Chemical Probes for the Functionalization of Polyketide Intermediates**

A library of functionalized chemical probes capable of reacting with ketosynthase-bound biosynthetic intermediates was prepared and utilized to explore in vivo polyketide diversification. Fermentation of ACP mutants of S. lasaliensis in the presence of the probes generated a range of unnatural polyketide derivatives, including novel putative lasalocid A derivatives characterized by variable aryl ketone moieties and linear polyketide chains (bearing alkyne/azide handles and fluorine) flanking the polyether scaffold. By providing direct information on microorganism tolerance and enzyme processing of unnatural malonyl-ACP analogues, as well as on the amenability of unnatural polyketides to further structural modifications, the chemical probes constitute invaluable tools for the development of novel mutasynthesis and synthetic biology.


Methyl 2-methyl-3-oxo-6-pent-4-ynamidohexanoate (9)
Compound 5 (1.11 g, 4.64 mmol) was dissolved in THF (150 mL) and potassium carbonate (1.66 g, 12.06 mmol) was added. Methyl iodide (0.29 mL, 4.64 mmol) was added dropwise at 0 °C. After one hour the reaction mixture was allowed to warm at room temperature and was left stirring overnight. The reaction was quenched by addition of water (20 mL). THF and water were removed by evaporation/freeze-drying. The crude residue was purified by preparative HPLC (elution gradient starting from 100% H 2 O and linearly increasing to 100% MeOH over 30 minutes) and 9 was yielded as white solid (238 mg, 20%, R t = 10.9 min).

Methyl 4-(4-azidobutanamido)butanoate (60)
Sodium azide (1.06 g, 16.3 mmol) was added to a solution of methyl 4-(4chlorobutanamido)butanoate (59) (1.81 g, 8.15 mmol) in dimethyl sulfoxide (50 mL). The reaction mixture was stirred at 70 °C for 18 h. The reaction mixture was allowed to cool to room temperature and water (100 mL) was added. The aqueous solution was extracted with ethyl acetate (4 x 50 mL). The combined organic phases were washed with water (2 x 100 mL) and brine (100 mL) and dried over MgSO 4 . The MgSO 4 was filtered off and the ethyl acetate was removed in vacuo to yield the crude product. The crude product was purified by column chromatography

Synthesis of probes 7 and 13 2.4.1 1 4-decanamidobutanoic acid (63)
γ-Aminobutyric acid (49, 1.00 g, 9.7 mmol) was suspended in dry methanol (60 mL) in the presence of triethylamine (5.75 mL, 29 mmol). The mixture was cooled to 0 °C and decanoyl chloride (62, 2.1 mL, 9.9 mmol) was added dropwise. The reaction was stirred at 0 °C for 1 h and at room temperature for 16 h. The solvent was evaporated and the crude N-decanoyl-γ-butyric acid (triethylammonium salt) was suspended in water (100 mL), a solution of aqueous 1 M HCl was added to adjust the pH to 2. The mixture was then extracted with CHCl 3 (3 x 100 mL). The organic phase was dried over MgSO 4 , filtered and evaporated to afford 63 as white powder (2.00 g, 80%).
The organic solvent was removed under reduced pressure and the remaining solution was acidified to pH 1 with hydrochloric acid (1M). The obtained suspension was extracted with ethyl acetate (3 x 20 mL). The organic phase was dried over MgSO 4 , filtered and the solvent was removed under reduced pressure. 4-(10-azidodecanamido)butanoic acid was obtained as white solid and used without further purification for the next step (1.24 g).
The reaction was stirred at room temperature for 48 h.

Synthesis of tert-butyl 5-azidopentylcarbamate (44)
Sodium azide (2.03 g, 31.2 mmol) was dissolved in a solvent mixture of water (9 mL) and dichloromethane (18 mL). [7] Trifluoromethansulphonic anhydride (1.05 mL, 6.24 mmol) was added to the mixture at 0 °C and stirred at room temperature for 2 hours. The aqueous layer was separated and extracted with dichloromethane (2 x 5 mL). The pooled organic layers were washed with brine (1 x 5 mL) and then the trifluoromethansulphonic azide solution was added to a solution of tert-butyl (5-aminopentyl)carbamate (0.65 mL, 3.12 mmol), potassium carbonate (0.98 g, 7.10 mmol) and copper sulphate pentahydrate (5 mg) in methanol (22.5 mL) and water (15 mL). The reaction mixture was stirred overnight. The organic layer was washed with water (2 x 5 mL) and the aqueous layers were extracted with dichloromethane (2 x 10 mL). The pooled organic layers were dried over Na 2 SO 4 , filtered and the solvent was removed in vacuo. 44 was yielded as colorless oil (0.69 g, 91%). Data correspond to literature. [8]

Microbiology methods
All media and glassware were sterilized prior to use by autoclave (Astell). Liquid cultures were grown with shaking in Innova 44 incubator/shaker (New Brunswick scientific).
MYM medium: 1.0 g maltose, 1.0 g yeast extract, 2.5 g malt extract in 250 ml of tap water adjusted to pH 7.1.

Construction of mutant strains of Streptomyces lasaliensis
The cultivation of the wild type lasalocid-producing strain S. lasaliensis NRRL3382 was carried out as previously described. 12 The construction of S. lasaliensis ACP12 (S970A) and ΔlasB ACP12 (S970A) mutant strains was previously reported. 12 The construction of S. lasaliensis ACP5 (S3799A) was similarly accomplished by site-directed mutagenesis utilising primer pairs O5_SDMF, oP5R, O5_SDMR and oP5L (Table 1S) by overlap extension PCR and cloning into pYH7 to give plasmid pA5M. 12 The ligated plasmid was transformed into E. coli strain DH10B and positive colonies were tested by restriction mapping and sequencing before a correct clone was transferred to E. coli ET12567/pUZ8002. Conjugation was carried out with the S. lasaliensis wild type as previously described. [12] Candidate mutants were confirmed by sequencing of the mutant region utilising oligonucleotides oA5SF and oA5SR (Table 1S). The mutant was fermented and LC-MS analysis of the ethyl acetate extracts of each culture showed that lasalocid production was completely abolished.  and 1b has been previously reported. [12] 3

LC-HRMS and MS/MS analysis of off-loading of 27 and 38, generated from
S. lasaliensis ACP12 (S970A) and probe 7    extracted ion trace for the off-loaded polyketide 28 is shown (B). Its stereochemistry is yet to be separately determined; further work is in progress to establish this, as well as the origin of two peaks for 28 (possibly arising from the presence of isomers/conformers). These species have also been detected as ammonium adduct (data not shown) and were absent in all the control samples (e.g. in absence of the probe, A, and in extracts obtained from the use of different probes). The high resolution masses and isotopic abundances for 28 are shown (C and D).    are shown (B and C, respectively, Analysis Method 3). Their stereochemistry is yet to be separately determined; further work is in progress to establish this, as well as the origin of two peaks for 28 and 30

LC-HRMS and MS/MS
(possibly arising from the presence of isomers/conformers). These species have been also detected as ammonium adducts (data not shown) and were absent in all the control samples (e.g. in absence of the probe, A, and in extracts obtained from the use of different probes). The high resolution masses and isotopic abundances for 30 (D and E) are shown.