1. Synthesis of Iodo Ester Azide
Weston Manter
Department of Natural and Environmental Sciences, WSCU
Gunnison CO, 81231
Intro: L-Glutamate is the major excitatory amino acid in the mammalian nervous system. Its
presence contributes to many neurological processes such as memory, learning and the
development or elimination of synaptic pathways (Danbolt, 2001). Glutamate controls these
functions by extracellular receptors on the surface of the cell, so the concentration of glutamate
in the extracellular fluid is what determines how much or if these receptors are stimulated. While
it is necessary to have glutamate in the extracellular fluid, too much of it is toxic and can
overstimulate these receptors causing neurological damage resulting in diseases such as
Alzheimer’s or Huntington’s (Danbolt, 2001). With this in mind it makes sense that the majority
of glutamate present in the brain is located in intracellular space and not the extracellular fluid,
“The normal (resting) concentration of glutamate in this fluid is low, in the order of a few
micromolar. In contrast, the glutamate concentration inside the cells is several thousand times
higher, at around 1 - 10 millimolar.”(Danbolt, 2001). Since high concentrations of glutamate
outside the cell are toxic, there are mechanisms in place to regulate the concentration of
glutamate outside the cell. There has been no enzyme found that can efficiently metabolize
extracellular glutamate so the main source of glutamate regulation comes from excitatory amino
acid transporters that uptake glutamate into the cell where it is relatively harmless. There are
known to be 5 subtypes of transporters, but they all follow the same general trend. These
transporters work by creating sodium and potassium concentration gradients across the cellular
membrane in order to bring glutamate into the cell. The selectivity of these transporters to
different conformations of glutamate has been looked into briefly and what has been discovered

2. is that there are a variety of receptors that will bind to many different conformations of glutamate
(Danbolt, 2001). The binding sites of these transporters are so flexible likely due to the many
different low-energy conformations glutamate can take, since these different conformations are
still energetically favorable, binding sites must be present to interact with them. For this
experiment a pathway for the synthesis of a rotationally restricted analogue of L-glutamate was
explored. The goal was to find a novel method by which variants of a rotationally restricted
analogue of glutamate called 2-CPG could be synthesized and then used in later experiments to
study the cellular uptake of different structures of glutamate.
NH2
COOH
COOH

3. Methods:
CO2CH3
CO2CH3
COOH
COOH
CH3OH
H2SO4
CO2CH3
CO2CH3
CO2CH3
CO2CH3I
H5IO6
KI
CH3 CH3
CH3
CH3 CH3
CH3
SO2Cl
CH3 CH3
CH3
CH3 CH3
CH3
SO2N3
NaN3
90% Acetone/H2O
CO2CH3
CO2CH3I
CO2CH3
CO2CH3I
N3
KHMDS, Tris Azide
THF @ -78 °C
The first reaction for this pathway (Scheme 1) is the synthesis of homophthalic ester from
homopthalic acid. Homophthalic acid was weighed out and placed into a round bottom flask
along with methanol and a stir bar. The solution was heated to 80 degrees Celsius in an oil bath,
while heating sulfuric acid was added to the flask dropwise. When all the sulfuric was added

4. reaction was allowed to proceed at 80 degrees Celsius for 48 hours while stirring. After workup
the homophthalic ester was obtained as a golden oil. A percent yield was not calculated for this
reaction.
The next step in the pathway (Scheme 2) is to halogenate the aromatic ring of
homophthalic ester with iodine. Iodine was chosen due to its usefulness in potential future
reactions such as a Suzuki-Miyaura cross-coupling. To iodinate homophthalic ester sulfuric acid,
periodic acid and KI were added to a round bottom flask and placed in an icebath. This turned
the solution a dark purple color. Next the homophthalic ester was added dropwise after all other
reagents dissolved and the reaction was allowed to proceed on ice for one hour while stirring.
Once reaction was complete a workup was done and a yellow/white solid was obtained. The
solid was recrystallized from isopropanol to afford a white crystalline solid iodinated ester
product. A percent yield was calculated and found to be 28.9% and product was verified by
NMR.
Before continuing on with the modification of this molecule, a reagent for the next
reaction had to be synthesized. Tris Azide was synthesized (Scheme 3) by adding 90%
acetone/DI water, sodium azide and triisopropylbenzenesulfonylchloride to a round bottom flask
and this reaction was allowed to proceed for 48 hours while stirring. Once product was obtained
it was ran through a column using 25% ethylacetate/hexane as the mobile phase. This yielded a
clear/white crystalline product. A percent yield was not calculated for this reaction
Now that all reagents are prepared the next step (Scheme 4) is to azidate the iodo-ester
molecule previously synthesized. All equipment was dried prior to the experiment and the
procedure was carried out in an inert environment of N2 gas. Iodo ester, THF, KHMDS and tris
azide were added to a round bottom flask in a dry ice and acetone bath and the reaction was

5. allowed to proceed for two hours while stirring. Product was collected and ran through 10%
ethyl acetate/hexanes column for purification and a fine white crystalline powder was obtained.
After this the purified product was analyzed by NMR and a percent yield was calculated that
came out to 43%.
Discussion: Homophthalic acid was successfully taken all the way to an iodo-azide-
homophthalic ester product, this was verified by TLCs throughout the procedure and a NMR of
the final molecule confirms it was successfully synthesized with a percent yield of 43% and no
contaminants. Sources of error for this experiment were mostly from human error, although it
should be noted that the KHMDS is getting old and it appears that the concentration in the bottle
is significantly lower than what the label says since we had to bump KHMDS concentration up
in our iodo-ester azidation reaction (Scheme 4). For future experiments this iodo-azide-
homophthalic ester molecule would be taken all the way to its 2-CPG variant and then a Suzuki
Cross-coupling reaction would be performed to add another ring to the structure, once a viable
path for this synthesis was established this cross-coupled molecule could be tested for glutamate
uptake rates in different cells surrounding neurons in the brain.

6. Experimental:
WM-I-3
.501g homophtalic acid was added to a 50mL round bottom flask. 8mL of ethanol was then
added to the homophthalic acid. Solution was placed in an 80° C oil bath and stirred
continuously. Once solution was heated .46ml of H2SO4 was added dropwise and the reaction
was allowed to proceed for 48 hours. After 48 hours the flask was taken off the heat and allowed
to cool to room temperature, after cooling sodium carbonate was used to neutralize the solution,
pH paper was used to determine when solution was neutralized. The neutralized solution was
then poured into a 125mL separatory funnel along with 10mL of ethyl acetate and shaken
vigorously to extract organic molecules from the inorganic layer. The organic layer was collected
in a separate container and the inorganic layer was poured back into the separatory funnel and
two more ethyl acetate extractions were performed collecting all organic layers in the same
container. Inorganic solution was discarded and the organic solution was dried using sodium
sulfate. After drying solution was cotton filtered and then rotovapped down using a Buchi
Homophthalic Acid H2SO4 Homophthalic ester
Amount 0.501g .46mL
MW 180.26 98.07 208.21
mM 2.78 8.34
Ratio 1 3 1

7. Rotovap RII. TLC was then ran in 25% ethyl acetate/ hexanes to verify synthesis of
homophthalic ester.
WM-I-47
6mL of H2SO4 was added to a 50mL round bottom flask and placed in an ice bath, next .276g of
H5IO6 was added while being stirred. Once cooled .398g of KI were added incrementally over a
10 minute period. Once all KI was dissolved .496g of homophthalic ester were added dropwise
and the reaction was allowed to take place for 2 hours. When the two hours were over the
solution was poured over ice and then into a 125mL separatory funnel, where an extraction was
performed 3 times using 20mL of dichloromethane and collecting the organic layer each time.
The collected organic solution was then placed back into the separatory funnel and 20mL of
sodium thiosulfate was added in order to quench any excess iodine, this turned the solution from
a dark purple to a clear white color. The organic layer was kept in the separatory funnel and the
inorganic was removed and discarded. The organic solution was then brined with 20mL of
sodium chloride, collected and then dried using sodium sulfate. This was then cotton filtered,
Homophthalic ester KI H5 IO6 Iodo Ester
Amount .509g 0.398g 0.276g
MW 208.21 166 228 334
mM 2.4 2.4 1.2
Ratio 1 1 0.5 1

8. rotovapped and allowed to dry. Once dry the product was recrystallized in isopropanol,
recrystallized product weighed .232g and gave a percent yield of 29%.
CH3 CH3
CH3
CH3 CH3
CH3
SO2Cl
CH3 CH3
CH3
CH3 CH3
CH3
SO2N3
NaN3
90% Acetone/H2O
WM-I-43
10mL of 90% acetone/H2O were placed into a 25mL round bottom flask, next .117g NaN3 were
added while being stirred continuously. After dissolved .519g 2,4,6-
triisopropylbenzenesulfonylchloride were added and the reaction was allowed to proceed for 24
hours. After 24 hours 10mL of DI H2O were added to the round bottom. This solution was
poured into a 125mL separatory funnel and 3 10mL diethyl ether extractions were conducted and
then combined and placed in an empty 125mL separatory funnel and brined with 10mL of NaCl.
The organic layer was collected and sodium sulfate was added to get rid of any remaining water.
Solution was then cotton filtered and rotovapped. Once dry the Tris azide was re-dissolved and
loaded onto a chromatography column and ran with a mobile phase of 25%ethyl acetate/hexane.
2,4,6 triisopropylbenzenesulfonylchloride NaN3 Tris Azide
Amount 0.519g .117g
MW 302.87 65.01 309.43
mM 1.714 1.799
Ratio 1 1.05 1

9. The purified Tris Azide was collected and verified by TLC but a product weight was not
obtained.
CO2CH3
CO2CH3I
CO2CH3
CO2CH3I
N3
KHMDS, Tris Azide
THF @ -78 °C
WM-I-63
All equipment for this experiment was flame dried prior to use to eliminate all possible H2O
present on the equipment. All procedures were carried in an inert environment of N2 gas to
eliminate the possibility of the anion reacting with any H2O in the air. Placed .25g of iodo ester
into a 50mL round bottom flask and placed in dry ice and acetone bath and cooled to -70° C.
Once cool 5mL of THF were added followed by 1.12mL of KHMDS, this caused the solution to
turn a bright orange color. In a separate flask .278g of tris azide were dissolved in 2mL of THF.
Waited 30 min for the anion to form in the round bottom and then added the dissolved tris azide
to the round bottom flask. Once tris azide was added the reaction was allowed to proceed for 2
hours, after this time .11mL of acetic acid was added to neutralize the solution. The product was
then placed in a chromatography column with a mobile phase of 10% ethylacetate/hexane,
fractions were collected and TLCs were ran for each fraction to determine the location of the
iodo-azide-homophthalic ester product in the fractions. It was found that fractions 5-11 all
Iodo Ester KHMDS Tris Azide Acetic Acid Iodo-azide-homphthalic ester
Amount 0.25g 1.12mL .278g .11mL
MW 334.2 199.49 309.43 17.5 M 361.2
mM 0.748 1.12 0.897 1.87
Ratio 1 1.5 1.2 2.5 1

10. contained the product so these were combined in a 50mL round bottom flask and rotovapped
down. The final product weighed .116g giving a percent yield of 43%. NMR was ran on this
product and the spectra came out very clean with no signs of contamination from other
compounds.
1H NMR results:

12. References:
"A Variety of Transporters with an Affinity for Glutamate." The Neurotransporter Group.
Center for Molecular Biology and Neuroscience, n.d. Web.
Danbolt, Neils C. "Glutamate Uptake." Progress in Neurobiology 65.1 (2001): 1-105. Science
Direct. Web.
Kotha, Sambasivarao, Kakali Lahiri, and Dhurke Kashinath. "Recent Applications of the
Suzuki–Miyaura Cross-coupling Reaction in Organic Synthesis." Tetrahedron 58.48 (2002):
9633-695. Print.