NMR goes bananas

A new type of multiply functionalized natural tetrapyrrole, known as Ma-FCC-61, is found in ripening or senescent parts of the banana plant as the breakdown of chlorophyll takes place. An NMR spectroscopic study reveals details and shows that these unique tetrapyrroles carry complex ester functions at the propionyl side chain. These colourful degradation products may act to attract fruit-eating animals that then spread the plant's seeds.

Over the last twenty years, chemists have studied the break down products of the chlorophyll in plants and fruits and had come to the conclusion that the "de-greening" of leaves, for instance, leads to colourless and non-fluorescent chlorophyll catabolites (NCCs). In apples and pears, the chlorophyll molecules catabolise to NCCs identical to those in the senescent leaves of the fruit tree from which they come. Senescence and fruit ripening to yield NCCs as the "final" product seemed to be an altogether colourless process.

But, then it was found that bananas are different. Fluorescent chlorophyll catabolites (FCCs) accumulate in senescent, yellow banana leaves, and their extracts contain only trace amounts of NCCs. In ripening bananas, "persistent" [long-lived] FCCs, such as Mc-FCC-56, have been discovered that explain the peculiar blue luminescence of senescent banana leaves under ultraviolet light.

Now, Srinivas Banala, Simone Moser, Thomas Mueller, Christoph Kreutz, Andreas Holzinger, Cornelius Luetz, and Bernhard Kräutler of the University of Innsbruck, Austria, have used NMR and other techniques to investigate the FCCs causing the blue luminescence they discovered previously in detail. They suggest that the decomposition products of chlorophyll in this plant become appended with special sugar molecules in a unique fashion (hypermodified), and accumulate in ageing leaves.

The team recorded the 600 MHz proton NMR spectrum of the FCC Ma-FCC-61, which displayed a characteristic set of signals indicative of the tetrapyrrole moiety, together with a low-field signal due to a formyl and a vinyl group, three singlets and one doublet of four methyl groups at high field, as well as a sharp singlet of a methyl ester at 3.71 ppm. They then carried out more detailed multidimensional NMR spectroscopy (proton,proton COSY and ROESY), as well as proton,carbon-13 HSQC and HMBC spectroscopy to assign all 35 of the non-exchangeable proton signals due to the tetrapyrrole unit. They thus established the core structure of the functionalized tetrapyrrole. The team further explains that additional signals due to 19 hydrogen atoms were observed in the intermediate field region of the proton NMR spectrum of Ma-FCC-61, and these too were investigated using extensive correlation experiments.

"In this way we could identify (two) pyranose units and a glycerol moiety as the intricate modification of Ma-FCC-61," the team explains. A correlation between the carbonyl carbon and one of the 6'-hydrogens of the directly bound pyranose unit indicated that the propionyl side chain of the tetrapyrrole core is the attachment site of the novel functionalized disaccharide unit, they add.

The compounds in the banana peels and leaves differ from each other, but they are both long-lived (persistent). In addition, the compounds have a structural similarity: a unique, complex ester function on a specific side chain. This group has a chemical stabilizing effect and explains the unusually long life of the fluorescing intermediates in the banana and its leaves. The discovery of the details of the leaf decomposition product, Ma-FCC-61, now revealed as a pyrrole pigment with a previously unknown sugar unit attachment, hints at the possibility that there are other novel chlorophyll decomposition products that might even be building blocks for previously undetected and even more complex pigments.

"When leaves of plants de-green and when fruits ripen, they develop fascinating colours," says Kräutler. "Bright colours of fruit are believed to have evolved as valuable signals to attract frugivores, which are needed for seed dispersal. Indeed, the blue luminescence of ripe bananas may fulfil such a role," he says. The additional advertising of fruit with colourful and possibly luminescent leaves could be a further optical signal from fruiting plants. "In either case, we need to reconsider the previous assumption that chlorophylls in ageing leaves are always disposed of by a general route leading to non-fluorescent decomposition products," he says.