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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

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)

outline
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??
slide4

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).

slide5

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.

nucleophilic substitution reaction
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
slide7

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

alkyl halides
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

s n 2 reaction
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

s n 2 reaction10

..

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

s n 2 reaction11
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

)

slide12

CONCEPTUAL ANALOGY

INVERSION OF AN UMBRELLA IN THE WIND

Inversion of the

umbrella is

similar in concept

to the inversion of

an SN2 atom.

slide14

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

example s n 2
Example… SN2

CH3CH2Br + NaOH

δ-

δ+

Transition state

bromoethane

ethanol

s n 1 reaction
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
slide17

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

s n 1 reaction18
SN1 Reaction

• results in a racemic mixture:

  • nucleophile can attack from either side of the carbocation
  • mixture of R / S configuration of products
slide19

SN1 MECHANISM

50%

sp2

+

-

planar

carbocation

attacks top

and bottom

equally

(R)

50%

RACEMIZATION

(S)

enantiomers

(R)

racemic mixture

s n 1 reaction20
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

+

slide21

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

slide23

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

example s n 1

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)

slide25

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)

outline of the lab
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
reflux
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

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

roto evaporation
Roto-evaporation

(rullindunstning)

distillation
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
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
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 ester33
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
ethyl phenyl ether
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 ether35
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
slide36
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
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 issues38
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

Safety continued
lab reports
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
calculation of 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 → c

2 mol 1 mol 0.8 mol

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

day of the lab
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 . . .
slide44

..

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

slide45

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