CHEMICAL SYNTHESIS

ABSTRACT

Since non‑natural amino acids are expensive in comparison to natural amino acids we searched for a high yield synthesis method for O‑methyl‑L‑tyrosine. Problems with chemical alterations of amino acids to form non‑natural derivates often lie in the higher reactivity of amino and carboxyl groups compared to other reactive groups. For this reason both groups need to be kept in mind while searching for a possible reaction for the desired synthesis.
For the protection of the amino group an acetylation reaction was carried out to form N‑acetyl‑L‑tyrosine. The tested method used N‑acetyl‑L‑tyrosine as a reagent which was then methylated at the carboxyl group and at the hydroxyl group using dimethyl sulfate by Williamson ether synthesis. To finally form the non‑natural amino acid, an acidic hydrolysis using hydrochloric acid was performed.

Theoretical background

In order to methylate the p‑hydroxy group at the benzene ring of tyrosine, the amino group which is more reactive, has to be protected. As protective group acetic anhydride is used to acetylate the amino group (figure 1).

The second step is a Williamson ether synthesis. Sodium hydroxide (NaOH) was added in order to deprotonate both hydroxyl groups of the amino acid. Dimethyl sulfate (DMS) transfers its methyl group to the now hydrolisated hydroxyl group at the benzene ring of tyrosine. Simultaneously the other methyl group is added to the carboxyl group as a methoxyl group (figure 2).

In order to eliminate the protecting groups hydrochloric acid (HCl) was added (ficture 3), both protective groups can be easily removed by hydrolysation to achieve O‑Methyl‑L‑tyrosine.

Implementation

The chemical synthesis of O‑Methyl‑L‑tyrosine (OMT) includes 4 steps which are listed in the scheme below.

Equipment consisting of a three-necked flask, a Dimroth condensor, a dropping funnel and a thermometer.

Sample A

5 g tyrosine was dissolved in 100 ml H₂O. The solution was heated up to 65 °C. Acetic anhydride was added to the solution and then the solution was heated up for 2 hours, afterwards the heating process was stopped. There was a mulky white precipitation. 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. Following that, the solution was heated up to 100 °C for 30 min and set to cool down. The solution was used for a TLC to prove if primary amino groups (→ results) were present.

Sample B

5 g tyrosine was dissolved in 100 ml H₂O. The solution was heated up to 65 °C. Acetic anhydrite and sodium hydroxide (NaOH) were added simultaneously to the solution so the pH value was constantly 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, so that's ensured, that 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. Following that the solution was heated up to 100 °C for 30 min and set to cool down.

Sample B1

MTBE (Methyl tert‑butyl ether) was added to the now 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 value = 1) 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 is saponified shifting the balance towards the product. During the reaction, a yellow colored substance appeared on the solution surface 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.

Sample B2

MTBE (Methyl tert‑butyl ether) was added to the now 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 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) then heated up to 100 °C for 30 min. While cooling off hydrochloric acid (HCl) 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.

Results & Conclusion

Run of sample A

In sample A a suspension formed instead of a solution. This is due to a mistake in the first step, because the acetic anhydride was added to early while the temperature of the solvent was to low. A ninhydrin test showed primary amino groups what proves that no reaction occured. Therefore sample A was discarded and considered a failure.

Run of sample B1

Sample	name	color	migration distance [mm]	R_f
S	solvent	‑	34 mm	1
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 the 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 are a sign for using an unsuitable solvent (propanol, ammoniac, H₂O). Both samples have migrated a similar distance distance at the end of the screening, which proves 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 (CAS: 6230‑11‑1).
As a result Sample B1 contains 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 (Melting point apparatus)

Run of sample B2

Sample	name	color	migration distance [mm]	R_f
S	solvent	‑	34 mm	1
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 migrated the same distance as tyrosine after the run of the TLC which proves a relation of the chemical structure between the samples.
Additionally a melting point determination was performed. The melting point of sample B2 was determined 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 (CAS: 6230‑11‑1).
As a result Sample B2 contains 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 (Melting point apparatus)

Conclusion

The melting point of the samples B1 and B2 are respectively 268 °C and 266 °C, even considering the uncertainty of the melting point apparatus to be 0,1-2,5 °C.
To be absolutely sure that the targeted molecule was synthesized, further analysis of the samples should be following. Analytical methods like Nuclear Magnetic Resonance (NMR), Gas Chromatography (GC), IR spectroscopy, or Mass Spectrometry (MS) are capable of identifying the structure of the molecules represented by sample B1 and B2. Sadly the department of chemistry gave us only 8 days to use their faculties, which was a way too short to successfully run all the experiments and therefore to run the final tests.
However it is a great success to have achieved very positive results after running the very first experiment. For sure we can proudly display our exertions as a co-working team that has learned a lot and achieved results beyond the initial expectations. In coming iGEM projects, more in‑depth cooperations with the chemistry department are conceivable. Also projects with a heavier 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.

Methods

Recrystallization

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 pH value of tyrosine is 5.66 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, not mixed, liquid phases. For separation, both effects play a role.
The stationary phase needs 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 horizontal, the saturation with fumes in the separating chamber should be optimal for best results. Leveling agents flow across the stationary phase because of 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

Sample apply

Chromatogram development

After development, the solvent front is immediately marked by a pencil. The chamber shouldn't be put into the air stream of a deduction. 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 Thin‑Layer‑plate. Some substances prevent fluorescence and don't appear under UV‑Light. Also coloring chemicals (p.e. ninhydrin) which are sprayed on the Thin‑Layer‑plates after chromatography are used.

Quantitative analysis

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.

Melting point determination

Melting point determination is a standard identification method in chemistry for solid products. A substance's melting point depends on its crystal construction. If there is a contamination, the melting point decreases. Eviations 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.

References

[1]
[2]
[3]

Orthogonal Pair
Reporter
KILL(switch)
Genomic Integration
Chemical Synthesis

Team:TU Darmstadt/Lab/ChemicalSynthesis