Investigation of Au SAMs Photoclick Derivatization by PM-IRRAS Wilson Luoa, Sydney M. Leggea, Johnny Luob,c, François Lagugné-Labarthet*a, and Mark S. Wor- kentin*a aDepartment of Chemistry and the Centre for Materials and Biomaterials Research, Western University, 1151 Rich- mond Street, London, ON, N6A 5B7, Canada. bDepartment of Biochemistry, Western University, London, Ontario N6A 5C1, Canada. cLawson Health Research Institute, London, Ontario N6C 2R5, Canada. ABSTRACT: In this work we present a clean one-step process for modifying headgroups of self-assembled monolayers (SAMs) on gold using photo-enabled click chemistry. A thiolated, cyclopropenone-caged strained alkyne precursor was first functionalized onto a flat gold substrate through self-assembly. Exposure of the cyclopropenone SAM to UV-A light initi- ated the efficient photochemical decarbonylation of the cyclopropenone moiety, revealing the strained alkyne capable of undergoing the interfacial strain-promoted alkyne-azide cycloaddition (SPAAC). Irradiated SAMs were derivatized with a series of model azides with varied hydrophobicity to demonstrate the generality of this chemical system for the modification and fine-tuning of the surface chemistry on gold substrates. SAMs were characterized at each step with polarization-mod- ulation infrared reflection-absorption spectroscopy (PM-IRRAS) to confirm successful functionalization and reactivity. Fur- thermore, to showcase the compatibility of this approach with biochemical applications, cyclopropenone SAMs were irra- diated and modified with azide-bearing cell adhesion peptides to promote human fibroblast cell adhesion, then imaged by live cell fluorescence microscopy. Thus, the “photoclick” methodology reported here represents an improved, versatile, catalyst-free protocol that allows for a high degree of control over the modification of material surfaces, with applicability in materials science as well as biochemistry. INTRODUCTION surface that may have an effect on biorecognition. An al- Self-assembled monolayers (SAMs) of thiolates on gold ternative route to target immobilization requires a small, represent a convenient, bottom-up approach towards pre- exogenous functional group to be installed into the target paring chemically and structurally well-defined organic in- molecule, which chemically reacts with the SAM head- terfaces. These SAMs are formed easily and rapidly, exhibit group to form a robust, covalent bond. high stability and reproducibility, and allow a high degree In this context, click chemistry, such as the copper-as- of control over the macroscopic interfacial properties of sisted alkyne-azide cycloaddition (CuAAC),9-10 can provide surfaces through specific tailoring of their microscopic the means to SAM derivatization with biomolecules via the structure and composition.1-3 Due to their controllable sur- formation of stable triazole linkages. For example, Murphy face properties — accessible either through the choice of and coworkers showed that an alkyne-bearing RGDSP pep- thiol during self-assembly or chemical modifications of the tide can be immobilized using the CuAAC on azide-termi- head group post-assembly — SAMs have emerged as key nated SAMs, which provided a modified platform for adhe- elements for biochemical research. Namely, they have sion studies of human mesenchymal stem cells.11-12 Yeo and been used in the development of biosensors, as substrates coworkers demonstrated on-demand electrochemical acti- for culturing cells, and as biorecognition interfaces.4-8 vation of the CuAAC on SAMs to afford a dynamic sub- 13 Substrates designed for studying biochemical events, strate to study cell migration. Lee and coworkers modi- such as protein interactions or cell adhesion, often require fied SAMs to display terminal maleimide groups using Cu- the presentation of specific ligands or biomolecules on the AAC, which was subsequently used for thiol-Michael addi- SAM interface in order to be effective.4-8 Although these tion (as a secondary click reaction) to immobilize poly(L- 14 target molecules can usually be thiolated and introduced lysine) as a model for polypeptide surfaces. More recently, to the surface during the self-assembly process, this Rubinstein and coworkers reported reactivity between method suffers from several drawbacks: 1) the target thiol SAM azide headgroups with terminal alkyne-containing must be stable and custom-synthesized for each system biological receptors via the CuAAC and demonstrated lo- used, which can be synthetically challenging and time-con- calized surface plasmon resonance (LSPR) biosensing on 15 suming since the target biomolecules are often large and the modified substrates. Although effective, these meth- complex; 2) target molecules with multiple thiol groups, as ods suffer from the cytotoxicity of Cu(I), which can have biomolecules often contain, can exhibit different binding an effect on cell viability even in trace amounts if not 16-18 modes with different molecular conformations at the properly removed. Furthermore, the introduction of al- kynes into target molecules, although synthetically simpler than thiolation, can still be challenging and time-consum- method will allow for faster and simpler preparation of ing. To circumvent these drawbacks, we propose a comple- functionalized and biofunctionalized SAMs for the study of mentary system that utilizes a strained cyclooctyne as the biochemical events. SAM headgroup, which can undergo strain-promoted al- kyne-azide cycloaddition (SPAAC) with azido molecules to afford the triazole linkage.19 The associated advantages are two-fold: 1) cytotoxic Cu(I) salts are no longer required, which makes this method suitable for use even with live samples, and 2) azido molecules feature high synthetic ac- cessibility and commercial availability; azide-bearing bio- molecules in particular are readily commercially available. The introduction of strained cyclooctynes onto gold sur- faces, however, is nontrivial. Their high reactivity means that special care needs to be taken to successfully incorpo- rate these moieties onto gold. In particular, a thiolated strained cyclooctyne molecule, which represents the most obvious route towards functionalizing gold, cannot be sta- bly obtained due to self-reactivity via thiol-yne addition.20- Scheme 1. General strategy for incorporation of 22 To overcome this synthetic challenge, our group previ- strained alkyne onto flat gold substrates. ously developed the use of cyclopropenones as photo- chemical alkyne precursors on gold nanoparticles EXPERIMENTAL (AuNPs)23-24, a chemical system adapted from Popik et al. Materials and Methods. All reagents, unless otherwise who first utilized cyclopropenone-masked dibenzocy- stated, were purchased from Sigma-Aldrich and used as re- clooctynes to label living cells,25 and then to functionalize ceived. All common solvents, triethylamine (TEA), sodium polymer brushes and immobilize azido molecules via sulfate anhydrous, and trifluoroacetic acid were purchased SPAAC.26 This method allowed us to synthesize a thiol pre- from Caledon. Cyclo[Arg-Gly-Asp-D-Phe-Lys(Azide)] peptide cursor and assemble it onto AuNPs directly.23 Subsequent was purchased from Peptides International (RGD-3749-PI). uncaging using UV irradiation cleanly afforded the Calcein AM was purchased from Abcam (ab141420). Azides A strained cyclooctyne headgroup, which was used as a ver- and B were synthesized according to a literature procedure.29- 30 satile reactive template to immobilize a number of azido Azide C was synthesized according to a protocol previously 31 molecules onto AuNPs. We envision that this “photoclick” developed in our group. system could be applied to SAMs on flat gold, enabling a 1H, 13C{1H}, and 19F{1H} NMR spectra were recorded on Var- similar approach to more effectively introduce new func- ian INOVA 400 (or 600) or Bruker AvIII HD 400 spectrome- tionalities. As the surface chemistry, preparation, and ap- ters using CDCl3 or CD3OD as solvent. FTIR spectra were rec- plications of SAMs on flat, solid-state Au substrates differ orded using an attenuated total reflectance (ATR) attachment significantly from solution-dispersible colloidal Au, we using a Bruker Vector 33 FTIR spectrometer. Ultraviolet (UV)- sought to investigate the feasibility of implementing this visible spectra were recorded using a Varian Cary 300 Bio spec- trometer. Photolyses were conducted in a Luzchem LZC-4V cyclopropenone-based strategy on this new material, as photoreactor equipped with 14 UVA (350 nm) 8W lamps. well as explore methods of validating the molecular reac- tivity. Synthesis of thiol precursor 3. To a solution of 1 (1.186 g, 3.70 mmol) in DMF (40 mL) was added compound 2 (3.37 g, Thus, we report herein the assembly of cyclopropenone- 5.55 mmol). Next, portionwise was added K2CO3 (0.512 g, 3.70 terminated SAMs on flat Au substrates, which were used as mmol), then the solution was stirred and heated to 80°C for 5 single versatile templates from which facile SAM head- hours (Scheme 2). The reaction was cooled to room tempera- group modifications can be achieved via photo-enabled ture, diluted with ethyl acetate (400 mL), washed 5 times with SPAAC with various azide reagents, as illustrated in water (75 ml), brine (100 mL), and dried over MgSO4. The or- Scheme 1. Furthermore, as a proof of concept for SAM deri- ganic layer was then filtered, concentrated in vacuo, and puri- vatization with large, commercially available biomole- fied via silica gel chromatography (hexanes: