Substitution Lab

October 31st, 2008Craig Wheelock

craig.wheelock@ki.sehttp://www.metabolomics.se/

(slides can be downloaded from my homepage)

Karolinska InstitutetDepartment of Medical Biochemistry and BiophysicsBiomedical candidate program, H08

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??

Nu:- + R X R Nu + :X-

NUCLEOPHILIC SUBSTITUTIONNUCLEOPHILIC SUBSTITUTION

nucleophile

substrate

product

leavinggroup

NUCLEOPHILIC DISPLACEMENT

The nucleophile “displaces” the leaving group.

This is a “substitution” reaction : Nu substitutes for X (takes its place).

IMPORTANT:IMPORTANT:

This is a reaction at sp3 (tetrahedral) carbon atoms.

CX

yes

C C

X X

C C X

no

Compounds that have sp2 or sp carbons generallydo not give nucleophilic substitution reactions.

sp3 sp2 sp

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

Nu:- + R X R Nu + :X-

NUCLEOPHILIC SUBSTITUTIONNUCLEOPHILIC SUBSTITUTIONMANY FACTORS INFLUENCE SN1 AND SN2 REACTIONS

a) structure

b) atom used

c) concentration

d) base strength

a) structure of R, stereochemistry

a) nature of X

b) concentration

a) solvent

b) temp.

c) pH

SOME PARAMETERS :

b) atom used

c) base strengthc) bond strength

a) bond strength

e) solubility

f) size

d) H

Alkyl halides• 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

R – X

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

CH3 X

Nu

CNu X

H

H

CH3 Nu + X

H

SN2 ReactionReactivity of alkyl halides

Methyl > primary > secondary >> tertiary

H3C XH2CR X

CH

R1

R2

X C

R1

R2

R3

X

C

R

Br:

H O:....

RR

easy accessno steric hindrance

large groupsintroduce steric

hindrance

C Br:

H O:.... H

H

C Br:

H O:....

H O:.... H

H

-

R

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

C

HH3C

R

XOH C

H

R

CH3

OH X C

H CH3

R

HO + X

(S) (R)

INVERSION OF AN UMBRELLA IN THE WINDINVERSION OF AN UMBRELLA IN THE WIND

Inversion of theumbrella is similar in conceptto the inversion ofan SN2 atom.

CONCEPTUAL ANALOGY

BrCH3 CH2 BrCH3 CH BrCH3

CH3

C BrCH3

CH3

CH3

150 1 0.01 0.001

decreasing rate

EFFECT OF DEGREE OF SUBSTITUTION - SEFFECT OF DEGREE OF SUBSTITUTION - SNN22

methyl primary secondary tertiary

rel rate = rate EtBr

rate

EFFECT OF SUBSTRATE ON RATE

RBr + NaOH ROH + NaBracetone

H2O

Example… SN2

C

H3C

Br

H

H

+OH C

H3C H

H

BrHO CH

CH3H

H

HO Br+

Transition state

CH3CH2Br + NaOH

δ-δ+

bromoethane ethanol

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

C XR

R

R

CHR3 X R3C + X

R3C R3COH2 OH

H

R3C OH

H

1st step: cleavage of alkyl halide in polar solvent

Transition state 1

2nd step: attack by the nucleophile and formation of the protonated product

Transition state 2

R3C OH

H

+ H2O R3C OH + H3O

3rd step: deprotonation of the product, an acid-base reaction

Unstable carbocationintermediate

[ ]

[ ]

RATE LIMITING!

SN1 Reaction

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

C

R3

R1R2

Nu Nu

R

HCH3 Br

CH

RCH3 O H

OH

HCH3 R

OH

HR CH3

+

+

enantiomers

SSNN1 MECHANISM1 MECHANISM

planarcarbocation

sp2

(S) (R)

(R)

50%

50%

racemic mixture

RACEMIZATION

-

attacks topand bottomequally

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 solvating polar solvents (protic) e.g., water • stabilizes the carbocation

C

CH3

H3C

CH3

+

CARBOCATION STABILITYCARBOCATION STABILITYHYPERCONJUGATION

CC

H..

H

H

+

R

R

electrons in an adjacent

C-H bond help to stabilizethe positive charge of thecarbocation by proximity(overlap)

CR

R

R+R CH R+

R CH2+

tertiary secondary primary

<< <lowestenergy

highestenergy

CH

H

H

Br CCH3

H

H

Br CCH3

H

CH3

Br CCH3

CH3

CH3

Br

relativerate

1.0 1.7 45

RBr + H2O ROH + HBr100%HCOOH

increasing rate

rel rate = rate CH3Br

rate

EFFECT OF INCREASING SUBSTITUTION - SEFFECT OF INCREASING SUBSTITUTION - SNN11

methyl primary secondary tertiary

Guess ?108

EFFECT OF SUBSTRATE ON RATE

Example… SN1

C

H3C CH3

H3C

Br C

H3C CH3

H3C

Br C

H3C CH3

H3C

+ CH3OH-Br-

Step 1, ionization

Transition state 1 Carbocation intermediate

C

H3C CH3

H3C

OCH3

H

C

H3C CH3

H3C

OCH3

H

C

H3C CH3

H3C

OCH3

-H+

Step 2, nucleophilic attack Step 3, deprotonation

Transition state 2 Final product

Tert-butylbromide + methanol (MeOH)

SN1 SN2tertiary methyl**

benzyl benzyl

allyl allyl

secondary primary

primary secondary

tertiary

neopentyl

Notice that benzyland allyl are goodfor both SN1 and SN2

BESTBEST

WORSTWORST

(fastest)

(slowest)

bridgehead

bridgehead

(bicyclic)

(bicyclic)APPROXIMATEAPPROXIMATERATE ORDERSRATE ORDERS

BESTBEST(fastest)

WORSTWORST(slowest)

SUMMARYSUMMARY

** In SN2 reactions benzyl is actually better than methyl, but allyl is not.

Outline of the lab1. Substitution reaction (1 of 3 reactions)

2. Reflux to increase reaction rate

3. Monitor progress by TLC (for ethyl phenyl ether)

4. Extract the product from the reaction mix

5. Wash and dry the organic phase

6. Remove the solvent by roto-evaporation

7. Purify the product by vacuum distillation and record its boiling point

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

Separatory Funnel

- organic phase on top- watch out for gas formation

organic

aqueous

Dry 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-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

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

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% EtOH to quench bromobutane

• test ether for peroxides

• bromobutane and diethyl malonate in hood

• use syringe to transfer bromobutane

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

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

• 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

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)

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

Safety continued• 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

Lab reports1. Abstract– experiment aim, what did you do? what did you see?

2. Introduction– experimental theory, pertinant chemical reactions,

reaction mechanisms, SN1 / SN2? Draw the transition state

3. Materials and Methods– what did you do? include an extraction scheme,

include lots and lots of observations!

4. 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

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 → c2 mol 1 mol 0.8 mol

% yield = 100 x 0.8 mol / 1 mol = 80%

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 . . .

Questions? Concerns? Comments?

PLEASE ASK!

Good luck!!!

C

R

HCH3

Br:C

R

HCH3

HO :

C

HCH3

R

BrHO

H O:....

activated complexis trigonal planar (sp2 )

(R)-configuration(S)-configuration

configurationis inverted

Ea

HO C B

partial bonding

2pTHE INVERSIONTHE INVERSIONPROCESSPROCESS

sp3

sp3

sp2

BENZYL ( GOOD FOR SBENZYL ( GOOD FOR SNN1 ) 1 ) IS ALSO A GOOD SIS ALSO A GOOD SNN2 SUBSTRATE2 SUBSTRATE

CH2 Br + NaI CH2 I + NaBr

primary, but faster than other primary

overlap inthe activatedcomplexlowers theactivationenergy

I

Br

H

H

criticaloverlap