Diverted Total Synthesis of Carolacton-Inspired Analogs Yields Three Distinct Phenotypes in Streptococcus mutans Biofilms

Abstract

The oral microbiome is a dynamic environment inhabited by both commensals and pathogens. Among these is Streptococcus mutans, the causative agent of dental caries, the most prevalent childhood disease. Carolacton has remarkably specific activity against S. mutans, causing acid-mediated cell death during biofilm formation; however, its complex structure limits its utility. Herein, we report the diverted total synthesis and biological evaluation of a rationally designed library of simplified analogs that unveiled three unique biofilm phenotypes further validating the role of natural product synthesis in the discovery of new biological phenomena.

Figure 1

S. mutans biofilm lifecycle (top); Kirschning Analog Library (middle); overview of the diverted total synthesis (DTS) of aryl-analogs; and modeling of simplified analog and carolacton (bottom).

Figure 2

Carolacton analogs elicit a range of phenotypic responses. (A) C3 inhibits biofilm formation with an MBIC₅₀ of 63 μM, “B” = Blank wells; “C” = S. mutans control. (B) Confocal microscopy highlights the effect of C1 and D2 on the growth of S. mutans biofilm at 500 μM (top, field view; bottom, 4× zoom). (C) Carolacton and D4 (top, field view; bottom, 16× zoom) display similar phenotypes at 500 nM. White circles indicate spherical cell morphology and ovals highlight chains of cells. See Figures S3 and S4 for additional representative images.

Figure 3

Summary of the unique phenotypes, and proposed stages of activity, of the carolacton-inspired analogs during the maturation of S. mutans biofilms. C3 inhibits biofilm growth at 63 μM (left). D4 kills cells during the growth of the biofilm similar to that of the natural product, carolacton (center). C1 inhibits biofilm maturation (bottom right).

Scheme 1. Diverted Total Synthesis of Carolacton Analogs^a

^a(a) K₂CO₃, MeOH/THF, >99% (b) DMSO, (COCl)₂, CH₂Cl₂, 94% (c) CuCN, Allyl-MgBr, THF, 90% (d) 4 Å MS, NaOH, PhMe, 83– 85% (e) EDCI, DMAP, CH₂Cl₂, 89–96% (f) Grubbs 2nd generation catalyst, CH₂Cl₂, 78–99% (g) Pd/C, H₂, 55-69% (h) TBAF, 64–94% (i) HCl, THF/MeOH, 50–82% (j) DMSO, SO₃·pyr, CH₂Cl₂, 63–89% (k) t-BuOH, 2-methyl-2-butene, NaH₂PO₄, NaClO₂, MeCN, H₂O, 65-96% (l) TFA/H₂O, 59–79%.