Substitution Lab October 31st, 2008Craig Wheelockcraig.wheelock@ki.sehttp://www.metabolomics.se/(slides can be downloaded from my homepage)Karolinska InstitutetDepartment of Medical Biochemistry and BiophysicsBiomedical candidate program, H08

OutlineTheory Understand substitution reactions (SN1 vs SN2)Experimental equipmentFamiliarity with the necessary equipmentSpecific tips on each experimentTips for conducting each experimentSafety issuesPotential hazards associated with this labLab reportsWhat do you need to include in your lab report??

NUCLEOPHILIC SUBSTITUTIONnucleophilesubstrateproductleavinggroupNUCLEOPHILIC DISPLACEMENTThe nucleophile displaces the leaving group.This is a substitution reaction : Nu substitutes for X (takes its place).

IMPORTANT:This is a reaction at sp3 (tetrahedral) carbon atoms.yesnoCompounds that have sp2 or sp carbons generallydo not give nucleophilic substitution reactions.sp3sp2sp

Nucleophilicsubstitution-reactionA displacement reaction of one chemical group to another

R X + Nu- R Nu + X-

Nucleophilic substitution can occur by two mechanisms: SN1 and SN2Substitution Nucleophilic uni / bimolecular

4 main factorsLeaving group: weak bases are better (X)Attacking group: strong bases are better (Nu-) Solvent: protic vs. aproticSterics: steric interactions affect reaction mechanism

NUCLEOPHILIC SUBSTITUTIONMANY FACTORS INFLUENCE SN1 AND SN2 REACTIONSa) structureb) atom usedc) concentrationd) base strengtha) structure of R, stereochemistrya) nature of Xb) concentrationa) solventb) temp.c) pHSOME PARAMETERS :b) atom usedc) base strengthc) bond strengtha) bond strengthe) solubilityf) sized) DH

Alkyl halidesHalides (X-) are electronegative groups that pull electrons through the C-X bond good leaving groups for substitution rxns C X reactivity of halides: I > Br > Cl > F basicityR X

SN2 ReactionBimolecular substitution= 2 molecules in the transition state- 2nd order reaction: both reactants affect the reaction rate v = k [Nu] [R-X] , where v = rate of reaction k = reaction constant [Nu], [RX] = concentration of nucleophile, alkyl halide Single step reaction: bond breaking/forming simultaneouslyTransition state

SN2 ReactionReactivity of alkyl halides Methyl > primary > secondary >> tertiaryeasy accessno steric hindrancelarge groupsintroduce steric hindranceR

SN2 Reaction Results in inversion of configuration if there is a chiral center, then R S

Supported by polar solvents that do not solvate the nucleophile (aprotic solvents), e.g., DMSO CHH3CRXOHCHRCH3OHXCHCH3RHO+ X(S)(R)

INVERSION OF AN UMBRELLA IN THE WINDInversion of theumbrella is similar in conceptto the inversion ofan SN2 atom.CONCEPTUAL ANALOGY

decreasing rateEFFECT OF DEGREE OF SUBSTITUTION - SN2methyl primary secondary tertiaryEFFECT OF SUBSTRATE ON RATERBr+NaOHROH+NaBracetoneH2O

Example SN2Transition stateCH3CH2Br + NaOH-+bromoethaneethanol

SN1 Reactionunimolecular = one molecule in the transition state 1st order: only concentration of the alkyl halide affects the rate of reaction v = k [R3CX]occurs via an unstable carbocation intermediate [R3C+]reaction occurs in several steps: two substitution reactions and an acid-base reaction, deprotonation

1st step: cleavage of alkyl halide in polar solvent Transition state 12nd step: attack by the nucleophile and formation of the protonated productTransition state 23rd step: deprotonation of the product, an acid-base reactionUnstable carbocationintermediate[ ][ ]RATE LIMITING!

SN1 Reaction results in a racemic mixture: nucleophile can attack from either side of the carbocationmixture of R / S configuration of products

+enantiomersSN1 MECHANISMplanarcarbocationsp2(S)(R)(R)50%50%racemic mixtureRACEMIZATION-attacks topand bottomequally

SN1 Reactionactivity order of alkyl halides

tertiary > secondary > primary > methyl

in practice only occurs with tertiary & secondary more stable carbocation more atoms share the positive charge

activated by solvating polar solvents (protic) e.g., water stabilizes the carbocation+

CARBOCATION STABILITYHYPERCONJUGATIONCCH..HH+RRelectrons in an adjacentC-H s bond help to stabilizethe positive charge of thecarbocation by proximity(overlap)tertiarysecondaryprimary

relativerate1.01.745RBr + H2OROH + HBr100%HCOOHincreasing raterel rate = rate CH3BrrateEFFECT OF INCREASING SUBSTITUTION - SN1methyl primary secondary tertiaryGuess ?EFFECT OF SUBSTRATE ON RATE

Example SN1Tert-butylbromide + methanol (MeOH)

SN1SN2tertiarymethyl**benzylbenzylallylallylsecondaryprimaryprimarysecondarytertiaryneopentylNotice that benzyland allyl are goodfor both SN1 and SN2BESTWORST(fastest)(slowest)bridgeheadbridgehead(bicyclic)(bicyclic)APPROXIMATERATE ORDERSBEST(fastest)WORST(slowest)SUMMARY** In SN2 reactions benzyl is actually better than methyl, but allyl is not.

Outline of the labSubstitution reaction (1 of 3 reactions)Reflux to increase reaction rateMonitor progress by TLC (for ethyl phenyl ether) Extract the product from the reaction mixWash and dry the organic phaseRemove the solvent by roto-evaporationPurify the product by vacuum distillation and record its boiling point

Reflux Do NOT preheat the peg-bath Use CaCl2 in the drying tube, torkrr Use gloves with glass wool mix well, use large magnetic stirrer Do not let sttkoka (bounce) Use 2 neck roundbottom flask, tvhalsad kolv

Separatory Funnel- organic phase on top- watch out for gas formationDry with Na2SO4- 1-2 spoons cover the flask 15-30 min- filter

Roto-evaporation(rullindunstning)

Distillation- do not use vacuum grease- measure vacuum- start at low vacuum to prevent bouncing- foil around the neck improves heating- use magnetic stirrer in oil bath- weigh the flasks to determine yield!!!!!!

1-BromooctaneHBr, H2SO4TLC not necessarylong reflux time of 4h, so get going!!!watch for gas formation during extractionuse syringe with HBr and octanol

n-Butylmalonic acid diethyl esterfill 2 neck round bottom flask with N2use ice-bath to cool when mixing diethyl malonate, bromobutane, THF and NaHafter gas evolution stops, then reflux for 3hmix welllong experiment, 3h reflux, so get going! no TLC needed

n-Butylmalonic acid diethyl esterNaH, bromobutane (butylbromide) NaH reacts strongly with water!!!!releases H2 gasbe careful when using ice-bathdry equipment!!!quench with acetoneuse NH3 / 95% EtOH to quench bromobutanetest ether for peroxidesbromobutane and diethyl malonate in hooduse syringe to transfer bromobutane

Ethyl phenyl etherphenol, iodoethane (etyljodide)dry equipment!!!measure phenol in hood, no open containersfill 2 necked round bottom flask with N2 make sure that sodium ethoxide is fully dissolved in abs EtOH before adding phenol (~30 min)prepare brine (saturated solution of NaCl) (for 500 ml, ~36g/100ml)one bottle for the whole lab is sufficient

Ethyl phenyl etherFollow reaction by TLC: collect sample prior to refluxing!!run TLC after 30 minif reaction has gone to completion, stop refluxingTLC mobile phase: heptane:ethyl acetate 9:1

For some reagents need to calculate volume from density . . . = m / V V = m / where = density V = volume m = mass

densities: diethyl malonate: 1.055 g/ml 1-bromobutane: 1.276 g/ml HBr: 1.49 g/ml 1-octanol: 0.827 g/ml iodoethane: 1.95 g/ml

Safety Issues . . .Peroxide-test ether (with strips), mark bottle when testedEther is explosive do not heat!!!Let ether evaporate in the hood (dragskp), do not put in organic wasteDo not preheat the PEG bathBe careful extracting: gas formationDry equipment (dry overnight in drying oven)

Safety Issues...use gloves with alkyl halidesdo not put them in the sink, measure in the hoodNH3/EtOH (1:1) as quenching solution (motmedel) for alkyl halidesprepare your own solution in the labrinse all glassware that has been in contact with RXreuse the same solutionafter rinsing wash with water

Safety continuedweigh chemicals in hood (dragskp)rinse all glassware in the hood first!check for residual smell from previous labdo not carry around open containers with chemicals (stinks and is dangerous)!can use aluminum foil to cover containersweigh phenol in the hood

Lab reportsAbstractexperiment aim, what did you do? what did you see?Introductionexperimental theory, pertinant chemical reactions, reaction mechanisms, SN1 / SN2? Draw the transition stateMaterials and Methodswhat did you do? include an extraction scheme, include lots and lots of observations!Results and Discussionhow did your experiment work? what went wrong? what went right? draw TLC-plates with Rf-values, boiling points, yield (include reactant amounts), demonstrate understanding of experiment YOU ARE NOT GRADED BASED UPON YIELD

Calculation of % yieldcalculate from the limiting compound least amount of compound in the reaction% yield = 100 x n(product) / n(limiting compound) where n = amount in moles

Example: a + b c2 mol 1 mol 0.8 mol% yield = 100 x 0.8 mol / 1 mol = 80%

Day of the lab . . . .Come preparedRead laboratory protocol thoroughlyTime-consuming, so important to be familiar with laboratory protocolPerform calculations in advanceMust wear goggles (safety glasses)Dont even think of eating/drinking in the labHave fun . . .

Questions? Concerns? Comments?

PLEASE ASK!

Good luck!!!

CHCH3RBrHOactivated complexis trigonal planar (sp2 )(R)-configuration(S)-configurationconfigurationis invertedEaHO C Bpartial bonding2pTHE INVERSIONPROCESSsp3sp3sp2

BENZYL ( GOOD FOR SN1 ) IS ALSO A GOOD SN2 SUBSTRATEprimary, but faster than other primaryoverlap inthe activatedcomplexlowers theactivationenergyIBrHHcriticaloverlap