Mayotlide: synthetic approaches and structural elucidation

Abstract

Mayotlide is a marine peptide isolated by PharmaMar S.A. from Spongia sp.. The sequence of the aminoacids were achieved by MS-MS spectrometry, where two of them were tryptophans. The NMR spectroscopy revealed the presence of N1-C3a bond between the tryptophans, which means that one of them was cyclized. On the first structure proposal, the aminoacids were forming two macrocyclic rings: on the ring B, all the aminoacids of molecule were tied by amide bond, remarking the presence of the cyclized tryptophan as a hexahydropyrroloindole unit (HPI). The ring A was established by three aminoacids of the ring B: one tryptophan, one isoleucine and the HPI, which became closed by the N1-C3a bond between the two tryptophans. The aminoacid analysis demonstrated the L configuration of all of them, just remaining the HPI with unknown stereochemistry. Due to the configuration of the HPI system, there can be four diasteromers. To the best of our knowledge, there were not empirical evidences about which one could be more favorable. Hence, a computational study of the ring A through the MOE program was performed, evidencing that the rings A with the exo HPI adducts (L and D) were more stable than the endo ones. Among the exo ones, another computational study with the whole molecule revealed that the mayotlide with the exo-L adduct of the HPI was more stable. Once that there was established a starting point with the HPI, it was necessary to find out a synthetic strategy that could fulfill the necessary requirements. A methodology developed by Baran and coworkers for the synthesis of the Trp-HPI fragment on molecules related to mayotlide was adapted to our proposal. On the first step of this strategy, under reaction conditions, the starting tryptophan cyclizes, providing exclusively the HPI with the exo configuration, at the same time that the N1-C3a bond between the HPI and 2-iodoaniline is formed. The 2-iodoaniline, along with a disubstituted alkyne, condense together yielding the upper tryptophan without loss of the former stereochemistry, reaching the Trp-HPI framework in two steps with high yields. Taking advantage of this methodology and an appropriate use of the protecting groups in order to perform the corresponding cyclizations of the two macrocyclic rings, the mayotlide with the exo-L HPI was accomplished. Nevertheless, nor the NMR neither the MS-MS fragmentation pattern of the final compound coincided with the natural product ones. Thus, the next alternative would consist on repeating the synthesis but with the exo-D adduct of the HPI instead. During the last synthesis, the bibliography and the analytical data of the natural product were extensively revised. There were no documented precedents of natural peptides with the Trp- HPI motif, and the initial structure did not justify the most important data extracted from the MS-MS spectrometry. the main fragmentation concerned to the isoleucine, and on the first proposal such aminoacid was not forming part of a conflictive scaffold. Likewise, among the eight proposed linear sequences for the ring B, the isoleucine just appear in one as C-terminus of the b-ions, when it is one of the aminoacids that forms part of both macrocycles. The most related family of peptides to mayotlide are the kapakahines. Kapakahines, instead of having a Trp-HPI moiety as the central part of the molecule, exhibit a Trp-α carboline, with an

aminoacid establishing the bridge for a very tensioned tetracyclic system. Such structure may justify the isoleucine fragmentation pattern, but on the other hand the sequence of the aminoacids did not fix. It was necessary to invert the central sequence of the aminoacids to reach out a final structure proposal which justifies all the requirements, relabeled as “kapakahine H”.

La mayotlida es un producto natural de origen marino aislado por PharmaMar. SA.. En la primera propuesta que se hizo, se elucidó como un péptido con dos anillos macrocíclicos: en el anillo B estaban contenidos todos los aminoácidos de la molécula, destacando la presencia de una unidad de HPI, que procede de la ciclación intramolecular del extremo amino de un triptófano con el C2 del anillo de indol. El anillo A está formado por tres aminoácidos del anillo B: un triptófano, una isoleucina y el HPI, quedando cerrado por la formación de un enlace entre el N1 del triptófano y el C3a del HPI. El análisis de aminoácidos demostró la configuración L de todos ellos, quedando desconocida la estereoquímica del HPI. El HPI posee tres estereocentros: los C3a y C8a, que siempre están en cis por la propia configuración del anillo y se pueden considerar como un conjunto, y el estereocentro del Cα. Por tanto, son cuatro el número total de diasterómeros posibles. El anillo A con las cuatro posibilidades de HPI fue estudiado energéticamente con el programa MOE, llegando a la conclusión que los aductos exo son más estables que los endo, y que el aducto exo-L es más estable que el exo-D. Para abordar la síntesis de la mayotlida exo-L se adaptó una metodología desarrollada por Phil Baran y colaboradores para moléculas con una estructura relacionada a la mayotlida. Tras conseguir la síntesis de la mayotlida con el aducto exo-L del HPI, se comprueba que los espectros de RMN y de MS-MS presentan grandes divergencias. Ante la incapacidad de interpretar las diferencias existentes entre el producto natural y la mayotlida exo-L, se tomó la decisión de abordar la síntesis de la mayotlida con el aducto exo-D del HPI. En el transcurso de la síntesis exo-D se revisó extensivamente la bibliografía y los datos analíticos relacionados con la mayotlida: no existían antecedentes de productos naturales peptídicos con estructura Trp-HPI, y la estructura no encajaba con los datos más transcendentales del MS-MS. La estructura fue revisada, llegando a la conclusión de que podía pertenecer a la familia de las kapakahines, con una estructura central Trp-α carbolina y con la secuencia central de los aminoácidos invertida, rebautizada como kapakahina H.