Synthesis and application of a radical trapping agent

Synthesis and application of a radical trapping agentChemistry:Techniques:Catalyzed oxidation with hydrogen peroxideTrapping of a free radicalAcquisition and interpretation of ESR spectraTime Required:Ca. Two lab periods.IntroductionReactions that are believed to involve radical intermediatesare often difficult to investigate because most odd-electronspecies are highly reactive and very often cannot be detecteddirectly. It is possible however to convert reactive radicals tospecies whose structures can be determined and related to theradical. One way in which this can be accomplished isconversion of the reactive radical to a persistent radical,whose identity can be determined by ESR (electron spinresonance spectroscopy), 1 through the use of moleculesknown to react with radicals in predictable way. Suchmolecules are referred to as radical traps or spin traps. Oneclass of molecules that reacts with radicals to give persistentradicals is the nitrones, 1, which are also known as alkylideneamine N-oxides.RH1NOR'One advantage of ESR as a detection tool is its very highsensitivity (as few as 10 15 unpaired spins can be detected),which makes the use of radical traps especially useful inbiological systems where radical intermediates are suspected.The objectives of this experiments are the synthesis of 2and its application as a radical trap.Ph+ R"H2NOThe persistent nitroxide radical generated in the trappingreaction will be examined by ESR spectroscopy.Preparation of 2 will be done by the Na 2 WO 4·2H 2 Ocatalyzed oxidation of N-tert-butyl benzylamine by H 2 O 2 .The oxidation of secondary amines to nitrones involves theintermediate formation of the corresponding hydroxylamine asshown in Scheme 1. In this scheme the nitrone is formed by amulti-step process that begins with conversion of thesecondary amine to the hydroxylamine where “O” refers to aspecies capable of transferring an oxygen atom derived fromhydrogen peroxide to a nucleophilic nitrogen, first in thesecondary amine and then in the hydroxyl amine. Secondaryamine oxides are intrinsically unstable with respect to tautomerizationto the hydroxylamine as in (2). The hydroxylamineN-oxide formed in reaction (3) eliminates water to form thenitrone. In at least one case reaction (4) has been shown to bethe rate determining step in forming the nitrone. As a resultRR"HNOR'careful control of conditions and stoichiometry maysometimes allow synthesis of hydroxylamines. 2Ph O"O"PhCH (1)2 NHC(CH 3 ) 3NH HHPhHHPhHHPhHHNNNOOHOHOH"O"-H 2 OScheme 1The nature of “O” in Scheme 1 for the WO 2- 4 catalyzedoxidation is not entirely certain, but the available evidencesuggests that it is most likely a species that contains a “sideon”bound peroxo species, i.e.,OOWHHPeroxo species such as this are well known to undergonucleophilic attack at oxygen with transfer of an oxygen atomto the nucleophile. 3Schematically, the overall oxidation reaction can be viewedas follows:H 2 O 2 + N(III) ÷ H 2 O + N(V)-OThe oxidation state change for each oxygen is formally from-1 to -2. Although H 2 O 2 is a fairly strong oxidant from athermodynamic viewpoint, it often reacts only slowly, if at all,in the absence of a catalyst. While not all steps in thecatalyzed reaction have been elucidated, the availableevidence suggests the following steps are likely:WO 2- 4 + H 2 O 2 ÷ O 3 W(O 2 ) 2- + H 2 OO 3 W(O 2 ) 2- + N(III) ÷ WO 2- 4 + N(V)-OPhPhHHPhHNNNOOOHOH(2)(3)(4)

It is possible that species involving more than one peroxoligand are (also) formed. The proposed species O 3 W(O 2 ) 2-corresponds to “O” is Scheme 1.Radical spin trappingDirect detection of radicals (by ESR) is usually extremelydifficult because most are highly reactive and (often) presentin low concentrations. As a result indirect methods must beutilized to obtain evidence for their presence and structure.There are two common indirect methods. The first is CIDNP 4(chemically induced dynamic nuclear polarization), which isan NMR technique in which resonances for radical speciesmay be observed because the intensities of their resonancesare much greater than expected for the concentration of thespecies present. This results from preferential polarization ofnuclear spins by unpaired electron spins. Because thesepolarizations may be positive or negative the anomalouslyintense resonances may reflect absorption or emission. Thesecond technique is spin trapping. 5 Spin trapping reagentsreact with radicals to give a long-lived (persistent) radicalwhich can be detected by ESR. The structure of the trappedradical is inferred on the basis of electron spin-nuclear spincoupling (hyperfine coupling) patterns in the persistent radicaland/or by independent synthesis.To successfully trap a highly reactive radical requires thatthe spin trap have a high rate of reaction (low activationenergy) toward radicals, but not with other reactioncomponents 6 and that the product be sufficiently long-livedunder reaction conditions for detection. It is usually necessaryto use a large concentration of the trapping reagent to generateenough product for detection. Nitrones are efficient radicaltraps, but are otherwise relatively unreactive both chemicallyand photochemically. Certain derivatives, including 2, havebeen used for in vivo biological applications.In this experiment you will utilize 2 to trap a radicalgenerated from a common initiator for radical polymerizationsand detect the persistent nitroxide radical product by ESR.ExperimentalN-Benzylidene-tert-butylamine N-Oxide (2). In a 50-mLflask equipped with a stirring bar place N-tert-butylbenzylamine (5 mmol), Na 2 WO 4 .2H 2 O (0.066 g, 0.20 mmol),and methanol (10 mL). Cool the solution in an ice bath andadd to the stirred solution 30% aqueous hydrogen peroxide(1.70 g, 15.0 mmol). After addition is complete, remove theice bath and stir the reaction mixture for at least one h, thenstopper the flask and let it stand unstirred until the next labperiod. Remove the methanol using a rotary evaporator andthen transfer the residue to a separatory funnel using 25 mL ofmethylene chloride. Wash the methylene chloride solutiontwice with 10 mL portions of saturated aqueous sodiumchloride, then dry the methylene chloride solution overanhydrous sodium sulfate. Remove the drying agent byfiltration and evaporate the methylene chloride on a steambath. The residue should crystallize upon cooling. Theproduct should be recrystallized from a small amount ofhexane. Collect the purified product by filtration and air drythe product. Obtain a melting point and 1 H NMR spectrum ofyour purified product.Trapping a radical using nitrone 2. Place 10 mg of nitroneand 15 mg of benzoyl peroxide, or 10 mg 2,2'-azobisisobutylronitrile in a vial. Add 1 mL of benzene andthen carefully sparge the solution with nitrogen for 2-3 min.Transfer about 0.5 mL of the solution to a serum-capped,nitrogen-flushed ESR sample tube. Heat the sample tube in a60-70 EC water bath for about 5 min and immediately obtainthe ESR spectrum.Calculate the g value and hyperfine coupling constants foryour spectrum. For information about ESR spectroscopy seereference 1. In preparing your report you should be careful toprovide the structure of the radical whose spectrum you haverecorded and to compare your results with a literaturespectrum (give the literature reference to the data that youcite).References and footnotes(1) ESR is the equivalent of NMR for electrons. A physicalmodel for the phenomenon is a spin flip of the electron in themagnetic field under resonance conditions. Electron spinnuclearspin coupling occurs (hyperfine coupling) whichprovides structural information. A satisfactory introduction tothe fundamentals to ESR spectral interpretation can be foundin Atkins, P. Physical Chemistry, 6 th Ed, W.H. Freeman, NewYork, NY, 1997, Chapter 18, sections 10-11(2) Yamazaki, S. Bull. Chem. Soc. Jpn. 1997, 70, 877.(3) Espenson, J.H. Chem. Commun. 1999, 479 and referencescited.(4) For an explanation of CIDNP see Pine, S.H. J. Chem. Ed.1972, 49, 664. Ward, H.R. Acc. Chem Res. 1972, 5, 18.Lawler, R.G. Acc. Chem. Res. 1972, 5, 25.(5) A lucid article on this topic is Janzen, E.G. Acc. Chem.Res. 1971, 4, 31.(6) This includes electromagnetic radiation since manyradicals are generated by photolysis.2