series of antiparasitic natural products
2.6 NMR characterization
2.6.2 Structure determination
Structural modelling using the NOESY NMR data was carried out using ARIA (Ambiguous Restraints for Iterative Assignment).53 ARIA iteratively reassigns ambiguously assigned NOESY cross-peaks during the structure calculation, thus allowing automatic assignment of overlapping signals (such as phenyl group aromatic resonances). The software uses CNS (Crystallography &
NMR System)54,55 for structure refinement against NOE-derived distance restraints using a modified version of the PARALLHDG force field.56 Typical simulation runs involve a combination of simulated annealing from a high initial temperature using fast torsion angle dynamics followed by conventional Cartesian dynamics structure refinement.57 Peak reassignment occurs following each iteration.
Peak picking and assignment of the 1H-1H NOESY spectrum was carried out using the CCPN (Collaborative Computing Project for NMR) software.58 Initial distance restraints were then calculated using the software, and restraint and peak lists were exported to ARIA using the inbuilt interface. Due to the presence ofD-amino acid residues, the N-terminal phenylacetyl capping
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Table 2.2: NMR spectroscopic data for chaiyaphumine 10a, 600 MHz (1H) and 150 MHz (13C) in d6-DMSO.
Synthetic peptide10a Peptide10a literaturea δ13C /ppm δ1H /ppm, mult (J /Hz) δ13C /ppm δ1H /ppm, mult (J /Hz)
Indole 3 109.9 109.9
Indole 3a 127.2 126.9
Indole 4 118.10 7.50 , d (8.0) 118.0 7.54, d (7.9)
Indole 5 118.07 6.97, ddd (8.0, 7.0, 1.0) 118.5 6.97, t (7.4) Indole 6 120.9 7.05, ddd (8.0, 7.0, 1.2) 121.0 7.03, t (7.7)
Indole 7 111.3 7.29-7.32, m 111.4 7.31, d (8.1)
Indole 7a 136.0 136.2e
C=O 170.3 171.3
aLiterature values for the naturally isolated compound as published previously.43
bOne peak with an integral for two carbons.
c,d,eAssignments are interchangeable (second assignment not specified for d).
Chapter 2. Synthesis of the antiparasitic chaiyaphumines
Figure 2.11: Overlay of1H NMR spectra of 10a (black) and natural isolate43 (red).
Chemical shift differences are observed.
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group and a depsipeptide bond in the structure, the ARIA run parameters and scripts generated by default had to be heavily modified (as described in the Experimental section).
Torsion angle dynamics simulations attempted using the default param-eters were found to cause instabilities due to the cyclization restraint and relatively small molecular size, which were not resolved upon decreasing high temperature values by more than half. Simulated annealing runs were therefore carried out using Cartesian dynamics only (with the default simulation total time period quadrupled), and were found to be stable when using the same high temperature value (10 000 K). An overlay of the final converging structural ensemble is given in Figure 2.12.
Figure 2.12: Overlay of 10 lowest energy conformers of 10a following molecular dynamics energy minimization using NOE-derived distance restraints (see text).
Chapter 2. Synthesis of the antiparasitic chaiyaphumines
2.7 Conclusions and future work
A synthetic route to the chaiyaphumines, a newly discovered class of antiparasitic cyclic depsipeptide natural products, has been successfully developed. Correct stereochemistry was confirmed via Marfey’s analysis, and the synthetic peptides exhibited antiparasitic bioactivity comparable to the naturally isolated peptides. In addition, a series of chaiyaphumine analogues were synthesized in which the ester bond was replaced by an amide linkage, and these exhibited a similar potency of antiparasitic action (<3 µM for11a against P. falciparum), although interestingly with respect to the N-terminal capping moiety the activity profile was reversed (see above).
Future work will involve assaying stability of the synthetic chaiyaphumines and analogues under different pH conditions and with regards to enzymatic degradation, to demonstrate the increased stability of the amide-linked analogues over the natural ester structures. Structure-activity studies will also be critical in further elucidating those structural features essential for activity.
This will involve the synthesis of analogues bearing amino acid substitutions, such as the replacement of D- with L-amino residues to evaluate the effect of stereochemistry. In particular the role of the capping group functionality (previously demonstrated to be important) should be probed – for example further studies of chain length with regards to the phenyl-substituted version, to contrast that for the aliphatic capping moiety already investigated.
Of particular interest are those modifications likely to increase hydropho-bicity and reduce hydrogen bonding ability, potentially resulting in increased cell permeability. Examples of this type, such as the halogenation of aromatic side chains or the introduction of the Dpr side chain methylation discussed in section 2.5, are given in Scheme 2.4a. These can be incorporated during Fmoc SPPS using the appropriate commercially available building blocks. In addition, a route to the methylated Dpr building block required for the latter modification is proposed in Scheme 2.4b. Although some difficulty is anticipated during cyclizations involving the relatively hindered methylated Dpr side chain, the already demonstrated propensity of the linear chaiyaphumines to readily undergo macrocyclizations at room temperature suggests that this will be feasible given the correct choice of activator and optimization of reaction conditions.
The elucidation of the mechanism of action of the chaiyaphumines is also necessary, and the identification of protein targets of this class of antiparasitics
37
NH
Scheme 2.4: (a) Suggested modifications to the chaiyaphumine scaffold, potentially introducing enhanced cell permeability. N-Methylation of the isopeptide bond can be incorporated using the appropriate methylated Dpr building block (see below).
Commercially available halogenated Phe and Trp building blocks facilitate introduction of chloro and fluoro substitutions on aromatic side chains. (b) A two-step route from Fmoc-Dpr(Boc)-OH (21) to methylated intermediate 23 recently published by Lindahl et al.59 23 can be Boc protected to give the methylated Dpr building block required above.
Chapter 2. Synthesis of the antiparasitic chaiyaphumines
is vital. The work described provides access to much larger amounts of peptidic material than was previously available, thus paving the way for target validation studies using affinity chromatography-based techniques (in which the active peptide is immobilized on a stationary phase followed by washing with protein extracts to identify binders).60 Drug affinity responsive target stability (DARTS) has also been used relatively recently to confirm or identify a number of small molecule-protein interactions, and selects proteins based on their induced stability with respect to proteolysis upon binding of the ligand of interest.60 Finally, the most valuable advantage of obtaining natural products via a chemical route is the potential for chemical modification. The introduction of reactive electrophilic functionality into the chaiyaphumine scaffold would allow covalent attachment to catalytic residues in the active site of a potential target enzyme, and could be used in a target-based screen.60
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