Information for Stereospecific prenylation of tryptophan by a cyanobacterial post-translational modification enzyme

Prenylation is a key post-translational reaction to increase the structural diversity and bioactivity of peptides and proteins. Until now, only one post-translational modification enzyme, ComQ, has been identified to mediate the prenylation of a tryptophan residue in ribosomally synthesized peptides. Here, we report the in vitro characterization of KgpF, a novel prenyltransferase which transfers dimethylallyl moieties to tryptophan residues during kawaguchipeptin A biosynthesis. The stereospecific prenylation by KgpF was determined by a combination of in vitro dimethylallylation of Fmoc-tryptophan by KgpF and chemical synthesis of dimethylallylated Fmoc-tryptophan diastereomers. KgpF modified the tryptophan derivative with a dimethylallyl group at the 3 position of its indole ring, resulting in the formation of a tricyclic structure with the same scaffold as prenylation by ComQ, but with the opposite stereochemistry.


Synthesis of 4a and 4b
According to our previous reports, 1, 2 compound 3 was prepared.To a 0.1 M solution of 3 (1.13 g, 3.54 mmol) in THF at 0 °C under Argon was added sodium hydride (225 mg, 5.63 mmol).After stirring at 0 °C for 1 h, dimethylallyl bromide (0.542 g, 3.64 mmol) was added to the mixture.After stirring for 3 h at 0 °C, the reaction mixture was quenched and neutralized with 0.1 M phosphate buffer (pH 7).The reaction mixture was extracted with diethyl ether, washed with saturated aqueous sodium chloride, and dried with anhydrous sodium sulfate.After filtration through a cotton plug, the solution was evaporated.The residue was purified by silica gel column chromatography (hexane/ethyl acetate) to give a mixture of 4a and 4b (1.16 g, 2.98 mmol, 84%, α : β = 1.0 : 0.6) as pale yellow oil.The two diastereoisomers could not be separated by column chromatography. 1 H-NMR spectrum of the mixture was shown in Figure S2.

Synthesis of 6a and 6b
To a 0.1 M solution of 4 (1.16 g, 2.98 mmol) in THF was slowly added a 1.0 M solution of diisobutylaluminium hydride (DIBAL) (3.50 mL) in THF under Argon at -80 °C.After stirring for 3 h at -80 °C, the reaction mixture was poured into 0.1 M phosphate buffer solution (pH 7).The reaction mixture was extracted with ethyl acetate, washed with saturated aqueous sodium chloride, and dried with anhydrous sodium sulfate.After filtration through a celite pad, the solution was evaporated.The residue was purified by silica gel column chromatography (chloroform/methanol) to give a mixture of 5a and 5b as a pale yellow oil.
The mixture was immediately used in next step without further purification because of instability of 5a.
For NMR analysis, the mixture was separated by silica gel column chromatography (hexane/acetone), and each solution was carefully evaporated, but not dried, to replace the solvent by deutrated chloroform.The stereochemistry of 5a was confirmed by NOEs between the a proton and the olefin and methylene protons of the dimethylalyl group, but no NOEs in 5b.Unfortunately, conclusive NOEs for determinatiob of the stereochemistry were not observed in 5b.
NMR spectra were shown in Figure S3-S8.
To a 0.1 M solution of a mixture of 5a and 5b in CH 3 CN was added a 1.0 M solution of aqueous sodium carbonate (30 mL) and N-(9-fluorenylmethoxycarbonyl)succinimide (Fmoc-OSu) (1.01 g, 2.98 mmol) at room temperature.After stirring for 1 h, the reaction mixture was neutralized with 0.1 M phosphate buffer (pH 7).The reaction mixture was extracted with ethyl acetate, washed with saturated aqueous sodium chloride, and dried with anhydrous sodium sulfate.After filtration through a cotton plug, the solution was evaporated.
The residue was purified by silica gel column chromatography (hexane/acetone) to give a mixture of 6a and 6b (1.06 g, 2.09 mmol, 70 % in 2 steps).The mixture was immediately used in next step without further purification because of instability. 1 H-NMR spectrum of the mixture of 6a and 6b was shown in Figure S9.

Synthesis of 7a and 7b
To a solution of the mixture of 6a and 6b in THF (20 mL) was added lithium borohydride (26 mg, 1.07 mmol) under Argon at 0 °C.After stirring for 2 h, the reaction mixture was quenched with 1 % solution of aqueous sodium carbonate.The reaction mixture was extracted with ethyl acetate, washed with saturated aqueous sodium chloride, and dried with anhydrous sodium sulfate.After filtration through a cotton plug, the solution was evaporated.
1 H-NMR spectrum of the mixture of 7a and 7b was shown in Figure S10.

Synthesis of 8a and 8b
To a solution of the mixture of 7a and 7b (0.422 g, 0.830 mmol) in acetonitrile (7.5 mL) was added piperidine (2.5 mL) at room temperature.After stirring for 2 h, the reaction mixture was quenched and neutralized with 0.1 M phosphate buffer (pH 7).The reaction mixture was extracted with ethyl acetate, washed with saturated aqueous sodium chloride, and dried with anhydrous sodium sulfate.After filtration through a cotton plug, the solution wass evaporated.
NMR spectra were shown in Figure S11-S14.

S5
To a 0.1 M solution of 8b (88.0 mg, 0.307 mmol) in THF (5 mL) and MeOH (2mL) was added a 1.0 M solution of aqueous lithium hydroxide (2.0 mL) at room temperature.After stirring for 1 h, the reaction mixture was neutralized with 5 % KHSO 4 .The reaction mixture was extracted with ethyl acetate, washed with saturated aqueous sodium chloride, and dried with anhydrous sodium sulfate.After filtration through a cotton plug, the solution was evaporated.To a solution of the residue in dioxane (5 mL) was added a 1.0 M solution of aqueous sodium carbonate (2.5 mL) and N-(9-fluorenylmethoxycarbonyl)succinimide (Fmoc-OSu) (117 mg, 0.347 mmol) at room temperature.After stirring for 2 h, the reaction mixture was neutralized with 5 % KHSO 4 .The reaction mixture was extracted with ethyl acetate, washed with saturated aqueous sodium chloride, and dried with anhydrous sodium sulfate.After filtration through a cotton plug, the solution was evaporated.The residue was purified by silica gel column chromatography (hexane/acetone) three times to give 2b (56.0 mg, 0.113 mmol, 37 % in 2 step) as pale yellow oil.By a similar method, 2a (28.0 mg, 0.0566 mmol, 58% in 2 step) was obtained from 8a (28.0 mg, 0.0978 mmol). 1 H-NMR spectra of 2a and 2b was observed as 1 : 1 equilibrium mixture between N-conformers, as shown in Figure S15 and S16, respectively.

HFigure S1 .
Figure S1.The biosynthetic gene cluster of kawaguchipeptins from Microcystis aeruginosa NIES-88.The 7.4 kb biosynthetic gene cluster consists of six ORFs.The red bars (A and G) indicate genes that encode N-terminal and C-terminal proteases.The blue bar (E) indicates genes that encode precursor peptide of kawaguchipeptins.The amino acid sequences of kawaguchipeptins are shown in blue triangles.The green bar (F) indicates genes that encode prenyltransferases belonging to the ABBA family.The orange bars (B and C) indicate genes that encode unknown proteins.