Total Synthesis and Functional Evaluation of Fourteen Derivatives of Lysocin E: Importance of Cationic, Hydrophobic, and Aromatic Moieties for Antibacterial Activity

Article abstract of DOI:10.1002/chem.201604022

Lysocin E (1) is a structurally complex 37-membered depsipeptide comprising 12 amino-acid residues with an N-methylated amide and an ester linkage. Compound 1 binds to menaquinone (MK) in the bacterial membrane to exert its potent bactericidal activity. To decipher the biologically important functionalities within this unique antibiotic, we performed a comprehensive structure-activity relationship (SAR) study by systematically changing the side-chain structures of l-Thr-1, d-Arg-2, N-Me-d-Phe-5, d-Arg-7, l-Glu-8, and d-Trp-10. First, we achieved total synthesis of the 14 new side-chain analogues of 1 by employing a solid-phase strategy. We then evaluated the MK-dependent liposomal disruption and antimicrobial activity against Staphylococcus aureus by 1 and its analogues. Correlating data between the liposome and bacteria experiments revealed that membrane lysis was mainly responsible for the antibacterial functions. Altering the cationic guanidine moiety of d-Arg-2/7 to a neutral amide, and the C7-acyl group of l-Thr-1 to the C2 or C11 counterpart decreased the antimicrobial activities four- or eight-fold. More drastically, chemical mutation of d-Trp-10 to d-Ala-10 totally abolished the bioactivities. These important findings led us to propose the biological roles of the side-chain functionalities.

Full text of DOI:10.1002/chem.201604022

DOI: 10.1002/chem.201604022
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Natural Products
Total Synthesis and Functional Evaluation of Fourteen Derivatives
of Lysocin E: Importance of Cationic, Hydrophobic, and Aromatic
Moieties for Antibacterial Activity
Takuya Kaji,[a] Motoki Murai,[a] Hiroaki Itoh,[a] Jyunichiro Yasukawa,[a, b] Hiroshi Hamamoto,[a, c]
Kazuhisa Sekimizu,[a, c] and Masayuki Inoue*[a]
Abstract: Lysocin E (1) is a structurally complex 37-mem-
bered depsipeptide comprising 12 amino-acid residues with
an N-methylated amide and an ester linkage. Compound
1 binds to menaquinone (MK) in the bacterial membrane to
exert its potent bactericidal activity. To decipher the biologi-
cally important functionalities within this unique antibiotic,
we performed a comprehensive structure-activity relation-
ship (SAR) study by systematically changing the side-chain
structures of l-Thr-1, d-Arg-2, N-Me-d-Phe-5, d-Arg-7, l-Glu-
8, and d-Trp-10. First, we achieved total synthesis of the 14
new side-chain analogues of 1 by employing a solid-phase
strategy. We then evaluated the MK-dependent liposomal
disruption and antimicrobial activity against Staphylococcus
aureus by 1 and its analogues. Correlating data between the
liposome and bacteria experiments revealed that membrane
lysis was mainly responsible for the antibacterial functions.
Altering the cationic guanidine moiety of d-Arg-2/7 to a neu-
tral amide, and the C7-acyl group of l-Thr-1 to the C2 or C11
counterpart decreased the antimicrobial activities four- or
eight-fold. More drastically, chemical mutation of d-Trp-10 to
d-Ala-10 totally abolished the bioactivities. These important
findings led us to propose the biological roles of the side-
chain functionalities.
weight=1618 Da).[5] Compound 1 exhibits potent antimicrobi-
al activity against MRSA with a minimum inhibitory concentra-
tion (MIC) of 4 mgmLꢀ1 (2.5 mm). Moreover, treatment of S.
aureus infected mice with 1 markedly increased their survival
rate compared with that of untreated mice (ED50 =0.5 mgkgꢀ1
body weight). Remarkably, this therapeutic effect of 1 was
even stronger than that of vancomycin (ED50 =5.8 mgkgꢀ1),
which is widely used to treat MRSA infections.
Introduction
Bacterial infections increasingly evade standard treatments
with growing resistance to multiple antibiotics.[1] Antibiotic-re-
sistant infectious diseases thus pose a threat to public health
around the world.[2] In particular, nosocomial infections caused
by methicillin-resistant Staphylococcus aureus (MRSA) in hospi-
tals are a serious clinical problem. The emergence of multidrug
resistance has created an urgent need for the development of
effective antibiotics with new modes of action against such re-
sistant strains.[3,4] Lysocin E (1, Figure 1) was isolated from Lyso-
bacter sp. RH2180-5 strain as a novel antibiotic, and structurally
determined to be a 37-membered depsipeptide (molecular
The molecular target of 1 is distinct from that of any other
reported antibiotic. A series of mutational analyses revealed
that 1 directly binds to menaquinone (MK) within the bacterial
membrane. MK is an essential factor for electron transfer in the
bacterial respiratory chain.[6] Formation of the 1-MK complex is
considered to disrupt the functional integrity of the bacterial
membrane, resulting in rapid bacteriolysis.[5] Isothermal titra-
tion calorimetry experiments further demonstrated that
1 forms a 1:1 complex with MK, with a dissociation constant of
4.5 mm. In sharp contrast, no complexation occurs between
1 and ubiquinone (UQ), a coenzyme in the mammalian respira-
tory chain. The selectivity of 1 toward MK over UQ is attributa-
ble to the bacterial/mammalian cell selectivity of 1. Generally,
antibiotics that disrupt membranes are less likely to induce
drug resistance than antibiotics that target other bacterial sys-
tems due to the relatively conserved molecular composition of
lipids.[7] The potent bactericidal activity and advantageous
mode of action make naturally occurring 1 a useful lead com-
pound for developing new antibiotics.[8]
[a] T. Kaji, Dr. M. Murai, Dr. H. Itoh, Dr. J. Yasukawa, Dr. H. Hamamoto,
Prof. Dr. K. Sekimizu, Prof. Dr. M. Inoue
Graduate School of Pharmaceutical Sciences
The University of Tokyo, Hongo, Bunkyo-ku
Tokyo 113-0033 (Japan)
E-mail: inoue@mol.f.u-tokyo.ac.jp
[b] Dr. J. Yasukawa
Present address: Faculty of Pharmaceutical Sciences
Doshisha Women’s College of Liberal Arts
Kohdo, Kyotanabe, Kyoto 610-0395 (Japan)
[c] Dr. H. Hamamoto, Prof. Dr. K. Sekimizu
Present address: Teikyo University Institute of Medical Mycology
359 Otsuka, Hachioji, Tokyo, 192-0395 (Japan)
Supporting information containing experimental procedures, characteriza-
tion of the compounds, assay data, and spectroscopic data for this article
is available on the WWW under http://dx.doi.org/10.1002/chem.201604022.
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quence contains one Gly-4, five types of l-configured amino
acids (l-Thr-1/12, l-Ser-3, l-Leu-6, l-Glu-8, l-Ile-11), and four
types of d-configured amino acids (d-Arg-2/7, N-Me-d-Phe-5,
d-Gln-9, d-Trp-10), and (R)-3-hydroxy-5-methylhexanamide is
appended to l-Thr-1 of the macrocyclic core. We recently re-
ported the first total synthesis of this structurally complex pep-
tidic natural product 1 by applying a full solid-phase strat-
egy.[9,10] The approach we developed allowed for synthetic
access to the enantiomeric (ent-1), epimeric (2), and N-deme-
thylated (3) analogues of 1. Synthesized 1 and its three ana-
logues were biologically evaluated in an antimicrobial assay
against S. aureus. The enantiomer ent-1 exhibited activity
(4 mgmLꢀ1) comparable to that of 1, and a single stereocenter
change in the acyl chain of Thr-1 (1!2) did not largely affect
the potency (8 mgmLꢀ1). In contrast, the antibacterial property
of N-demethylated analogue 3 (32 mgmLꢀ1), which has a differ-
ent macrocycle conformation from that of 1 based on NMR
analysis, was decreased eight-fold.[11] Indifference of the abso-
lute configuration of 1 corroborated the importance of the rec-
ognition of achiral MK, and not that of chiral biomolecules
such as proteins for the activity, and the decreased activity of
3 suggested the significance of the main-chain structure for or-
ganizing the bioactive three-dimensional shape.
The preliminary structure-activity relationship (SAR) study
prompted us to perform another set of SAR studies on the
side-chains of 1 (Figure 1).[12–14] Specifically, the 15 newly de-
signed analogues 4–18 all have the same main-chain structure
but the side-chain structures are diversified. Herein we report
the highly efficient total syntheses of the 15 lysocin analogues
4–18 by utilizing two solid-phase strategies. The functions of
1 and its analogues were systematically evaluated based on
the MK-dependent membrane lysis of liposomes and antibac-
terial activity against S. aureus. Comprehensive SAR studies of
the analogues shed light on the side-chain functionalities rele-
vant to the molecular mode of action of 1, thereby providing
valuable information for the search of optimized antibiotics.
Results and Discussion
Before planning the sites for the chemical modification of 1,
we hypothesized the following three potentially attractive in-
teractions among 1, MK, and phospholipids (Figure 1): 1) an
electrostatic interaction of the anionic carboxylate group of l-
Glu-8 or the cationic guanidine moieties of d-Arg-2/7 with the
polar head group of phospholipids or the carbonyl groups of
MK; 2) a hydrophobic interaction of the acyl chain of l-Thr-
1 with the lipid chains of MK or phospholipids; and 3) an aro-
matic–aromatic interaction of the phenyl group of N-Me-d-
Phe-5 or the indole ring of d-Trp-10 with the naphthoquinone
structure of MK. To systematically investigate the significance
of each of these interactions, the functional groups of l-Thr-1,
d-Arg-2, N-Me-d-Phe-5, d-Arg-7, l-Glu-8, and d-Trp-10 were
planned to be replaced in analogues 4–18. In 4 and 5, we sub-
stituted a neutral amide for the carboxylic acid of 1, and in 6–
9 we altered the guanidine groups of d-Arg-2/7. In 10, we re-
moved the acyl chain of 1; in 11–16, we exchanged the C7-
Figure 1. Structures of lysocin E (1), analogues 2-18, menaquinone-4 (MK-4,
19), and ubiquinone-10 (UQ-10, 20). The names of the amino acid residues
of 1 are written in the structure. Substituted moieties of the analogues are
displayed in red. Boc=tert-butoxycarbonyl; TFA=trifluoroacetic acid.
The 37-membered macrocycle of 1 comprises 12 amino acid
residues with an N-methylated amide at N-Me-d-Phe-5 and an
ester linkage between l-Thr-1 and l-Thr-12 (Figure 1). The se-
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Figure 2. Building blocks for total syntheses of 1 and analogues 4–18. Circled numbers are residue numbers (gray: common amino acids, red: alternative
amino acids for the synthetic analogues). TBS=tert-butyldimethylsilyl; Fmoc=9-fluorenylmethoxycarbonyl; Pbf=2,2,4,6,7-pentamethyldihydrobenzofuran-5-
sulfonyl; tBu=tert-butyl; Trt=triphenylmethyl.
acyl chain of 1 into the C2- to C11-counterparts; and in 17 and
18, we deleted the phenyl and indole rings, respectively.
The total synthesis of 1 was previously achieved by using
the full solid-phase strategy without tedious purification of the
intermediates.[9] The route to 1 employed 21–31 as monomers
(Figure 2) and involved the following operations: 1) stepwise
solid-phase assembly of the linear 12-mer sequence using
Fmoc-based solid-phase peptide synthesis (SPPS)[15,16] from l-
Glu-8 (24), the side-chain of which was anchored to Wang
resin;[17] 2) orthogonal deprotection of the allyl group of l-Glu-
8 to selectively liberate a Ca-carboxyl group; 3) intramolecular
amidation between the free Ca-carboxyl and Na-amino groups,
Scheme 1. Synthesis of ester 33. DIC=N,N’-diisopropylcarbodiimide;
DMAP=N,N-dimethyl-4-aminopyridine; DMF=N,N-dimethylformamide.
taking advantage of the pseudo high dilution phenomenon of
the resin-bound molecule;[18] and 4) simultaneous acid-promot-
ed cleavage (Wang resin) and global deprotection of the side-
chain protective groups (Pbf,[19] tBu, TBS, Boc, and Trt) to re-
lease 1.[20] This first-generation strategy was utilized to synthe-
size 4–9 with the same acyl group, and was modified to the
second-generation strategy to prepare another series of ana-
logues, 10–18. Specifically, the macrocyclic amine 10 was de-
signed as a common intermediate for the alternative strategy
to enable efficient synthetic access to 11–16 by a single-step
acylation. To construct 10, the ester linkage within the macro-
cycle was envisioned to be preformed by solution-phase
chemistry to avoid ester condensation, which is less efficient
than the amide counterpart on the solid matrices. Therefore,
the second-generation strategy was planned to utilize N-Boc-
protected ester 33 instead of the acyl chain-linked 27 and 28.
Among all the components 21–35 necessary for the assem-
bly of 4–18, 27 and 33 required synthetic preparation
(Figure 2). The route to 27 was described previously.[9]
Scheme 1 depicts the three-step synthesis of the building
block 33 from Boc-l-Thr-OH (36) for the second-generation
synthesis. Treatment of carboxylic acid 36 with BnBr and K2CO3
afforded benzyl ester 37. The hydroxy group of 37 was in turn
condensed with the carboxylic acid of Fmoc-l-Thr(tBu)-OH (28)
in the presence of DIC and DMAP to produce 38. Hydrogenoly-
sis of the benzyl group of 38 with Pd/C gave rise to the requi-
site Boc-protected ester 33 in 88% yield.
With all the building blocks in hand, we began the first-gen-
eration solid-phase synthesis of lysocin E (1) and 6 from allyl
glutamate-loaded resin 24 (Scheme 2A). Natural 1 and its ana-
logue 6 were assembled from components 21-31, and 21-28,
30, 31, and 32, respectively. Based on our previous report,[9]
the peptide chain of 1 or 6 was elaborated at 408C under mi-
crowave (MW)-assisted conditions[21] to facilitate the reactions.
Cycles of piperidine-promoted Na-deprotection and HOBt/
HBTU[22]-mediated amide coupling were applied to 24 using
29, 23, 30, 22, 21, and 29 for 1; or 32, 23, 30, 22, 21, and 32
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Scheme 2. A) First-generation solid-phase synthesis of 1 and 6, and preparation of 4–9. B) Second-generation solid-phase synthesis of 10 and 45b/c, and
preparation of 11–18. MW-assisted SPPS: i) 20% piperidine/DMF; ii) Na-Fmoc-protected amino acid, HBTU, HOBt, iPr2NEt, DMF, NMP, 408C. HBTU=O-benzotria-
zole-N,N,N’,N’-tetramethyl-uronium-hexafluoro-phosphate; HOBt=1-hydroxybenzotriazole; IBCF=isobutylchloroformate; NMM=N-methylmorpholine;
NMP=N-methyl-2-pyrrolidone; PyBOP=(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate.
for 6. This sequence resulted in the formation of the resin-
bound heptapeptide 39a or 39b after removing the Fmoc
group at the N-terminus. To prevent epimerization at the Ca-
position of amide 27, condensation of amine 39a/b with (R)-3-
hydroxy-5-methylhexanamide-substituted 27 was preemptively
conducted at room temperature without MW irradiation to
produce the octapeptide. The subsequent DIC/DMAP-promot-
ed esterification of the obtained peptide with 28 gave rise to
nonapeptide 40a/b. The MW-assisted Na-deprotection/conden-
sation protocol was re-applied to 40a/b for stepwise elonga-
tion of the three residues (26, 31, and 25) to deliver the linear
dodecapeptide. The Fmoc group of the N-terminus and the
allyl group of the C-terminus of the product were then re-
moved by treatment with piperidine, and subsequently with
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[23]
catalytic Pd(PPh3)4 and an excess of morpholine to provide
Remarkably, the post-SPPS acylation of non-protected 10
chemoselectively occurred at the Na-amino group of l-Thr-1,
permitting rapid access to the six acyl-chain analogues.
Namely, TFA salt 10 was treated with activated carboxylic
acids, which were prepared from carboxylic acids 50, 51, 52,
53, 54, and 55 in the presence of IBCF and NMM, leading to
analogues 11, 12, 13, 14, 15, and 16 in 34, 51, 29, 33, 41, and
25% yields, respectively.
the macrolactam precursor 41a/b. Facile on-resin cyclization of
41a/b was then effected using PyBOP[24]/2,4,6-collidine, leading
to the 37-membered macrolactam. Finally, treatment of the
macrolactam with 95% aqueous TFA simultaneously realized
cleavage from the Wang resin and global deprotection of the
acid-labile protective groups (Pbf, tBu, Boc, Trt, and TBS
groups), releasing 1/6 into solution. After purification by re-
versed-phase HPLC, lysocin E (1) and its derivative 6 were ob-
tained in 8.0 and 6.1% yields, respectively, over 25 steps from
24.
The thus-obtained fully deprotected 1 and 6 were further
transformed into the five analogues 4, 5, and 7–9, by applying
chemoselective single-step reactions. The carboxylic acid of l-
Glu-8 of 1 was condensed with benzylamine 46 or mono-Boc-
protected diamine 47 by using PyBOP to furnish the amide an-
alogues 4 and 5 in 48 and 44% yields, respectively. Alternative-
ly, the two primary amines of 6 were functionalized to gener-
ate 7–9. Treatment of 6 with 1H-pyrazole-N,N-dimethyl-1-car-
boxamidine (48),[25] nitrourea (49), and Ac2O resulted in the for-
mation of dimethylguanidine (7, 46%), urea (8, 58%), and
acetyl (9, 27%) analogues, respectively.
The second-generation strategy was applicable for the total
syntheses of N-Me-d-Phe-5-substituted 17 and d-Trp-10-substi-
tuted 18. By use of monomers 34 and 35 in place of the origi-
nal 30 and 31, the linear dodecapeptides 44b and 44c were
obtained by the Fmoc-based SPPS (24!42!43b!44b, and
24!39a!43a!44c, respectively). Macrolactamization and
subsequent TFA treatment transformed 44b and 44c into 45b
in 6.5% overall yield, and 45c in 12% overall yield, respective-
ly. Lastly, the targeted aromatic ring analogues 17 and 18 were
synthesized from amine-TFA salts 45b and 45c using the IBCF-
activated ester of 56 and NMM in 22 and 34% yields, respec-
tively.[26] The total syntheses of the 15 structurally complex and
diverse analogues 4–18 clearly demonstrated the robustness
and generality of the present synthetic strategies.
Next, the second-generation strategy was utilized to con-
struct the common macrocyclic amine 10 (Scheme 2B). Hepta-
peptide 39a, which was also used in the first-generation syn-
thesis, was prepared from the Wang-resin tethered 24 by re-
peated cycles of piperidine-promoted Fmoc removal and MW-
assisted condensation with the six Fmoc-amino acids. N-Boc-
protected ester 33 was in turn attached using HBTU/HOBt,
giving rise to nonapeptide 43a. Three additional cycles of Na-
deprotection and MW-promoted chain-elongation (26, 31, and
25), followed by detachment of the Fmoc and allyl groups,
provided the macrolactamization precursor 44a. Finally, the
on-resin macrolactamization by the action of PyBOP, and the
TFA-promoted global deprotection and resin cleavage fur-
nished amine 10 as its TFA salt (26% yield over 24 steps from
24). The excellent overall yield of 10 compared with that of
1 (8.0%) verified the advantage of direct attachment of the
pre-esterified 33 over separate application of the two mono-
mers 27 and 28. Despite its stability under acidic conditions,
the 37-membered lactone of 10 readily converted into 36-
membered lactam 58 via an O to N acyl transfer in solution at
pH 7 or above (Scheme 3, see also Supporting Information).
Having successfully prepared all of the analogues, the MK-
dependent membrane disruption and antimicrobial activities
of the 14 analogues (4–9 and 11–18) were assessed along with
the parent compound 1. To estimate the MK-dependent mem-
brane lytic activity without the interference of other biological
molecules, the first assay employed liposomes containing only
MK or UQ and lipids.[5] In general, bacterial membranes are
negatively charged with lipids bearing phospholipid head
groups such as phosphatidylglycerol (PG) and cardiolipin,
whereas mammalian membranes are enriched in zwitterionic
phospholipids (neutral in net charge) such as phosphatidylcho-
line (PC) sphingomyelin.[27] To mimic the bacterial membranes,
large unilamellar vesicles (LUVs) containing PGs were used as
liposomes. Specifically, LUVs comprising a 1:1 ratio of egg yolk
PC (EYPC)/egg yolk PG (EYPG) were prepared in the presence
of 1.25 mol% of MK-4 (19)[28] or UQ-10 (20). Carboxyfluorescein
(CF) was encapsulated as a fluorescent indicator in the LUVs.
Although fluorescence of the CF molecules within the LUVs is
self-quenched due to the high concentration, membrane dis-
ruption by peptides causes the CF to leak from LUVs, resulting
in dilution of the CF molecules and increase of fluorescence in-
tensity. Therefore, fluorescence was measured as an indicator
of the LUV membrane disruption. The fluorescence change
caused by lysis in the presence of each peptide was standar-
dized according to the maximum intensity induced by adding
Triton X-100.
When 2.5 mm of lysocin E (1) was added to the LUVs with
either 1.25 mol% of MK (19) or UQ (20) (Figure 3), only the
MK-containing LUVs showed increased fluorescence, corrobo-
rating the MK-dependency of the membrane rupture by 1. The
non-activity toward the UQ-containing LUVs was consistent for
all of the analogues, confirming their non-disruption activities
without MK (red lines). Interestingly, the ability to disrupt the
MK-doped membrane varied significantly. The fluorescence
changes (%) of all the analogues after reaching their plateaus
Scheme 3. Undesired O to N acyl transfer of 10 in pH 7 buffer.
Based on this specific physicochemical feature of 10, we decid-
ed not to use 10 for the functional evaluation, and it was in-
stead directly subjected to acylation reactions.
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Table 1. Membrane-disrupting and antimicrobial activities of the natural
1, analogues 4–9 and 11–18.
Compounds
Membrane disruption [%][a]
MIC[e]
[mgmLꢀ1
]
1.25 mol% 19
1.25 mol% 20
2.5 mm[b,d] 10 mm[c,d] 2.5 mm[c,d] 10 mm[c,d]
1
4
5
6
7
8
9
11
12
13
14
15
16
17
18
62
64
93
62
55
1.0
0
0
2.2
5.1
0.9
0
0
0
0
0
4
4
2
4
4
8
16
16
4
2
4
4
32
7.4
0
51
48
65
62
42
20
9.3
0
0.7
0
0
0.8
0
65
0
0
0
8
0
>128
[a] The membrane disruption activities (%) were determined from fluores-
cence intensity between 600–650 s. The LUVs consisted of EYPC/EYPG
(1:1) containing 1.25 mol% 19 or 20. [b] The mean values of the duplicate
experiments. [c] The values of the single experiment. [d] Final concentra-
tion of peptides. [e] Minimal concentration required to inhibit cell growth
of methicillin-susceptible S. aureus. 4 mgmLꢀ1 of lysocin E (1) corresponds
to 2.5 mm.
the MK-dependent membrane rupture (9.3%). The assay data
of 4–9 revealed the lack of impact of the anionic character of
l-Glu-8 and the importance of the cationic character of d-Arg-
2/7 for the activity. Accordingly, it is likely that the guanidine
(1), amine (6), and dimethylguanidine (7) structures play an im-
portant role in binding to the anionic lipid head groups of PG
through attractive electrostatic interactions to induce mem-
brane rupture.
The C2- (11), C4- (12) C6- (13), C7- (14), and C9- (15) acyl
chain-modified analogues exhibited similar membrane-disrupt-
ing activities to that of the parent 1 with the hydroxylated C7-
acyl group at l-Thr-1 (51, 48, 65, 62, and 42% for 11, 12, 13,
14, and 15, respectively). Although the more hydrophobic mol-
ecule 16 with the C11-acyl chain was expected to more favora-
bly insert into the hydrophobic membrane, 16 turned out to
have lower activity (20%). The lower water solubility of 16
likely reduced the effective concentration of 16, thereby de-
creasing its activity.
Figure 3. Time-course of CF leakage from LUVs [EYPC/EYPG (1:1) containing
1.25 mol% 19 or 20] caused by a) 2.5 mm of 1, 4, and 5, b) 2.5 mm of 6–8
and 10 mm of 9, c) 2.5 mm of 11–16, and d) 10 mm of 17 and 18. Each ana-
logue was added at 50 s. CF leakage caused by each analogue was deter-
mined by the fluorescence intensity of leaked CF standardized against the
maximum leakage by Triton X-100. CF=carboxyfluorescein; EYPC=egg yolk
phosphatidylcholine; EYPG=egg yolk phosphatidylglycerol; LUV=large uni-
lamellar vesicle.
(600–650 s) were quantified for comparison of the activities
(Table 1). In these experiments, the natural 1 exhibited 62%
disruption at 2.5 mm. Exchange of the anionic carboxylate of l-
Glu-8 of 1 with the neutral amides of 4 and 5 did not decrease
the membrane-disrupting activity compared to 1 (62% for 1,
64% for 4, and 93% for 5). When the cationic guanidine moiet-
ies of d-Arg-2/7 of 1 were substituted with the other cationic
amine (6) and dimethyl guanidine (7) functionalities, the po-
tency was retained (62% for 6 and 55% for 7) despite the dif-
ference in steric sizes of the functional groups. In sharp con-
trast, applying the neutral urea (8) and amide (9) analogues di-
minished the fluorescence change (7.4% for 8 and 0% for 9).
A higher concentration of 9 (10 mm) was required to observe
Drastic changes in the membrane-disrupting activities were
observed upon assay of the aromatic ring-modified analogues
17 and 18. Removing the phenyl group of N-Me-d-Phe-5 (17)
or the indole ring of d-Trp-10 (18) abolished the activities
(0.8% for 17 and 0% for 18). When the concentrations of the
peptides were increased from 2.5 mm to 10 mm, the des-phenyl
analogue 17 regained its activity (65%), but the des-indole an-
alogue 18 was inert. Because all 14 analogues except for 18
possessed the activity, the indole ring appeared to be the
most essential structural unit of 1 for the MK-dependent mem-
brane-disruption.
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Finally, an antibacterial assay for the 14 lysocin derivatives
4–9 and 11–18 was carried out using S. aureus (Table 1). All 14
analogues, except for 18, were found to possess antimicrobial
activitity. The MIC values of the parent 1 and 10 analogues (4,
5, 6, 7, 8, 12, 13, 14, 15, and 17) were within single-digit mi-
cromolar range (2-8 mgmLꢀ1), and those of the other three an-
alogues (9, 11, and 16) were double-digit micromolar (16-
32 mgmLꢀ1). The des-indole analogue 18 showed no antimicro-
bial activity up to 128 mgmLꢀ1. The effect of the polar function-
al groups of 1 on the antibacterial activities was well-correlated
with the membrane-disrupting activities. The low MIC values (2
or 4 mgmLꢀ1) were preserved upon alteration of the anionic
carboxylate group of l-Glu-8 of 1 to the neutral amides (4, 5),
or exchange of the cationic guanidines of d-Arg-2/7 to the cat-
ionic amines (6) and dimethyl guanidines (7). On the other
hand, deletion of the cationic character increased the MIC
values (8 mgmLꢀ1 for 8, and 16 mgmLꢀ1 for 9), again emphasiz-
ing the significance of the cationic functions of 1 for the
potent activities.
tively. On the membrane surface, the electron-rich indole of d-
Trp-10 and the electron-deficient naphthoquinone of MK bind
as a result of the aromatic-aromatic interaction,[30] leading to
the formation of the equimolar 1-MK complex. Finally, the
complexation causes membrane damage and eventual cell
death. This new hypothetical mechanism of action provides us
with valuable information for designing more active and selec-
tive antibiotics based on the lysocin structure.
Conclusion
In summary, we performed comprehensive SAR studies of the
14 new analogues of a structurally complex 37-membered
depsipeptide, lysocin E (1). The 14 analogues 4–9 and 11–18
were designed to decipher the biological roles of the anionic
residue l-Glu-8, the cationic residues d-Arg-2/7, the hydropho-
bic N-acyl group of l-Thr-1, and the aromatic residues d-Trp-
10/N-Me-d-Phe-5. The two different full solid-phase synthetic
routes allowed for the efficient access to 1 and the 14 structur-
ally diverse analogues 4–9 and 11–18. The first- and second-
generation SPPS strategies assembled 1, 6, 10, 45b, and 45c,
which were further derivatized into 4/5, 7–9, 11–16, 17, and
18, respectively, by chemoselective post-SPPS modifications.
MK-dependent membrane-disrupting activity using LUVs and
antibacterial activity using S. aureus were evaluated for the 14
synthesized analogues, 4–9 and 11–18. The prominent func-
tionalities that were necessary for the potent liposomal and
antimicrobial activities were established to be the cationic gua-
nidines of d-Arg-2/7, the hydrophobic acyl group of four to
nine carbons at l-Thr-1, and the indole ring at d-Trp-10. These
novel findings provide the first evidence for the hypothetical
roles of these functionalities. After the cationic guanidine moi-
eties and the hydrophobic acyl chain effectively bind to the
negatively charged bacterial membrane, the aromatic-aromatic
interaction between the indole ring and the naphthoquinone
of MK within the membrane leads to critical membrane
damage. The present achievements highlight the power of
robust and divergent solid-phase strategies for systematic SAR
studies, and chart a rational path forward for development of
lysocin-based next-generation antibiotics for treating various
infectious diseases.
Despite their relative unimportance in the liposome experi-
ments, the lengths of the acyl chains at l-Thr-1 influenced the
MIC values of 11–16. The antimicrobial activity of analogues
12–15 possessing C4- to C9-acyl chains was comparable (2-
4 mgmLꢀ1) to that of 1, whereas that of C2-acyl analogue 11
and C11-acyl analogue 16 was weaker (16 and 32 mgmLꢀ1, re-
spectively). The discrepancies between the liposome and bac-
teria assays could originate from the differences in the surface
structures between the simple model membranes and the bac-
terial plasma membrane/peptidoglycan layer containing many
other components. Nevertheless, these antimicrobial data indi-
cated the importance of the appropriate hydrophobicity of
this moiety for the bioactivity. Due to the likelihood that most
hydrophilic 11 does not partition into hydrophobic lipids, and
most hydrophobic 16 does not dissolve in aqueous media in
effective fashions, the lower activities of 11 and 16 are attribut-
able to the changed physicochemical properties.
As expected from the membrane-disrupting activities of 17
and 18, deletion of the phenyl ring or the indole ring reduced
the antimicrobial activity. The MIC value of the N-Me-d-Phe-5-
substituted analogue 17 was 2-fold higher (8 mgmLꢀ1) than
that of 1. On the other hand, the d-Trp-10-substituted ana-
logue 18 totally lost its activity (>128 mgmLꢀ1). The activities
of 17 and 18 proved that the indole structure of 1 is more bio-
logically essential than the phenyl ring. As all the lysocin-based
structures with the indole ring (1, 4–9 and 11–17) retained
both the MK-dependent membrane-disrupting and antimicro- Acknowledgements
bial activities, the indole ring could serve as the requisite rec-
ognition unit of the MK structure.
This research was financially supported by Grant-in-Aids for
Overall, the key structural features that are crucial for the
two types of activities were established to be 1) the cationic
functionalities of d-Arg-2 and -7, 2) the hydrophobic acyl
group of four to nine carbons at l-Thr-1, and 3) the indole ring
of d-Trp-10. These results offered a clearer picture of the mode
of action of the original 1. The cationic guanidine moieties of
d-Arg-2/7 and the hydrophobic (R)-3-hydroxy-5-methylhexana-
mide of l-Thr-1 help 1 bind through the anionic polar heads[29]
and hydrophobic lipid tails of the bacterial membrane, respec-
Scientific Research (A) (JSPS KAKENHI Grant Number
JP26253003) and Scientific Research on Innovative Areas (JSPS
KAKENHI Grant Number JP16H01130) to M.I., for Research Ac-
tivity Start-up (JSPS KAKENHI Grant Number JP15H06156) to
H.I., and for Scientific Research (S) (JSPS KAKENHI Grant
Number JP15H05783) and JST A-STEP to K.S. A JSPS fellowship
to T.K. is gratefully acknowledged. We thank Drs. T. Kuranaga
(Hokkaido University) and M. Urai (National Institute of Infec-
tious Diseases) for valuable discussion.
Chem. Eur. J. 2016, 22, 1 – 9
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Keywords: antibiotics · peptides · solid-phase synthesis ·
structure-activity relationships · total synthesis
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Received: August 24, 2016
Published online on && &&, 0000
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FULL PAPER
What parts work best? Antibiotic lyso-
cin E (1), a 37-membered depsipeptide,
binds to menaquinone in the bacterial
membrane to exert its potent bacterici-
dal activity (see figure). Comprehensive
SAR studies of 1 were performed by sys-
tematically changing the side-chain
structures. The key structural features
for the antimicrobial activities were es-
tablished to be the cationic functionali-
ties at d-Arg-2/7, the hydrophobic acyl
group at l-Thr-1, and the indole ring at
d-Trp-10.
&
Natural Products
T. Kaji, M. Murai, H. Itoh, J. Yasukawa,
H. Hamamoto, K. Sekimizu, M. Inoue*
&& – &&
Total Synthesis and Functional
Evaluation of Fourteen Derivatives of
Lysocin E: Importance of Cationic,
Hydrophobic, and Aromatic Moieties
for Antibacterial Activity
Chem. Eur. J. 2016, 22, 1 – 9
www.chemeurj.org
9
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ