Naturally Inspired Peptide Leads: Alanine Scanning Reveals an Actin‐Targeting Thiazole Analogue of Bisebromoamide

Abstract Systematic alanine scanning of the linear peptide bisebromoamide (BBA), isolated from a marine cyanobacterium, was enabled by solid‐phase peptide synthesis of thiazole analogues. The analogues have comparable cytotoxicity (nanomolar) to that of BBA, and cellular morphology assays indicated that they target the actin cytoskeleton. Pathway inhibition in human colon tumour (HCT116) cells was explored by reverse phase protein array (RPPA) analysis, which showed a dose‐dependent response in IRS‐1 expression. Alanine scanning reveals a structural dependence to the cytotoxicity, actin targeting and pathway inhibition, and allows a new readily synthesised lead to be proposed.


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Section Title S1

Methyl (2R)-3-(3-Bromo-4-tert-butoxy-phenyl)-2-(tert-butoxycarbonylmethylamino)-propanoate 12
Carbonate 11 (2.32 g, 5.40 mmol) was dissolved in dry THF (50 mL) and stirred at 0 ºC. NaH (0.320 g, 8.10 mmol, 60% dispersion in mineral oil) was added in small portions and the reaction was stirred for 15 min at 0 ºC. A solution of MeI (2.69 mL, 43.2 mmol) in DMF (10 mL) was then added and the reaction was stirred for a further 60 min at 0 ºC before being allowed to return to rt and stirred for 18 h. The reaction mixture was diluted with EtOAc (100 mL) then quenched with NaHCO3 (100 mL; sat aq). The aqueous phase was extracted with EtOAc (3 × 100 mL) and the combined organic phases were washed with brine (100 mL; sat aq) then dried with anhydrous MgSO4. The solvent was removed in vacuo and the crude product was purified by column chromatography (DCM:MeOH, 98:2) to give the desired product 12 as a colourless oil (

SPPS -Rink Amide Resin
Attachment of first amino acid to Rink Amide: Rink Amide resin (200-400 mesh, 0.69 mmol/g) (0.20 g, 0.14 mmol, 1.00 eq.) was swollen in DCM (5 cm 3 ) for 30 min, then washed with DMF (3 × 3 cm 3 ). The resin was purchased in Fmoc protected form so the general procedure for Fmoc deprotection was followed before attachment of the first amino acid.
The Fmoc-protected amino acid residue (0.41 mmol, 3.00 eq.) and Oxyma (0.06 g, 0.41 mmol, 3.00 eq.) were dissolved in DMF (2 cm 3 ) and DIC (64.1 L, 0.41 mmol, 3.00 eq.) was added. The reaction mixture was agitated for 5 min before addition to the resin, which was then agitated for 1 h at rt. The reaction mixture was removed and the resin washed with DMF (3 × 3 cm 3

Fmoc deprotection:
20% Piperidine in DMF (3 mL) was added to the resin and the resin was agitated for 10 min at rt. This was repeated with fresh solution for a further 10 min, then the resin was washed with DMF (3 × 3 cm 3 ), DCM (3 × 3 cm 3

Boc Deprotection:
A 1:1 mixture of TFA and DCM (4 mL) was added to the resin and the resin was agitated for 5 min. This was repeated with fresh solution for a further 20 min, then the resin was washed with DCM (3 × 3 cm 3 ), MeOH (3 ×

cm ) and DCM (× cm ). The Chloranil test was performed and the coupling was repeated if any colourless
beads were observed.

Selective Boc deprotection:
The resin was solvated in dry DCM (5 mL) and chilled (dry ice) before addition of 2,6-lutidine (242 L, 2.10 mmol, 15 eq.) and TMSOTf (304 L, 1.68 mmol, 12 eq.). The resin was agitated at 78 ºC for 15 min then for a further 90 min at rt. The reaction mixture was removed and the resin washed with DCM (

Amino Acid coupling:
The Boc or Fmoc-protected amino acid residue (0.41 mmol, 3.00 eq.) and Oxyma (0.06 g, 0.41 mmol, 3.00 eq.) were dissolved in DMF (2 cm 3 ) and DIC (64.1 L, 0.41 mmol, 3.00 eq.) was added. The reaction mixture was agitated for 5 min before addition to the resin,* and the resin was then agitated for 1 h at rt. The reaction mixture was removed and the resin washed with DMF (3 × 3
* If the coupling step followed a Boc deprotection then DIPEA (36.6 L, 0.210 mmol, 1.50 eq.) was also added to the resin at this point.

Cleavage of Peptide from Rink Amide:
A mixture of TFA:TIS:H2O (3 cm 3 , 95:2.5:2.5) was added to the resin and the resin was agitated at rt for 3 h. The reaction solution was filtered and collected, then evaporated to dryness under nitrogen. The resulting residue was cooled in an ice bath and cold ether was added until a colourless precipitate formed. The suspension was cooled (dry ice) to ensure complete precipitation, then centrifuged (5000 rpm, 5 min), and the ether poured off before fresh ether (15 cm 3 ) was added. Sonication was used to re-mobilise the precipitate and the suspension was centrifuged again (5000 rpm, 5 min). The ether was then decanted and the resulting white pellet purified to obtain the desired product.

A.
Confluence (cell growth) Apoptosis (caspase activity)   Only 24 h time point data from selected analogues is shown.