Total Synthesis and Biological Mode of Action of WAP-8294A2: A Menaquinone-Targeting Antibiotic

Article abstract of DOI:10.1021/acs.joc.7b02318

WAP-8294A2 (lotilibcin, 1) is a potent antibiotic with superior in vivo efficacy to vancomycin against methicillin-resistant Staphylococcus aureus (MRSA). Despite the great medical importance, its molecular mode of action remains unknown. Here we report the total synthesis of complex macrocyclic peptide 1 comprised of 12 amino acids with a β-hydroxy fatty-acid chain, and its deoxy analogue 2. A full solid-phase synthesis of 1 and 2 enabled their rapid assembly and the first detailed investigation of their functions. Compounds 1 and 2 were equipotent against various strains of Gram-positive bacteria including MRSA. We present evidence that the antimicrobial activities of 1 and 2 are due to lysis of the bacterial membrane, and their membrane-disrupting effects depend on the presence of menaquinone, an essential factor for the bacterial respiratory chain. The established synthetic routes and the menaquinone-targeting mechanisms provide valuable information for designing and developing new antibiotics based on their structures.

Full text of DOI:10.1021/acs.joc.7b02318

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Article
Total Synthesis and Biological Mode of Action of
WAP-8294A2: A Menaquinone-Targeting Antibiotic
Hiroaki Itoh, Kotaro Tokumoto, Takuya Kaji, Atmika Paudel, Suresh
Panthee, Hiroshi Hamamoto, Kazuhisa Sekimizu, and Masayuki Inoue
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02318 • Publication Date (Web): 11 Oct 2017
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Total Synthesis and Biological Mode of Action of
WAP-8294A2: A Menaquinone-Targeting Antibiotic
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Hiroaki Itoh,† Kotaro Tokumoto,† Takuya Kaji,† Atmika Paudel,‡ Suresh Panthee,‡ Hiroshi
Hamamoto,‡ Kazuhisa Sekimizu,‡ and Masayuki Inoue*†
†Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7‐3‐1 Hongo, Bunkyo‐ku, Tokyo 113‐0033, Japan.
‡Teikyo University Institute of Medical Mycology, 359 Otsuka, Hachioji, Tokyo 192‐0395, Japan.
ABSTRACT: WAP‐8294A2 (lotilibcin, 1) is a potent antibiotic with superior in vivo efficacy to vancomycin against methi‐
cillin‐resistant Staphylococcus aureus (MRSA). Despite the great medical importance, its molecular mode of action re‐
mains unknown. Here we report the total synthesis of complex macrocyclic peptide 1 comprised of 12 amino acids with a
‐hydroxy fatty‐acid chain, and its deoxy analogue 2. A full solid‐phase synthesis of 1 and 2 enabled their rapid assembly
and the first detailed investigation of their functions. Compounds 1 and 2 were equipotent against various strains of
Gram‐positive bacteria including MRSA. We present evidence that the antimicrobial activities of 1 and 2 are due to lysis
of the bacterial membrane, and their membrane‐disrupting effects depend on the presence of menaquinone, an essential
factor for the bacterial respiratory chain. The established synthetic routes and the menaquinone‐targeting mechanisms
provide valuable information for designing and developing new antibiotics based on their structures.
S. aureus (MRSA) clinical isolates (minimum inhibitory
concentration [MIC] = 0.78 g/mL). Assessment of the in
INTRODUCTION
Antibiotics are crucial in modern medicine for the treat‐
ment of infectious diseases and invasive surgery, and their
use has increased life expectancy. The number of infec‐
tions caused by multidrug‐resistant bacteria is increasing‐
ly globally, however, and nosocomial infections caused by
methicillin‐resistant Staphylococcus aureus (MRSA) in
hospitals have become an especially serious clinical prob‐
lem.1,2 Therefore, antibiotics that belong to new struc‐
tural classes and manifest their biological activity via nov‐
el mechanisms are urgently needed.3,4,5,6
vivo efficacy of 1 in an experimental systemic MRSA
mouse infection model revealed that 1 is 10 times more
active than vancomycin, which is a last line of resort in
treating MRSA infections (median effective dose [ED50] of
1 = 0.38 mg/kg; ED50 of vancomycin = 5.3 mg/kg). Conse‐
quently, 1 is considered a promising lead structure for the
development of new antibiotics against multidrug‐
resistant human pathogenic bacteria, and has entered
phase I clinical trials against systemic MRSA infection. 16
Despite the medical importance of 1, its antibacterial
mechanism has not been investigated in detail, and its
chemical synthesis has only been recorded in a PhD the‐
sis.17 In 1998, the antimicrobial activity of 1 was reported‐
ly decreased by addition of the typical negatively‐charged
lipids of bacterial membranes, phosphatidylglycerol (PG)
and cardiolipin (CL).12 Because the net charge of 1 is posi‐
tive, the antagonistic activities of PG and CL are likely due
to non‐specific electrostatic effects rather than a specific
Among the underutilized antibiotic candidate groups,
cyclic peptides represent a promising class of natural
products with potent antibacterial activity. 7,8,9,10 In 1997,
WAP‐8294A2 (1, Figure 1), which is also known as lotilib‐
cin, was isolated from a culture broth of Lysobacter sp.
strain, and structurally determined to be an antimicrobial
cyclic depsipeptide.11,12,13,14,15 The antibiotic 1 has potent
bactericidal activity against otherwise antibiotic‐resistant
Gram‐positive pathogens, including methicillin‐resistant
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interaction of 1 with the anionic lipids. Accordingly, the
molecular target of 1 remains to be clarified.
N‐Me‐D‐Phe‐5, L‐Leu‐6, L‐Glu‐8, and D‐Trp‐10). Moreo‐
ver, 1 and 3 have a hydrophobic acyl chain at L‐Ser‐1 and
L‐Thr‐1, respectively, and two cationic groups at D‐Orn‐
7/11 and D‐Arg‐2/7, respectively. The analogous patterns
of the functional groups of 1 and 3 raised the possibility
that these two peptides have related mechanisms of ac‐
tion.
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The mode of action of 3 has been shown to be distinct
from those of any other reported antibiotics.18 The mo‐
lecular target of 3 is menaquinone (MK), which is an es‐
sential factor for electron transfer in the bacterial respira‐
tory chain.20,21 While 3 forms a 1:1 complex with MK (KD =
4.5 M), no apparent complexation occurs between 3 and
ubiquinone (UQ), a coenzyme in the mammalian respira‐
tory chain. Formation of the 3‐MK complex is considered
to disrupt the membrane integrity of bacterial cells in the
presence of mammalian cells, resulting in rapid and selec‐
tive bacteriolysis. This intriguing function of 3 motivated
us to conduct solid‐phase total synthesis and comprehen‐
sive structure‐activity relationship (SAR) studies of 3 in
201522 and 2016,23 respectively. As a result, we established
the biologically important functionalities of 3 to be the
cationic functional groups at D‐Arg‐2/7 (highlighted in
cyan), the hydrophobic acyl chain at L‐Thr‐1 (pink), and
the indole ring at D‐Trp‐10 (yellow). Since 1 possesses all
of these characteristic features of 3, we hypothesized that
1 also displays antibacterial activity by targeting MK.
In this manuscript, we first report the full solid‐phase
total synthesis of WAP‐8294A2 (1) as well as its more syn‐
thetically accessible deoxy analogue 2. Rapid and efficient
synthetic preparation of 1 and 2 allowed us to perform a
series of detailed functional analyses. Consequently, we
corroborated that 1 and 2 indeed exert their antimicrobial
activities by MK‐dependent membrane lysis. These find‐
ings decipher the molecular mode of action of 1 for the
first time.
RESULTS AND DISCUSSION
Figure 1. Structures of WAP‐8294A2 (lotilibcin, 1), deoxy
analogue 2, lysocin E (3), menaquinone‐4 (MK‐4, 4), and
ubiquinone‐10 (UQ‐10, 5). Amino acids common to 1 and 3
are highlighted in red. Other amino acids are highlighted in
dark grey. The hydrophobic acyl chains, cationic functional
groups, and indole moieties of 1 and 3 are highlighted in pink,
cyan, and yellow, respectively.
Structurally, 1 has a 40‐membered macrocycle com‐
prising (R)‐3‐hydroxy‐7‐methyloctanoic acid and 12 amino
acid residues with an ester linkage (Figure 1). There are
five proteinogenic (L‐Ser‐1, L‐Ser‐3, Gly‐4, L‐Leu‐6, L‐Glu‐
8) and seven non‐proteinogenic amino acid residues (D‐
OHAsn‐2, N‐Me‐D‐Phe‐5, D‐Orn‐7, D‐Asn‐9, D‐Trp‐10, D‐
Orn‐11, N‐Me‐L‐Val‐12) within the sequence. By close
inspection of the structure of 1, we identified striking
structural similarity between 1 and the antibiotic lysocin E
(3), although the sizes of their macrocycles differ (40‐ vs.
37‐membered).18,19 Six amino acid residues (colored in
red) are shared by the sequences of 1 and 3 (L‐Ser‐3, Gly‐4,
Figure 2. Building blocks for 1 and 2. Boc = tert‐
butoxycarbonyl; Fmoc = 9‐fluorenylmethoxycarbonyl; TBS =
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tert‐butyldimethylsilyl; t‐Bu = tert‐butyl; Tr = triphenylme‐
thyl.
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Scheme 1. Synthesis of 14 and 15a
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aBINAP = 2,2’‐bis(diphenylphosphino)‐1,1’‐binaphthyl; DMAP = 4‐(
bis(trimethylsilyl)amide.
N
,
N
‐dimethylamino)pyridine; LiN(TMS)2 = lithium
Synthetic strategy. We adopted a full solid‐phase
Preparation of building blocks 14 and 15. The syn‐
strategy for the total synthesis of WAP‐8294A2 (1) be‐
cause it omits multiple chromatographic purifications
and ensures rapid preparation of a wider variation of ana‐
logues for future SAR studies compared with the solution‐
phase counterpart. Accordingly, the structure of 1 was
retrosynthetically disassembled into 10 different building
blocks 6‐15 (Figure 2). Fmoc‐based solid‐phase peptide
synthesis (SPPS) was planned to be applied beginning
with 11,24,25 the side‐chain of which was anchored to
Wang‐ChemMatrix resin.26 The ester linkage between N‐
Me‐L‐Val‐12 and (R)‐3‐hydroxy‐7‐methyloctanoic acid was
envisioned to be preformed as 14 to avoid inefficient ester
condensation on the solid matrices. The full solid‐phase
strategy would therefore involve the following sequential
operations:22,23,27,28,29,30,31 (1) stepwise solid‐phase assembly
of the linear dodecapeptide from 11; (2) chemoselective
removal of the allyl group of L‐Glu‐8 under palladium‐
catalyzed neutral conditions; (3) intramolecular ami‐
dation between the C‐carboxyl group of L‐Glu‐8 and the
N‐amino group of D‐Asn‐9, taking advantage of the
pseudo‐dilution phenomenon that favors intramolecular
reactions of the resin‐bound molecules;32,33 and (4) tri‐
fluoroacetic acid (TFA) treatment for simultaneous cleav‐
age from the Wang‐ChemMatrix resin and global depro‐
tection of the acid‐labile side‐chain protective groups
(TBS, Boc, and Tr) to release 1. While N‐Fmoc‐protected
amino acids 6‐13 were commercially available, ester 14
and TBS‐protected D‐OHAsn‐2 15 required synthetic
preparation.34,35,36 Another target molecule, deoxy WAP‐
8294A2 (2), was designed as a more synthetically accessi‐
ble analogue of 1, because 12 would be used instead of 15
as a common building block for both D‐Asn‐2 and D‐Asn‐
9. From a biological perspective, it would be more useful
if the activity assays of synthesized 1 and 2 shed light on
the importance of the ‐hydroxy group of D‐OHAsn‐2.37,38
thesis of ester 14 started from 5‐methylhexanoic acid 16
(Scheme 1). After 16 was subjected to thionyl chloride,
the resultant acid chloride was treated with ester enolate
generated from AcOEt and LiN(TMS)2, resulting in the
formation of ‐keto ester 17. Asymmetric Noyori hydro‐
genation of 17 using RuCl2[(R)‐BINAP] as the catalyst gave
rise to 18.39 Hydrolysis of ethyl ester 18 by aqueous NaOH
and subsequent protection of the carboxylic acid as the
benzyl ester generated benzyl (R)‐3‐hydroxy‐7‐
methyloctanoate 19. The enantiomeric excess of 19 was
determined to be 97% by chiral HPLC analysis (Figure S1).
Secondary alcohol 19 and Fmoc‐N‐Me‐L‐Val‐OH 20 were
then condensed by the action of diisopropylcarbodiimide
and DMAP, leading to 21. Finally, hydrogenolysis in the
presence of Pd/C under H2 atmosphere transformed the
benzyl ester of 21 to the carboxylic acid of 14.
Compound 15 was prepared from the known D‐
aspartate derivative 22.40 Ester‐amide exchange reaction
of 22 using NH3 was followed by treatment with BnBr and
NaHCO3 to provide 23.41,42 Then, three‐step protective
group manipulations from 23 furnished N‐Fmoc‐, C‐
CO2Bn‐, and CN‐Tr‐protected ‐hydroxy D‐asparagine 24.
The secondary hydroxy group of 24 was protected as its
TBS ether using TBSOTf and 2,6‐lutidine to generate 25.
The benzyl group of 25 was in turn removed by applying
H2 and Pd/C, giving rise to the requisite 15 with the ap‐
propriate protective groups for the next SPPS.
Solid‐phase total synthesis of WAP‐8294A2 (1) and
deoxy analogue 2. The SPPS of 1 and 2 started from allyl
glutamate‐loaded resin 11 (Scheme 2). First, N‐Fmoc of
11 was removed by treatment with 20% piperidine/NMP
to give a free amine. The peptide chains of 1 and 2 were
elongated at 40 °C under microwave‐assisted conditions
to facilitate the condensation.43,44 Cycles of 20% piperi‐
dine/NMP‐promoted N‐deprotection (10 min, rt) and
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Scheme 2. Solid‐phase total syntheses of 1 and 2a
1. 20% piperidine/NMP, 10 min, rt
2. 10, HATU, HOAt, i-Pr2NEt, NMP, 40 °C, 20 min
3. 20% piperidine/NMP, 10 min, rt
R1
O
O
O
6
OAllyl
7
8
9
4. 9, HATU, HOAt, i-Pr2NEt, NMP, 40 °C, 20 min
5. 20% piperidine/NMP, 10 min, rt
6. 8, HATU, HOAt, i-Pr2NEt, NMP, 40 °C, 20 min
7. 20% piperidine/NMP, 10 min, rt
HN
HN
NH
O
O
1. 10, HATU, HOAt, i-Pr2NEt, NMP
40 °C, 90 min (2 cycles)
2. 20% piperidine/NMP, rt, 1 min
O
O
O
NHBoc
8. 7, HATU, HOAt, i-Pr2NEt, NMP, 40 °C, 20 min
10
11
12
13
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NH
O
11
9. 20% piperidine/NMP, 10 min, rt
10. 6, HATU, HOAt, i-Pr2NEt, NMP, 40 °C, 20 min
11. 20% piperidine/NMP, 10 min, rt
12. 15 or 12, HATU, HOAt, i-Pr2NEt, NMP, 40 °C, 20 min
13. 20% piperidine/NMP, 10 min, rt
14. 6, HATU, HOAt, i-Pr2NEt, NMP, 40 °C, 20 min
15. 20% piperidine/NMP, 10 min, rt
HN
HN
N
O
O
Ot-Bu
O
HN
O
X
NH
O
Ot-Bu
NHTr
26a: X = OTBS, R1 = Wang-ChemMatrix resin
26b: X = H, R1 = Wang-ChemMatrix resin
16. 14, HATU, HOAt, i-Pr2NEt, NMP, 40 °C, 20 min
17. 20% piperidine/NMP, 10 min, rt
TrHN
H2N
O
R1
O
O
O
H2N
O
N
1. 13, HATU, HOAt, i-Pr2NEt, NMP
40 °C, 20 min
2. 20% piperidine/NMP, rt, 10 min
3. 12, HATU, HOAt, i-Pr2NEt, NMP
40 °C, 20 min
O
OAllyl
NBoc
O
HN
R1
O
O
O
NH
NH
O
O
OR2
O
O
NHBoc
NHBoc
O
N
O
4. 20% piperidine/NMP, rt, 10 min
NH
O
O
NH
O
O
O
NHBoc
NHBoc
O
HN
HN
NH
O
N
O
O
HN
Ot-Bu
HN
HN
X
O
HN
O
N
O
NH
O
X
O
O
HN
Ot-Bu
HN
O
Ot-Bu
NHTr
NH
O
27a: X = OTBS, R1 = Wang-ChemMatrix resin
27b: X = H, R1 = Wang-ChemMatrix resin
Ot-Bu
NHTr
28a: X = OTBS, R1 = Wang-ChemMatrix resin, R2 = Allyl
28b: X = H, R1 = Wang-ChemMatrix resin, R2 = Allyl
Pd(PPh3)4, morpholine
CH2Cl2, rt
30a: X = OTBS
R
R
1 = Wang-ChemMatrix resin
3 = Boc, R4 = t-Bu, R5 = Tr
29a: X = OTBS, R1 = Wang-ChemMatrix resin, R2 = H
29b: X = H, R1 = Wang-ChemMatrix resin, R2 = H
1. TBAF, AcOH, THF, rt
2. 95% TFA/H2O, rt
95% TFA/H2O, rt
1: X = OH
R1, 3-5 = H
8.4% overall yield
30b: X = H
R1 = Wang-ChemMatrix resin
3 = Boc, R4 = t-Bu, R5 = Tr
R5HN
O
HN
NR3
R1
O
O
O
NH
O
NH
O
O
R
N
2: X = R1, 3-5 = H
6.6% overall yield
NH
O
O
PyBOP, 2,4,6-collidine
10% NMP/CH2Cl2, rt
O
O
O
NHR3
N
N
N
NH
O
NHR3
N+
HN
HN
-
–
N
PF6
PF6
O
N
O
N+
N
N
N
N
P+
N
O
O
HN
OR4
N
N
HN
O
X
N
N
N
NH
O
O–
HATU
OH
OR4
NHR5
HOAt
PyBOP
aHATU
=
O‐(7‐aza‐1H‐benzotriazol‐1‐yl)‐N,N,N',N'‐tetramethyluronium hexafluorophosphate; HOAt
azabenzotriazole; NMP = N‐methyl‐2‐pyrrolidone; PyBOP = (benzotriazol‐1‐yloxy)tripyrrolidinophosphonium hexafluorophos‐
phate; TBAF = tetrabutylammonium fluoride; TFA = trifluoroacetic acid.
=
1‐hydroxy‐7‐
HATU/HOAt45‐mediated amide coupling (20 min, 40 °C)
were applied to 11 using 10, 9, 8, 7, 6, 15 for 1 or 12 for 2, 6,
and 14. These repeated reactions resulted in the for‐
mation of the resin‐bound nonapeptide 26a/b including
the ester linkage after removal of the Fmoc group at the
N‐terminus under basic conditions. The next reaction of
10 with the sterically demanding N‐methyl amine of
26a/b necessitated a double coupling sequence with a
longer reaction time (90 min). Then, the piperidine
treatment for the subsequent N‐deprotection was short‐
ened to 1 min to prevent diketopiperazine formation via
attack of the N‐terminal amine of D‐Orn‐11 of 27a/b to the
proximal ester carbonyl group. The thus obtained
decapeptide 27a/b was again subjected to two cycles of
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Table 1. Antimicrobial activities of 1‐3
MIC (
the condensation and N‐deprotection reactions using 13
and 12 to furnish the linear dodecapeptide 28a/b.

g/mL)a
Next, the allyl group of the C‐terminus of 28a/b was
chemoselectively removed by catalysis of Pd(PPh3)4 in the
presence of excess morpholine,46 generating the precursor
29a/b for macrolactamization. Amide formation between
the N‐ and C‐termini of 29a/b was attained by the action
of PyBOP/2,4,6‐collidine47 under pseudo high‐dilution
conditions, realizing the on‐resin cyclization of the 40‐
membered ring of 30a/b. To complete the solid‐phase
total synthesis of 1 and 2, conditions for the last global
deprotection were individually optimized for 30a and 30b.
Treatment of the fully protected deoxy analogue 30b with
95% aqueous TFA at rt simultaneously realized cleavage
from the resin and removal of the t‐Bu, Boc, and Tr
groups, releasing 2 into solution. After purification by
reversed‐phase HPLC, deoxy analogue 2 was obtained in
6.6% yield in 26 steps from 11. In contrast to this success‐
ful result, application of the same TFA conditions to 30a
did not effect full detachment of the extra TBS group at D‐
OHAsn‐2 to yield the parent natural product WAP‐
8294A2 (1). Higher temperature only resulted in low
yielding generation of the desired 1. Thus, we adopted a
two‐step approach to improve the deprotection efficacy.
First, the reagent combination of TBAF and AcOH was
used to remove the TBS group from 30a, and then the
resultant compound was subjected to 95% aqueous TFA
at rt to deliver 1, which was purified by reversed‐phase
HPLC. Consequently, the total yield of WAP‐8294A2 (1)
from 11 in 27 steps was 8.4%. The 91% and 90% average
yields per step for assembly of 1 and 2, respectively,
demonstrated the high efficiency of the present full solid‐
phase synthetic strategy. All the spectral and physical
6
7
8
9
strains
1
2
3
(ref 18)
Gram‐positive bacteria
Methicillin‐susceptible
S. aureus MSSA1
(clinical isolate)
10
11
12
13
14
15
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26
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2
2
2
2
4
4
Methicillin‐resistant
S. aureus MRSA4
(clinical isolate)
Staphylococcus simulans
JCM2424
2
2
2
2
4
4
Staphylococcus
pseudintermedius JCM17571
Bacillus subtilis JCM2499
Bacillus cereus JCM20037
2
2
2
2
4
2
Streptococcus pyogenes
SS1‐9
64
128
>128
>128
Streptococcus pneumoniae
(clinical isolate)
>128
>128
Gram‐negative bacteria
Serratia marcescens
(clinical isolate)
>128
>128
>128
>128
>128
>128
>128
>128
>128
Escherichia coli W3110
Pseudomonas aeruginosa
PAO1
True fungi
1
Candida albicans
ATCC10231
data of 1, including H NMR, 13C NMR, []D, and HRMS
>128
>128
64
>128
>128
64
>128
>128
>128
data, matched those of naturally occurring 1 (Tables S1
1
and S2).11,12 In addition, the H and 13C NMR chemical
Candida tropicalis pK233
shifts of 2 were in excellent agreement with those of 1,
and thus the deletion of the one hydroxy group from 1
was inconsequential to the three‐dimensional core struc‐
ture.
Cryptococcus neoformans
H99
aAntimicrobial activities against various bacteria and true
fungi were determined by the microdilution method.
Antimicrobial activity. We next evaluated the anti‐
bacterial activities of the synthetically prepared 1 and 2
(Table 1). The MIC values were determined using the
standard microdilution procedure against eight strains of
Gram‐positive bacteria, three strains of Gram‐negative
bacteria, and three strains of true fungi. The comparable
MIC values of natural 1 (0.78 g/mL) and synthetic 1 (2
g/mL) on Methicillin‐resistant S. aureus (MRSA) further
confirmed their chemical equivalence. Compound 1 ex‐
hibited potent antimicrobial activity (2 g/mL) against
the six strains of Gram‐positive bacteria (Methicillin‐
susceptible S. aureus, MRSA, Staphylococcus simulans,
Staphylococcus pseudintermedius, Bacillus subtilis, and
Bacillus cereus), whereas 1 was ineffective against the two
strains of Gram‐positive bacteria (Streptococcus pyogenes
and Streptococcus pneumoniae), and all strains of Gram‐
negative bacteria (Serratia marcescens, Escherichia coli,
and Pseudomonas aeruginosa) and true fungi
Table 2.
Antimicrobial activities against MK‐
deficient deletion mutants of S. aureus
MIC (g/mL)a
strains
1
2
3
(ref 18)
Wild‐type (RN4220)
menA
4
128
128
4
4
64
64
>128
>128
menB
aAntimicrobial activities against various bacteria were de‐
termined by the microdilution method.
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cation of the solutions of 1 (blue) and 2 (green) in a con‐
centration range similar to their MIC values (2‐8 g/mL)
induced a rapid increase in the fluorescence of DiSC3(5),
indicating loss of the membrane potential. These results
strongly suggested that the antimicrobial activities of 1
and 2 are due to their effects to alter the functional integ‐
rity of the bacterial membrane.
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Menaquinone‐dependent membrane lysis. MK‐
dependent membrane‐lysis of 1 and 2 was investigated
using four types of liposomes, and compared with that of
3. Large unilamellar vesicles (LUVs) comprising egg yolk
phosphatidylcholine (PC)/egg yolk phosphatidylglycerol
(PG) (50:50 ratio) and PC/PG/cardiolipin (CL) (50:40:10
ratio) were prepared to mimic the negatively‐charged
surface of bacterial membranes.51 The PC/PG LUVs were
doped with 1.25 mol% of MK‐4 (4),52,53 or with 1.25 mol%
of UQ‐10 (5) to access the selectivity of 1 and 2 toward MK
over UQ. The total lipid concentration of each LUV solu‐
tion was adjusted to 8 M, and carboxyfluorescein (CF)
was encapsulated as a fluorescent indicator in all four
LUVs.54 While fluorescence of the CF molecules within
the LUVs is self‐quenched due to the high concentration,
an increase in fluorescence intensity can be observed
when membrane disruption by peptides causes the CF to
leak from LUVs, resulting in dilution of the CF molecules.
Therefore, fluorescence was measured as an indicator of
LUV membrane disruption. The fluorescence change (%)
in the presence of each peptide was standardized accord‐
ing to the maximum intensity (100%) induced by adding
Triton X‐100. The membrane‐disrupting activities of 1‐3
toward the four types of LUVs were quantified as half‐
maximal response (EC50) values by measuring fluores‐
cence changes (%) of varied concentrations (0.0015 nM to
2970 nM) of 1‐3 after reaching their plateaus (30 min).
Figure 3. Change in the membrane potential of S. aureus
after the addition of 1 and 2. The change in membrane po‐
tential was monitored by the fluorescence of DiSC3(5) in the
presence of 2, 4, or 8 g/mL of 1 and 2. Compound 3 (16
g/mL) and chloramphenicol (50 g/mL) were used as a
positive control and a negative control, respectively. a.u.:
arbitrary unit; DiSC3(5): 3,3'‐dipropylthiadicarbocyanine io‐
dide.
(Candida albicans, Candida tropicalis, and Cryptococcus
neoformans). Intriguingly, deoxy analogue 2 had the
same MIC values as 1 for all the tested strains except for S.
pyogenes. These data uncovered the dispensability of the
additional hydroxy group at D‐OHAsn‐2 for the antimi‐
crobial activity.
The observed potency and spectrum of the activity of 1
and 2 were in accordance with those of lysocin E (3) (Ta‐
ble 1). Importantly, all of the three compounds displayed
significantly low activities toward Gram‐positive S. py‐
ogenes and S. pneumoniae that lack MK within the mem‐
brane.48 To further verify that the presence of MK affects
the antimicrobial activities of 1 and 2, we next used two
MK‐deficient mutants (menA and menB) of S. aureus
RN4220 (Table 2).19,49 MenB and MenA are involved in
the formation of naphthoquinone and the attachment of
an isoprenyl chain to naphthoquinone, respectively, in
the MK biosynthesis pathway, and their deletion results
in a complete loss of MK in S. aureus. Consistent with the
substantial activity loss of the MK‐targeting antibiotic 3
toward the deletion mutants, 1 and 2 had negligible ac‐
tivities against both the menA and menB mutants (128
and >128 g/mL for 1 and 2, respectively) compared to the
wild‐type (4 g/mL). These findings indicated that the
antimicrobial actions of 1 and 2 correlate with MK pro‐
duction.
Although all four LUVs could be ruptured dose‐
dependently by 1 and 2 (Figures 4a and b), the MK‐4 (4)‐
containing LUVs were most responsive toward the addi‐
tion of 1 and 2 (blue lines vs. red, black, and green lines).
It therefore demonstrated a key role of MK in the mem‐
brane rupturing activities of 1 and 2. The same MK‐
sensitive behavior was observed upon subjecting 3 to the
four LUVs (Figure 4c). The EC50 values of 1, 2, and 3 for
the 4‐containing LUVs were calculated to be 14.2, 22.1,
and 15.9 nM (Table 3), respectively. In contrast, 27 to 59
times higher concentrations were necessary to cause 50%
leakage of the LUVs containing PC/PG (EC50 = 503, 640,
and 435 nM for 1, 2, and 3, respectively) and PC/PG/CL
(EC50 = 833, 781, and 501 nM for 1, 2, and 3, respectively).
Furthermore, the EC50 values of 1, 2, and 3 for UQ‐10 (5)‐
containing‐LUVs were 9.3‐, 12‐, and 3.6‐fold larger than
those for 4‐containing‐LUVs (EC50 = 132, 261, and 56.5 nM
for 1, 2, and 3, respectively).
Evaluation of membrane potential. The potent
bactericidal function of lysocin E (3) originates from its
membrane‐disrupting activity.18 For example, 3 caused a
loss of membrane potential in S. aureus in the presence of
3,3'‐dipropylthiadicarbocyanine iodide [DiSC3(5)] as a
fluorescent probe (Figure 3, red),50 while the antibiotic
chloramphenicol had no effect (gray). In this assay, both
1 and 2 were found to have the same function as 3. Appli‐
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Figure 4. Comparison of dose‐dependent membrane disrupting activities of 1 (a), 2 (b), and 3 (c) against the four LUVs [PC/PG
= 50:50, PC/PG/CL = 50:40:10, PC/PG = 50:50 containing 1.25 mol% MK‐4 (4) or UQ‐10 (5)]. Egg yolk PC and egg yolk PG were
used as PC and PG, respectively. The total lipid concentration of each LUV solution was adjusted to 8 M. Mean values ± SD of
three independent experiments are shown.
Table 3.
EC50 values (nM) for the membrane‐
CONCLUSION
disrupting activities of 1, 2, and 3 against the four
types of LUVs
Here we report the first successful solid‐phase total syn‐
thesis of the antibiotic peptide WAP‐8294A2 (1) and its
deoxy analogue (2), and their MK‐dependent mode of
action. After preparing preformed ester 14 and D‐OHAsn‐
2 15, stepwise‐elongation of the peptide chain from the
side‐chain anchored 11 gave rise to the dodecapeptide 28a.
Chemoselective deprotection of the C‐terminus of 28a,
on‐resin macrolactamization, and global deprotection
delivered the complex 40‐membered macrolactam 1. Ap‐
plication of the same strategy except for the use of com‐
mercially available 12 in place of 15 realized the chemical
construction of the more synthetically accessible 2. The
rapid and efficient access to 1 and 2 enabled detailed in‐
vestigations of their biological functions. The equipotent
antimicrobial activities of the parent natural product 1
and its deoxy analogue 2 indicate the biological insignifi‐
cance of the hydroxy group on D‐OHAsn‐2. While 1 and 2
displayed the low MIC values toward the six strains of
Gram‐positive bacteria (2 g/mL), the MK‐deficient spe‐
cies (S. pyogenes and S. pneumoniae) and mutants
(menA and menB) were highly resistant to 1 and 2, sug‐
gesting a strong correlation between their antimicrobial
activities and MK production. The membrane‐disrupting
effects of 1 and 2 were then validated by the loss of the
membrane potential of S. aureus, and by the leakage of
the encapsulated fluorescent molecules from the LUVs.
Further, we determined the EC50 values of 1 and 2 for lysis
of four types of LUVs that differed in their membrane
components. As a result, the activities of 1 and 2 was sub‐
stantially enhanced by MK compared with the anionic
lipids (PG and CL) or the structurally related quinone UQ.
These data along with the similar assay results of lysocin
E (3) supported that 1 and 2 selectively recognize MK pre‐
sent in the cytoplasmic membrane of Gram‐positive bac‐
teria, and kill the cells by disrupting their membranes.
Thus, we present 1 and 2 as the first series of compounds
that share the unique MK‐targeting mode of action of 3.
As 1 and 2 exhibited higher selectivity toward MK over
UQ than 3 in the LUV experiments, these compounds are
potentially superior to 3 as the lead structures for drugs
EC50 (nM)
com‐
poun
ds
PC/PG
PC/PG
/MKc
PC/PG
/UQd
PC/PGa
/CLb
1
2
3
503 ± 165
640 ± 39
435 ± 83
b
833 ± 68
14.2 ± 3.1
132 ± 14
261 ± 47
781 ± 54 22.1 ± 12.9
501 ± 57
15.9 ± 7.3 56.5 ± 11.9
aPC/PG = 50:50, PC/PG/CL = 50:40:10, PC/PG = 50:50 con‐
c
d
taining 1.25 mol% MK‐4 (4), PC/PG = 50:50 containing 1.25
mol% UQ‐10 (5). Egg yolk PC and egg yolk PG were used as
PC and PG, respectively. The total lipid concentration of
each LUV solution was adjusted to 8 M. EC50 values are
displayed as mean ± SD of three independent experiments.
These liposome experiments validated the MK‐
dependency of the potent membrane lytic activities of 1
and 2. The electrostatic interactions of positively‐charged
1 and 2 with the negatively charged PG or CL did not lead
to membrane disruption at micromolar concentrations of
1 and 2, clarifying that PG and CL were not their specific
targets. Addition of only 1.25 mol% of 4 to PC/PG en‐
hanced the membrane lysis activities of 1 and 2 29‐ to 35‐
fold, while addition of 1.25 mol% of the structurally relat‐
ed quinone 5 was much less effective for increasing the
activities (2.5‐ to 3.8‐fold). These membrane‐disrupting
and antimicrobial activities together strongly supported
the MK‐dependent mode of action by 1 and 2. The selec‐
tive molecular recognition of MK by 1 and 2 presumably
leads to membrane damage and eventual bacterial death.
Hence, we present WAP‐8294A2 (1) and its deoxy ana‐
logue 2 as the first series of antibiotics that share the
unique mechanism of lysocin E (3). It is worth noting
that the MK/UQ‐selectivity of 1 and 2 is higher than that
of 3 (9.2 for 1, 12 for 2, and 3.6 for 3), suggesting that 1 and
2 are superior to 3 as lead structures for developing opti‐
mal candidates of novel antibiotics.
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with maximized antimicrobial activities and fewer unde‐
was stirred at rt under H2 (10 bar) for 3 d. After venting
sirable side effects. Consequently, the present findings
provide a new chemical basis for the development of MK‐
targeting next‐generation antibiotics for the treatment of
MRSA and various infectious diseases.
H2 at rt, the solution was concentrated. The residue was
purified by flash column chromatography on silica gel (20
g, pentane/Et2O 25/1 to 5/1) to give 18 (451 mg, 35% over 3
steps): yellow oil; []D27 = −19.4 (c = 1.37, CHCl3); IR (film)
 3446, 2954, 2931, 2870, 1733, 1458, 1372, 1174, 1036 cm‐1; 1H
NMR (500 MHz, CDCl3)  0.87 (6H, d, J = 6.3 Hz), 1.15‐1.58
(10H, m), 2.40 (1H, dd, J = 16.6, 9.2 Hz), 2.50 (1H, dd, J =
16.6, 2.9 Hz), 2.94 (1H, d, J = 4.1 Hz), 3.90 (1H, m), 4.17 (2H,
q, J = 7.5 Hz); 13C NMR (125 M Hz, CDCl3) 14.2, 22.6 (2C),
23.2, 27.9, 36.7, 38.8, 41.3, 60.7, 68.0, 173.2; HRMS (ESI‐
TOF) calcd for C11H22O3Na [M+Na]+ 225.1461, found
225.1469. The configuration of ‐hydroxy group of 18 was
determined to be R by the modified Mosher method.55
EXPERIMENTAL SECTION
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General Methods. All reactions sensitive to air and/or
moisture were carried out under argon atmosphere in dry
solvents, unless otherwise noted. THF, CH2Cl2, DMF, and
Et2O were purified by a Glass Contour solvent dispensing
system. All other reagents were used as supplied unless
otherwise stated. Analytical thin‐layer chromatography
(TLC) was performed using 0.25 mm plates. Flash col‐
umn chromatography was performed using 40–50 m
silica gel. Solid‐phase peptide synthesis (SPPS) was per‐
formed on a microwave‐assisted peptide synthesizer us‐
ing a sealed reaction vessel, a reaction temperature of
which was monitored by an internal temperature probe.
Infrared (IR) spectra were recorded as a thin film on a KBr,
NaCl, or CaF2. 1H NMR spectra were recorded at 400 or
500 MHz. 13C NMR spectra were recorded at 100 or 125
MHz. Chemical shifts are denoted in  (ppm) relative to
Benzyl ester 19. To a solution of 18 (327 mg, 1.62 mmol)
in THF (9.34 mL) was added a solution of NaOH (747 mg,
18.7 mmol) in H2O (9.34 mL) at 0 °C. After being stirred
at rt for 3.5 h, the solution was concentrated. The residue
was dissolved in H2O (30 mL). The aqueous layer was
washed with Et2O (20 mL). The aqueous layer was acidi‐
fied (pH 1) with 4 M aqueous HCl (6 mL). The resultant
solution was extracted with Et2O (20 mL ×3). The com‐
bined organic layers were washed with brine (40 mL),
dried over anhydrous Na2SO4, filtered, and concentrated
to give the crude carboxylic acid (268 mg), which was
used in the next reaction without further purification.
1
residual solvent peaks as internal standard (CDCl3, H 
1
7.26, 13C  77.0; CD3OD, H  3.31, 13C  49.0; DMSO‐d6,
13
1H2.50, C  39.5). HRMS spectra were recorded on an
electrospray ionization time‐of‐flight (ESI‐TOF) mass
spectrometer.
To a solution of the above crude carboxylic acid (253
mg) in DMF (3.03 mL) were added K2CO3 (220 mg, 1.59
mmol) and benzyl bromide (189 L, 1.59 mmol) at 0 °C.
The reaction mixture was stirred at rt for 10 h, and then
saturated aqueous NH4Cl (5 mL) was added. The result‐
ant mixture was extracted with Et2O (10 mL ×3). The
combined organic layers were washed with brine (15 mL),
dried over anhydrous Na2SO4, filtered, and concentrated.
The residue was purified by flash column chromatog‐
raphy on silica gel (20 g, hexane/AcOEt 10/1 to 5/1) to give

‐keto ester 17. To 5‐methylhexanoic acid (16, 1.04 g,
7.98 mmol) was added thionyl chloride (754 L, 10.4
mmol) at rt. The solution was heated to reflux, and
stirred for 3 h. The reaction mixture was concentrated to
give the crude acid chloride (810 mg), which was used in
the next reaction without further purification.
To a solution of LiN(TMS)2 (12.0 mmol) in THF (12.0
mL) was added AcOEt (642 L, 6.54 mmol) over 5 min at
−78 °C. The resultant solution was stirred at −78 °C for 30
min. A solution of the above crude acid chloride (810 mg)
in THF (1.09 mL) was added to the solution over 7 min at
−78 °C. The mixuture was stirred at −78 °C for 1 h, and
then saturated aqueous NH4Cl (15 mL) was added. The
resultant mixture was extracted with Et2O (30 mL ×1, 10
mL ×2). The combined organic layers were washed with
brine (30 mL), dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was roughly purified by flash
column chromatography on silica gel (20 g, pentane/Et2O
40/1 to 10/1) to give the crude 17 (807 mg), which was used
in the next reaction without further purification.
23
19 (346 mg, 90%): yellow oil; []D = −14.4 (c = 1.15,
CHCl3); IR (film)  3446, 2953, 2931, 2869, 1733, 1457, 1384,
1
1261, 1166 cm‐1; H NMR (500 MHz, CDCl3)  0.87 (6H, d, J
= 6.3 Hz), 1.12‐1.66 (7H, m), 2.47 (1H, dd, J = 16.6, 9.2 Hz),
2.56 (1H, dd, J = 16.6, 3.5 Hz), 4.03 (1H, m) 5.16 (2H, s),
13
7.31‐7.40 (5H, m); C NMR (125 M Hz, CDCl3) 22.5 (2C),
23.2, 27.9, 36.7, 38.8, 41.3, 66.5, 68.0, 128.3 (2C), 128.4, 128.6
(2C), 135.6, 172.9; HRMS (ESI‐TOF) calcd for C16H24O3Na
[M+Na]+ 287.1618, found 287.1619. Enantiomeric excess of
19 was determined to be 97% ee of the R‐enantiomer (tR =
20.3 min, Figure S1)39 by normal phase chiral HPLC analy‐
sis (column: Chiralcel OD‐H 4.6 ×250 mm, eluent A: hex‐
ane; eluent B: i‐PrOH; 95% A/5% B, flow rate: 0.5 mL/min,
detection: UV 210 nm).

‐hydroxy ester 18. A solution of benzenerutheni‐
um(II) chloride dimer (49.9 mg, 99.9 mol) and (R)‐
BINAP (124 mg, 0.200 mmol) in DMF (499 L) was stirred
at 100 °C for 10 min. The reaction mixture was concen‐
trated to give the crude catalyst RuCl2[(R)‐BINAP](DMF)n,
which was used in the next reaction without further puri‐
fication.
Ester 21. To a solution of 19 (346 mg, 1.31 mmol) in
CH2Cl2
(6.55
mL)
were
added
N,N’‐
diisopropylcarbodiimide (DIC, 307 L, 1.96 mmol), N,N‐
dimethyl‐4‐aminopyridine (DMAP, 48.0 mg, 0.552 mmol),
and Fmoc‐L‐N‐MeVal‐OH (20, 509 mg, 1.44 mmol) at rt.
The mixture was stirred at rt for 3 h. Then, 20 (231 mg,
0.655 mmol) and DIC (205 L, 1.31 mmol) was added to
the mixture. The solution was stirred at rt for 3 h, and
A solution of the above crude 17 (532 mg) and the
above crude catalyst RuCl2[(R)‐BINAP](DMF)n in MeOH
(1.00 mL) was transferred to an autoclave. The solution
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then saturated aqueous NH4Cl (6 mL) was added. The
127.62 (A or B), 127.64 (2C of A or B), 141.26 (A or B), 141.29
(A or B), 141.34 (A or B), 141.4 (A or B), 143.8 (A or B), 143.9
(A or B), 144.0 (A or B), 144.3 (A or B), 156.3 (A), 156.9 (B),
169.4 (A), 170.2 (B), 174.6 (A), 175.4 (B); HRMS (ESI‐TOF)
calcd for C30H39NO6Na [M+Na]+ 532.2670, found 532.2683.
resultant mixture was extracted with Et2O (25 mL). The
combined organic layers were washed with brine (20 mL),
dried over anhydrous Na2SO4, filtered, and concentrated.
The residue was purified by flash column chromatog‐
raphy on silica gel (30 g, hexane/AcOEt 20/1 to 10/1) to
Benzyl ester 23. A solution of monomethyl ester 22
(4.85 g, 18.4 mmol) in MeOH (136 mL) was bubbled with
NH3 at 0 °C for 4 h under sonication in a pressure tube.
The tube was sealed and warmed to rt. After being stirred
at rt for 2 d, the solution was concentrated to give the
crude material, which was used in the next reaction with‐
out further purification.
20
give 21 (716 mg, 91%): colorless oil; []D = −46.5 (c =
10
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0.714, CHCl3); IR (film)  2957, 2870, 1739, 1703, 1452, 1307,
1
1197, 1169, 1149, 988 cm‐1; H NMR (500 MHz, CDCl3) Sig‐
nals derived from rotamers A and B (1:1) were observed. 
0.72 (1.5H, d, J = 6.9 Hz), 0.77‐1.01 (10.5H, m), 1.07‐1.35
(4.0H, m), 1.40‐1.65 (3.0H, m), 2.04‐2.21 (1.0H, m), 2.48‐
2.71 (2.0H, m), 2.82 (1.5H, s), 2.86 (1.5H, s), 4.06 (0.5H, d, J
= 10.9 Hz), 4.21‐4.27 (1.0H, m), 4.30‐4.55 (2.5H, m), 5.01‐
5.12 (2.0H, m), 7.55‐7.64 (2.0H, m), 7.73‐7.79 (2.0H, m); 13C
NMR (125 MHz, CDCl3) Signals derived from rotamers A
and B were observed.  18.5 (A), 18.8 (B), 19.5 (A), 19.6 (B),
22.5 (2C of A and B), 22.9 (A and B), 27.0 (A), 27.4 (B),
27.8 (A and B), 34.1 (A and B), 38.5 (A and B), 39.1 (A and
B), 47.3 (A and B), 64.0 (B), 64.3 (A), 66.4 (A or B), 66.6
(A or B), 67.5 (A), 67.6 (B), 67.6 (B), 71.1 (A and B), 119.9
(2C of A and B), 124.9 (2C of A or B), 125.0 (2C of A or B),
126.96 (A or B), 127.02 (2C of A or B), 127.1 (A or B), 127.7
(2C of A and B), 128.2 (2C of A and B), 128.3 (A and B),
128.5 (2C of A and B), 135.6 (A and B), 141.3 (2C of A or B),
141.4 (2C of A or B), 143.85 (A or B), 143.91 (A or B), 144.0
(A or B), 144.3 (A or B), 156.1 (A), 156.8 (B), 169.7 (A or B),
169.9 (A or B), 170.0 (A or B), 170.3 (A or B); HRMS (ESI‐
TOF) calcd for C37H45NO6Na [M+Na]+ 622.3139, found
622.3147.
To a solution of the above crude material in DMF (94.0
mL) were added NaHCO3 (6.79 g, 80.8 mmol) and BnBr
(6.92 mL, 58.3 mmol) at 0 °C. The mixture was stirred at
rt for 21 h. Then, NaHCO3 (1.58 g, 18.8 mmol) and BnBr
(2.68 mL, 22.6 mmol) were added to the solution at 0 °C.
The solution was stirred at rt for 19 h, and then H2O (250
mL) was added at 0 °C. The resultant mixture was ex‐
tracted with AcOEt (150 mL ×3). The combined organic
layers were washed with brine (150 mL), dried over anhy‐
drous Na2SO4, filtered, and concentrated. The residue
was purified by flash column chromatography on silica
gel (200 g, CH2Cl2/MeOH 1/0 to 25/1) to give 23 (3.83 g,
24
60% over 2 steps): white foam; []D = +24.6 (c = 0.765,
CHCl3); IR (film)  3354, 2979, 1694, 1682, 1504, 1368, 1254,
1
1162, 1106, 1060 cm‐1; H NMR (500 MHz, CD3OD) Signals
derived from rotamers A and B (4:1) were observed.  1.34
(1.8H, s), 1.41 (7.2H, s), 4.53 (0.2H, s), 4.59 (0.8H, d, J = 2.3
Hz), 4.63 (0.2H, s), 4.70 (0.8H, d, J = 2.3 Hz), 5.17‐5.26
(2.0H, m), 7.29‐7.42 (5.0H, m); 13C NMR (125 MHz,
CD3OD)  28.6 (3C), 58.2, 68.3, 72.8, 80.9, 129.1 (2C), 129.3,
129.5 (2C), 137.1, 158.1, 172.0, 176.6; HRMS (ESI‐TOF) calcd
Ester 14. To a solution of 21 (1.02 g, 1.70 mmol) in Ac‐
OEt (34.0 mL) was added 5 wt% Pd/C (203 mg, 95.3 mol)
at rt. The flask equipped with a balloon was evacuated
and recharged with H2 (×3). After being stirred at rt for 3
h under H2 atmosphere, the reaction mixture was filtered
through a pad of Celite with AcOEt (30 mL). The filtrate
was concentrated. The residue was purified by flash col‐
umn chromatography on silica gel (60 g, hexane/AcOEt =
4/1 to 1/1) to give 14 (756 mg, 85%): white solid; m.p. 118‐
1
for C16H22N2O6Na [M+Na]+ 361.1370, found 361.1373; H
NMR spectrum was identical with that of ent‐23.41,42
N‐Trityl amide 24. To a solid of 23 (3.83 g, 11.3 mmol)
was added 4 M HCl in 1,4‐dioxane (113 mL) at 0 °C. The
resultant mixture was stirred at rt for 3.5 h, and concen‐
trated to give the crude material, which was used in the
next reaction without further purification.
23
120 °C; []D = −66.1 (c = 1.09, CHCl3); IR (film) 2959,
1
2929, 2870, 1739, 1708, 1451, 1260, 1199, 1033 cm‐1; H NMR
To a solution of the above crude material in 1,4‐
dioxane/H2O (1/1, 113 mL) were added NaHCO3 (5.71 g,
67.9 mmol) and FmocCl (2.93 g, 11.3 mmol) at 0 °C. After
being stirred at rt for 2 h, the resultant suspension was
diluted with H2O (100 mL) and AcOEt (800 mL). The
organic layer was washed with 1 M aqueous HCl (400 mL
×2), saturated aqueous NaHCO3 (400 mL) and brine (400
mL), dried over anhydrous Na2SO4, filtered, and concen‐
trated. The residue was purified by flash column chroma‐
tography on silica gel (100 g, CH2Cl2/MeOH 1/0 to 25/2) to
give Fmoc‐protected amine, which was used in the next
reaction without further purification.
(500 MHz, CDCl3) Signals derived from rotamer A and B
(1:1) were observed.  0.67 (1.5H, d, J = 6.9 Hz), 0.79‐0.90
(9.0H, m), 0.99 (1.5H, d, J = 6.3 Hz), 1.08‐1.38 (4.0H, m),
1.40‐1.69 (3.0H, m), 2.08 (0.5H, m), 2.19 (0.5H, m), 2.41‐
2.67 (2.0H, m), 2.79 (1.5H, s), 2.87 (1.5H, s), 3.98 (0.5H, d, J
= 11.5 Hz), 4.20‐4.29 (1.0H, m), 4.37‐4.56 (2.5H, m), 5.17‐
5.29 (1.0H, m), 7.28‐7.34 (2.0H, m), 7.37‐7.44 (2.0H, m),
7.57‐7.68 (2.0H, m), 7.76 (2.0H, d, J = 7.5 Hz), 9.32 (1.0H,
13
br); C NMR (125 M Hz) Signals derived from rotamers A
and B were observed.  18.3 (A), 18.7 (B), 19.4 (A), 19.6 (B),
22.4 (2C of A or B), 22.5 (2C of A or B), 22.9 (A and B),
26.6 (A), 27.3 (B), 27.8 (A and B), 29.6 (A), 30.0 (B), 34.05
(A or B), 34.14 (A or B), 38.40 (A or B), 38.44 (A or B),
38.77 (A or B), 38.79 (A or B), 47.2 (A and B), 64.0 (B),
64.2 (A), 67.5 (A), 67.7 (B), 70.8 (A or B), 70.9 (A or B),
119.85 (2C of A or B), 119.93 (2C of A or B), 124.8 (A or B),
124.9 (A or B), 124.98 (A or B), 125.02 (A or B), 126.9 (A or
B), 127.01 (2C of A or B), 127.05 (A or B), 127.56 (A or B),
To a solution of the above Fmoc‐protected amine (2.07
g, 4.49 mmol) in AcOH (15.6 mL) were added TrOH (11.7
g, 44.9 mmol), H2SO4 (144 L, 2.69 mmol), and Ac2O (1.06
mL, 11.2 mmol) at 50 °C. After being stirred at 50 °C for
2.5 h, the resultant suspension was diluted with AcOEt
(200 mL) at rt. The resultant mixture was poured into
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saturated aqueous NaHCO3 (350 mL) at 0 °C with vigor‐
being stirred at rt for 40 min under H2 atmosphere, the
ous stirring. The organic layer was separated, and the
aqueous layer was extracted with AcOEt (300 mL ×2).
The combined organic layers were washed with brine
(200 mL), dried over anhydrous Na2SO4, filtered, and
concentrated. The residue was purified by flash column
chromatography on silica gel (200 g, hexane/AcOEt 20/1
to 2/1) to give 24 (2.50 g, 35% over 3 steps): white foam;
reaction mixture was filtered through a pad of Celite with
AcOEt (30 mL) and EtOH (30 mL). The filtrate was con‐
centrated. The residue was purified by flash column
chromatography on silica gel (50 g, CH2Cl2/MeOH = 1/0
25
to 10/1) to give 15 (2.14 g, 91%): white foam; []D = +15.8
(c = 0.628, CHCl3); IR (film)  3405, 2928, 1722, 1692, 1493,
1448, 1256, 1211, 1105, 1056 cm‐1; 1H NMR (400 MHz, DMSO‐
d6) Signals derived from rotamer A and B (7:3) were ob‐
served.  −0.08‐0.12 (6.0H, m), 0.79‐0.95 (9.0H, m), 4.00‐
4.71 (5.0H, m), 6.96‐7.94 (24.0H, m), 8.20 (0.7H, s), 8.32
(0.3H, s); 13C NMR (100 M Hz, DMSO‐d6) −5.3, −4.9, 17.8,
25.7 (3C), 46.7, 57.6, 66.3, 69.6, 73.9, 120.1 (2C), 125.4, 125.7,
126.8 (3C), 127.1, 127.2, 127.6 (6C), 127.7 (2C), 128.5 (6C),
140.7, 140.8, 143.7 (2C), 144.3 (3C), 156.6, 168.8, 171.5; HRMS
(ESI‐TOF) calcd for C44H46N2O6SiNa [M+Na]+ 749.3017,
found 749.3009.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
24
[]D = +12.5 (c = 0.303, CHCl3); IR (film)  3370, 3061,
1
2921, 2851, 1726, 1666, 1513, 1494, 1448, 1207 cm‐1; H NMR
(400 MHz, CDCl3)  4.17 (1H, t, J = 7.3 Hz), 4.26 (1H, dd, J
= 10.5, 7.3 Hz), 4.38 (1H, dd, J = 10.5, 7.3 Hz), 4.57 (2H, m),
4.84 (1H, d, J = 9.6 Hz), 5.19 (2H, s), 5.96 (1H, d, J = 9.2
Hz), 7.14‐7.30 (22H, m), 7.39 (2H, m), 7.54 (2H, m), 7.75
(2H, m), 7.94 (1H, s); 13C NMR (125 MHz, CDCl3)  46.9,
56.4, 67.78, 67.82, 70.5, 72.9, 119.9 (2C), 125.2 (2C), 127.1
(2C), 127.2 (3C), 127.7 (2C), 128.0 (6C), 128.1 (2C), 128.5,
128.55 (6C), 128.60 (2C), 134.7, 141.2 (2C), 143.6 (2C), 144.1
(3C), 157.0, 170.3, 170.6; HRMS (ESI‐TOF) calcd for
Determination of loading rate of 11. Resin 11 was treat‐
ed with piperidine/NMP (1/4, 1.00 mL) at rt for 15 min.
The supernatant was collected. The resin was again
treated with piperidine/NMP (1/4, 1.00 mL) at rt for 15
min. The supernatant was collected. The resin was
washed with NMP (2 mL ×4), and then the supernatant
was collected. UV absorption at 301 nm of the combined
supernatants was measured. The background absorbance
was canceled by subtracting the control absorbance ob‐
tained from a solution of piperidine/NMP (1/24). The
loading rate (x mmol/g) was determined by the following
equation, where a is the weight of 11 (mg), and b is ab‐
sorbance at 301 nm.
1
C45H38N2O6Na [M+Na]+ 725.2622, found 725.2611; H NMR
spectrum was identical with that of ent‐24.41,42
Silyl ether 25. To a solution of 24 (2.45 g, 3.49 mmol)
and 2,6‐lutidine (1.62 mL, 14.0 mmol) in CH2Cl2 (34.9 mL)
was added TBSOTf (1.60 mL, 6.98 mmol) at 0 °C. After
being stirred at 0 °C for 0.5 h, the reaction mixture was
diluted with Et2O (150 mL), washed with 0.1 M aqueous
HCl (150 mL ×3), saturated aqueous NaHCO3 (150 mL)
and brine (150 mL), dried over anhydrous Na2SO4, filtered,
and concentrated. The residue was purified by flash col‐
umn chromatography on silica gel (50 g, hexane/AcOEt
26
10/1 to 3/1) to give 25 (2.71 g, 95%): white foam; []D
=
x = (10000 × b) / (7800 × a)
+13.1 (c = 2.53, CHCl3); IR (film)  3410, 2927, 2856, 1730,
Procedures for solid‐phase peptide synthesis. Peptides 1
and 2 were prepared on a peptide synthesizer. Standard
operation was shown as follows:
1
1693, 1494, 1448, 1254, 1197, 1104 cm‐1; H NMR (500 MHz,
CDCl3) Signals derived from rotamers A and B (4:1) were
observed.  −0.02 (2.4H, s), 0.00 (1.2H, s), 0.05 (2.4H, s),
0.82 (7.2H, s), 0.84 (1.8H, s), 4.10 (0.2H, m), 4.17‐4.26
(1.6H, m), 4.31 (0.2H, m), 4.47 (0.8H, dd, J = 9.7, 6.3 Hz),
4.54 (0.8H, d, J = 2.3 Hz), 4.70 (0.2H, s), 4.88 (0.8H, dd, J
= 10.0, 2.6 Hz), 4.97 (0.2H, d, J = 10.3 Hz), 5.13 (1.0 H, d, J =
12.6 Hz), 5.18 (1.0 H, d, J = 12.6 Hz), 5.42 (0.2H, d, J = 10.9
Hz), 5.95 (0.8H, d, J = 10.3 Hz), 7.11‐7.60 (28.0H, m), 7.71‐
7.84 (3.0H, m); 13C NMR (125 MHz, CDCl3) Signals derived
from rotamer A and B were observed.  −5.4 (B), −5.3 (A),
−5.1 (A), −4.8 (B), 17.7 (A and B), 25.5 (3C of A and B), 47.0
(A and B), 57.3 (A), 57.9 (B), 67.5 (A), 67.6 (A), 67.7 (B),
67.9 (B), 70.5 (A and B), 73.4 (A) 73.6 (B), 119.9 (2C of A
and B), 125.1 (A and B), 125.3 (A and B), 127.0 (2C of A and
B), 127.2 (3C of A and B), 127.6 (2C of A and B), 128.0 (8C
of A and B), 128.4 (A and B), 128.5 (2C of A and B), 128.6
(6C of A and B), 134.6 (B), 134.7 (A), 141.1 (A and B), 141.2
(A and B), 143.6 (A and B), 143.9 (A and B), 144.1 (3C of A
and B), 155.4 (B), 155.9 (A), 168.5 (B), 169.0 (A), 169.9 (A),
170.1 (B); HRMS (ESI‐TOF) calcd for C51H52N2O6SiNa
[M+Na]+ 839.3487, found 839.3511.
Step 1: The solid supported N‐Fmoc peptide was
deprotected with piperidine/NMP (1/4) at rt for 10 min.
Step 2: The reaction vessel (LibraTube) containing the
resin was washed with NMP (5 mL ×6 for synthesis of 1, 2
mL ×6 for synthesis of 2).
Step 3: A Fmoc‐amino acid (4.0 eq) was activated by a
solution
of
O‐(7‐azabenzotriazole‐1‐yl)‐N,N,N’,N’‐
tetramethyluronium hexafluorophosphate (HATU)/1‐
hydroxy‐7‐azabenzotriazole (HOAt) (4.0 eq, 0.45 M) in
NMP. To the solution of activated Fmoc‐amino acid was
added a solution of i‐Pr2NEt (8.0 eq, 2.0 M) in NMP. The
resultant mixture was transferred to the reaction vessel.
Step 4: The activated Fmoc‐amino acid was coupled
with the peptide on the resin (40 °C, 200 W; 20 min) and
the reaction vessel containing the resin was washed with
NMP (5 mL ×6 for synthesis of 1, 2 mL ×6 for synthesis of
2).
Steps 1–4 were repeated and amino acids were con‐
densed on the solid support.
Carboxylic acid 15. To a solution of 25 (2.64 g, 3.23
mmol) in AcOEt/EtOH (1/1, 60.0 mL) was added a sus‐
pension of 10 wt% Pd/C (227 mg, 0.213 mmol) in AcO‐
Et/EtOH (1/1, 4.60 mL) at rt. The flask equipped with a
balloon was evacuated and recharged with H2 (×3). After
WAP‐8294A2 (lotilibcin, 1). To a solution of Fmoc‐L‐
Glu‐OAllyl (1.05 g, 2.57 mmol) in CH2Cl2/NMP (30/1, 38.9
mL) was added N‐N’‐diisopropylcarbodiimide (DIC, 413
L, 2.67 mmol) at rt. After being stirred at rt for 20 min,
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the solution was concentrated to give the crude acid an‐
hydride, which was used in the next reaction without fur‐
ther purification.
concentrated. The residue was purified by 1st reversed‐
phase HPLC (column: Inertsil ODS‐3 20 ×250 mm, eluent
A: MeOH + 0.05% TFA, eluent B: H2O + 0.05% TFA, linear
gradient A/B = 72/28 to 82/18 over 40 min, flow rate: 4.0
mL/min, detection: UV 280 nm), 2nd reversed‐phase
HPLC (column: Inertsil ODS‐3 20 ×250 mm, eluent A:
MeCN + 0.05% TFA, eluent B: H2O + 0.05% TFA, A/B =
43/57, flow rate: 4.0 mL/min, detection: UV 280 nm), and
3rd reversed‐phase HPLC (column: Inertsil C8‐3 10 ×250
mm, eluent A: MeOH + 0.05% TFA, eluent B: H2O +
0.05% TFA, linear gradient A/B = 60/40 to 80/20 over 50
min, flow rate: 3.0 mL/min, detection: UV 280 nm) to give
1 (tR = 35.7 min, 14.7 mg, 8.4 % over 27 steps): white solid;
Wang‐ChemMatrix resin (1.01 g, 0.513 mmol) in 20 mL
LibraTube was washed with CH2Cl2 (5 mL ×3) and NMP (5
mL ×5). To the resin were added a solution of the above
crude acid anhydride in NMP (6.88 mL) and a solution of
DMAP (6.27 mg, 51.3 mol) in NMP (0.627 mL). After
being stirred at rt for 1 h, the reaction mixture was filtered,
and washed with NMP (5 mL ×5) and CH2Cl2 (5 mL ×5).
To the resin was added Ac2O/CH2Cl2 (1/3, 6 mL). After
being stirred at rt for 20 min, the reaction mixture was
filtered, washed with CH2Cl2 (5 mL ×5), MeOH (5 mL ×5),
and Et2O (5 mL ×5), and dried under vacuum to give 11
(1.19 g, 72%). The loading rate was determined to be
0.265 mmol/g by the above method.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
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43
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45
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48
49
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26
[]D = +29.8 (c = 0.303, MeOH); IR (film)  3277, 2956,
1
1730, 1672, 1632, 1540, 1436, 1203, 1137 cm‐1; H NMR (500
MHz, DMSO‐d6) 3‐OH‐7‐Me‐octanoic acid  2.33 (H2),
2.56 (H2'), 4.95 (H3), 1.52 (H4), 1.24 (H5), 1.13 (H6), 1.48 (H7),
0.84 (H8, H8'); L‐Ser‐1  4.79 (H), 3.49 (H), 3.60 (H'),
7.93 (NH); D‐OHAsn‐2  4.95 (H), 4.28 (H), 7.34 (NH),
7.37 (NH'), 8.04 (NH), 5.72 (OH); L‐Ser‐3  4.80 (H),
3.64 (H), 8.23 (NH); Gly‐4  3.89 (H), 8.15 (NH); N‐Me‐
D‐Phe‐5  4.48 (H), 2.95 (H), 3.27 (H'), 7.17 (H2, H6),
7.28 (H3, H5), 7.19 (H4), 2.61 (NCH3); L‐Leu‐6  4.30 (H),
1.39 (H), 1.58 (H'), 1.40 (H), 0.71 (H), 0.73 (H), 7.94
(NH); D‐Orn‐7  4.63 (H), 1.66 (H), 1.71 (H'), 1.54 (H),
2.86 (H), 7.81 (NH2), 7.49 (NH); L‐Glu‐8  4.62 (H), 1.73
(H), 1.86 (H'), 2.20 (H), 8.46 (NH), 12.09 (OH); D‐Asn‐9
 4.78 (H), 2.45 (H), 2.62 (H'), 6.92 (NH), 7.33 (NH'),
8.16 (NH); D‐Trp‐10  4.72 (H), 2.81 (H), 3.13 (H'), 7.14
(H2), 7.46 (H4), 6.91 (H5), 6.97 (H6), 7.31 (H7), 10.30 (NH1),
8.65 (NH); D‐Orn‐11  4.60 (H), 1.58 (H), 1.70 (H'), 1.45
(H), 2.82 (H), 7.81 (NH2), 7.98 (NH); N‐Me‐L‐Val‐12 
The above resin 11 (391 mg, 0.104 mmol) in 20 mL Li‐
braTube was washed with CH2Cl2 (5 mL ×3) and NMP (5
mL ×6). The resin was subjected to 8 cycles (10, 9, 8, 7, 6,
15, 6, and 14) of the microwave‐assisted SPPS protocol to
give peptide 26a.
The resin‐bound peptide 26a was subjected to steps 3
and 4 of the microwave‐assisted SPPS protocol using 10
(188 mg, 0.414 mmol), which was performed by the dou‐
ble coupling method (90 min ×2, steps 3 and 4 were re‐
peated before step 1). After the 2 cycles of condensation
of 10, deprotection of N‐Fmoc group was performed for 1
min. The reaction mixture was washed with NMP (5 mL
×6) to give peptide 27a.
The resin‐bound peptide 27a was subjected to 2 cycles
(13 and 12) of the microwave‐assisted SPPS protocol. Af‐
ter the final Fmoc deprotection, the reaction mixture was
washed with CH2Cl2 (5 mL ×6) to give peptide 28a.




13
4.62 (H ), 2.08 (H ), 0.74 (H ), 0.88 (H '), 2.57 (NCH3); C
NMR (125 MHz, DMSO‐d6)  18.0, 19.1, 20.8, 22.0, 22.2,
22.9, 23.0, 23.2, 24.1, 25.7, 27.2, 27.4, 28.1, 28.4, 29.4, 29.8,
30.2, 33.1, 34.0, 34.7, 38.1, 38.1, 38.4, 38.5, 38.6, 39.3 (over‐
lapped with CHD2S(O)CD3, determined from 1H‐13C
HMQC correlation), 42.0, 48.3, 49.2, 50.8, 51.2, 51.3, 52.7,
54.1, 54.8, 54.9, 61.0, 62.0, 62.7, 62.8, 71.0, 71.2, 109.4, 111.3,
118.0, 118.1, 120.6, 123.3, 126.2, 127.2, 128.2, 129.0, 135.9, 138.2,
169.0, 169.1, 169.2, 169.3, 169.3, 169.6, 169.8, 169.9, 170.4,
170.5, 170.7, 171.0, 171.5, 171.8, 173.5, 173.9; HRMS (ESI‐TOF)
calcd for C73H112N17O21+ [M+H]+ 1562.8213, found 1562.8232.
To the resin‐bound peptide 28a in 20 mL LibraTube
was added a solution of Pd(PPh3)4 (31.1 mg, 26.9 mol)
and morpholine (179 L, 2.07 mmol) in CH2Cl2 (3.45 mL).
After being stirred at rt for 0.5 h, the reaction mixture was
washed with CH2Cl2 (5 mL ×6), NMP (5 mL ×6), and
NMP/CH2Cl2 (1/9, 5 mL ×6) to give peptide 29a.
To the resin‐bound peptide 29a in 20 mL LibraTube
was added a solution of PyBOP (226 mg, 0.435 mmol) and
2,4,6‐collidine (112 L, 0.850 mmol) in NMP/CH2Cl2 (1/9,
2.59 mL). After being stirred at rt for 12 h, the reaction
mixture was washed with CH2Cl2 (5 mL ×6), NMP (5 mL
×6), MeOH (5 mL ×6), and Et2O (5 mL ×6), and dried un‐
der vacuum to give peptide 30a.
Peptide 2. To a solution of Fmoc‐L‐Glu‐OAllyl (261 mg,
0.637 mmol) in CH2Cl2/NMP (30/1, 9.64 mL) was added
DIC (98.7 L, 0.637 mmol) at rt. After being stirred at rt
for 20 min, the solution was concentrated to give the
crude acid anhydride, which was used in the next reaction
without further purification.
The resin‐bound peptide 30a was washed with THF (5
mL ×6). To the resin‐bound peptide was added a solution
of TBAF (81.2 mg, 0.311 mmol) and AcOH (17.8 L, 0.311
mmol) in THF (3.45 mL). After being stirred at rt for 0.5
h, the reaction mixture was washed with THF (5 mL ×6)
and Et2O (5 mL ×6), and dried under vacuum.
Wang‐ChemMatrix resin (209 mg, 0.127 mmol) in 5 mL
LibraTube was washed with CH2Cl2 (2 mL ×3) and NMP (2
mL ×3). To the resin were added a solution of the above
crude acid anhydride in NMP (1.72 mL) and a solution of
DMAP (1.56 mg, 12.7 mol) in NMP (0.156 mL). After be‐
ing stirred at rt for 1 h, the reaction mixture was filtered,
and washed with NMP (2 mL ×3) and CH2Cl2 (2 mL ×3).
To the resin was added Ac2O/CH2Cl2 (1/3, 2 mL). After
being stirred at rt for 20 min, the reaction mixture was
To the resin‐bound peptide was added 95% aqueous
TFA (8.0 mL). After being stirred for 2 h, the reaction
mixture was filtered, and washed with 95% aqueous TFA
(5.0 mL ×6). The filtrate was concentrated to give the
crude 1. The crude 1 was dissolved in MeOH, filtered, and
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 4.87 (H), 2.51 (H), 2.58 (H') 6.96 (NH), 7.39 (NH'),
filtered, washed with CH2Cl2 (2 mL ×3), MeOH (2 mL ×3)
8.23 (NH); L‐Ser‐3  4.80 (H), 3.62 (H), 8.34 (NH); Gly‐4
 3.88 (H), 8.16 (NH); N‐Me‐D‐Phe‐5  4.43 (H), 2.96
(H), 3.27 (H'), 7.17 (H2, H6), 7.27 (H3, H5), 7.19 (H4), 2.59
(NCH3); L‐Leu‐6  4.28 (H), 1.38 (H), 1.51 (H'), 1.40 (H),
0.69 (H), 0.72 (H), 7.89 (NH); D‐Orn‐7  4.62 (H), 1.64
(H), 1.70 (H'), 1.56 (H), 2.84 (H), 7.81 (NH2), 7.53 (NH);
L‐Glu‐8  4.67 (H), 1.74 (H), 1.86 (H'), 2.19 (H), 8.49
(NH), 12.10 (OH); D‐Asn‐9  4.76 (H), 2.46 (H), 2.57
(H'), 6.92 (NH), 7.39 (NH'), 8.29 (NH); D‐Trp‐10  4.73
(H), 2.82 (H), 3.14 (H'), 7.12 (H2), 7.47 (H4), 6.91 (H5),
6.97 (H6), 7.32 (H7), 10.33 (NH1), 8.62 (NH); D‐Orn‐11 
4.60 (H), 1.55 (H), 1.71 (H'), 1.45 (H), 2.80 (H), 7.81
(NH2), 7.99 (NH); N‐Me‐L‐Val‐12  4.63 (H), 2.08 (H),
0.72 (H), 0.87 (H'), 2.56 (NCH3); 13C NMR (125 MHz,
DMSO‐d6)  18.0, 19.1, 20.8, 22.0, 22.2, 22.9, 23.0, 23.2, 24.1,
25.7, 27.2, 27.4, 28.1, 28.3, 29.4, 29.8, 30.1, 33.1, 33.9, 34.9,
38.1, 38.1, 38.4, 38.4, 38.4, 38.6, 39.3 (overlapped with
and Et2O (2 mL ×6), and dried under vacuum to give 11
(254 mg, 73%). The loading rate was determined to be
0.366 mmol/g by the above method.
The above resin 11 (108 mg, 39.6 mol) in 5 mL Li‐
braTube was washed with CH2Cl2 (2 mL ×3) and NMP (2
mL ×3). The resin was was subjected to 5 cycles (10, 9, 8,
7, and 6) of the microwave‐assisted SPPS protocol and
washed with NMP (2 mL ×6), CH2Cl2 (2 mL ×6), MeOH(2
mL ×6), and Et2O (2 mL ×6), and dried under vacuum to
give the resin‐bound peptide (217 mg).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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36
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The resin‐bound peptide (131 mg, 23.9 mol) in 5 mL
LibraTube was washed with CH2Cl2 (2 mL ×3) and NMP (2
mL ×3). The resin‐bound peptide was subjected to 3 cy‐
cles (12, 6, and 14) of the microwave‐assisted SPPS proto‐
col to give peptide 26b.
The resin‐bound peptide 26b was subjected to steps 3
and 4 of the microwave‐assisted SPPS protocol using 10
(43.5 mg, 95.6 mol), which was performed by the double
coupling method (90 min ×2, steps 3 and 4 were repeated
before step 1). After the 2 cycles of condensation of 10,
deprotection of N‐Fmoc group was performed for 1 min.
The reaction mixture was washed with NMP (2 mL ×6) to
give peptide 27b.
1
CHD2S(O)CD3, determined from H‐13C HMQC correla‐
tion), 42.0, 48.4, 49.3, 49.4, 50.8, 51.2, 51.3, 52.7, 54.1, 54.8,
61.0, 62.1, 62.4, 63.0, 71.0, 109.4, 111.3, 118.0, 118.0, 120.6,
123.2, 126.2, 127.2, 128.2, 129.0, 135.9, 138.3, 169.1, 169.1, 169.3,
169.4, 169.5, 169.7, 170.0, 170.4, 170.4, 170.6, 170.7, 171.0,
171.5, 171.7, 171.9, 173.8; HRMS (ESI‐TOF) calcd for
C73H112N17O20+ [M+H]+ 1546.8264, found 1546.8236.
The resin‐bound peptide 27b was subjected to 2 cycles
(13 and 12) of the microwave‐assisted SPPS protocol. Af‐
ter the final Fmoc deprotection, the reaction mixture was
washed with CH2Cl2 (2 mL ×6) to give peptide 28b.
Antimicrobial Activity Assay. The MIC assay was per‐
formed according to the Clinical and Laboratory Stand‐
ards Institute protocols. The antimicrobial and antifungal
activities of each compound were measured using the
microdilution method.56,57
To the resin‐bound peptide 28b in 5 mL LibraTube was
added a solution of Pd(PPh3)4 (7.18 mg, 6.22 mol) and
morpholine (41.2 L, 0.478 mmol) in CH2Cl2 (0.797 mL).
After being stirred at rt for 30 min, the reaction mixture
was washed with CH2Cl2 (2 mL ×6), NMP (2 mL ×6), and
NMP/CH2Cl2 (1/9, 2 mL ×6) to give peptide 29b.
Evaluation of Membrane Potential. Membrane poten‐
tial was measured using the fluorescence assay.58 S. aure‐
us MSSA1 strain was grown to the mid‐log phase in cation
adjusted Mueller‐Hinton broth medium. Cells were
washed with 5 mM HEPES buffer, pH 7.0, 50 mM glucose,
5 mM EDTA, and resuspended in the same buffer to ob‐
To the resin‐bound peptide 29b in 5 mL LibraTube was
added a solution of PyBOP (52.0 mg, 0.100 mmol) and
2,4,6‐collidine (25.9 L, 0.196 mmol) in NMP/CH2Cl2 (1/9,
0.598 mL). After being stirred at rt for 18 h, the reaction
mixture was washed with CH2Cl2 (2 mL ×6), NMP (2 mL
×6), CH2Cl2 (2 mL ×6), MeOH (2 mL ×6), and Et2O (2 mL
×6), and dried under vacuum to give peptide 30b.
tain an A600 of 0.05.
DiSC3(5) (3,3 ′ ‐
dipropylthiadicarbocyanine iodide) dye was added to a
final concentration of 250 nM and cells were incubated in
a water bath maintained at 37˚C for 15 min. The fluores‐
cence emission was measured at 670 nm with the excita‐
tion at 622 nm using a spectrofluorometer equipped with
a chamber heated at 37 °C. Data were collected for 400 s
after addition of each compound.
To the resin‐bound peptide 30b was added 95% aque‐
ous TFA (3.0 mL). After being stirred for 2 h, the reaction
mixture was filtered, and washed with 95% aqueous TFA
(2.0 mL ×6). The filtrate was concentrated to give the
crude 2. The crude 2 was dissolved in MeOH, filtered,
and concentrated. The residue was purified by reversed‐
phase HPLC (column: Inertsil ODS‐4 10 ×250 mm, eluent
A: MeOH + 0.05% TFA, eluent B: H2O + 0.05% TFA, linear
gradient A/B = 70/30 to 85/15 over 40 min, flow rate: 3.0
mL/min, detection: UV 280 nm) to give 2 (tR = 25.2 min,
2.62 mg, 6.6 % over 26 steps): white solid; []D25 = +36.7 (c
= 0.169, MeOH); IR (film)  3277, 2957, 2359, 1722, 1631,
Preparation of CF‐encapsulated LUVs consisting of
PC/PG.
Carboxyfluorescein (CF)‐encapsulated large
unilamellar vesicles (LUVs) were prepared according to
the thin‐film hydration method, followed by extrusion
through polycarbonate filters mounted in the extrusion
apparatus. A solution of egg yolk phosphatidylcholine
(EYPC, 9.60 mg, 12.5 µmol) and egg yolk phosphatidyl‐
glycerol (EYPG, 9.78 mg, 12.5 µmol) in CHCl3 (0.505 mL)
was concentrated, and dried under vacuum to form a li‐
pid thin film. The thin film was hydrated and suspended
in CF‐containing buffer solution (0.500 mL, 5 mM HEPES,
20 mM CF, pH 7.5) by vortexing and sonication. After
five‐times freeze‐thaw cycles, the lipid suspension was
extruded 19 times through a polycarbonate filter with
1
1547, 1440, 1204, 1138 cm‐1; H NMR (500 MHz, DMSO‐d6)
3‐OH‐7‐Me‐octanoic acid  2.32 (H2), 2.55 (H2'), 4.95 (H3),
1.49 (H4), 1.24 (H5), 1.13 (H6), 1.48 (H7), 0.82 (H8, H8'); L‐
Ser‐1  4.66 (H), 3.50 (H), 3.57 (H'), 7.92 (NH); D‐Asn‐2
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0.40 µm of pore size in the diameter. The external CF‐
100% lysis. The LUV suspension and 40% aqueous MeOH
(2 L) and were mixed in each 96‐well plate to determine
0% lysis. The membrane disruption activities of tested
peptides (I) were normalized against 100% lysis (Imax) and
0% lysis (I0) as following: I = 100 × (Ix – I0)/ (Imax – I0),
where Ix is the fluorescence intensity. The membrane‐
disrupting activities were evaluated as EC50 (nM) by
means of three replicates. Sigmoidal curve fittings were
performed on R60 with drc package (Figure S2).61 Four‐
parameter logistic model was applied for the fitting. The
obtained EC50 values with S.D. (average of 3 independent
containing buffer was replaced with a CF‐free buffer solu‐
tion, (20 mM HEPES, 1 mM EDTA, pH 7.5) through size
exclusion chromatography using disposable PD‐10 col‐
umn. Concentration of EYPC was determined by using
Phospholipid C‐Test Wako. The final concentration of
EYPC was adjusted to 4.0 µM with the CF‐free buffer so‐
lution, and used for membrane disruption assay.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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Preparation of CF‐encapsulated LUVs consisting of
PC/PG/CL. To a solution of EYPC (4.80 mg, 6.25 µmol)
and EYPG (3.91 mg, 5.00 µmol) in CHCl3 (0.140 mL) was
added a solution of cardiolipin sodium salt from bovine
heart (CL, 1.86 mg, 1.25 mol) in CHCl3 (0.186 mL). The
solution was concentrated, and dried under vacuum to
form a lipid thin film. The thin film was hydrated and
suspended in CF‐containing buffer solution (0.250 mL, 5
mM HEPES, 20 mM CF, pH 7.5) by vortexing and soni‐
cation. After five‐times freeze‐thaw cycles, the lipid sus‐
pension was extruded 19 times through a polycarbonate
filter with 0.40 µm of pore size in the diameter. The ex‐
ternal CF‐containing buffer was replaced with a CF‐free
buffer solution (20 mM HEPES, 1 mM EDTA, pH 7.5)
through size exclusion chromatography using disposable
PD‐10 column. Concentration of EYPC was determined
by using Phospholipid C‐Test Wako. The final concentra‐
tion of EYPC was adjusted to 4.0 µM with the CF‐free
buffer solution, and used for membrane disruption assay.
Averaged molecular weight of CL (1489) was calculated
from the reported data.59
experiments) were 503
± 165 (PC/PG), 833 ± 68
(PC/PG/CL), 14.2 ± 3.1 (PC/PG/MK), and 132 ± 14
(PC/PG/UQ) nM for 1, 640 ± 39 (PC/PG), 781 ± 54
(PC/PG/CL), 22.1 ± 12.9 (PC/PG/MK), and 261 ± 47
(PC/PG/UQ) nM for 2, and 435 ± 83 (PC/PG), 501 ± 57
(PC/PG/CL), 15.9 ± 7.3 (PC/PG/MK), and 56.5 ± 11.9
(PC/PG/UQ) nM for 3, respectively (Figure S3).
ASSOCIATED CONTENT
Supporting Information. Determination of the configura‐
1
tion of 18, chiral HPLC analysis of 19, assay data, H and 13C
1
1
NMR spectra for all new compounds, H‐1H COSY, H‐1H
TOCSY, H‐1H NOESY, H‐13C HMBC, H‐13C HMQC spectra,
and HPLC charts for peptides 1 and 2. This material is avail‐
able free of charge via the Internet at http://pubs.acs.org.
1
1
1
AUTHOR INFORMATION
Corresponding Author
*E‐mail: inoue@mol.f.u‐tokyo.ac.jp
Preparation of CF‐encapsulated LUVs consisting of
PC/PG/MK or PC/PG/UQ. To a solution of EYPC (9.48
mg, 12.4 µmol) and EYPG (9.66 mg, 12.4 µmol) in CHCl3
(0.208 mL) was added a solution of 4 or 5 (0.313 mol) in
CHCl3 (21.8 µL). The solution was concentrated, and
dried under vacuum to form a lipid thin film. The thin
film was hydrated and suspended in CF‐containing buffer
solution (0.500 mL, 5 mM HEPES, 20 mM CF, pH 7.5) by
vortexing and sonication. After five‐times freeze‐thaw
cycles, the lipid suspension was extruded 19 times
through a polycarbonate filter with 0.40 µm of pore size
in the diameter. The external CF‐containing buffer was
replaced with a CF‐free buffer solution (20 mM HEPES, 1
mM EDTA, pH 7.5) through size exclusion chromatog‐
raphy using disposable PD‐10 column. Concentration of
EYPC was determined by using Phospholipid C‐Test
Wako. The final concentration of EYPC was adjusted to
4.0 µM with the CF‐free buffer solution, and used for
membrane disruption assay.
ORCID
Hiroaki Itoh: 0000‐0002‐1329‐6109
Masayuki Inoue: 0000‐0003‐3274‐551X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This research was financially supported by Grant‐in‐Aids for
Scientific Research (S) (JP17H06110) and Scientific Research
on Innovative Areas (JP16H01130) to M.I., for Young Scien‐
tists (B) (JP17K15421) to H.I., Scientific Research (S)
(JP15H05783) to K.S., and by Takeda Science Foundation to
H.H. Fellowships from JSPS to T.K. and from Tokyo Bio‐
chemical Research Foundation to A.P. are gratefully
acknowledged.
REFERENCES
Membrane disruption assay. Peptides 1‐3 were diluted
with 40% aqueous MeOH to various concentrations as 5‐
fold serial dilutions. The solution of peptides (2 L) and
the CF‐encapsulated LUV suspension (200 L) were
mixed in 96‐well plates. The final concentration of pep‐
tides ranged from 2970 nM to 0.00152 nM. The 96‐well
plates were vortexed at rt for 30 min, and then fluores‐
cence emission of each well was measured at 490 nm with
the excitation at 517 nm using a fluorescence microplate
reader. The LUV suspension and 5% aqueous Trion X‐100
(2 L) were mixed in each 96‐well plate to determine
(1)
Blair, J. M. A.; Webber, M. A.; Baylay, A. J.; Ogbolu, D. O.;
Piddock, L. J. V. Nat. Rev. Microbiol. 2015, 13, 42.
(2)
(3)
Brown, E. D.; Wright, G. D. Nature 2016, 529, 336.
von Nussbaum, F.; Brands, M.; Hinzen, B.; Weigand, S.;
Häbich, D. Angew. Chem., Int. Ed. 2006, 45, 5072.
(4) Butler, M. S.; Blaskovich, M. A.; Cooper, M. A. J. Antibiot.
2013, 66, 571.
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The Journal of Organic Chemistry
Page 14 of 14
1
2
3
4
5
6
7
8
(5)
O’Connell, K. M. G.; Hodgkinson, J. T.; Sore, H. F.; Welch,
(36) The TBS protection of D‐OHAsn was important for in‐
creasing the overall yield of 1.
M.; Salmond, G. P. C.; Spring, D. R. Angew. Chem., Int. Ed. 2013,
52, 10706.
(6)
Ed. 2014, 53, 8840; Angew. Chem. 2014, 126, 8984.
(7) Schneider, T.; Müller, A.; Miess, H.; Gross, H. Int. J. Med.
Microbiol. 2014, 304, 37.
(37) Itoh, H.; Matsuoka, S.; Kreir, M.; Inoue, M. J. Am. Chem.
Soc. 2012, 134, 14011.
(38) Itoh, H.; Inoue, M. Acc. Chem. Res. 2013, 46, 1567.
(39) Noyori, R.; Ohkuma, T.; Kitamura, M.; Takaya, H.; Sayo,
N.; Kumobayashi, H.; Akutagawa, S. J. Am. Chem. Soc. 1987, 109,
5856.
Wright, P. M.; Seiple, I. B.; Myers, A. G. Angew. Chem., Int.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(8)
Harvey, A. L.; Edrada‐Ebel, R.; Quinn, R. J. Nat. Rev. Drug
Discov. 2015, 14, 111.
(40) Sang, F.; Ding, Y.; Wang, J.; Sun, B.; Sun, J.; Geng, Y.;
Zhang, Z.; Ding, K.; Wu, L.‐L.; Liu, J.‐W.; Bai, C.; Yang, G.; Zhang,
Q.; Li L.‐Y.; Chen, Y. J. Med. Chem. 2016, 59, 1184.
(41) Boger, D. L.; Lee, R. J.; Bounaud, P.‐Y.; Meier, P. J. Org.
Chem. 2000, 65, 6770.
(42) Guzmán‐Martínez, A.; VanNieuwenhze, M. Synlett 2007,
1513.
(43) Yu, H.‐M.; Chen, S.‐T.; Wang, K.‐T. J. Org. Chem. 1992, 57,
4781.
(44) Bacsa, B.; Horváti, K.; Bõsze, S.; Andreae, F.; Kappe, C. O. J.
Org. Chem. 2008, 73, 7532.
(45) Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397.
(46) Friedrich‐Bochnitschek, S.; Waldmann, H.; Kunz, H. J.
Org. Chem. 1989, 54, 751.
(9)
Panthee, S.; Hamamoto, H.; Paudel, A.; Sekimizu, K. Arch.
Microbiol. 2016, 198, 839.
(10) Wencewicz, T. A. Bioorg. Med. Chem. 2016, 24, 6227.
(11) Kato, A.; Nakaya, S.; Ohashi, Y.; Hirata, H.; Fujii, K.;
Harada, K. J. Am. Chem. Soc. 1997, 119, 6680.
(12) Kato, A.; Nakaya, S.; Kokubo, N.; Aiba, Y.; Ohashi, Y.;
Hirata, H. J. Antibiot. 1998, 51, 929.
(13) Kato, A.; Hirata, H.; Ohashi, Y.; Fujii, K.; Mori, K.; Harada,
K. J. Antibiot. 2011, 64, 373.
(14) Zhang, W.; Li, Y.; Qian, G.; Wang, Y.; Chen, H.; Li, Y.‐Z.;
Liu, F.; Shen, Y.; Du, L. Antimicrob. Agents Chemother. 2011, 55,
5581.
(15) Wang, Y.; Qian, G.; Liu, F.; Li, Y.‐Z.; Shen, Y.; Du, L. ACS
Synth. Biol. 2013, 2, 670.
(16) Butler, M. S.; Cooper, M. A. J. Antibiot. 2011, 64, 413.
(17) Wohlrab, A. M. PhD thesis, University of California (2007).
(18) Hamamoto, H.; Urai, M.; Ishii, K.; Yasukawa, J.; Paudel,
A.; Murai, M.; Kaji, T.; Kuranaga, T.; Hamase, K.; Katsu, T.; Su, J.;
Adachi, T.; Uchida, R.; Tomoda, H.; Yamada, M.; Souma, M.;
Kurihara, H.; Inoue, M.; Sekimizu, K. Nat. Chem. Biol. 2015, 11,
127.
(19) Panthee, S.; Hamamoto, H.; Suzuki, Y.; Sekimizu, K. J.
Antibiot. 2017, 70, 204.
(20) Bentley, R.; Meganathan, R. Microbiol. Rev. 1982, 46, 241.
(21) Tynecka, Z.; Szcześniak, Z.; Malm, A.; Los, R. Res. Micro‐
biol. 1999, 150, 555.
(22) Murai, M.; Kaji, T.; Kuranaga, T.; Hamamoto, H.;
Sekimizu, K.; Inoue, M. Angew. Chem., Int. Ed. 2015, 54, 1556;
Angew. Chem. 2015, 127, 1576.
(23) Kaji, T.; Murai, M.; Itoh, H.; Yasukawa, J.; Hamamoto, H.;
Sekimizu, K.; Inoue, M. Chem. Eur. J. 2016, 22, 16912.
(24) Carpino, L. A.; Han, G. Y. J. Am. Chem. Soc. 1970, 92, 5748.
(25) Chan, W. C.; White, P. D. Fmoc Solid Phase Peptide Syn‐
thesis—A Practical Approach (Oxford Univ. Press, 2000).
(26) Garcia‐Martin, F.; Quintanar‐Audelo, M.; Garcia‐Ramos,
Y.; Cruz, L. J.; Gravel, C.; Furic, R.; Côte, S.; Tulla‐Puche, J.;
Albericio, F. J. Comb. Chem. 2006, 8, 213.
(27) Kates, S. A.; Solé, N. A.; Johnson, C. R.; Hudson, D.;
Barany, G.; Albericio, F. Tetrahedron Lett. 1993, 34, 1549.
(28) Lee, J.; Griffin, J. H.; Nicas, T. I. J. Org. Chem. 1996, 61,
3983.
(47) Coste, J.; Le‐Nguyen, D.; Castro, B. Tetrahedron Lett. 1990,
31, 205.
(48) Collins, M. D.; Jones, D. J. Gen. Microbiol. 1979, 114, 27.
(49) Wakeman, C. A.; Hammer, N. D.; Stauff, D. L.; Attia, A. S.;
Anzaldi, L. L.; Dikalov, S. I.; Calcutt, M. W.; Skaar, E. P. Mol.
Microbiol. 2012, 86, 1376.
(50) Sims, P. J.; Waggoner, A. S.; Wang, C.‐H.; Hoffman, J. F.
Biochemistry 1974, 13, 3315.
(51) Epand, R. M.; Epand, R. F. Mol. BioSyst. 2009, 5, 580.
(52) Frerman, F. E.; White, D. C. J. Bacteriol. 1967, 94, 1868.
(53) Joyce, G. H.; Hammond, R. K.; White, D. C. J. Bacteriol.
1970, 104, 323.
(54) Weinstein, J. N.; Blumenthal, R.; Klausner, R. D. Methods
Enzymol. 1986, 128, 657.
(55) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am.
Chem. Soc. 1991, 113, 4092.
(56) Clinical and Laboratory Standards Institute. Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria that
Grow Aerobically; Approved Standard—Eighth Edition (CLSI
document M07–A8) (Clinical and Laboratory Standards Institute,
Wayne, PA, 2009).
(57) Clinical and Laboratory Standards Institute. Reference
Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts; Approved Standard (CLSI document M27‐A) (Clinical
and Laboratory Standards Institute, Wayne, PA, 1997).
(58) Breeuwer, P.; Abee, T. Assessment of the Membrane Po‐
tential, Intracellular pH and Respiration of Bacteria Employing
Fluorescence Techniques (Kluwer Academic Publishers, Nether‐
lands, 2004).
(59) Minkler, P. E.; Hoppel, C. L. J. Lipid Res. 2010, 51, 856.
(60) R Core Team (2016). R: A language and environment for
statistical computing. R Foundation for Statistical Computing,
Vienna, Austria. URL https://www.R‐project.org/.
(61) Ritz, C.; Streibig, J. C. J. Stat. Softw. 2005, 12, DOI:
10.18637/jss.v012.i05.
(29) Walther, T.; Arndt, H.‐D.; Waldmann, H. Org. Lett. 2008,
10, 3199.
(30) Dixon, M. J.; Nathubhai, A.; Andersen, O. A.; van Aalten,
D. M. F.; Eggleston, I. M. Org. Biomol. Chem. 2009, 7, 259.
(31) Pelay‐Gimeno, M.; García‐Ramos, Y.; Martin, M. J.;
Spengler, J.; Molina‐Guijarro, J. M.; Munt, S.; Francesch, A. M.;
Cuevas, C.; Tulla‐Puche, J.; Albericio, F. Nat. Commun. 2013, 4,
2352.
(32) Mazur, S.; Jayalekshmy, P. J. Am. Chem. Soc. 1979, 101, 677.
(33) White, C. J.; Yudin, A. K. Nat. Chem. 2011, 3, 509.
(34) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97,
2243.
(35) Stolze, S. C.; Kaiser, M. Synthesis 2012, 44, 1755.
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