Molecularly Imprinted Porous Monolithic Materials from Melamine

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Molecularly Imprinted Porous Monolithic Materials from MelamineFormaldehyde for Selective Trapping of Phosphopeptides Mingquan Liu, Tri Minh Tran, Ahmed Awad Abbas Elhaj, Silje Bøen Torsetnes, Ole Nørregaard Jensen, Börje Sellergren, and Knut Irgum Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02470 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 12, 2017

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Analytical Chemistry

Molecularly Imprinted Porous Monolithic Materials from MelamineFormaldehyde for Selective Trapping of Phosphopeptides Mingquan Liua, Tri Minh Trana†, Ahmed Awad Abbas Elhaja‡, Silje Bøen Torsetnesb¶, Ole N. Jensenb, Börje Sellergrenc, Knut Irguma* a) Umeå University, Department of Chemistry, S-901 87 Umeå, Sweden. b) University of Southern Denmark, Department of Biochemistry & Molecular Biology and VILLUM Center for Bioanalytical Sciences, Campusvej 55, DK-5230 Odense M, Denmark. c) Malmö University, Department of Biomedical Sciences, Faculty of Health and Society, S-205 06 Malmö, Sweden.

Abstract Thirty-five melamine-formaldehyde (MF) monolithic materials with bimodal pore distributions were synthesized in fused silica capillaries by catalyst-free polycondensation, starting with an aqueous MF precondensate, using acetonitrile as macroporogen and a variety of aliphatic polyethers and triblock copolymeric surfactants as porogens and mesoporogens, respectively. By varying the prepolymer composition and the type and molecular weights of the polymeric porogen components, a library of porous monolithic materials were produced, covering a range of meso- and macroporous properties. A multivariate evaluation revealed that the amount of surfactant was the strongest contributor to specific surface area and pore volume, and to the inversely related mesopore size, whereas the macropore dimension was mainly controlled by the amount of aliphatic polyether porogen. One of these capillary monoliths, chosen based on the combination of meso- and macropores providing optimal percolative flow and accessible surface area, was synthesized in the presence of N-Fmoc and O-Et protected phosphoserine and phosphotyrosine, in order to prepare molecularly imprinted monoliths with surface layers selective for phosphopeptides. These imprinted monoliths were characterized alongside non-imprinted monoliths by a variety of techniques and finally evaluated by liquid chromatography-mass spectrometry in the capillary format to assess their abilities to trap and release phosphorylated amino acids and peptides from partly aqueous media. Selective enrichment of phosphorylated targets was demonstrated, suggesting that these materials could be useful as trapping media in affinity-based phosphoproteomics.

Keywords Melamine-formaldehyde,

step-growth

polymerization,

phosphorylated

peptides,

polymeric

monolithic supports, mesoporosity, liquid chromatography-mass spectrometry.

* Corresponding author. Phone: +46 90 7865997; e-mail: [email protected] Present addresses: † VISSAN Joint Stock Company, Quality Assurance Department, 717 707 Ho Chi Minh City, Vietnam; ‡ ALcontrol AB, Mariehemsvägen 6, Box 3080, 906 54 Umeå, Sweden; ¶ Department of Neurology, Akershus University Hospital, Sykehusveien 25, 1478 Lørenskog, Norway.

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Introduction Monoliths have become an important part of separation science and as flow-through supports in many catalysis, reaction, and heterogeneous interaction schemes due to the combination of high permeability and potential for good mass transfer compared to packed particle beds.1 Most polymeric monoliths are made by chain-growth (addition) polymerization of vinylics2 and ring-opening metathesis polymerization,3 yet there are examples in the literature using urea/formaldehyde4 and epoxide/amine5,6 systems, showing that monoliths with suitable porous structures can also be made by step-growth (condensation) reactions. In our search for alternative routes to prepare monolithic carrier materials, we are in this work using a step-growth polymerization of melamine and formaldehyde (MF) as a means of preparing hydrophilic porous supports with structures and pore size distributions suitable for use in flow-through interaction and reaction schemes. Melamine (2,4,6-triamino-1,3,5-triazine) is an aromatic heterocycle capable of acting as a hexafunctional monomer by means of its three amino groups, each capable of reacting with up to two bi- or oligofunctional electrophiles, leading to highly crosslinked products. Cured MF is hydrophilic and known for its chemical resistance, heat durability, and toughness,7 but its uncured prepolymers are typically suspensions.8 This allows the preparation of porous foams and particles from MF by first synthesizing a precondensate in aqueous solution, followed by a separate curing step.9 Melamine is capable of reacting with up to six equivalents of formaldehyde in the precondensation step, which leads to a variety of hydroxymethylated monomers and oligomeric pre-polymers. The melamine:formaldehyde (M:F) molar ratio in precondensates can be from 1:2 up to 1:6, but is usually around 1:3.10 Gelling of the prepolymer to form a porous polymer is generally initiated by tuning the pH of the water-soluble MF precondensate dispersed in an organic phase in the presence of an emulsifier/antifoaming agent.11 This triggers polycondensation into an intermediate gel, which is finally cured by heating (Scheme 1). Phosphorylation is a post-translational modification (PTM) involved in numerous human diseases of widespread impact, such as cancer,12 diabetes,13 cardiovascular,14 and Alzheimer’s diseases.15 Analysis of phosphopeptides is therefore essential in the search of new biomarkers, to gain a better understanding of the causes and progression of diseases, and in monitoring of responses to clinical treatment.16 Mass spectrometry (MS) is indispensable in phosphorylation analysis, but still far from routine because of low abundance of phosphopeptides, the low ionization efficiency, and the signal suppression by abundant non-phosphorylated peptides. Several phospho-specific enrichment techniques have been developed, e.g., immunoaffinity,17 chemical coupling,18 and immobilized metal ion affinity chromatography (IMAC)19 using Fe3+, Ga3+, or Ti4+ as chelated metal ions.20–22 Antibodies are susceptible to denaturation, and show substantial cross-selectivity and an undesirable selectivity based on the amino acids flanking the phosphorylated tyrosine (pY) sites.23 IMAC is quite efficient but suffers from trapping of non-phosphorylated peptides with multiple carboxylic residues and metal ion loss during elution.18 Metal oxide affinity (MOAC)24 is currently preferred for phosphopeptide trapping, mainly on TiO2 and ZrO2, but also using insoluble oxides of Al, Ga, Fe, Nb, Sn, Hf, and Ta.25 The efficiency depends, however, on the morphology and surface structure of the metal oxides, and 2 – Environment ACS Paragon –Plus

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MOAC is also, similar to IMAC, hampered by significant co-extraction of acidic non-phosphorylated peptides.26 Molecularly imprinted polymers (MIPs) have been applied in areas such as separation,27 sensing,28 disease diagnostics,29 catalysis,30 and bioimaging.31 We have found monoliths to work well as grafting substrates for MIPs,32,33 and vinylic monomers with urea functionality can offer selectivity in MIPs prepared for screening of specific phosphorylation.34 The current work is inspired by the polar groups formed in the backbones of most polymers prepared by step-growth polymerization. These polar main chain moieties will be more rigidly locked in the structure, as opposed to polar groups located on side chains, such as, e.g., in MIPs prepared from esters and amides of (meth)acrylic acid. There are moreover reasons to assume that concurrent, slow polymerization will promote the formation of recognition sites with enhanced binding capacity and low heterogeneity, seen in MIPs based on controlled radical polymerization.35,36 In crosslinking step-growth polymerization systems, the viscosity increases only slowly at the onset of polymerization, followed by a sudden and rapid gelation.37 The template–monomer–polymer system will therefore have ample opportunities to relax to its lowest overall energy state as new polar groups are formed in the monomer conversion process. Recognition sites of optimal binding energy should therefore not be “locked” until the final stages of monomer conversion.37 To test this concept, we have attempted to create selectivity against phosphopeptides by preparing mesoporous monolithic MF MIPs by an organic sol-gel process.38,39 Our first step was to optimize the synthesis of rigid MF monoliths with a suitable mix of meso- and macropores using homo- and block aliphatic polyethers as porogens, which served as starting point for the preparation of MIPs, using phosphorylated N-Fmoc protected ethyl esters of serine and tyrosine as templates.

MATERIALS AND METHODS Reagents and Materials. The monomers used were 1,3,5-triazine-2,4,6-triamine (melamine; 99 %) from Sigma-Aldrich (Schnelldorf, Germany) and 37 % aqueous formaldehyde from BDH Chemicals (Poole, England), with ≈ 10 % MeOH added as stabilizer. The acetonitrile (ACN) used as diluent was of analytical grade from Merck (Darmstadt, Germany). The polymeric porogens were α-hydro-ωhydroxy-poly(oxyethylene) [PEG; poly(ethylene glycol)] and α-hydro-ω-hydroxypoly[oxy(1methylethylene)] [PPG; poly(propylene glycol)] of average molecular weights (MW) between 200 and 20,000 Da, all obtained from Sigma-Aldrich. The triblock α,ω-hydroxy-poly(oxyethylene)-blockpoly[oxy(1-methylethylene)]-block-poly(oxyethylene), i.e., hydroxy-terminated [poly(ethylene oxide)block-poly(propylene oxide)-block-poly(ethylene oxide); EOxPOyEOx] surfactants L61 (nominally EO2PO31EO2; MW ≈ 2000), L121 (EO5PO68EO5; MW ≈ 4400), P123 (EO20PO69EO20; MW ≈ 5750) and F127 (EO106PO69EO106; MW ≈ 13300) were from BASF (Ludwigshafen, Germany). L-Serine ethyl ester hydrochloride, L-tyrosine ethyl ester hydrochloride, and the Fmoc amino acids Fmoc-S-OH, Fmoc-YOH, Fmoc-pS-OH, and Fmoc-pY-OH, were from Bachem (Bubendorf, Switzerland). The human Angiotensin II octapeptide DRVYIHPF was from Fluka (Buchs, Switzerland) and its phosphorylated modifications DRVpSIHPF and DRVpYIHPF were custom synthesized by LifeTein LLC (Hillsborough, NJ, USA). All chemicals were used as received, unless otherwise noted. The water used was prepared

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by Milli-Q or Ultra-Q equipment from Merck Millipore (Bedford, MA, USA) and had a resistivity of > 18 MΩ∙cm–1. Syntheses of the Fmoc-pS-OEt and Fmoc-pY-OEt templates are described in the Supporting Material, as are the sources of chemicals used in those syntheses and in the characterizations. Capillary pretreatment. Prior to synthesizing the monolithic packing, the inner surface of the 100 μm i.d. × 360 μm o.d. polyimide-coated fused-silica capillaries obtained from Polymicro Technologies (Phoenix, AZ, USA) was covalently modified with amino groups to ascertain that the MF monolith became anchored to the capillary wall. The procedure was based on previous reports,40,41 with the final protocol as follows: 1) The capillaries were rinsed in sequence with methanol and deionized water in order to remove impurities, followed by; 2) filling with aqueous 1 M NaOH for 1 h, then rinsing with deionized water until neutral, thereafter; 3) washing with 1 M aqueous HCl for 1 h followed rinsing with deionized water until neutral; 4) filling with methanol for 1 h, followed by drying by a flow of nitrogen at 60 °C overnight. The pretreated capillaries were amino-functionalized by 5) reacting with a solution of 8 % (v/v) (3-aminopropyl)triethoxysilane (APTES) in methanol at 60 °C for 12 h, pumped through the capillary at a flow rate of 1 µL/min; followed by 6) a rinse with methanol to flush out the residual reagent and finally drying as under (4). The amino-functionalized capillaries were cooled to room temperature and stored in a desiccator for further use in polymerization of MF monolithic columns. Synthesis of Porous Melamine-Formaldehyde Monoliths. Formaldehyde (27.1 g of the 37 % aqueous solution) was added under stirring to 70 mL water in a 100 mL round bottom flask. Ten gram aliquots of this solution (containing ≈ 34 mmol formaldehyde) were thereafter transferred into five individual 25 mL round bottom flasks, followed by addition of melamine powder under agitation, to yield the formaldehyde:melamine (F/M) molar ratios listed in Table 1. The flasks containing the melamine-formaldehyde suspensions were immediately placed on a water bath set at 80 °C and heated under stirring until the solutions appeared clear, which took ≈ 30 min. The MF precondensates were then allowed cool to room temperature and thereafter stored in a refrigerator at +4 °C for a maximum of four weeks until next step. Precondensates prepared according to above were used to prepare a library of thirty-five monolithic non-imprinted MF polymers (NIPs) by the one-pot synthesis route shown in Scheme 1, using variable parameters given in Table 1. In this procedure, the specified amounts of porogens (aliphatic polyethers PEG or PPG) and mesoporogens (triblock polyether surfactants) were mixed in 2 mL GC vials, followed by addition of 300 µL MF precondensate. The acetonitrile diluent was thereafter added to the vials, followed by ultrasonic agitation for five minutes in an Emmi 30 Eco ultrasonic cleaning bath (EMAG Technologies, Walldorf, Germany) to prepare homogeneous precursor solutions. The sonicated vials were capped with PTFE-lined septa, shaken vigorously, and given a 30 s purge with N2(g). Amino-functionalized fused silica capillary was cut into 70 mm long pieces that were filled with monolith precursor solution by inserting one end through the pre-pierced septum of the precursorcontaining vial and the other end likewise into an empty septum-capped vial. Capillary force and a slight N2 overpressure applied to the vial containing the precursor solutions caused a slow flow, while 4 – Environment ACS Paragon –Plus

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maintaining the capillary end submerged so that no gas bubbles were trapped in the capillary. After several drops of precursor solution had been transferred to the empty vial, the N2 pressure was released from the vial containing the precursor solution and both vials taken to atmospheric pressure by momentarily piercing their septa by a hypodermic needle. The filled capillary/vial assemblies were thereafter immediately transferred to the oven of an HP 5890A GC instrument (Agilent Technologies, Palo Alto, CA, USA), ensuring that both ends of the capillaries were fully submerged in precursor solution in both vials. The programming feature of the GC was used to program the heating at a rate of 5 °C/min from ambient to 80 °C, where it was kept for 4 h to allow the polymerization to take place under static conditions, followed by cooling to ambient temperature at –5 °C/min. Molecularly imprinted MF monoliths (MIPs) were prepared in the same way as the NIPs, with the difference that varying amounts of Fmoc-pS-OEt or Fmoc-pY-OEt were added as templates along with two equivalents of PMP (a strong, non-nucleophilic base) to form the corresponding bis-PMP salts in situ. The vials containing the MIP precursor solutions were stirred for 30 minutes at 40 °C to allow initial self-assembly of the recognition sites. The capillaries were thereafter filled and the polymerization proceeded as above. After polymerization, the capillaries were detached from the vials, which were disintegrated with minimal force to render the bulk monolithic materials formed there as intact as possible. The recovered bulk materials were cut into roughly cubiform pieces with approximately 2-3 mm sides and transferred to cellulose extraction thimbles. Unreacted monomers, polymeric surfactants and porogens, diluent solvents, and templates in the case of MIPs, were removed from the capillaries by flushing for 6-18 h at 20 µL/min, and from the recovered bulk monoliths by Soxhlet extraction for 24 h, using pure methanol for the NIPs and methanol with 0.1 % TFA for the MIPs. The cleaning of the capillary monoliths was followed by flushing with at least five column volumes of 70:30 % (v/v) acetonitrile/water, which was also used for storing the capillary monoliths in a refrigerator at + 4 °C. The Soxhlet-extracted bulk monolithic materials were finally dried under reduced pressure (≈ 100 Pa) in a Gallenkamp (Loughborough, UK) vacuum oven at 60 °C for at least 6 h prior to further characterization. Characterization and Evaluation. Procedures, background information, and results from characterization and evaluation of porosity by nitrogen cryosorption and mercury intrusion, multivariate data analysis, capillary LC, field emission scanning electron microscopy (FE-SEM), Fourier-transform infrared (FT-IR) spectroscopy, solid-state nuclear magnetic resonance (NMR) spectroscopy, X-ray photoelectron spectroscopy (XPS), suspended-state and saturation transfer difference high-resolution magic-angle-spinning nuclear magnetic resonance (STD HR/MAS NMR), and thermogravimetric analysis (TGA) are found in the Supporting Information.

Results and Discussion Optimization of the MF Monoliths Synthesis. It is well known1 from previous work with addition polymerization, that monolithic supports with tailor-made distributions of macro- and mesopores, pore volume, and specific surface area can be prepared, provided appropriate amounts of monomers, crosslinkers, initiator, and porogenic solvents are paired with well-controlled polymerization 5 – Environment ACS Paragon –Plus


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conditions. Therefore, the idea that underpinned the present work was to investigate the possibility of preparing MF monoliths with porous properties suitable for molecular imprinting by dispersing aqueous MF precondensate solutions into an organic phase by a nonionic surfactant, mixed with aliphatic polyethers of varying MW that were intended to interact with the non-ionic surfactant, and an organic diluent to establish a ternary porogen system.42 In other words, in this system we expected the molar ratio of melamine to formaldehyde and the ternary porogen system to be the main factors for adjusting the structure and porosity of the MF monoliths produced. After considering numerous water-miscible solvent candidates based on their boiling points, lack of reactivity with monomers and other components, and their ability to dissolve the monomers, the polyether porogens and mesoporogens, and the templates, we carried out a preliminary screening involving DMF, THF, methanol, 2-propanol, and acetonitrile. Among these, acetonitrile was selected based on its compliance with the above-mentioned criteria, its ability to induce phase separation leading to monolith formation, and the porous properties of the monoliths produced in the initial screening (data not shown). The sequential step-growth mechanism used to prepare these melamine-formaldehyde polymers is radically different from the addition polymerization schemes commonly used in preparing porous organic monoliths. In these experiments we started by first preparing aqueous precondensates of melamine and formaldehyde under slightly alkaline conditions in bulk phase and thereafter mixing these with porogens and diluents, filling it in silica capillaries followed by curing. The compositions used in the screening experiments are found in Table 1, which also shows the qualitative morphology assessments along with specific surface areas, total pore volumes, and average mesopore sizes determined by multipoint nitrogen cryoadsorption-desorption, and median macropore sizes corresponding to the most prominent peak in differential mercury intrusion measurements of the monolithic bulk materials synthesized alongside the capillary monoliths. Most of the materials were rigid and opaque monoliths in the dry state. Formaldehyde:Melamine (F/M) molar ratio. The steps in the polymerization of melamine and formaldehyde are shown in Scheme 1.43 The first step (a) is a methylolation of melamine by formaldehyde forming prepolymers in aqueous phase at a weakly alkaline pH, where the distribution of monomers and low MW oligomers is determined by the ratio of the reactants, and the temperature and duration of the prepolymerization step. The second step is a curing (b), which took place in the capillaries under moderate heating after the prepolymers had been mixed with diluent and porogens (and in the case of MIPs also with templates), producing crosslinked monolith networks by condensation reactions leading to methylene ether and methylene bridges. The formaldehyde:melamine molar ratio should be one of the most important factors in the preparation of MF monoliths, since formaldehyde is capable of forming dense crosslinks with its potentially hexafunctional comonomer melamine. This is expected to affect the rigidity and porous properties of the final monoliths. Therefore, as shown in Table 1, we initially investigated five formaldehyde:melamine (F/M) molar ratios in monoliths MF01 to MF05. When the F/M ratio was increased stepwise from 0.5:1 to 3:1, the specific surface area increased as expected, accompanied by correspondingly smaller pore sizes, owing to an increase in degree of crosslinking and rigidity. The


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increment in F/M ratio from 3:1 to 6:1 led to substantially smaller mesopores and lower pore volume, which we ascribe to having exceeded the maximum degree of crosslinking (F/M ratio of 3:1). The weakly basic properties of melamine seemed to promote the polymerization process, as it proceeded without separate addition of acid or base as catalyst, which could have affected the quality of the molecular imprinting.44 Based on this we fixed the F/M molar ratio at 3:1 in the continued study, while the volume of diluent, and the type and amount of polymeric porogens and surfactants were varied. Variation in the polymeric porogen. We then went on to optimize the polymeric coporogens. Based on previous results42 we chose a series of hydroxy-terminated poly(ethylene glycols) (PEG) and poly(propylene glycols) (PPG) with average MWs spanning from 200 to 20,000 Da, represented by MF04 and MF10-MF24 in Table 1; MF25 serving as a control without coporogen added. The first observation to be noted is that both PPG and PEG led to more resilient monolithic materials. The coporogens also had significant impact on the pore formation processes, evident from the increases of meso- and macropore sizes and correspondingly lower specific surface areas with coporogen MW. When comparing PEG and PPG of similar MWs, PEG gave significantly lower specific surface areas and total pore volumes as coporogen, compared to PPG. Another interesting observation is that the decrease in specific surface area levelled off at around 2,000 Da for PPG, beyond which the total pore area started to increase. In contrast, increasing the MW of PEG led to strong and monotonic decreases in both specific surface area and total pore volume and corresponding increases in both meso- and macropore sizes. In finding the balance between a relatively high specific surface area, mesopores that are sufficiently wide to be accessed by peptides, and macropores that are capable of supporting a through-flow without excessively high back-pressure, we therefore settled on PPG4000 as coporogen for the continued experiments. This experiment series sheds some light on the roles of the porogens and surfactant in this rather complicated system. The Pluronic F127 block copolymeric surfactant EO106PO69EO106 chosen has a central PO block closely matching PPG4000, with EO blocks slightly longer than PEG4000. The lower polarity of the central PO block forces it to associate with the acetonitrile diluent phase in the partly aqueous polymerization mixture. The tendency of identical polymers to associate consequently attracts the PPG coporogen into the diluent phase, widening the macropores and leaving the EO blocks of the surfactant free to orchestrate formation of the mesopore system. If the more hydrophilic PEO is used as coporogen, it will not associate itself with the organic diluent phase but instead with the aqueous phase containing the prepolymer, where it risks being trapped as the system crosslinks. This reasoning explains the evolution of the porosity with both EO and PO as coporogens, and the significant decrease in total pore volume observed when PEO of increasing length was used as coporogen. We then studied the effect varying amounts of PPG4000 by synthesizing MF25-MF28, a series of experiments where MF04 is also a part. Although the specific surface areas were higher for MF25 and MF26, synthesized without, and with only half the amount of PPG4000 used in MF04, this was at the expense of the meso- and macropore sizes and the total pore volumes. The mechanical strengths of MF25 and MF26 were also clearly inferior to MF04. We did not devise an objective measurement of 7 – Environment ACS Paragon –Plus


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physical strength for the scouting experiments; instead the materials showing promising porous properties were subjected to back-pressure tests in capillaries, which is a realistic quality test for their intended use. We find it quite remarkable that the coporogens not only influenced the pore properties, but also had a substantial effect on their physical strength. It cannot be entirely ruled out that the coporogens are being incorporated in the monolith matrix, either reactively by transetherification, or simply by mechanical trapping. However, the almost identical C:N ratios of about 1.04:1 in the XPS measurements accounted for in Table S-1 match the 3:1 F/M-ratio well and therefore contradict that significant amounts of polyalkyleneoxide polymers should have been incorporated in the monoliths. We therefore attribute the superior mechanical properties at higher coporogen MWs to better pore forming abilities of the combined porogen/diluent system. Effect of the triblock copolymeric surfactant. Preparation of MF monoliths with bimodal pore structure from aqueous prepolymers combined with acetonitrile as diluent, PEG/PPG coporogens, and amphiphilic triblock copolymers orchestrating the pore formation is an uncharted area of polymer science. We therefore had no guidance in choosing the block ratio, the MW, and concentration of the amphiphilic triblock copolymer. After some initial scouting, we designed two series of experiments where we first evaluated Pluronics with varying MW and block lengths, and thereafter optimized the concentration of triblock copolymer at four different levels for the Pluronic with the most promising surface area and bimodal pore properties. Pluronics L61 and L121 have identical EO:PO:EO mass ratios of 5:90:5 and share “hydrophilic-lipophilic balance” (HLB)45 values in the range 1-7, typical of water-in-oil (W/O) surfactants, but differ in MW (L61 ≈ 2,000 Da and L121 ≈ 4,400 Da). Pluronics P123 and F127 share the length of the middle PPO segment (≈ 4,000 Da) with L121, but the PEO:PPO:PEO mass ratios are 15:70:15 and 35:30:35, respectively,46 resulting in MWs of ≈ 5,750 Da for P123 and ≈ 12,600 Da for F127. Their HLB values are 7-12 and >24, respectively, and they therefore qualify as oil-in-water (O/W) surfactants or solubilization agents. The set of Pluronics chosen thus represent a wide span in both MW and relative block lengths (and hence HLB values). Monoliths MF29-MF31 and MF26 show the effect of adding Pluronics L61, L121, P123, and F127 as mesoporogens. MF32, of identical composition except that the Pluronic triblock copolymer was omitted, serves as a reference for the effect of adding block copolymic surfactants. As seen in Table 1, the specific surface areas of MF29 and MF30 synthesized with Pluronic L61 and L121 were only slightly higher than MF32, the material prepared without triblock copolymer. The nitrogen cryosorption isotherms for these three materials were also practically identical (cf. Figure S-1). The main effect of increasing the MW more than two-fold from L61 to L121 was an increased total pore volume. For MF 30, MF 31, and MF26, prepared with Pluronic L121, P123, and F127, sharing the same PPO block length, the specific surface area increased with increasing PEO block length (and consequently total MW). This was accompanied by a modest decrease in both meso- and macropore size. The increase in specific surface area without significant decrease in average mesopore sizes is interpreted as formation of a more homogeneous cohort of mesopores. Since MF26 also had a rigid structure we therefore chose Pluronic F127, since its high MW and HLB value appeared to promote its meso-structure-directing ability.47,48


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We finally varied the amount of Pluronic F127 by preparing MF33, MF34 and MF35, including in this series MF26 and MF32, to arrive at block copolymer surfactant loadings of 0, 3, 6, 9, and 12 mg with other parameters kept fixed. The results listed in Table 1 show that increased F127 loading resulted in a steep increase in specific surface areas and decreased median macropore sizes. The specific surface area started to level off at 9 mg, and on increasing to 12 mg the total pore volume and the sizes of the meso- as well as the macropores began to decrease sharply. From the cumulative surface area plots (Figure S-1) it is also evident, that in spite of MF35 having a larger total surface area, the fraction of the surface area present in pores > 10 nm was larger in MF34. Our decision to select MF34 as the base recipe for preparing the MIPs was therefore based on a more suitable pore size distribution for molecular imprinting of phosphopeptides, but also on a considerably lower back-pressure of MF34, compared to MF35. Multivariate assessment. The interaction and correlation of the synthesis parameters with key materials properties, all presented in Table 1, were modelled using projection against latent structures (PLS) and visualized as a “biplot” of the scores and loadings in Figure 1, with all materials contributing to the model included. This biplot shows the two first extracted components, where the first, as usual, had better correlation than the second with the X and Y data blocks. This first component, plotted along the abscissa, pinpoints the factors contributing to larger macropore size (MPS) to the right and higher specific surface area (SSA) to the left, factors that are obviously inversely related. The synthesis factors contributing most strongly to this dimension are the amounts of block copolymeric surfactant (SurfAmt) and coporogen (PorAmt). The second dimension mainly explains the total pore volume (TPV) and mesopore size (AMS), which are inversely related to the F/M molar ratio (FM_Ratio). The coporogen type had an effect on both dimensions, where PPG is associated with higher specific surface area and total pore volume, with the opposite for PEG. It should be noted that the overall significance of the model just discussed, which attempts to model all response variables simultaneously, was not very high. Repeated PLS models were therefore made with each response variable modeled separately. These biplots, shown in Figure S-3, all had better correlations than the combined model and confirmed that the controlled variables contributing to the measured responses were those pinpointed in the discussion above. Influence of the melamine:template ratio. In the process of choosing templates, we considered that the epitopes to be imprinted for selectivity against peptides phosphorylated on serine and tyrosine are the O-phosphorylated side chains, as well as the main chain amide groups of the amino acids serine and tyrosine. To orient these polar group towards the surface of the MF polymer during polymerization in a partly aqueous monolith precursor solution rich in acetonitrile, we chose to protect the N- and C-terminals of the amino acids by Fmoc and ethyl groups, respectively. This was based on a hypothesis that the templates would preferentially arrange their characteristic epitopes towards the aqueous MF prepolymer phase, with their non-polar protecting groups oriented towards the acetonitrile diluent, which will form the pore space after polymerization. The molar ratio of functional monomer:template is a central factor for reaching strong affinity and selectivity of molecularly imprinted materials. A series of monolithic MIP capillaries were therefore polymerized based on the MF34 NIP recipe with melamine:template (M/T) molar ratios of 1:1, 2:1, 9 – Environment ACS Paragon –Plus


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5:1, 10:1, and 15:1. The isocratic elution profiles of injected templates on these MIPs are shown in Figure 2, revealing that the imprinting factors increased strongly with a gradual increase in M/T ratio up to 5:1 for both Fmoc-pS-OEt and Fmoc-pY-OEt, and then significantly weakened when additional template was added. We therefore settled on a M/T molar ratio of 5:1. Materials Characterization. The non-imprinted MF34 and the pS and pY MIPs based on this monolith were characterized by N2 cryosorption and Hg intrusion to chart their porosity, FE-SEM for visualizing the morphology, FT-IR, XPS, and solid state NMR for determining chemical composition, STD-NMR to probe interaction patterns, TGA to verify curing and probing the thermal stability, and flow-dependent back pressure in three different solvents. For space reason, these results along with the accompanying experimental descriptions are shown Figures S-1 to S-10 and Tables S-1 and S-2 in Supporting Information. References to Figures and Tables numerated by an initial S in the following text refer to the data located there. Chromatographic Evaluation. We used LC-MS for the final evaluation of the ability of the imprinted capillary monoliths to trap and release phosphorylated entities. Phosphorylated and nonphosphorylated Fmoc amino acids, as well as variants of the human Angiotensin II octapeptide were loaded onto the capillary monolith columns and gradient eluted by increasing concentrations of water in the acidic mixed acetonitrile/water eluent, using an LTQ Orbitrap XL MS instrument to monitor the column effluents ionized by the nESI source. Extracted ion chromatograms (EICs) showing the affinities of non-imprinted (NIP), and Fmoc-pS-OEt (pS MIP) imprinted, and Fmoc-pY-OEt (pY MIP) imprinted capillary monoliths are found in Figures 3 and 4. We first consider the NIP and MIP affinities for the N-Fmoc protected amino acids; Fmoc-S-OH, FmocY-OH, Fmoc-pS-OH, and Fmoc-pY-OH, by their retention under a gradient of increasing concentration of water (Figure 3). Neither of the non-phosphorylated Fmoc amino acids (Fmoc-S-OH or Fmoc-Y-OH) had significant retention, evident from their elution as sharp peaks close to the void (Figure 3a). The phosphorylated counterparts (Fmoc-pS-OH and Fmoc-pY-OH) also produced some signals close to the void on the NIP, but in addition we also saw wide and tailing signals characteristic of MIPs peaking at around 20 minutes retention time. The integrated ion count ratios for the sharp peaks eluting close to the void and the wide peaks were about 3:7. The latter peaks for the phosphorylated Fmoc-protected amino acids did, however, appear earlier in the NIP chromatograms (cf. the two lower chromatograms in Figure 3a) than the corresponding phosphorylated AAs on the pS MIP, and pY MIP (the corresponding chromatograms in Figures 3b and c, respectively). The Fmoc-pS-OEt imprinted monolith (Figure 3b) showed a clear imprinting effect, with the nonphosphorylated Fmoc-S-OH and Fmoc-Y-OH eluting close to the void, whereas their phosphorylated counterparts produced peaks with similar and characteristic “MIP shapes”, peaking at 30 minutes retention time for both Fmoc-pS-OH and Fmoc-pY-OH, with very little breakthrough close to the void volume. We interpret this as an effect of imprinting, selectively targeting the phosphoric ester group, without signs of the Fmoc group contributing to the retention (compare Figures 3a and b). Interestingly, the Fmoc-pY-OEt imprinted monolith (Figure 3c) showed significant retention for the non-phosphorylated Fmoc-protected amino acids, with Fmoc-S-OH and Fmoc-Y-OH both eluting at

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around 11 minutes as relatively sharp peaks. This is clearly different from the behavior of these probes on the Fmoc-pS-OEt imprinted monolith, where these compounds both eluted as sharp peaks close to the void volume. A rational explanation to this could be a combined effect of the aromatic groups of the Fmoc and tyrosine moieties, causing a retention for this motif. Yet the phosphorylated probes had significantly higher retention than their non-phosphorylated counterparts, in this case peaking as late as around 40-45 minutes, showing the additive effect of the aromatic and phosphate esters on the binding strength of the imprinted monoliths. Turning to the intended use of these imprinted monoliths in phosphopeptides trapping, we used human Angiotensin II with (DRVpYIHPF) and without (DRVYIHPF) phosphorylation on tyrosine, and a homolog (DRVpSIHPF) with pS substituted for Y as probes. The EICs in Figure 4 show the affinities of the NIP (a), pS MIP (b), and pY MIP (c) for these octapeptides. The retention patterns of all three peptides were remarkably similar on the NIP, shown by their EICs in Figure 4a, all with a large peak eluting at ≈ 12 minutes. When injected on the pS MIP (Figure 4b), both phosphorylated probes (DRVpSIHPF and DRVpYIHPF) eluted in the span from 22 and 40 minutes and appeared to have been trapped almost quantitatively, since there were no signs of breakthrough close to the void volume. The peak shapes of DRVpSIHPF and DRVpYIHPF were also complementary, with the front of DRVpYIHPF rising sharply, after which it dropped when DRVpSIHPF overtook the elution profile. Both the sharp rise and the initial rapid decrease of the DRVpYIHPF signal can be explained by a higher affinity of the pS MIP for DRVpSIHPF, which hence displaced DRVpYIHPF in a multicomponent displacement type elution scheme, where DRVpYIHPF in turn displaced DRVYIHPF. This implies that the imprinting of Fmoc-pS-OEt had established an improved affinity for the peptide phosphorylated on Ser compared its Tyr phosphorylated analog, but also that the pS MIP was capable of effectively trapping and releasing both the serine and tyrosine phosphorylated peptides when elution conditions were altered. Referring finally to Figure 4c, we notice that the pY MIP also had an affinity for both phosphorylated peptides (DRVpSIHPF and DRVpYIHPF). In this case there were signals close to the void indicating minor bleed-through, where about 1.5 % (based on the integrated EIC signals) of the injected phosphopeptides appeared. More puzzling was the apparent affinity for the non-phosphorylated peptide (DRVYIHPF), evident from the peaks eluting later in the top chromatograms of Figures 4b and c. We cannot pinpoint the reason why the imprinting produced an affinity for the non-phosphorylated peptide, but a hypothesis could be an imprinting of the aromatic moieties of the Fmoc-OEt protected amino acid templates, resulting in a non-specific retention for in DRVYIHPF which has two aromatic side chains (Tyr and Phe). Imprinting of the Fmoc group was seen also in the STD-NMR experiments accounted for in the Supporting Information. Such imprinting of aromaticity would explain why the pY MIP monolith showed a higher retention for all peptides (35-40 minutes), due to three imprintable aromatic centers (Fmoc, Tyr, and Phe), whereas the pS MIP monolith, which retained DRVYIHPF for 22-26 minutes had contributions to imprinting of aromatics only from the Fmoc and Phe. It is not unlikely that these receptors could also be active against the tyrosine side chains of DRVYIHPF, which would thereby have better opportunities of interacting with sites imprinted against aromaticity on the pY MIP than on the pS MIP. For future experiments using this scheme, it may therefore be worthwhile



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to test non-aromatic alternatives to Fmoc (for instance t-BOC) as N-protective group of the amino acid templates if this unintentional selectivity for aromatic groups is to be avoided.

Conclusions A catalyst-free and facile single-step route featuring a tunable ternary porogen system was explored for the synthesis of robust porous melamine-formaldehyde (MF) capillary monoliths by a self-initiated condensation polymerization via a one-pot organic sol-gel route under mild condition. Successful recognition and separations of phosphorylated peptides suggests that imprinted hydrophilic MF monolithic columns could useful in complex phosphoproteomic analysis, clinical diagnosis, and other areas of biological science.

Conflict of interest statement The authors declare no competing financial interest.

Acknowledgments This work has been performed as part of the Marie Curie ITN project “Robust affinity materials for applications in proteomics and diagnostics” project PEPMIP, supported by European Commission grant PITN-GA-2010-569 264699. The authors are indebted to Julien Courtois, Wen Jiang, Celina Wierzbicka, Phuoc Dinh, Peter A. G. Cormack, Sudhirkumar Shinde, Michał Szumski, and Sebastiaan Eeltink for discussions throughout this work. The authors also deeply acknowledge the technical assistance of Cheng Choo Lee, Beatriz Galindo Prieto, Tobias Sparrman, Andras Gorzsas, Andrey Shchukarev, Robin Sandström, and Thomas Wågberg.

Supporting Information Additional reagents and synthesis description for templates; experimental details, background information, and results for the characterization experiments in Figures S-1 to S-10 and Tables S-1 and S-2.


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Legends to Figures Scheme 1. Polymerization of melamine and formaldehyde in two stages; (a) methylolation of melamine by formaldehyde in bulk aqueous phase at a weakly alkaline pH to form prepolymers, and (b) the curing which took place after the prepolymer had been mixed with diluent and porogens (and in the case of MIPs also templates) and transferred to the capillaries. Figure 1. A “biplot” of scores and loadings of a Projections against Latent Structures (PLS) model using all variables and responses. Monoliths are shown as brown hexagons, synthesis factors as green circles, and responses as blue circles. Factor abbreviations: F:M Ratio, formaldehyde:melamine molar ratio; PorType & PorAmt, type and amount of coporogen; DilVol, volume of acetonitrile; SurfAmt, amount of block copolymer surfactant. Block copolymer surfactant types are indicated by their designations. See Table 1 for a legend to response variable abbreviations. Figure 2. Elution profiles of 35 nL of Fmoc-pS-OEt and Fmoc-pY-OEt templates at 0.04 mM concentration dissolved in acetonitrile, injected on a MF34 NIP and on pS (a) and pY (b) MIPs based on MF34 with varying M:T ratio using ACN/H2O (0.1% TFA) (95/5) as eluent at a flow rate of 4 µL/min. UV detection at 254 nm. The inserts show the imprinting factor as a function of the M:T ratio. Figure 3. Extracted ion chromatograms from injections of a mixture of Fmoc-S-OH, Fmoc-pS-OH, Fmoc-Y-OH, and Fmoc-pY-OH, on (a) the NIP, (b) the pS MIP and (c) the pY MIP based on the MF34 recipe. The gradient profiles as percent water (v/v) in the aqueous acetonitrile acidic eluent are overlaid as dashed lines in the extracted ion chromatograms. See experimental section for eluent compositions. Figure 4. Extracted ion chromatograms from injection of Angiotensin II without (DRVYIHPF) and with (DRVpYIHPF) phosphorylation on Tyr, and a homolog (DRVpSIHPF) with pS substituted for Y on (a) the MF34 NIP, (b) the pS MIP and (c) the pY MIP based on this recipe. For eluents, see Figure 3. *A single noise spike with side band artifacts caused by the Sawitsky-Golay smoothing filter.



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Table 1. Synthetic composition and pore characterization of MF monoliths. F/M Sample molar ratio MF01 MF02 MF03 MF04 MF05 MF06 MF07 MF08 MF09 MF10 MF11 MF12 MF13 MF14 MF15 MF16 MF17 MF18 MF19 MF20 MF21 MF22 MF23 MF24 MF25 MF26 MF27 MF28 MF29 MF30 MF31 MF32 MF33 MF34 MF35

0.5 1 1.5 3 6 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

ACN volume

Porogen

Surfactant

mL

Type, amount

Type, amount

0.3 PPG4000, 6 mg 0.3 PPG4000, 6 mg 0.3 PPG4000, 6 mg 0.3 PPG4000, 6 mg 0.3 PPG4000, 6 mg None added PPG4000, 6 mg 0.05 PPG4000, 6 mg 0.15 PPG4000, 6 mg 0.6 PPG4000, 6 mg 0.3 PPG425, 6 mg 0.3 PPG725, 6 mg 0.3 PPG1000, 6 mg 0.3 PPG2000, 6 mg 0.3 PPG2700, 6 mg 0.3 PPG3000, 6 mg 0.3 PEG200, 6 mg 0.3 PEG400, 6 mg 0.3 PEG600, 6 mg 0.3 PEG1000, 6 mg 0.3 PEG1500, 6 mg 0.3 PEG2000, 6 mg 0.3 PEG4000, 6 mg 0.3 PEG10000, 6 mg 0.3 PEG20000, 6 mg 0.3 None added 0.3 PPG4000, 3 mg 0.3 PPG4000, 9 mg 0.3 PPG4000, 12 mg 0.3 PPG4000, 3 mg 0.3 PPG4000, 3 mg 0.3 PPG4000, 3 mg 0.3 PPG4000, 3 mg 0.3 PPG4000, 3 mg 0.3 PPG4000, 3 mg 0.3 PPG4000, 3 mg

F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg F127, 3 mg L61, 3 mg L121, 3 mg P123, 3 mg None added F127, 6 mg F127, 9 mg F127, 12 mg

State a)

OG WF WG WR WR GR WG WR WF WF WF WG WR WR WR WF WF WF WG WG WG WR WR WR WG WG WR WR WR WR WR WR WR WR WR

SSAb)

TPV c)

AMS d)

MPS e)

m2/g

cm³/g

nm

µm

16.2 ± 0.16 17.8 ± 0.12 43.9 ± 0.22 48.6 ± 0.20 50.4 ± 0.16 5.7 ± 0.04 17.2 ± 0.07 32.3 ± 0.10 31.8 ± 0.15 82.6 ± 0.25 78.6 ± 0.28 60.5 ± 0.27 53.7 ± 0.21 53.1 ± 0.21 49.9 ± 0.20 82.0 ± 0.24 51.1 ± 0.19 46.8 ± 0.21 41.1 ± 0.14 29.4 ± 0.10 20.8 ± 0.09 17.6 ± 0.06 13.0 ± 0.05 12.9 ± 0.08 98.6 ± 0.37 71.8 ± 0.23 41.0 ± 0.20 23.3 ± 0.09 33.6 ± 0.12 34.3 ± 0.12 54.9 ± 0.25 27.4 ± 0.13 98.4 ± 0.25 131.8 ± 0.38 147.5 ± 0.39

0.487 0.276 0.429 0.255 0.108 0.021 0.055 0.099 0.168 0.156 0.158 0.153 0.141 0.169 0.193 0.186 0.122 0.129 0.121 0.092 0.067 0.085 0.070 0.073 0.173 0.227 0.242 0.137 0.094 0.134 0.184 0.119 0.272 0.304 0.216

85.8 61.0 38.8 20.8 8.4 15.5 12.5 12.2 21.3 7.3 8.3 10.0 10.5 12.5 15.3 8.8 9.6 11.1 12.0 12.4 12.8 19.4 21.3 22.8 6.9 12.6 23.5 23.9 11.3 15.5 13.1 17.6 10.9 9.3 5.8

3.47 3.04 2.37 2.24 2.06 4.23 3.09 2.51 2.52 1.59 1.74 1.89 1.91 1.94 2.10 1.73 1.97 2.30 2.38 2.73 2.89 3.07 3.53 3.68 1.55 1.85 2.44 2.85 2.47 2.48 1.90 2.74 1.56 1.29 0.80

a) Mechanical/physical properties and appearances of the recovered materials: OG = opaque glass-like solid; WF = white fluffy solid; WG = white granular solid; WR = white robust solid; GR = grey-white resilient solid; b) Specific surface area measured by nitrogen cryoadsorption according to the BET principle; c) Total pore volume determined by single point adsorption at P/P0 ≈ 0.992; d) Average mesopore size measured by nitrogen cryodesorption according to the BJH principle; e) Median macropore size (volume) measured by mercury intrusion porosimetry.


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B., Möbius, D., Miller, R., Surfactants: Chemistry, Interfacial Properties, Applications; Elsevier: Amsterdam, 2001, pp. 3−18. (48) Goodwin, J. W., Colloids and Interfaces with Surfactants and Polymers. 2nd Ed.; John Wiley & Sons: New York, 2009; pp. 49.



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for TOC only


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Scheme 1. Polymerization of melamine and formaldehyde in two stages; (a) methylolation of melamine by formaldehyde in bulk aqueous phase at a weakly alkaline pH to form prepolymers, and (b) the curing which took place after the prepolymer had been mixed with diluent and porogens (and in the case of MIPs also templates) and transferred to the capillaries. 65x50mm (300 x 300 DPI)



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Figure 1. A “biplot” of scores and loadings of a Projections against Latent Structures (PLS) model using all variables and responses. Monoliths are shown as brown hexagons, synthesis factors as green circles, and responses as blue circles. Factor abbreviations: F:M Ratio, formaldehyde:melamine molar ratio; PorType & PorAmt, type and amount of coporogen; DilVol, volume of acetonitrile; SurfAmt, amount of block copolymer surfactant. Block copolymer surfactant types are indicated by their designations. See Table 1 for a legend to response variable abbreviations. 84x84mm (300 x 300 DPI)


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Figure 2. Elution profiles of 35 nL of Fmoc-pS-OEt and Fmoc-pY-OEt templates at 0.04 mM concentration dissolved in acetonitrile, injected on a MF34 NIP and on pS (a) and pY (b) MIPs based on MF34 with varying M:T ratio using ACN/H2O (0.1% TFA) (95/5) as eluent at a flow rate of 4 µL/min. UV detection at 254 nm. The inserts show the imprinting factor as a function of the M:T ratio. 43x22mm (300 x 300 DPI)



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Figure 3. Extracted ion chromatograms from injections of a mixture of Fmoc-S-OH, Fmoc-pS-OH, Fmoc-YOH, and Fmoc-pY-OH, on (a) the NIP, (b) the pS MIP and (c) the pY MIP based on the MF34 recipe. The gradient profiles as percent water (v/v) in the aqueous acetonitrile acidic eluent are overlaid as dashed lines in the extracted ion chromatograms. See experimental section for eluent compositions. 94x104mm (300 x 300 DPI)


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Figure 4. Extracted ion chromatograms from injection of Angiotensin II without (DRVYIHPF) and with (DRVpYIHPF) phosphorylation on Tyr, and a homolog (DRVpSIHPF) with pS substituted for Y on (a) the MF34 NIP, (b) the pS MIP and (c) the pY MIP based on this recipe. For eluents, see Figure 3. *A single noise spike with side band artifacts caused by the Sawitsky-Golay smoothing filter. 71x60mm (300 x 300 DPI)


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