Fluostatins, benzofluorene-containing aromatic polyketides in the atypical angucycline family, conjugate into dimeric and even trimeric compounds in the post-biosynthesis. The formation of the C-C bond involves a non-enzymatic stereospecific coupling reaction. In this work, the unusual regio- and enantioselectivities were rationalized by density functional theory calculations with the M06-2X (SMD, water)/6-311 + G(d,p)//6-31G(d) method. These DFT calculations reproduce the lowest energy C1-(R)-C10'-(S) coupling pathway observed in a nonenzymatic reaction. Bonding of the reactive carbon atoms (C1 and C10') of the two reactant molecules maximizes the HOMO-LUMO interactions and Fukui function involving the highest occupied molecular orbital (HOMO) of nucleophile p-QM and lowest unoccupied molecular orbital (LUMO) of electrophile FST2- anion. In particular, the significant π-π stacking interactions of the low-energy pre-reaction state are retained in the lowest energy pathway for C-C coupling. The distortion/interaction-activation strain analysis indicates that the transition state (TScp-I) of the lowest energy pathway involves the highest stabilizing interactions and small distortion among all possible C-C coupling reactions. One of the two chiral centers generated in this step is lost upon aromatization of the phenol ring in the final difluostatin products. Thus, the π-π stacking interactions between the fluostatin 6-5-6 aromatic ring system play a critical role in the stereoselectivity of the nonenzymatic fluostatin conjugation.

Iodine-induced cleavage at phosphorothioate DNA (PT-DNA) is characterized by extremely high sensitivity (∼1 phosphorothioate link per 106 nucleotides), which has been used for detecting and sequencing PT-DNA in bacteria. Despite its foreseeable potential for wide applications, the cleavage mechanism at the PT-modified site has not been well established, and it remains unknown as to whether or not cleavage of the bridging P-O occurs at every PT-modified site. In this work, we conducted accurate ωB97X-D calculations and high-performance liquid chromatography-mass spectrometry to investigate the process of PT-DNA cleavage at the atomic and molecular levels. We have found that iodine chemoselectively binds to the sulfur atom of the phosphorothioate link via a strong halogen-chalcogen interaction (a type of halogen bond, with binding affinity as high as 14.9 kcal/mol) and thus triggers P-O bond cleavage via phosphotriester-like hydrolysis. Additionally, aside from cleavage of the bridging P-O bond, the downstream hydrolyses lead to unwanted P-S/P-O conversions and a loss of the phosphorothioate handle. The mechanism we outline helps to explain specific selectivity at the PT-modified site but also predicts the dynamic stoichiometry of P-S and P-O bond breaking. For instance, Tris is involved in the cascade derivation of S-iodo-phosphorothioate to S-amino-phosphorothioate, suppressing the S-iodo-phosphorothioate hydrolysis to a phosphate diester. However, hydrolysis of one-third of the Tris-O-grafting phosphotriester results in unwanted P-S/P-O conversions. Our study suggests that bacterial DNA phosphorothioation may more frequently occur than previous bioinformatic estimations have predicted from iodine-induced deep sequencing data.