Vaugh, Jeremy

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670Efforts Towards the Synthesis of a Molecularly Imprinted Matrix as a PhosphonatedAcetylcholine MimicJeremy F. VaughnC500 Project ReportSpring 2006Advisor: Theodore WidlanskiAbstract The goal of my research has been to develop a molecularly imprinted polymermatrix that mimics the transition state of acetylcholinesterase binding to a phosphonate.The monomer is based around a modified styrene ‘anchor’ attached to a ‘linker’ segment,which is then attached to choline-bound phosphonate.  Upon polymerization, thephosphonate will be removed and the exposed pockets of the matrix will be converted to ahydrated aldeyhyde to facilitate future bindings to substrates.671Acetylcholine, Acetylcholinesterase, and OrganophosphonatesAcetylcholine (ACh) is a neurotransmitter responsible for muscular contractionand is located in the brain, spinal cord, and parts of the peripheral nervous system.Acetylcholine is synthesized in the body by choline acetyltransferase (ChAT) reacting withacetyl-coenzyme A (Acetyl-CoA).  Acetylcholine is deactivated by the enzymeacetylcholinesterase (AChE), which is located in the post-synaptic membrane.  Themanipulation of acetylcholine and acetylcholinesterase is a key operating function of manytoxins.  Additionally, several medical conditions exist wherein acetylcholinesteraseinhibitors become necessary for normal function.1Figure 1: Nerve Synapse Diagram2672Acetylcholinesterase in normal biological systems acts by deactivatingacetylcholine via rapid hydrolysis.  In addition to the regulation of acetylcholine,acetylcholinesterase also affects cell proliferation, differentiation, as well as cellular stressresponse.3As a class of chemicals, organophosphonates have been successfully applied asacetycholinesterase inhibitors.  Practical applications include use as pesticides andchemical warfare agents, as well as implementation as therapeutic agents for neurologicalconditions such as Alzheimer’s disease, Glaucoma, and Myasthenia Gravis.4The inhibition of acetylcholinesterase results in unnaturally high levels ofacetylcholine.  This in turn causes an over-stimulation of the cholinergic receptors.Symptoms of organophosphonate exposure are muscarinic, nicotinic, central nervoussystem disruption, as well as adverse reproductive effects (Table 1).  It is precisely thistoxicity from over-stimulation that is exploited in the use of organophosphate agents aspesticides and chemical warfare agents (Sarin, VX).4The commonly agreed upon function of inhibition for organophosphate pesticides(and by association, chemical warfare agents) is the phosphorylation ofacetylcholinesterase at the serine 200 residue via fluoride replacement.3,4  Figure 2illustrates the key amino acid residues of acetylcholinesterase.  Figure 3 demonstrates thephosphorylation of acetylcholinesterase at the serine 200 residue by cyclosarin, a potentorganophosphate classified as a biochemical weapon.673Table 1: Organophosphate Exposure Effects4,5Muscarinic Effects- Nausea- Vomiting- Heart Block- PulmonaryEdema- AbdominalCrampsNicotinic Effects- Hypertension- Weakness- MuscleFasciculationCentral Nervous SystemEffects- Confusion- Headache- Tremor- Convulsions- Coma- RespiratoryDepressionVisible PhysicalSymptoms- Salivation- Lacrimation- Miosis- Hypothermia- Mild Tremors- Mouthasmacking- Lowered MotorActivity- Decreased Tail-Pinch Response- AlteredNeuromuscularFunction674Figure 2: Key Acetylcholinesterase Residues and Bound Acetylcholine3+POH3C OFP OOCH3+ H FHN CH CCH2OOO{}HN CH CCH2OOOH{}675Figure 3: Cyclosarin Binding to Acetylcholinesterase Serine 200 Residue5Dougherty and coworkers have studied the binding of acetylcholine to both natural andsynthetic receptors.6,7  They have determined that the primary binding action involves a pi-cationinteraction between the receptor and the quaternary trimethyl ammonium of choline (See Figure4).Figure 4: Acetylcholine binding to key Residues of Immunoglobin Fab McPC6037676Solid Phase Organic Synthesis, JandaJel, and Molecularly Imprinted PolymersPolystyrene-based resins have proved to be a useful field for the development of solidphase organic synthesis (SPOS). JandaJelTM continues to be a platform of interest; it is believedthat the presence of ether linkages results in the observed superior swelling characteristics uponexposure to a variety of solvents.  This results in reaction kinetics superior to that observed inMerrifield resins.8-11 Molecularly imprinted polymers (MIP) are an area of increasing interest to syntheticchemists.  Rather than simply serving as a media within which to conduct reactions, a polymermatrix forms around a template molecule.  When the template is subsequently removed, ‘pockets’remain within the matrix corresponding to the shape of interest dictated by the template.  Appliedas such, imprinted polymers have been successfully employed as coenzyme analogs.12Research Interests and Project Planning The primary goal of this research project is to develop an imprinted polymer that mimicsthe transition state of phosphorylated acetylcholinesterase.  The target molecule will exhibit apolymerizable anchor, a ‘linker’ and a phosphonate-choline group (see Figure 5).  Success willrequire the use of a polymer base that can provide the required pi-cation interaction between thequaternary trimethyl ammonia of choline.  Furthermore, the end polymer matrix must exhibitsuitable swelling properties.677PolymerAnchorLinkerOPOOCH3NFigure 5: General Molecule Scheme A styrene anchor is a logical choice, as it will be readily polymerized to form the imprintedmatrix upon completion of target molecule.  Multiple ether linkages within the ‘linker’ shouldprovide enhanced swelling properties in the final matrix.OO OPOONFigure 6: Target Molecule678OO OPOONOBrHO OPOONCHOCHOHONClPOClHO OH + ++Figure 7: RetrosynthesisScheme 1: Synthesis of 2-(4-vinylbenzyloxy)ethanolCHOCHO CHOOH OH Br OOHJOC 2001, 66, 4476 Tetrahedron, 2005, 61, 12160 Macromolecules, 2002, 35, 946655% 99% 68% 58%NaBH4EtOH, 0oC1hrNaH, DMSO+PPh3CH3I-20 hrs, RTPBr3Et2O0oC, 2 hrs(CH2OH)2,NaHTHF, 7.5 hrs70.0% Average Yield21.5% Process Yield2-(4-vinylbenzyloxy)ethanol1-(bromomethyl)-4-vinylbenzene(4-vinylphenyl)methanol4-(hydroxymethyl)benzaldehydeTerephthalaldehyde Previous syntheses of (4-vinylphenyl) methanol have been conducted using styrene as astarting material.  However, multiple steps are involved and the yield suffers accordingly.13,14Thus, starting from inexpensive and readily available terephthalaldehyde appeared to be679advantageous.  Initial attempts at a mono-reduction to 4-(hydroxymethyl)benzaldehyde employedlithium aluminum hydride.  Despite varying equivalents of the reagent (1.00, 0.50, and 0.25 eq),the reactions all resulted in over-reduction.  Employing sodium borohydride in tetrahydrofuran15afforded a mixture of the monohydroxy and dihydroxy products.  Employing ethanol as a solventafforded a mixture of starting material and the monohydroxy product in a satisfactory yield. (4-vinylphenyl)methanol was synthesized by performing a Wittig reaction upon  4-(hydroxymethyl)benzaldehyde.16,17  The ylide was prepared by reacting triphenylphosphine withmethyl iodine in benzene.  Reaction optimizations lead to the use of two equivalents of theactivated ylide.  Presumably, the hydroxyl group is deprotonated by the first equivalent, with thesecond reacting normally with the substrate.  Upon purification, the desired compound wasobtained in very high yield.  It is interesting to note that attempts to conduct a Wittig reactionwith terephthalaldehyde failed to react. Use of freshly distilled tribromophosphine resulted in the formation of the desiredbromine17 in a fair yield, which was then reacted with ethylene glycol to afford 2-(4-vinylbenzyloxy)ethanol.18Future Endeavors Focus is now shifting to the formation of the phosphonate ester fragment.Methylphosphonic dichloride has been successfully reacted to form phosphonate esters with avariety of alcohols.19-21  Additionally, recent literature indicates that the process may be facilitatedby catalysts such as triazole.  Upon completion, this fragment will be attached to the styrene-linker previously developed.22,23  Polymerization will be induced utilizing techniques establishedfor styrene polymerization.24680ExperimentalGeneral Comments: Chemicals employed were obtained from commercial sources.  Reactionswere typically monitored at bench via thin-layer chromatography.  Routine structure analysis wasconducted utilizing 400MHz 1H-NMR to confirm agreement with reported literature data.CHOOH4-(hydroxymethyl)benzaldehyde:  2.000g of terephthalaldehyde (14.92 mmol) was placed in aflame-dried, nitrogen-purged round bottom flask with a stir bar.  The compound was dissolved in44.8 mL of 100% ethanol and chilled to 0oC.  After cooling, 0.202g of sodium borohydride (5.22mmol) was added to the flask.  After one hour the reaction was quenched with water, worked upin ethyl acetate, dried with magnesium sulfate, and the solvent removed.  Purification via silicacolumn chromatography utilizing a 50/50 mixture of ethyl acetate and hexanes afforded 1.110grams (55% yield) of the desired product as white crystals. 1H-NMR was in accordance withpreviously reported data.15681OH(4-vinylphenyl)methanol:  2.20 mL of distilled DMSO was placed in a flame-dried nitrogen-purged 5mL flask, into which 0.035g of dried sodium hydride was added.  0.594g (1.470 mmol)of methyl ylide was then added to the mixture and allowed to stir for 30 minutes.  100mg (0.735mmol) of 4-(hydroxymethyl)benzaldehyde was then added to the mixture.  After 22 hours, thereaction appeared to have run to completion as indicated by bench-top TLC.  The reaction wasquenched using a saturated solution of ammonium chloride, worked up in ethyl acetate, driedusing magnesium sulfate, and the solvent removed via rotary evaporation.  Silica gel columnchromatography utilizing a 50/50 mixture of ethyl acetate and hexanes resulted in the recovery of98mg of product (99% yield) in the form of an light-colored oil. 1H-NMR was in accordancewith previously reported data.17682Br1-(bromomethyl)-4-vinylbenzene:  50mg (0.373mmol) of (4-vinylphenyl)methanol was placedin a flame-dried nitrogen-purged 10mL flask.  4.14mL of diethyl ether was added and the mixturechilled to 0oC.  After cooling, 0.055mL of tribromophosphine (0.5595 mmol) was added andallowed to react for one hour, after which another 055mL of tribromophosphine (0.5595 mmol)was added and stirred for another hour.  The reaction was then quenched with water and asaturated solution of sodium bicarbonate, worked up in diethyl ether, rinsed with brine, dried withmagnesium sulfate and the solvent eliminated via rotary evaporation.  Silica gel columnchromatography was employed to purify the compound using an 80/20 mixture of hexanes andethyl acetate.  50mg of the desired product was recovered (68% yield) as a reddish oil. 1H-NMRwas in accordance with previously reported data.17683OOH2-(4-vinylbenzyloxy)ethanol:  1.18mL of ethylene glycol was added to a 5mL flame-dried,nitrogen purged round bottom flask with a magnetic stir bar.  A mixture of THF and 0.010g ofsodium hydride was then injected. A mixture of 0.021g of 1-(bromomethyl)-4-vinylbenzene(0.1065mmol) and the remaining THF (total of 0.11mL) was injected into the flask.  Thetemperature was increased to 60oC via an oil bath.  After 7.5 hours the reaction was cooled,quenched with water, worked up in chloroform, dried with magnesium sulfate, and the solventremoved via rotary evaporation.  Silica gel column chromatography was performed using an80/20 mixture of hexanes and ethylacetate and afforded 11mg of the product (58% yield) as a clear, colorless oil. 1H-NMR was inaccordance with previously reported data.18684References1. Carlson, N. R. Physiology of Behavior, 6th Edition. 1998, Allyn and Bacon, NeedhamHeights, MA2. Campbell, N. A., Reece, J. B., Mitchell, L. G. Biology, 5th Edition. 1999,Benjamin/Cummings, Menlo Park, CA3. Tougu, V. Curr. Med. Chem., 2001, 1, 155-1704. Pope, C. Karantha, S., Liu, J. Environmental Toxicology and Pharmacology, 2005, 19,433-4665. Krejcova, G., Kuca, K., Sevelova, L. Defence Science Journal, 2005, 55, 105-1156. Peterson, E. J., Choi, A., Dahan, D. S., Lester, H. A., Dougherty, D. A. J. Am. Chem.Soc. 2002, 124, 12662-126637. Dougherty, D. A., Staufer, D. A. Science, 1990, 250, 1558-15608. Toy, P. H., Janda, K. D. Tetrahedron Letters, 1999, 40, 6329-63329. Moss, J. A., Dickerson, T. J., Janda, K. D. Tetrahedron Letters, 2002, 43, 37-4010. Gambs C., Dickerson, T. J., Mahajan, S., Pasternack, L. B., Janda, K. D. J. Org. Chem.2003, 68, 3673-367811. Shimomura, O., Lee, B. S., Meth, S., Suzuki, H., Mahajan, S., Nomura, R., Janda, K. D.Tetrahedron, 2005, 61, 12160-1216712. Alexander, C., Davidson, L., Hayes, W. Tetrahedron, 2003, 59, 2025-205713. Bamford, C. H., Lindsay, H. Polymer, 1973, 14, 330-33214. Arshady, R., Ledwith, A. Makromol. Chem. 1978, 179, 819-82215. Mak, C. C., Bampos, N., Darling, S. L., Montalti, M., Prodi, L., Sanders, J. K. J. Org.Chem. 2001, 66, 4476-448616. Greenwald, R., Chaykovsky, M., Corey, E. J. J. Org. Chem. 1963, 28, 112817. Osamu, S., Lee, B. S., Meth, S., Suzuki, H., Mahajan, S., Nomura, R., Janda, K. D.Tetrahedron, 2005, 61, 12160-1216718. Hill, D. E., Lin, Y., Rao, A. M., Allard, L. F., Sun, Y-P. Macromolecules, 2002, 35,9466-947168519. Sadikov, O. S., Abduvakhobov, A. A., Kosimov, S. K., Dobenkov, A. G. UzbekskiiKhimicheskii Zhurnal, 1983, 5, 25-2820. 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Efforts Towards the Synthesis of a Molecularly Imprinted Matrixas a Phosphonated Acetylcholine Mimic

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Efforts Towards the Synthesis of a Molecularly Imprinted Matrixas a Phosphonated Acetylcholine Mimic

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