Synthesis and Stability of Aspartame - Schweizer Jugend Forscht Study week February 2015
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Schweizer Jugend Forscht
Study week February 2015
Synthesis and Stability of Aspartame
Author: Supervisor:
Jérémie Reusser Peter Kirschner
March 4, 2015
Synthesis and Stability of Aspartame (H-Asp-Phe-OMe) Contents 1 Abstract 2 2 Introduction 3 3 Equipment and Methods 4 3.1 Synthetization Method . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.2 Hydrogenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.3 Separation of the Boc protective group . . . . . . . . . . . . . . . . . 5 3.4 Stability testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4 Results 8 5 Discussion 10 5.1 Products of the synthesis . . . . . . . . . . . . . . . . . . . . . . . . 10 5.2 Stability of Aspartame . . . . . . . . . . . . . . . . . . . . . . . . . . 11 6 Conclusion 12 7 Aknowledgements 13 References 14 Chapter 0 Jérémie Reusser 1
Synthesis and Stability of Aspartame (H-Asp-Phe-OMe) 1 Abstract This projects aims to synthesize the artificial sweetener Aspartame using the pre- cursors Boc-Asp(OBzl)-OH and H-Phe-OMe, bonding these to oneanother using classical protein synthesis. Additionally, the stability of the molecule under every- day circumstances was studied by taking advantage of HPLC. The final product was of very high purity (99.8 percent or higher), the synthesis therefore a success. Stability studies confirm that Aspartame decays very easily under high temperature conditions (regarless of the environment, be it aqueous or as a solid powder). In addition, it appears that some sort of different decay occurs in acidic solutions. This phenomenon needs more detailed analysis prior to further conclusions. Chapter 1 Jérémie Reusser 2
Synthesis and Stability of Aspartame (H-Asp-Phe-OMe) 2 Introduction The goal of this project was to study and synthesize the molecule of Methyl L-α- aspartyl-L-phenylalaninate, more commonly known under the name of Aspartame. It is a common sugar substitute which was first synthesized by accident in 1965. It has since then found use in many products such as soft drinks, chewing gums, drug additives or simply used to sweaten coffee or tea. It is considered to be around 200 times more powerful than sucrose, greatly decreasing the necessary amount and therefore calories of a product. [1] Due to its reputation of being instable, a study of the stability of the molecule under various conditions was also object of this project. In alkaline or high temperature conditons Aspartame can react from H-Asp-Phe-OMe to a cyclic molecule, Cyclo(Asp-Phe) and MeOH (Methanol). Chapter 2 Jérémie Reusser 3
Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
3 Equipment and Methods
3.1 Synthetization Method
The synsthesis of Aspartame for this project was executed using a classic proteinic
synthesis. Due to time constraints, the synthesis of the two main reactants was done
prior to this synthesis. The precursor Boc-Asp(OBzl)-Phe-OMe was therefore used
as the primary educt of the synthesis.
Figure 1: Synthesis of the primary precursor (Boc-Asp(OBzl)-Phe-OMe)
3.2 Hydrogenation
The goal of the first step of this synthesis is to separate the OBzl protecting group
from the Aspartic acid (Asp). To achieve this, 48.5g of the precursor is dissolved in
a mixture of Acetic acid (AcOH, 225ml) and water (25ml). 1.5g of Palladium-coated
active coal powder is soaked in water to prevent spontaneous ignition and added to
the mixture. The suspension is kept in a four-necked round-bottom flask, where it
is stirred continuously. The athmosphere inside the flask is then neutralised with
Nitrogen gas, to prevent the ignition of the reactants after Hydrogen is added. Once
the athmosphere is inert (after about two minutes), the flow of Nitrogen is replaced
by Hydrogen - the reaction is then left alone overnight. A TLC analysis of the prod-
ucts is done: the educt, reference product and a mixture of the latter are compared
to the product of the reaction. Since the product still is in the AcOH solution, it
must be cristallized under vaccum in a rotary evaporator. Water is also added to
homogenize the slurry. The powder left over is constituted of Boc-Asp-Phe-OMe,
the OBzl protective group having been replaced by a single Hydrogen atom. One
molecule of Toluene per molecule of product is therefore dissolved in the acid.
The resulting powder is then washed in water and filtered in a Büchner funnel
three times. The effectiveness of the washing process can be checked by performing
a TLC of the water used for cleaning and comparing it to the purified substance.
The purified powder is put back into the rotary evaporator under vacuum and dried
at a temperature of 40◦ C overnight.
Chapter 3 Jérémie Reusser 4Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
Figure 2: Separation of the OBzl protective group (Boc-Asp-Phe-OMe)
3.3 Separation of the Boc protective group
Next, Boc-Asp-Phe-OMe must be reacted to H-Asp-Phe-OMe. In order to do this,
the powder is dissolved in 180ml of 3.4N Hydrochloric acid in Ethyl acetate solution.
The fumes are aspirated by a water vacuum pump, which dissolves the HCl fumes
and is then neutralized in a water treatment facility. The mixture must be kept
under constant stirring. Since the Boc group is instable in acidic environment, it is
separated from the molecule and replaced by Hydrogen. Large amounts of carbon
dioxide gas are generated by this reaction, so the fumes must be sucked out of the
reaction vessel.
Figure 3: Chlorinated H-Asp-Phe-OMe is left over after the reaction
As pictured, the product remains chlorinated, which means, that it must go
through additional purification reactions. Isopropyl ether is then added to the solu-
tion, which is poured into a Büchner funnel, so that the product can be filtered. It is
then cleaned and filtered three more times in Isopropyl alcohol. Again, the resulting
liquid is analysed using TLC, to check for the effectiveness of the filtration.
Chapter 3 Jérémie Reusser 5Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
To remove the leftover Chlorine from the H-Asp-Phe-OMe, the substance is dis-
solved in a mixture of 500ml of Ethyl alcohol and 200ml of water and approximately
11.7ml of Triethylamine is added drop by drop. The pH must be constantly moni-
tored and the solution cooled in an ice bath. Around pH 5.4, the isoelectric point
should be reached and the product should abruptly cristallize and form a thick
suspension. The product is washed and filtered in Ethyl alcohol five times, while
samples of the washing solution are taken for analysis by TLC. Also, silver nitrate is
added to a small sample of the remaining filtrate. If chloride ions are present, it will
form very poorly soluble silver chloride, instantly disclosing chloride’s presence. If
the result of the test is negative, the product can be dried in the rotary evaporator
and should be constituted of pure H-Asp-Phe-OMe (Aspartame). The product is
now ready for use or further studies of the compound.
Figure 4: After removal of the hydrochloric acid, pure Aspartame (H-Asp-Phe-OMe)
is left over
Chapter 3 Jérémie Reusser 6Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
3.4 Stability testing
Since Aspartame is notoriously unstable, several scenarios were simulated. For in-
stance, under high temperatures or alkaline environments, the molecule can sponta-
neously react to its cyclic form of Cyclo(Asp-Phe) by separation of the OMe group.
This happens because the amine group of Alanine can bind to the carboxylic group
of the Phenylalanine, resulting in the cyclic shape of the molecule and release of
OMe. With adition of a proton, OMe essentially turns into MeOH - Methyl alco-
hol/Methanol, which is notoriously toxic to human beings. [1]
To test the behavior of Aspartame, commercially available samples were chosen,
in order to create a close approximation of what might happen in reality.
Table 1: Setup of the stability samples
1 2 3 4
Simulated Hot coffee Used in an Baked into Reference
situation acidic diet- a cake
soft drink
Test setup Heated up Dissolved in Heated Dissolved
in aqueous 5% solution powder to in water at
solution at of Phospho- 140◦ C room tem-
80◦ C ric acid perature
All the samples are then analyzed through High Performance Liquid Chromatog-
raphy (HPLC).
Chapter 3 Jérémie Reusser 7Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
4 Results
The first step of synthesis (hydrogenation) appears to have left almost no traces of
precursor, the reaction can therefore be called complete. Similar TLC analysis were
performed after various reactions and filtrations - all showing flawless results.
Figure 5: TLC of the product comapared to reference substances
The HPLC chromatogram of the finished products shows no signs of impurities,
nor leftover reactants. The synthesis can be declared a great success.
Figure 6: HPLC of H-Asp-Phe-OMe/REJ1
Chapter 4 Jérémie Reusser 8Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
The chromatogram of the stability study clearly shows, that H-Asp-Phe-OMe
has decomposed to Cyclo(Asp-Phe) in some cases. However, resolution is insuficient
and references missing, to determine what exactly consitutes the peak located near
the decay product in the case of the acidic environment sample. This matter will
be futher discussed in following sections. It can however be deduced, that the
environment (especially heat) has an effect on the decay of Aspartame, since the
control sample shows no signs of decay whatsoever.
Figure 7: Chromatogram of the decay products of Aspartame
Chapter 4 Jérémie Reusser 9Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
5 Discussion
5.1 Products of the synthesis
Considsering the HPLC data, it appears that the synthesization process works ef-
fectively and produces a very pure output. The exact purity cannot be determined,
since no notable peaks can be distinguished in the chromatogram, other than injec-
tion peaks and influences from the MeOH-solvant itself. It is to assume purity is
somewhere close to 100 percent. Integration of the graph leads to a value of 99.8%,
without showing any traces of distinguishable impurities. The HPLC data does not
give any representative evidence of Palladium traces in the final product, though
the possibility of its presence can not be excluded. The numerous purification steps
likely have eliminated significant amounts of the catalyst. Further investigation
would have to be performed to qualify the purity of the product as sufficient for use
in hypothetical nutritional applications.
Figure 8: The completed and purified product
Chapter 5 Jérémie Reusser 10Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
5.2 Stability of Aspartame
The results show that Aspartame can decompose in high temperature environments.
This is not a surprise and has been documented numerous times in the past. The
chromatograms show a very consistens overlap, which leads to the assumption that
the retention times can be trusted. Therefore it becomes clear that the second
(or in case of the sample in Phosphoric acid, the shifted) peak must be attributed
to some other molecule. This is supported by the fact, that due to its structure,
Aspartame should not spontaneously decay in acidic environment - at least not in
a way analogous to the way this occurs under high temperature. The reason for
this lies in the chemical structure itself: For it to reach a cyclic shape, H+ must
be released from H-Asp-Phe-OMe, something unlikely to occur spontaneously in
acidic conditions, where the concentration of H+ is higher than usual. Following
the law of Le Chatelier, the reaction equilibrium must be shifted left. Data about
Aspartame seems to confirm this: it’s most stable state is reported to be at pH
4.3. For the reaction to occur, the equilibrium has to be shifted right by either
temperature increase or higher pH. This does however not exclude a different reaction
with the acid, which might produce a decay molecule different from Cyclo(-Asp-
Phe). This second decay reaction can not be attributed to the acid exclusively, since
it appears to have occured in a neutral but high temperature aqueous environment as
well. Determining the exact nature of the compound would require further analysis,
possibly of the structural properties of the molecule in question.
Phe)
ance
Aso-
edsubst
o(
Cycl -
i
dent
uni i
f
Figure 9: Two separate decay products can be produced
Chapter 5 Jérémie Reusser 11Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
6 Conclusion
The synthesis of Aspartame by proteinic synthesis, based on the precursors Boc-
Asp(OBzl)-Oh and H-Phe-OMe can be considered a success, though not proven
viable for use in nutritional applications, due to the inability to confirm or exclude
the presence of Palladium in the final product. It must still be noted, that this should
probably not be an issue, due to numerous filtrations and purifications along the way.
The issue could also be resolved with the use of a scavenger-compound, which could
eliminate traces of the metal. Industrial methods notably use a different process,
which is simpler in practice, than optimizing the method used for the project.
Also Aspartame is confirmed to be unstable under eveveryday conditions (dis-
solving it in hot beverages for example). Acid does not appear to affect it much,
although surprisingly, unexpected decomposition occured regardless. The nature of
this would have to be further investigated.
Chapter 6 Jérémie Reusser 12Synthesis and Stability of Aspartame (H-Asp-Phe-OMe) 7 Aknowledgements I greatly thank Peter Kirschner and Marc for their support, insight, assistance and interesting conversations over the course of my stay at the lab B-127. Along with them I thank the rest of the team of lab B-127 and all the employees of Bachem otherwise related to this project. Special thanks of course go to Bachem AG for hosting and enabling this project, as well as to Schweizer Jugend forscht for the organization of this great week of new experiences. Chapter 7 Jérémie Reusser 13
Synthesis and Stability of Aspartame (H-Asp-Phe-OMe)
References
[1] Wikipedia. Aspartame — Wikipedia, the free encyclopedia, 2015. URL http:
//en.wikipedia.org/wiki/Aspartame. [10.02.2015].
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