The Journal of Antibiotics

https://doi.org/10.1038/s41429-019-0276-7

ARTICLE

Polyketide glycosides phialotides A to H, new potentiators

of amphotericin B activity, produced by Pseudophialophora
sp. BF-0158
Akiho Yagi1 Ryuji Uchida
●
1 ●
Keisuke Kobayashi2 Hiroshi Tomoda2
●

Received: 10 October 2019 / Revised: 13 December 2019 / Accepted: 19 December 2019

© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2020

Abstract
Eight new potentiators of antifungal amphotericin B (AmB) activity, phialotides A to H, were isolated from the fermentation
broths of the rare fungus Pseudophialophora sp. BF-0158. The structures of phialotides were elucidated by spectroscopic analyses,
including NMR and MS, and degradation studies. Phialotides were novel polyketide glycosides consisting of a 1,3-dimethylbut-1-
ene (C6-unit) repeating substructure and one to three hexopyranoses. None of the phialotides exhibited antifungal activity, whereas
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all potentiated AmB activity against several fungi. Phialotide F was the most effective potentiator of AmB activity against Candida
albicans, with a decrease in the MIC from 0.50 to 0.016 µg ml−1 being observed in combination with phialotide F at 1.0 µg ml−1.

Introduction fungi. However, AmB may interact with cholesterol in

Since the 1950s, amphotericin B (AmB), a polyene mac-
rolide antibiotic with antifungal activity produced by
Streptomyces nodosus [1], has been clinically used in the
treatment of systemic fungal infections, such as candidiasis,
aspergillosis, cryptococcosis, and zygomycosis. AmB is
regarded as the most reliable antifungal agent among those
in clinical use because of its broad and potent fungicidal
activity. Liposomal AmB was developed in the 1990s to
improve tolerability [2, 3]. The mode of action of AmB
involves the formation of pores in fungal membranes
through interactions with ergosterol, the main sterol of
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0276-7) contains supplementary
material, which is available to authorized users.
* Ryuji Uchida
uchidar@tohoku-mpu.ac.jp
* Hiroshi Tomoda
tomodah@pharm.kitasato-u.ac.jp
mammalian cell membranes, resulting in cytotoxicity
against human cells and serious side effects including
nephrotoxicity, hypokalemia, fever, chills, and vomiting.
Therefore, therapeutic interventions are required in some
cases. In an attempt to offset the side effects associated with
AmB therapy, we screened microbial potentiators of AmB
activity against Candida albicans, which may reduce the
AmB dosage. We previously discovered simpotentin [4]
and nectriatide [5] as on-target metabolites from fungal
culture broths. These potentiators have diverse structures:
simpotentin, a glycolipid composed of a mannose with two
medium-chain fatty acids, and nectriatide, a 13-membered
cyclic tetrapeptide with an anthranilic acid residue. During
the course of our continuous screening for new AmB
potentiators of microbial origin, eight new compounds,
designated phialotides A (1) to H (8) (Fig. 1), were isolated
from the culture broth of the fungal strain Pseudophialo-
phora sp. BF-0158. The fermentation, isolation, physico-
chemical properties, structural elucidation, and AmB-
potentiating activity of 1–8 are described herein.

1
Department of Natural Product Chemistry, Faculty of Results
Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical
University. 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-
8558, Japan Isolation
2
Microbial Chemistry and Medicinal Research Laboratories,
Graduate School of Pharmaceutical Sciences, Kitasato University. A 68-hour-old culture broth (1.0 l) was centrifuged to
5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan separate the mycelium and supernatant, and the mycelium

Fig. 1 Structures of phialotides
A (1) to H (8)
was treated with acetone (1.0 l) for 1 h. After filtration,
acetone solution was concentrated to remove acetone in
vacuo. The remaining aqueous solution (200 ml) was
adjusted at pH 3.0 with 1.0 M HCl and extracted with
EtOAc (200 ml) twice. The EtOAc layer was dried over
Na2SO4 and concentrated in vacuo to give crude materials
(404 mg). These materials were purified by preparative
HPLC (column, CAPCELL PAK C18 MGII (Osaka soda,
Osaka, Japan, i.d. 20 × 250 mm); mobile phase, 70%
CH3CN containing 0.05% H3PO4. isocratic; detection, UV
at 210 nm; flow rate, 8.0 ml min−1). Under these conditions,
1–8 were eluted as peaks with retention times of 14, 16, 19,
20, 22, 31, 33, and 36 min, respectively (Supplementary
Fig. S1). Each fraction was concentrated in vacuo to
remove CH3CN, and the aqueous solution was then
extracted with EtOAc twice. The EtOAc layer was
dried over Na2SO4 and concentrated in vacuo to yield pure
1 (10.4 mg), 2 (14.0 mg), 3 (2.49 mg), 4 (1.83 mg),
5 (4.88 mg), 6 (4.58 mg), 7 (7.20 mg), and 8 (5.05 mg) as a
white powder.
Structural elucidation of phialotides
The physicochemical properties of 1–8 are summarized in
Table 1. Compounds 1–8 showed characteristic absorption
maxima at 201–203 nm in UV spectra. Common IR
absorption at 3399–3425, 2928–2930, and 1645–1717 cm−1
suggested the presence of alcohol, alkyl, and carbonyl
moieties, respectively, in the structures. These spectral data
showed that 1–8 have a similar core structure.
A. Yagi et al.
Phialotide A (1): Its molecular formula was elucidated as
C47H80O19 based on HR-ESI-MS measurements. The 13C
NMR spectrum (in CD3OD) showed 47 signals, which were
classified into 12 methyl carbons, one sp3 methylene car-
bon, one oxygenated sp3 methylene carbon, five sp3
methine carbons, 16 oxygenated sp3 methine carbons, three
anomeric carbons, four sp2 methine carbons, four sp2 qua-
ternary carbons, and one carbonyl carbon from an analysis
of the HSQC spectrum. Among them, the chemical shifts
observed in four methyl groups, 2-CH3 (δH 1.86), 6-CH3 (δH
1.63), 10-CH3 (δH 1.60), and 14-CH3 (δH 1.62), in a lower
magnetic field suggested that they attached to the olefinic
carbons. The 1H NMR spectrum (in CD3OD) showed 42
proton signals, which were classified into 12 methyl pro-
tons, one sp3 methylene proton, five sp3 methine protons, 16
oxygenated sp3 methine protons, one oxygenated sp3
methylene proton, four sp2 methine protons, and three
anomeric protons. The connectivity of proton and carbon
atoms was established by the HSQC spectrum (Table 2).
The structure of the aglycone part in 1 was elucidated by
2D NMR experiments including 1H–1H COSY and HMBC
spectra. The spin systems of 1H–1H COSY correlations
between sp3 methine proton H-4 (δ 2.81) and oxygenated
sp3 methine proton H-5 (δ 3.78), between H-4 and methyl
proton 4-CH3 (δ 0.87), between sp2 methine proton H-3 (δ
6.65) and H-4, and between H-3 and methyl proton 2-CH3
(long-range correlation) are shown in Fig. 2, suggesting the
presence of a 1,3-dimethylbut-1-ene (C6-unit) moiety (par-
tial structure I). Moreover, 1H–13C long-range couplings of
2
J and 3J were observed in the HMBC spectrum from H-3
Polyketide glycosides phialotides A to H, new potentiators of amphotericin B activity, produced by. . .

Table 1 Physicochemical properties of 1–8

1 2 3 4

Appearance White powder White powder White powder White powder

Molecular weight 948 802 786 640
Molecular formula C47H80O19 C41H70O15 C41H70O14 C35H60O10
HR-ESI-MS (m/z)
Calcd 971.5192 [M + Na]+ 825.4612 [M + Na]+ 809.4663 [M + Na]+ 663.4084 [M + Na]+
Found 971.5143 [M + Na]+ 825.4569 [M+Na]+ 809.4613 [M+Na]+ 663.4053 [M+Na]+
UV (MeOH) λmax (log ε) 202 (4.57) 203 (4.52) 202 (4.54) 202 (4.56)
[a]D25 (c = 0.1, MeOH) −5.10 −16.0 −23.0 −9.8
IR (KBr) νmax (cm−1) 3425, 2964, 2929, 2874, 3424, 2965, 2929, 2880, 3424, 2965, 2928, 2868, 3408, 2964, 2928, 2878,
1645, 1637, 1449, 1398, 1694, 1646, 1450, 1388, 1703, 1454, 1387, 1270, 1650, 1547, 1452, 1385,
1260, 1234, 1070 1266, 1235, 1070 1232, 1132, 1074, 1046 1304, 1267, 1072, 1043

5 6 7 8

Appearance White powder White powder White powder White powder

Molecular weight 860 900 1046 884
Molecular formula C44H76O16 C47H80O16 C53H90O20 C47H80O15
HR-ESI-MS (m/z)
Calcd 883.5031 [M + Na]+ 923.5344 [M + Na]+ 1069.5923 [M + Na]+ 907.5395 [M+Na]+
+ + +
Found 883.4991 [M+Na] 923.5287 [M + Na] 1069.5829 [M + Na] 907.5308 [M+Na]+
UV (MeOH) λmax (log ε) 201 (4.63) 203 (4.67) 202 (4.68) 203 (4.68)
[a]D25 (c = 0.1, MeOH) +6.98 +5.02 −13.8 −18.4
IR (KBr) νmax (cm−1) 3411, 2966, 2930, 2880, 3421, 2965, 2929, 2878, 3399, 2966, 2929, 2880, 3417, 2966, 2928, 2880,
1717, 1455, 1385, 1301, 1691, 1642, 1453, 1381, 1686, 1451, 1386, 1264, 1688, 1647, 1451, 1384,
1252, 1069, 1036 1239, 1071, 1034 1233, 1128, 1071, 1265, 1044, 992

and methyl proton 2-CH3 to sp2 quaternary carbon C-2
(δ 129.2), from H-3 to methyl carbon 2-CH3 (δ 12.9), and
from 2-CH3 to sp3 methine carbon C-3 (δ 147.3), supporting
the presence of partial structure I. Three sets of similar
correlations in 1H-1H COSY and HMBC spectra revealed
partial structures II to IV, all consisting of the C6-unit. Key
HMBC correlations from sp2 methine proton H-7 (δ 5.36)
and methyl proton 6-CH3 (δ 1.63) to oxygenated sp2
methine carbon C-5 (δ 87.4), from sp2 methine proton H-11
(δ 5.26) and methyl proton 10-CH3 (δ 1.60) to oxygenated
sp2 methine carbon C-9 (δ 88.3), and from sp2 methine
proton H-15 (δ 5.46) and methyl proton 14-CH3 (δ 1.62) to
oxygenated sp2 methine carbon C-13 (δ 90.4) exhibited the
consecutive connectivity of partial structures I–IV. More-
over, both ends of the aglycone part assembly at C-2 and
C-17 were elucidated by 2D NMR spectra. 1H–1H COSY
correlations through oxygenated sp3 methine proton H-17
(δ 3.24) to methyl proton H3-20 (δ 0.92) including methyl
proton 18-CH3 (δ 0.95) showed the presence of a 1-methyl
propyl group at C-17, and HMBC correlations from H-3
and 2-CH3 to carboxy carbon C-1 (δ 171.7) showed the
presence of a carboxy group at C-1. Furthermore, ROESY
correlations (Fig. 3) were observed between 2-CH3 and H-4,
between 6-CH3 and H-8, between 10-CH3 and H-12, and
between 14-CH3 and H-16, indicating that the geometry of
all olefins at C-2/C-3, C-6 (δ 132.1)/C-7 (δ 139.2), C-10
(δ 132.4)/C-11 (δ 139.1), and C-14 (δ 133.1)/C-15 (δ 136.6)
in 1 was the E-configuration. Based on these results, the
aglycone of 1 was elucidated as shown in Fig. 2.
The structures of three sugar moieties in 1 were then
examined. As shown by the bold lines in Fig. 2, three partial
structures V–VII were elucidated by a selective 1D
HOHAHA spectrum (Supplementary Figs. S11 to S13).
HMBC correlations from three anomeric protons, H-1′
(δ 4.70), H-1″ (δ 5.10), and H-1‴ (δ 4.40), to their
respective oxygenated sp3 methine carbons, C-5′ (δ 70.5),
C-5″ (δ 70.3), and C-5‴ (δ 78.3), indicated the presence of
two deoxyhexopyranoses (sugars A and B containing partial
structures V and VI) and one hexopyranose (sugar C con-
taining partial structure VII). Furthermore, the cross peaks
from H-1′ to C-5, from H-1″ to C-9, and from H-1‴ to C-13
indicated that sugars A to C were connected to the aglycone
at C-5, C-9, and C-13, respectively, via a glycosidic bond.
Consequently, the planar structure of 1 was elucidated as
shown in Fig. 2, which fulfilled the molecular formula and
degree of unsaturation.
The relative configuration of sugars A to C in 1 was
elucidated from an analysis of the 1H–1H coupling
 13
Table 2 1H and C NMR chemical shifts in 1 to 8
position 1 2 3 4
δC (ppm) δH (ppm) JH-H (Hz) δC (ppm) δH (ppm) JH-H (Hz) δC δH JH-H (Hz) δC δH JH-H (Hz)

2''''
1''''
1 171.7 s – 171.7 s – 172.7 s – 172.8 s –
2 129.2 s – 128.9 s – 130.2 s – 130.0 s –
3 147.3 d 6.65 dd 1.5, 9.8 147.8 d 6.66 dd 1.0, 9.7 146.1 d 6.58 dd 1.0, 9.5 146.4 d 6.60 dd 1.0, 9.8
4 37.0 d 2.81 m 36.4 d 2.81 m 36.6 d 2.80 m 37.0 d 2.78 m
5 87.4 d 3.78 d 9.1 88.1 d 3.74 d 9.3 87.3 d 3.77 d 9.5 87.7 d 3.76 d 9.4
6 132.1 s – 131.9 s – 132.3 s – 132.82 s –
7 139.2 d 5.36 dd 1.0, 9.2 139.3 d 5.30 dd 1.0, 9.8 138.8 d 5.36 dd 1.0, 9.0 137.9 d 5.40 dd 1.0, 10.0
8 35.4 d 2.75 m 35.4 d 2.75 m 35.9 d 2.76 m 37.1 d 2.66 m
9 88.3 d 3.73 d 9.7 90.3 d 3.78 d 9.3 87.8 d 3.75 b 9.5 84.0 d 3.73 b 8.6
10 132.4 s – 133.3 s – 132.9 s – 137.8 s –
11 139.1 d 5.26 dd 1.0, 9.7 138.3 d 5.34 dd 1.0, 9.2 138.4 d 5.38 dd 1.0, 9.0 133.5 d 5.29 dd 1.0, 9.0
12 35.43 d 2.75 m 37.3 d 2.64 m 37.5 d 2.68 m 37.0 d 2.64 m
13 90.4 d 3.75 d 9.7 84.3 d 3.69 d 9.3 83.8 d 3.73 d 9.5 84.1 d 3.71 d 8.8
14 133.1 s – 137.4 s – 137.2 s – 137.2 s –
15 136.6 d 5.46 dd 1.0, 9.9 132.8 d 5.36 dd 1.0, 9.2 132.7 d 5.36 dd 1.0, 9.0 132.79 d 5.36 dd 1.0, 10.0
16 36.6 d 2.70 m 36.6 d 2.64 m 36.9 d 2.64 m 36.7 d 2.63 m
17 79.4 d 3.24 t 5.3 79.4 d 3.24 dd 4.7, 6.3 79.4 d 3.24 dd 4.5, 6.5 79.4 d 3.23 dd 4.5, 6.7
18 39.4 d 1.44 m 38.3 d 1.51 m 38.3 d 1.51 m 38.2 d 1.51 m
19 27.3 t 1.46, 1.18 m, m 27.7 t 1.48, 1.23 m, m 27.7 t 1.48, 1.23 m, m 27.8 t 1.51, 1.24 m, m
20 11.9 q 0.92 t 7.6 11.9 q 0.91 t 7.0 12.0 q 0.91 dd 7.1 12.0 q 0.91 dd 7.0
21
22
23
24
1''''-CH3
2-CH3 12.9 q 1.86 d 1.5 12.9 q 1.86 d 1.0 13.2 q 1.86 d 1.0 13.2 q 1.86 d 1.0
4-CH3 16.8 q 0.87 d 7.1 16.7 q 0.86 d 6.9 16.9 q 0.87 d 7.0 16.9 q 0.86 d 7.0
6-CH3 10.79 q 1.63 d 1.0 11 q 1.62 d 1.0 10.8 q 1.63 d 1.0 11.2 q 1.63 d 1.0
8-CH3 17.75 q 0.81 d 6.9 17.5 q 0.79 d 7.2 17.9 q 0.81 d 7.0 17.8 q 0.81 d 7.1
10-CH3 11.0 q 1.60 d 1.0 10.9 q 1.63 d 1.0 11.1 q 1.61 d 1.0 11.4 q 1.68 d 1.0
12-CH3 17.4 q 0.78 d 7.2 17.7 q 0.79 d 6.2 17.9 q 0.79 d 6.6 17.86 q 0.79 d 7.1
14-CH3 10.82 q 1.62 d 1.0 11.4 q 1.68 d 1.0 11.6 q 1.67 d 1.0 11.6 q 1.66 d 1.2
16-CH3 18.3 q 0.98 d 7.1 18.2 q 0.93 d 7.2 18.1 q 0.93 d 6.7 18.0 q 0.93 d 6.5
18-CH3 14.5 q 0.95 d 6.7 13.8 q 0.92 d 6.5 13.7 q 0.92 d 6.4 13.6 q 0.92 d 6.7
20-CH3
22-CH3
1' 97.5 d 4.70 d 1.3 97.0 d 5.05 d 1.5 97.4 d 4.70 d 1.5 97.9 d 4.70 d 1.4
2' 72.8 d 3.71 m 72.9 d 3.66 m 73.0 d 3.70 m 72.8 d 3.69 dd 1.0, 3.0
A. Yagi et al.
Table 2 (continued)
position 1 2 3 4
δC (ppm) δH (ppm) JH-H (Hz) δC (ppm) δH (ppm) JH-H (Hz) δC δH JH-H (Hz) δC δH JH-H (Hz)

3' 72.6 d 3.51 dd 3.0, 9.5 72.4 d 3.52 dd 3.5, 9.0 72.4 d 3.54 dd 3.5, 9.5 72.5 d 3.56 dd 3.4, 9.6
4' 73.8 d 3.31 t 9.5 73.7 d 3.33 t 9.0 73.9 d 3.29 dd 9.5 73.9 d 3.32 dd 9.8
5' 70.5 d 3.62 m 70.2 d 3.62 m 70.5 d 3.63 m 3.63 d 3.63 m
6' 17.9 q 1.24 d 6.2 17.8 q 1.23 d 6.2 17.8 q 1.23 d 6.2 17.96 q 1.23 d 6.4
1'' 96.9 d 5.10 d 1.7 98.1 d 4.52 s 97.5 d 4.74 s 1.5
2'' 73.2 d 3.69 m 75.6 d 3.71 d 3.5 72.8 d 3.71 d
3'' 72.3 d 3.64 dd 3.5, 9.5 73.3 d 3.40 dd 3.5, 9.5 72.5 d 3.65 dd 3.5, 9.5
4'' 74.6 d 3.31 t 9.5 68.2 d 3.60 t 9.5 74.5 d 3.30 dd 9.5
5'' 70.3 d 3.69 m 78.1 d 3.11 ddd 2.5, 4.0, 9.5 70.4 d 3.66 ddd
6'' 17.79 q 1.31 d 6.3 62.6 q 3.80 m 18.1 q 1.29 m 6.5
1''' 98.3 d 4.40 s
2''' 73.4 d 3.66 d 3.0
3''' 75.8 d 3.35 dd 3.0, 9.5
4''' 68.1 d 3.61 t 9.5
5''' 78.3 d 3.07 ddd 3.0, 3.5, 9.5
6''' 62.4 t 3.80 m

position 5 6 7 8
δC (ppm) δH (ppm) JH-H (Hz) δC (ppm) δH (ppm) JH-H (Hz) δC (ppm) δH (ppm) JH-H (Hz) δC (ppm) δH (ppm) JH-H (Hz)

2'''' 180.2 s –
1'''' 44.8 d 2.53 dd 7.0, 9.5
1 81.7 d 4.05 d 9.5 172.4 s – 171.8 s – 171.7 s –
2 136.2 s – 129.5 s – 129.2 s – 129.3 s –
3 135 d 5.20 dd 1.0, 9.5 147.1 d 6.62 dd 1.0, 9.5 147.2 d 6.65 dd 1.0, 9.5 147.3 d 6.65 dd 1.5, 9.8
4 35.1 d 2.73 m 36.3 d 2.80 m 36.9 d 2.82 m 37.0 d 2.81 m
Polyketide glycosides phialotides A to H, new potentiators of amphotericin B activity, produced by. . .

5 87 d 3.61 d 9.5 88.1 d 3.74 d 9.5 87.4 d 3.78 d 9.5 87.4 d 3.78 d 9.5
6 131.9 s – 132.0 s – 132.1 s – 132.2 s –
7 139 d 5.26 dd 1.0, 9.5 139.4 d 5.30 dd 1.0, 9.0 139.2 d 5.36 dd 1.0, 9.0 138.9 d 5.36 dd 1.0, 9.0
8 35.4 d 2.75 m 35.4 d 2.76 m 35.4 d 2.76 m 36.0 d 2.76 m
9 90.3 d 3.77 d 9.5 90.3 d 3.78 b 9.5 88.3 d 3.74 d 9.5 87.8 d 3.77 d 9.3
10 133.3 s – 133.3 s – 132.4 s – 132.8 s –
11 138.4 d 5.34 dd 1.0, 9.0 138.2 d 5.35 dd 1.0, 9.5 139.2 d 5.27 dd 1.0, 9.5 138.3 d 5.39 dd 1.0, 9.0
12 37.3 d 2.66 m 37.0 d 2.64 m 35.4 d 2.75 m 37.0 d 2.66 m
13 84.3 d 3.69 d 9.5 84.5 d 3.72 d 9.5 90.2 d 3.78 d 9.5 84.0 d 3.73 d 9.3
14 137.4 s – 138.0 s – 133.2 s – 137.8 s –
15 132.8 d 5.36 dd 1.0, 9.5 133.7 d 5.29 dd 1.0, 9.0 138.5 d 5.34 dd 1.0, 9.0 133.5 d 5.29 dd 1.0, 9.0
16 36.6 d 2.64 m 36.9 d 2.65 m 37.3 d 2.65 m 37.1 d 2.65 m
17 79.5 d 3.24 dd 4.5, 6.5 84.2 d 3.70 d 9.5 84.3 d 3.69 d 9.5 84.1 d 3.71 d 9
18 38.4 d 1.51 m 137.1 s – 137.4 s – 137.2 s –
Table 2 (continued)
position 5 6 7 8
δC (ppm) δH (ppm) JH-H (Hz) δC (ppm) δH (ppm) JH-H (Hz) δC (ppm) δH (ppm) JH-H (Hz) δC (ppm) δH (ppm) JH-H (Hz)

19 27.7 t 1.48, 1.26 m, m 132.9 d 5.37 dd 1.0, 9.5 132.8 d 5.36 dd 1.0, 9.5 132.8 d 5.36 dd 1.0, 9.0
20 12.0 q 0.91 t 7.0 36.6 d 2.64 m 36.6 d 2.64 m 36.6 d 2.64 m
21 79.4 d 3.24 dd 4.5, 6.5 79.5 d 3.24 dd 4.5, 6.5 79.4 d 3.24 dd 4.5, 6.5
22 38.2 d 1.52 m 38.4 d 1.51 m 38.2 d 1.51 m
23 27.8 t 1.48, 1.24 m 27.7 t 1.48, 1.23 m 27.8 t 1.48, 1.22 m
24 12.0 q 0.91 t 7.0 11.9 q 0.91 t 7.0 12.0 q 0.91 t 6.5
1''''-CH3 15.3 q 0.98 d 7.0
2-CH3 11.1 q 1.66 d 1.0 13.1 q 1.86 d 1.0 13.0 q 1.86 d 1.0 13 q 1.86 d 1.5
4-CH3 17.5 q 0.81 d 6.5 16.7 q 0.86 d 6.5 16.9 q 0.88 d 6.5 16.9 q 0.87 d 7.0
6-CH3 10.9 q 1.61 d 1.0 10.9 q 1.62 d 1.0 10.9 q 1.63 d 1.0 10.8 q 1.63 d 1.0
8-CH3 17.3 q 0.78 d 7.0 17.5 q 0.79 d 6.5 17.8 q 0.81 d 6.5 17.9 q 0.81 d 6.5
10-CH3 11.0 q 1.63 d 1.0 11.0 q 1.63 d 1.0 11.0 q 1.61 d 1.0 11.1 q 1.61 d 1.0
12-CH3 17.8 q 0.79 d 6.5 17.7 q 0.78 d 6.5 17.5 q 0.79 d 6.5 17.9 q 0.80 d 6.5
14-CH3 11.3 q 1.68 d 1.0 11.2 q 1.69 d 1.0 10.8 q 1.63 d 1.0 11.6 q 1.69 d 1.0
16-CH3 18.2 q 0.93 d 6.8 17.8 q 0.80 d 6.5 17.8 q 0.79 d 6.5 17.7 q 0.80 d 6.5
18-CH3 13.8 q 0.92 d 6.5 11.5 q 1.67 d 1.0 11.3 q 1.68 d 1.0 11.4 q 1.67 d 1.0
20-CH3 18.0 q 0.93 d 6.8 27.7 q 0.93 d 6.8 18.0 q 0.93 d 6.5
22-CH3 13.6 q 0.92 d 6.5 13.8 q 0.92 d 6.5 13.6 q 0.92 d 6.5
1' 96.0 d 5.09 d 1.5 97.0 d 5.05 d 1.0 97.4 d 4.70 d 1.2 97.5 d 4.71 d 1.5
2' 69.4 d 3.77 m 72.9 d 3.66 m 73.2 d 3.71 m 73.0 d 3.71 m
3' 72.7 d 3.65 dd 3.5, 9.5 72.4 d 3.54 dd 3.0, 9.5 72.6 d 3.52 dd 3.0, 9.5 72.5 d 3.52 dd 3.5, 9.5
4' 74.5 d 3.31 t 9.5 73.8 d 3.34 t 9.5 3.31 d 5.51 t 9.5 73.7 d 3.31 t 9.5
5' 72.1 d 3.62 m 70.2 d 3.63 m 3.62 d 5.62 m 70.5 d 3.63 m
6' 17.7 q 1.28 d 6.5 17.7 q 1.23 d 6.5 17.9 q 1.24 d 6.5 17.9 q 1.24 d 6.0
1'' 98.1 d 4.53 s 98.1 d 4.54 s 96.8 d 5.10 d 1.5 97.6 d 4.76 d 1.5
2'' 73.3 d 3.71 m 73.3 d 3.71 m 73.3 d 3.70 m 72.8 d 3.71 m
3'' 75.6 d 3.40 dd 3.5, 9.5 75.6 d 3.41 dd 3.0, 9.5 72.8 d 3.64 dd 3.0, 9.5 72.4 d 3.65 dd 3.5, 9.5
4'' 68.2 d 3.60 t 9.5 68.2 d 3.60 t 9.5 74.6 d 3.31 t 9.5 74.6 d 3.31 t 9.5
5'' 78.1 d 3.10 m 78.1 d 3.12 m 70.3 d 3.69 m 70.4 d 3.66 m
6'' 62.4 q 3.80 m 62.6 q 3.80 m 17.8 q 1.31 d 6.5 18.1 q 1.24 d 6.0
1''' 98 d 4.55 s
2''' 72.3 d 3.65 d 3.0
3''' 75.6 d 3.40 dd 3.0, 9.5
4''' 68.2 d 3.60 t 9.5
5''' 78.1 d 3.11 ddd 3.0, 3.5, 9.5
6''' 62.5 t 3.80 m
13
C (100 MHz) and 1H (400 MHz) spectra were taken on the Inova 400 system (Agilent) in methanol-d4, and the solvent peaks were used as internal standards at 3.31 ppm for 1H NMR and at 49.0
ppm for 13C NMR. Coupling constants (Hz) were determined by the 1H-1H decoupling experiments
A. Yagi et al.
Polyketide glycosides phialotides A to H, new potentiators of amphotericin B activity, produced by. . .
constant (Table 2), and one-bond 13C–1H coupling con-
stants (1JC-H) (Supplementary Fig. S6) are summarized in
Fig. 4. In sugar A, the large vicinal coupling constants
between H-3′ and H-4′ (9.5 Hz) and between H-4′ and H-5′
(9.5 Hz) indicated that these protons were axial, while the
small vicinal coupling between H-2′ and H-3′ (3.0 Hz)
showed that H-2′ was equatorial. Thus, sugar A was iden-
tified as a rhamnopyranoside. Furthermore, the one-bond
13
C–1H coupling constant between C-1′ and H-1′ was
165.6 Hz, indicating that the anomeric configuration of
sugar A was oriented in an α form (literature values α-
anomer, >160 Hz; β-anomer, <160 Hz) [6]. In sugar B, the
large vicinal coupling constants between H-3′′ and H-4′′
(9.5 Hz) and between H-4′′ and H-5′′ (9.5 Hz) indicated that
these protons were axial, while the small vicinal coupling
between H-2′′ and H-3′′ (3.5 Hz) showed that H-2′′ was
equatorial. Thus, sugar B was identified as a rhamnopyr-
anoside. Furthermore, the coupling constant between C-1′′
and H-1′′ (170.3 Hz) indicates that sugar B was oriented in
an α form. In sugar C, the large vicinal coupling
constants between H-3′′′ and H-4′′′ (9.5 Hz) and between
H-4′′′ and H-5′′′ (9.5 Hz) indicated that these protons were
axial, while the small vicinal coupling between H-2′′′ and
H-3′′′ (3.0 Hz) showed that H-2′′′ was equatorial. Thus,
sugar C was identified as a mannopyranoside. Furthermore,
the coupling constant between C-1′′′ and H-1′′′ (153.3 Hz)
indicated that sugar C was oriented in a β form. Additional
Fig. 2 2D NMR experiments on 1
Fig. 3 Rotamer of stereogenic
centers of 1 with 1H-1H coupling
constants and ROESY
correlations
ROESY
ROESY correlations (Fig. 4) confirmed the relative con-
figurations of sugars A to C as described above.
The stereochemistry of the aglycone of 1 was elucidated
by the 1H–1H coupling constant and ROESY experiments
(Figs. 3 and 5). The large vicinal coupling constant (9.1 Hz)
observed between H-4 and H-5 in partial structure I indi-
cated that they were in an anti-relationship. Moreover,
ROESY correlations between H-4 and 2-CH3, and between
4-CH3 and H-5 also supported the anti-relationship. In a
similar manner, the corresponding protons (H-8/H-9 and
H-12/H-13) in the two repeating C6-units of partial
structures II and III also possessed the anti-relationship.
Thus, the stereochemistries of the six chiral centers,
except for C-16, C-17, and C-18 in the aglycone, were
assigned as 4 S*, 5 S*, 8 S*, 9 S*, 12 S*, and 13 S*, which
were similar to the aglycone in the known TMC-151
compounds [7].
Phialotide B (2): Its molecular formula was elucidated as
C41H70O15 based on HR-ESI-MS measurements, indicating
that 2 lacked the C6H10O4 (one deoxyhexopyranose unit)
present in 1. In comparisons of the 1H and 13C NMR spectra
of 2 with those of 1 (Table 2), chemical shifts in the agly-
cone (C-1 to C-20 including methyl signals) and α-
rhamnopyranoside at C-5 in 2 closely resembled those in
1. On the other hand, the signals of β-mannopyranoside
observed at C-13 in 1 were missing in 2, and the chemical
shift in C-13 was shifted to a higher field in the 13C NMR
spectra (δC 90.4 in 1, δC 84.3 in 2), suggesting the presence
of a hydroxy group at C-13.
HOHAHA and HMBC experiments showed the presence
of a hexopyranose unit, as shown in Fig. 6. The cross peak
from anomeric proton H-1′′ (δ 4.52) to oxygenated sp3
methine carbon C-9 (δ 90.3) in the HMBC experiment
indicated that the sugar was connected to the aglycone at
C-9 via a glycosidic bond. Moreover, the sugar was iden-
tified as a mannopyranoside by 1H–1H coupling constants
(Table 2) and ROESY spectra, and the anomeric config-
uration of mannopyranoside was elucidated to be in a
β form because of the 13C–1H coupling constant between
C-1′′ and H-1′′ (1JC′′-1/H-1′′ = 155.8 Hz) (Supplementary
Fig. S17). The structure of 2 was finally confirmed by 2D
1H-1H coupling constant
 A. Yagi et al.

(a) JH4’-H5’ = 9.5 Hz (b) JH4’’-H5’’ = 9.5 Hz

6’ 6’’

4’ JC1’-H1’ = 165.6 Hz 4’’ JC1’’-H1’’ = 170.3 Hz

5’ 5’’

2’ 2’’
3’ 1’ 3’’ 1’’

JH1’-H2’ = 1.3 Hz JH1’’-H2’’ = 1.7 Hz

JH3’-H4’ = 9.5 Hz JH3’’-H4’’ = 9.5 Hz
JH2’-H3’ = 3.0 Hz JH2’’-H3’’ = 3.5 Hz

(c) JH4’’’-H5’’’ = 9.5 Hz

6’’’
4’’’ 5’’’ JC1’’’-H1’’’ = 153.3 Hz ROESY
1H-1H coupling constant
3’’’ 2’’’
1’’’

JH3’’’-H4’’’ = 9.5 Hz
JH2’'’-H3’’’ = 3.0 Hz

Fig. 4 Stereochemistry of sugar A (a), B (b), and C (c) moieties of 1

Fig. 5 ROESY experiments of 1

NMR experiments, as shown in Fig. 6, which fulfilled the
molecular formula and degrees of unsaturation.
Phialotide C (3): Its molecular formula was elucidated as
C41H70O14 based on HR-ESI-MS measurements, indicating
that 3 lacked the C6H10O5 (one hexopyranose unit) present
in 1. Comparisons of the 1H and 13C NMR spectra of 3 with
those of 1 (Table 2) showed that the chemical shifts in 3,
except for the oxymethine signal at C-13 in the aglycone,
closely resembled those in 1. Moreover, the signals of
mannopyranoside observed at C-13 in 1 were missing in 3,
and the chemical shift in C-13 was shifted to a higher field
in the 13C NMR spectra (δC 90.4 in 1, δC 83.8 in 3), sug-
gesting the presence of a hydroxy group at C-13 in 3. The
structure of 3 was finally confirmed by 2D NMR experi-
ments, as shown in Fig. 6, which fulfilled the molecular
formula and degrees of unsaturation.
Phialotide D (4): Its molecular formula was elucidated as
C35H60O10 based on HR-ESI-MS measurements, indicating
that 4 lacked the C6H10O4 (one deoxyhexopyranose unit)
present in 3. In comparisons of the 1H and 13C NMR spectra
of 4 with those of 3 (Table 2), the chemical shifts in 4,
except for the oxymethine signal at C-9 in the aglycone,
closely resembled those in 3. Moreover, the signals of
rhamnopyranoside observed at C-9 in 3 were missing in 4,
and the chemical shift in C-9 in 4 was shifted to a higher
field in the 13C NMR spectra (δC 87.8 in 3, δC 84.0 in 4),
suggesting the presence of a hydroxy group at C-9 in 4. The
structure of 4 was finally confirmed by 2D NMR experi-
ments, as shown in Fig. 6, which fulfilled the molecular
formula and degrees of unsaturation.
Phialotide E (5): Its molecular formula was elucidated as
C44H76O16 based on HR-ESI-MS measurements, indicating
Polyketide glycosides phialotides A to H, new potentiators of amphotericin B activity, produced by. . .
Fig. 6 2D NMR experiments of 2–8
that 5 was bigger by C3H6O than 2. Comparisons of the 1H
and 13C NMR spectra of 5 with those of 2 (Table 2) showed
that the chemical shifts in 5, except for those in C-1, C-2,
C-3, and 2-CH3 in the aglycone, closely resembled those in
2. Additionally, a methyl signal (1′′′′-CH3: δC 15.3, δH
0.98), sp3 methine signal (C-1′′′′: δC 44.8, δH 2.53), and
oxygenated sp3 methine signal (C-1: δC 81.7, δH 4.05)
were observed in 5 (Table 2). As shown by the bold lines in
Fig. 6, partial structure I′ was elucidated by the TOCSY
spectrum. Moreover, HMBC correlations were observed
from H-1, H-1′′′′, and 1′′′′-CH3 to carbonyl carbon C-2′′′′
(δ 180.2), indicating the presence of a 3-hydroxy-2-
methylpropionic acid moiety including partial structure I′.
Furthermore, the cross peaks from methyl proton
2-CH3 (δ 1.66) and sp2 methine proton H-3 (δ 5.20) to C-1
indicated that the 3-hydroxy-2-methylpropionic acid
moiety was connected to C-2. The structure of 5 was
finally confirmed by 2D NMR, as shown in Fig. 6,
which fulfilled the molecular formula and degrees of
unsaturation.

Phialotide F (6): Its molecular formula was elucidated as
C47H80O16 based on HR-ESI-MS measurements, indicating
that 6 was bigger by C6H10O than 2. In comparisons of the
1
H and 13C NMR spectra of 6 with those of 2 (Table 2),
chemical shifts in 6, except for those in C-18, C-19, and
C-20 in the aglycone, closely resembled those in 2. More-
over, an additional two methyl signals (18-CH3: δC 11.5, δH
1.67, 20-CH3: δC 18.0, δH 0.93), sp3 methine signal (C-20:
δC 36.6, δH 2.64), sp2 methine signal (C-19: δC 132.9, δH
5.37), sp2 quaternary signal (C-18: δC 137.1), and oxyge-
nated sp3 methine signal (C-21: δC 79.4, δH 3.24) were
observed in 6 (Table 2). TOCSY correlations as shown by
the bold lines between H-20 and H-21, between H-20 and
20-CH3, between H-19 and H-20, and between H-19 and
18-CH3 (long-range correlation) (Fig. 6) were observed,
indicating the presence of one more C6-unit moiety (partial
structure IV′). Moreover, TOCSY correlations through
H-22 to H-23, including 22-CH3, showed the presence of a
1-methyl propyl group at C-21. The structure of 6 was
finally confirmed by 2D NMR, as shown in Fig. 6, which
fulfilled the molecular formula and degrees of unsaturation.
Phialotide G (7): Its molecular formula was elucidated as
C53H90O20 based on HR-ESI-MS measurements, indicating
that 7 was bigger by C6H10O (C6-unit) than 1. Additionally,
in comparisons of the 1H and 13C NMR spectra of the
aglycone in 7 with those in 6 (Table 2), chemical shifts in 7
closely resembled those in 6. Therefore, similar to 6, the
presence of one more C6-unit moiety (partial structure IV′)
in the aglycone of 7 was suggested. The structure of 7 was
finally confirmed by 2D NMR, as shown in Fig. 6, which
fulfilled the molecular formula and degrees of unsaturation.
Phialotide H (8): Its molecular formula was elucidated as
C47H80O15 based on HR-ESI-MS measurements, indicating
that 8 lacked the C6H10O5 (one hexopyranose unit) present
in 7. In comparisons of the 1H and 13C NMR spectra of
8 with those of 7 (Table 2), chemical shifts in 8, except for
that in C-13 in the aglycone, closely resembled those in 7.
Moreover, the signals of β-mannopyranoside observed at C-
13 in 7 were missing in 8, and the chemical shift in C-13
was shifted to a higher field in the 13C NMR spectra (δC
90.2 in 7, δC 84.0 in 8), suggesting the presence of a
hydroxy group at C-13 in 8. The structure of 8 was finally
confirmed by 2D NMR experiments, as shown in Fig. 6,
which fulfilled the molecular formula and degrees of
unsaturation.
Absolute configuration of sugar units
The absolute configuration of three sugar units in 1 was
elucidated by degradation studies and subsequent HPLC
analyses of their O-tolyl isothiocyannate derivatives [8].
After the acid hydrolysis of 1 (200 µg in 100% MeOH:
100 µl) with 4 M HCl (100 µl) at 60 °C for 8 h, the reaction
A. Yagi et al.
mixture was lyophilized and treated with EtOAc-H2O
(400 µl) three times. The aqueous layer containing sugars
was lyophilized, 10 mM L-cysteine methyl ester (100 µl in
pyridine) was added, and the mixture was heated at 60 °C
for 60 min. In the next step, 10 mM O-tolyl isothiocyanate
(100 µl in pyridine) was added to the mixture, which was
heated at 60 °C for 60 min. After evaporation of the solvent,
the residue was analyzed by HPLC (column, CAPCELL
PAK C18 MGII (Osaka soda, Osaka, Japan, i.d. 4.6 × 250
mm); mobile phase, 25% CH3CN containing 50 mM
H3PO4; detection, UV at 250 nm; flow rate, 0.8 ml min−1).
The results obtained showed two peaks at retention times of
12 and 31 min, which corresponded to the authentic
D-mannopyranoside and L-rhamnopyranoside derivatives,
respectively (Supplementary Fig. S2). Thus, we confirmed
that 1 contained one D-mannopyranoside and two
L-rhamnopyranosides in its structure. Accordingly, the
absolute stereochemistry of the aglycone in 1 was proven to
be 4 S, 5 S, 8 S, 9 S, 12 S, and 13 S. Thus, the total structure
including the absolute stereochemistry of 1 was elucidated
as shown in Fig. 1.
Antimicrobial activity
In the microdilution method, 1–8 did not exhibit anti-
microbial activity even at 128 µg ml-1 against ten test
microorganisms: C. albicans, Cryptococcus neoformans,
Aspergillus fumigatus, Rhizopus oryzae, Bacillus subtilis,
Staphylococcus aureus, Micrococcus luteus, Escherichia
coli, Pseudomonas aeruginosa, and Mycobacterium
smegmatis.
Amphotericin B-potentiating activity
In the microdilution method, the MIC values of AmB alone
for C. albicans, C. neoformans, A. fumigatus, and R. oryzae
were 0.50, 2.0, 4.0, and 1.0 µg ml−1, respectively, which
were within the MIC ranges recommended in the guidelines
of the CLSI documents M27-A3 [9] and M38-A2 [10]. As
described above, 1–8 alone exhibited no antifungal activity
against the four tested fungi, even at 128 µg ml−1.
Regarding anti-C. albicans activity, 1–8 (0.25 to 4.0 µg
ml−1) enhanced AmB activity against C. albicans in a
concentration-dependent manner, reducing the MIC value
from 0.50 µg ml−1 (alone) to 0.25–0.016 µg ml−1 (in com-
bination) (Table 3). Among the phialotides, 6 (1.0 µg ml−1)
showed the most potent efficacy with 32-fold potentiation
of AmB activity against C. albicans. Similarly, the anti-C.
neoformans, -A. fumigatus, and -R. oryzae activities of
AmB were investigated in combination with phialotides.
Consequently, compounds 1–8 (0.25–4.0 µg ml−1) also
exhibited AmB-potentiating activity against C. neoformans
and R. oryzae in a concentration-dependent manner, but
Polyketide glycosides phialotides A to H, new potentiators of amphotericin B activity, produced by. . .

Table 3 MIC values of AmB against C. albicans, C. neoformans, and R. oryzae in the presence or absence of 1–8
Fungus Combination with MIC (µg ml−1) of AmB (Ratio = MICAmB/MICAmB + phialotide)
1 2 3 4 5 6 7 8

C. albicans AmB alone 0.50 (1) 0.50 (1) 0.50 (1) 0.50 (1) 0.50 (1) 0.50 (1) 0.50 (1) 0.50 (1)
−1
+ (0.25 µg ml ) 0.25 (2) 0.50 (1) 0.25 (1) 0.50 (1) 0.25 (2) 0.25 (2) 0.25 (2) 0.25 (2)
(0.50 µg ml−1) 0.13 (4) 0.13 (4) 0.063 (8) 0.25 (2) 0.13 (4) 0.063 (8) 0.13 (4) 0.13 (4)
(1.0 µg ml−1) 0.031 (16) 0.063 (8) 0.031 (16) 0.063 (8) 0.063 (8) 0.016 (32) 0.031 (16) 0.031 (16)
(2.0 µg ml−1) 0.031 (16) 0.031 (16) 0.031 (16) 0.031 (16) 0.031 (16) 0.016 (32) 0.031 (16) 0.031 (16)
−1
(4.0 µg ml ) 0.031 (16) 0.031 (16) 0.031 (16) 0.031 (16) 0.031 (16) 0.016 (32) 0.031 (16) 0.031 (16)
C. neoformans AmB alone 2.0 (1) 2.0 (1) 2.0 (1) 2.0 (1) 2.0 (1) 2.0 (1) 2.0 (1) 2.0 (1)
+ (0.25 µg ml−1) 2.0 (1) 2.0 (1) 1.0 (2) 1.0 (2) 1.0 (2) 1.0 (2) 1.0 (2) 1.0 (2)
(0.50 µg ml−1) 1.0 (2) 1.0 (2) 1.0 (2) 0.50 (4) 0.50 (4) 0.25 (8) 0.50 (4) 0.50 (4)
(1.0 µg ml−1) 0.50 (4) 0.50 (4) 0.25 (8) 0.50 (4) 0.25 (8) 0.13 (16) 0.13 (16) 0.25 (8)
(2.0 µg ml−1) 0.50 (4) 0.50 (4) 0.13 (16) 0.13 (16) 0.13 (16) 0.13 (16) 0.13 (16) 0.25 (8)
(4.0 µg ml−1) 0.25 (8) 0.25 (8) 0.13 (16) 0.13 (16) 0.13 (16) 0.13 (16) 0.13 (16) 0.25 (8)
R. oryzae AmB alone 1.0 (1) 1.0 (1) 1.0 (1) 1.0 (1) 1.0 (1) 1.0 (1) 1.0 (1) 1.0 (1)
+ (0.25 µg ml−1) 1.0 (1) 0.50 (2) 0.50 (2) 0.50 (2) 1.0 (1) 0.50 (2) 1.0 (1) 0.50 (2)
(0.50 µg ml−1) 0.50 (2) 0.13 (8) 0.25 (4) 0.13 (8) 0.25 (4) 0.25 (4) 0.50 (2) 0.13 (8)
(1.0 µg ml−1) 0.25 (4) 0.13 (8) 0.13 (8) 0.13 (8) 0.25 (4) 0.25 (4) 0.25 (4) 0.13 (8)
(2.0 µg ml−1) 0.25 (4) 0.13 (8) 0.13 (8) 0.13 (8) 0.25 (4) 0.13 (8) 0.25 (4) 0.13 (8)
(4.0 µg ml−1) 0.13 (8) 0.13 (8) 0.13 (8) 0.13 (8) 0.25 (4) 0.13 (8) 0.13 (8) 0.13 (8)

showed no potentiation against A. fumigatus. The MIC
value of AmB against C. neoformans and R. oryzae in
combination with 6 decreased from 2.0 and 1.0 µg ml−1 to
0.13 and 0.13 µg ml−1, respectively, yielding 16- and 8-
fold potentiation of AmB activity, respectively (Table 3).
Discussion
As described in the present study, phialotides A to H (1–8)
are new potentiators of AmB activity against yeast-like
fungi, including C. albicans and C. neoformans and the
zygomycete R. oryzae. The phialotide-producing fungal
strain BF-0158 was isolated from soil collected at Sho-
doshima Island, Kagawa, Japan. The rDNA sequence of the
internal transcribed spacer (ITS) region clarified that
the fungus belongs to the genus Pseudophialophora in the
Magnaporthaceae. Members of the genus Pseudophialo-
phora are rare consisting of only eight species [11], and
secondary metabolites derived from this genus have not
been reported to date.
The chemical structures of 1–8 belong to a polyketide
glycoside family composed of repeating common C6-unit
substructures with one to three sugar moieties including α-
rhamnopyranoside and β-mannopyranoside. Structurally
related compounds to phialotides, bionectriols [12], cla-
dionol A [13], TMC-151s [7], TMC-154s [14], TMC-171s
[14], MK7924 [15], and roselipins [16, 17] were all isolated
from fungi, such as Gliocladium sp., Bionictria sp., and
Coronophora sp. They have a highly methylated polyketide
skeleton with one or two sugar moieties, and the sugar is
limited to mannose. Moreover, the carboxy termini of the
aglycones are often modified with a sugar alcohol, pentitol
or hexitol. Phialotides produced by Pseudophialophora sp.
BF-0158 have a unique rhamnose in addition to mannose,
and no sugar alcohol at the carboxy termini of the agly-
cones. The sugar alcohol derivatives may have no poten-
tiated AmB activity or were not biosynthesized by the
fungus. To clarify this issue, a precise analysis of the
metabolites in the culture broth using LCMS is required.
Regarding biological activity, 1–8, which do not exhibit
antimicrobial activity, even at 128 µg ml−1, potentiated
AmB activity against C. albicans by 8- to 32-fold at
1.0 µg ml−1. Although 6 exhibited the most potent activity,
no clear structure-activity relationships were observed.
However, it is plausible that phialotides possess amphi-
pathic characteristics to interact with AmB in fungal
membranes. TMC-151s and bionectriols have been reported
to potentiate AmB activity (4-fold) against C. albicans
caused by the inhibition of biofilm formation [12]. Among
them, TMC-151s C and D and bionectriols B and D with an
acetylated mannose in their structures exhibited a weak
antifungal activity [12]. Therefore, phialotides may affect
biofilm formation and their acetylated mannose derivatives
may also inhibit the growth of C. albicans.
In our previous study, glycolipid simpotentin [4] and
peptide nectriatide [5] were isolated as new potentiators of
AmB activity from the fungal strains Simplicillium

minatense FKI-4981 [4] and Nectriaceae sp. BF-0114,
respectively. In the case of nectriatide, the MIC of AmB
decreased from 0.50 to 0.031 µg ml−1 in combination with
the compound at a high dose (32 µg ml−1), showing 16-fold
potentiation of AmB activity. More importantly, 6 poten-
tiated AmB activity by 32-fold in the presence of the
compound at a lower dose (1.0 µg ml−1), and is the stron-
gest potentiator of AmB activity ever discovered in the
course of our screening. Therefore, further studies are
warranted to clarify the mechanisms of action of phialotides
for the potentiation of AmB activity.
Matrerials and methods
Microorganisms
The phialotide-producing fungus BF-0158 was isolated
from soil collected at Shodoshima Island, Kagawa, Japan. A
taxonomic study and analysis of the rDNA sequence of
the ITS region of this fungus showed high levels of simi-
larity to members of the genus Pseudophialophora (99.9%).
Therefore, the producing strain was designated as Pseudo-
phialophora sp. BF-0158.
The following fungal strains were used in the micro-
dilution assay: C. albicans ATCC 90029, C. neoformans
ATCC 90113, A. fumigatus NBRC 33022, and R. oryzae
NBRC 4705, for antifungal activity; B. subtilis PCI 219,
S. aureus FDA 209 P, M. luteus ATCC 9341, E. coli JM
109, P. aeruginosa IFO 12689, and M. smegmatis M341 for
antibacterial activity.
General experimental procedures
UV spectra were recorded on a spectrophotometer (U-2800
UV-Visible Double Beam spectrophotometer; Hitachi
High-Technologies, Tokyo, Japan). IR spectra were recor-
ded on a Fourier transform infrared spectrometer (FT/IR-
460; JASCO Co., Tokyo, Japan). Optical rotations were
measured with a digital polarimeter (DIP-370; JASCO Co.,
Tokyo, Japan). HREI-MS spectra were recorded on a mass
spectrometer (JMS-T100 LP, JEOL, Tokyo, Japan). Var-
ious NMR spectra were measured with a spectrometer
(Inova 600; Agilent Technologies, Santa Clara, CA, USA).
Materials
Amphotericin B was purchased from Sigma–Aldrich
(St. Louis, MO, USA). RPMI1640 medium was purchased
from Thermo Fisher Scientific (Waltham, MA, USA).
3-Morpholinopropanesulphonic acid (MOPS) was pur-
chased from DOJINDO (Kumamoto, Japan). L-Cysteine
methyl ester was purchased from Nacalai Tesque (Kyoto,
A. Yagi et al.
Japan). O-Tolyl isothiocyanate was purchased from Tokyo
Chemical Industry Co., Ltd. (Tokyo, Japan). Potato dex-
trose agar (PDA) and potato dextrose broth (PDB) were
purchased from Becton, Dickinson and Company (Franklin
Lakes, NJ, USA). Ehrlich meat extract was purchased from
Kyokuto Pharmaceutical Co. (Tokyo, Japan). Solulys 095E
was purchased from Oriental Yeast Co. (Tokyo, Japan).
PDA, PDB, Ehrlich meat extract, and Solulys 095E were
used for fermentation. The other compounds used were of
special grade.
Fermentation
A slant culture of strain BF-0158 grown on a slant medium
(PDA 2.4%) was used to inoculate three 500-ml Erlenmeyer
flasks each containing 100 ml of the seed medium (PDB
2.4% and agar 0.1%. pH 6.0). The flasks were shaken on a
rotary shaker (170 rpm) at 27 °C for 3 days. The seed cul-
ture (300 ml) was transferred into a 10-l jar fermenter
containing 5 l of production medium (sucrose 2.0%, glucose
1.0%, Solulys 0.5%, Ehrlich meat extract 0.5%, CaCO3
0.3%, Mg2SO4·7H2O 0.1%, KH2PO4 0.1%, FeSO4·7H2O
0.001%, ZnSO4·7H2O 0.001%, MnCl2·4H2O 0.001%,
CuSO4·5H2O 0.001%, CoCl2·6H2O 0.001%, and agar 0.1%.
adjusted to pH 6.5). Fermentation was performed at 27 °C
for 68 h with aeration of 5 l min−1 and agitation of 300 rpm.
Assay for antifungal AmB activity in combination
with phialotides
Two yeasts, a filamentous fungus and a zygomycetous
fungus, were used for antifungal activity assays [18]. The
broth microdilution method using 96-well microplates
(Corning, New York, USA) was performed according to the
guidelines of the CLSI documents M27-A3 [9] and M38-A2
[10]. Regarding yeasts, five colonies with diameters of 1
mm were suspended in sterile 0.85% saline to adjust to a 0.5
McFarland standard by spectrophotometric measurements.
The seed of yeast was diluted 2000 times with RPMI1640
medium (165 mM MOPS buffer (pH 7.0)). Regarding the
filamentous fungus and the zygomycetous fungus, spores
were suspended in sterile 0.85% saline. After allowing
heavy particles to settle for 5 min, the supernatant was
transferred to a sterile tube and adjusted to an optical den-
sity at 550 nm (OD550) of 0.0621 for the filamentous fungus
and 0.0765 for the zygomycetous fungus. This seed was
diluted 50 times with RPMI1640 medium. The diluted seed
(100 µl) and RPMI1640 medium (100 µl) were added to
each well of a 96-well microplate with or without serial
concentrations of test compounds. These 96-well plates
were incubated at 35 °C for 24 h (Candida sp. and zygo-
mycetous fungus), 48 h (filamentous fungus), or 72 h
(C. neoformans). After the incubation, OD550 was measured
Polyketide glycosides phialotides A to H, new potentiators of amphotericin B activity, produced by. . .
with a microplate reader (Elx808, Bio-Tek Instruments,
VT, USA) to assess the minimum inhibitory concentration
(MIC). The antifungal activities of 1–8 and AmB
were tested at concentrations ranging between 0.13 and
128 µg ml−1. Antifungal AmB (0.016 to 4.0 µg ml−1)
potentiation activities were tested in combination with 1–8
(0.13–4.0 µg ml−1). MIC was defined as the lowest con-
centration of AmB at which fungal growth was inhibited by
90% from control growth (no AmB).
Acknowledgements We express our thanks to Dr. Kenichiro Nagai
and Ms. Noriko Sato of the School of Pharmacy, Kitasato University
for measurements of NMR and mass spectra. This work was supported
by JSPS KAKENHI Grant Numbers 16H05095 (to RU) and 21310146
(to HT).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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