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Substitution Lab

Karolinska Institutet Department of Medical Biochemistry and Biophysics Biomedical candidate program, H08. Substitution Lab. October 31 st , 2008 Craig Wheelock craig.wheelock@ki.se http://www.metabolomics.se/ (slides can be downloaded from my homepage). Outline. Theory

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Substitution Lab

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  1. Karolinska Institutet Department of Medical Biochemistry and Biophysics Biomedical candidate program, H08 Substitution Lab October 31st, 2008 Craig Wheelock craig.wheelock@ki.se http://www.metabolomics.se/ (slides can be downloaded from my homepage)

  2. Outline • Theory • Understand substitution reactions (SN1 vs SN2) • Experimental equipment • Familiarity with the necessary equipment • Specific tips on each experiment • Tips for conducting each experiment • Safety issues • Potential hazards associated with this lab • Lab reports • What do you need to include in your lab report??

  3. NUCLEOPHILIC SUBSTITUTION NUCLEOPHILIC DISPLACEMENT leaving group substrate product nucleophile The nucleophile “displaces” the leaving group. This is a “substitution” reaction : Nu substitutes for X (takes its place).

  4. IMPORTANT: This is a reaction at sp3 (tetrahedral) carbon atoms. sp3 sp2 sp yes no Compounds that have sp2 or sp carbons generally do not give nucleophilic substitution reactions.

  5. Nucleophilicsubstitution-reaction • A “displacement”reaction of one chemical group to another R – X + Nu-→ R – Nu + X- • Nucleophilic substitution can occur by two mechanisms: SN1 and SN2 • Substitution Nucleophilic uni / bimolecular • 4 main factors • Leaving group: weak bases are better (X) • Attacking group: strong bases are better (Nu-) • Solvent: protic vs. aprotic • Sterics: steric interactions affect reaction mechanism

  6. NUCLEOPHILIC SUBSTITUTION MANY FACTORS INFLUENCE SN1 AND SN2 REACTIONS SOME PARAMETERS : a) solvent b) temp. c) pH d) DH a) structure a) structure of R, stereochemistry a) bond strength a) nature of X b) atom used b) atom used b) concentration c) concentration c) base strength c) bond strength d) base strength e) solubility f) size

  7. Alkyl halides R – X • Halides (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 basicity

  8. SN2 Reaction • Bimolecular 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 simultaneously Transition state

  9. .. R : H O .. : C Br R R - SN2 Reaction Reactivity of alkyl halides Methyl > primary > secondary >> tertiary large groups introduce steric hindrance easy access no steric hindrance R

  10. 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 H C H H C C H H 3 3 3 H + X O H C X O H H O C C X R R R ( S ) ( R )

  11. CONCEPTUAL ANALOGY INVERSION OF AN UMBRELLA IN THE WIND Inversion of the umbrella is similar in concept to the inversion of an SN2 atom.

  12. 150 1 0.01 0.001 rate rel rate = rate EtBr EFFECT OF DEGREE OF SUBSTITUTION - SN2 acetone R B r + N a N a B r OH R OH + H O 2 methyl primary secondary tertiary decreasing rate EFFECT OF SUBSTRATE ON RATE

  13. Example… SN2 CH3CH2Br + NaOH δ- δ+ Transition state bromoethane ethanol

  14. SN1 Reaction • unimolecular = 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

  15. 1st step: cleavage of alkyl halide in polar solvent RATE LIMITING! [ ] Unstable carbocation intermediate Transition state 1 2nd step: attack by the nucleophile and formation of the protonated product [ ] Transition state 2 3rd step: deprotonation of the product, an acid-base reaction

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

  17. SN1 MECHANISM 50% sp2 + - planar carbocation attacks top and bottom equally (R) 50% RACEMIZATION (S) enantiomers (R) racemic mixture

  18. SN1 Reaction • activity 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 solvatingpolarsolvents (protic) e.g., water • stabilizes the carbocation +

  19. CARBOCATION STABILITY HYPERCONJUGATION H electrons in an adjacent C-H s bond help to stabilize the positive charge of the carbocation by proximity (overlap) .. + R C C H R H lowest energy highest energy << < tertiary secondary primary

  20. 108 EFFECT OF INCREASING SUBSTITUTION - SN1 100% RBr + H2O ROH + HBr HCOOH methyl primary secondary tertiary 1.0 1.7 45 Guess ? relative rate increasing rate rate rel rate = rate CH3Br EFFECT OF SUBSTRATE ON RATE

  21. Step 1,ionization Carbocation intermediate Transition state 1 Step 2, nucleophilic attack Step 3, deprotonation Final product Transition state 2 Example… SN1 Tert-butylbromide + methanol (MeOH)

  22. SUMMARY Notice that benzyl and allyl are good for both SN1 and SN2 SN1 SN2 (fastest) (fastest) tertiary methyl** BEST BEST benzyl benzyl allyl allyl ** In SN2 reactions benzyl is actually better than methyl, but allyl is not. secondary primary primary secondary WORST bridgehead tertiary (slowest) (bicyclic) neopentyl APPROXIMATE RATE ORDERS bridgehead WORST (bicyclic) (slowest)

  23. Outline of the lab • Substitution reaction (1 of 3 reactions) • Reflux to increase reaction rate • Monitor progress by TLC (for ethyl phenyl ether) • Extract the product from the reaction mix • Wash and dry the organic phase • Remove the solvent by roto-evaporation • Purify the product by vacuum distillation and record its boiling point

  24. Reflux • Do NOT preheat the peg-bath • Use CaCl2 in the drying tube, torkrör • Use gloves with glass wool • mix well, use large magnetic stirrer • Do not let “stötkoka” (bounce) • Use 2 neck roundbottom flask, tvåhalsad kolv

  25. organic aqueous Separatory Funnel • Dry with Na2SO4 • - 1-2 spoons • cover the flask • 15-30 min • - filter - organic phase on top - watch out for gas formation

  26. Roto-evaporation (rullindunstning)

  27. 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!!!!!!

  28. 1-Bromooctane • HBr, H2SO4 • TLC not necessary • long reflux time of 4h, so get going!!! • watch for gas formation during extraction • use syringe with HBr and octanol

  29. n-Butylmalonic acid diethyl ester • fill 2 neck round bottom flask with N2 • use ice-bath to cool when mixing diethyl malonate, bromobutane, THF and NaH • after gas evolution stops, then reflux for 3h • mix well • long experiment, 3h reflux, so get going! • no TLC needed

  30. n-Butylmalonic acid diethyl ester • NaH, bromobutane (butylbromide) • NaH reacts strongly with water!!!! • releases H2 gas • be careful when using ice-bath • dry equipment!!! • quench with acetone • use NH3 / 95% EtOHto quenchbromobutane • test ether for peroxides • bromobutane and diethyl malonate in hood • use syringe to transfer bromobutane

  31. Ethyl phenyl ether • phenol, iodoethane (etyljodide) • dry equipment!!! • measure phenol in hood, no open containers • fill 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

  32. Ethyl phenyl ether • Follow reaction by TLC: • collect sample prior to refluxing!! • run TLC after 30 min • if reaction has gone to completion, stop refluxing • TLC mobile phase: • heptane:ethyl acetate 9:1

  33. For some reagents need to calculate volume from density . . . σ = m / V → V = m / σ where σ = 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

  34. Safety Issues . . . • Peroxide-test ether (with strips), mark bottle when tested • Ether is explosive – do not heat!!! • Let ether evaporate in the hood (dragskåp), do not put in organic waste • Do not preheat the PEG bath • Be careful extracting: gas formation • Dry equipment(dry overnight in drying oven)

  35. Safety Issues... • use gloves with alkyl halides • do not put them in the sink, measure in the hood • NH3/EtOH (1:1) as quenching solution (motmedel) for alkyl halides • prepare your own solution in the lab • rinse all glassware that has been in contact with RX • reuse the same solution • after rinsing wash with water

  36. weigh chemicals in hood (dragskåp) rinse all glassware in the hood first! check for residual ”smell” from previous lab do not carry around open containers with chemicals (stinks and is dangerous)! can use aluminum foil to cover containers weigh phenol in the hood Safety continued

  37. Lab reports • Abstract • experiment aim, what did you do? what did you see? • Introduction • experimental theory, pertinant chemical reactions, reaction mechanisms, SN1 / SN2? Draw the transition state • Materials and Methods • what did you do? include an extraction scheme, include lots and lots of observations! • Results and Discussion • how 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

  38. Calculation of % yield • calculate 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 → c 2 mol 1 mol 0.8 mol % yield = 100 x 0.8 mol / 1 mol = 80%

  39. Day of the lab . . . . • Come prepared • Read laboratory protocol thoroughly • Time-consuming, so important to be familiar with laboratory protocol • Perform calculations in advance • Must wear goggles (safety glasses) • Don’t even think of eating/drinking in the lab • Have fun . . .

  40. Questions? Concerns? Comments?PLEASE ASK!Good luck!!!

  41. .. R : H O R .. : C Br : HO C CH3 H CH3 H R THE INVERSION PROCESS 2p HO C B sp2 partial bonding HO Br C activated complex is trigonal planar (sp2 ) CH3 H configuration is inverted sp3 sp3 Ea (R)-configuration (S)-configuration

  42. BENZYL ( GOOD FOR SN1 ) IS ALSO A GOOD SN2 SUBSTRATE primary, but faster than other primary I overlap in the activated complex lowers the activation energy H H Br critical overlap

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