Sonogashira diversification of unprotected halotryptophans, halotryptophan containing tripeptides; and generation of a new to nature bromo-natural product and its diversification in water

Aqueous Sonogashira cross-coupling of unprotected bromotryptophan, tripeptides and a new to nature natural product (accessed through biosynthetic manipulation) is reported.


Sonogashira diversification of unprotected halotryptophans, halotryptophan containing tripeptides; and generation of a new to nature bromo-natural product and its diversification in water
M. J. Corr, a S. V. Sharma, a C. Pubill-Ulldemolins, a R. T. Bown, a P. Poirot, b D. R. Smith, a C. Cartmell, a A. Abou Fayad, c R. J. M. Goss a*

F NMR analysis of cross-coupled cystargamide
Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2016

General Chemical Experimental
All reagents were purchased from commercial suppliers and were used without further purification unless otherwise stated. Dry dichloromethane was dried and deoxygenated with an MBraun SPS-800 solvent purification system and the moisture content of the solvent was analysed using a Karl Fischer coulometer (Metler Toledo DL32).
Purification of selected peptides was carried out on a Biotage Isolera Four using reverse-phase SNAP C18 12 g column cartridges. The purification was carried out using water/methanol on the following gradient: 12 mL/min elution, 2% methanol/water for 1.15 min, 215% methanol/water for 1.15 min, 15% methanol/water for 5.15 min, 1595% methanol/water for 15 min, 95% methanol/water for 3.45 min. The collection wavelength was set a 254 nm.
UPLC analysis was carried out on a Waters Acquity H-Class UPLC system.
A 1:1 mixture of water:acetonitrile was freshly prepared before each set of cross coupling reactions. The solvent was degassed by bubbling nitrogen through the solutions for at least 15 minutes, then storing under nitrogen.

Sodium 2'-(Dicyclohexylphosphanyl)-2,6-diisopropyl-(1,1-biphenyl)-4-sulfonate (sXPhos) 12 2
Sulfuric acid (1.0 mL) and fuming sulfuric acid (3.0 mL) were added to a suspension of XPhos (433 mg, 0.9 mmol, 1.0 eq) in dry DCM (3 mL) at 0 C under nitrogen. The reaction was allowed to warm to r.t. and stirred for 24 h. The reaction mixture was cooled to 0 C and crushed ice (10 g) was added. The reaction mixture was neutralised to pH 7 using sodium hydroxide solution (6 N, ~25 mL). The reaction mixture was extracted with DCM (3 x 50 mL) and the solvent was removed in vacuo. The residue was dissolved in cold methanol (20 mL), filtered, the filtrate collected and the solvent removed in vacuo to give sodium 2'-
Degassed water/acetonitrile (1:1, 2 mL) was added to the flask, followed by the liquid alkyne substrate (variable, see Table 1 in main text). The reaction mixture was heated at 100 C for 18 h. The reaction mixture was then cooled to r.t.
For analysis of the reaction conversions, 0.2 mL of reaction mixture was removed from the well-stirred reaction mixture and the solvent was removed in vacuo. The residue was dissolved in d4-MeOH (0.7 mL) and analysed by 1 H NMR.
The flask was evacuated and flushed with nitrogen three times. Degassed water/acetonitrile (1:1, 2 mL) and acetonitrile (1.0 mL) were added to the microwave tube, followed by the liquid alkyne substrate (33 L, 0.3 mmol, 3.0 eq). The tube was sealed and heated at 100 C for 2 h using a microwave.
For analysis of the reaction conversion, 0.2 mL of reaction mixture was removed from the wellstirred microwave tube and the solvent was removed in vacuo. The residue was dissolved in d4-MeOH (0.7 mL) and analysed by 1 H NMR.
For analysis of the reaction conversions, 0.2 mL of reaction mixture was removed from the well-stirred microwave tube and the solvent was removed in vacuo. The residue was dissolved in d4-MeOH (0.7 mL) and analysed by 1 H NMR.
For purification of specified reactions, the reaction mixture was cooled to r.t. and diluted with water (6.0 mL). The reaction mixture was centrifuged (13,000 rpm, 16060 g, for 5 min). The ultrafiltrate was collected and purified by HPLC purification, as described above, in 1 mL injections. The appropriate HPLC fractions (generally eluted at 18-20 minutes) were collected and the solvent removed in vacuo. The residue was re-suspended in water (50-100 mL) and lyophilised to give the product.
Aqueous KOH (1 M, 0.45 mL, 0.45 mmol, 1.25 eq) was added dropwise. The mixture was stirred for 4 h while warming to room temperature. The reaction was diluted with water (10 mL) and extracted with diethyl ether (2 x 10 mL). The aqueous layer was cooled in an ice-bath and the pH was adjusted to 2 using 1 M HCl. The resultant white suspension was extracted using ethyl acetate (3 x 10 mL). The combined organic layers were dried over anhydrous Na2SO4 and the solvent was removed in vacuo to give N-Boc-7-bromo-S-tryptophan 66 (120 mg, 89%) as a white, waxy solid that was used without further purification; 1 H NMR (300

7-Bromo-S-tryptophan methyl ester hydrochloride 67
Thionyl chloride (290 µL, 4 mmol, 4.0 eq) was added to a flask containing dry methanol (10 mL) at 0 C under argon. After stirring for 10 min, 7-bromo-S-tryptophan 22 (285 mg, 1 mmol, 1.0 eq) was added. The reaction was stirred overnight while allowing it to warm to r.t. The solvent was removed in vacuo to give 7-bromo-S-tryptophan methyl ester hydrochloride 67 (330 mg, 100%) as an off white hydrochloride salt that was used without further purification.
NMR on the HCl salt (d6-DMSO) revealed broad signals. Hence, an analytical sample was obtained by desalting with dilute NaHCO3 solution and extraction with ethyl acetate. Drying

N-Boc-Ala-Trp-(7-Br)-Phe-OMe 72
Water ( DIPEA (0.2 mL, 1.2 mmol, 3.5 eq) was added dropwise and the reaction mixture was warmed to r.t. and stirred for 16 h. The reaction mixture was diluted with ethyl acetate (20 mL) and washed successively with water (3 x 10 mL) and brine (10 mL). The combined organic layers were dried over anhydrous MgSO4 and the solvent was removed in vacuo to give N-Boc-Ala-   mol, 5 mol%) was injected into the microwave tube, followed by degassed H2O:CH3CN (1:1, 0.1 mL). 3-Fluorophenylacetylene 24 (10 L, 80 mol, 10.0 eq) was then injected into the microwave tube. The reaction mixture was well stirred, then heated at 100 C for 2 h in a microwave. The reaction mixture was cooled to r.t. and diluted to 3 mL with H2O:CH3CN
The UPLC analysis was carried out in acetonitrile/0.1% TFA in water at a gradient of 20% acetonitrile to 90% acetonitrile over 4.7 minutes. A 3:1 mixture of L-tryptophan to Dtryptophan was used as a standard. Two blank runs using acetone were conducted between each tryptophan sample. In all of the tryptophans analysed, only a single peak for the Lenantiomer was observed.

General Biological Experimental
Biological reagents and components for media, buffers and stock solutions were purchased from commercial suppliers, used without further purification and stored according to the supplier's instructions. Microorganisms were stored at -80 C for long and short-term storage. Genlab incubator (static). pH measurements were taken using a Fisherbrand Hydrus 300 pH meter. Centrifugation was carried out using a Thermo Scientific IEC CL30R centrifuge.

Kitasatospora cystargenia culturing, and precursor directed biosynthesis of new to nature bromo-cystargamide
Kitasatospora cystargenia NRRL-B16505 was obtained from the USDA agriculture research service culture collection. The strain was fermented in GY medium (10 g/L glucose, 10 g/L yeast extract) for 48 h at 28 °C (as previously reported by Kerr and coworkers) 3 and 200 rpm, in an incubator with a 2.54 cm throw. The mycelium from this culture was stored in 20% glycerol at -80 °C.

Small scale feeding experiments
In order to explore whether the halotryptophans could be incorporated too, we carried out small scale feeding experiments with 7, 6 and 5-chlorotryptophan and 7, 6 and5 -bromotryptophan Results demonstrated that all of the halotryptophans incorporated into cystargamide, generating new to nature halogenated analogues. As the concentration of tryptophan had no major impacted the amount of halo-cystargamide produced (see tables below) , we selected to scale up of the feeding of 6-bromotryptophan at 0.25 mM . Cystargamide and bromocystargamide, both in their linear and cyclised forms, eluted between 120 -180 mL. These fractions were further fractionated by RP-HPLC, using a Waters XBridge

Scaled up feeding experiment with 6-bromotryptophan
Prep Phenyl 10 x 250 mm 5 µm column with an isocratic gradient of 70% aqueous methanol and 0.1% formic acid and a flow rate of 6 mL/min, monitoring UV absorbance at 280 nm.
Cystargamide was found to elute at 10.5 minutes, followed by bromocystargamide at 11 minutes. Solvent was removed from fractions using a Genevac centrifugal vacuum concentrator.
Throughout the purification, fractions were analysed by UPLC (95% acetonitrile and 0.1% trifluoroacetic acid with a flow rate of 0.6 ml/min, monitoring between 220 and 400 nm) or by The cystargamide and bromocystargamide were produced in linear and cyclic forms.
Extraction and purification resulted in a pure fraction containing 1.0 mg 6-bromocystargamide 9. By comparison of the absorbance peak areas it could be estimated that the parent unhalogenated cystargamide 62, a bromo-cystargamide analogue that lacked the 5-hydroxy on the tryptophan 63 and bromocystargamide 9, were present in an approximate 10:0.2:1.4 ratio.

Name
Retention Time Area % Area