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
• 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??
NUCLEOPHILIC SUBSTITUTION
NUCLEOPHILIC DISPLACEMENT
leaving
group
substrate
-
Nu:
+
nucleophile
R
X
R Nu +
product
The nucleophile “displaces” the leaving group.
This is a “substitution” reaction :
Nu substitutes for X (takes its place).
:X
-
IMPORTANT:
This is a reaction at sp3 (tetrahedral) carbon atoms.
sp3
sp2
C
yes
sp
C C
X
C C X
X
X
no
Compounds that have sp2 or sp carbons generally
do not give nucleophilic substitution reactions.
Nucleophilic
substitution-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
NUCLEOPHILIC SUBSTITUTION
MANY FACTORS INFLUENCE SN1 AND SN2 REACTIONS
SOME PARAMETERS :
-
Nu:
+
R
X
a) solvent
b) temp.
c) pH
R Nu +
:X
-
d) DH
a) structure
b) atom used
c) concentration
d) base strength
e) solubility
f) size
a) structure of R,
stereochemistry
b) concentration
c) bond strength
a) bond strength
a) nature of X
b) atom used
c) base strength
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
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
H
CH3
Nu
X
Nu
X
C
H
H
Transition state
CH3
Nu
+X
SN2 Reaction
Reactivity of alkyl halides
R1
R1
H3C
X
R
H2
C
CH
X
R2
X
R2
C
X
R3
Methyl > primary > secondary >> tertiary
easy access
no steric hindrance
.. H O
..:
..
H O
..:
H
C
H
large groups
introduce steric hindrance
R
C
: Br
R
R
R
: Br
SN2 Reaction
• Results in inversion of configuration
if there is a chiral center, then R S
H3C
OH
C
R
(S)
H
H
X
C
OH
CH3
X
H
HO
CH3
+X
C
R
R
(R)
• Supported by polar solvents that do not solvate
the nucleophile (aprotic solvents), e.g., DMSO
CONCEPTUAL ANALOGY
INVERSION OF AN UMBRELLA IN THE WIND
Inversion of the
umbrella is
similar in concept
to the inversion of
an SN2 atom.
EFFECT OF DEGREE OF SUBSTITUTION - SN2
RBr + Na OH
methyl
primary
acetone
H2O
R OH + NaBr
secondary
tertiary
CH3
CH3 Br
CH3 CH2 Br CH3 CH Br CH3
CH3
150
1
0.01
C Br
CH3
0.001
decreasing rate
EFFECT OF SUBSTRATE ON RATE
rate
rel rate =
rate EtBr
Example… SN2
CH3CH2Br + NaOH
H3C
OH
+
H3C
H
C
δ+
δ-
Br
H
HO
H
C
CH3
H
Br
H
HO
CH
H
Transition state
bromoethane
ethanol
+
Br
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
1st step: cleavage of alkyl halide in polar solvent
R
R
CHR3
X
C
R
[
X
Transition state 1
R3 C
]
+X
Unstable carbocation
intermediate
RATE
LIMITING!
2nd step: attack by the nucleophile and formation of the protonated product
H
H
[ R3 C ]
R3C
OH2
R3 C
OH
OH
Transition state 2
3rd step: deprotonation of the product, an acid-base reaction
H
R3C
OH
+ H2O
R3C
OH
+ H3O
SN1 Reaction
• results in a racemic mixture:
– nucleophile can attack from either side of the carbocation
– mixture of R / S configuration of products
R2
R1
Nu
C
R3
Nu
SN1 MECHANISM
R
CH3
H
sp2
CH3
Br
50%
+
C
CH3
(S)
+
H
R
attacks top
and bottom
50% equally
OH
OH
RACEMIZATION
-O H
H
planar
carbocation
(R)
R
R
H
enantiomers
racemic mixture
CH3
(R)
• activity order of alkyl halides
SN1 Reaction
tertiary > secondary > primary > methyl
in practice only occurs with tertiary & secondary
– more stable carbocation
– more atoms share the positive charge
CH3
H3C
C+
CH3
• activated by solvating polar solvents (protic) e.g., water
• stabilizes the carbocation
CARBOCATION STABILITY
HYPERCONJUGATION
H
..
electrons in an adjacent
+
R
C C
H
R
C-H s bond help to stabilize
the positive charge of the
carbocation by proximity
(overlap)
H
R
lowest
energy
R C
+ R
tertiary
<<
R CH R
+
secondary
<
R CH2
+
primary
highest
energy
EFFECT OF INCREASING SUBSTITUTION - SN1
RBr + H2O
methyl
H
H C Br
relative
rate
100%
HCOOH
primary
H
ROH + HBr
secondary
H
CH3 C Br
tertiary
CH3
CH3 C Br
CH3 C Br
H
H
CH3
CH3
1.0
1.7
45
Guess
108 ?
increasing rate
EFFECT OF SUBSTRATE ON RATE
rate
rel rate =
rate CH3Br
Example… SN1
Tert-butylbromide + methanol (MeOH)
Step 1, ionization
H3C
CH3
C
H3C
Br
CH3
C
H3C
-Br-
CH3
C
H3C
Carbocation intermediate
Step 3, deprotonation
H3C
OCH3
H
Transition state 2
+ CH3OH
H3C
Transition state 1
H3C
CH3
C
Br
H3C
Step 2, nucleophilic attack
H3C
CH3
C
H3C
-H+
H3C
C
OCH3
H
CH3
OCH3
H3C
Final product
SUMMARY
SN1
(fastest)
BEST
WORST
(slowest)
SN2
tertiary
methyl**
benzyl
benzyl
allyl
allyl
secondary
primary
primary
secondary
bridgehead
tertiary
(bicyclic)
APPROXIMATE
RATE ORDERS
Notice that benzyl
and allyl are good
for both SN1 and SN2
(fastest)
BEST
** In SN2
reactions
benzyl is
actually better
than methyl,
but allyl is not.
neopentyl
bridgehead
(bicyclic)
WORST
(slowest)
Outline of the lab
1. 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
Dry with Na2SO4
- 1-2 spoons
- cover the flask
- 15-30 min
- filter
Separatory Funnel
organic
aqueous
- organic phase on top
- watch out for gas formation
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
1. Abstract
–
Lab reports
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 → c
2 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!!!
THE INVERSION
PROCESS
sp2
HO
R
C
CH3 H
..
H O
..:
R
C
2p
HO
Br
C
B
partial bonding
activated complex
is trigonal planar (sp2 )
configuration
is inverted
sp3
R
: Br
Ea
CH3
H
(R)-configuration
HO :
sp3
C
H
CH3
(S)-configuration
BENZYL ( GOOD FOR SN1 )
IS ALSO A GOOD SN2 SUBSTRATE
primary, but faster
than other primary
CH2 I + NaBr
CH2 Br + NaI
I
H
H
critical
overlap
Br
overlap in
the activated
complex
lowers the
activation
energy