Synthesis of octapeptin C4 and biological profiling against NDM-1 and polymyxin-resistant bacteria.

The first synthesis of octapeptin C4 was achieved using a combination of solid phase synthesis and off-resin cyclisation. Octapeptin C4 displayed antibiotic activity against multi-drug resistant, NDM-1 and polymyxin-resistant Gram-negative bacteria, with moderate activity against Staphylococcus aureus. The linear analogue of octapeptin C4 was also prepared, which showed reduced activity.

polymyxin B and colistin (also termed polymyxin E and generally administered as colistimethate, a polymethanesulfonic acid prodrug). These 'forgotten' antibiotics were first identified in the late 1940s, but until recently were only used sparingly due to potential nephrotoxic and neurotoxic side effects. [6][7][8] Today they are seeing a resurgence in use as 'antibiotics of last resort' to treat these MDR Gram-negative infections.
Octapeptins are 8-residue peptides, consisting of a cyclic heptapeptide core with a lipophilic acyl monopeptide tail (Fig. 1). [9][10][11] They are similar in structure to the polymyxins, sharing the same 2,4-diaminobutyric acid (Dab)-rich cyclic core but with two fewer amino acids in the tail and an inversion in stereochemistry in the exocyclic Dab residue. They possess potent, broad spectrum activity against Gram-negative bacteria, but weak activity against Gram-positive bacteria and fungi. 12 Notably, mixtures of natural product octapeptins were reported 13,14 to display activity against polymyxin-resistant strains of E. coli and showed a low propensity for resistance generation. 15 Most of the octapeptins were isolated and investigated as mixtures in the 1970s and 1980s and were grouped into four sub-classes, octapeptins A, B, C, and D, based variations in amino acid (AA) sequence at positions AA4, AA5 and AA8. Individual members of each class differ in the N-terminal lipophilic acid component, having straight chain, iso and anteiso fatty acids and C8-C10 chain lengths. 16 The absolute configuration of the 3-hydroxy and anteiso methyl groups were both assigned as R using optical rotations of fatty acids derived from a mixture of octapeptins A1, A2, A3 and/or B1, B2, B3. 17 Interestingly, the stereochemistry of AA4 and AA5 in the recently reported octapeptin B5 (battacin) was reversed compared to other members of the same subclass, featuring L-Phe4 and D-Leu5 instead of D-Phe4 and L-Leu5. 18 The structure of octapeptin B5 was recently confirmed by synthesis. 19 Several reports have described synthetic modifications of the octapeptins, replacing the 3-hydroxy fatty acid with simple, long-chain aliphatic acids 20 and non-selective poly-acylation and poly-alkylation of the Dab groups. 21 Given their structural similarity to the polymyxins and our work on the structure-activity and structure-toxicity relationships of this class, 22 we wished to determine if a synthetic, pure octapeptin would retain the intriguing antimicrobial activity seen for the natural product mixtures. We thus embarked upon the total synthesis of octapeptin C4, as a representative of this class of lipopeptide antibiotics, and evaluated its biological activity against a panel of polymyxin-sensitive and -resistant strains and for mammalian cell cytotoxicity. A noncyclised linear analogue of octapeptin C4 was also synthesised and assayed.
Octapeptin C4 was synthesised using manual Fmoc-based solid phase chemistry according to the procedure outlined in Scheme 1 and the Supplementary data). A completely protected linear peptide was prepared on a 2-chlorotrityl resin using Boc side chain protection on Dab residues 1,3,6 and 7, and either ivDde (1a) or Boc (1b) protection on Dab-2. Sequential resin cleavage of (1b) followed by global deprotection with TFA gave linearised octapeptin C4 (2b). Alternatively, the Dab-2 ivDde protecting group of (1a) was selectively removed using 4% hydrazine; subsequent cleavage of the resin-bound intermediate was effected with 20% hexafluoroisopropanol (HFIP) in DCM without the risk of premature Boc cleavage. 23 Cyclisation of (2a) between the deprotected Dab-2 side chain and unmasked C-terminal carboxyl group was achieved using diphenylphosphoryl azide (DPPA)/NaHCO 3 in DMF under high dilution conditions, (Scheme 1). Final deprotection with TFA and HPLC purification provided octapeptin C4, with the structure confirmed using HRMS, MS/MS fragmentation and NMR (Supplementary Data, Fig. S1-S13).
Octapeptin C4 and its linear analogue 2b were screened against a representative panel of sensitive and resistant Gram-negative ESKAPE strains, against the Gram-positive S. aureus, and counter-screened for mammalian cell cytotoxicity against the kidney cell lines HK2 (human kidney 2) and hRPTEC (primary human renal proximal tubule epithelial cells) ( Table 1). Octapeptin C4 displayed activity against all Gram-negative pathogens, albeit with less potency compared to polymyxin B, while the linear analogue 2b displayed weaker activity, demonstrating the importance of cyclisation. More importantly, octapeptin C4 was active (MIC 2 μg/mL) against two polymyxin-resistant (Pmx R ) P. aeruginosa strains, in which polymyxin B activity was reduced to 32 and 128 μg/mL and the linear analogue 2b was ≥32 μg/mL. Octapeptin C4 showed four-fold greater cytotoxicity than polymyxin B against the HK-2 human kidney cell line (CC 50 = 31 μM vs 135 μM), with a similar level of toxicity against primary proximal tubule hRPTEC cells (CC 50 = 39 μM), which may be more predictive of nephrotoxic potential. 24 The linear analogue 2b showed similar toxicity as polymyxin B to HK2 cells and intermediate toxicity to hRPTEC cells (CC 50 = 139 μM and 71 μM, respectively).
In conclusion, a discrete component of the naturally-occurring mixtures of octapeptins, octapeptin C4, has been chemically synthesised for the first time, and remained active against polymyxin-resistant strains of P. aeruginosa. Work is ongoing to investigate the mechanistic differences between octapeptins and polymyxins in their action against MDR bacteria, and to synthesise analogues with a broad spectrum Gram-negative activity against polymyxin-resistant and -sensitive strains with improved toxicity profiles.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.