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iGEM Tübingen 2017

Team Inspiration Results Human Practice Lab Attributions

Chemistry

Introduction

In the process of modifying our aminocoumarin in vivo, the need arose to create a novel 4-hydroxybenzoic acid derivative, that might be recognized by the CloL enzyme and contains the possibility to form a β-lactam ring. Therefore, we chose to substitute the isoprenyl moiety of the native aminocoumarins with a 3-amino butanoic acid. As shown in Fig.1, multiple synthesis pathways could easily be imagined to yield the desired compound.

Fig. 1: Various reactions that would result in the desired compound.

Unfortunately, many of those options had limited use for the synthesis of our compound, due to overly high costs (nucleophilic substitution) or poor expected yields in combination with a high probability of side reactions (Mannich). Therefore, we focused our efforts on the Aza-Michael and reductive amination reactions. Due to budget limitations, we chose to manufacture the 3-formyl-4-hydroxybenzoic acid needed for the reductive amination pathway ourselves. The inclined chemist may recognize many suitable reactions that are able to create this particular compound. However most of them either project low yields (Reimer-Tiemann) or deal with toxic compounds (Gattermann-Koch). Remaining pathways include the Vilsmeier-Haack and much less known Duff reaction, for which the synthesis of this specific intermediate has already been published. (Fu et al. 2015) The reaction mechanism can be seen in Fig. 2.

Fig. 2: Reaction mechanism of the Duff reaction. As a first step the Urotropin is protonated, which causes a rearrangement to yield an iminium-ion. This ion is then attacked by the α-C atom of the phenolic compound in a nucleophilic substitution. After further protonation of the Urotropin-moiety, followed by formation of another iminium ion, a series of hydride shifts will result in a conjugated system. This compound is then hydrolysed by addition of aqueous acid to yield the formyl-derivative of the phenolic compound. In the same step the urotropin moiety decomposes into ammonia and formaldehyde.

Although not necessary for ortho-selectivity in our reaction, we elected to use the copper catalyzed Duff reaction hoping it might result in an increased reaction rate. Additionally to the Duff-Reaction, we elected to use an Aza-Michael reaction to yield a methylated derivative of our originally designed compound. The reaction mechanism can be seen in Fig.3

Fig. 3: Reaction mechanism of the Aza-Michael reaction. This reaction occurs congruently to a normal Michael-reaction, the only difference being, that the attacking nucleophile is not a deprotonated carbon, but a deprotonated amine.

Results

The outcome of the chemical synthesis was analyzed by LC-MS. Because the acidified substance did not crystallize it was stored in methanol. Fig 4. shows us how the substance was mono-, di-, and trimethylated over time.

Fig. 4: Our desired product is in the top left corner, the other molecules are the mono (top right corner)-, di (bottom left corner)- and tri (bottom right corner)-methylated products.

The spectra in Fig 5-10 reflect the methylation over the time.

Fig. 5: MS-Chromatogram of the product at 260 nm

Fig. 6: MS chromatogram of the product. Red indicates more signal than can be processed

Fig. 7: MS spectrum at minute 5.0. Red indicates more signal that can be processed.

Fig. 8: MS spectrum at minute 5.4. Red indicates more signal that can be processed

Fig. 10: MS spectrum at minute 7.6. Red indicates more signal that can be processed.

The synthesis was successful. In the ring closure experiment we used HPLC-purified unmethylated substance. Results are further explained in the discussion.

Discussion

As can be seen in the results (Figure 2-7) the synthesis of our intermediate compound was successful. The chromatograms show the expected mass and additionally the mono-, di-, and trimethylated form. First the methylation appeared to be a problem since most purification methods wouldn’t apply to our problem. Since we needed to separate our product from the methylated form as they could be poisonous to the colonies. Cristallisation proved impossible in the remaining time but we were able to purify it by using a HPLC. The Duff reaction can be recommended as an efficient way to synthesize 3-formyl-4-hydroxybenzoic acid.

References

Fu, X. W., Pu, W. C., Zhang, G. L., & Wang, C. (2015). Synthesis of salicylaldehydes from phenols via copper-mediated duff reaction. Research on Chemical Intermediates, 41(11), 8147-8158.