Functionalized in Triplicate: A Ring-By-Ring Approach to Tailored Prodiginine Derivatives for Site-Specific Conjugation Through Click Chemistry

Abstract

The tripyrrole prototype alkaloid prodigiosin and members of the prodigiosin family are structurally diverse bacterial secondary metabolites. The privileged scaffold accounts for versatile biological activities, for example, antimicrobial, antitumoral, and immunosuppressive. Its Lewis-basic lipophilic tripyrrole core and the aliphatic side chains allow for passive membrane diffusion, thereby trespassing the natural permeability barrier. However, diffusion-controlled uptake is accompanied by low target specificity, hampering the development of tailored prodigiosin therapeutics and their selective delivery to target sites. To address this downside, this work focuses on providing the chemical methodology to synthesize prodiginines that are amenable to click chemistry on each of the three pyrrole moieties and facilitate the development of prodigiosin conjugates. Installing reactive azides and maleimides in the A-, B-, and C-ring of the cytotoxic scaffold enables further functionalization per azide-alkyne cycloaddition (CuAAC and SPAAC) and thiol-maleimide addition, giving rise to protein-conjugable prodiginines for the first time. The presented synthetic routes comprise yields of 3.24.7% over 1215 steps and grant access to valuable synthetic elements for expanding the toolbox of click chemistry to other pyrrole-, pyrrolidinone-, and tetramic acid-containing natural products. Together, the devised methodology for prodiginine derivatization will collectively advance the development of alkaloid-based conjugate therapeutics, eligible for target-selective delivery.

Keywords: CuAAC; alkaloids; click chemistry; prodigiosin; pyrrole.

Scheme 1

The cytotoxic tripyrrole alkaloid prodigiosin (1).

Scheme 2

The retrosynthetic analysis of the prodiginine scaffold (shown as free base) uncovers essential synthetic equivalents for transferring organic azides into the natural product family.

Scheme 3

Synthesis scheme of prodigiosin A‐ring azide 2·HCl. Omitted synthetic milestones from the precursor synthesis of the azide boronic acid 17 can be found in the Supporting Information.

Scheme 4

Synthesis scheme of prodigiosin B‐ring azide 3·HCl.

Scheme 5

Synthesis scheme of prodigiosin C‐ring azide 4·HCl.

Scheme 6

Click functionalization of prodigiosin azides and manufacturing of small‐molecule and protein conjugates. Top: Preparation and CuAAC‐functionalization of homo‐dimeric prodigiosin zinc complexes 43, 44, and 45 with the bifunctional alkyne‐maleimide linker 42 uncover the maleimide decorated A‐, B‐, and C‐ring prodiginines 46, 47, and 48, respectively. Bottom left: Clickable prodigiosin F‐BODIPY A‐ring azide 49 with bright‐red fluorescence [here shown as an 80 µm solution in THF and n‐heptane/CH₂Cl₂ (99:1)]. Bottom middle: Prodigiosin‐d‐biotin conjugate facilitates selective binding to streptavidin, useful in pull‐down assays and target fishing approaches. Bottom right: Generic protein functionalization per thiol‐Michael addition with the novel prodiginine maleimides 46, 47, and 48.