge resin-bound trityl cations). Aer purication by chromatography(either SiO2 or C18), the pure LCAT–OEGs 11a, 11b, 13, 15, 17and 18 were obtained in good total yields, based on the loadingof 2-chlorotrityl chloride resin 8.‡Next, we analysed the quality of SAMs assembled from ourfunctionalised LCAT–OEGs on sputtered gold surfaces. Using X-ray photoelectron spectroscopy (XPS), we found S 2p peaks ofSAMs of 11a, 11b, and 13 to have binding energies consistentwith the formation of thiolate bonds (Fig. S2.1†). The C 1s regionconsisted of three peaks for all SAMs, corresponding to the alkylchain, the OEG chain, and carbonyls (Fig. 2A). The analysis of theN 1s region for SAMs of 13 was complicated due to rapid degra-dation of the azide group under XPS conditions (see the ESI†).The azide-terminated SAM 13 resulted in a surface that was ofslightly lower surface energy than SAMs of 11a or 11b. Thisenabled the degree of mixing in the SAMs between diluents 11aor 11b and azide 13 to be estimated by contact angle analysis byapplying the Cassie equation18 (Fig. 2B). The insulating proper-ties of SAMs containing different ratios of 11a : 15 or 11a : 17awere assessed by electrochemical impedance spectroscopy(Fig. S3.1 and S3.2†) The minimum phase angles of88 to83for SAMs of 11a : 15 and 88 to 87 for SAMs of 11a : 17a,measured at 0.1 Hz, correspond to the formation of well-packed,insulating monolayers that are almost free of pinholes andcollapsed sites effects. Furthermore, atomic force microscopyFig. 2 Characterisation of LCAT–OEG SAMs. (A) XPS data of C 1s regionfor reference SAMs of 11a (dotted), 11b (dashed) and 13 (solid) normalisedto the alkyl chain peak at 284.9 eV. Spectra reflect similar carbonyl (288.3eV), but varying amount of OEG (286.9 eV) carbon. (B) A linear change incosine of the contact angle with molar ratio of azide 13 in mixed SAMswas observed. Triangles facing down represent 11a : 13; facing up –11b : 13 SAMs; advancing and receding angles are filled and opensymbols, respectively. Dotted and solid lines represent the line of best fitfor the Cassie equation for contact angles for 11a : 13 and 11b : 13 SAMs,respectively. (C) Typical cyclic voltammograms (left) for the 2-electronoxidation/reduction of SAM-bound methylene blue (right) at scan ratesof 200 (black), 400 (red), 600 (blue) and 800 mV s1 (green). The insetshows the linear relationships between current and scan rate.This journal is © The Royal Society of Chemistry 2014(AFM) of a SAM of 11a : 17a (1 : 1) revealed uniform surfacesabsent of macroscopic islands resulting from phase separation(Fig. S5.1†). Cyclic voltammetry (CV) measurements of mixedSAMs formed from different ratios of 11b : 15 (Fig. S6.1–S6.3†)revealed SAM capacitance values of 4.4  106 F cm1, whichcorresponds well with the capacitance of simple LCAT mono-layers.19 Furthermore, the absence of redox peaks due to electrontransfer to solution phase redox probe conrms the absence ofpin holes within the LCAT–OEG monolayers.To demonstrate the potential suitability of MB-functional-ised LCAT–OEGs for molecular electronics applications, weassessed the redox behaviour of surface-bound MB by CV. CVmeasurements on SAMs of 18 showed clear oxidation andreduction peaks associated with the MB moiety around 130mV vs. Ag/AgCl (Fig. 2C). No redox peaks were observed inequivalent CV measurements performed on non-redox activeLCAT–OEG SAMs (compounds 15 and 17a, Fig. S6.1–S6.3†). Thepeaks are reasonably symmetric with only a small peak sepa-ration (9 mV at a scan rate of 200 mV s1), typical of the elec-trochemical behaviour of a fully reversible redox-activemonolayer. Furthermore, the peak anodic and cathodic currentwas found to increase linearly with scan rate (see inset ofFig. 2C), again characteristic of a surface-tethered redox-activegroup. From the gradient of the straight line t to peak currentvs. scan rate we calculated a surface coverage, G ¼ 8.8  1013molecules per cm2. This is slightly lower than the theoreticaldensity predicted for a perfect SAM formed from LCATs only (4 1014 molecules per cm2) and is likely due to the sterichindrance of the bulky OEG and MB groups that inhibit densermonolayer assembly. Finally, the FWHM of the oxidation andreduction peaks was found to be 37 mV (at 200 mV s1). Whilethis is lower than the theoretical ideal (45.3 mV for a 2 electronprocess) we note that deviations in the FWHM are notuncommon in densely packed redox-active monolayers due toelectrostatic interactions between adjacent charged species.Condent that our molecules formed well-dened SAMs, wedemonstrated 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 attachedcovalently to the surface using a colorimetric assay (Fig. 3A).Briey, we introduced streptavidin-alkaline phosphatase fusionprotein to the surface and used western blue as a stain. Onlyspots where biotin had been successfully linked to the surfacewere stained purple (Fig. 3A, S7.1 and S7.2†). Finally, we testedthe suitability of SAMs containing amine 15 for immobilisationof the clinically relevant human chorionic gonadotropin anti-body (anti-hCG).20 A SAM of 11a : 15 (1 : 1) was formed andactivated using the bis-succinimide crosslinker BS3 on a Bia-core SPR chip (Scheme S8.1†). Injection of anti-hCG gave amuch stronger signal in the channel pre-treated with BS3 thanin the control channel (no BS3), indicating successful covalentimmobilisation of anti-hCG (Fig. 3B). The lack of bindingobserved on the control channel suggests the LCAT–OEGmonolayer is efficient at minimising non-specic adsorption.J. Mater. Chem. B, 2014, 2, 3741–3744 | 3743Fig. 3 Post-assembly conjugation: (A) biotin immobilised throughCuAAC on SAMs of 11a : 13 (1 : 1; left) was detected colorimetrically, aspurple spots (right), upon incubation of the gold slide with streptavi-din-alkaline phosphatase (SA-AP) and western blue. (B) Immobilisationof anti-hCG on a BS3-activated amine SAM (11a : 15, 1 : 1; left) wasdetected by SPR (right).Journal of Materials Chemistry B CommunicationOpen Access Article. Published on 23 April 2014. Downloaded on 03/07/2014 12:43:36.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineIn conclusion, we have established a robust and versatilesolid phase synthesis of LCAT–OEGs functionalised with alco-hols, azides, amines, peptides, and the redox probe methyleneblue, thereby signicantly expanding the range of LCAT–OEGsthat are easily accessible through solid phase approaches suchas those reported by Albericio.6b,7a All LCAT–OEGs could beisolated in good yields as pure materials, and were used to formdense and well-dened, high quality mixed SAMs absent ofmacroscopic island formation (AFM data) and pinholes (EISand CV data). In addition, the ratios of functionalised LCAT–OEGs and diluents on the surface were directly proportional tothe ratios of these molecules in the applied solutions. Finally,we demonstrated that in addition to total synthesis of func-tional LCAT–OEGs for pre-assembly conjugations, our solidphase methodology also allows the use of bioorthogonal post-assembly conjugation techniques.AcknowledgementsThis research was funded by the Biomedical andHealth ResearchCentre of the University of Leeds (RSB and SJ), a Henry EllisonPhD Studentship (JM), EPSRC Grants EP/J010731/1 (RSB, DN andSJ), EP/J01513X/1 (KC), and EP/I014039/1 (LP), the Centre forChronic Diseases and Disorders (C2D2; RA), the University ofYork (SJ), and WELMEC, a Centre of Excellence in MedicalEngineering funded by the Wellcome Trust and EPSRC, undergrant number WT 088908/Z/09/Z (DN and CW). We thank Dr.Simon White for assistance with SPR measurements and Prof.Stephen Evans for assistance with interpretation of XPS data.Notes and references‡ We routinely perform the synthesis of 11a, 11b, 13, 15, 17 and 18 on a 0.1–0.6mmol scale per SPE tube, which provides plenty material for optimisation studieswith SAMs. Instead of using chromatography, LCAT–OEGs incorporating basic3744 | J. Mater. Chem. B, 2014, 2, 3741–3744amines can also be puried by precipitation (as their TFA salts) into mixtures ofdiethyl ether and hexane.1 J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo andG. M. Whitesides, Chem. Rev., 2005, 105, 1103–1169.2 (a) P. Jonkheijm, D. Weinrich, H. Schröder, C. M. Niemeyerand H. Waldmann, Angew. Chem., Int. Ed., 2008, 47, 9618–9647; (b) I. Zaccari, B. G. Catchpole, S. X. Laurenson,A. G. Davies and C. Wälti, Langmuir, 2014, 30, 1321–1326.3 X. Zhou, F. Boey, F. Huo, L. Huang and H. Zhang, Small,2011, 7, 2273–2289.4 K. L. Prime and G. M. 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Richardson, E. Bat, C. J. Stevens,D. P. Parra, J. K. Dozier, M. D. Distefano, B. Dunn andH. D. Maynard, ChemBioChem, 2013, 14, 2464.10 M. Kleinert, N. Röckendorf and T. K. Lindhorst, Eur. J. Org.Chem., 2004, 18, 3931–3940.11 D. Ryan, B. A. Parviz, V. Linder, V. Semetey, S. K. Sia, J. Su,M. Mrksich and G. M. Whitesides, Langmuir, 2004, 20,9080–9088.12 (a) S.-W. Tam-Chang, H. A. Biebuyck, G. M. Whitesides,N. Jeon and R. G. Nuzzo, Langmuir, 1995, 11, 4371–4382;(b) S. Svedhem, S. C. T. Svensson and B. Liedberg,Langmuir, 1999, 15, 3390–3394.13 A. Bouzide and G. Sauve, Org. Lett., 2002, 4, 8781–8783.14 S. Svedhem, C. A. Hollander, J. Shi, P. Konradsson,B. Liedberg and S. C. Svensson, J. Org. Chem., 2001, 66,4494–4503.15 M. Bartra, P. Romea, F. Urpi and J. Vilarrasa, Tetrahedron,1990, 46, 587–594.16 D. Kang, X. Zuo, R. Yang, F. Xia, K. W. Plaxco and R. J. White,Anal. Chem., 2009, 81, 9109–9113.17 C. G. Pheeney and J. K. Barton, Langmuir, 2012, 28, 7063–7070.18 A. B. D. Cassie and S. 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Solid phase synthesis of functionalised SAM-forming alkanethiol-oligoethyleneglycols

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Solid phase synthesis of functionalised SAM-forming alkanethiol-oligoethyleneglycols

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