Diprenylated cyclodipeptide production by changing the prenylation sequence of the nature’s synthetic machinery

Abstract

Ascomycetous fungi are often found in agricultural products and foods as contaminants. They produce hazardous mycotoxins for human and animals. On the other hand, the fungal metabolites including mycotoxins are important drug candidates and the enzymes involved in the biosynthesis of these compounds are valuable biocatalysts for production of designed compounds. One of the enzyme groups are members of the dimethylallyl tryptophan synthase superfamily, which mainly catalyze prenylations of tryptophan and tryptophan-containing cyclodipeptides (CDPs). Decoration of CDPs in the biosynthesis of multiple prenylated metabolites in nature is usually initiated by regiospecific C2-prenylation at the indole ring, followed by second and third ones as well as by other modifications. However, the strict substrate specificity can prohibit the further prenylation of unnatural C2-prenylated compounds. To overcome this, we firstly obtained C4-, C5-, C6-, and C7-prenylated cyclo-l-Trp-l-Pro. These products were then used as substrates for the promiscuous C2-prenyltransferase EchPT1, which normally uses the unprenylated CDPs as substrates. Four unnatural diprenylated cyclo-l-Trp-l-Pro including the unique unexpected N1,C6-diprenylated derivative with significant yields were obtained in this way. Our study provides an excellent example for increasing structural diversity by reprogramming the reaction orders of natural biosynthetic pathways. Furthermore, this is the first report that EchPT1 can also catalyze N1-prenylation at the indole ring. Key points • Prenyltransferases as biocatalysts for unnatural substrates. • Chemoenzymatic synthesis of designed molecules. • A cyclodipeptide prenyltransferase as prenylating enzyme of already prenylated products. Graphical Abstract

Colletotriauxins A–D, New Plant Growth Inhibitors from the Phytopathogenic Fungus Colletotrichum gloeosporioides

“…These include signals for two carbonyl groups at δ c 174.6 and 170.5 ppm, eight indole carbons at δ c 122.4, 113.9, 118.7, 118.4, 121.0, 111.4, 136.2, and 126.1 ppm, two methyl carbons at δ c 18.0 and 20.8 ppm, two oxymethylene at δ c 67.2 and 66.9 ppm, four oxymethine carbons at δ c 68.3, 69.2, 69.0, and 68.1 ppm, and a methine carbon at δ c 36.6 ppm. The signal of H-2 at δ H 7.19 clearly showed HMBC correlations to the aromatic carbon C-3a at δ c 126.1 and the methine carbon C-2′ at δ c 36.6, proving the C3-substitution of the indole moiety . The HMBC spectrum also showed clear correlations from the methyl group at δ H 1.47 ppm (d, 3H-3′) with the quaternary olefinic carbon C-3 at δ c 113.9 and the carbonyl carbon C-1′ at δ c 174.6 ppm.…”

“…Chemical shifts and coupling constants of five protons in the aromatic range of the 1 H NMR spectrum of 1 (Table S1, Figure S1) at 7.12 (d, J = 2.5, H-2), 7.50 (d, J = 7.0, H-4), 6.95 (ddd, J = 8.0, 7.0, 1.0, H-5), 7.04 (ddd, J = 8.0, 7.0, 1.0, H-6), and 7.31 ppm (d, J = 7.0, H-7) suggested the presence of a typical 3-substituted indole moiety. 24 Signals for an OCH 2 −CH 2 group were observed at 3.59 (m, H-1′), 3.77 (dt, J = 9.5, 7.0, H-1′), and 2.91 ppm (td, J = 7.0, 2.5, 2H-2′). In addition, resonances of a rhamnosyl moiety were also detected at 4.59 (d, J = 1.5, H-1″), 3.59 (m, H-2″), 3.43 (dd, J = 9.5, 3.5, H-3″), 3.16 (t, J = 9.5, H-4″), 3.37 (dq, J = 9.5, 6.0, H-5″), and 1.08 ppm (d, J = 6.0, 3H-6″).…”

“…Colletotriauxin A ( 1 ) was obtained as a yellow oil with a molecular formula of C 16 H 21 NO 5 according to its [M + H] + ion at m / z 308.1495 in the positive HRESIMS, suggesting 7 degrees of unsaturation. Chemical shifts and coupling constants of five protons in the aromatic range of the 1 H NMR spectrum of 1 (Table S1, Figure S1) at 7.12 (d, J = 2.5, H-2), 7.50 (d, J = 7.0, H-4), 6.95 (ddd, J = 8.0, 7.0, 1.0, H-5), 7.04 (ddd, J = 8.0, 7.0, 1.0, H-6), and 7.31 ppm (d, J = 7.0, H-7) suggested the presence of a typical 3-substituted indole moiety . Signals for an OCH 2 –CH 2 group were observed at 3.59 (m, H-1′), 3.77 (dt, J = 9.5, 7.0, H-1′), and 2.91 ppm (td, J = 7.0, 2.5, 2H-2′).…”

“…Colletotriauxin C ( 3 ) was obtained as a colorless oil, and its molecular formula of C 19 H 25 NO 8 was deduced from [M + H] + ion at m / z 396.1653, implying 8 degrees of unsaturation. The 1 H NMR spectrum of 3 (Table S2 and Figure S12) also displays signals for a C3-substituted indole nucleus at 7.19 (d, J = 2.5, H-2), 7.55 (d, J = 7.0, H-4), 6.94 (ddd, J = 8.0, 7.0, 1.0, H-5), 7.03 (ddd, J = 8.0, 7.5, 1.0, H-6), and 7.31 ppm (d, J = 7.5, H-7) . In addition, signals for one methine proton at 3.96 (q, J = 7.0, H-2′), one methyl group at 1.47 (d, J = 7.0, H-3′), four oxymethines at 3.66 (m, H-2″/H-5″) and 3.55 (dd, H-3″/H-4″), two oxymethylenes at 3.87 (dd, J = 11.5, 7.0, H-1″), 4.37 (dd, J = 11.5, 2.5, H-1″), 3.93 (dd, J = 11.0, 6.5, H-6″) and 4.23 (dd, J = 11.0, 2.5, H-6″), and an acetyl methyl at 1.98 ppm (s, H-8″) were also detected.…”

Structural insights into the diverse prenylating capabilities of DMATS prenyltransferases

“…Their unusually broad substrate scopes (Table 1), established utility in gaining access to hard-tomodify positions of NP scaffolds, and regio-and stereoselective chemistries have made DMATSs attractive candidates as biocatalysts for late-stage derivatization of several structurally diverse aromatic scaffolds. 24,25,65,67,69,86,88,89 Furthermore, as prenylation is oen a precursor to scaffold modications such as spirocyclization in vivo, it is also feasible to consider the use of DMATSs in biologically inspired synthetic workows. 41,[90][91][92] Prenylation is known to favorably modulate bioactivities, and prenylated aromatic NPs possess a wide range of desirable bioactivities, making DMATSs potential tools for use in drug discovery efforts.…”