Phytosterol Recognition Via Rationally Designed Molecularly Imprinted Polymers

Journal of C Carbon Research

Article Phytosterol Recognition via Rationally Designed Molecularly Imprinted Polymers

Lachlan J. Schwarz †, Brenda K. Y. Leung, Basil Danylec, Simon J. Harris, Reinhard I. Boysen ID and Milton T. W. Hearn * Centre for Green Chemistry and Australian Centre for Research on Separation Science, School of Chemistry, Monash University, Melbourne, VIC 3800, Australia; [email protected] (L.J.S.); [email protected] (B.K.Y.L.); [email protected] (B.D.); [email protected] (S.J.H.); [email protected] (R.I.B.) * Correspondence: [email protected]; Tel.: +61-3-9905-4547 † Current Address: School of Agricultural and Wine Sciences, Faculty of Science, Charles Sturt University, Wagga Wagga, NSW 2678, Australia.

Received: 29 December 2017; Accepted: 4 February 2018; Published: 12 February 2018

Abstract: Molecularly imprinted polymers (MIPs) prepared via a semi-covalent imprinting strategy using stigmasteryl methacrylate as a polymerisable template have been evaluated by static binding methods for their ability to selectively capture other valuable phytosterol targets, including campesterol and brassicasterol. Design criteria based on molecular modelling procedures and interaction energy calculations were employed to aid the selection of the co-monomer type, as well as the choice of co-monomer:template ratios for the formation of the pre-polymerisation complex. These novel hybrid semi-covalently imprinted polymers employed N,N0-dimethylacryl-amide (N,N0-DMAAM) as the functional co-monomer and displayed speciﬁc binding capacities in the range 5.2–5.9 mg sterol/g MIP resin. Their binding attributes and selectivities towards phytosterol compounds were signiﬁcantly different to the corresponding MIPs prepared via non-covalent procedures or when compared to non-imprinted polymers. Cross-reactivity studies using stigmasterol, ergosterol, cholesterol, campesterol, and brassicasterol as single analytes revealed the importance of the A-ring C-3-β-hydroxyl group and the orientational preferences of the D-ring alkyl chain structures in their interaction in the templated cavity with the N,N0-dimethylamide functional groups of the MIP. Finally, to obtain useful quantities of both campersterol and brassicasterol for these investigations, improved synthetic routes have been developed to permit the conversion of the more abundant, lower cost stigmasterol via a reactive aldehyde intermediate to these other sterols.

Keywords: phytosterols; molecular modelling; interaction energies; molecularly imprinted polymers; selectivity

1. Introduction Phytosterols are naturally occurring bioactive compounds, which have attracted wide-spread interest in recent years within the biomedical research and pharmaceutical/nutraceutical communities due largely for their ability to (i) lower blood serum levels of LDL-cholesterol; (ii) inhibit oxidative stress and cellular deterioration; (iii) act as anti-inﬂammatory compounds; and (iv) for their role as steroidal intermediates and precursors in the production of several pharmaceuticals [1–3]. Although feasible, the production of phytosterols by total chemical synthesis methods is both technically challenging and expensive. Further, current methods for sourcing these compounds from plant extracts typically involve multi-step procedures based on a combination of distillation, liquid-liquid or supercritical ﬂuid extractions, and crystallization methods. Often, these procedures lead to products

C 2018, 4, 13; doi:10.3390/c4010013 www.mdpi.com/journal/carbon C 2018, 4, 13 2 of 13 in relatively low yields or poor quality [2,4–6]. An opportunity exists to explore alternative, more cost-effective technologies of lower energy consumption for the separation and puriﬁcation of these valuable compounds from natural sources, including food and oil processing waste streams. In this context, molecularly imprinted polymers (MIPs) represent an attractive option for the isolation of phytosterols, since these functional polymers have been employed for other classes of compounds as re-usable adsorbents in solid-phase tank batch extraction procedures or packed-bed chromatographic formats [7–9]. MIPs are porous materials, designed to contain complimentary ‘receptor-like’ binding sites capable of recognising a specific molecular template [10]. Such MIPs can exhibit a ‘molecular memory’ for the template or structurally related molecular analogues [11–13]. To date, most studies involving sterol-related molecularly imprinted polymers, usually involving non-covalent self-assembly approaches, have focused on cholesterol [14–23] or estradiol [24–27]. The focus of this investigation was to explore an alternative method for the generation of a covalently imprinted MIP based on the use of the cleavable stigmasterol methacrylate as template, in silico determination of optimal co-monomer-template ratios, batch binding studies with several sterol analogues, and molecular modeling of the binding interactions. The studies reported herein document an alternative way to design and develop phytosterol-selective MIP adsorbents with the potential for more generic applications.

2. Results and Discussion In this investigation, stigmasterol was utilised as a suitable molecular template due to its relative abundance and affordability, and also due to its capability to act as a common precursor for the synthesis of significantly more valuable sterols, such as campesterol and brassicasterol (Schemes1–3). Molecularly imprinted polymers are often prepared by a self-assembly approach, wherein the molecular template and functional monomers are allowed to self-assemble in solution prior to polymerisation. The advantage of this approach is its technical simplicity. However, when attempts to make stigmasterol-imprinted polymers via self-assembly approaches with N,N0-dimethylacrylamide (N,N0-DMAAM), methacrylic acid (MAA), or 4-vinylpyridine (4-VP) were initially pursued, upon evaluation of their binding properties, the derived polymers failed to demonstrate any significant imprinting effect for stigmasterol when compared to their corresponding non-imprinted control counterparts. The observed binding was largely associated with non-specific interactions. These studies confirmed an earlier report on the failure of the monomers MAA and 4-VP to generate by non-covalent self-assembly approaches stigmasterol-imprinted polymers with acceptable imprinting factors [28]. This outcome can be attributed to an inability to form a stable pre-polymerisation complex, involving the functional monomers and the sterol template, owing to the presence of only a single A-ring C-3β-hydroxyl group of the sterol capable of forming a hydrogen bond with, for example, the N,N0-dimethylamido group of the N,N0-DMAAM monomer. Thermal disruption of this hydrogen bond during polymerisation would account for the low specific binding levels of the derived polymer. Analogous experiences have been reported for cholesterol as the template with self-assembling imprinting procedures [17,20]. Therefore, an alternative strategy was adopted based on the use of a semi-covalent MIP approach with the functional monomer and template combined via a formal covalent linkage.

2.1. Selection of the Molecular Template Upon polymerisation with N,N0-dimethylacrylamide (N,N0-DMAAM) as the co-monomer, the molecular template, stigmasteryl-3-O-methacrylate, generated a MIP which can be hydrolyzed to afford a complimentary cavity housing carboxylic acid and N,N0-dimethylamido group functionalities as hydrogen bonding recognition units [17,28]. On the basis of molecular modelling investigations, it was anticipated that this MIP would be able to recognize not only stigmasterol but also the more valuable campesterol and brassicasterol. It was thus envisaged that the use of the more abundant and C 2018, 4, 13 3 of 13 affordable stigmasterol would lead to a more cost-effective development of MIP technologies capable of recognizing similar steryl core backbones for both analytical and industrial-scale applications. C 2018, 4, x FOR PEER REVIEW 3 of 13 2.2. Molecularly Imprinted Polymer Design technologies capable of recognizing similar steryl core backbones for both analytical and industrial- Forscale MIPs applications. prepared by non-covalent methods, the design procedures typically involve in silico modelling of the self-assembled pre-polymerisation complexes, which aids in the selection of suitable 2.2. Molecularly Imprinted Polymer Design functional monomers and the determination of appropriate template to monomer ratios [13,29]. In this current investigation,For MIPs prepared this modelling by non-covalent approach methods, has been the design adapted procedures in a novel typically three-stage involve manner in silico to suit the designmodelling of hybrid of the self MIPs,-assembled accommodating pre-polymerisation imprinted complexes, sites prepared which viaaids a in semi-covalent the selection of methodology suitable (i.e., usingfunctional a polymerisable monomers and template) the determination in combination of appropriate with atemplate co-monomer to monomer that canratios interact [13,29] through. In this current investigation, this modelling approach has been adapted in a novel three-stage manner both hydrogen bonding and other non-covalent processes. As a first step in this in silico modelling to suit the design of hybrid MIPs, accommodating imprinted sites prepared via a semi-covalent procedure, the impact of solvent was excluded in order to reduce the complexity of the system methodology (i.e., using a polymerisable template) in combination with a co-monomer that can and theinteract computation through both time. hydrogen This bonding step was and achieved other non by-covalent conducting processes. in silicoAs a first modelling step in thi titrationss in of thesilico stigmasteryl modelling procedure, methacrylate the impact functional of solvent template was excluded with increasing in order to equivalentsreduce the complexity of the different of co-monomersthe system in and order the to computation form ‘virtual’ time. pre-polymerisation This step was achieved complexes by conducting using semi-empirical in silico modelling equilibrium geometrytitrations calculations of the stigmasteryl based on a methacrylate parameterized functional model te numbermplate with 3 (PM3) increasing force field equivalents to derive of the heat of formationdifferent valuesco-monomers (∆Hf) andin order the to energy form ‘virtual’ of interaction pre-polymerisation (∆Ei) of the template,complexes theusing co-monomer semi-empirical clusters, and theequilibrium template:co-monomer geometry calculations cluster based complexes. on a parameterized As a second model step, numberall polymerisable 3 (PM3) force atomsfield to were thenderive ‘frozen’ the to heat mimic of formation polymerisation values (ΔH usingf) and the the Merckenergy Molecularof interaction Force (ΔEi) Fieldof the (MMFF)template, procedures,the co- monomer clusters, and the template:co-monomer cluster complexes. As a second step, all permitting any free monomer units to be excluded but including solvent and performing a ‘virtual’ polymerisable atoms were then ‘frozen’ to mimic polymerisation using the Merck Molecular Force base hydrolysis of the ester linkage, whereby the covalent bond between the steryl moiety and Field (MMFF) procedures, permitting any free monomer units to be excluded but including solvent the methacrylateand performing was a ‘virtual’ cleaved. base This hydrolysis step resulted of the ester in thelinkage, ester whereby being replacedthe covalent by bond a carboxylic between acid functionality.the steryl moiety Finally, and as the a methacrylate third step, was stigmasterol cleaved. This was step then resulted re-introduced in the ester being into replaced the cavity by and semi-empiricala carboxylic PM3 acid equilibriumfunctionality. geometryFinally, as calculationsa third step, stigmasterol performed again,was then including re-introduced all of into the solvatedthe ‘frozen’cavity polymethacrylic and semi-empirical acid PM3 units. equilibrium This rebinding geometry of stigmasterolcalculations performed to these ‘virtual’ again, including cavities all (Figure of 1) revealedthe s thatolvated the ‘frozen’ use of 2 polymethacrylic mole equivalents acid of units. the functional This rebinding co-monomer of stigmasterolN,N0-DMAAM to these ‘virtual’ resulted in cavities (Figure 1) revealed that the use of 2 mole equivalents of the functional co-monomer N,N′- silico in the thermodynamically most stable association, with a ∆Ei of −37.1 KJ/mol. For comparative purposes,DMAAM a non-covalent resulted in silico titration in the (at thermodynamically 2 monomer equivalents) most stable was alsoassociation, conducted with and a ΔE revealedi of −37.1 much KJ/mol. For comparative purposes, a non-covalent titration (at 2 monomer equivalents) was also smaller in silico-generated interaction energies (∆Ei values < −5.0 kJ/mol) for the same monomer conducted and revealed much smaller in silico-generated interaction energies (ΔEi values < −5.0 type when employing a purely non-covalent self-assembly approach. In order to investigate the extent kJ/mol) for the same monomer type when employing a purely non-covalent self-assembly approach. to whichIn order non-specific to investigate binding the extent may contribute to which non to-specific the observed binding interaction may contribute energies, to the non-imprinted observed polymerinteraction systems energies, werealso non- modelled.imprinted polymer systems were also modelled.

Figure 1. Modelling titration simulations for stigmasterol against three commercially available Figure 1. Modelling titration simulations for stigmasterol against three commercially available functional functional monomers, namely 4-vinylpyridine (4VP), methacrylic acid (MAA), and N,N′- monomers, namely 4-vinylpyridine (4VP), methacrylic acid (MAA), and N,N0-dimethylacrylamide dimethylacrylamide (N,N′-DMAAM), with the change in interaction energy (ΔEi) calculated for N N0 E ( , stigmast-DMAAM),erol within with the pre change-polymerisation in interaction complex energy leading (∆ toi) calculateda covalently for produced stigmasterol cavity based within the pre-polymerisationon the deployment complex of 1, leading 2, or 3 to free a covalently functional produced monomer cavity mole based equivalents. on the Alsodeployment shown isof the1, 2, or 3 free functionalcorresponding monomer simulated mole energy equivalents. (ΔEi) for Also the non shown-imprinted is the correspondingpolymer (NIP) simulatedwith 2 monomer energy mole (∆E i) for the non-imprintedequivalents of DMAAM. polymer (NIP) with 2 monomer mole equivalents of DMAAM. C 2018, 4, 13 4 of 13

In the case of the non-imprinted polymer (NIP), no pre-association between the template and the monomers occurs and no pre-polymerisation template–monomer cluster complexes exist, with the consequence that the co-monomer molecules, e.g., DMAAM, can only initially interact with each other, and only after being polymerised (frozen) can the derived NIP polymer interact with stigmasterol in a non-speciﬁc fashion. For this reason, the ∆Ei values for DMAAM in the MIP and NIP are different (Figure1). These results indicated a clear preference in terms of the energetics of the binding interactions with the N,N0-dimethylamino groups of the DMAAM monomers that were able to position around the template molecule, i.e., imprinted site, with the hybrid semi-covalent technique predicted to have the greatest differential between MIPs and NIPs at 2 mole equivalents of monomer.

2.3. Preparation of the Molecularly Imprinted Polymers To conﬁrm that the in silico modelling predictions associated with the use of a free functional co-monomer in combination with this semi-covalent imprinting approach resulted in a more effective MIP for the recognition of the stigmasterol, two MIP preparations, P1 and P2, respectively, were investigated, one with and the other without the functional co-monomer, N,N0-DMAAM (2 mmol), present but with the cross-linker, ethyleneglycol dimethacrylate (EGDMA) (10 mmol), retained (Table1).

Table 1. Summary of the molecularly imprinted polymer (MIP), P1 and P2, and non-imprinted polymer (NIP), P3–P5, synthetic preparations used in this investigation 1.

Polymer Template DMAAM (FM) MAA (FM) EGDMA (Cross-Linker) Porogen Code (mmol) (mmol) (mmol) (mmol) (6 mL)

P1 1 2 - 10 CHCl3 P2 1 - - 10 CHCl3 P3 - 2 - 10 CHCl3 P4 - - 2 10 CHCl3 P5 - - - 10 CHCl3 1 All polymerisations were initiated with 2,20-azobis(2-methyl-propionitrile) (AIBN) and performed at 60 ◦C for 24 h. FM: functional monomer.

Also, three different NIPs were prepared to examine the extent of non-specific binding that contributes to the overall interaction performance, and in particular the role that the functional and cross-linking monomers play with respect to these interactions. In each case, the non-imprinted polymers (NIPs) were prepared in the same manner as the imprinted polymers (MIPs) with the exception that the stigmasterol template (1 mmol) was not included. For comparison, the non-imprinted polymer, P5, using only the crosslinker EGDMA was also included. To accommodate the possibility that non-specific interactions occur due to residual co-monomer functionalities residing within the binding cavity of the MIP post template extraction, NIPs were prepared with randomly dispersed MAA functional monomers (P4) or N,N0-DMAAM monomers (P3). In this manner, it was possible to examine the extent to which these functionalities themselves in the absence of the stigmasterol imprinting template may contribute to non-specific adsorption.

2.4. Evaluation of the Molecularly Imprinted Polymers Evaluation of the binding of stigmasterol to each of the imprinted and non-imprinted polymers was conducted via static batch binding methods with stigmasterol (0.1 mM) dissolved in acetonitrile:water (9:1, v/v). These assays revealed that polymer P1, e.g., the hybrid ‘semi-covalent MIP’ containing N,N0-DMAAM co-monomers, exhibited under these experimental conditions a greater binding capacity (BP1, max − BP2, max ≈ 8%) (Figure2) with an amount bound for the P1 MIP of ca. 5.6 mg stigmasterol/g MIP polymer. Depending on the choice of co-monomer, differences in binding capacity and selectivity can be anticipated, which reﬂect subtle differences in the way the C 2018, 4, 13 5 of 13

C 2018, 4, x FOR PEER REVIEW 5 of 13 pre-polymerisation complex assembles with the same template. Clearly, not all of the observed ofbinding stigmasterol of stigmasterol to P1 or P2 to may P1 orhave P2 been may due have to beenthe imprinting due to the process imprinting per se as process the nonper-imprinted se as the polymersnon-imprinted P3 and polymers P5 also P3displayed and P5 also some displayed degree of some binding. degree The of binding. binding Theto P5 binding represents to P5 the represents extent tothe which extent the to cross which-linker, the cross-linker,poly-EGDMA, poly-EGDMA, alone influences alone the influences overall binding, the overall which, binding, as expec which,ted, wasas expected found to, was be minimal found to (< be5 minimal%). The binding (<5%). The to the binding NIP P3, to thewhich NIP was P3, whichgenerated was generatedfrom the N from,N′- DMAAMthe N,N0-DMAAM co-monomer co-monomer and the EGDMA and the EGDMAcross-linker cross-linker,, represented represented a ‘worst acase ‘worst scenario’ case scenario’ with respect with torespect non-specific to non-specific binding. binding. The binding The binding result result for P3 for incorporates P3 incorporates the the extent extent to to which which non non-specific-specific interactionsinteractions arise arise from from DMAAM DMAAM co co-monomer-monomer units units that that were were not not involved involved in in the the formation formation of of the the prepre-polymerization-polymerization complex complex and and therefo thereforere were were not not incorporated incorporated as part as part of the of binding the binding cavities, cavities, but insteadbut instead werewere dispersed dispersed randomly randomly throughout throughout the the polymer polymer matrix. matrix. Similarly, Similarly, the the binding binding to to P4 P4 providesprovides a a useful useful insight insight into into the the possible possible interactions interactions of of stigmasterol, stigmasterol, or or indeed indeed other other sterol sterol analy analytes,tes, withwith randomly randomly dispersed dispersed MAA MAA functionalities functionalities rather rather than than those those solely solely localized localized within within binding binding cavities. cavities.Although Although interactions interactions of stigmasterol of stigmasterol with MAA with functionalities MAA functionalities localized localized within bindingwithin binding cavities cavitieswould bewould extremely be extremely unlikely unlikely due to the due nature to the of nature the semi-covalent of the semi imprinting-covalent imprinting protocol employed protocol employedin this investigation, in this investigation, it is interesting it is tointeresting note that to the note selective that the binding selective (BMIP binding− BNIP ()B wasMIP − statistically BNIP) was statisticallythe same, regardless the same, regardless of whether of the whether hybrid the semi-covalent hybrid semi- imprintingcovalent imprinting protocol protocol or a semi-covalent or a semi- 0 covalentimprinting impri protocolnting protocol in the absence in the absence of N,N -DMAAMof N,N′-DMAAM was employed was employed with 17% with (B 17%P1, max (BP1−, maxBP3, − maxBP3,) maxand) and 18% 18% (BP2, (B maxP2, max− −B P5,BP5, max max)) selectiveselective binding,binding, respectively. Since Since these these values values are are based based on on ‘the ‘the worstworst-case-case scenario’ scenario’ with with respect respect to to non non-specific-specific binding binding ( (i.e.,i.e., bin bindingding of of the the template template to to the the NIP NIP P3), P3), thethe actual actual selective selective binding binding and and associated associated interaction interaction affinity affinity of of the the ‘hybrid’ ‘hybrid’ adsorbent adsorbent made made by by the the semisemi-covalent-covalent approach approach will will be be higher. higher.

FigureFigure 2. StigmasterolStigmasterol binding binding evaluation evaluation for forthe imprintedthe imprinted (P1 and (P1 P2, and black) P2, andblack non-imprinted) and non- imprintedpolymers (P3–P5, polymers grey) under (P3–P5 static, grey batch) binding under conditions. static batch Binding binding conditions conditions. are described Binding in the conditionsMaterials and are Methods. described The in error the Materials bars indicate and the Methods. standard The error error of the bars mean. indicate the standard error of the mean. The ability of the stigmasterol-templated MIPs to recognize other 3β-hydroxy sterol analogues,The ability such of as the ergosterol stigmasterol (a fungal-templated sterol), MIPs cholesterol to recognize (an animal other 3 derivedβ-hydroxy sterol), sterol campesterol, analogues, suchand brassicasterol as ergosterol, was (a fungal documented sterol), via cholesterol single analyte (an animal binding derived and multiple sterol), analyte campesterol competitive, and brassicasterolbinding studies., was Since, documented in all cases, via single similar analyte binding binding patterns and weremultiple obtained analyte with competitive a higher binding binding studies.capacity Since for the, in P1all MIPcases, compared similar binding to the P2patterns MIP, only were the obtained data for with the a P1 higher MIP havebinding been capacity discussed for thebelow. P1 MIP The compared single analyte to the binding P2 MIP, studies only revealedthe data for that the the P1 P1 MIP MIP have preferentially been discussed ‘recognized’ below. eachThe singleof these analyte sterols binding with respect studies to the revealed non-imprinted that the P1 polymer MIP preferentially P3 with imprinting ‘recognized’ factors each of 4.0, of 2.1, these 5.1, sterols10.5, and with 7.1, respect respectively to the (Figurenon-imprinted3), concordant polymer with P3 the with A-ring imprinting C-3 β-hydroxyl factors of group 4.0, 2.1, acting 5.1, 10.5 as a, headand 7.1,group respectively for binding (Figure via hydrogen 3), concordant bond interactions with the A- withring theC-3 carboxylβ-hydroxyl and groupN,N0 -dimethylamidoacting as a head groupsgroup forin thebinding imprinted via hydrogen cavity of bo thend MIP. interactions with the carboxyl and N,N′-dimethylamido groups in the imprintedWhen cavity the molecular of the MIP. structures of these phytosterols are taken into consideration, a rationale emergesWhen with the regard molecular to the structures rank order of ofthese binding phytosterols afﬁnities observedare taken forinto these consideration, compounds a (Figurerationale4). emerges with regard to the rank order of binding affinities observed for these compounds (Figure 4). Although only small variations occur in the structures of these sterols, the most prominent are associated with (a) the level of olefinic unsaturation at the ∆22,23 position; (b) the pattern of side chain C 2018, 4, 13 6 of 13

Although only small variations occur in the structures of these sterols, the most prominent are associatedC 2018, 4 with, x FOR (a) PEER the REVIEW level of oleﬁnic unsaturation at the ∆22,23 position; (b) the pattern of6 side of 13 chain substitution/stereochemistry of the D-ring alkyl chain tail groups, and (c) the extent of puckering of substitutionC 2018, 4, x FOR/stereochemistry PEER REVIEW of the D-ring alkyl chain tail groups, and (c) the extent of puckering6 of of 13 the C and D sterol rings due to the presence of the B-ring ∆5,6 double bond. Greater binding differences the C and D sterol rings due to the presence of the B-ring ∆5,6 double bond. Greater binding differences substitution/stereochemistry of the D-ring alkyl chain tail groups, and (c) the extent of puckering of werewere apparent apparent between between brassicasterol brassicasterol and and stigmasterol, stigmasterol, which which structurally structurally differ differby replacement by replacement of the C24S and D sterol rings due to the presence of the B-ring24S ∆5,6 double bond. Greater binding differences of thethe C C24S--methylmethyl group group in brassicasterol in brassicasterol by a C by24S-ethyl a C group-ethyl in groupstigmasterol, in stigmasterol, than those that than occurred those that were apparent between brassicasterol and stigmasterol, which structurally differ by replacement of occurredbetween between campesterol campesterol and stigmasterol, and stigmasterol, which structurally which structurally differ due differ to the due replacement to the replacement of the C24S- of the the C24S-methyl group in brassicasterol by a C24S-ethyl group in stigmasterol, than those that occurred C24S-ethylethyl substituent substituent in instigmasterol stigmasterol by a by C24R a- Cmethyl24R-methyl group groupas well asas wellthe lack as theof the lack ∆22,23 of double the ∆22,23 bonddouble between campesterol and stigmasterol, which structurally differ due to the replacement of the C24S- bondin in campesterol. campesterol. Besides Besides documenting documenting the role the that role the that single, the common single, commonA-ring C-3 A-ringβ-hydroxyl C-3 βgroup-hydroxyl ethyl substituent in stigmasterol by a C24R-methyl group as well as the lack of the ∆22,23 double bond plays in the molecular recognition of these phytosterols by the semi-covalently imprinted MIP, these groupin plays campesterol. in the molecular Besides documenting recognition the of role these that phytosterols the single, common by the semi-covalentlyA-ring C-3β-hydroxyl imprinted group MIP, results confirm the importance of shape determinants, mediated by van der Waals and Lifshitz force theseplays results in the conﬁrm molecular the importancerecognition of of these shape phytosterols determinants, by the mediated semi-covalently by van imprinted der Waals MIP, and these Lifshitz interactions, in determining the overall selectivity of these molecular imprinted polymers with these forceresults interactions, confirm in the determining importance of the shape overall determinants, selectivity mediated of these by molecular van der Waals imprinted and Lifshitz polymers force with sterols. theseinteractions, sterols. in determining the overall selectivity of these molecular imprinted polymers with these sterols.

Figure 3. Static cross-reactivity binding evaluation based on single analyte binding studies with the Figure 3. Static cross-reactivity binding evaluation based on single analyte binding studies with the semi-covalentlysemi-covalently imprinted imprinted stigmasterol stigmasterol MIP polymer polymer P1 P1 and and the the NIP NIP polymer polymer P3 with P3 withthe sterols the sterols Figure 3. Static cross-reactivity binding evaluation based on single analyte binding studies with the stigmasterol,stigmasterol, ergosterol, ergosterol, cholesterol, cholesterol, campesterol, campesterol and, and brassicasterol. brassicasterol. Binding Binding conditions conditions are described are semi-covalently imprinted stigmasterol MIP polymer P1 and the NIP polymer P3 with the sterols in thedescribed Materials in the and Materials Methods. and The Methods. error bars The error represent bars represent the standard the standard error of error the mean.of the mean. stigmasterol, ergosterol, cholesterol, campesterol, and brassicasterol. Binding conditions are described in the Materials and Methods. The error bars represent the standard error of the mean. 1 . (c) top 21. . view (c) top 2. 3. view 3. 4.

4. (d) Offset 5. top view (d) Offset 5. top view

(a) side view (b) end view

Figure (4.a )Density side view functional equilibrium(b) end geometry view models of five different sterol compounds shown in (a) side view; (b) end view; (c) top view; and (d) an offset top view, where the sterol compounds Figure 4. Density functional equilibrium geometry models of five different sterol compounds shown Figureare: 4. 1.Density stigmasterol, functional 2. ergosterol, equilibrium 3. cholesterol, geometry 4. campestero models ofl, and ﬁve 5. different brassicasterol sterol, illustrating compounds their shown in (a) side view; (b) end view; (c) top view; and (d) an offset top view, where the sterol compounds in (astructural) side view; similarities (b) end and view; differences. (c) top view; All models and ( dwere) an generated offset top using view, SPARTAN where the ‘10 sterol for Windows compounds are: 1. stigmasterol, 2. ergosterol, 3. cholesterol, 4. campesterol, and 5. brassicasterol, illustrating their with the density functional equilibrium geometry calculation at the 3–21 G level. are: 1.structural stigmasterol, similarities 2. ergosterol, and differences. 3. cholesterol, All models 4. campesterol,were generated and using 5. brassicasterol,SPARTAN ‘10 for illustrating Windows their structuralwith the similarities density functional and differences. equilibrium All geometry models calculation were generated at the 3 using–21 G SPARTANlevel. ‘10 for Windows with the density functional equilibrium geometry calculation at the 3–21 G level. C 2018, 4, 13 7 of 13

3. Materials and Methods

3.1. LC Equipment and Methods An Agilent Technologies 1100 LC system (Waldbronn, Germany), consisting of a binary pump with vacuum degasser, an auto-sampler with a 900 µL sample loop, and a thermostated column compartment, was employed for the analysis of the different sterol samples with an Agilent Technologies 1100 Series UV-Vis G1315C diode array detector SL (80 Hz) set to the absorbance wavelengths of 210, 280, and 330 nm. Injected samples (5 µL) were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Zorbax Eclipse XDB-C18 double end-capped column (4.6 × 150 mm, 5 µm particle size) (Agilent Technologies, Melbourne, Australia) at 23 ◦C. Isocratic elution conditions were used with a mobile phase generated from a 5:95 ratio of solvent A (0.1% (v/v) AcOH in H2O) and solvent B (0.1% (v/v) AcOH in EtOH), respectively, at a ﬂow rate of 0.5 mL min−1.

3.2. Molecular Modelling All in silico modelling calculations were conducted using the Spartan ‘10 for Windows version 1.1.0 software package (Wavefunction, Inc., Irvine, CA, USA) on a Pentium IV 2.0 GHz computer. Modelling procedures were based on our previously described methods [29], whereby semi-empirical equilibrium geometry level theory was applied using a PM3 force field to calculate the heat of formation values (∆Hf) and the energy of interaction (∆Ei) for the template, the monomer clusters, and the template:monomer cluster complexes in the gas phase, initially without consideration of solvent effects. The impact of these solvent effects was then determined by inserting the template file into each monomer cluster file, without any pre-defined orientation imposed upon either the template or the monomer cluster, using an iterative approach. Moreover, using methodologies established in our previous modelling titration experiments, the template molecule stigmasterol was titrated in silico with the three commercially available functional monomers (FM) of different characteristics, namely, methacrylic acid (MAA), 4-vinylpyridine (4VP), and N,N0-dimethylacrylamide (N,N0-DMAAM). A minimum of three iterations were carried out to derive the average net interaction energy (∆Ei) for the template:monomer cluster complex, as determined from Equation (1):

∆Ei = ∆Hf_template:monomer cluster complex − (∆Hf_template + ∆Hf_monomer cluster). (1)

Modelling of the stigmasteryl methacrylate cavities (containing multiple equivalents of the free FM) post hydrolysis (i.e., after removal of the template) was achieved using the following procedure. Initially, as a ﬁrst step, the stigmasteryl methacrylate template was modelled (using semi-empirical PM3 equilibrium geometry calculations with the MMFF) against increasing molar equivalents of the free functional monomer, e.g., MAA. The polymerisable groups were then ‘frozen’ (i.e., ﬁxed in space with regard to all other adjacent atoms) to simulate the process of polymerisation, and semi-empirical PM3 equilibrium geometry calculations conducted again employing MMFF procedures. A virtual in silico ‘base hydrolysis’ was then performed, whereby the steryl portion of the template was removed, leaving the polymethacrylic acid functionalities (and additional free monomer units) within the cavity. As a second step, the polymerised methacrylic acid units were also ‘frozen’ to simulate the process of polymerisation using the MMFF procedures, permitting the free monomer units to be excluded and solvent included. Finally, as a third step, stigmasterol was then re-introduced into the cavity and semi-empirical PM3 equilibrium geometry calculations performed again, including all of the ‘frozen’ polymethacrylic acid atoms. The relevant average net interaction energy (∆Ei) for these MIP systems was determined from Equation (2):

∆Ei = ∆Hf_Binding complex − (∆Hf_target + ∆Hf_cavity) (2) C 2018, 4, 13 8 of 13

where ∆Hf_Binding complex equates to the ∆Hf of the complex between the cavity and the target (i.e., stigmasterol); ∆Hf_target equates to the ∆Hf of the target; and ∆Hf_cavity equates to the ∆Hf of the cavityC 2018, generated4, x FOR PEER by REVIEW modeling the template (stigmasteryl methacrylate) in the presence of8 of the 13 free co-monomer/s (e.g., N,N0-DMAAM). Angenerated additional by modeling set of modelling the template experiments (stigmasteryl related methacrylate) to non-specific in the binding presence of of the the template free co- was also undertakenmonomer/s (e.g., to simulate N,N′-DMAAM). the interaction with the non-imprinted polymer (NIP). This task was An additional set of modelling experiments related to non-specific binding of the template was performed by modeling the monomer cluster (as done for the previous MIP modeling titrations) but in also undertaken to simulate the interaction with the non-imprinted polymer (NIP). This task was the absenceperformed of theby modeling template the with monomer the atoms cluster of the(as done monomers, for the previous e.g., acrylic MIP modeling acid groups, titrations) subsequently but ‘frozen’in the in placeabsence to of simulate the template the process with the of atoms polymerisation. of the monomers, The e.g., template acrylic file acid was groups, then subsequently inserted into the monomer‘frozen’ cluster in place file to and simulate the semi-empirical the process of PM3polymerisation. equilibrium The geometry template calculationsfile was then againinserted carried into out startingthe from monome MMFF.r cluster The file∆E i andvalues the for semi this-empirical NIP systems PM3 equilibrium were then calculated geometry calculations based on procedures again similarcarried to those out starting described from above MMFF. for The the Δ MIP.Ei values for this NIP systems were then calculated based on procedures similar to those described above for the MIP. 3.3. Synthesis of Molecular Targets and Templates 3.3. Synthesis of Molecular Targets and Templates Cholesterol (Sigma-Aldrich, Sydney, Australia, 95% purity), ergosterol (Fluka Chemicals, Melbourne,Cholesterol Australia, (Sigma >95%-Aldrich, purity), Sydney, and stigmasterol Australia, (Sigma 95% purity), Aldrich, ergosterol Sydney, (Fluka Australia Chemicals, and Acros Melbourne, Australia, >95% purity), and stigmasterol (Sigma Aldrich, Sydney, Australia and Acros Organics, NJ, USA, 95% purity) were obtained from the vendors and their purity confirmed Organics, NJ, USA, 95% purity) were obtained from the vendors and their purity confirmed by RP- by RP-HPLCHPLC methods. methods. Stigmasterol Stigmasterol was converted was in converted high yield to in the high reactive yield aldehyde to the (20 reactiveS)-20-formyl aldehyde- (20S)-20-formyl-66β-methoxy-3α,5β-methoxy-3-cyclo-5α-pregnaneα,5-cyclo-5 (4) α(Scheme-pregnane 1). (4) (Scheme1).

Scheme 1. Synthesis of (20S)-20-formyl-6β-methoxy-3α,5-cyclo-5α-pregnane (4) from stigmasterol. Scheme 1. Synthesis of (20S)-20-formyl-6β-methoxy-3α,5-cyclo-5α-pregnane (4) from stigmasterol. Reagents, conditions, and yield: (i) TsCl, pyridine, 22 °C, 72 h, 96%; (ii) MeOH, pyridine, reflux, 6 h, Reagents, conditions, and yield: (i) TsCl, pyridine, 22 ◦C, 72 h, 96%; (ii) MeOH, pyridine, reﬂux, 6 h, 69%; (iii) (a) O3, DCM/pyridine, −78 °C; (b) Zn, AcOH, 22 °C, 3 h, ~100% crude. Nomenclature: − ◦ ◦ 69%;stigmasteryl (iii) (a) O3, tosylate DCM/pyridine, (1), (22E)-6β78-methoxyC; (b)-3,5α Zn,-cyclo AcOH,-5α-stigmasta 22 C, 3- h,22- ~100%ene (stigmasterol crude. Nomenclature: i-methyl stigmasterylether) (2), tosylate and 3-O- (methylstigmasterol1), (22E)-6β-methoxy-3,5 (3). α-cyclo-5α-stigmasta-22-ene (stigmasterol i-methyl ether) (2), and 3-O-methylstigmasterol (3). 3.4. Synthesis of Campesterol and Brassicasterol Targets 3.4. SynthesisThe ofaldehyde Campesterol (4) was and then Brassicasterol used as the Targets synthetic precursor to prepare both campesterol (12) The(Scheme aldehyde 2) and ( 4 brassicasterol) was then used (17) (Scheme as the synthetic 3) via strategies precursor similar to prepare to those both reported campesterol for the (12) preparation of cholesterol and cholestadiene/cholestatriene derivatives [30–34]. Further experimental (Scheme2) and brassicasterol ( 17) (Scheme3) via strategies similar to those reported for the preparation details related to the synthesis of the reactive intermediate (4) and its conversion to campersterol (12) of cholesterol and cholestadiene/cholestatriene derivatives [30–34]. Further experimental details and brassicasterol (17) are included in the Supplementary Materials section, please see S1: General relatedexperimental to the synthesis procedures of and the S2: reactive Experimental intermediate details. (4) and its conversion to campersterol (12) and brassicasterol (17) are included in the Supplementary Materials section, please see S1: General experimental procedures and S2: Experimental details. C 2018, 4, 13 9 of 13

C 2018, 4, x FOR PEER REVIEW 9 of 13 C 2018, 4, x FOR PEER REVIEW 9 of 13

Scheme 2. Synthesis of campesterol (12) from (20S)-20-formyl-6β-methoxy-3α,5-cyclo-5α-pregnane β α α Scheme(4 2.). Synthesis Reagents, conditions of campesterol, and yield: (12) from(i) n-propynylmagnesium (20S)-20-formyl-6 bromide,-methoxy-3 THF, ,5-cyclo-5 0 °C, 1 h, 17%;-pregnane (ii) (4). Reagents, conditions, and yield: (i) n-propynylmagnesium bromide, THF, 0 ◦C, 1 h, 17%; (ii) Pd/CaCO , SchemePd/CaCO 2. Synthesis3, Pb, quinoline, of campesterol EtOAc, H 2(, 123 h,) from 98%; (20(iii)S triethyl)-20-formyl-orthopropion-6β-methoxyate, -propionic3α,5-cyclo acid,-5α- xylene,pregnane 3 Pb, quinoline,(4).reflux, Reagents, 1 h, EtOAc, ~100% conditions crude; H2,, 3 (iv)and h, 10% 98%; yield: Pd/C, (iii) (i) EtOAc,n triethyl-orthopropionate,-propynylmagnesium H2, 20 h, 96%; (v) LiAlH bromide,4,propionic THF, THF, 22 °C, 0 °C, acid,2 h, 172%; h, xylene, 17%;(vi) a. (ii) reﬂux, MsCl, pyridine, 22 °C, 2 h, 94%, b. LiAlH4, THF, 22 °C, 24 h, 94%; (vii) p-TsOH, dioxane/H◦ 2O, reflux, 1 h, ~100%Pd/CaCO crude;3, Pb, (iv)quinoline, 10% Pd/C,EtOAc, EtOAc,H2, 3 h, 98%; H2, 20(iii) h, triethyl 96%;-orthopropion (v) LiAlH4,ate, THF, propionic 22 C, acid, 2 h, 72%;xylene, (vi) a. 1 h, 87%. Nomenclatu◦ re: (22R)-6β-methoxy-3α,5-cyclo-26,27◦ -dinor-5α-cholest-23-yn-22-ol (5), (22S)- MsCl,reflux, pyridine, 1 h, ~100% 22 C,crude; 2 h, (iv) 94%, 10%b. Pd/C, LiAlH EtOAc,4, THF, H2, 20 22 h, 96%;C, 24 (v)h, LiAlH 94%;4, THF, (vii) 22p-TsOH, °C, 2 h, 72%; dioxane/H (vi) a. 2O, 6β-methoxy-3α,5-cyclo-26,27-dinor-5α-cholest-23-yn-22-ol (6), (22S,23Z)-6β-methoxy-3α,5-cyclo- reﬂux,MsCl, 1 h, pyridine, 87%. Nomenclature: 22 °C, 2 h, 94%, b. (22 LiAlHR)-64β, -methoxy-3THF, 22 °C, 24α,5-cyclo-26,27-dinor-5 h, 94%; (vii) p-TsOH, dioxane/Hα-cholest-23-yn-22-ol2O, reflux, 26,27-dinor-5α-cholest-23-en-22-ol (7), (22E,24R)-ethyl-6β-methoxy-24-methyl-3α,5-cyclo-5α-cholest- (5), (221 h,S)-6 87%.β-methoxy-3 Nomenclatuαre:,5-cyclo-26,27-dinor-5 (22R)-6β-methoxy-3α,5α-cholest-23-yn-22-ol-cyclo-26,27-dinor-5α-cholest (6), (22-23S-,23yn-Z22)-6-olβ (-methoxy-35), (22S)- α, 22-en-26-oate (8), (24R)-ethyl-6β-methoxy-24-methyl-3α,5-cyclo-5α-cholestan-26-oate (9), (24R)-6β- 6β-methoxy-3α,5-cyclo-26,27-dinor-5α-cholest-23-yn-22-ol (6), (22S,23Z)-6β-methoxy-3α,5-cyclo- 5-cyclo-26,27-dinor-5methoxy-24-methylα-cholest-23-en-22-ol-3α,5-cyclo-5α-cholestan (7),-26 (22-olE (,2410),R and)-ethyl-6 (24R)-β6β-methoxy-24-methyl-3-methoxy-24-methyl-3α,5-αcyclo,5-cyclo-5- α- cholest-22-en-26-oate26,275α--dinorcholestane-5α-cholest (campesterol (8),-23 (24-enR- i22-)-ethyl-6methyl-ol (7), ether) (22β-methoxy-24-methyl-3E,24 (11R).) -ethyl-6β-methoxyα-24,5-cyclo-5-methyl-3α,5α-cholestan-26-oate-cyclo-5α-cholest- (9), (24R)-622β-en-methoxy-24-methyl-3-26-oate (8), (24R)-ethylα,5-cyclo-5-6β-methoxyα-cholestan-26-ol-24-methyl-3α,5-cyclo (10),- and5α-cholestan (24R)-6β-26-methoxy-24-methyl-3-oate (9), (24R)-6β- α, methoxy-24-methyl-3α,5-cyclo-5α-cholestan-26-ol (10), and (24R)-6β-methoxy-24-methyl-3α,5-cyclo- 5-cyclo-5α-cholestane (campesterol i-methyl ether) (11). 5α-cholestane (campesterol i-methyl ether) (11).

Scheme 3. Synthesis of brassicasterol (17) from (20S)-20-formyl-6β-methoxy-3α,5-cyclo-5α-pregnane (4). Reagents, conditions, and yield: (i) n-propynylmagnesium bromide, THF, 0 °C, 1 h, 13%; (ii)

Pd/CaCO3, Pb, quinoline, H2, EtOAc, 3 h, 99%; (iii) triethylorthopropionate, propionic acid, xylene,

reflux, 1 h, 100% crude; (iv) LiAlH4, THF, 22 °C, 2 h, 98%; (v) (a) MsCl, pyridine, 22 °C, 2 h, 100% Schemecrude, 3. (b) Synthesis LiAlH4, of THF, brassicasterol 22 °C, 21 ( h,17 100%) from crude; (20S)- 20 (vi)-formyl p-TsOH,-6β -methoxy dioxane/H-3α,52O, - reflux,cyclo- 5α 1 - h,pregnane 73%. Scheme 3. Synthesis of brassicasterol (17) from (20S)-20-formyl-6β-methoxy-3α,5-cyclo-5α-pregnane (4).Nomenclature: Reagents, conditions (22S)-6β, -andmethoxy yield:-3α,5 (i)- cyclon-propynylmagnesium-26,27-dinor-5α-cholest bromide,-23-yn- 22 THF,-ol ( 06 ), °C, (22 1R h,,23 Z 13%;)-6β - (ii) (4). Reagents, conditions, and yield: (i) n-propynylmagnesium bromide, THF, 0 ◦C, 1 h, 13%; Pd/CaCOmethoxy3, -Pb,3α,5 quinoline,-cyclo-26,27 H-dinor2, EtOAc,-5α-cholest 3 h, 99%;-23-en (iii)-22 -trieol (thylorthopropionate,13), (22E,24S)-ethyl-6β propionic-methoxy- 24acid,-methyl xylene,- (ii) Pd/CaCO , Pb, quinoline, H , EtOAc, 3 h, 99%; (iii) triethylorthopropionate, propionic acid, reflux,3α,5 -1cyclo h,3 100%-5α-cholest crude;-22 (iv)-en -LiAlH26-2oate4, (THF,14), (22 22E °C,,24S 2)- 6βh, -98%;methoxy (v) -(a)24- methylMsCl, -pyridine,3α,5-cyclo -225α °C,-cholest 2 h,- 22100%- ◦ ◦ xylene,crude,en reflux,-26 (b)-ol 1LiAlH (15 h,), 100% and4, THF, (22 crude;E ,24 22 R °C,)-6β (iv) - 21methoxy LiAlH h, 100%-424, - THF,methyl crude; 22 -3α,5 (vi)C, -cyclop-TsOH, 2 h,-5α 98%;- cholest dioxane/H (v)-22 (a)-ene2O, MsCl, (brassicasterol reflux, pyridine, 1 h, 73%.i- 22 C, ◦ 2 h, 100%Nomenclature:methyl crude, ether) (b) ( (2216). LiAlHS )-6β-methoxy4, THF,-3α,5 22 -cycloC, 21-26,27 h,- 100%dinor-5α crude;-cholest (vi)-23-pyn-TsOH,-22-ol dioxane/H(6), (22R,232ZO,)-6β reflux,- 1 h,methoxy 73%.-3α,5 Nomenclature:-cyclo-26,27-dinor (22-5αS-)-6cholestβ-methoxy-3-23-en-22-αol,5-cyclo-26,27-dinor-5 (13), (22E,24S)-ethyl-6βα-cholest-23-yn-22-ol-methoxy-24-methyl- (6), (22R,233α,5Z)-6-cycloβ-methoxy-3-5α-cholestα-22,5-cyclo-26,27-dinor-5-en-26-oate (14), (22Eα,24-cholest-23-en-22-olS)-6β-methoxy-24-methyl (13),- (223α,5E,24-cycSlo)-ethyl-6-5α-cholestβ-methoxy--22- 24-methyl-3en-26-olα ,5-cyclo-5 (15), andα -cholest-22-en-26-oate (22E,24R)-6β-methoxy-24 (14-methyl), (22E,24-3α,5S)-6-cycloβ-methoxy-24-methyl-3-5α-cholest-22-ene (brassicasterolα,5-cyclo-5α -cholesti- -22-en-26-olmethyl ether) (15), ( and16). (22E,24R)-6β-methoxy-24-methyl-3α,5-cyclo-5α-cholest-22-ene (brassicasterol i-methyl ether) (16). C 2018, 4, 13 10 of 13

C 2018, 4, x FOR PEER REVIEW 10 of 13 3.5. Synthesis of the Polymerisable Molecular Template 3.5. Synthesis of the Polymerisable Molecular Template StigmasterolC 2018, 4, x FOR PEER was REVIEW converted to the polymerisable template stigmasteryl methacrylate10 of ( 1813 ) via a dicyclohexylcarbodiimideStigmasterol was converted (DCC)-mediated to the polymerisable reaction template [28] between stigmasteryl stigmasterol methacrylate and (18 methacrylic) via a acid3.5.dicyclohexylcarbodiimide (Scheme Synthesis4) basedof the Polymerisable on conditions (DCC) Molecular-mediated similar Template reaction to those [28] between used for stigmasterol generation and of methacrylic cholesterol-based acid (Scheme 4) based on conditions similar to those used for generation of cholesterol-based templates templatesStigmasterol [15]. Further was details converted of the to the synthesis polymerisable of (18) template are included stigmasteryl in the Supplementarymethacrylate (18) Materialsvia a [15]. Further details of the synthesis of (18) are included in the Supplementary Materials section, dicyclohexylcarbodiimide (DCC)-mediated reaction [28] between stigmasterol and methacrylic acid section,Figures Figures S1 and S1 S2 and. S2. (Scheme 4) based on conditions similar to those used for generation of cholesterol-based templates [15]. Further details of the synthesis of (18) are included in the Supplementary Materials section, Figures S1 and S2.

Scheme 4. The preparation of the stigmasteryl methacrylate (18). Reagents, conditions, and yield: (i) Scheme 4. The preparation of the stigmasteryl methacrylate (18). Reagents, conditions, and yield: DCC, 4-(dimethylamino)pyridine (DMAP), dichloromethane (DCM), room temp, 18 h, 74%. (i) DCC, 4-(dimethylamino)pyridine (DMAP), dichloromethane (DCM), room temp, 18 h, 74%. 3.6. MIPScheme Preparation 4. The preparation of the stigmasteryl methacrylate (18). Reagents, conditions, and yield: (i) 3.6. MIP PreparationDCC, 4-(dimethylamino)pyridine (DMAP), dichloromethane (DCM), room temp, 18 h, 74%. Stigmasteryl methacrylate (0.480 g, 1 mmol), N,N′-DMAAM (206 μL, 2 mmol), and CHCl3 (6 mL) 0 Stigmasteryl3.6.were MIP placed Preparation in methacrylate 15 mL test tubes (0.480 fitted g, with 1 mmol), a subaN seal.,N -DMAAM The mixture (206 wasµ L,cooled 2 mmol), on ice, and purged CHCl with3 (6 mL) wereN placed2(gas) for in 2 15min mL, and test left tubes on ice fitted for a withfurther a suba28 min. seal. Ethyleneglycol The mixture dimethacrylate was cooled on(EGDMA) ice, purged (1.89 with Stigmasteryl methacrylate (0.480 g, 1 mmol), N,N′-DMAAM (206 μL, 2 mmol), and CHCl3 (6 mL) mL, 10 mmol) and 2,2′-azo-bis(2-methyl-propionitrile) (AIBN) (50 mg, 0.31 mmol) were then added. N2(gas)werefor placed 2 min, in and 15 leftmL ontest ice tubes for afitted further with 28 a min.suba Ethyleneglycolseal. The mixture dimethacrylate was cooled on ice, (EGDMA) purged with (1.89 mL, The mixture was0 again purged with N2(gas) for a further 5 min. This pre-polymerisation mixture was 10 mmol)N2(gas) for and 2 min 2,2 ,-azo-bis(2-methyl-propionitrile) and left on ice for a further 28 min. (AIBN)Ethyleneglycol (50 mg, dimethacrylate 0.31 mmol) (EGDMA) were then (1.89 added. placed in a thermostated water bath at 60 °C for 24 h. A non-imprinted polymer (NIP) was prepared The mixturemL, 10 mmol)was again and 2,2 purged′-azo-bis(2 with-methyl N2(gas)-propionitrile)for a further (AIBN) 5 min. (50 This mg, pre-polymerisation0.31 mmol) were then mixture added. was in a similar manner with the exception ◦that the polymerisable template (stigmasteryl methacrylate) placedThe in mixture a thermostated was again water purged bath with at 60N2(gas)C for a 24 further h. A non-imprinted5 min. This pre- polymerpolymerisation (NIP) mixture was prepared was in was not included. Additional non-imprinted polymers were prepared without stigmasteryl placed in a thermostated water bath at 60 °C for 24 h. A non-imprinted polymer (NIP) was prepared a similarmethacrylate manner with or a theN,N exception′-DMAAM that functional the polymerisable monomer; however template, MAA (stigmasteryl (84.4 μL, methacrylate) 1 mmol) was was in a similar manner with the exception that the polymerisable template (stigmasteryl methacrylate) not included.included in Additional the pre-polymerisation non-imprinted mixture. polymers The p wereolymers prepared were removed without from stigmasteryl the reaction methacrylate tubes, was 0 not included. Additional non-imprinted polymers were prepared without stigmasteryl or a Nthen,N crushed-DMAAM and functionalground using monomer; a Retsch PM however, 200 ball mill MAA (Haan, (84.4 Germany)µL, 1 mmol) operated was at included300 rpm for in the methacrylate or a N,N′-DMAAM functional monomer; however, MAA (84.4 μL, 1 mmol) was pre-polymerisation6 min in intervals mixture. of 2 min Thebursts. polymers The template were was removed removed from from the the reaction MIP resin tubes, by base then hydrolysis crushed and included in the pre-polymerisation mixture. The polymers were removed from the reaction tubes, in methanolic NaOH (1.5 M, 150 mL) under reflux overnight. The MIP was then extensively extracted groundthen using crushed a Retsch and ground PM 200 using ball a mill Retsch (Haan, PM 200 Germany) ball mill operated(Haan, Germany) at 300 rpm operated for 6 minat 300 in rpm intervals for of with methanol via Soxhlet procedures, filtered, and dried in vacuo. A summary of various conditions 2 min6 bursts.min in intervals The template of 2 min was bursts. removed The template from the was MIP removed resin byfrom base the hydrolysisMIP resin by in base methanolic hydrolysis NaOH used for the MIP preparation, schematically shown in Scheme 5, and the NIP is given in Table 1. (1.5 M,in methanolic 150 mL) under NaOH reflux (1.5 M, overnight. 150 mL) under The reflux MIP overnight. was then extensivelyThe MIP was extractedthen extensively with extracted methanol via Soxhletwith procedures, methanol via filtered, Soxhlet andprocedures, dried in filtered vacuo., and A dried summary in vacuo. of various A summary conditions of various used conditions for the MIP preparation,used for schematicallythe MIP preparation, shown schematically in Scheme5 shown, and thein Scheme NIP is 5, given and the in TableNIP is1 .given in Table 1.

H H N O H O O H AIBN, CHCl3 H O H o H 60 C, 24 h H O N O O O H O O O N N O H O AIBN, CHCl3 H 2 O 10 O H 60 oC, 24 h O 1 M NaOH in MeOH reflux, Adjust pH with 3.2% HCl O O O Wash with MeOH via Soxhlet O N N O 2 O 10

1 M NaOH in MeOH reflux, Adjust pH with 3.2% HCl Wash with MeOH via Soxhlet

N N H H O Non-covalent rebinding O H O HO O

OHN OHN H O H O O N Non-covalent rebinding O N H O HO O OH OH O O Scheme 5. The preparationN of a hybrid semi-covalent MIP for stigmasterolN using the stigmasterol methacrylate template (18) and the N,N′-DMAAM co-monomer.

SchemeScheme 5. The 5. The preparation preparation of of a a hybrid hybrid semi-covalentsemi-covalent MIP MIP for for stigmasterol stigmasterol using using the thestigmasterol stigmasterol methacrylatemethacrylate template template (18 ()18 and) and the theN N,N,N0-DMAAM′-DMAAM co co-monomer.-monomer. C 2018, 4, 13 11 of 13

3.7. MIP Batch Binding Evaluation Batch binding studies were conducted in triplicate to evaluate the binding attributes of the stigmasterol-imprinted polymer and the non-imprinted polymer using stigmasterol (0.1 mM) in acetonitrile/H2O (9:1 v/v). The MIP and NIP polymers (20 mg) were weighed into sealable Eppendorf tubes (1.7 mL) and incubated at 20 ◦C with the analyte solution (1.0 mL, 0.1 mM) on a rotary mixer spinning at 40 rpm for 18 h. The mixture was then centrifuged at 16,060× g for 30 min to pellet the analyte-bound polymer complex. An aliquot (100 µL) of the supernatant was removed and analysed by RP-HPLC with UV detection at 210 nm and 280 nm. The amount of analyte bound (B) was determined as the difference between the initial analyte concentration and the test concentration. Cross-reactivity studies using solutions (0.1 mM) of ergosterol, cholesterol, campesterol, or brassicasterol in acetonitrile/H2O (9:1 v/v) were also performed in a similar manner.

4. Conclusions In this investigation, several molecularly imprinted polymers were designed and prepared using the template stigmasteryl methacrylate (18) via the semi-covalent-imprinting approach. The derived MIPs were found to efﬁciently bind stigmasterol and several other sterol targets. The advantages of utilising design criteria based on molecular modelling procedures and interaction energy calculations to guide the selection of the monomer type, as well as the choice of the monomer:template ratios for the formation of the pre-polymerisation complex with these semi-covalently imprinted polymers, was documented. The formation of a stable pre-polymerization complex requires a close proximity of template and functional monomer. This can be achieved through the interplay of hydrogen bonding between the template and the monomer if that is possible, but other effects (electrostatic, van derWaals, Lipshitz, dipolar, etc.) can also participate. What the current investigation demonstrates is that at the stage of formation of the pre-polymerization complex, hydrogen bond interactions are not required under covalent imprinting conditions to form a stable complex. However, post cleavage and removal of the stigmasterol template, hydrogen bond capabilities are regenerated in the MIP cavity, and hydrogen bond interactions with the 3-hydroxy group of the sterol can then occur. Moreover, the importance of shape determinants in controlling the binding selectivity with templates that contain only a single hydrogen bonding site was established. Collectively, these results have provided additional insights into the unique binding propensities of molecularly imprinted polymers which involve only a single hydrogen bonding site, but are capable of molecular recognition through shape, van der Waals, and non-polar Lifshitz force interactions. In addition, these investigations have provided a basis for our ongoing research into the use of MIP separation technologies with a focus on the development of alternative routes for the recovery of phytosterols from plant waste sources, compared to current techniques involving protracted extraction procedures or costly synthetic methods.

Supplementary Materials: The following are available online at http://www.mdpi.com/2311-5629/4/1/13/s1, 1 S1: General experimental procedures, S2: experimental details, Figure S1: H NMR (400 MHz, CDCl3) of 13 stigmasteryl methacrylate (18), Figure S2: C NMR (100 MHz, CDCl3) of stigmasteryl methacrylate (18). Acknowledgments: The authors gratefully acknowledge financial support from the CSIRO National Research Food Futures Flagship. Author Contributions: L.J.S., B.D., and M.T.W.H. conceived and designed the experiments; L.J.S., B.K.Y.L., and B.D. performed the experiments; L.J.S., S.J.H., R.I.B., and M.T.W.H. analyzed the data; B.D. and R.I.B. contributed reagents/materials/analysis tools; L.J.S., R.I.B., and M.T.W.H. wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. C 2018, 4, 13 12 of 13

References

1. Abidi, S.L. Chromatographic analysis of plant sterols in foods and vegetable oils. J. Chromatogr. A 2001, 935, 173–201. [CrossRef] 2. Fernandes, P.; Cabral, J.M.S. Phytosterols: Applications and recovery methods. Bioresour. Technol. 2007, 98, 2335–2350. [CrossRef][PubMed] 3. Schröder, M.; Vetter, W. High-speed counter-current chromatographic separation of phytosterols. Anal. Bioanal. Chem. 2011, 400, 3615–3623. [CrossRef][PubMed] 4. Yan, F.; Yang, H.; Li, J.; Wang, H. Optimization of phytosterols recovery from soybean oil deodorizer distillate. J. Am. Oil Chem. Soc. 2012, 89, 1363–1370. [CrossRef] 5. Rosello-Soto, E.; Koubaa, M.; Moubarik, A.; Lopes, R.P.; Saraiva, J.A.; Boussetta, N.; Grimi, N.; Barba, F.J. Emerging opportunities for the effective valorization of wastes and by-products generated during olive oil production process: Non-conventional methods for the recovery of high-added value compounds. Trends Food Sci. Technol. 2015, 45, 296–310. [CrossRef] 6. Uddin, M.S.; Sarker, M.Z.I.; Ferdosh, S.; Akanda, M.J.H.; Easmin, S.; Bt Shamsudin, S.H.; Yunus, K.B. Phytosterols and their extraction from various plant matrices using supercritical carbon dioxide: A review. J. Sci. Food Agric. 2015, 95, 1385–1394. [CrossRef][PubMed] 7. Cheong, W.J.; Yang, S.H.; Ali, F. Molecular imprinted polymers for separation science: A review of reviews. J. Sep. Sci. 2013, 36, 609–628. [CrossRef][PubMed] 8. Schwarz, L.J.; Danylec, B.; Harris, S.J.; Boysen, R.I.; Hearn, M.T.W. Sequential molecularly imprinted solid-phase extraction methods for the analysis of resveratrol and other polyphenols. J. Chromatogr. A 2016, 1438, 22–30. [CrossRef][PubMed] 9. Hashim, S.N.N.S.; Schwarz, L.J.; Danylec, B.; Mitri, K.; Yang, Y.; Boysen, R.I.; Hearn, M.T.W. Recovery of ergosterol from the medicinal mushroom, Ganoderma tsugae var. Janniae, with a molecularly imprinted polymer derived from a cleavable monomer-template composite. J. Chromatogr. A 2016, 1468, 1–9. [CrossRef] [PubMed] 10. Danylec, B.; Schwarz, L.J.; Harris, S.J.; Boysen, R.I.; Hearn, M.T.W. The application of template selectophores for the preparation of molecularly imprinted polymers. Molecules 2015, 20, 17601–17613. [CrossRef] [PubMed] 11. Dickert, F.L.; Hayden, O. Molecular imprinting in chemical sensing. TrAC Trends Anal. Chem. 1999, 18, 192–199. [CrossRef] 12. Whitcombe, M.J.; Kirsch, N.; Nicholls, I.A. Molecular imprinting science and technology: A survey of the literature for the years 2004–2011. J. Mol. Recognit. 2014, 27, 297–401. [PubMed] 13. Boysen, R.I.; Schwarz, L.J.; Nicolau, D.V.; Hearn, M.T.W. Molecularly imprinted polymer membranes and thin ﬁlms for the separation and sensing of biomacromolecules. J. Sep. Sci. 2017, 40, 314–335. [CrossRef] [PubMed] 14. Whitcombe, M.J.; Rodriguez, M.E.; Villar, P.; Vulfson, E.N. A new method for the introduction of recognition site functionality into polymers prepared by molecular imprinting: Synthesis and characterisation of polymeric receptors for cholesterol. J. Am. Chem. Soc. 1995, 117, 7105–7111. [CrossRef] 15. Sellergren, B.; Wieschemeyer, J.; Boos, K.S.; Seidel, D. Imprinted polymers for selective adsorption of cholesterol from gastrointestinal ﬂuids. Chem. Mater. 1998, 10, 4037–4046. [CrossRef] 16. Kim, J.M.; Ahn, K.D.; Wulff, G. Cholesterol esterase activity of a molecularly imprinted polymer. Macromol. Chem. Phys. 2001, 202, 1105–1108. [CrossRef] 17. Hwang, C.C.; Lee, W.C. Chromatographic characteristics of cholesterol-imprinted polymers prepared by covalent and non-covalent imprinting methods. J. Chromatogr. A 2002, 962, 69–78. [CrossRef] 18. Gore, M.A.; Karmalkar, R.N.; Kulkarni, M.G. Enhanced capacities and selectivities for cholesterol in aqueous media by molecular imprinting: Role of novel cross-linkers. J. Chromatogr. B 2004, 804, 211–221. [CrossRef] [PubMed] 19. Ciardelli, G.; Borrelli, C.; Silvestri, D.; Cristallini, C.; Barbani, N.; Giusti, P. Supported imprinted nanospheres for the selective recognition of cholesterol. Biosens. Bioelectron. 2006, 21, 2329–2338. [CrossRef][PubMed] 20. Puoci, F.; Curcio, M.; Cirillo, G.; Iemma, F.; Spizzirri, U.G.; Picci, N. Molecularly imprinted solid-phase extraction for cholesterol determination in cheese products. Food Chem. 2008, 106, 836–842. [CrossRef] C 2018, 4, 13 13 of 13

21. Aghaei, A.; Milani Hosseini, M.R.; Najafi, M. A novel capacitive biosensor for cholesterol assay that uses an electropolymerized molecularly imprinted polymer. Electrochim. Acta 2010, 55, 1503–1508. [CrossRef] 22. Kitahara, K.I.; Yoshihama, I.; Hanada, T.; Kokuba, H.; Arai, S. Synthesis of monodispersed molecularly imprinted polymer particles for high-performance liquid chromatographic separation of cholesterol using templating polymerization in porous silica gel bound with cholesterol molecules on its surface. J. Chromatogr. A 2010, 1217, 7249–7254. [CrossRef][PubMed] 23. Li, J.; Zhang, Z.; Xu, S.; Chen, L.; Zhou, N.; Xiong, H.; Peng, H. Label-free colorimetric detection of trace cholesterol based on molecularly imprinted photonic hydrogels. J. Mater. Chem. 2011, 21, 19267–19274. [CrossRef] 24. Ye, L.; Weiss, R.; Mosbach, K. Synthesis and characterization of molecularly imprinted microspheres. Macromolecules 2000, 33, 8239–8245. [CrossRef] 25. Ye, L.; Yu, Y.H.; Mosbach, K. Towards the development of molecularly imprinted artificial receptors for the screening of estrogenic chemicals. Analyst 2001, 126, 760–765. [CrossRef][PubMed] 26. Szumski, M.; Buszewski, B. Molecularly imprinted polymers: A new tool for separation of steroid isomers. J. Sep. Sci. 2004, 27, 837–842. [CrossRef][PubMed] 27. Zhu, Q.; Wang, L.; Wu, S.; Joseph, W.; Gu, X.; Tang, J. Selectivity of molecularly imprinted solid phase extraction for sterol compounds. Food Chem. 2009, 113, 608–615. [CrossRef] 28. Hashim, S.N.N.S.; Boysen, R.I.; Schwarz, L.J.; Danylec, B.; Hearn, M.T.W. A comparison of covalent and non-covalent imprinting strategies for the synthesis of stigmasterol imprinted polymers. J. Chromatogr. A 2014, 1359, 35–43. [CrossRef][PubMed] 29. Boysen, R.I.; Schwarz, L.J.; Li, S.; Chowdhury, J.; Hearn, M.T.W. Photolithographic patterning of biomimetic molecularly imprinted polymer thin films onto silicon wafers. Microsyst. Technol. 2014, 20, 2037–2043. [CrossRef] 30. Chung, S.K.; Ryoo, C.H.; Yang, H.W.; Shim, J.Y.; Kang, M.G.; Lee, K.W.; Kang, H.I. Synthesis and bioactivities of steroid derivatives as antifungal agents. Tetrahedron 1998, 54, 15899–15914. [CrossRef] 31. Fujimoto, Y.; Kimura, M.; Khalifa, F.A.M.; Ikekawa, N. Side chain structural requirement for utilization of sterols by the silkworm for growth and development. Non-stereoselective utilization of the 24-stereoisomeric pairs of 24-alkylsterols. Chem. Pharm. Bull. 1984, 32, 4372–4381. [CrossRef] 32. Hutchins, R.F.N.; Thompson, M.J.; Svoboda, J.A. The synthesis and the mass and nuclear magnetic resonance spectra of side chain isomers of cholesta-5,22-dien-3β-ol and cholesta-5,22,24-trien-3β-ol. Steroids 1970, 15, 113–130. [CrossRef] 33. Partridge, J.J.; Faber, S.; Uskoković,M.R. Vitamin D3 metabolites I. Synthesis of 25-hydroxycholesterol. Helv. Chim. Acta 1974, 57, 764–771. [CrossRef][PubMed] 34. Steele, J.A.; Mosettig, E. The solvolysis of stigmasteryl tosylate. J. Org. Chem. 1963, 28, 571–572. [CrossRef]

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