Supplementary Note: Biosynthesis and characterization of U-13C-Amphotericin
Biosynthesis
U-13C-Amphotericin (U-13C-AmB) (was prepared biosynthetically from cultures of the bacterium Streptomyces nodosus (American Type Culture Collection [ATCC], Manassas, VA, cell line 14899) using a modified version of the protocol previously reported.18 In our modified protocol, the “FCA” medium66 was used, with substitution of U-13C-Glucose (U-13C-Glc) for natural abundance fructose. Inoculation of culture medium and pulse feeding U-13C-Glc were carried out in a Labconco Clean Bench (Labconco, Kansas City, MO) sterilized with 70% EtOH before and after each inoculation/feeding. S. nodosus starter cultures were prepared from ATCC 14899 as previously described.66 Nine 250 mL baffled culture flasks, each containing 20 mL of the U-13C-Glc-enriched medium in 250 mL baffled flasks were inoculated with S. nodosus starter culture. Pulse feeding of cultures was done according to the schedule previously described,65 but with140 µL of aqueous 50% w/v U-13C-Glc added to each baffled flask.
Purification
After work-up of the U-13C-AmB as previously described,65 if there remained any sign of residual Aliquat 336 (strong odor of Aliquat 336, dark yellow solid, sticky solid, not free flowing), then this material was further purified via C18 reverse phase flash chromatography as follows: AmB was dissolved in a minimum amount of DMF and Celite 545 was added to form a slurry. This slurry was concentrated in vacuo, thus adsorbing the AmB onto the Celite. This solid was loaded onto a flash column loaded with Silicycle C18 silica gel and equilibrated with 20% MeCN / 5 mM ammonium acetate. Purification proceeded with a gradient of 20% MeCN / 5 mM NH4OAc to 100% MeCN followed by 100% Optima MeOH (Fisher Scientific). The resulting yellow fractions were concentrated in vacuo and analyzed via 1H NMR in DMF-d7 for presence of residual Aliquat 336. If Aliquat 336 was still present, the material was purified again by C18 flash column using the above protocol. Once removal of Aliquat 336 was confirmed, U-13C-AmB was purified by preparative scale HPLC as described in General Methods above (Section I). Prior to each experiment involving U-13C-AmB, the purity of the compound was confirmed by analytical HPLC (see representative chromatogram below).
Isotopic enrichment
For natural abundance AmB, the calculated mass for C47H74NO17 (M+H)+ is 924.49. Average isotopic enrichment of U-13C-AmB was calculated from ESI-MS peak intensities. The average m/z (M+H)+ was 965.8. Thus, on average, 41 of the 47 carbon atoms of AmB were 13C-enriched and the overall enrichment was thus approx. 87% (41/47 ≈ 0.87).
18Matsuoka, S., Ikeuchi, H., Umegawa, Y., Matsumori, N. & Murata, M. Membrane interaction of amphotericin B as single-length assembly examined by solid state NMR for uniformly 13C-enriched agent. Bioorg. Med. Chem. 14, 6608-6614 (2006).