Chemical Biology 3OA3
P.H. Harrison
SEPTEMBER 2009
CHEM BIO 3OA3
TABLE OF CONTENTS
Page
Schedule.......................................................................................................................................... 3
Evaluation ....................................................................................................................................... 4
Safety Bulletin................................................................................................................................. 5
Check In/Out Procedures .............................................................................................................. 12
Objectives ..................................................................................................................................... 15
Experiment 1: Isolation of Caffeine from Tea............................................................................ 22
Experiment 2: 1-Benzyldihydronicotinamide from 1-Benzylnicotinamide Chloride…………..25
Experiment 3: Sugar Anomers: α and β–D-Glucose penta-acetate............................................ 28
Experiment 4: Catalysis by Yeast: reduction of 1-phenyl-1,2propanedione…………………...33
Experiment 5: Solid Phase Synthesis of a Peptide ..................................................................... 38
Experiment 6: Synthesis of the Antiepileptic Drug Dilantin, by a Biomimetic Synthesis ......... 47
Experiment 7: Synthesis of Diesters, and Cleavage by Orange Peel Esterase…...……………..52
Experiment 8: Claisen Condensation: Microscale Synthesis of 4-Hydroxycoumarin................ 60
Appendix 1:
Microscale Laboratory Techniques..................................................................... 63
Appendix 2:
Spectra of Products from Labs………………………………………………….71
NOTE: page numbers are approximate and will depend on your printer
Chemical Biology 3OA3, September 2009
Page 2
CHEM BIO 3OA3 SCHEDULE – SEPTEMBER 2009
All labs take place in ABB-217. This is an approximate schedule only. Work to
your own pace. Your TA will inform you when reports are due.
NOTE: in case of sickness for 1 week, you should still be able to complete all labs,
and therefore do not need a Deans’ note. In case of a second absence from the lab,
a Deans’ note must be obtained, and you should discuss how to proceed with the
instructor.
Date
Experiment
Brief Title
September 17/18
Check-in & 1
Check-in and Caffeine from Tea
September 24/25
2
Sugar Anomers
October ½
3
1-Benzyl-dihydronicotinamide
October 8/9
4
Baker’s Yeast Reduction
October 15/16
5
Peptide Synthesis
October 22/23
5
Peptide Synthesis
October 29/30
5
Peptide Synthesis
November 5/6
6
Synthesis of Dilantin
November 12/13
6/7
November 19/20
7
Orange Peel Esterase
November 26/27
8
Microscale Claisen Condensation
December 3/4
-
Completion and Check out
Check-out
extra session if necessary
TBA
Chemical Biology 3OA3, September 2009
Synthesis of Dilantin/Orange Peel Esterase
Page 3
EVALUATION
The final grade in CHEM BIO 3OA3 is normally determined on the basis of the
following scheme. However, the instructor and university reserve the right to modify elements of
the course during the term. The university may change the dates and deadlines for any or all
courses in extreme circumstances. If either type of modification becomes necessary, reasonable
notice and communication with the students will be given with explanation and the opportunity
to comment on changes. It is the responsibility of the student to check their McMaster email and
course websites weekly during the term and to note any changes.
Marking Scheme
Midterm Test
20 %
Assignments
10 %
Laboratory
20 %
lab reports 15%
practical mark 5%
safety
0%
(negative marks may be awarded)
Final Exam
50 %
100 %
The written laboratory reports are worth 15%. The practical mark is worth 5% and is
assigned by the teaching assistants on the basis of each student's performance in the laboratory,
based on preparation, understanding of the material, organization in the laboratory and technique.
The third part of the mark is for safety practices. Any safety violations (such as not wearing
goggles) will result in lost marks, up to the full 20% of the laboratory component.
Laboratory
Marks
Performance
Experiment 1
10
5
Experiment 2
10
5
Experiment 3
10
5
Experiment 4
10
5
Experiment 5
30
15
Experiment 6
20
10
Experiment 7
20
10
Experiment 8
10
5
NMR spectrum report
10
N/A
Total
130
60
Final lab mark(/20) = Σ(Marks X 15/130) + Σ(Performance/12)
Chemical Biology 3OA3, September 2009
Page 4
SAFETY BULLETIN
A chemical laboratory is potentially a dangerous place, and safety precautions should be the
concern of everyone who works in one. The following notes are provided to assist you in avoiding
unnecessary accidents, and to indicate the action to take should an accident occur. Study this
material carefully.
SAFETY EQUIPMENT
1.
FIRE ALARMS - PULL STATIONS
Are located in all corridors near corridor and building exits.
2.
FIRE EXTINGUISHERS
Know the location of the fire extinguishers in your laboratory and read the directions for
their operation. They are very effective for fires involving organic liquids and electrical
wiring.
3.
SHOWERS
There is an emergency shower in each laboratory. These are for use when corrosive liquids
have spilled over large areas of clothes and skin, and when clothing is afire. Become
familiar with the location of these showers.
4.
EYE-WASH FOUNTAINS
Learn the location of the eye-wash in your lab. In the event that a chemical has been
splashed into the eyes, immediate action must be taken to prevent damage to the sensitive
tissues in the eyes. The person (with assistance if it is available) should wash the eyes with
water for at least 10 minutes. Roll the eyeballs to ensure thorough washing. Seek medical
attention immediately.
5.
PANIC ALARMS
Are in all lab rooms. Know their location. These are used to alert Security to an emergency
within the lab room. When activated, a continuous low pitch alarm will sound and an
emergency light in the corridor outside of the lab room will flash.
6.
WATER
Chemical burns do not often become serious if the affected part is washed promptly and
thoroughly with water. Water -- lots of it -- is the first treatment for all accidents in which
corrosive chemicals have been spilled or splashed on external skin surfaces.
7.
SAFETY GOGGLES
Safety goggles approved by the Chemistry Department must be worn by all persons while
working in all laboratories (Instrumental Labs exempt).
Chemical Biology 3OA3, September 2009
Page 5
This includes persons wearing prescription glasses.
Contact Lens MUST NOT be worn in the Laboratory
8.
CLOTHING
Lab coats are strongly recommended.
Shorts and Sandals are not to be worn in the lab; all loose or long hair must be tied back.
Clothing or unnecessary books must be placed in designated ‘coat’ drawers.
9.
FACE SHIELDS
The use of face shields is recommended when high vacuum distillations are being done in
glass equipment and when there is a high possibility that a violent reaction can occur.
These shields can be obtained from the Technician.
10.
GLOVES
Plastic gloves should be worn at all times when handling chemicals. It is important to note
that gloves provide an immediate protective effect; however, some chemicals can permeate
or even dissolve gloves. Therefore, if chemicals are spilled on the gloves, they should be
removed immediately and replaced.
10.
FIRST AID
First Aid Kits are in each laboratory. Become familiar with their location and contents. In
the event of an injury report to a TA who will take the necessary action. Do not neglect
even small cuts or burns. These can become very serious if they are not treated promptly
and properly. The first treatment for burns is ice or cold water.
11.
SAND PAILS
The sand, in pails in each of the laboratories, is for smothering small fires. It is particularly
useful for sodium or potassium fires.
12.
SPILL KITS
Acid, Base and Solvent Spill kits are provided in each lab room for use in the event of a
chemical spill. Know their location and how to use them.
13.
AIR PACK (BREATHING APPARATUS)
(TO BE USED BY TRAINED PERSONNEL ONLY)
ABB. - 1st floor, beside Room 113
14.
MSDS
Material Safety Data Sheets are kept in the Technician’s prep lab and are available for your
review.
Chemical Biology 3OA3, September 2009
Page 6
SAFETY IN LABORATORY OPERATIONS
1.
Accidents occur every year from the careless handling of glassware. These result in
considerable inconvenience and loss of time. These kinds of accidents could be avoided if
the following rules are observed.
(a)
Before inserting a thermometer or glass tube into a stopper or rubber tube:
i)
Be certain the hole is large enough to accommodate the glass.
ii)
Lubricate the glass and the rubber with glycerol or stopcock grease.
iii)
Protect your hands by holding the stopper and the glass in towels. Hold
the stopper by your fingers NOT IN THE PALM OF YOUR HAND.
iv)
Grasp the glass close to the end that is to fit into the stopper and twist
with an even pressure.
(b)
Do not attempt to push or pull glass tubing or thermometers from rubber tubing,
corks or stoppers which have become hardened. Cut the rubber or cork from the
glass.
(c)
Fire-polish the ends of all glass tubing and glass rods.
(d)
Do not try to force an oversized stopper into a flask or bottle. Use the proper
size stopper.
(e)
When picking up a beaker, the fingers should be placed around the outside, not
over the rim. If the beaker is hot, use tongs.
(f)
Never point test tubes at your neighbour or yourself when heating substances.
2.
ALL BOTTLES CONTAINING CHEMICALS should be plainly labelled. Materials
found in unlabelled bottles should be returned to the Assistant.
3.
FLAMMABLE VOLATILE LIQUIDS such as alcohol, ether, etc., must never be
distilled or evaporated over an open flame. Furthermore, there must not be an open
flame in the neighbourhood of such operations. A column of flammable vapour can be
wafted towards a lighted burner ten feet or more away and become ignited to strike back
to the flask or bottle containing the flammable liquid and set it afire.
Be careful always to keep all bottles and flasks, containing flammable and volatile liquids,
well stoppered, and never open them near a flame. Care should also be exercised in
preventing these flasks or bottles from becoming heated by steam pipes or other sources of
heat, since the internal pressure may become sufficient to blow out the stopper.
Careful attention should be given to such sources of ignition as sparking brushes from
electric motors and exposed heating elements.
Chemical Biology 3OA3, September 2009
Page 7
4.
WASTE CHEMICALS or BROKEN GLASS placed in regular garbage bins can cause
injury to the persons required to dispose of that garbage. For this reason all laboratory
waste must be put in the appropriate container.
(a)
CLEAN WASTE (bin near each large sink). Items such as paper towels,
J-cloths, corks, rubber stoppers, plastic vials and filter papers can be put in the
regular garbage AFTER THEY HAVE BEEN CLEANED OF ALL
CHEMICALS.
(b)
GLASS (plastic pails labelled for glass disposal). All waste glass (with the
exception of thermometers containing mercury, unknown sample vials and
melting point capillaries and slides) must be placed in glass disposal bins. THE
GLASS MUST BE CLEAN.
(c)
BROKEN THERMOMETERS (fume hoods). Labelled containers are
provided in a fume hood at the front of the lab for broken thermometers which
contain mercury.
(d)
SAMPLE VIALS (fume cupboards). Dispose of any chemical remaining in the
vial (use the proper waste container) and place the empty vial in the labelled
container located in the waste fume hood.
(e)
MELTING POINT WASTE (fume cupboards). Labelled containers are
provided in the fume cupboards for used melting point capillaries and cover
slides.
(f)
CHEMICAL WASTE (fume cupboards). Labelled bottles are provided in the
fume cupboards for the disposal of all chemical waste. The bottles are labelled
for the following categories of chemical:
ORGANIC
ORGANIC HALOGENATED
INORGANIC ACIDS AND SALTS
ACID SENSITIVE SALTS
INORGANIC BASES
SOLIDS
It is extremely important that all waste go into the proper container. Injuries
have resulted in the past from the careless mixing of chemical waste. If you
have any questions consult your T.A. before proceeding.
Chemical Biology 3OA3, September 2009
Page 8
5.
EATING, DRINKING AND SMOKING are not allowed in the laboratory.
6.
HORSEPLAY, stunt experiments, or experiments not assigned are strictly forbidden
in all laboratories. The Department has the right to banish an offender from the
laboratories.
7.
CLEANING SOLUTION of the sulfuric-dichromate type should be treated with
respect. Any splattering will destroy clothing and quickly produce painful skin
burns. Under no circumstances should nitric acid be used to clean vessels that
contain organic material, because an explosion or a fire may ensue.
8.
WATER OR CHEMICALS spilled on the floor must be mopped or cleaned up
promptly. Spill Kits are provided in each laboratory for the neutralization of spilled
chemicals. Never leave Apparatus containing corrosive materials around where
someone might be burned. When diluting
an acid, pour the acid into water.
NEVER POUR WATER INTO AN ACID.
9
GOOD TECHNIQUE in assembling apparatus and in proceeding methodically greatly
reduces the possibility of accidents. An important aspect of good technique is
cleanliness. If the student who used your work station before you put away the clamps,
hot plates, heating mantles, etc., you would find it much easier to perform your
experiment and there would be less chance of an accident occurring. Keep this in mind
as you clean up your area before leaving the lab.
10.
MOST CHEMICAL REAGENTS ARE POTENTIAL HAZARDS. Every chemical is
injurious if a sufficient quantity enters the body. Proper methods of handling, adequate
precautions, and acknowledge of protective measures and first aid should be learned
NOW. Otherwise, the penalty may be impaired health and a shortened life. Material
Safety Data Sheets are provided in the lab rooms. Refer to these books if you have
questions about the toxicity or characteristics of a chemical.
11.
NO UNDERGRADUATE STUDENT is permitted to work in any laboratory unless a
teaching assistant is present.
12.
ALWAYS WASH YOUR HANDS before leaving the laboratory.
13.
Sign and complete last page of this bulletin and place it in the box before you leave on
the first lab day.
Chemical Biology 3OA3, September 2009
Page 9
COMPLETE THIS PAGE AND KEEP IT FOR YOUR INFORMATION
The location of the Fire Alarm Pull Station nearest to my locker is:
List four (4) pieces of Emergency Safety Equipment located by the lab door:
1.
2.
3.
4.
The location of the First Aid Kit nearest to my locker is:
Locate and list the three (3) different types of Chemical Spill Kits:
1.
2.
3.
Be prepared! Know the location and proper use of emergency safety equipment
and be familiar with safety rules and emergency procedures.
Chemical Biology 3OA3, September 2009
Page 10
COMPLETE THIS PAGE AND RETURN IT TO YOUR TEACHING ASSISTANT
BEFORE YOU LEAVE THE ROOM
Please note the location of all room, hallway and building exits.
The location of the Fire Alarm Pull Stations nearest to my locker is:
A Red Panic Alarm Button is located by at least one lab room door. List three (3) other pieces of
Emergency Safety Equipment located at the lab door.
1.
2.
3.
The location of the First Aid Kit nearest to my locker is:
List the 3 types of Spill Kits and their location:
1.
2.
3.
STUDENT INFORMATION: (please print clearly)
NAME:
_________________________
STUDENT ID NO: ________________________
EMAIL ADDRESS: ________________________
LAB COURSE:
________________LAB DAY:________________
ROOM NO:
________________LOCKER NO:
____________
Name and phone number of friend or relative to be called in case of an emergency:
Phone No:
(please include the area code)
I ACKNOWLEDGE HAVING READ THE SAFETY BULLETIN AND I UNDERSTAND
THE SAFETY INFORMATION DESCRIBED THEREIN.
I AGREE TO WEAR
PROTECTIVE EYE GOGGLES AT ALL TIMES IN THE LABORATORY.
Signature:
Chemical Biology 3OA3, September 2009
Date:
Page 11
PROCEDURES
A.
Procedure for the Checking of Laboratory Locker Equipment
1.
The student reports to the appropriate laboratory and at each station will find:
(i)
Locker equipment sheet.
(ii)
Locker number and key.
PLEASE NOTE: 50 cent cash fine to have locker opened when key is forgotten.
(iii)
Safety Bulletin, if not included in Lab Manual.
2.
Sign and complete last page of Safety Bulletin and place in box in lab before you leave the lab on the
first day.
3.
Print name, I.D. No., and date on locker equipment sheet.
4.
Place all items from the locker on the bench and after checking them against the locker equipment
sheet, return to the locker only those items which are listed on the locker equipment sheet. Any
surplus equipment noticed in locker, during the course, should be returned to the extra equipment
box.
5.
Report all discrepancies or damage, to the Assistant. If the Assistant decides that a damaged item is
still usable or a deficiency is acceptable, then the assistant should record the details and initial it on
your equipment sheet.
6.
The Assistant will send you to the Technician if replacement items are needed.
7.
If a replacement item is not available the technician will record the details and initial it on your
equipment sheet. Outstanding items will be replaced as soon as possible
Sign the equipment sheet ONLY AFTER your assistant has signed that he/she considers it complete.
Chemical Biology 3OA3, September 2009
Page 12
You are responsible for all the chargeable items in your locker after you sign the sheet.
Place your signed equipment sheet in the box in your room.
CLAIMS FOR SHORTAGES WILL NOT BE RECOGNIZED AFTER THIS TIME.
B.
Procedure for Breakage etc., During Course
When the cost of replacement is $1.50 or more (see equipment sheet for prices -- posted in
lab), the student is to obtain replacements from the Technician
1st year:
ABB 127
3rd Floor
ABB 308
2nd Floor:
ABB 222,
4th Floor
ABB 412
(you must present your ID Card). You will be billed at the conclusion of the term. Any item
charged out of the store is non-returnable. Less expensive items can be obtained from the
Technician at no charge. If a student breaks any item, which is used in common, this student
will be charged for such, to a maximum of $30.00 per item.
C.
Procedure when student withdraws during course.
At that time, the student must arrange with the technician for check-out of locker equipment:
In the case where a student withdraws because of illness and is unable to attend to check-out,
such a student must inform in writing, one of the above so that the locker equipment can be
checked-out by our personnel. The locker key(s), if any, must be returned. Failure to
comply with the above will result in the following charges being made:
NOT CHECKING-OUT:
$10.00
KEY:
$ 5.00
CLEANING OF DIRTY LOCKER EQUIPMENT:
$10.00
PLUS COST OF REPLACEMENT OF BROKEN OR MISSING EQUIPMENT.
D.
Safety Goggles.
If safety goggles are forgotten, the Technician will provide such on a temporary loan by
charging $2.00 cash and a deposit of your student ID card.
Chemical Biology 3OA3, September 2009
Page 13
E.
Checking-out Laboratory Lockers
The following procedures are to be used for the "checking-out" of lockers on the last day of
the laboratory course, or when withdrawing from the course.
1.
Remove everything from your locker and wipe the locker clean.
2.
Make certain you have all the items on your locker list (including non-chargeables) and that
everything is clean and not chipped, cracked or broken.
3.
Return to the locker only those items listed on your equipment sheet.
4.
Ask your teaching assistant to inspect the equipment and to sign the sheet when the locker is
complete.
5.
Lock your locker.
6.
If your locker key is not stamped with the room and locker no. - label it and attach it to your
equipment sheet.
7.
Give the completed equipment sheet to your TA.
THE LOCKER KEY must be returned or you will be charged $3.00 for each key
All students must "CHECK-OUT" their lockers on the scheduled day or on withdrawal from course or they
will be fined $10.00. An additional charge of $10.00 will be levied for the cleaning of dirty locker
equipment.
The onus is on the STUDENT to obtain the teaching assistant's signature on the equipment sheet, indicating
that "CHECK-OUT" is complete and satisfactory.
Updated: September 11,2009
Chemical Biology 3OA3, September 2009
Page 14
Chemical Biology 3OA3, September 2009
Page 15
Chemical Biology 3OA3, September 2009
Page 16
CHEMICAL BIOLOGY 3OA3
Objectives
The purpose of these laboratory experiments is to illustrate and reinforce some of the
lecture material of Chemical Biology 3OA3, as well as to improve and extend your practical
ability. You will carry out a sequence of technique and synthetic experiments. Other
experiments illustrate particular effects, such as the anomeric effect (Lab 2), and allow you to
explore the nature of catalysis, both in chemistry and in biology.
Preparation
Do not come into a laboratory unprepared. Read and understand the experiment ahead of
time. If you do not do this you will be unable to appreciate fully what is going on and be unable
to plan and use your time efficiently.
Safety and Cleanliness
Many of the compounds and solvents that you will be handling are highly inflammable
and/or highly toxic. Stay alert to these hazards by checking for flames before pouring volatile
solvents and, conversely, by checking for fumes before lighting a burner. In case of a fire, DO
NOT PANIC, retreat to a safe position and call a demonstrator. Avoid inhalation of vapours.
Wash quickly and thoroughly with lots of water if you get chemicals in contact with the skin.
Glassware can generally be cleaned with a brush, soap, and water. Stubborn gums and tars
may respond to acetone or other solvents. The demonstrators may be able to advise you with
respect to the best solvent for a given job. Use these solvents sparingly, washing several times
with a small volume rather than once with a large amount. Keep your apparatus and your
working area clean. In general, the longer you leave apparatus dirty, the harder it is to clean! Do
your share of the tidying up of communal areas like reagent shelves, hoods and weighing
stations.
Chemical Biology 3OA3, September 2009
Page 17
Notebooks
Use a bound hard cover notebook.
Duplicate notebooks, such as used in CHEM
2OA3/2BA3/2OB3/2BB3, are REQUIRED. The instructor will not be responsible for lost lab
books; therefore failure to use a duplicate book may result in (a) mark(s) of 0 for laboratories.
A laboratory notebook is a diary of the laboratory work. IT SHOULD BE WRITTEN AS THE
EXPERIMENT IS CARRIED OUT AND SHOULD BE AS BRIEF AS POSSIBLE. Remember,
it must be precise and contain enough information to trace a mistake or allow another person to
repeat the work.
Record in your notebook everything which you do and observe as you carry out the experiment.
Write in the past tense. Do not copy out the laboratory instructions.
TLC plates: Do not tape TLC plates into your lab notebook. Trace/sketch out the plate into your
notebook showing all spots, their positions and intensity, then dispose of the TLC plate.
It is a waste of time to spend hours decorating a laboratory notebook, nor does it impress the
instructor. A notebook should be neat but need not be a work of art. Clarity of procedure and
results are what count. A general outline for a report as well as a specific example follow.
Note: This type of laboratory report is written at least as far as the conclusion during the lab
period. You have enough to do as it is without spending evenings writing up labs. In some
cases, you may wish to record a yield, melting point, or spectrum in the lab period after the one
in which the preparation was carried out. In this case, be prepared to write these results up in the
lab, and hand your report in.
Chemical Biology 3OA3, September 2009
Page 18
General Outline
Title
Date
References:
Only include sources you have examined.
Purpose:
A one sentence description of the experiment being performed.
*Equations:
If any. It is often convenient to indicate amounts of materials, etc. under
the equation.
*Procedure:
See next page.
*Results:
Yield, properties of product (m.p., b.p., colour, etc.) Calculation of %
yield. Draw TLC plates.
Conclusions:
Only some laboratories require conclusions. In preparative experiments,
usually results speak for themselves. In problem-based labs, give a one-totwo sentence conclusion, e.g. the product was the α-anomer (expt.2).
Often problems are posed at the end of the instruction sheets and these
should be briefly answered at this point.
Rough Work:
One way of organizing your notebook is to use the left-hand page for
weighings, calculation of quantities, etc., and the right-hand page for the
main description of each experiment.
*If the laboratory involves a sequence of reactions, it is usually best to write out the equation,
procedure and results of each separately.
Chemical Biology 3OA3, September 2009
Page 19
Sample Write Up from a Research Notebook:
Attempted Reaction of 8,8-Dimethylbicyclo[5.1.0]octa-3, 5-dien-2-one with Methyl Lithium
Procedure
The methyl lithium solution was added dropwise to a stirred solution of the ketone in
ether at -78 oC under Ar. The mixture was kept at -78 oC for 1 h. after addition, then
warmed to 0 oC and H2O (3 mL) was added through a syringe.
The ether layer was separated, and the water layer was washed with Et2O (2 x 3 mL).
The combined ether layers were dried (K2 CO3) and ether was evaporated to give an oily
solid.
The solid was recryst. from pentane, then sublimed (50 oC/10 mm) to give a colourless
solid.
Results
Yield 45 mg; 45/164 = 0.274 mmol;
0.274/0.5 = 55%
mp 61-64 oC
1
H nmr (CS2 , 60 MHz) δ 5.95 (t, J=7 Hz, 1H, C=CH), 5.50 (m, 3H, C=CH), 1.70 (d, J=9 Hz,
1H, cyclopropyl-H), 1.30 (m, 2H, OH and cyclopropyl-H), 1.30 (s, 3H, CH3 ), 1.19 (s, 3H, CH3
), 1.08 (s, 3H, CH3 ).
Conclusion
Sample appears pure (mp and 1H nmr). Seems to be desired tertiary alcohol.
Need infrared and C/H analysis. OK to try next step. Stereochemistry?
Chemical Biology 3OA3, September 2009
Page 20
Experiments
Work in an organic chemistry laboratory falls generally into one of two categories: preparative
(synthesis) and physical organic experiments.
(A)
Organic Preparations:
Preparative work usually involves 3 steps:
1.
Performing the reaction;
2.
Isolation and separation of the product; and
3.
Establishing the identity and purity of the product (characterization).
The first experiment involves steps 2 and 3: caffeine in tea leaves is separated and
characterized.
You will then perform some one or two step reactions, and then perform the
preparation of a peptide by chemical synthesis. The later sequences involve several steps,
and it is important that you take great care with the early steps, since if these are not done
properly the later steps will also fail. In this course, the structure of the products will be
compared by spectroscopy.
(B)
Physical Organic Experiments
Physical organic chemists measure properties of molecules; for example, the rate
of a chemical reaction may be correlated with the structures of a range of similar starting
materials to gain insight into the reaction mechanism.
In this course, you will investigate various physical effects, such as the anomeric
effect (Lab 2), which are relevant to enzyme catalysis, as we will see in class; and the
effect of substituents on the rate of the enzyme orange peel esterase (Lab 7).
Chemical Biology 3OA3, September 2009
Page 21
EXPERIMENT 1
ISOLATION OF CAFFEINE FROM TEA
In this experiment, a common alkaloid natural product, caffeine, responsible for the mild
stimulating action of tea and coffee, will be isolated from tea leaves, purified and characterized
by preparation of a derivative. Caffeine is a derivative of one the bases found in nucleic acids,
and provides a good illustration of the chemistry of these bases.
The caffeine will be extracted by hot water from tea leaves, where it is present to the extent of
about 5%. This treatment also extracts the tannins, another class of compounds present in tea. It
is therefore necessary to separate the caffeine from the tannins by adding lead acetate to the
extract. The tannins are acidic and form insoluble lead salts, whereas the caffeine remains in
solution. The caffeine will be extracted from the aqueous solution with chloroform. It can be
purified by recrystallization or sublimation.
Safety Caution:
Lead is toxic: handle carefully and be environmentally friendly: dispose
as per instructions.
USE PROTECTIVE GLOVES WHILE HANDLING THE LEAD SOLUTIONS.
Procedure
Add 20 g of dry tea leaves to 150 mL of water contained in a 400 mL beaker, cover the beaker
with a watch glass and boil the suspension for 10 minutes. [NOTE: it is fastest to start heating
the water as soon as you arrive in the lab.] Remove the heat source, add 40 mL of a 10% lead
acetate solution, and 30 mL water. Stir thoroughly to precipitate the lead salts of the tannins.
Allow the leaves and precipitate to settle, and decant the hot supernatant liquid through a
Buchner filter (with paper and vacuum). To facilitate the filtration prepare a Celite pad 5 - 6 mm
in thickness on top of the filter paper. To do this make a slurry of Celite in H2O (make a rough
calculation based on the size of your Buchner funnel) and carefully filter it under vacuum so that
you end up with a pad of uniform thickness. Now carefully filter the tea-leaf mixture through the
Celite pad under vacuum. Discard the leaves and precipitate into the waste pail provided.
At this point, the filtrate should be clear; if solid has passed through the filter, be sure to filter
again, otherwise there are problems later. Transfer the filtrate to a clean 400 mL beaker and
concentrate the solution to a volume of 50 mL by heating on a hot-plate.
Cool the concentrate to room temperature, transfer it to a 125 mL separatory funnel, and
extract the aqueous solution three times with three 20 mL portions of methylene chloride
(CH2Cl2). Methylene chloride is more dense than water and forms the lower layer. Swirl the
funnel to break up any slight emulsion that may form. The addition of 5-10 mL of sat. aqueous
sodium chloride (brine) may be required to “break” the emulsion. Discard the aqueous layer,
transfer the combined CH2C12 extracts to the separatory funnel, and once again withdraw the
clear CH2Cl2 layer (leaving behind 2-3 mL of a tan-brown aqueous layer; make sure you take
ONLY the organic layer here) into a dry 125 mL distilling flask. Remove most of the CH2Cl2 on
the rotary evaporator. Your TA will demonstrate how to use this equipment. The crude caffeine
will spontaneously crystallize as a colorless solid. The yield is about 200-400 mg.
Chemical Biology 3OA3, September 2009
Page 22
Recrystallization.
[NOTE: review the process of recrystallization here: it is NOT a question of dissolving the
compound in solvent then letting the solvent evaporate: this simply returns all the impurities to
the sample!]
Transfer about one third of the crude caffeine to a clean 50 mL beaker, add 5 mL of toluene,
and heat on a steam or water bath (HOOD!) to dissolve the caffeine. Remove the beaker from
the heat source, add 10 mL of petroleum ether (boiling range 60-90o), COVER with a watch
glass, and allow the caffeine to crystallize. If the solution is appreciably coloured, add a small
amount of charcoal ("norit")-sufficient to give a dark suspension. This should remove the
coloured impurities, and after filtration, colourless crystals should be obtained. Collect the
product by vacuum filtration (Hirsch funnel), wash it with 1 mL of petroleum ether, allow it to
air dry, and determine its melting point.
NMR spectra:
Your objective in this course is to obtain at least one NMR spectrum of a product that you made
yourself. You might choose caffeine, and run the spectrum now, or you might choose a (perhaps
more interesting) sample later in the course. Your TA will help you choose when to run your
sample.
When you run your own sample, you should write a SEPARATE brief report. This will not be
due at the same time as the lab from which the sample came. Consult your TA for when this
NMR report is due. It will be marked separately from the lab report, and will contribute to your
overall lab mark, as indicated in the introduction.
In this report, you should provide a thorough interpretation of the NMR spectrum you obtained.
Is the spectrum consistent with the proposed structure, or not? Which resonances, and their
chemical shifts, integrals, multiplicities and coupling constants, support your interpretation? Is
the sample pure? Does the spectrum correspond to the standard spectrum provided in the
Appendix? What resonances correspond to any impurities? E.g. “the proton NMR spectrum
shown exhibits the expected doublet at δ 6.5, corresponding to the olefinic proton….. However,
the triplet at 1.2 ppm, the singlet at 2.0 ppm and the quartet at 4.0 ppm indicate that ethyl acetate
is present. This is not unexpected, since the compound was recrystallized from ethyl acetate, and
was only dried for 1 hour prior to recording the NMR spectrum.”
HOWEVER, spectra from some of the labs that were run by students in previous years are
provided in Appendix 1. You should interpret these spectra as part of your write-up for every
experiment for which these spectra are provided.
Questions
1. Draw the Kekule structure of caffeine showing all lone pair electrons and the hybridization of
the nitrogen atoms.
2. Caffeine has a ring structure in common with nucleoside bases.
(a) Draw and name the parent compound (i.e. the simplest compound with this ring
Chemical Biology 3OA3, September 2009
Page 23
structure).
(b) Draw and name the two common nucleosides containing this ring structure that occur in
nucleic acids.
(c) Draw the structures of each of these bases, hydrogen – bonded to their (Watson-Crick)
complement.
3. Caffeine forms salts with carboxylic acids. Deduce which is the most basic site of caffeine
and account for your choice (hint - take into account resonance stabilization and the molecular
orbitals involved).
4. Is the NMR spectrum (either your own or the one in the Appendix) consistent with the
structure of caffeine? Is the sample pure by NMR? (It is not necessary (or easy) to assign the
resonances to each of the specific methyl groups).
Chemical Biology 3OA3, September 2009
Page 24
EXPERIMENT 2
1-BENZYLDIHYDRONICOTINAMIDE FROM 1-BENZYLNICOTINAMIDE CHLORIDE
H
O
H
H
NH2
N+
Cl-
O
NH2
N
dithionite
CH2
CH2
Introduction to Experiments 2 and 4
As we have seen in sugar chemistry, many natural compounds are adorned with oxygen–
containing functionality, e.g. alcohols, aldehydes, ketones, and carboxylic acids. Nature has
evolved enzymes to inter-convert these functional groups with exquisite regio-, diastereo- and
enantio-selectivity. These oxidations and reductions require a redox reagent, and the redox
couple between nicotinamide adenine dinucleotide (phosphate) (NAD(P)+) and its reduced form,
NAD(P)H, provides the “co-factor” for many of these redox reactions, while the enzyme
provides catalysis.
Consider the reduction of a ketose to an alditol:
OH
O
OH
"[H-]"
OH
OH
1
OH
+
HO
OH
2
OH
3
Sodium borohydride (NaBH4) would be the Chemist’s first choice of reagent. It is cheap, but
would give a mixture of isomers (2 and 3). What type of isomers are these? NAD(P)H and an
enzyme (alcohol dehydrogenase – the name describes the reverse reaction) would be selective,
but hugely expensive for reactions on the gram scale.
In experiment 2, we study the simpler and more readily available analog of NADH,
benzyldihydronicotinamide. Just like NAD(P)H, this compound is formed by reduction of the
oxidized compound benzylnicotinamide (cf. NAD⊕). Although the reduction destroys the
aromaticity of the pyridine ring, the positive charge on the nitrogen cation is neutralized. These
two effects counter-balance each other making both forms reasonably stable, with the redox
potential between the two suited to performing both oxidation of alcohols and reduction of
aldehydes and ketones.
Thus, in the second part of the experiment, the reduced form gives up a “hydride ion”
Chemical Biology 3OA3, September 2009
Page 25
equivalent to malachite green. The dihydronicotinamide returns to the oxidized form.
In experiment 4, whole cells of yeast are used as a reducing agent to reduce a carbonyl
compound. This application of biology to chemistry avoids the expense of NADH and purified
enzymes discussed above, but still proceeds with very high selectivity (the product is a single
enantiomer produced in >98% enantiomeric excess (e.e.)), unlike the product using the chemical
reagent, NaBH4. So what’s the downside? Unfortunately, not all carbonyl compounds will enter
the yeast cells, and of those that do, some will not be substrates for the yeast enzyme. In that
case, it’s back to sodium borohydride, or else a more sophisticated chemical reagent. As with all
of organic chemistry, the choice of reagent is not as obvious as it might appear!
Procedure
Prepare a solution of 2.1 g of sodium carbonate monohydrate and 2.6 g (about 0.015 mole) of
sodium dithionite (also known as sodium sulfoxylate) in 20 mL of water in a 125mL Erlenmeyer
flask. Place a magnetic stirring bar in the flask and arrange for the solution to be stirred by a
magnetic stirrer. Dissolve 1.0 g (4.0 mmol) of 1-benzylnicotinamide chloride in 5 mL of water
and add this solution all at once to the well-stirred solution of sodium carbonate and sodium
dithionite. The resulting yellow-orange solution should produce a yellow precipitate in several
minutes. If the product oils out, do not cool the oil in ice-water but continue to stir the mixture
vigorously until it solidifies and collect the yellow solid by suction filtration using several small
portions of water for rinsing and washing.
The product obtained from the dithionite reduction can be used directly for the reduction of
malachite green, or it can be recrystallized to give beautiful yellow spars. To recrystallize,
dissolve the crude product (save a few crystals) in about 3 mL of hot 95% ethanol and filter the
solution through a Pasteur pipette plugged with a small ball of cotton to remove a small amount
of insoluble impurity. Dilute the filtrate with 2.5 mL of warm water, and, after swirling to mix,
allow the solution to stand undisturbed for several hours. (To induce crystallization add the
crystals of the crude product you saved.) Collect the crystals by suction filtration and wash them
with a small amount of ice-cold 50% aqueous ethanol. The literature melting point of the
product is 119-121°C. Record your melting point and compare with the literature value.
Reduction of Malachite Green by 1-Benzyldihydronicotinamide
Procedure
Dissolve about 10 milligrams (0.03 mmole) of malachite green in 1 mL of 95% ethanol in a
small test tube - this solution may be provided. To this add 20 - 40 milligrams of 1benzyldihydronicotinamide (0.1 to 0.2 mmole). Swirl the mixture gently to dissolve the crystals.
The intense green-blue colour of the malachite green will give way to the yellow colour of the
excess reducing agent in 2 to 4 minutes.
Chemical Biology 3OA3, September 2009
Page 26
Questions
1. 1-Benzylnicotinamide chloride can be prepared by heating nicotinamide with benzyl chloride
in DMSO. Give a mechanism for the reaction and explain why alkylation occurs at the
pyridine nitrogen. Why is DMSO a good solvent for this reaction?
2. 1-Benzylnicotinamide is similar to a naturally occuring enzyme cofactor that is used
reduction/oxidation reactions. What is the name and structure?
3. Ethyl benzoylformate is not reduced by 1-benzyldihydronicotinamide alone. In the presence
of magnesium chloride, however, racemic ethyl mandelate is formed in quantitative yield.
Explain how MgCl2 accelerates the reaction.
φ
O
O
C
C
1-benzyl dihydronicotinamide
O
Et
ethyl benzoyl formate
MgCl2
HO
φ
O
CH C
O
Et
ethyl mandelate
H
4. When ethyl benzoylformate is treated with (R)-N-(α-methylbenzyl)dihydronicotinamide in the presence of an equimolar amount of
magnesium chloride in aqueous acetonitrile solution, the ethyl
mandelate produced is 60% R and 40% S.
H
O
NH
N
N-(α-methylbenzyl)-dihydronicotinamide
a.
Draw formulae indicating the structure and configuration of (R)-N-(α-methylbenzyl)dihydronicotinamide and of the two enantiomers of ethyl mandelate.
b.
Explain why the product is not a racemic mixture.
c.
What is the enantiomeric excess of the ethyl mandelate?
Chemical Biology 3OA3, September 2009
Page 27
EXPERIMENT 3
SUGAR ANOMERS: α- AND β-D-GLUCOSE PENTA-ACETATE
Introduction:
D-Glucose is the most abundant sugar on the planet. It has been suggested that this is because
all the substituents on the 6-membered ring are equatorial, making glucose the most stable
structure possible. Notably, however, the stabilities of the two anomers, (isomers at the chiral C1 acetal centre) are very similar. Thus, at equilibrium, the equatorial (β) and axial (α) anomers of
glucose are present at similar concentrations. This unusual stability of the axial anomer has been
ascribed to the “:anomoric effect”. In the axial anomer, the ring oxygen possesses one lone pair
of electrons that is anti-periplanar to the C-OH σ bond. However, the in the β-anomer, there is
no lone pair that is anti-periplanar to the C-OH bond:
HO
6
H+, H2O
O
HO
HO
3
C-1
OH
OH
AXIAL OH @ C-1:
alpha ANOMER
stabilized by
anomeric effect
HO
HO
HO
O
OH
OH
EQUATORIAL OH @ C-1:
beta ANOMER
not stabilized by
anomeric effect
Thus, the α-anomer is stabilized through a resonance interaction as shown in the figure. This
effect is observed, for example, as a lengthening of the C-1-OH bond. The resonance effect
compensates for the unfavourable 1, 3-diaxial interactions, making both anomers equally stable.
In this course, we will see that the anomeric effect is important in determining reactivity of
sugars, e.g. in catalysis of reactions at the anomeric centre, both in chemistry and in enzymatic
processes.
In this experiment, you will investigate the acetylation of glucose, and prepare both the α- and
β-anomers of glucose penta-acetate. The conversion of the alcohol groups of glucose into esters
in an important process, and is used for protection of the alcohols at C-2 to C-6 prior to
performing chemistry at the anomeric centre.
Alcohols react only very slowly with carboxylic acids, and thus an activated form of the acid
is needed – we use acetic anhydride in this experiment (why are anhydrides more reactive than
acids?). These ideas of protection and activation will recur many times in this course, e.g. in
forming peptides from acids and amines.
To further accelerate the reaction, a catalyst is used. Catalysts accelerate reactions but are not
consumed. In this course, we will be comparing chemical and enzymatic catalysis many times.
Chemical Biology 3OA3, September 2009
Page 28
One of you will use zinc chloride as a catalyst, giving the α-anomer of glucose penta-acetate,
while your partner will prepare the β-anomer using sodium acetate. Why does the choice of
catalyst alter the stereochemical outcome of the reaction? It has been shown that BOTH catalysts
promote interconversion of the two anomers of glucose. The β-anomer reacts rapidly with acetic
anhydride, but the α-anomer only slowly. As the β-anomer is consumed, the α-and β-anomers
keep re-equilibrating and so α-D-glucose is converted to the β-anomer in preference to reacting.
The result is that BOTH catalysts initially form the β-D-glucose penta-acetate. This is the fastest
formed product, and we say that it is formed under kinetic control. However, the α-D-glucose
penta-acetate is in fact the most stable of the two products: it will be formed under
thermodynamic control, i.e. if the two product species can equilibrate. Zinc chloride catalyses
this equilibration, but sodium acetate does not. Thus, different anomers are formed depending on
the catalyst. Note that different enzymes may form different anomers in reactions of sugar
derivatives, and this also depends on the mechanism of catalysis.
HO
HO
HO
O
OH
OH
FAST
HO
HO
O
OH
OH
catalysis
by NaOAc
or ZnCl2
SLOW
catalysis
by NaOAc
or ZnCl2
FAST
AcO
AcO
AcO
AcO
HO
O
OAc
OAc
AcO
AcO
catalysis
by ZnCl2
ONLY
O
OAc
OAc
Finally, in this experiment, you will catalyze the reaction using N-methyl imidazole and
determine which anomer is produced in this case, by comparison with your α and β-standards.
Later in the course, we will see that imidazole can be an effective chemical catalyst, and that it
performs critical roles in enzymatic catalysis, in the form of the amino acid histidine (His).
In the present experiment, N-methyl imidazole (N-MeIm) causes a faster acetylation of
glucose and requires a lower temperature; the yield is >90%. The reaction proceeds through: (i)
the reaction of N-MeIm with Ac2O; (ii) Reaction of the resulting acetyl-N-MeIm with glucose.
Both these steps are faster than the reaction of Ac2O with glucose, and the last step must be
exothermic. Note that the last step RE-GENERATES N-MeIm so the process is catalytic.
Chemical Biology 3OA3, September 2009
Page 29
AcO
Me
N
AcO
AcO
N
O
OAc
OAc
Ac2O
FAST
O
Me
N
+
FAST
N
O
Me
+
N
N
Ac2O,
no cat.
SLOW
HO
HO
HO
O
OH
OH
Chemical Biology 3OA3, September 2009
Page 30
Procedure
1. α -and β-D-Glucose penta-acetate.
Weigh your catalyst into a 50 mL round bottom flask:
Or
Zinc chloride:
0.5 g
Sodium acetate:
1.2 g
(NOTE: the catalysts need to be reasonably anhydrous: keep the lids of the bottles tightly closed,
and cap your flask)
Add 12 mL of acetic anhydride (Lachrymator: Hood!) and 2 g glucose. Add a boiling stone
and attach a condenser in the reflux position. Heat slowly until the mixture begins to boil. Use a
heating mantle. To avoid charring the sugar, touch the flask onto the hot surface of the mantle,
then remove. Swirl the contents to help mixing. Once the reaction begins, remove the heat
source, and allow the exothermic reaction to sustain the reflux. Boil the mixture for a further two
minutes, by gently touching the flask to the mantle. Pour the hot mixture into 200 mL of an icewater slush, while rapidly stirring with a glass rod. Stir until all the oil has solidified: this may
take a few minutes. Break up any lumps, and then filter through a Buchner funnel using vacuum
aspiration. Collect the solid, and recrystallize your product from methanol. Allow your crystals
to dry completely then record the yield, calculate the % yield, and determine the melting point.
Typical yields are: 50% (α) and 62% (β).
Melting points: α: 112-113oC; β: 132-134oC
2. Catalysis by N-methyl-imidazole.
Start this part during the recrystallization in Part 1
Add 0.25 g glucose to 2.5 mL N-methylimidazole in a large test tube. Warm gently to
dissolve, using the heating mantle. Add 0.75 mL acetic anhydride dropwise and shake to mix.
After 15 min, add 2.5 mL of water. Allow to crystallize (5-10 min), and collect the solid. NOTE
that you may have to scratch and/or cool the tube to induce crystallization: be patient! Dry and
record the yield. If necessary, recrystallize from ethanol (but do NOT leave to crystallize until
next week).
Determine the melting point, and compare with that for the authentic α- and β- products.
Which anomer has been formed?
You can choose to record you own NMR data or use the data provided in the Appendix for
samples of the α-, the β- and the unknown anomer.
In your report, assign the anomeric proton in each isomer. Determine its coupling constant to
the proton at C-2. How does the coupling constant tell you which anomer is which? Use
Newman projections along the C-1 to C-2 bond to explain. Use the coupling constant and shift
for the unknown anomer to determine which anomer has formed with N-methyl-imidazole. Does
your conclusion agree with the results of the melting points?
Chemical Biology 3OA3, September 2009
Page 31
Questions:
1. Explain why acetic anhydride is a much more reactive acetylating reagent then acetic
acid.
2. Suggest a reason why the β-anomer of glucose is acetylated faster than the α-anomer.
3. How would you establish that zinc chloride but not sodium acetate inter-converts the two
product anomers?
4. Suggest a reason why zinc chloride catalyzes inter-conversion of the α- and β-anomers of
glucose penta-acetate, but sodium acetate does not.
Reference to catalysis by N-methyl-imidazole: R. Wachowiak and K.A. Connors, Anal. Chem.
51, 27 (1979).
Chemical Biology 3OA3, September 2009
Page 32
EXPERIMENT 4
CATALYSIS BY YEAST: REDUCTION OF 1-PHENYL-1,2-PROPANEDIONE
Introduction
As noted in the introduction to Experiment 2, the use of biological methods to effect
chemical transformations has proved useful in chemical synthesis, not least because of the
spectacular control of selectivity that can be achieved. This is one example of integrating
biology with chemistry, a field that has become known as chemical biology.
In this experiment, you will use baker’s yeast (Saccharomyces cerevisiae) to reduce both
carbonyl groups of 1-phenyl-1,2-propanedione (1), leading to 1-phenyl-1,2-propanediol (2) as the
major product. In this process, two new stereogenic centres are produced from the achiral
starting material. The major product is a single isomer, which is formed in > 98% enantiomeric
excess, and in high diastereomeric excess. It has the (R) configuration at C-1. You will establish
the absolute configuration at C-2.
OH
O
O
1
2
OH
3
1
O
2
OH
O
1
2
OH
4
Start your report by drawing out the reaction scheme, showing all the possible
stereoisomers of diol 2, and labelling each chiral centre. Indicate which isomers have the R
configuration at C-1.
This reaction can proceed through two possible intermediates, ketols 3 and 4. You need
to bear this in mind when identifying your product How can the structure and stereochemistry of
the product be confirmed? How is enantiomeric excess determined? These and other questions
form an open-ended discussion that enables you to explore this aspect of chemical biology.
Chemical Biology 3OA3, September 2009
Page 33
NMR analysis:
Your TA will collect one or more samples of the diol 2 and you will obtain the NMR
spectra of these samples in deuterated chloroform (CDCl3). You will first use these spectra to
confirm the identity and purity of the diol. Typical spectra are also provided in this manual in
Appendix 2, but you should also interpret your own results.
IR and NMR data for 1, 2, 3 ,and 4 are given below:
δ 4.9
(1H, s)
δ ca. 4.0
(1H, s)
OH
H
δ 2.0
(3H, s)
1680 cm-1
δ ca. 3.2
(2H, s)
O
O
δ 7.7-7.9
(m, 2H)
δ 7.2
(s, 5H)
δ 2.4
(3H, s)
OH
OH
1710 cm -1
H
O
δ 7.3-7.5
(m, 3H)
δ 3.9-4.1
H (m, 1H)
3
1680 cm -1
1710 cm-1
O
δ 7.6-8.0
(m, 2H)
1
δ 1.4
(3H, d)
δ 7.3
(s, 5H)
δ 4.7 δ 1.1
(1H, d) (3H, d)
2
H δ 5.0
OH (1H, q)
δ 7.2-7.6
(m, 3H)
4
δ ca. 3.6
(1H, s)
Analysis of the coupling constants in the diol can be used to determine which
diastereomer is formed. NMR spectroscopy in an achiral environment cannot distinguish pairs of
enantiomers, e.g. (1R,2R) and its enantiomer (1S,2S), but a pair of diastereomers such as (1R,2R)
and (1R,2S) give different spectra. In an aprotic solvent such as CDCl3, the two hydroxyl groups
of the diols form a strong intramolecular hydrogen bond. This bonding can only occur in the
conformations in which the two OH groups are gauche; the anti arrangement cannot hydrogen
bond. The two possible diastereomers of the diol give rise to different arrangements of the
protons at C-1 and C-2, as shown in the figure below: H-1 and H-2 are gauche in BOTH major
conformers in the (1R,2S) isomer, but gauche in one and anti in the other in the (1R,2R)
diastereomer.
The Karplus equation relates proton-proton coupling constants (J) to dihedral angles (pHC-C-H) between two protons on adjacent carbon atoms. An anti arrangement gives a large value
of J (typically 10 Hz), whereas a gauche arrangement gives a small J, around 3 Hz. Thus, the
value of J predicted for the (1R,2S) isomer is approximately the average of the two dominant
conformers, i.e. (3 + 3)/2, or approx. 3 Hz, while that for the (1R,2R) diastereomer is the average
of 3 and 10 Hz, i.e. approx. 7.5 Hz. Therefore, by measuring the coupling constant between the
C-1 and C-2 protons in the diol, it is possible to determine which diastereomer has been formed.
In practice, the coupling is most easily observed at the signal for H-1, around 4.7 ppm. In this
experiment, you will measure this coupling, to determine which diastereomer is formed in the
reaction.
Chemical Biology 3OA3, September 2009
Page 34
1R, 2S isomer:
Ph
Me
H
OH
Ph
OH
Me
OH
OH
Ph
H
HO
H
H
Major conformer 1:
OH and OH gauche
H and H gauche
H
OH
Me
H
Major conformer 2:
OH and OH gauche
H and H gauche
Minor conformer:
OH and OH anti
H and H anti
1R, 2R isomer:
Ph
H
Me
Ph
OH
OH
OH
H
H
OH
Ph
Me
HO
Major conformer 2:
OH and OH gauche
H and H gauche
OH
H
H
H
Major conformer 1:
OH and OH gauche
H and H anti
Me
Minor conformer:
OH and OH anti
H and H gauche
At the end of this experiment, you should know the stereochemistry of the diol product 2.
Optical rotation:
Optical activity is a property of chiral compounds. It can be used to determine which
enantiomer of the diol has been obtained. In this experiment, time and equipment permitting,
you will combine several samples of diol, and the optical rotation will be measured by your TA
on a polarimeter. You should convert the observed rotation, α, into a specific rotation [α] (see
the 2OA3/2OB3 textbook for the procedure).
The literature melting points and optical rotations for the stereoisomers of the diol are
shown in the Table below:
Isomer
mp
[α]D
(1R,2S)
89-91oC
-40o
(1S,2R)
89-91oC
+40o
(1R,2R)
51-53oC
-20o
(1S,2S)
51-53oC
+20o
Chemical Biology 3OA3, September 2009
Page 35
Procedure:
1.
Reduction of 1-phenyl-1,2-propanedione with baker’s yeast
Weigh 3 g of dry active yeast into a 125 mL Erlenmeyer flask. Add 20 mL of distilled
water, add a magnetic stir bar, and place the flask into a beaker of warm water at 30oC. Place the
beaker on a stirrer, and stir at medium speed.
IMPORTANT NOTE: use the hot and cold tap water to set the temperature to 30oC: this
experiment uses live yeast, so the temperature must be maintained within a couple of degrees
throughout the experiment: too hot and the yeast will die, too cold and the reaction will be too
slow.
Add 115 µL of 1-phenyl-1,2-propanedione to the flask. Take aliquots of approximately
0.2 mL with a pasteur pipette immediately after addition of the diketone and every 15-30 mins
for at least 90 mins. In the meantime, between TLC runs, obtain the IR spectrum of the starting
diketone. The NMR spectrum of the starting ketone will be recorded by your TA.
Transfer each aliquot to a 1-dram vial, and add approx. 0.4 mL of tert-butyl methyl ether
(BME). Shake the vial and vent immediately; repeat shaking and venting for about 30 seconds.
Allow the layers to separate, and then spot the upper layer onto a silica gel TLC plate. Develop
the plate using a mixture of cyclohexane-BME (6:4).
Visualize the spots using a UV lamp.
CAUTION: BME AND CYCLOHEXANE ARE FLAMMABLE!
After the TLC shows the presence of diol (normally approximately 90 minutes of
reaction), transfer the reaction mixture to a 125 mL separatory funnel. Add 25 mL of BME, then
GENTLY invert the funnel, and vent the pressure by opening the tap. Keep inverting and
venting for 5 minutes. DO NOT SHAKE because the layers will emulsify. Let the layers
separate for a few minutes, and then collect the lower aqueous layer in the original Erlenmeyer
flask. The interface can be made clearer by applying a gentle rotational motion to the separatory
funnel in a vertical position while holding it from the top and lifting it from the ring. Try to drain
most of the emulsion into the aqueous layer. Collect the upper layer in a dry 125 mL Erlenmeyer
flask. Label it “organic”. Transfer the aqueous layer back to the separatory funnel, add 20 mL of
BME and repeat the extraction, collecting the BME layer in the same flask as the 1st time. Repeat
once more.
If there is a lot of aqueous layer in the organics at this point, decant the organic layer
carefully into a fresh, DRY flask. To the combined organic layers add about 2 g anhydrous
magnesium sulfate, and swirl. After about 5 minutes, filter using a glass funnel and fluted filter
paper into a pre-weighed and DRY 50 mL round-bottomed flask. Evaporate the solvent by
placing the flask on a rotary evaporator (you can also perform a classical distillation if the rotary
evaporator space is limited). Remove the flask once the solvent stops distilling. Dry the outside
Chemical Biology 3OA3, September 2009
Page 36
of the flask, and then use a stream of air to remove the last traces of solvent. Make sure all the
solvent is gone, and that no water enters the flask. Weigh the flask and calculate the mass of
product by difference.
Obtain the IR spectrum of your product. Your TA will collect one or more samples of
the diol, and obtain NMR spectra, and optical rotation. Measure the melting point of the diol.
Questions
1.
2.
3.
4.
5.
In your report, clearly show which isomer of the diol is produced during reduction of
1-phenylpropane-dione. Describe your evidence (how do the melting point, IR,
NMR, and optical rotation data combine to substantiate your claim?).
Indicate which face of each carbonyl group, re or si, is attacked by hydride from
NAD(P)H, in order to generate the observed diol enantiomer.
What influences the Rf of the various compounds on the TLC plate? Do the dione,
any ketol intermediate(s) you observe, and diol elute in the order you expect? Why?
How would you isolate and purify the ketol?
How would you further explore this reaction?
Chemical Biology 3OA3, September 2009
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EXPERIMENT 5
SOLID PHASE SYNTHESIS OF A PEPTIDE AMIDE
1. INTRODUCTION
1.1
Peptides and Peptide Amides
Peptides are polymers derived from amino acids and have molecular weights less than 5,00010,000; similar polymers with molecular weights greater than this are called proteins. The
component amino acids are joined by amide bonds from the amino group of one unit to the
carboxyl group of the next.
In writing structures of polypeptides, the convention is to place the N-terminal amino acid on
the left and the C-terminal amino acid on the right. Then the amino acids are named left to right
using either their common names or their standardized three-letter or one-letter codes.
During this course, we will see how peptides are made in vivo from the constituent amino
acids, both on the ribosome and by non-ribosomal peptide synthetases, and relate these processes
to chemical synthesis. Peptides have a plethora of biological activities, and are also the
precursors for a range of drugs such as the cyclic immunosuppressant cyclosporine. Slight
variations on linear peptides are common, and one such example is the peptide amides, where the
carboxy terminus is amidated, i.e. the molecule ends in C(=O)NH2 rather than C(=O)O-.
Amongst other things, this terminus changes from an anionic functionality to a neutral one,
thereby altering the total net charge on the peptide, and consequently altering the biological
activity of the compound drastically. Examples of peptide amides include: enkephalinamide, a
super-active analog of Met-enkephalin; and dermorphin, a hepta-peptide amide first isolated
from the skin of South American frogs which is a natural opiate. It binds as an agonist with high
potency and selectivity to mu Opioid receptors. Dermorphin is about 30-40 times more potent
than morphine but less likely to produce drug tolerance and addiction. Dermorphin has the
structure H2N-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-C(=O)NH2.
Chemical Biology 3OA3, September 2009
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In this experiment, you will synthesize a simple peptide amide.
1.2
Peptide Synthesis: Activation and Protection
Because amines only react with carboxylic acids under extreme conditions, the standard
strategy for forming amide bonds is to prepare a chemically active functional derivative of the
carboxylic acid and allow it to react with an amine. For example, a carboxylic acid can be
activated by converting to an acyl chloride, which, on treatment with an amine, gives an amide:
This simple strategy, so effective for monofunctional components, fails miserably when
applied to making amide bonds between amino acids. With such bifunctional compounds, the
initial activation step produces an intermediate that reacts with itself to produce a polymer before
the amine component is added. For example, the amino acid glycine, on conversion to glycyl
chloride, polymerizes to polyglycine and other condensation products, such as the
diketopiperazine discussed in class:
To avoid this unwanted reaction it is necessary to "protect" the amino group temporarily, that
is, to convert the NH2 group to some other unreactive functional group. After the desired
coupling has occurred, the protecting group can be removed.
The repeated sequence of amino protection, acid activation, condensation reaction, and
deprotection, terminated by final removal of all protective groups is time-consuming and fraught
with many subtle difficulties. Still, this method is used to this day for very small peptides.
However, the ability to synthesize any arbitrary amino acid polymer is so important to modern
biochemical research that a great effort has been invested in making the process practical.
1.3
Solid Phase Peptide Synthesis (SPPS)
A brilliant achievement in peptide synthesis was Merrifield's step-by-step synthesis of the
enzyme bovine pancreatic ribonuclease, a protein with a chain of 124 amino acid units. He was
awarded the Nobel Prize in 1984. A total of 369 consecutive chemical reactions was achieved
with an overall yield of 17% which corresponds to an average yield of better than 99% for each
step. The key to success was to use a solid support; the polypeptide chain grows on the support,
while reagents, solvents, etc. can be added to the resin, then easily removed by filtration.
Chemical Biology 3OA3, September 2009
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Many advances on the original Merrifield procedure have been developed. In this
experiment, you will use the Rink Tentagel amide resin to synthesize your peptide amide. This
methodology uses fluorenylmethoxycarbonyl (Fmoc) groups to protect the amino groups of each
amino acid, rather than the tBoc group used by Merrifield; these Fmoc groups are more quickly
and efficiently removed when needed, using piperidine in DMF. The Rink resin arrives with an
Fmoc group attached, so this reaction is the first chemistry you’ll perform on your resin, after
washing and swelling. Removal of the Fmoc group on the resin yields a free amine attached to
the polymer support, and you are ready to attach the first amino acid.
Rink Resin as Supplied
O
OMe
O
N
H
Resin Bead
(Polystyrene)
OMe
NH
O
H
H
N
Piperidine,
DMF
}
O
Fmoc group
O
OMe
O
N
H
R1
NH2
R1
N
N
mix
HO
OMe
O
N
mix
NHFmoc
O
+
N+Me 2
OMe
O
N
+
Me2N
O
N
H
NHFmoc
O
Amino acid #1
DIPEA
(a base)
PF6-
O
N
N
N
OMe
NH
(1) Piperidine, DMF
(2) Amino acid
#2 plus HBTU, etc.
(3) Piperidine, DMF
HBTU
NHFmoc
O
R1
O
OMe
O
N
H
NH
CF3CO2H
O
(TFA)
R1
OMe R2
H
N
O
O
N
H
etc....NH2
R3
O
N
H
OMe
O
NH2
+
OH
Spent Resin
Chemical Biology 3OA3, September 2009
OMe
O
R1
R2
H
N
O
O
N
H
etc....NH2
R3
Peptide Amide
Page 40
The next step is to take your first desired amino acid, protected as the Fmoc derivative. The acid
must be activated, and you’ll use HBTU (short for O-(benzotriazol-1-yl)-N,N,N’,N’tetramethyluronium hexafluorophosphate; see why abbreviations are useful?) to activate the acid.
Once mixed, this solution is applied to the resin, where the activated carboxylic acid of amino
acid #1 condenses with the amino group of the resin to generate an amide bond. The excess
reagent is then washed away, and another cycle of deprotection with piperidine can occur. The
next amino acid is activated with HBTU, and added to the resin, generating a dipeptide, still
bound to the resin. This cycle can continue until the correct length of peptide has been built up;
the final product is removed from the resin by reaction with trifluoroacetic acid (TFA), which
reacts specifically with the functionality directly adjacent to the original Fmoc-amine on the
tentagel resin.
1.4 Combinatorial Libraries of Peptides
One important aspect of solid phase synthesis is its adaptability to combinatorial synthesis.
In the present experiment, each student will prepare one specific peptide, but the class as a whole
will prepare a library of peptides, based on a selection of 3 possible amino acids (Gly, Ala and
Phe) at each stage. Your TA will tell you which sequence to prepare. In principle, each
student could do each reaction separately from one another; however, it is much more efficient if
a single reaction is used to generate all the peptides that start with e.g. Gly. The resin with Gly
loaded can be split into several pools, and each of the next amino acids desired in the sequence
can then be added. This type of combinatorial approach is used in research to generate very large
libraries of related structures such as peptides.
To illustrate this technique, you and a partner will work together to attach the first
amino acid, then you’ll divide the resin into two and each of you will continue individually
to add the next amino acids in your individual sequences.
2.
PRE-LAB:
You will be using 100 mg of Rink resin with your partner for the first step, then you’ll split
the resin into two equal portions. The loading is 0.24 meq/g resin (milli-equivalents per gram);
this means that 1 g of resin has 0.24 mmol of functional groups that are available for the
chemistry you’ll perform.
Based on this information, calculate the number of moles and number of milligrams of each
reagent you’ll require for the steps below. NOTE that an excess of each reagent is used, so
READ the instructions through before doing this (see step 8). Remember to calculate not only
the Fmoc-amino acid quantities, but also the HBTU (MW = 379) and di-isopropylethylamine
(DIPEA, MW = 129, density = 0.742). The weights of Fmoc-amino acids depend on the amino
acid you are using, Gly, Phe or Ala. Your calculation should give you quantities of approx. 3040 mg Fmoc amino acid, 40 mg HBTU, and 30 µL DIPEA for the first coupling.
Remember to halve the quantities for each subsequent amino acid coupling step, as you will
have split the resin into two portions, one for you and one for your partner.
Draw structures for all of your reagents, the specific Fmoc-amino acids you are planning to
use, and your peptide product. You should also prepare for this lab by considering the
questions posed at the end.
Chemical Biology 3OA3, September 2009
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3.
EXPERIMENTAL PROCEDURE:
a. Washing and swelling the resin:
Work as a pair for the first few steps.
You have been given a tube containing 100 mg of the Rink resin on top of a porous frit. The frit
allows solvent to pass through, while retaining the resin beads. Beneath the frit is a tap, and an
exit tube. The resin is quite expensive: do not attempt to open the tube. Attach the exit tube to
the rubber bung provided (CAUTION: use the correct procedure to insert the glass tube into the
bung). Place the bung into a 250 mL Buchner flask, and attach the side arm to the laboratory
vacuum supply. Your TA will demonstrate how to set up this apparatus initially. Close the tap,
and turn on the vacuum SLIGHTLY to provide a weak suction.
1. Wash the resin with 3 separate aliquots of 1.5 mL of dichloromethane (DCM).
Throughout this lab, the term “Wash” means add the solution to the resin, swirl gently
with the rod provided, wait 60 seconds, swirl again, wait another 60 sec, then turn the tap
on and allow the vacuum to draw all the liquid off the resin beads. Then, turn the tap
OFF. GENTLE mixing is needed to avoid damaging the beads.
2. Wash the resin with 3 separate aliquots of 1.5 mL of dimethylformamide (DMF).
b. Deprotecting the Fmoc group:
3. Add 1.5 mL of a solution of 20% piperidine in DMF to the resin. Swirl as above with the
rod. Allow this reaction to take place for 10 minutes. Swirl occasionally to mix the reagent with
the resin.
NOTE: Piperidine has an unpleasant smell: use ONLY in the hood.
NOTE: Start weighing out reagents for step 8 while waiting for this step.
4. Remove the liquid by aspiration, as above.
NOTE: do not aspirate the resin to dryness after these deprotection steps, just lower the solution
to just above the resin surface, then add the next reagent.
5. Add a further 1.5 mL of the solution of 20% piperidine in DMF to the resin. Swirl as above
with the rod. Allow this reaction to take place for 10 minutes. Swirl occasionally to mix the
reagent with the resin.
6. Remove the liquid by aspiration, as above.
7. Wash the resin with 4 separate aliquots of 1.5 mL of dimethylformamide (DMF).
c. Activation of amino acid #1:
8. Into a glass vial weigh 4 equivalents (relative to milli-equivalents of resin, see PRE-LAB) of
your selected Fmoc-amino acid. Separately, weigh out 4 equivalents of HBTU, then add this to
Chemical Biology 3OA3, September 2009
Page 42
the vial. Add a solution of di-isopropylethylamine (DIPEA, 8 equivalents) in DMF (0.5 mL).
Stir thoroughly to dissolve the ingredients.
9. Add this solution to the resin in the column. Swirl as above with the rod. Allow this reaction
to take place for 15 minutes. Swirl occasionally to mix the reagent with the resin.
10. Remove the liquid by aspiration, as above.
11. Wash the resin with 4 separate aliquots of 1.5 mL of dimethylformamide (DMF).
OPTIONAL: it is possible to check for complete coupling at this point by transferring a few
beads of resin into a test tube containing approx. 1 mL of 0.5% ninhydrin in ethanol solution.
Heat for 10 min at 75-80 degrees. A blue colour indicates free amino groups on the resin,
implying incomplete coupling.
d. Splitting the resin:
12. Add 1.5 mL of dimethylformamide (DMF) to the resin. Stir, and use a Pasteur pipette to
transfer half of the beads into a new apparatus. Use more DMF if needed. You do not need to be
exact, just estimate half the resin by comparing the heights of the resin column in the two
apparati. Once separated, use the vacuum to remove the DMF you used for the transfer.
Each student will now proceed separately to attach the second and any subsequent amino
acids.
e. Removal of Fmoc and coupling of second amino acid:
13. Repeat steps 3-7 above, to remove the Fmoc group from amino acid #1. Remember to halve
all the quantities of solvents, reagents, washes, etc, as you only have half the resin. For example,
only 0.8 mL aliquots of wash solvents are needed.
NOTE: do not aspirate the resin to dryness after these deprotection steps, just lower the solution
to just above the resin surface, then add the next reagent.
14. Follow steps 8-11 above to activate your second amino acid (remember, the second amino
acid may be different from the first, so you may need a different weight), and couple it onto the
first one on the resin.
15. The cycle of deprotection, washing, coupling and washing can be repeated a third time to
generate a tripeptide. However, REMEMBER that you MUST have time to complete a full cycle
of steps 3 to 11 to do this (approx. 1 hour).
16. Wash the resin 4 times with DCM (1 mL). This is a good point to stop after week 1. To
stop here, use a Pasteur pipette to gently remove the resin beads from the column, along with the
DCM. Use more DCM as needed to rinse the beads from the column. Store the beads in the
DCM in a test tube, stopper TIGHTLY, parafilm, and leave until next week.
Week 2:
OPTIONAL: you can add another amino acid at this point: see steps 13-14 above.
Chemical Biology 3OA3, September 2009
Page 43
1. Load your resin in DCM from last week back into the reaction tube. Use a Pasteur pipette,
and gently transfer. Use more DCM to rinse the tube that the resin was stored in.
2. Remove the DCM by aspiration.
3. Re-swell the resin by washing with 1 mL of DMF twice. NOTE: recall the meaning of
“wash” from last week).
f. Final Fmoc removal and washing:
4. Remove the Fmoc group with 20% piperidine in DMF (as for steps 3-7 above, but remember
half the quantities; remember not to let the resin go completely dry).
5. Wash the resin with 0.8 mL of DMF three times.
6. Wash the resin with 0.8 mL of methanol.
7. Wash the resin with 0.8 mL of DMF three times.
8. Wash the resin with 0.8 mL of DCM four times.
g. Cleavage of product from the resin:
[NOTE: do not start this part unless you can complete it at least to the end of step 13]
9. Make SURE the tap is closed, and stop the vacuum! Add 0.8 mL of 95% aqueous TFA to the
resin. CAUTION: TFA fumes, smells bad, and is corrosive. WEAR GLOVES and handle
ONLY in the FUME HOOD.
10. Allow to react for 60 mins, swirling gently on occasion using the mixing rod.
11. Remove the bung from the Buchner flask. Place the column over a dry 25 mL round bottom
flask. Open the tap, and allow the TFA solution of peptide to drain by gravity into the flask.
12. Add a further 0.8 mL 95% TFA to the resin, mix for 1 min, then drain that liquid into the
receiver flask as well. Repeat with one last wash of the resin.
h. Purification of the product:
13. Place the flask, containing your peptide product in TFA, on the rotary evaporator, and
evaporate almost all of the TFA. Heat at 40 degrees. Do not exceed this temperature!
14. Add 5 mL of diethyl ether to the flask. Swirl. The peptide may form a fine film on the flask
walls at this point, or may form a fine precipitate. If nothing forms, cool the flask in dry ice,
swirl and scratch to induce crystallization. The exact optimal conditions at this point depend on
the specific sequence you have synthesized. If no solid forms, evaporate the solution on the
rotary evaporator again to remove the last of the TFA, and add ether as above. Consult your TA
if you have further problems.
15. Remember, your expected yield on this small scale synthesis is about 2-5 mg: watch this step
carefully, as it is easy not to see your product!
16. Carefully remove the ether by decanting or pipetting it out of the flask; if needed, your TA
may help you to use a centrifuge at this point.
17. Wash your product with a further aliquot of ether, approx. 5 mL, cooling again to induce
crystallization if necessary.
Chemical Biology 3OA3, September 2009
Page 44
i. Characterization of product peptide:
TLC:
In this experiment, TLC will be used to check the purity of the peptide. Fine capillaries for
applying spots will be provided, or else can be made from Pasteur pipettes. Your T.A. will
demonstrate the technique. You can also review the CHEM 20A3 Lab Manual at this point.
1. Place 1-2 mL of the developing solvent (chloroform : methanol : acetic acid (30:15:5 v/v)) in
the chromatography jar, cover tightly and allow to stand 5-10 minutes before using.
2. Remove a small sample of your peptide (enough to see) and dissolve in 2-3 drops of
methanol in a vial.
3. Make up a similar solution of the Gly-Ala peptide standard provided.
4. Use the silica gel TLC sheets provided. With a pencil mark two dots about 6 mm apart, and
8-10 mm from the bottom of the plate. On the first mark, place a spot of your control peptide
solution. On the second mark, place a spot of your product solution. When each plate has been
spotted, place the plate, spotted end down, in the developing jar. Make sure that the solvent
pool begins below the spots. Close the jar tightly, set it aside and do not disturb it. Allow the
solvent to rise to within about 1 cm from the top of the sheet. Remove the sheet and immediately
mark the solvent front. Allow the sheet to dry in the hood.
5. For peptides containing Phe, the plate can be visualized under UV light because the aromatic
ring shows up. Circle the spots with a pencil. However, the standard will not show under these
conditions.
6. Treat the TLC plate with a solution of 0.5% ninhydrin in 95% ethanol.
7. Heat the TLC plate, either with the heat gun or on a hot plate at 100 degrees.
8. Compounds containing a free amine give rise to blue spots.
9. If needed, adjust the TLC solvent by changing the chloroform : methanol ratio (more
methanol to increase the Rf).
10. Sketch your TLC plate in your lab book. Discuss the results you obtain in your report.
Mass spectrometry:
1. Remove a tiny sample (just enough to see) of your peptide and place in a vial.
2. Label the vial with your name, e-mail, the proposed peptide sequence, and the expected
molecular weight of the peptide. Use the molecular weight of the cation that is generated in the
peptide TFA salt (i.e. the MH+ ion. E.g. H3N+Gly-GlyC(=O)NH2 is the ion that will be observed
in the mass spectrum of the salt of the peptide you have isolated with TFA anion).
3. Hand your product to your TA. Mass spectra of each student’s product will be recorded for
you, and returned either by e-mail or in the following lab period.
4. Interpret the mass spectrum when you receive it, and include this in your write-up. Is there
an ion at the expected molecular weight? If not, are any other ions identifiable? Do they
correspond to your peptide that is missing one amino acid? If so, what does that mean for your
synthesis?
NMR:
Because of the small scale of your synthesis, it is not easy to obtain an nmr spectrum of each
student’s sample. However, other students in the class may have made the same peptide as you.
If you can identify these students, you can pool your samples and obtain an nmr spectrum of the
Chemical Biology 3OA3, September 2009
Page 45
combined products. To do this, dissolve the sample in 1 mL of deuterated TFA (TFA-d), and
transfer to an nmr tube. Take the sample to the TA in charge of the nmr spectrometer, and obtain
the spectrum. Share this spectrum amongst the students who generated the sample, and interpret
the spectrum in your report.
[NOTE: the product peptides are not stable in TFA overnight; if you do this, you need to record
the spectrum within an hour or two].
4.
QUESTIONS:
1. Proton nmr data and a COSY spectrum for a peptide are given in the Appendix. Interpret this
data to assign resonances as far as is possible.
2. Give mechanisms for each of the chemical steps involved in the synthesis of your peptide.
Where needed, some helpful hints are provided in the Scheme in the introduction.
3. Use the appropriate mechanism above to explain why the Rink resin gives a peptide amide,
rather than a peptide. What feature(s) of the resin would you modify to turn it into a resin that
could be used to make regular peptides?
5.
REFERENCES:
This laboratory was adapted from Truran, G.A.; Aiken, K.S.; Fleming, T.R.; Webb, P.J; Hodge
Markgraf, J., J. Chem. Educ., 79, 85 (2002).
Chemical Biology 3OA3, September 2009
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EXPERIMENT 6
SYNTHESIS OF THE ANTIEPILEPTIC DRUG DILANTIN, BY A BIOMIMETIC SYNTHESIS
Introduction
This experiment describes the synthesis of the antiepilepsy drug dilantin. First however, a
few words about the causes of epilepsy and its control.
In the body a signal from a sensory organ is transmitted along a nerve fiber until it reaches the
end of the cell, called the synapse. At the synapse the arriving electrical signal induces the
release of a chemical neurotransmitter that diffuses across the synaptic gulf, a short distance of
about 100Å that separates the end of one nerve cell and the beginning of another, where it
triggers another electrical signal in the next nerve fiber. Thus nerve transmission is a
combination of chemical and electrical signals.
In the brain many simultaneous signals are involved and the signal flow is controlled, in part,
by the plasma level of γ-aminobutyric acid (GABA). The GABA level, to offer a simplistic
analogy, is like the gain control on a public address system in an auditorium. If the GABA level
is high, the gain of the nervous system is low; low GABA levels produce high gains and
increased sensitivity to small signals. In the public address system, if the gain is too high, even
the smallest output feeds back to the input microphone and the system breaks into uncontrolled
squeals. In animals the equivalent to the squeal is a random, uncontrolled firing of neurons that
induces violent spastic movements. This apparently is what happens in at least some forms of
epileptic seizures and is associated with lower than normal levels of GABA in the brain. One
might hope to control the seizures by administering GABA but this fails because GABA does not
pass the blood-brain barrier. Instead, one can take advantage of the fact that much of the natural
GABA is absorbed on cell walls; if it is displaced by some agent, the level of GABA in the
plasma can rise to an adequate control level.
In 1938 a compound that controls epileptic convulsions in this fashion was discovered. This
material, now called dilantin, proved particularly valuable because in ordinary doses it is not a
sedative and does not impair consciousness, unlike phenobarbital that had been used earlier.
You will be preparing dilantin by the three-step synthesis shown below. As it happens, each
of these steps contains some particularly pretty chemistry that will be described separately in the
next few sections.
Chemical Biology 3OA3, September 2009
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The Benzoin Condensation
Two molecules of an aromatic aldehyde, when heated with a catalytic amount of sodium or
potassium cyanide in aqueous ethanol, react to form a new carbon-carbon bond between the
carbonyl carbons. The product is an α-hydroxy ketone (a class of compounds with the generic
name benzoin).
The mechanism for cyanide-catalyzed benzoin formation involves a rather long sequence of
steps. (The mechanism can be found in most of the introductory organic chemistry texts.) It starts
with reversible cyanide ion addition to the carbonyl group of one benzaldehyde to form the anion
of the cyanohydrin, which in aqueous ethanol rapidly equilibrates with the neutral cyanohydrin.
The acidity of the C-H bond adjacent to the cyano group is enhanced by resonance stabilization
of the anion and under the basic conditions of the reaction (NaCN is basic) the isomeric
carbanion is formed. This adds to a second molecule of benzaldehyde; proton interchange and
loss of cyanide ion lead to benzoin.
There are two requirements for an effective catalyst of the benzoin condensation. First, the
catalyst must give significant amounts of carbonyl adduct (steps 1 and 2), but not form such a
strong bond that the catalyst is not easily lost in the last step. Second, the catalyst must stabilize
the anion sufficiently to allow the C-H bond to be broken readily, but not so much that the anion
becomes unreactive. For more than 100 years, the only species that had been found that satisfied
these requirements was the cyanide ion. However, in 1958 Breslow discovered that the
conjugate base of a thiazolium salt also was an effective catalyst; it added reversibly to aldehydes
and stabilized the α-anion by resonance.
Chemical Biology 3OA3, September 2009
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What gave Breslow's study broader significance was his recognition that thiamine (vitamin
B1) contains a thiazole unit and that a number of important biochemical reactions requiring it as a
coenzyme could be understood as analogs of the benzoin condensation.
Oxidation of Benzoin to Benzil
Benzoin can be oxidized to the diketone benzil in a number of ways, of which the most
interesting is by a "coupled oxidation" that uses Cu2+ as the catalytic-transfer oxidant. In a
coupled oxidation the overall oxidation proceeds in two distinct stages. In the present procedure,
cupric acetate is used in catalytic amount (less than 1% of the stoichiometric requirement) and is
continuously reoxidized from the reduced (cuprous) state by ammonium nitrate, which is present
in excess. The latter is reduced to ammonium nitrite, which decomposes in the reaction mixture
into nitrogen and water. It is convenient to represent this two-stage oxidation in the manner used
by biochemists, who commonly deal with multiple coupled reactions.
Cupric salts are mild oxidizing agents that do not attack the diketone product. In the absence
of Cu2+, ammonium nitrate will not oxidize benzoin (or benzil) at a significant rate. The reaction
is general for α-hydroxyketones (acyloins) and is the basis for the Fehling's test for reducing
sugars.
Chemical Biology 3OA3, September 2009
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Condensation of Benzil with Urea to Form Dilantin
Benzil and urea when heated together with base as catalyst condense to form dilantin. One
step of the process involves a phenyl shift so that both phenyls end up on the same carbon atom.
While 1,2 shifts occur readily in the case of carbocations, they are rare in the case of anions.
The rearrangement is analogous to the benzilic acid rearrangement. It is not clear in this case
why the rearrangement occurs. Perhaps it is the stability of the imide (-CO-NH-CO-) group that
drives it.
Procedures:
Benzoin
In a 50-mL Erlenmeyer flask equipped with a stirring bar prepare a solution of 1.04 g (0.003
mole) of thiamine hydrochloride in 3 mL of water. When all of the thiamine hydrochloride has
been dissolved, add 8 mL of 95% ethanol, 3 mL of 10% sodium hydroxide (0.006 mole), and 3
mL (3.2 g, 0.03 mole) of benzaldehyde, with vigorous stirring during each addition. Continue
the stirring for 2 hr, remove the stirring bar from the yellow solution, stopper the flask with a
cork and allow it to stand at room temperature in your drawer until the next lab period.
At the beginning of the next lab period, cool the flask in an ice-water bath to complete the
crystallization of the fine crystals. Collect the product on a Hirsch funnel and wash the crystals
thoroughly with two 20-mL portions of cold 50% ethanol until the filtrate is clear. Press the
colorless crystals as dry as possible and spread them on a fresh filter paper to dry in the air. The
yield is 1.8-2.2 g (dry weight).
The product may be used, without careful drying or recrystallization, for the preparation of
derivatives or for conversion to benzil. Benzoin may be purified, with a loss of 10-15%, by
recrystallization from methanol (12 mL/g of benzoin) or from ethanol (8 mL/g).
Oxidation of Benzoin by Cupric Salts.
In a 50-mL round-bottomed flask equipped with a magnetic stirring bar place 1.75 g (0.008
mole) of unrecrystallized benzoin, 5 mL of glacial acetic acid, 0.8 g (0.01 mole) of pulverized
ammonium nitrate, and 1 mL of a 2% solution of cupric acetate. Attach a reflux condenser and
bring the solution to a gentle boil while it is being stirred. As the reactants dissolve, evolution of
nitrogen begins. Boil the blue solution for 45 min to complete the reaction.
Cool the brownish green solution to 50-60o - it turns green - and pour it into 10 mL of icewater, while stirring. After crystallization of the benzil is complete, collect the pale yellow
crystals on a suction filter and wash them thoroughly with water. Press the product as dry as
possible on the filter. The yield is 1.4-1.6 g (dry weight). Benzil obtained in this way is
sufficiently pure for conversion to dilantin. If desired, it may be purified by recrystallization
from methanol or 75% aqueous ethanol.
Chemical Biology 3OA3, September 2009
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Dilantin
In a 25 mL round-bottomed flask equipped with a stirring bar place 400 mg of
unrecrystallized benzil, 200 mg of urea, 6.0 mL of ethanol, and 1.2 mL of 30% aqueous sodium
hydroxide. Attach an upright condenser (to avoid "freezing" the ground glass joint use a dab of
stopcock grease), and boil the brownish mixture gently for 1 h with stirring. Cool the reaction
mixture, add 10 mL of water and if a ppt is present, filter the solution to remove a sparingly
soluble side product that sometimes forms. Acidify the clear brownish filtrate with dilute
hydrochloric acid, collect the product on a suction filter, and wash it thoroughly with water. The
product may be recrystallized from ethanol. The yield is 0.28-0.40 g. The recorded melting point
is 286-295 °C; do not attempt to determine the melting point with an oil bath. Collect an IR
spectrum. (Caution-Dilantin is a powerful therapeutic agent and must be taken only on the
advice and supervision of a physician!)
Questions
1. Draw the structure of the product formed by addition of vitamin B1 and benzaldehyde.
2. Ammonia adds to carbonyl groups but it is ineffective as a catalyst for the benzoin
condensation. Explain.
3. Devise a very simple method for studying the kinetics of the oxidation reaction.
4. Write a detailed step-by-step mechanism for the reaction of benzil with urea in the presence
of hydroxide ion.
5. In dilantin sodium, it is the imide proton that is abstracted rather than the amide proton.
Why?
Chemical Biology 3OA3, September 2009
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EXPERIMENT 7
STUDYING THE REGIOSELECTIVITY OF ORANGE PEEL ESTERASE
1.
OVERVIEW
In Part 1 of this lab, you will be given one of 6 possible monoesters containing a phenolic
group, and acylate this compound with one of 5 possible acyl chlorides to prepare one of 30
possible diesters. Then, in Part 2, you will examine the enzyme orange peel esterase (OPE) and
determine which of the two distinct ester groups in your synthetic substrate is hydrolyzed by this
enzyme. You will also estimate the rate of ester hydrolysis for your substrate, and finally you
will use the entire class data set of rates for all the substrates to construct a model of the esterase
active site.
2.
INTRODUCTION
A highly active area of research in organic chemistry involves using enzymes to perform
organic transformations (1-4). Enzymes have the potential advantages of (1) catalyzing reactions
under mild conditions (aqueous solution, neutral pH, room temperature) and (2) high selectivity.
In this context, selectivity can mean chemical selectivity, i.e., reacting with only one type of
functional group, or it can mean regioselectivity, reacting with only one of multiple sites
containing similar functional groups in a single molecule. It can also mean stereoselectivity,
reacting with only one stereoisomer in a racemic mixture. However, high selectivity can be a
disadvantage if an enzyme does not act on the molecule and/or site you want. Chemists
examining a new enzyme for its potential synthetic utility will test it against a panel of potential
substrates to characterize its activity. In this lab, you will generate such a panel by using
combinatorial synthesis: each student will contribute one compound to the panel.
Biotransformations using enzymes have recently been used in environmental clean-up
processes, and are also routinely carried out on large scales in industrial processes. Examples
include: the conversion of glucose to the sweetener fructose in the food and beverage industry;
industrial fermentation to produce ethanol in the fine chemical and biofuel industries; and the use
of penicillin acylase to generate different members of the penicillin family in the pharmaceutical
industry.
Esterases are enzymes that hydrolyze ester functional groups (Scheme 1). They are used
widely in organic synthesis to remove ester functionalities that would otherwise have to be
hydrolyzed under harsh conditions (either acid or base, plus heat). A number of esterases are
also routinely used in industrial processes, often to take advantage of one or more of the abovementioned selectivities.
Scheme 1.
O
1
R
O
R2
esterase
R1 COOH
R2
HO
H2O
Chemical Biology 3OA3, September 2009
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3.
OBJECTIVES
In this lab, we will examine orange peel esterase's (OPE's) regioselectivity against a series
of diester substrates. You will be organized into groups by your TA, and each group member
will synthesize a unique diester compound to test as an esterase substrate. You will then
determine both the site and rate of esterase activity. By combining the results of your activity
assays, your group will devise a model of the esterase active site that explains the activity you
observe.
Week 1
R3O
O
O
R3O
O
R4
Cl
O
O
OH
R4
R3 = Me, Et or CH2CF3
OH = meta or para
R4 = Me, Et, C9H19, Ph or t-Bu
Week 2
R3O
HO
O
O
R3O
O
Orange Peel
O
O
Esterase
&/or
O
O
R4
OH
R4
(1) Site of cleavage?
(2) Rate of cleavage?
4.
SAFETY NOTES
Many acid chlorides are lachrymators: they induce tears. Keep the acid chlorides in the
hood, and in CAPPED vials while weighing. Keep all containers closed wherever possible: this
will avoid vapourization, and also prevent spontaneous hydrolysis of the acid chloride by
moisture in the air. Wear gloves when handling these compounds.
Chemical Biology 3OA3, September 2009
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5.
PRE-LAB
In week 1, you will have to calculate, beforehand, how much of each reagent to add to the
reaction. Most reagents will be present in excess in order to drive the reaction toward
completion. Complete Table 1, then calculate the number of moles of alkyl m- or
p-hydroxybenzoate you will be using, based on the mass you start with (250 mg). Then calculate
the number of moles of the other reagents, as well as the weight and volume, using Table 2, and
enter your calculated masses and volumes in Table 3.
Table 1. Your target compound.
Alkyl m- or p-hydroxybenzoate?
Molecular weight of starting material:
Molecular weight of target diester:
R4-(C=O)Cl:
Table 2. Molecular weights and densities of acyl chlorides
Reagent
Molecular Weight
(g/mol)
Acetyl Chloride
78
Propionyl Chloride
93
Benzoyl Chloride
141
Pivaloyl Chloride (t-BuCOCl)
121
Decanoyl Chloride
191
Table 3. Complete this table before the lab.
molar
Reagent
equivalents Moles
Alkyl
mor
p- 1
Hydroxybenzoate
Triethylamine
2
Acyl chloride
2
Chemical Biology 3OA3, September 2009
Density (g/mL)
1.10
1.06
1.21
0.98
0.93
mol. wt.
(g/mol)
density
(g/mL)
N/A
weight (g)
0.25
volume (mL)
N/A
0.73
Page 54
6.
EXPERIMENTAL PROCEDURE
Week One: Diester synthesis.
You will be organized into groups; your TA will tell you the structure you are to synthesize
ahead of time. Make sure you have selected the appropriate starting alkyl meta- or
para-hydroxybenzoate and acyl chloride to give the correct product.
Diester Synthesis
 Place the appropriate monoester (250 mg) in a 25 mL round-bottom flask, and add 5 mL of
dichloromethane (DCM), then 2 equivalents of triethylamine and finally 2 equivalents of acyl
chloride.
 Attach a reflux condenser, and heat at reflux gently for 15 min.
 Remove from heat and, when cool, check the reaction by TLC, using the saved starting monoester, diluted in DCM, as a standard. If a significant amount of starting material is still
present, heat for a further 30 min., then test again by TLC.
 Place the reaction mixture in a separatory funnel. Wash with 2 × 10 mL of 2 M HCl. (If there
is precipitate before washing, add DCM, 1 mL at a time, until it is dissolved.)
 Wash the organic layer twice with 5% aqueous NaHCO3.
 Check the product organic layer by TLC, comparing with the starting mono-ester. You MUST
ensure that all starting material has been removed at this point. If any is detected, repeat the
wash(es) described above.
 Dry the organic layer with anhydrous sodium sulphate, then filter the mixture, and wash the
solid sodium sulphate with excess DCM.
 Transfer the solution of product to a clean, dry round bottom flask, and evaporate the DCM
layer using a rotary evaporator (a steam bath can also be used here). There should be less than
0.5 mL of liquid after evaporation.
 Place the product in a vial labeled with the diester structure, your name, and date.
 Keep your product, and a sample of the saved starting monoester until the next lab.
Thin layer chromatography (TLC)
Draw a pencil line ~1 cm from the bottom of a TLC plate. Spot a sample of the starting material, and the
reaction mixture, and develop using 7:3 hexanes:ethyl acetate. Use a UV lamp to visualize the spots, and
draw an outline of the spots in pencil. Sketch the TLC in your lab book, and report the Rf value of each
spot.
Important Note: It is important to spot approximately the correct amount of material on the plate: too
little and the spot(s) are faint or not detected; too much and you see a giant spot with poor definition,
along with every trace of impurity. For UV detection, you need about 1-10 µg of material: this
corresponds to about 1-5 µL of a 1-10% solution.
The TLC plates contain a fluorophore that fluoresces green under UV light. If UV-absorbing
compounds, such as the aromatic rings of your mono- and di-esters, are present, they will absorb the UV
light and block fluorescence. Non-UV absorbing compounds are not visible by this method.
Chemical Biology 3OA3, September 2009
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Week Two
The goal of this week's experiments is to test each diester as an OPE substrate. The
reactions will be followed by TLC, which will be performed as in week 1. By pooling the results
of each group member and answering the questions below, it should be possible to create a model
of the OPE active site. Work together with the other members of your group to complete Table
4, which contains the site and rate of reaction for each diester. As a group, create a model of the
active site. Each group will submit a single group lab report at the end of the lab period.
Reaction mixtures and standards
 Dissolve 50 mg of the diester in 1 mL of acetone. If your diester is a liquid, place a graduated
vial on the balance and tare it. Then add drops of diester until you have 50 mg.
 Dilute 6 drops of your mono-ester and diester standard solutions (approx. 1%) into 0.8 mL of
acetone for use as TLC standards. Spot them onto a TLC plate.
 Add 0.1 mL (3 drops) of the diester solution from the step above to 0.8 mL of the provided
OPE solution in citrate buffer.
 Shake gently until the diester dissolves.
 Immediately spot a sample of the reaction mixture (RM) onto the TLC plate. Let it dry 5 min.,
then develop it in 7:3 hexanes:ethyl acetate. Failing to allow the RM spot to dry will result in
streaky, poorly resolved spots. If the RM spots are too faint, spot it on the TLC plate twice,
waiting one minute between applications.
 Use three lanes for each TLC, one each for the reaction mixture (RM), diester standard and
monoester standard.∗
 Run TLC's immediately after adding substrate to the OPE solution (t = 0), then at t = 15, 30, 60
and 120 min. If there is hydrolysis, compare the Rf's of the new spot(s) in the reaction mixture
samples to the standards to determine the cleavage site(s).
For our purposes, reactions will be classified as:
Rate
Fast
Medium
Slow
None
Criterion
- hydrolysis to monoester complete in ≤ 30 min
- hydrolysis to monoester complete in ≤ 120 min
- hydrolysis evident, but not complete within 120 min
- no hydrolysis visible after 120 min
NOTE: Clean the glassware for expt. 8 and rinse with acetone so it can be perfectly dry for next
lab period. Label your equipment, and place it in the oven.
∗ Ideally, you should also use the other possible monoester product as a TLC standard. For the purposes of this lab,
however, we will assume that any new spot that appears on the TLC during the course of the reaction that has a
different Rf than starting diester or the known monoester is this compound.
Chemical Biology 3OA3, September 2009
Page 56
7.
INTERPRETING YOUR DATA
The following pieces of information will help you interpret your data.
(1) Active site serine
Esterases use a Ser residue as an active site nucleophile to form a covalent acyl-enzyme
intermediate, and release the first product, HO-R2 (Scheme 2). In the second step, water acts as a
nucleophile to complete the reaction, releasing R1-COOH. Thus, we know that if a diester
substrate is cleaved at a particular site, then that ester group was located near the active site Ser
during catalysis.
Scheme 2.
HO
O
R1
O
HO
R2
R2
O
R1
esterase
R1
O
esterase
H2O
COOH
HO
esterase
(2) Hydrophobic substrates
Esterases tend to favour hydrophobic (non-polar) substrates.
The hydrophobic
interactions between non-polar groups are relatively non-specific - the tightest binding (and
highest rate) is achieved with substrates that fill the active site to the greatest extent without
causing steric clashes. Substituents that are too large or too small will be worse substrates.
Substituents with polar functional groups may require you to consider electronic effects (electron
withdrawing / donating) as well.
(3) Substrate variation and types of interactions
Each variable site in the substrates provides some information on specificity. Each varied
group (R3, R4, meta- vs para-) may interact favourably to increase the catalytic rate, unfavourably
to slow it, or not interact at all with the active site.
(4) Active site models
Jones and co-workers used very effective and simple "box" models of enzyme active sites
to explain specificity (4). They simply drew appropriately sized rectangles to represent the active
site, and labeled each site as "hydrophobic", "polar", "negative", "hydrogen bond donor", etc
(Fig. 1).
Hydrolysis rate
Box model
polar
small,
hydrophobic
medium
O
HO
medium,
hydrophobic
O
active site Ser
O
fast
HO
slow
HO
none
HO
O
O
O
O
O
Figure 1. Hypothetical box model for an esterase, showing how interactions with the active site
would be expected to affect rates of hydrolysis
Chemical Biology 3OA3, September 2009
Page 57
8.
QUESTIONS:
1. Complete Table 4 to record OPE's activity against all the diester substrates synthesized by
your group.
Table 4. Regioselectivity of OPE with diester substrates. In each cell, record the rate (fast, medium, slow,
none) in the top left corner, and site (R4 or R3) of cleavage in the bottom right corner. Use the top Table
(A) for meta substrates, and the bottom one (B) for para substrates.
A: meta
4
R
R3
Me
Et
CH2CF3
R3
Me
Et
CH2CF3
Me
Et
C9H19
Ph
tBu
B: para
B: para
R4
Me
Et
C9H19
Ph
tBu
(a) Compare your completed Table 4 to another group's. Are there any differences?
(b) In cases where there are discrepancies, it may be possible to make an educated guess which
is the correct result, based on the other results in Table 4. Can you make an educated guess
to resolve any discrepancies?
(c) Compare the completed Table 4A for meta substituents with 4B for the para ones. Can you
deduce any trends?
Chemical Biology 3OA3, September 2009
Page 58
2.
(a)
(b)
Which is the overall preferred hydrolysis site?
Are there any exceptions?
3.
(a)
Create a "box model" of OPE's active site based on the results in Table 4. Indicate
the relative sizes of the hydrophobic sites, and rationalize your model.
Do the aromatic ring, R3 and R4 interact favourably / unfavourably / not at all with
OPE?
Did the electron withdrawing character of R3 = CH2CF3 affect the rates relative to
Et?
What effect would you expect an electron-withdrawing R3 group to have on the
reaction rate if the reaction occurred at R3?
(b)
(c)
(d)
4.
9.
(a)
(b)
Are there any exceptions to the generalizations made above?
Can you rationalize these exceptions?
REFERENCES
1.
Koeller, K. M., and Wong, C.-H. (2000) Synthesis of Complex Carbohydrates and
Glycoconjugates: Enzyme-Based and Programmable One-Pot Strategies, Chemical Reviews
(Washington, D. C.) 100, 4465-4493.
2.
Wong, C. H., and Whitesides, G. M. (1994) Enzymes in synthetic organic chemistry,
Pergamon, Oxford.
3.
Jones, J. B. (1993) Probing the specificity of synthetically useful enzymes, Aldrichimica
Acta 26, 105-112.
4.
Jones, J. B., and Desantis, G. (1999) Toward understanding and tailoring the specificity
of synthetically useful enzymes, Acc. Chem. Res. 32, 99-107.
Chemical Biology 3OA3, September 2009
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EXPERIMENT 8
CLAISEN CONDENSATION: MICROSCALE SYNTHESIS OF 4-HYDROXYCOUMARIN
Lab by W. J. Leigh, edited by PH
One of the important enolate condensation reactions is the Claisen condensation, which
involves reaction of an enolate with the carbonyl compound of an ester. The enolate is generated
by removal of a slightly acidic hydrogen from the α-carbon of a ketone, nitrile or ester using a
relatively strong base (Step 1). The enolate then attacks the carbonyl carbon of an ester to yield
an intermediate which undergoes rapid loss of the alkoxide leaving group to yield a β-dicarbonyl
compound (Step 2). As usual, both steps in the sequence are completely reversible; the reaction is
driven to the product side by deprotonation of the relatively acidic product by the alkoxide ion
produced in the condensation.
Claisen-like condensations are also found in vivo, e.g. the condensation of 2 molecules of
acetyl-Coenzyme A to give acetoacetyl-CoA, a process catalyzed by acetoacetyl-CoA thiolase
(named for the reverse reaction). This process uses an enzyme base to produce an enolate from
one acetyl-CoA molecule; this enolate then attacks the other acetyl-CoA substrate molecule.
(More details on this mechanism can be found in a biochemistry text).
O
STEP 1
X
O
X
O
+
X
O
STEP 2
O
X
X = OR: ester Claisen reaction. X = SCoA: acetoacetyl-CoA thiolase reaction
The synthesis of 4-hydroxycoumarin involves reaction of o-hydroxyacetophenone, which
has two nucleophilic sites (the methyl carbon and the phenol oxygen), with diethyl carbonate,
which has two leaving groups attached to its carbonyl carbon. Because of the second nucleophile
and leaving group, the initial Claisen condensation product (Steps 1 and 2 below) undergoes an
intramolecular transesterification reaction to yield the cyclic product. Thus, the phenolate anion
acts as an internal nucleophile, and it participates in an intramolecular acyl substitution reaction
(Step 3). The resulting β-ketoester is highly acidic. The final product is therefore a new enolate,
which can be extracted from the reaction mixture by washing with water, in which it is soluble
because it is a salt. It is finally liberated by acidifying the aqueous extract (Step 4).
Chemical Biology 3OA3, September 2009
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OH
O- Na+
-OEt
1.
H
O
O- Na+
O
O- Na+
2.
O
O- Na+
O
O
O- Na+
O
OEt
OEt
O- Na+
O
O
O- Na+
3.
O
O
OEt
OEt
O
O
O
O
OEt
O
O
O
-OEt
O
O
O
O
O- Na+
4.
H
O
H3O+
during
work-up
O- Na+
O
This experiment will be done on milligram scale, using microscale equipment and techniques.
Before coming to the lab, be sure to read the sections on microscale distillations, extractions, and
recrystallizations in "Microscale Laboratory Techniques" after this lab.
Microscale Preparation of 4-Hydroxycoumarin
Note: It is very important that all equipment used in this reaction be thoroughly dried in an
oven at 110 oC for 30 minutes just prior to use. Upon removal from the drying oven, it should be
allowed to cool to room temperature in a desiccator.
Weigh and place 85 mg (2.13 mmol) of sodium hydride (60% dispersion in mineral oil) in a 10mL round bottom vial containing a magnetic stirring bar. Now add 3.0 mL of dry toluene. To the
flask attach a Hickman still fitted (at the top) with a condenser protected (again, at the top) by a
calcium chloride drying tube.
CAUTION: Sodium hydride (NaH) is a flammable solid which reacts violently with water
to produce hydrogen gas. Dispense in the hood. Toluene is distilled and stored over
molecular sieves. Dispense this solvent in the hood as well.
IMPORTANT NOTE: Use the right stuff: sodium hydride is in the SMALL BOTTLE
inside the larger bottle. The large bottle is a dessicant to keep the NaH dry. This reaction
Chemical Biology 3OA3, September 2009
Page 61
does not work with dessicant! It also doesn’t work with sodium hydrOXide (NaOH). Both
these variations were proven to fail last year!
In rapid order, place 133 µl (150 mg, 1.1 mmol) of o-hydroxyacetophenone, 3.0 mL of dry
toluene, and 333 µl (324 mg, 2.75 mmol) of diethyl carbonate in a stoppered 10-mL cylindrical
vial, using dried syringes to dispense the liquids. Remove the condenser from the distilling head
and add this solution, with stirring, to the reaction flask as rapidly as possible using a Pasteur
pipette. The resulting solution turns yellow. Immediately reattach the air condenser.
Place the reassembled apparatus in a sand bath and rapidly raise the temperature of the bath to
about 175 °C.
Collect the ethanol and toluene distillate (1.0 mL in 2 or 3 fractions) in the collar of the Hickman
still, and then remove the apparatus from the heat source. Allow the reaction solution to cool to
room temperature, and then add 3.0 mL of water with stirring.
Transfer the resulting two-phase solution to a 15 mL centrifuge tube using a Pasteur pipet. Rinse
the reaction flask with an additional 2 mL of water and add this to the centrifuge tube. Separate
the toluene layer using a Pasteur filter pipette and transfer it to a second 15-mL centrifuge tube.
Extract the toluene phase with 3 mL of water, and add the aqueous extract to the original water
phase. This aqueous solution contains the water-soluble sodium enolate of 4-hydroxycoumarin.
Cool the combined aqueous layers in an ice bath and acidify them by dropwise addition of
concentrated HCl using a Pasteur pipette. The solid product should precipitate from the acidic
aqueous phase. Add acid until the yellow color of the solution disappears (~10 drops). Collect the
product by vacuum filtration using a Hirsch funnel.
Recrystallize the crude 4-hydroxycoumarin from 50% aqueous ethanol using a Craig tube. Dry
the material on a piece of filter paper. Weigh the dried product and calculate the percent yield.
Determine the melting point and obtain an IR spectrum.
Questions:
1.)
Assign the major bands of the IR spectrum of 4-hydroxycoumarin.
2.)
Why does the nucleophilic attack in the first step occur through the enolate carbon, rather
than through the enolate oxygen or the aryloxide oxygen?
3.)
Why does 4-hydroxycoumarin exist primarily in the enol form?
Chemical Biology 3OA3, September 2009
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APPENDIX 1: MICROSCALE LABORATORY TECHNIQUES
By W. J. Leigh
Traditionally, experiments in organic chemistry are carried out on a macroscale level, employing
quantities of chemicals on the order of 5-100 g, using glassware designed to contain between 25
and 500 mL of liquids. For quantities of materials in the 0.005-0.5 gram range, one employs
different, "microscale" techniques and equipment in order to carry out the various standard
organic laboratory operations. In the following, the student is introduced to the special
equipment used in microscale experiments, as well as the somewhat different methods which are
used.
Basic Equipment
The glassware used for microscale experiments is contained in a kit. Some of the contents of the
microscale kit are illustrated in the drawing below.
The conical vial is used as a reaction
vessel, for extractions, and as a storage
container. Its flat base allows it to stand
upright on the laboratory bench. The
interior of the vial tapers to a narrow
bottom, making it possible to withdraw
liquids completely from the vial using a
disposable Pasteur pipette. The vial has
a screw cap which tightens by means of
threads cast into the top of the vial.
These threads also allow attachment of
various other pieces such as a condenser
or distillation head, using the doublecaps provided in the kit.
Liquids Handling
Since one rarely works with volumes larger than 2-3 mL, graduated cylinders are rarely used in
microscale experiments. Instead, one uses smaller scale volumetric devices such as syringes,
automatic pipettes, and calibrated disposable Pasteur pipettes.
Automatic pipettes are commonly used in microscale organic and biochemistry laboratories.
They are available in different sizes, and can deliver accurate volumes of aqueous solutions from
0.10 mL to 1.0 mL. They were not designed for use with organic solvents and are generally less
accurate depending on the liquid. Automatic pipettes are very expensive, and it is critical that
the student handle them carefully and responsibly. Follow the steps outlined below to use an
automatic pipette:
1. Select the desired volume by adjusting the micrometer control on the pipette handle.
2. Place a plastic tip on the pipette. Be certain that the tip is attached securely.
Chemical Biology 3OA3, September 2009
Page 63
3. Push the plunger down to the first detent position. Do not press the plunger to the second
position. (If the plunger is pressed to the second detent, an incorrect volume of liquid will be
delivered).
4. Dip the tip of the pipette into the liquid sample. Do not immerse the entire length of the plastic
tip in the liquid. It is best to dip the tip only to a depth of about one centimetre.
5. Release the plunger slowly. Do not allow the plunger to snap back, or liquid may splash up
into the plunger mechanism and ruin the pipette. Furthermore, rapid release of the plunger may
cause air bubbles to be drawn into the pipette. At this point the pipette has been filled.
6. Move the pipette to the receiving vessel. Touch the tip of the pipette to an interior wall of the
container, and slowly push the plunger down to the first detent. This action will dispense the
liquid into the container.
7. Pause one or two seconds and then depress the plunger to its second detent position to expel
the last drop of liquid. The action of the plunger may be stiffer in this range than it was up to the
first detent.
8. Withdraw the pipette from the receiver. If the pipette is to be used with a different liquid,
remove the pipette tip and discard it.
Syringes are especially useful when anhydrous conditions must be maintained during an
experiment. The needle can be inserted through a rubber septum sealing the reaction vessel, and
the liquid added to the reaction mixture. We use plastic (polyethylene) syringes which, although
they are called "disposable", can be cleaned and re-used. While the polyethylene barrels are
impervious to most solvents, the plungers are made of a less inert material; thus, these syringes
cannot be used with THF or halogenated solvents. To fill the syringe, insert the needle into the
liquid and draw in the required volume. Withdraw the syringe and pull the barrel back ever so
slightly to draw any liquid remaining in the needle into the syringe.
Disposable Pasteur pipettes are used for dispensing small quantities of liquids, as filtration
devices, and as columns for small-scale column chromatography. Although they are considered
disposable, you should be able to clean them for reuse as long as the tip remains unchipped.
Pasteur pipettes may be calibrated for use in operations where the volume does not need to be
known precisely, such as for measurement of solvents need for extraction and for washing a solid
obtained following crystallization. To calibrate a Pasteur pipette, weigh 0.5 g (0.5 mL) of water
into a small test tube on a balance. Attach a rubber bulb to a short Pasteur pipette. Squeeze the
rubber bulb before inserting the tip of the
pipette into the water. Try to control how
much you depress the bulb so that, when
the pipette is placed into the water and
the bulb is completely released, only the
desired amount of liquid is drawn into the
pipette. When the water has been drawn
up, place a mark with an indelible
marking pen at the position of the
meniscus. A more durable mark can be
made by scoring the pipette with a file.
Repeat this procedure with 1.0 g of water,
and make a 1-mL mark on the same
pipette.
Chemical Biology 3OA3, September 2009
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A filtering pipette is used to remove solid impurities from a liquid with a volume less than 10mL. To prepare it, a small piece of cotton is inserted into the top of a Pasteur pipette and pushed
down to the beginning of the lower constriction in the pipette. It is important that enough cotton
is used to collect all the solid being filtered; however, the amount used should not be so large that
the flow rate through the pipette is significantly restricted. The cotton plug can be pushed down
with a long thin object such as a glass stirring rod or a wooden applicator stick. In some cases,
such as when filtering a strongly acidic mixture or when performing a very rapid filtration, it may
be better to use glass wool in place of the cotton, even though it is not quite as good as a filtering
aid. To conduct a filtration, the filtering pipette is clamped so that the filtrate will drain into an
appropriate container. The mixture to be filtered is transferred to the filtering pipette with another
Pasteur pipette. If the volume of the mixture being filtered is less than 1-2 mL, you should rinse
the filter and plug with a small amount of solvent after the last of the filtrate has passed through
the filter. If desired, the rate of filtration can be increased by gently applying pressure to the top
of the pipette using a pipette bulb.
A filter-tip pipette is useful for transferring volatile solvents during extractions and in filtering
very small amounts of solid impurities from solutions. It is made by loosely shaping a tiny piece
of cotton into a ball, and pushing it to the bottom of the pipette using a wire with a diameter
slightly smaller than the inside diameter of the narrow end of the pipette. If it is difficult to push
the cotton into the tip, you've probably used too much cotton. To use the filter-tip pipette, simply
draw the mixture to be filtered into the pipette using a pipette bulb and then expelling it. With
this procedure, small amounts of solid will be captured by the cotton.
Solids Handling
Microscale experiments involve quantities on the order of 200-300 mg at most, and it is thus
important to be able to weigh solid substances to the nearest milligram. This requires use of a
sensitive top-loading balance protected against drafts with a shield, or an analytical balance.
All weighings must be made into a previously weighed ("tared") container. The tare weight is
then subtracted from the total weight of container plus sample to give the weight of the sample.
Solid samples are manipulated using microspatulas similar to those shown below. The larger
style is more useful when relatively large quantities of solid must be dispensed.
Carrying out Reactions
A typical assembly for heating a reaction mixture under reflux is shown below. While an air
condenser is adequate for most applications, a water-jacketed condenser is also supplied, for
cases where the solvent is very volatile or where the ambient air temperature is very high. A
"spin vane" might also be included for magnetic stirring of the reaction mixture - this is a
triangular device coated with Teflon, which is shaped to fit the bottom of the conical flask.
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Note that the apparatus is clamped at the condenser
rather than at the flask, as one would do for a
macroscale experiment using conventional groundglass joint glassware. The apparatus can be
clamped in this way because of the screw-cap
connection between the condenser and reaction
vial, which prevents the connection from falling
apart.
Heating is provided by a sandbath atop a magnetic
stirrer/heater. A thermometer should be clamped in
contact with the sand so as to allow monitoring of
the bath temperature. The bath contains slightly
more than 1 cm of sand - it is important to have
enough to ensure good thermal contact with the
reaction vial, but not so much that it is difficult to
see the contents.
Extractions
In microscale experiments, the conical reaction vial
is the glassware item used for extractions. The two
immiscible liquid layers are placed in the vial, and
the top is sealed with a cap and a Teflon insert
(with the Teflon side toward the inside of the vial).
The vial is shaken to provide thorough mixing between the two liquid phases. As the shaking
continues, the vial is vented periodically by loosening the cap and then tightening it again. After
about 5-10 seconds of shaking, the cap is loosened to vent the vial, retightened, and the vial is
allowed to stand upright in a beaker until the two liquid layers separate completely.
Two basic procedures are possible, depending on whether the solvent being used to extract the
desired product is heavier or lighter than water. Method A is employed for extractions where the
lower layer is a heavy solvent such as dichloromethane. Method B is employed for extraction
with a solvent which is lighter than water, such as diethyl ether.
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Method A - solvent heavier than water
Method B - solvent lighter than water
Note that in this technique, one draws both phases into the pipette and then returns the heavy
(aqueous) phase to the conical vial. Ether is so volatile that it is often difficult to hold it in the
pipette. Use of a filter-tip pipette for this procedure will help prevent the volatile organic layer
from squirting out in an uncontrolled way.
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Recrystallizations
Recrystallizations can be carried out using a conical reaction vial and conventional vacuum
filtration to collect the crystals on a small filter paper, or in a Craig tube, which is a device
designed specifically for recrystallization of very small quantities of materials.
In recrystallizations with a conical reaction vial, the conical vial simply takes the place of the
Erlenmeyer flask used for macroscale recrystallizations. The isolation of the crystals can be done
in a number of ways depending on their form:
(i) Once crystallization is complete, the mother liquors and crystals are vacuum-filtered through a
small Hirsch funnel. Most commonly, the material is transferred to the filter by pouring, using a
microspatula to help transfer the crystals from the vial to the filter. In cases where the crystals are
fairly small and fluffy, it may be more convenient to draw the entire mixture of crystals + mother
liquors into a Pasteur pipette and transfer them to the Hirsch funnel that way.
(ii) If the crystals adhere to the side of the flask, then filtration is unnecessary. Simply use a filtertip pipette to remove the mother liquors and transfer them to another flask. Fresh, cold solvent is
added to wash the crystals, and this is then removed with the pipette in the same way. The
crystals are then dried using a very light stream of air or nitrogen, but care must be taken to
ensure that the stream is light enough that the crystals don't get blown out of the vial.
Craig tube Recrystallizations
Craig tubes are particularly
useful for recrystallizing
amounts of solid less than
~100 mg, the main advantage
being that it minimizes the
number of transfers of solid
material and thus maximizes
the yield of crystals. The
separation of the crystals
from the mother liquor with
the Craig tube is very
efficient, and little time is
required for drying the
crystals. The steps involved are fundamentally the same as those performed in macroscale
crystallizations with an Erlenmeyer flask and a Hirsch funnel:
Step 1. In crystallizations where a filtration step is not required in order to remove insoluble
impurities such as dirt or activated charcoal, this step can be done directly in the Craig tube;
otherwise, a small test tube is used. The solid is placed in the Craig tube and the appropriate
solvent is heated to boiling in a test tube placed in a sand bath. Several drops of hot solvent is
added to the Craig tube, which is then heated in the sand bath while stirring continuously with a
microspatula using a twirling motion. This helps dissolve the solute and prevent the boiling
liquid from bumping. Additional portions of hot solvent are added until the solid is completely
dissolved. Do not add too much solvent, in order to maximize the yield.
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Step 2. When a hot filtration is necessary, the solid should be dissolved as much as possible in a
test tube as described above. Alternatively, the solid can be dissolved in the Craig tube and the
liquid transferred to the test tube using a Pasteur pipette preheated with hot solvent. To draw the
liquid into the pipette, expel the air from the pipette and then place the end of the pipette on the
bottom of the tube, being careful not to trap any solid in the pipette. The small space between the
pipette and the bottom of the tube should allow you to draw up the liquid without removing any
solid. Rinse the Craig tube with a few drops of hot solvent, and then add these to the test tube.
This procedure serves to perform a rough pre-filtration to remove larger pieces of solid. The
Craig tube is then washed and dried.
Step 3. The test tube containing the mixture is then heated in the sand bath, adding 5-10 drops of
solvent to ensure that premature crystallization doesn't occur during the filtration step. To filter
the mixture, take up the mixture in a filter tip pipette which has been preheated with hot solvent,
and quickly transfer the liquid to the clean Craig tube. Passing the liquid through the cotton plug
in the filter-tip pipette should remove the solid impurities. If this is unsuccessful, it may be
necessary to add more solvent to prevent crystallization and filter the mixture through a filtering
pipette. In either case, once the filtered solution has been returned to the Craig tube, it is
necessary to evaporate some solvent until the solution is saturated near the boiling point of the
liquid. This is best done by placing the Craig tube in the sand bath, and boiling the solution while
rapidly twirling the solution with a microspatula. When a trace of solid material coating the
spatula just above the level of the liquid is observed, the solution is near saturation and
evaporation should be stopped.
Step 4. The hot solution is cooled slowly in the Craig tube to room temperature. This is done by
inserting the inner plug into the outer part of the Craig tube, and then placing the whole thing into
a 10-mL Erlenmeyer flask. This provides some insulation to slow the cooling rate. The cooling
rate can be slowed even further by first filling the Erlenmeyer flask with ~8 mL of hot water at a
temperature below the boiling point of the solvent (be careful not to put so much water in the
Erlenmeyer that the Craig tube floats). The Erlenmeyer flask containing the Craig tube is then
placed on a few layers of paper and left alone to cool to room temperature. Once crystallization at
room temperature is complete, the Craig tube is then placed in an ice-water bath to maximize the
yield.
Step 5. Once crystallization is complete, a 3" piece of copper wire is wrapped around the barrel
of the inner plug of the Craig tube (see 'A' below), and a Centrifuge tube is placed over top of it.
After bending the copper wire back up the side of the centrifuge tube so that the Craig tube is
held securely inside it (see 'B' below), the centrifuge tube is inverted (see 'C' below). The solvent
should seep out of the Craig tube, leaving the crystals behind. The tube is then centrifuged for a
few minutes to complete the separation of the mother liquors from the crystals. Using a
microspatula, the crystals are then scraped off the end of the inner plug or from inside the Craig
tube onto a watch glass or piece of paper. Minimal drying will be necessary.
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Distillation
The key to successful microscale distillations is in
avoiding long distillation paths, since this is the main
factor leading to loss of material during distillation.
Short-path microscale distillations are carried out using
the Hickman distillation head as the receiving device
for the distilled liquid. Two types of Hickman head,
'ported' and 'unported', are shown in the figure below.
The complete apparatus consists of a flask or vial
containing the liquid and a magnetic spin vane or
boiling stone, attached to the bottom joint of the
Hickman head. If desired, a condenser is attached to the
top joint. A thermometer can be suspended down the
middle in order to record the distilling temperature,
with the bottom of the thermometer in the lower part of
the Hickman head just below the circular well. The
vapours of the heated liquid rise upward and are cooled
and condensed on either the inside walls of the
Hickman head or on the walls of the condenser. As
liquid drains downward, it collects in the circular well
at the bottom of the still. The well can contain as much
as 2-mL of liquid.
Collection of fractions is easiest with the ported
Hickman head; the port is opened and the liquid in the
well removed with a Pasteur pipette (see 'C'). With the
unported head, the liquid is drawn out from the top
with a plastic Pasteur pipette (see 'A'). If a condenser or
internal thermometer is used, the distilling apparatus
must be partially disassembled in order to do this. In
some stills the inner diameter of the head is so small
that it is difficult to reach in at an angle with the pipette
and make contact with the liquid. This problem may be
remedied by bending the tip of the pipette slightly in a
flame.
Once removed, the liquid is transferred to a small vial
and capped with a Teflon-sealed cap.
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