Mutanofactins are a family of natural products produced by Streptococcus mutans from the human oral microbiome. We report a unified approach to all mutanofactins by developing a total synthesis amenable to diversification. The key to success for the most complex members, mutanofactins 607 and 697, was an acyl ketene based strategy. Access to the family enabled comprehensive biological profiling, where we demonstrate that all mutanofactins are biofilm promoting in Streptococcus mutans. Experiments were extended to other inhabitants of the oral microbiome for the first time: Streptococcus gordonii and Streptococcus oralis, two early colonizers, were similarly affected with mutanofactins being biofilm promoting. Conversely, Veillonella dispar and Fusobacterium nucleatum showed little to no reaction to mutanofactins. Biophysical investigations based on quartz crystal microbalance with dissipation monitoring and atomic force microscopy reveal a previously unknown mucin-mutanofactin 697 interaction. Incubation of a mucin layer with mutanofactin 697 induces a morphology change within the mucin layer, which promotes bacterial adhesion and biofilm formation. This unique property of mutanofactin 697 might be key to early stages of biofilm formation in the human oral microbiome. Combined, an interdisciplinary approach consisting of total synthesis, microbiology and biophysical characterization provides insight into the roles of mutanofactins in the oral microbiome.

Mutanobactin D is an interkingdom communicator derived from the human oral microbiome. The lipopeptide prevents yeast-to-hyphae morphogenesis in Candida albicans, notably without fungicidal or fungistatic activity. The mode of action and structure-activity relationship of mutanobactin D are unknown and prompt an interdisciplinary program of study. Stereoselective synthesis of designed mutanobactin D analogs reveals that the C26 configuration is crucial for bioactivity associated with inhibition of pathogenesis, or yeast-to-hyphae transition, in C. albicans. To shed light on this finding, we employ molecular dynamics (MD) simulations of mutanobactin D and selected analogs in increasingly complex environments: Monophasic (water or CHCl3), interfacial (water/CHCl3), and explicit lipid membrane (phosphatidylcholine) models. Monophasic MD simulations do not distinguish between bioactive and inactive compounds. In contrast, at a polar/apolar interphase, a dominant, stable conformation emerges for mutanobactin D and bioactive analogs. Explicit lipid membrane simulations reinforce these results and further reveal the formation of a continuous, structured water cushion, which is not found for inactive analogs. Our studies collectively reveal how the stereodefined attachment of the lipid in the C26-C28 motif governs activity against C. albicans and provide a framework for understanding the membrane behavior of mutanobactin D, which may be coupled to its role in the human oral microbiome. The approach described herein, consisting of synthesis and evaluation of designed analogs complemented by MD simulations, provides a blueprint for the study of bioactive natural products in various contexts, including the human microbiome.

Key words (+)-candol A - (+)-powerol A - (–)-trachinol - ent-kaurane diterpenoids - ent-tachylobane diterpenoids - Conia–ene reaction - Luche reduction - Swern oxidation - Fe-catalyzed HAT - Wolff–Kishner–Huang reduction - Meerwein–Ponndorf–Verley reduction - Barton–McCombie deoxygenation - Mukaiyama hydration

Key words (+)-monensin - polyether antibiotics - Brown hydroboration - Prilezhaev reaction - Wittig olefination - aldol addition

Key words (–)-simplicissin - spiromeroterpenoids - Luche reduction - Suzuki–Miyaura coupling - Meerwein–Ponndorf–Verley reduction

Key words (±)-2‑isocyanoallopupukeanane - pupukeanane sesquiterpene isonitriles - Wharton transposition - Parikh–Doering oxidation - Alder-ene reaction - HAT azidation

Key words caranine alkaloids - (–)-γ-lycorane - asymmetric aziridination - hydrogen borrowing catalysis - Heck reaction

We report the first total syntheses of tricyclic mutanobactins A and B, lipopeptides incorporating a thiazepanone, isolated from Streptococcus mutans , a member of the human oral microbiome. A rapid, solid-phase peptide synthesis (SPPS) based route delivers these natural products from a cascade of cyclization reactions. This versatile process was also employed in a streamlined synthesis of mutanobactin D. Additionally, we provide an independent synthesis of a truncated mutanobactin A analog, utilizing a novel thiazepanone amino acid building block.

Key words (+)-decursivine - indole alkaloids - Ullmann coupling - Fischer indole synthesis - C–H insertion

Key words (±)-longifolene - sesquiterpenes - de Mayo reaction - Simmons–Smith cyclopropanation - enamine acylation

Key words (+)-mutilin - antibiotic diterpenoid - Peterson olefination - Claisen rearrangement - [2+2] cycloaddition - retro-aldol reaction

Key words (–)-triptonide - hydrogen atom transfer - Weitz–Scheffer epoxidation - Giese addition - Adler oxidation - olefin metathesis

Key words (+)-dysiherbol A - (+)-dysiherbol D - hydroquinone sesquiterpenoids - [1,2]-migration - cyclopropane fragmentation - Wittig olefination - [2+2] cycloaddition

Key words (–)-chrodrimanin C - (–)-verruculide A - enzymatic hydroxylation - Mukaiyama hydration - 6π-electrocyclization - aromatization

Key words (+)-ent-shagene A - (–)-ent-shagene B - Regitz diazo transfer - cyclopropanation - olefin metathesis

Key words thromboxane A2 - oxetane natural products - macrocyclic selectivity control - Mitsunobu displacement

Mutanobactin D is a non-ribosomal, cyclic peptide isolated from Streptococcus mutans and shows activity reducing yeast-to-hyphae transition as well as biofilm formation of the pathogenic yeast Candida albicans. We report the first total synthesis of this natural product, which relies on enantioselective, zinc-mediated 1,3-dipolar cycloaddition and a sequence of cascading reactions, providing the key lipidated γ-amino acid found in mutanobactin D. The synthesis enables configurational assignment, determination of the dominant solution-state structure, and studies to assess the stability of the lipopeptide substructure found in the natural product. The information stored in the fingerprint region of the IR spectra in combination with quantum chemical calculations proved key to distinguishing between epimers of the α-substituted β-keto amide. Synthetic mutanobactin D drives discovery and analysis of its effect on growth of other members of the human oral consortium. Our results showcase how total synthesis is central for elucidating the complex network of interspecies communications of human colonizers.

Key words divergent synthesis - cytochalasans - structural revision - carbonyl–ene reaction

Key words aconitine-type alkaloids - Giese reaction - semipinacol rearrangement - (–)-talatisamine - (–)-liljestrandisine