Cinnamosyn, a Cinnamoylated Synthetic-Bioinformatic Natural Product with Cytotoxic Activity

Abstract

Most biosynthetic gene clusters (BGCs) are functionally inaccessible by using fermentation methods. Bioinformatic-coupled total synthesis provides an alternative approach for accessing BGC-encoded bioactivities. To date, synthetic bioinformatic natural product (synBNP) methods have focused on lipopeptides containing simple lipids. Here we increase the bioinformatic and synthetic complexity of the synBNP approach by targeting BGCs that encode N-cinnamoyl lipids. This led to our synthesis of cinnamosyn, a 10-mer N-cinnamoyl-containing peptide that is cytotoxic to human cells.

Figure 1.

(a) Cinnamic-acid based lipids observed in CCPs. (b) Biosynthetic origin of the N-cinnamoyl substituent in CCPs. (c) A screen of the bioinformatically predicted peptide sequences of 192 CCP gene clusters resulted in the prioritization of the cnn BGC (only partially shown) for chemical synthesis. ISO = isomerase, ACP = acyl carrier protein, KS = ketosynthase, CLF = chain length factor, KR = ketoreductase, DH = dehydratase, CCP = cinnamoyl-containing peptide.

Figure 2.

The cnn cluster and the predicted structure of its metabolite, cinnamosyn (1). (a) The cnn cluster expanded to show its non-ribosomal peptide synthetase module and domain organization. Automated A domain substrate predictions from our screen (with mismatched residues to the closest known code highlighted in red) are shown. (b) Predicted cinnamoyl-ACP biosynthetic genes in cnn and their homologues in atr (atratumycin). (c) The predicted metabolite, cinnamosyn (1) produced by the cnn cluster

Scheme 1.. Chemical synthesis of cinnamosyn (1).

Conditions: (a) (i) Fmoc-AA-OH, HATU, DIPEA, 1h, (ii) 20% piperidine in DMF, 15 min; (b) Fmoc-L-Leu, BzCl, DIPEA, 72h; (c) (i) 20% piperidine in DMF, 15 min, (ii) Fmoc-L-Orn, HATU, DIPEA, 1h, (iii) 20% piperidine in DMF, 15 min, (iv) 20% HFIP in DCM, (v) PyAOP, DIPEA, 1h, (vi) 95:2.5:2.5 TFA:TIPS:H₂O, 2h. All reactions were performed at room temperature. Abbreviations: HATU = (1-[bis(dimethylamino)-methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide, DIPEA = N,N-diisopropylethylamine, DMF = N,N-dimethylformamide, HFIP = 1,1,1,3,3,3-hexafluoroisopropanol, DCM = dichloromethane, PyAOP = (7-Azabenzotriazol-1-yloxy)trispyrrolidinophosphonium hexafluorophosphate.