Since non‑natural amino acids are expensive in comparison to natural amino acids, we searched for a high yielding synthesis of O-methyl-l-tyrosine. When chemically altering an amino acid to a non‑naftural derivate the higher reactivity of the amino and carboxyl groups in comparison to the desired reactive group has to be considered. For this reason, amino and carboxyl groups need to be protected before carrying out the desired synthesis.
For the protection of the amino group an acetylation reaction was performed to form N‑acetyl‑L‑tyrosine, which was then methylated at the carboxyl group and the hydroxyl group using dimethyl sulfate in a Williamson ether synthesis. In order to finally form the non‑natural amino acid, an acidic hydrolysis using hydrochloric acid was performed.
In order to methylate the p‑hydroxy group at the benzene ring of tyrosine, the amino group which is more reactive needs to be protected. Acetic anhydride is used to acetylate the amino group for creating a prospective group (see figure 1) .
Figure 1: Reaction mechanismn of an acetylation of the amino group to protect the site from unwanted side reactions in later synthesis steps.
The second step was a Williamson ether synthesis. Sodium hydroxide (NaOH) is added in order to deprotonate both hydroxyl groups of the amino acid. Dimethyl sulfate (DMS) transfers its methyl group to the deprotonated hydroxyl group at the benzene ring of tyrosine. Simultaneously, the other methyl group is added to the carboxyl group as a methoxyl group (see figure 2) .
Figure 2: Reaction mechanism of a Williamson ether synthesis to add a methyl groups to both hydroxy functions of the amino acid.
In order to remove the protecting acetyl group and methoxy group, hydrochloric acid (HCl) was added (see figure 3). Both groups are removed by hydrolysis to achieve O‑methyl‑l‑tyrosine .
Figure 3: Reaction mechanism of the hydrolysis to remove remaining amino and methoxy groups.
The chemical synthesis of O‑methyl‑l‑tyrosine includs 4 steps, which are shown in the figure 4.
Figure 4: Overview of the four step synthesis with its educts, reagents, duration and conditions.
Figure 5: Experimental setup consisting of a three-necked flask, a Dimroth condensor, a dropping funnel and a thermometer.
5 g tyrosine were dissolved in 100 ml H2O. The solution was heated up to 65 °C before acetic anhydride was added to the solution. After a second heating step for 2 hours, the heating process was stopped and a milky white precipitation was visible. The pH value was adjusted to pH 12 by the addition of NaOH. The solution was heated up to reach 42 °C and then 5.22 ml DMS was added. Afterwards, the solution was heated up to 100 °C for 30 min and set to cool down. The solution was used for a thin-layer chromatography (TLC) in order to prove, if primary amino groups were present (see results).
5 g tyrosine were dissolved in 100 ml H2O and the solution was heated up to 65 °C. Acetic anhydrite and sodium hydroxide (NaOH) were added simultaneously to the solution to ensure a constant pH value between slightly acidic and neutral. The pH value needs to be neutral for the reaction of tyrosine and acetic anhydrite because of the weak solubility of tyrosine in H2O. Acetic anhydride works as a protective group at the amino group of tyrosine. The solution was heated up for 2 hours. As soon as the solution turned into a clear green colored solution, the heating process was stopped. The pH value was adjusted to pH 12 with addition of NaOH. The solution was heated up to reach 42 °C and then 5.22 ml DMS was added. Afterwards, the solution was heated up to 100 °C for 30 min and set to cool down.
MTBE (Methyl tert‑butyl ether) was added to the cooled down solution which was filled into a separating funnel for the extraction. This step guarantees that the acetylated amino acid stays in the organic phase while the by‑products remain in the aqueous phase.
The organic phase was set into a rotary evaporator with the help of negative pressure to distillate the solution in order to acquire the intermediate product (step 3; scheme). The product which was left in the evaporator was added to a three-necked flask with H2O and a small amount of hydrochloric acid (pH 1) and then heated up to 100 °C for 30 min. In order to increase the yield of the product, NaOH was added so that the solution was saponified shifting the equilibrium towards the product. During the reaction a yellow colored substance appeared on the surface of the solution, which was aspirated. The by-product was dried and the melting point was determined to 92 °C.
Further hydrochloric acid was added to the solution and heated up for 30 min. While cooling off, NaOH was added to the solution to adjust the pH value to 5.66 which is the isoelectric point of tyrosine.
Subsequently the solution was recrystallized with the help of activated carbon and cooled down in an ice bath in order to precipitate the product. The sediment was filtered and dried to measure the melting point.
After cooling MTBE (Methyl tert‑butyl ether) was added to the solution which was filled into a separating funnel for the extraction. This step guarantees that the acetylated amino acid stays in the organic phase while the by-products remain in the inorganic phase.
The organic phase was set into a rotary evaporator with the help of negative pressure to distillate the solution in order to acquire the intermediate product (step 3; scheme). The product which was left in the evaporator was added to a three-necked flask with H2O and a small amount of hydrochloric acid (pH value = 1). The solution was then heated up to 100 °C for 30 min. While cooling down, hydrochloric acid (HCl) was added to the solution to adjust the pH value to 5.66. Subsequently, the solution was recrystallized with the help of activated carbon and cooled down in an ice bath in order to precipitate the product. The sediment was filtered and dried to measure the melting point.
Results & Conclusion
Run of sample A
In sample A a suspension was formed instead of a solution what was due to a mistake in the first step. The acetic anhydride was added too early while the temperature of the solvent was too low. A ninhydrin test showed primary amino groups proving that no reaction occured. Therefore sample A was discarded and considered a failure.
Run of sample B1
|Sample||name||color||migration distance [mm]||Rf|
|O||product (sample B1)||purple||25 mm||0,735|
|T||educt (tyrosine)||purple||26 mm||0,765|
Sample B1 was taken after a full synthesis (scheme; Step 4) and a recrystallization.
Tyrosine, which was used as a control sample, and sample B1 were solved and applied on the Thin Layer Plate (TLP).
A ninhydrin-test was done after a Thin Layer Chromatography (TLC) to test whether primary amino groups were present.
The semicircles at the top of the bands were a sign for using an unsuitable solvent (propanol, ammoniac, H2O). Both samples had migrated a similar distance at the end of the screening, which proved a relation of the chemical structure between the two samples.
Additionally, a melting point determination was performed. The melting point of sample B1 was determined to be 266 °C. The determination of the product's melting point showed a significant deviation to the educt's (tyrosine) melting point of 342 °C. The melting point of sample B1 was also far from the melting point of O‑methyl‑l‑tyrosine which is 259‑261 °C .
As a result sample B1 assumed to contain residues of the educt (tyrosine) which increases the melting point of the product. Furthermore, the melting point apparatus has a deviation of 0.1 °C‑2,5 °C
Run of sample B2
|Sample||name||color||migration distance [mm]||Rf|
|T||educt (tyrosine)||purple||20 mm||0,588|
|B1||product (sample B1)||purple||20 mm||0,588|
|B2||product (sample B2)||purple||20 mm||0,588|
Sample B2 was taken after a full synthesis (scheme Step 4) and a recrystallization.
Tyrosine which was used as a control sample, a sample of B1 and a sample of B2 were solved and applied on the thin-layer plate (TLP).
A ninhydrin-test was done after a thin-layer chromatography (TLC) to test whether primary amino groups were present.
Sample B1 and sample B2 have covered the same distance as tyrosine after the run of the TLC which indicates a relation of the chemical structure between the samples.
Additionally a melting point determination was performed. The melting point of sample B2 was detected to be 268 °C. The determination of the product's melting point showed a significant deviation to the educt's (tyrosine) melting point of 342 °C. The melting point of sample B2 was also far from the melting point of O‑methyl‑l‑tyrosine which is 259‑261 °C .
As a result sample B2 was assumed to contain residues of the educt (tyrosine) which increases the melting point of the product. Furthermore, the melting point apparatus has a deviation of 0,1 °C‑2,5 °C
The melting points of the samples B1 and B2 are 268 °C and 266 °C, respectively, considering the uncertainty of the melting point apparatus to be 0.1-2.5 °C.
To ensure that the target molecule was synthesized, further analysis of the samples should be performed. Analytical methods like nuclear magnetic resonance (NMR), gas chromatography (GC), IR spectroscopy or mass spectrometry (MS) are capable to identify the structure of the molecules represented by sample B1 and B2. Due to temporal restrictions the successful synthesis of O‑methyl‑l‑tyrosine and the final analyses could not be perforemed.
However, it is a great success to have achieved positive results after running the very first experiment. In retrospect, we can proudly display our exertions as a co-working team that has learned a lot especially concerning preparative organic chemistry. In coming iGEM projects, more in‑depth cooperations with the chemistry department are conceivable and projects with a greater chemistry part are of potential future interest, so the established connections could be of high value to the next generations of iGEM teams in Darmstadt.
Recrystallization is a process for purifying a solid or crystalline substance.
The substance is dissolved and filtered to remove impurities and to regain the crystallized product. Recrystallization depends on temperature and solubility of the purified substance and its impurities. The substance is required to be soluble under high temperatures and less soluble if the temperature is lowered. Solvent and substance should have similar polarities and the solvent should be inert towards the recrystallized substance. Expected impurities should be highly or unremarkably soluble in the chosen solvent. The optimal solvent and its volume is usually determined in several experiments before the recrystallization process can be started.
During the conduction the isoelectric point of tyrosine was used for precipitation. The isoelectric point of tyrosine is 5.66 , the pH value where tyrosine possess the lowest solubility in H2O.
Thin-layer Chromatography (TLC)
Thin-layer chromatography (TLC) is based on two principles called adsorption and distribution. Adsorption means dissolved substances in leveling agents adsorb on stationary phase (e.g. silica gel) and distribution describes different solubility of substances in different and non-mixed liquid phases. For separation, both effects play a role.
The stationary phase has three characteristics: Chemical structure (cellulose, silica gel, poly amide, etc.), grain size (smaller and more uniform grains for higher separating efficiency) and film thickness (100‑250 μm). Leveling agents in the mobile phase also have influence on separation, they allow polar groups of the substance to be separated. Often a mixture of different leveling agents is used.
The main concepts of thin-layer chromatography (TLC) are separating efficiency, selectivity and saturation of the separating chamber. Separating efficiency can be observed by spot widening with increasing separation height. The general spot widening decreases when separating efficiency increases. Separating efficiency is influenced by selectivity, how well two similar substances can be separated from each other. In the separating chamber, the movement has to be vertical, the saturation with fumes in the separating chamber should be optimal for best results. Leveling agents flow across the stationary phase due capillary forces. The solvent front must not reach the upper edge of the thin layer. After removing the thin layer, the solvent front has to be marked and dried in a horizontal position.
Implementation and Development of TLC
- Sample preparation The sample has to be a homogeneous liquid. Typically purified samples are used. Comparative solutions of known concentrations are used as control samples.
- Sample apply The samples should be placed on a line 1 cm from the edge. The distance between the samples should be about 1 cm as well. Typical sample volume ranges between 1-10 μl.
- Chromatogram development The liquid level in the chamber must not touch the applied samples at any time. Furthermore, the TLC plate must not stand towards the wall with its coated surface because of capillary effects. The mobile phase moves across the TLC plate. The solvent front may not reach the end of the TLC plate.
After development, the solvent front is immediately marked by a pencil. The chamber should not be put into the air stream of the fume hood. For detection mostly either fluorescence or fluorescence-discharge is used. Fluorescence can help to visualize the sample via UV‑light, fluorescence‑discharge has a fluorescing layer integrated into the TLC plate. Some substances prevent fluorescence and do not appear under UV light. In addition, coloring chemicals (e.g. ninhydrin) may be sprayed on the TLC plates after chromatography.
The amount of the substances can be estimated by the size of the spot. TLC-scanners (densitometric evaluation) measure the intensity of reflected light with a certain wavelength.
Development of a TLC with the ninhydrin‑test
Ninhydrin targets primary amino groups. These amino groups are colored yellow on the TLC with the reaction in the scheme below.
Figure 6: Reaction scheme of the ninhydrin test for coloring primary amino groups after chromatography.
Melting point determination
Melting point determination is a standard identification method in chemistry for solid products. The melting point of a substance depends on its crystal construction. If there is a contamination, the melting point decreases. Deviations from literature values up to 1-2 centigrade are tolerable. The substance is placed in a capillary tube and heated up until the substance starts to melt.
- Wiley-VCH, Organikum, 24. Edition, ISBN:978‑3‑527‑33968‑6
- http://www.chemspider.com/Chemical-Structure.2006112.html 10/12/2016