Team:Manchester/Chemical Analysis

Chemical Analysis

We propose a non-enzymatic reaction that would convert homogentisic acid to hipposudoric acid
We argue that this reaction occurs spontaneously in the presence of oxygen

Chemical analysis

When RetroPath did not predict a complete reaction pathway towards Hipposudoric Acid (HA), we re-examined the structure of our intended product:

It can actually be seen quite easily that this molecule can be derived from two units of Homogentisic Acid (HGA), and we indeed found a number of papers speculating that Hipposudoric Acid could be produced by the oxidative dimerisation of Homogentisic Acid (HGA) (4,5,6).

This dimerisation reaction has to be oxidative, and might proceed in two oxidation steps via the oxidation product of HGA: benzoquinone acetate (BQA).

Step 1: 2 × C₈H₈O₄ + O₂ → 2 × C₈H₆O₄ + 2 × H₂O

Step 2: 2 × C₈H₆O₄ + O₂ → C₁₆H₈O₈ + 2 × H₂O

It is well known that the oxidation of HGA to BQA occurs spontaneously in the rare human metabolic disorder of Alkaptonuria (3), and that BQA polymerises in the presence of oxygen, resulting in the characteristic dark urine of affected individuals. In the presence of oxygen, in our bacterial cultures and on hippopotamus skin, we would expect the second step of our proposed reaction to occur spontaneously, the equilibrium favouring the extended system of conjugated bonds in the dimeric compound, Hipposudoric Acid.

The polymerisation of HGA to black pigment is sped up in alkaline conditions; this matched the observation that hippos produce and stabilise Hipposudoric Acid in an alkaline mucus secretion in their sweat (2,7). This compelling evidence strongly suggests that the proposed route to Hipposudoric Acid is indeed feasible.

The plausibility of the proposed spontaneous nature of the oxidative dimerisation of HGA to Hipposudoric Acid is further supported by the well-known observation that Hipposudoric Acid over time polymerises further into an insoluble brown-black polymer, as was also observed in our validation experiment using bacteria engineered to express our hypothesised pathway. The oxidative reaction might even be light-activated and might contribute to the UV-protective effect of Hipposudoric Acid,

Once we had identified a plausible precursor of Hipposudoric Acid, we re-run our retrosynthesis again, with HGA as our target compound. Retropath successfully identified a route from E. coli central metabolism to HGA, thus completing our new biosynthetic pathway.

References

Literature

Constantinou, L., Gani, R., (1994) New Group Contribution Method for Estimating Properties of Pure Compounds, American Institute of Chemical Engineers Journal, 40, 1697-1710

HTokuhura, Y., Shukua, K., Tanaka, M., Sogabe, K., Ejima, Y., Hosokawa. S., Ohsaki, H., Morinishi, T., Hirakawa, E., Yatomi, Y., Shimosawa, T., (2018) Absorbance measurements of oxidation of homogentisic acid accelerated by the addition of alkaline solution with sodium hypochlorite pentahydrate, Scientific Reports, 8, 1 - 9

Mistry, J.B., Bukhari, M., Taylor, A.M., (2013) Alkaptonuria, Rare Diseases, 1, 1 - 7

Saikawa, Y., Moriya, K., Hasimoto, K., Nakata, M., (2006) Synthesis of hipposudoric and norhipposudoric acids: the pigments responsible for the color reaction of the red sweat of Hippopotamus amphibious, Tetrahedron Letters, 47, 2535-2538

Hashimoto, K., Saikawa, Y., Nakata, M., (2009) Studies on the red sweat of the Hippopotamus amphibius, Pure and Applied Chemistry, 79, 507-517

Roberts, N.B., Curtis, S.A., Ranganath, M.L.R., (2015) The Pigment in Alkaptinuria Relationship to Melanin and Other Coloured Substances: A Review of Metabolism, Composition and Chemical Analysis, JIMD Reports, 24, 51-66

Saikawa, Y., Hashimoto, K., Nakata, M., Yoshihara, M., Nagai, K., Ida, M., Komiya, T., (2004) The red sweat of the hippopotamus, Pigment chemistry, 429, 363

    
     igem2020manchester@gmail.com