Experimental details and references
7.4 NMR structure calculation
1H-1H NOESY NMR data were processed separately using vnmrJ (version 4.2) and imported into CcpNmr Analysis58 (version 2.4) for manual peak picking and assignment to a molecular system. Custom residue definitions were created using CcpNmr ChemBuild (version 1.0). Initial distance restraints were calculated from assigned peaks using the Analysis software and split into ambiguous and unambiguous restraint lists. Complete peak lists including unassigned peaks were also generated. The inbuilt graphical interface was used to export peak and restraint lists to ARIA53 (version 2.3), and set up the run directory tree.
Structure refinement was carried out by ARIA using CNS54,55 (version 1.2 at patch level 1) and a modified version of the PARALLHDG force field56 (version 5.3). New force field topology definitions were added manually for D-amino acid residues, the phenylacetyl capping group and the ester linkage in topallhdg5.3.pro. Parameters for new bond length, angle, dihedral and improper torsion angle types were added to parallhdg5.3.pro.
Bond lengths and angles for similar cyclic depsipeptides were taken from the literature231–233 and dihedrals were generated using SwissParam.234 Phenylacetyl group parameters were taken from HIC-Up.235Greater flexibility was introduced into the force field by dividing all force constants for bond angles and impropers by 10, as described by Mareuil et al.236 Topology and parameter files were modified in both the run and ARIA distribution directories.
The CNS generate.inp script was modified to include the sequence (declared manually within the chain statement, incorporating nonstandard residues) and topology patch for the side-chain-to-tail ester bond. The linkage definitions in topallhdg5.3.pep were also modified to prevent terminal hydrogens being added to the capping residue, as an N-terminal amine group is not present in this case.
Simulated annealing was carried out using Cartesian dynamics only (torsion angle dynamics disabled) on an ensemble of 20 structures over 8 iterations each (without final refinement in explicit solvent). The Cartesian high temperature value was increased to 10 000 K (default value for torsion angle annealing) and the default simulation total time period was quadrupled (step length kept the same). The 10 lowest energy structures were chosen for the final ensemble.
Chapter 7. Experimental
7.5 Chaiyaphumines
7.5.1 Natural ester series
Chaiyaphumine 10a
The linear sequence was synthesized as described in section 7.1.2 and capped with 2-phenylacetyl chloride (procedure 7.1.5), before undergoing resin cleavage and deprotection as described in proce-dure 7.1.3. Solution-phase cyclization of 224 mg of the linear precursor according to procedure 7.1.6, followed by HPLC purification (procedure 7.2.1) yielded 10a (16 mg, 7%), HPLC Rt= 20.3 min (procedure 7.2.2); HRMS m/z (ESI) 721.3364, consistent with empirical formula C40H45N6O7 with an
The linear sequence was synthesized as described in section 7.1.2 and capped with butyryl chloride (procedure 7.1.5). Resin cleavage was carried out using a 1% TFA solution in DCM (5 × 30 s).237 The resulting combined filtrates were concentrated in vacuo and lyophilized. Global deprotection was carried out on the crude solid as described in procedure 7.1.3. A modification of solution-phase cyclization procedure 7.1.6 was carried out whereby the linear precursor was added under syringe pump control at a rate of 0.35 mL/h using EDCI as the activator (1.3 equiv) in the presence of DMAP (1.3 equiv) with HOBt (1.3 equiv).50,238 The procedure was carried out on 66 mg of the linear precursor, followed by HPLC purification (procedure 7.2.1), to yield10b (5 mg, 8%), HPLC Rt= 5.0 min (procedure 7.2.2, 10 min gradient); HRMS m/z (ESI)
143
673.3353, consistent with empirical formula C36H45N6O7 with an accuracy of
The linear sequence was synthesized as described in section 7.1.2 and capped with propionyl chloride (procedure 7.1.5), before undergoing resin cleavage and deprotection as described in procedure 7.1.3. Solution-phase cyclization of 27 mg of the linear precursor according to procedure 7.1.6, followed by HPLC purification (procedure 7.2.1) yielded 10c (2 mg, 8%), HPLC Rt = 18.3 min (procedure 7.2.2); HRMS m/z (ESI) 659.3174, consistent with empirical formula C35H43N6O7 with an accuracy of 2.9 ppm
The linear sequence was synthesized as described in section 7.1.2 and capped with acetic anhydride (procedure 7.1.5), before undergoing resin cleavage and deprotection as described in procedure 7.1.3. Solution-phase cyclization of 10 mg of the linear precursor according to procedure 7.1.6 using HATU as the activator, followed by HPLC purification (procedure 7.2.1) yielded 10d (2 mg, 21%), HPLC Rt= 16.8 min (procedure 7.2.2); HRMS m/z (ESI) 645.3044, consistent with empirical formula C34H41N6O7 with an accuracy of 1.1 ppm (accepted as [M + H]+).
Chapter 7. Experimental
The linear sequence was synthesized as described in section 7.1.2 and capped with 2-phenylacetyl chloride (procedure 7.1.5), before undergoing resin cleavage and deprotection as described in procedure 7.1.3. Solution-phase cyclization of 18 mg of the linear precursor according to procedure 7.1.6, followed by HPLC purification (proce-dure 7.2.1) yielded11a (4 mg, 23%), HPLC Rt= 16.3 min (procedure 7.2.2); HRMS m/z (ESI) 706.3355, consistent with empirical formula C39H44N7O6 with an accuracy of
The linear sequence was synthesized as described in section 7.1.2 and capped with butyryl chloride (procedure 7.1.5), before undergoing resin cleavage and deprotection as described in procedure 7.1.3. Solution-phase cyclization of 12 mg of the linear precursor according to procedure 7.1.6, followed by HPLC purification (procedure 7.2.1) yielded 11b (1 mg, 9%), HPLC Rt = 16.3 min (procedure 7.2.2); HRMS m/z (ESI) 658.3358, consistent with empirical formula C35H44N7O6 with an accuracy of 0.8 ppm (accepted as [M + H]+).
145
Chaiyaphumine 11c
The linear sequence was synthesized as described in section 7.1.2 and capped with propionyl chloride (procedure 7.1.5), before undergoing resin cleavage and deprotection as described in procedure 7.1.3. Solution-phase cyclization of 15 mg of the linear precursor according to procedure 7.1.6, followed by HPLC purification (procedure 7.2.1) yielded 11c (1 mg, 7%), HPLC Rt = 15.3 min (procedure 7.2.2); HRMS m/z (ESI) 644.3176, consistent with empirical formula C34H42N7O6 with an accuracy of 3.3 ppm
The linear sequence was synthesized as described in section 7.1.2 and capped with acetic anhydride (procedure 7.1.5), before undergoing resin cleavage and deprotection as described in procedure 7.1.3. Solution-phase cyclization of 23 mg of the linear precursor according to procedure 7.1.6, followed by HPLC purification (procedure 7.2.1) yielded 11d (2 mg, 9%), HPLC Rt = 14.1 min (procedure 7.2.2); HRMS m/z (ESI) 630.3034, consistent with empirical formula C33H40N7O6 with an accuracy of 1.0 ppm (accepted as [M + H]+).
Chapter 7. Experimental