Rate Determination of Azide Click Reactions onto Alkyne Polymer

Sep 25, 2012 - Chengsha Wei , Mingming Chen , Dong Liu , Weiming Zhou , Majid Khan , Xibo Wu , Ningdong Huang , Liangbin Li. RSC Advances 2015 5 ...
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Rate Determination of Azide Click Reactions onto Alkyne Polymer Brush Scaffolds: A Comparison of Conventional and Catalyst-Free Cycloadditions for Tunable Surface Modification Sara V. Orski, Gareth R. Sheppard, Selvanathan Arumugam, Rachelle M. Arnold, Vladimir V. Popik,* and Jason Locklin* Department of Chemistry, College of Engineering, and the Center for Nanoscale Science and Engineering, University of Georgia, Athens, Georgia 30602, United States S Supporting Information *

ABSTRACT: The postpolymerization functionalization of poly(N-hydroxysuccinimide 4-vinylbenzoate) brushes with reactive alkynes that differ in relative rates of activity of alkyne−azide cycloaddition reactions is described. The alkynederived polymer brushes undergo “click”-type cycloadditions with azido-containing compounds by two mechanisms: a strain-promoted alkyne−azide cycloaddition (SPAAC) with dibenzocyclooctyne (DIBO) and azadibenzocyclooctyne (ADIBO) or a copper-catalyzed alkyne−azide cycloaddition (CuAAC) to a propargyl group (PPG). Using a pseudo-first-order limited rate equation, rate constants for DIBO, ADIBO, and PPG-derivatized polymer brushes functionalized with an azide-functionalized dye were calculated as 7.7 × 10−4, 4.4 × 10−3, and 2.0 × 10−2 s−1, respectively. The SPAAC click reactions of the surface bound layers were determined to be slower than the equivalent reactions in solution, but the relative ratio of the reaction rates for the DIBO and ADIBO SPAAC reactions was consistent between solution and the polymer layer. The rate of functionalization was not influenced by the diffusion of azide into the polymer scaffold as long as the concentration of azide in solution was sufficiently high. The PPG functionalization by CuAAC had an extremely fast rate, which was comparable to other surface click reaction rates. Preliminary studies of dilute solution azide functionalization indicate that the diffusion-limited regime of brush functionalization impacts a 50 nm polymer brush layer and decreases the pseudo-first-order rate by a constant diffusion-limited factor of 0.233.

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metal catalysts.29 Several reports of other diaryl and highenergy cyclic alkynes have been developed that are aimed at improving the reaction rate and synthetic ease of these catalystfree cycloadditions with azides.30−37 Previous reports have described the catalyst-free cycloaddition by the cyclooctyne as “strain-promoted”, although a significant amount of energy is not released once ring strain is relieved.38−41 Instead, the driving force for copper-free click chemistry is bending of the acetylenic fragment of the cyclooctyne in a transition-state-like geometry, which lowers the activation energy of the cycloaddition by 8.2 kcal/mol.38 Different cyclooctyne derivatives have different rates of reaction based on substituents that change the polarization of the alkyne bond, thereby changing the activation energy.28,30,34,37,42,43 Copper-free cycloaddition and CuAAC have both been well studied for the functionalization of polymers in solution.1−6 The interfacial reaction, however, involving diffusion of one reactant to the reactive sites in the densely packed polymer brush is less understood and limited by the interaction between

INTRODUCTION The incorporation of Sharpless-type click chemistry into postpolymerization modification has recently attracted attention,1−6 especially in terms of polymer brush functionalization.7−12 Click chemistry has been used previously to modify both planar and nanostructured interfaces.13−18 The alkyne− azide Huisgen 1,3-dipolar cycloaddition is emerging as an ideal coupling approach because of its high selectivity, functional group tolerance, and straightforward reaction conditions. It is also compatible with protic, aprotic, and aqueous solvent conditions and has a rapid rate with high yields.3,18−20 Click chemistry reactions are especially appealing for biological labeling and coupling reactions due to their facile incorporation into biomolecules through postsynthetic modification,21,22 enzymatic transfer,23,24 and azide-modified nutrients for metabolic functionalization.25 While the conventional Cu(I)catalyzed alkyne−azide cycloaddition (CuAAC) is ideal for many applications,26,27 the cytotoxicity of the copper catalyst can limit bioorthogonal conjugation. Bertozzi and co-workers designed the first alternative to using CuAAC for alkyne−azide cycloaddition by synthesizing a difluoronated cyclooctyne (DIFO) derivative for in vivo imaging28 and to fluorescently label azide-functionalized glycans in live zebra fish without © 2012 American Chemical Society

Received: August 10, 2012 Revised: September 24, 2012 Published: September 25, 2012 14693

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topographic image of the monolayer was featureless, with a rootmean-square (rms) roughness of 1.2 nm. Synthesis of N-Hydroxysuccinimide 4-Vinylbenzoate (NHS4VB). NHS4VB was prepared in a three-step procedure from 4-bromobenzaldehyde. Briefly, 4-bromobenzaldehyde was converted to 4-bromostyrene using Wittig chemistry with triphenylphosphine methyl ylide.55 4-Bromostyrene was converted to 4-vinylbenzoic acid through Grignard formation and quenching with CO2. Finally, coupling of N-hydroxysuccinimide with 4-vinylbenzoic acid gave the active ester NHS4VB.56,57 Polymerization of NHS4VB. The initiator substrates, one quartz and one silicon wafer, were placed in a dry, flat bottom Schlenk flask in a glovebox. The NHS4VB monomer (1.65 g, 6.75 mmol) and 1.2 mL of DMSO were added to the Schlenk flask. Separately, a stock solution was made that consisted of 0.5 mL of DMSO, PMDETA (423 μL, 2.03 mmol), CuBr (39 mg, 0.27 mmol), and CuCl2 (7.26 mg, 0.05 mmol). An aliquot of 230 μL of the stock solution was added to the Schlenk flask, which was then sealed, brought outside the glovebox, and stirred in a 50 °C oil bath for 1 h to yield a polymer brush of ∼50 nm thickness. The flask was then opened to air, and the wafers were rinsed thoroughly with DMF and dried under a stream of nitrogen. Synthesis of DIBO Amine. The cyclopropenone amine precursor was prepared following procedures described in the literature.32 A solution of cyclopropenone amine (0.1 g, 0.298 mmol) in methanol (10 mL, 2.98 × 10−2 M) was irradiated (15 fluorescent tubes, 8 W each, 350 nm) for 5 min at room temperature. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (MeOH:DCM 1:20) to provide dibenzocyclooctyne amine (0.076 g, 83%) as a slightly yellow oil. 1H NMR (400 MHz, CDCl3): δ (ppm) 7.24−7.17 (dd, J = 8.3, 5.2 Hz, 2H), 6.90−6.86 (m, 2H), 6.79−6.72 (dt, J = 8.3, 2.3 Hz, 2H), 4.09− 3.99 (t, J = 6.1 Hz, 2H), 3.83 (s, 3H), 2.95−2.87 (t, J = 6.8 Hz, 2H), 2.51−2.35 (m, 2H), 1.98−1.86 (p, J = 6.5 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ (ppm) 159.26, 158.67, 155.09, 126.90, 116.89, 116.38, 112.02, 111.49, 110.62, 66.15, 55.54, 36.43, 36.86, 33.17. FW calcd ([C20H21NO2]H+): 308.1645. ESI-HRMS: 308.1644. Synthesis of ADIBO Amine. The ADIBO amine was prepared according to our previously reported procedures.37 Functionalization of Poly(NHS4VB) Brushes with Reactive Alkyne Primary Amines. Poly(NHS4VB) brushes were converted to functionalized amide derivatives of 4-vinylbenzoic acid using DIBO amine, ADIBO amine, and PPG amine (36.2 mM in dry DMF) at 40 °C for 2 h with triethylamine as an acid scavenger.44 Synthesis of 4-(N-3-Azidopropyl)sulfonylamido Lissamine Rhodamine B (Azido-RB). 3-Azidopropylamine (0.049 g, 0.49 mmol) was added to a solution of lissamine rhodamine B sulfonyl chloride (0.200 g, 0.347 mmol) in DMF (3 mL) followed by the addition of N,N-diisopropylethylamine (0.072 g, 0.628 mmol). The reaction mixture was stirred overnight at room temperature, followed by concentration of the product, which was purified by chromatography (CHCl3:MeOH 15:1) to provide dark red crystals. Determination of Polymer Brush Functionalization Kinetics of Copper-Free Click Alkynes: DIBO and ADIBO by UV−vis Spectroscopy. A quartz slide with either a DIBO or ADIBO cyclooctyne-functionalized polymer brush was submerged in a 40 mM solution of azido-RB in MeOH:DMF (approximately 10:1, v/v) for several seconds, removed, rinsed thoroughly with MeOH, and dried under a stream of nitrogen. A UV−vis spectrum was then recorded between 190 and 700 nm. This cycle was repeated several times until the absorbance of azido-RB attached to the brush no longer increased. An identical polymer brush on a silicon wafer was placed in the azidoRB solution for an equivalent amount of time as the quartz wafer to obtain an accurate measure of film thickness change, contact angle change, and to confirm chemical functionality by grazing angle attenuated total reflection Fourier transform infrared spectroscopy (GATR-FTIR). The kinetics experiment was repeated with a dilute solution of azido-RB (500 μM) in 10:1 MeOH:DMF (v/v) using an identical ADIBO-functionalized brush as directed above in order to determine the diffusion-limited rate of brush functionalization.

neighboring polymer chains. Also, the substrate prevents diffusion of solution analyte from underneath the brushes, restricting diffusion to above the plane of the surface to which the polymer chains are immobilized. The extent of polymer brush modification has been investigated previously for Nhydroxysuccinimide (NHS) ester-functionalized brushes.44−46 In these studies, low molecular weight amines achieve high reaction conversion with NHS esters,44,47 but a decrease in penetration depth into the brush is observed for larger, aminecapped polymer analytes, which limit reaction yields.45 In order to increase the scope of alkyne−azide clickable platforms both with and without the use of copper catalyst, the rate and extent of postpolymerization modification must be well understood in the surface confined environment.45,48−52 Previously, we have used poly(N-hydroxysuccinimide 4vinylbenzoate) (poly(NHS4VB)) polymer brush scaffolds to generate pendant alkyne functionality in high density through aminolysis with alkyne-functionalized amines.7,53 These pendant alkynes can then be used to couple azides to the surface. In this work, we compare the relative rates of click reactions on polymer brush scaffolds that contain different functional alkyne pendant groups, dibenzocyclooctyne (DIBO), azadibenzocyclooctyne (ADIBO), and a terminal alkyne group (propargyl, PPG). DIBO and ADIBO undergo cycloaddition without a metal catalyst while the alkyne pendant group requires a copper(I) catalyst. The rates of reaction for polymer brush coupling with azides are compared to the analogous small molecule reactions in solution as well as the relative rates of azide coupling on the brush scaffold between the three different alkynes studied. These investigations establish the extent of polymer brush functionalization, determine the rate of azide cycloaddition reactions within a densely packed polymer brush, and elucidate the influence of the polymer brush environment for SPAAC and CuAAC postpolymerization modification.

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EXPERIMENTAL SECTION

Materials. Solvents were distilled from sodium ketyl (tetrahydrofuran (THF)) or calcium hydride (toluene and dichloromethane (DCM)). Anhydrous dimethyl sulfoxide (DMSO) and dimethylformamide (DMF) (DriSolv, 99.8% by GC) were purchased from EMD and used as received. Methanol (MeOH) and chloroform (CHCl3) were purchased from VWR (ACS grade) and used as received. Silicon wafers (orientation ⟨100⟩, native oxide) were purchased from University Wafer. Quartz microscope slides were purchased from Technical Glass Products. N,N′,N′,N″,N″-Pentamethyldiethylenetriamine (PMDETA) was purchased from Alfa Aesar and distilled prior to use. All other chemicals were purchased from Sigma-Aldrich and used as received. Preparation of Initiator Layers for Surface-Initiated Atom Transfer Radical Polymerization (SI-ATRP). Silicon wafers and quartz slides were cut into square pieces (∼1 cm2) and sonicated for 5 min each in acetone, ethanol, and deionized water (18.2 MΩ). The wafers were dried under a stream of nitrogen and then subjected to argon plasma (Harrick Plasma, PDC-32-G, 0.8 mbar, 18 W) for 2 min. The initiator, 11-(2-bromo-2-methyl)propionyloxyundecenyl trichlorosilane, was synthesized following literature procedures.54 The substrates and all dry, degassed reagents were transferred into a nitrogen-filled glovebox. One drop of initiator was mixed with 20 mL of toluene (approximate concentration 10 mM), and the solution was filtered through a 0.45 μm poly(tetrafluoroethylene) (PTFE) filter and poured over the clean substrates in a glass slide holder. After 16 h, the substrates were rinsed individually with toluene. Initiator substrates were stored in freshly distilled toluene until used for polymerization. The self-assembled monolayer was 2.5 nm on average, as measured by spectroscopic ellipsometry. An atomic force microscopy (AFM) 14694

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Scheme 1. (a) Functionalization of Poly(NHS4VB) with Alkyne-Containing Amines: DIBO (1), ADIBO (2), and PPG (3); (b) Immobilization of Azido-rhodamine B on the Polymer Brush Backbone Using SPAAC (4 and 5) and CuAAC (6) to Form the Triazole

Determination of Polymer Brush Functionalization Kinetics of Alkyne Polymer Brushes via CuAAC by UV−vis Spectroscopy. A flat bottom Schlenk flask was placed under an argon blanket, and a 1 mL solution of 40 mM azido-RB (10:1 MeOH:DMF (v/v)) was added and allowed to equilibrate. Separately, a stock solution of CuBr (28.7 mg, 0.2 mmol), PMDETA (41.8 μL, 0.2 mmol), and sodium ascorbate (79.2 mg, 0.4 mmol) was prepared in a 10 mL DMF:water solution (9:1, v/v) and degassed with argon for 1 h. A 100 μL aliquot of the stock solution was added to the azido-RB solution and stirred to equilibrate the solution. An alkyne-functionalized polymer brush on quartz was immersed in the solution under a blanket of argon gas for 10 s. The quartz slide was removed, thoroughly rinsed with MeOH, and dried under nitrogen gas. A UV−vis spectrum was taken from 190 to 700 nm to observe the increase in absorbance of the covalently bound azide dye. This was repeated several times until absorbance measurements no longer changed after additional azido-RB deposition cycles. An identical polymer brush on a silicon wafer was placed in the azido-RB solution for the total amount of time the quartz wafer was in the solution to determine an accurate measure of film thickness changes, contact angle changes, and to confirm functional groups on the polymer brush surface using grazing angle attenuated total reflection Fourier transform infrared (GATR-FTIR) spectroscopy. Determination of Corresponding Kinetic Rate of DIBO and ADIBO in MeOH. The SPAAC reaction kinetics of benzyl azide and

cyclooctyne (DIBO or ADIBO, 0.06 mM) were conducted by UV−vis spectroscopy in MeOH at 25.0 ± 0.1 °C. The reactions of cyclooctynes with variable concentrations of benzyl azide were monitored by the disappearance of DIBO and ADIBO characteristic absorption bands at 326 and 315 nm, respectively. The pseudo-firstorder rate constants were obtained by least-squares regression analysis. The dependence of the observed rates on the concentration of azide produced the bimolecular rates of cycloaddition reaction. Characterization. Spectroscopic ellipsometry was performed on a J.A. Woollam M-2000 V with a white light source at 65°, 70°, and 75° angles of incidence to the silicon wafer normal. Delta (Δ) and psi (Ψ) were measured as a function of wavelength between 400 and 1000 nm. Polymer film thicknesses were determined using a three-layer model (Si, SiO2, and polymer), where the polymer’s refractive index was modeled using the Cauchy equation

n=a+

b λ2

(1)

where n is the refractive index, a and b are fitted parameters, and λ is the wavelength of light. The software allowed for simultaneous modeling of film thickness and refractive index for the polymer. Fitted parameters a and b were computed for each film with an average value for all samples being 1.47 and 0.009, respectively. Thickness measurements for all azido-RB functionalized brushes were measured 14695

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by profilometry (Veeco Dektak 150). Rhodamine B absorbs within the ellipsometer’s wavelength range, which led to an unacceptable fit using the Cauchy model. Static contact angle measurements were taken on a KRÜ SS DSA 100 using a 1 μL drop of 18 MΩ water. UV−vis spectroscopy was taken on a Varian 50 Bio spectrometer. GATR-FTIR measurements were taken with a Nicolet Model 6700 at 128 scans with 4 cm−1 resolution. UV−vis kinetic measurements were analyzed at λmax of azido-RB dye (570 nm) using linear regression software in MATLAB (MathWorks) for pseudo-first-order limited kinetics.

brushes functionalized with azido-RB decreases slightly from 79° to 78°. The contact angle for ADIBO increases from 64° to 74° upon functionalization with azido-RB. For the PPG brushes, the contact angle decreases from 72° to 62° upon functionalization. While the exact cause of the difference in contact angle of the resulting films is not known, we speculate that the overall contribution of the zwitterionic lissamine− rhodamine B to the surface energy of the film is greater with the lower molecular weight PPG than with the DIBO or ADIBO substituents, as the measured contact angle is similar to the contact angle of rhodamine B monolayers (69°).59 The RB-functionalized brush thicknesses were measured by spectroscopic ellipsometry but were difficult to model due to the strong absorbance of the azido-RB dye within the wavelength range used. Therefore, brush thicknesses for azido-RB functionalized brushes in Table 1 were confirmed using surface profilometry. The large increase in film thicknesses of the brushes observed after functionalization is due to the increase in the molar mass of the polymer brush pendant substituent upon azido-RB conjugation. Chemical functionality of the active ester polymer brush, derivatization with DIBO amine, and attachment of azido-RB to the polymer brush were all confirmed by GATR-FTIR. Figure 1 shows the FTIR spectra characterizing the films upon

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RESULTS AND DISCUSSION Aminolysis of Poly(NHS4VB) Brushes with Reactive Alkynes. Our previous studies demonstrated that the controlled polymerization of NHS4VB by atom transfer radical polymerization (ATRP) affords a linear increase in brush thickness with time for the first hour of polymerization and has an overall homogeneous morphology with a rms surface roughness of less than 2 nm.47 Activated ester polymer brushes undergo quantitative functionalization with small molecule amines within an hour of reaction time, confirmed by infrared spectroscopy and null ellipsometry for brushes before and after functionalization.44,47 In this study, poly(NHS4VB) brushes were functionalized with primary amines that contained two different dibenzocyclooctynes for SPAAC chemistry and a terminal alkyne for conventional copper alkyne−azide cycloaddition (CuAAC). A depiction of DIBO, ADIBO, and PPG functionalized brushes can be seen in Schemes 1a and 1b. An increase in brush thickness of ∼60% was observed for the cyclooctynes (71−110 nm for DIBO, 57−90 nm for ADIBO). For the low molecular weight PPG group, the brush thickness decreased by 24%, from 50 to 38 nm due to the smaller molecular weight and molar volume of PPG relative to the NHS ester. A summary of brush thicknesses and contact angles for all functionalized films can be found in Table 1. The DIBO Table 1. Summary of Thicknesses and Contact Angles for Polymer Brushes on Silicon Wafers

a

sample

av brush thickness (nm)

av contact angle (deg)

poly(NHS4VB) DIBO azido-RB poly(NHS4VB) ADIBO azido-RB poly(NHS4VB) PPG azido-RB

71.0 110.0 175.9a 56.8 89.8 199.3a 50.0 38.3 96.3a

65 79 78 65 64 74 65 72 62

Figure 1. GATR-FTIR spectra of (a) poly(NHS4VB), (b) DIBO amine-functionalized brushes, and (c) azido-RB-functionalized brushes. DIBO and azido-RB brush spectra are shown at 5 times the absorbance intensity.

conversion from an (a) active ester surface into an (c) azidofunctionalized scaffold by (b) DIBO functionalization. The spectrum of the polymer brush functionalized with DIBO is shown in Figure 1b. The peaks at 1801, 1769, and 1738 cm−1 are assigned to the carbonyl stretches of the NHS activated ester, while the C−O stretches from DIBO are visible at 1233 and 1035 cm−1. The peak at 2258 cm−1 is due to the CC stretch of the cyclooctyne. Functionalization of the DIBO brush was indicated by the disappearance of the CC stretch and appearance of triazole absorbances at 1538 and 1262 cm−1, representing the CN and NN stretches, respectively (Figure 1c). The sulfonate of rhodamine B is also visible at 1179 cm−1. A thorough assignment of the vibrations in the original and functionalized polymer brushes are highlighted in Table S1 of the Supporting Information. Figure 2 shows the functionalization of the (a) active ester brushes with (b) ADIBO amine, followed by functionalization with (c) azido-RB. Prominent absorbances for ADIBO are the

Brush thicknesses were measured using surface profilometry.

and PPG brushes become more hydrophobic upon functionalization, increasing static contact angle measurements by 10°. The ADIBO brushes, however, had the same contact angle as the original poly(NHS4VB) brush. The nitrogen atom in the cyclooctyne ring of ADIBO increases the hydrophilicity of ADIBO58 and most likely accounts for the lower contact angle change relative to the DIBO functionalized brushes. Characterization of Alkyne-Functionalized Brushes before and after Reaction with Azido-RB. The DIBO, ADIBO, and PPG-containing polymer brushes were functionalized with azido-rhodamine B conjugate (azido-RB, Scheme 1b) and monitored by UV−vis spectroscopy until the reaction was complete. The contact angles for azido-RB clicked brushes are shown in Table 1. The static contact angle for DIBO 14696

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Figure 2. GATR-FTIR of (a) poly(NHS4VB) brushes functionalized with (b) ADIBO amine and then (c) azido-RB. ADIBO is shown at 3 times the absorbance intensity.

Figure 4. UV−vis absorbance spectra of DIBO brush functionalization with time (spectra recorded every 15 min). Functionalization is complete after 60 min of reaction.

amide in the cyclooctyne ring at 1643 cm−1 and the CC stretch of the cyclooctyne at 2243 cm−1. Azido-RB functionalization is again observed by the disappearance of the CC stretch and formation of triazole absorbances at 1538 and 1241 cm−1 and the sulfonate at 1179 cm−1. Propargyl (PPG) functionalization and derivatization are shown in Figure 3. A terminal alkyne stretch is visible at 3280

decreases with time at 306 and 325 nm. Triazole formation is indirectly observed by monitoring the increase in the RB dye absorbance (575 nm) with reaction time in the brush layers. The spectra indicate that functionalization is complete after 60 min, after which the absorbance values of RB and DIBO are unchanged. A small amount of DIBO absorbance remains after functionalization, indicating that conversion may not be quantitative, as the zwitterionic RB dye may induce charge repulsion within the brush as more alkyne sites are functionalized along the backbone. Copper-free cycloaddition of azido-RB and the highly energized cyclooctyne were also recorded with ADIBO functionalized polymer brushes, which can be seen in Figure S2 of the Supporting Information. Absorbance maxima of ADIBO at 297 and 316 nm decrease with time and a prominent absorbance band at 575 nm increases with time from the attachment of azido-RB dye. The absorbances of ADIBO and RB remain constant after 15 min, indicating that the reaction is complete. Time-dependent functionalization of PPG derivatized polymer brushes with azido-RB using conventional coppercatalyzed alkyne−azide cycloaddition was monitored by UV− vis spectroscopy (Figure S3 in the Supporting Information). The PPG functionalized polymer brush shows two absorbance bands at 204 and 244 nm, which encompass both the absorbance of the alkyne and the styrenic polymer backbone. A measurable change in these absorbances is not observed. There is a broad, weak band at 330 nm that can be attributed to triazole formation. Absorbance of the RB attached to the polymer brush can is observed at 577 nm and increases with reaction time for 2 min and then remains constant. For consistency, the increase in RB dye absorbance was used for rate constant calculations for ADIBO, DIBO, and PPG. Kinetics of Copper-Free and Copper-Catalyzed Cycloaddition in Solution. In order to compare the copper-free cycloadditions onto the brush scaffold, the kinetics of the solution reaction were also investigated. The pseudo-first-order rate constant for copper free cycloadditions of DIBO (0.06 mM) with benzyl azide (25 mM) in solution (Figure 5) was (1.7 ± 0.03) × 10−3 s−1, which agrees with values previously reported.32 Rate measurements of ADIBO with benzyl azide were conducted with variable concentrations of azide (6.0 × 10−4−2.5 × 10−2 M). The dependence of the observed rates on the concentration of azides was linear, and the least-squares

Figure 3. GATR-FTIR spectra of (a) poly(NHS4VB), (b) active ester brush functionalized with PPG amine, and (c) subsequent derivatization with azido-RB. PPG and azido-RB brush spectra are shown at 5 times the absorbance intensity.

cm−1 as well as amide I, II, and III bands at 1640, 1535, and 1309 cm−1 (Figure 3b). Functionalization with azido-RB was confirmed by loss of the terminal alkyne band and the presence of additional absorbances at 1594 and 1258 cm−1 assigned to the triazole and the absorbances at 1176 cm−1 for the sulfonate group (Figure 3c). Time-Dependent Azido-RB Functionalization of Polymer Brushes Using Both Copper-Free and CuAAC Click Chemistries. Polymer brush functionalization with azido-RB was monitored by UV−vis spectroscopy, in which several spectra were taken after submerging the alkyne polymer brushes in 40 mM azido-RB for various time intervals. This method allows for the ex situ monitoring of the absorbance of the dye (λmax ≈ 570 nm), which indicates the alkyne−azide cycloaddition into the brush. The absorbance spectra of the functionalization of DIBO brushes can be seen in Figure 4. The DIBO absorbance 14697

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DIBO, ADIBO, and PPG functionalized brushes at λmax for azido-RB covalently attached to the brush scaffold at 575, 571, and 577 nm, respectively (spectra in Figure S4 of the Supporting Information). On the surface, one can assume pseudo-first-order conditions for alkyne−azide cycloadditions due to the large excess of azido-RB in solution (40 mM) relative to the nanomolar concentrations of alkyne immobilized on the surface. Functionalization of the brush is limited by either the number of alkyne sites on the polymer brush or by limited access to alkyne sites on the polymer chains by diffusion, giving a final absolute absorbance value. This is represented graphically in Figure 6 with the ADIBO kinetics.

Figure 5. Solution kinetic plot of copper-free click reactions of DIBO and ADIBO (0.06 mM) with benzyl azide (25 mM) in MeOH. Plots represent the consumption of DIBO and ADIBO during cycloaddition at a pseudo-first-order rate of 1.7 × 10−3 and 9.9 × 10−3 s−1, respectively.

fitting of the data produced a bimolecular rate of 0.428 ± 0.054 M−1 s−1. The reaction of 0.06 mM ADIBO with benzyl azide (25 mM) is shown in Figure 5, which produced a pseudo-firstorder rate constant of 9.9 × 10−3 s−1. The faster rate of ADIBO over DIBO is due to the polarized alkyne bond generated by the nitrogen in the cyclooctyne ring,34,37,43 which increases distortion energy of ADIBO and further lowers the activation energy of the cycloaddition reaction. In contrast to SPAAC, reaction rates of CuAAC in solution have been extensively studied under various reaction conditions.60−62 The reaction rate of copper-catalyzed cycloadditions can be tuned by solvent,63 addition of base,64 amine ligands,62 and other chelating agents.65 No single set of reaction conditions yield fast, quantitative rates for every type of CuAAC reactions. Instead, the conditions must be varied to best suit the reaction to be completed. In this study, the PPG polymer brushes underwent CuAAC using a reducing agent (sodium ascorbate) and a supporting ligand (PMDETA), in order to maintain a stable Cu(I) concentration during reaction. Use of a reducing agent and supporting ligand aids in reducing the reactive oxygen species generated by the reduction of Cu(II), which can cause unwanted side reactions.66−68 Reactions involving amine ligands have demonstrated up to a 2 orders of magnitude acceleration on the cycloaddition rate,69 although the accelerating nature of PMDETA ligand has varied among different reaction conditions.69,70 As a comparison of solution and brush kinetics, we attempted CuAAC solution experiments using identical conditions (Cu(I), sodium ascorbate, and PMDETA), and the solution reactions reached full conversion in less than 10 s for all concentrations studied. Further lowering the concentration of reactants to slow down the reaction to a measurable rate was not possible and beyond the limit of detection of our HPLC. However, prior studies of CuAAC in solution using similar reducing agents and chelating ligands have produced cycloaddition rates as high as 1 × 105 M−1 s−1 per mole of copper catalyst.62 This value, while not a direct comparison to the PPG brush system, illustrates the rapid rate of the CuAAC in solution relative to the brush substrate and to the SPAAC solution reactions. Kinetics of Alkyne Polymer Brush Functionalization. The absorbances as a function of time were measured for

Figure 6. Graphical representation of time lapse polymer brush functionalization when a pendant group (small green circles) is placed in a solution with an absorbing reactive species (large red circles). The functionalization occurs until a maximum amount of reactive species is incorporated into the brush within reaction time of t∞. The blue line is meant to guide the eye.

In order to determine the pseudo-first-order rate constant, the rate of reaction must be modeled as the change in absorbance relative to the total possible change in absorbance for the polymer brush. This model is termed the pseudo-firstorder limited rate equation ⎛ A − A∞ ⎞ k′t = ln⎜ 0 ⎟ ⎝ A t − A∞ ⎠

(2)

where k′ is the pseudo-first-order rate constant, and A0, At, and A∞ are the absorbance values at λmax initially, at time interval t, and at the end of functionalization, respectively. The absorbance data at λmax for azido-RB functionalization of the brushes was fit to eq 4 and is shown below in Figure 7. The rate of azide functionalization of DIBO polymer brushes is the slowest, with a rate constant of (7.7 ± 1.2) × 10−4 s−1 with standard deviation determined between multiple trials. The rate of ADIBO polymer brush functionalization was faster with a rate constant of (4.4 ± 0.8) × 10−3 s−1. The pseudo-firstorder rate constants for the cycloaddition of benzyl azide (25 mM) with DIBO and ADIBO in solution were 1.7 × 10−3 and 9.9 × 10−3 s−1. To directly compare the pseudo-first-order rates of strain promoted alkyne−azide coupling in solution and on the surface, the relationship between these rates is represented by the ratio of the pseudo-first-order brush functionalization rate (k′B) computed from eq 4 to the pseudo-first-order solution functionalization rate (k′S) as the term alpha (α) in eq 14698

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to measure the CuAAC reaction rate in solution under these conditions because of the extremely fast reaction. However, Collman and Chidsey have observed monolayer CuAAC reactions, in which the reaction was performed with a reducing agent and accelerating amine ligand, that demonstrate fast cycloaddition kinetics with a pseudo-first-order rate constant of kobs = 0.010 s−1.15 This surface rate is similar to the PPG brush functionalization rate of 0.02 s−1 we observed in this study. Collman and Chidsey contributed the limit of the CuAAC surface reaction to steric effects at the surface,15 and this is most likely the case with the polymer brush coated substrates as well. Analysis of Copper-Free Click Chemistry of ADIBO Polymer Brushes using Dilute Solution Azides. The pseudo-first-order rates of azide functionalization for DIBO, ADIBO, and PPG derivatized brushes discussed in this study were conducted using a concentrated solution (40 mM) of azido-RB. In addition to using the high concentration of azide to assume pseudo-first-order reaction rates with the polymer brush, the concentration was used to limit the possibility of small molecule mass transport within the brush layer, which can affect the rate of reaction. This large concentration of azide, however, represents an ideal scenario for functionalization of the polymer brush scaffold, where the availability (and cost) of the analyte at higher concentrations is not a significant concern. A direct comparison of polymer brush reaction kinetics under both concentrated and dilute azide conditions can be made to elucidate the diffusion effects into the polymer brush matrix deconvoluted from reaction at the polymer brush. The kinetic experiment was therefore repeated using similar ADIBO brushes, with a dilute azide solution (500 μM) for functionalization. In this experiment, the overall concentration of azide in solution is still much greater than the potential concentration of active sites on the surface, ∼25 nmol/cm2,47 which maintain pseudo-first-order reaction conditions within the polymer brush. The solution azide will first react with alkynes on the outer portions of the polymer brush (Figure 6) and continue to undergo diffusion into the extended polymer brush chains, reacting with alkynes as azide penetrates into the brush. As the azide functionalization adds molar mass to the pendant polymer brush side chains, the polymer backbone will begin to extend (stretch) further away from the surface to accommodate the increased polymer density, which increases film thickness. Azides must further diffuse into the brush matrix in order to functionalize the buried alkyne sites closest to the substrate surface, albeit at a diminished functionalization rate. The azide functionalization rate of the ADIBO brush remains pseudo-first-order, but the diminished concentration of azide molecules in solution has to diffuse into the 50 nm brush matrix for the reaction to occur. Figure 8 shows the kinetic absorbance at λmax = 571 nm versus time for the SPAAC reaction of the ADIBO functionalized brush with a dilute azide solution (500 μM). Fitting the data (from 0 to 16000 s) to a pseudo-first-order limited equation yielded a reaction curve with varying slopes and poor fit, indicating that the reaction rate decreased over time. To model an accurate representation of the rate, one must examine the reaction rate in a regime of strong diffusional resistance71 where reaction conversion decreases with time.

Figure 7. Pseudo-first-order limited plot of azide functionalization of polymer brushes containing reactive alkynes DIBO (red hexagons), ADIBO (green circles), and alkyne (blue triangles).

3. The relative rate of click reaction for all SPAAC surface and solution kinetics are summarized in Table 2. a=

k′B k′S

(3)

Table 2. Pseudo-First-Order Rate Constants of Azide Functionalization of dibenzocyclooctynes (DIBO and ADIBO) Studied on Polymer Brushes and in Solution DIBO ADIBO

polymer brush rate, k′B (s−1)a

solution rate, k′S (s−1)b

α

7.7 × 10−4 4.4 × 10−3

1.7 × 10−3 9.9 × 10−3

0.45 0.44

a

[Azide] = 40 mM. b[DIBO] and [ADIBO] = 0.06 mM, [azide] = 25 mM.

A 55% decrease in reaction rate was observed for polymer brush functionalization with the cyclooctynes, DIBO and ADIBO, relative to the rate of SPAAC in solution. The overall ratio of solution reaction rates for ADIBO and DIBO were 5.8:1 (Figure 5). In comparing that ratio to the 5.7:1 that was observed on the surface, the relative rates of ADIBO:DIBO on the surface and in solution are analogous, demonstrating no significant change in the distortion energy of the cycloalkyne of copper-free click chemistry when attached to the polymer brush backbone. The decrease in the rate of functionalization in the brushes relative to the solution reactions is attributed to the confinement of the polymer chains at the surface, where solvation of the brush and diffusion of the azide through the brush layer to the active sites slows the rate of reaction The rate constant for azide functionalization of the PPG polymer brushes using a copper(I) catalyst was (2.00 ± 0.06) × 10−2 s−1. This rate is faster than either of the catalyst-free click reactions on the polymer brush scaffold. The overall ratio of the rates of polymer brush functionalization was 1:5.7:26 for DIBO, ADIBO, and PPG functionalized brushes, respectively. Values of CuAAC rates reported in solution are 10-fold faster than the most active ADIBO reported in solution, with rate constants of kobs ≈ 10−100 M−1 s−1 per 10−100 μM Cu(I) and kobs ≈ 1 M−1 s−1, respectively.34,62,65 The PPG brush functionalization is 5-fold faster than ADIBO brush functionalization, indicating that the reaction rate of CuAAC at the brush surface is slowed to a greater extent when compared to solution. We were unable

−r′A = k′[A]a − 14699

da = kda = C dt

(4)

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CONCLUSIONS The utility of using alkyne functional polymer brushes rests on knowing the optimum conditions and limitations of reactions in dense polymer brush scaffolds. It has been demonstrated that immobilized alkynes with different structures and reactivities can be used to immobilize azides through both CuAAC and SPAAC reactions with reaction times ranging from seconds to hours. Click kinetics for ADIBO and DIBO brushes are both 55% slower than corresponding reaction rates in solution. Surface CuAAC of PPG brushes show pseudo-first-order reaction rates similar to rates of monolayer functionalization at the surface. Preliminary work on low concentrations of azides indicates that diffusion of azides within a thin (50 nm) polymer brush has a greater than 80% reduction in functionalization rate, where the diffusion-limited reaction slows the reaction rate to one-quarter of the non-diffusion-limited rate. The development of a tunable alkyne functional polymer brush scaffold directly from an active ester polymer brush provides alternative chemistry for postpolymerization modification at the surface. Alkyne−azide cycloaddition is a near ideal chemistry for the postpolymerization functionalization of polymer scaffolds due to their ease of derivatization, rapid rates, and increased tolerance to different reaction conditions. The noncompetitive nature of alkyne−azide cycloaddition and aminolysis also provides a pathway to more complex polymer scaffolds, in which both types of pendant functionality can be incorporated into a well-defined polymer scaffold through orthogonal reactions.

Figure 8. Kinetic fit of dilute azide/ADIBO brush functionalization. The red and blue lines represent two regimes of brush functionalization: pseudo-first-order limited regime from 0 to 2300 s (blue triangles) and a pseudo-first-order diffusion-limited regime from 2301 to 16 000 s (red diamonds) using the diffusion parameter, a.

As stated in eq 4, the rate of azido-RB functionalization (r′A) is the product of the pseudo-first-order rate constant (k′), the azide concentration ([A]), and a deactivation term (a), which accounts for the change in reaction kinetics by the rate of diffusion, kd. The assumption is made that the diffusion rate does not change with time and is a constant (C), which is plausible upon examination of the data in Figure 8. If the data is split into two regimes, the first from 0 to 2300 s, and the second from 2301 to 16 000 s, it is observed that the first regime is pseudo-first-order limited with k′ = 8.01 × 10−4 s−1, and the second regime is diffusion limited with a value of k′ = 1.87 × 10−4 s−1. Diffusion into the polymer brush retards the reaction rate, but at a constant factor, da/dt = C. The ratio of the pseudo-first-order limited rate constants for the two regimes yields the deactivation term, a = 0.233, which indicates that brush functionalization slows to a quarter of the initial rate due to diffusion limitations. The derived diffusion limited pseudo-first-order rate equation, including the deactivation constant, a, is shown in eq 5.

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ASSOCIATED CONTENT

S Supporting Information *

Table of FTIR assignments, time-dependent absorbance spectra of ADIBO and PPG, and RB absorbance increases of DIBO, ADIBO, and PPG functionalized brushes with time. This material is available free of charge via the Internet at http:// pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.L.); [email protected] (V.V.P.). Notes

⎛ A − A∞ ⎞ k′t = a ln⎜ 0 ⎟ ⎝ A t − A∞ ⎠ A∞ a= A′0 − A∞

Article

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS The authors gratefully acknowledge financial support from the National Science Foundation (ECCS 0901141 and DMR 0953112).

(5)

■

The term A′0 is the absorbance value where the SPAAC reaction on the brush enters a diffusion-limited regime, and the slope of the first-order limited equation begins to deviate. Dilute solution kinetics of ADIBO brushes are significantly slower than the concentrated reaction kinetics (4.4 × 10−3 s−1 for 40 mM azide), yielding a 82% slower rate for cycloaddition in the first regime and continuing to a rate reduction of 96% in the diffusion-limited regime. In order to fully evaluate diffusionlimited kinetics, however, more concentration studies of polymer brush reactions need to be performed using a variety of dilute solutions and brushes of varying thicknesses. These experiments will determine when the diffusion limited regime begins for small molecule polymer brush functionalization and are currently underway in our laboratory.

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