Total Synthesis of Laspartomycin C and Characterization of Its Antibacterial Mechanism of Action

Article abstract of DOI:10.1021/acs.jmedchem.6b00219

Laspartomycin C is a lipopeptide antibiotic with activity against a range of Gram-positive bacteria including drug-resistant pathogens. We report the first total synthesis of laspartomycin C as well as a series of structural variants. Laspartomycin C was found to specifically bind undecaprenyl phosphate (C₅₅-P) and inhibit formation of the bacterial cell wall precursor lipid II. While several clinically used antibiotics target the lipid II pathway, there are no approved drugs that act on its C₅₅-P precursor.

Full text of DOI:10.1021/acs.jmedchem.6b00219

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Brief Article
Total Synthesis of Laspartomycin C and
Characterization of its Antibacterial Mechanism of Action
Laurens HJ Kleijn, Sabine F. Oppedijk, Peter 't Hart, Roel M. van Harten, Leah
A. Martin-Visscher, Johan Kemmink, Eefjan Breukink, and Nathaniel I. Martin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00219 • Publication Date (Web): 11 Mar 2016
Downloaded from http://pubs.acs.org on March 13, 2016
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Total Synthesis of Laspartomycin C and Characterization of its
Antibacterial Mechanism of Action
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a
b
a
a
c
Laurens H.J. Kleijn, Sabine F. Oppedijk, Peter ‘t Hart, Roel M. van Harten, Leah A. Martin-Visscher, Johan
a
Kemmink, Eefjan Breukink, Nathaniel I. Martin *
b
a ,
a
Department of Medicinal Chemistry & Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University,
b
Universiteitsweg 99, 3584 CG Utrecht, The Netherlands. Membrane Biochemistry and Biophysics Group, Department of Chemistry,
c
Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. The King’s University, 9125 50th Street, Edmonton Alberta
T6B 2H3, Canada.
ABSTRACT: Laspartomycin C is a lipopeptide antibiotic with activity against a range of Gram-positive bacteria including drug-resistant
pathogens. We report the first total synthesis of laspartomycin C as well as a series of structural variants. Laspartomycin C was found to
specifically bind undecaprenyl phosphate (C55-P) and inhibit formation of the bacterial cell wall precursor lipid II. While several clinically-
used antibiotics target the lipid II pathway, there are no approved drugs that act on its C55-P precursor.
INTRODUCTION
O
OH
D
Laspartomycin C is a cyclic lipopeptide that belongs to the
family of calcium-dependent antibiotics (CDAs). Originally
isolated from Streptomyces viridochromogenes, laspartomycin C
has been found to possesses antibacterial activity against a variety of
N
11
N
1
H
O
HN
HN
O
O
H
N
H
O
N
1
N
H
O
2
O
O
OH
O
O
O
N
D
OH
3
NH
NH
O
Gram-positive
pathogens
including
methicillin-resistant
O
H
N
Staphylococcus aureus (MRSA), vancomycin-intermediate S.
aureus (VISA), vancomycin-resistant S. aureus (VRSA) and
N
H
O
Laspartomycin C
(1)
O
2
,3
OH
OH
vancomycin-resistant enterococci (VRE). The structure of
laspartomycin C (1, Figure 1) was elucidated in 2003 showing it to
O
1
,4
O
be a member of the CDA family . In the same year daptomycin
2) became the first CDA to receive FDA approval for clinical use
O
OH
NH2
13 H
(
NH2
N
D
N
H
5
O
H
N
and it is now a widely used antibiotic of last resort. In addition,
surotomycin, a semi-synthetic analog of daptomycin, is currently
undergoing phase 3 clinical trials for the treatment of Clostridium
O
O
H
N
O
O
O
O
HN
O
1
N
H
D
N
H
O
4
O
O
HN
NH
HN
OH
6
OH
5
O
difficile infections. In light of emerging bacterial resistance to
O
NH
O
O
NH
O
O
conventional antibiotics, interest in the CDAs has grown steadily
over the past two decades. This increased interest is driven by
findings which indicate that the various CDAs operate via modes of
H
N
D
N
H
O
Daptomycin
2)
(
NH2
7
,8
OH
action unlike those of conventional antibiotics.
Figure 1. Structures of laspartomycin C (1) and daptomycin (2)
indicating N-terminal lipids (red) and conserved Asp-X-Asp-Gly motif
Laspartomycin C, also known as glycinocin A, consists of a 10
amino acid cyclic core and an N-terminal exocyclic region. The
4
(
blue). The peptide macrocycles are formed biosynthetically via
macrocycle contains a number of nonproteinogenic and D-amino
acids including L-2,3-diaminopropionic acid (L-2,3-Dap), D-
pipecolic acid (D-Pip), and D-allo-threonine, as well as the Asp-X-
cyclization of the C-terminal residue with the side chain of L-2,3-Dap2
in laspartomycin C or Thr4 in daptomycin (linkages shown in green).
2
+
Asp-Gly motif, which is implicated in Ca binding and conserved
among all known CDAs. Laspartomycin C bears an unsaturated
and branched C15 fatty acid tail linked to the exocyclic N-terminal
aspartic acid residue. By comparison, daptomycin contains a larger
exocyclic tripeptide unit terminated with a straight chain, fully
saturated C10 lipid. In addition, while the 10 amino acid macrocycle
in daptomycin is formed via an ester linkage between its C-terminal
its C-terminal proline and the side chain of the L-2,3-Dap residue at
position 2.
2
+
Interestingly, while all CDAs require the presence of Ca to
achieve their antimicrobial activity, they do not all act via the same
7
antibacterial mechanism. Daptomycin is the most potent of the
known CDAs and acts on the bacterial membrane where the
proposed formation of daptomycin oligomers is believed to induce
residue (L-kynurenine) and
a threonine side chain, the
8
-10
laspartomycin C macrocycle is closed via an amide linkage between
membrane perturbation. Alternatively, the CDAs amphomycin
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and friulimicin B were recently shown to exert their bactericidal
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O
O
effect by inhibiting bacterial cell wall synthesis through complex
formation with the essential cell wall precursor undecaprenyl-
H
NHAlloc
N
Dmb
O
O
N
H
O
N
a
O
O
b
Dmb
O
OtBu
1
1,12
N
O
phosphate (C55-P).
While laspartomycin C shares structural
N
Fmoc
OtBu
O
NH
O
NH
O
similarities with amphomycin and friulimicin B (all three contain
amide-linked macrocycles flanked by D-Pip and Pro residues at
positions 3 and 11), the mechanistic details of its antibiotic mode of
action have not been reported. Furthermore, while multiple
H
N
3
N
4
O
Dmb
O
OtBu
1
3,14
O
OH
syntheses of daptomycin have been reported,
to date no other
N
O
N
H
CDAs have been prepared in synthetic fashion. In this study we
report the first total synthesis of laspartomycin C as well as the
preparation and evaluation of a series of chimeric lipopeptides that
also comprise elements of the daptomycin macrocycle. In addition,
we here describe the application of a range of biochemical and
biophysical approaches in characterizing the antibacterial
mechanism of laspartomycin C.
O
NH2
O
O
O
c
9
H
N
N
H
Laspartomycin C
(1)
0
1
2
3
4
5
6
7
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9
0
1
2
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0
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0
1
2
3
4
5
6
7
8
9
0
Dmb
N
H
O
N
O
O
OtBu
O
O
N
OtBu
NH
O
NH
O
O
H
N
N
5
O
Dmb
O
OtBu
Scheme 1. a) Fmoc SPPS; b) (i) Pd[(C
1h (ii) Fmoc SPPS; c) (i) HFIP, CH
CH Cl , 16h (iii) TFA, TIS, H O, 1h (Fmoc-D-Thr was employed
6
H
Cl
5
)
3
P]
4
, C
6
H
5
SiH
3
, CH
2
Cl ,
2
2
2
, 1h; (ii) BOP, DIPEA,
RESULTS AND DISCUSSION
2
2
2
without side chain protection and incorporated without incident).
Our synthetic route to laspartomycin C involved a strategy
wherein cyclization at Gly8 provided the laspartomycin C
macrocycle (Scheme 1). Ring closure at glycine offers the inherent
advantage of avoiding racemization upon activation and
cyclization, an approach we previously employed in the preparation
15
Figure 2). This modification led to a significant loss of
antimicrobial activity, likely due to the different conformational
restrictions associated with an amide linkage compared to an ester.
In this regard it is interesting to note that in laspartomycin C and
the structurally-related amphomycins and friulimicins,
conformationally restricted cyclic amino acids are found on either
side of ring-closing amide linkage. The presence of Pro and D-Pip
residues likely play an important role in establishing the biologically
relevant conformation of the peptide. The flexibility of our
synthetic route to laspartomycin C provides convenient access to
structurally related compounds. To this end we next prepared a
series of analogues to specifically investigate whether incorporating
conformationally restricting amino acids would improve the
antibacterial activity of our previously prepared daptomycin
“amide-analogues” 6 and 7. As illustrated in Figure 2, analogs 8 and
9 contain a Gly to D-Pip mutation in comparison with compounds
6 and 7. Analogue 10 bears a Pro in place of the Kyn normally
present in daptomycin and in analogue 11 both Pro and D-Pip
residues are introduced as in laspartomycin C. Analogue 12
represents a hybrid structure wherein the laspartomycin C
macrocyle is augmented with the daptomycin exocyclic tripeptide
unit and C10 lipid tail. The synthesis of analogues 8-12 proceeded
without incident and with no detectable racemization to provide
the target compounds in an average yield of 7.1% (30 reaction
steps) based on initial resin loading.
15
of various daptomycin analogues. The solid phase portion of the
laspartomycin synthesis began with immobilization of
C
Fmoc(Dmb)-Gly-OH on the 2-chlorotrityl resin (the Dmb
protecting group was employed at Gly6 and Gly8 to prevent
possible aspartamide formation). Interestingly, treatment of the
resin-bound Fmoc(Dmb)-Gly with piperidine:DMF did not result
in complete Fmoc deprotection nor did treatment with a more
potent mixture of DBU:piperidine:DMF. However, treatment with
ethanolamine:DMF did lead to full Fmoc removal indicating that
deprotection of resin-bound Fmoc(Dmb)-Gly requires a less
sterically-hindered base. After coupling of aspartic acid, the Fmoc
loading of the resin was determined spectrophotochemically (0.52
-
1
mmol g ). Standard Fmoc SPPS was then employed to obtain
intermediate 4 with N-terminal acylation achieved using (E)-13-
methyltetradec-2-enoic acid (synthesized in 6 steps from methyl
undec-10-enoate, see Supplemental Scheme S2). Removal of the
side chain Alloc protecting group in resin-bound 4, followed in
turn by addition of the remaining three amino acids, then provided
intermediate 5. Cleavage from the resin using mild acidic
conditions yielded the protected peptide, which was directly
subjected to solution phase cyclization. Complete conversion from
the linear to the cyclic peptide was achieved within 12 hours (as
evidenced by HPLC analysis) followed by global deprotection.
After purification by reverse phase HPLC, NMR analysis showed
that the synthetic peptide was identical to natural laspartomycin C,
The antibacterial activities of laspartomycin C, daptomycin, as
well as the chimeric analogues were determined against
Staphylococcus aureus and Staphylococcus simulans (Table 1, for
activity of all analogues see Supplemental Table S1). The MICs
were measured at various calcium concentrations to investigate the
influence this has on antibiotic potency. The antibacterial activity
of laspartomycin C and daptomycin increases significantly at higher
calcium concentrations, an effect that was also reported by Taylor
and coworkers who observed that elevated calcium concentrations
were required to achieve full potency with their daptomycin
1
,4
confirming the previously assigned chemical structure.
1
3
Characteristic C chemical shifts of Pro at positions β (29.1 ppm)
and γ (24.2 ppm) confirm a trans orientation as was also reported
1
6,17
1
Similarly, H and C chemical
13
for natural laspartomycin C.
1
13
1
shifts at D-Pip positions α ( H 4.80 ppm, C 55.8 ppm) and ε ( H
.88/4.36 ppm, C 39.5 ppm), along with strong NOESY
1
3
2
1
correlations between the α proton of D-pip and Dap ( H 4.66 ppm),
confirm the cis conformation of the D-Pip residue.
1
6
14
analogues. The influence of introducing conformationally
Previously in our group, daptomycin analogues were prepared in
which the ester linkage of the macrocycle was exchanged for an
amide as found in laspartomycin C (see analogues 6, and 7 in
restricted amino acids in the macrocycle was investigated with
analogues 8-12. In general, incorporation of D-Pip and Pro
residues was found to be detrimental, resulting in large increases in
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HO
O
1
2
3
4
5
6
7
8
9
O
OH
NH2
O
H
N
X2
N
H
O
O
H
N
NH
O
HN
HN
O
N
H
N
H
O
O
O
O
NH
OH
X1
O
OH
O
NH
O
NH
H
N
O
N
H
O
O
6
7
8
9
: X1 = Gly, X2 = Trp
: X1 = Gly, X2 = Kyn
: X1 = D-Pip, X2 = Trp
: X1 = D-Pip, X2 = Kyn
NH2
OH
10: X1 = Gly, X2 = Pro
11: X1 = D-Pip, X2 = Pro
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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29
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31
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
OH
Figure 3. HPLC traces for UDP-MurNAc-pentapeptide
accumulation assay: Treatment of S. aureus 29213 with laspartomycin
C results in accumulation of UDP-MurNAc-pentapeptide, an effect not
observed with daptomycin (full-length traces provided in the
Supplemental Figure S2).
NH2
O
H
N
N
N
H
O
HN
HN
O
O
H
O
N
H
O
N
N
N
H
O
H
O
O
O
NH
OH
O
O
O
N
NH
O
OH
NH
O
H
N
N
H
O
O
1
2
the subsequent membrane-associated steps involved in bacterial
cell wall biosynthesis.
OH
From
Daptomycin
From
Laspartomycin C
We then investigated whether the antimicrobial activities of
laspartomycin C, daptomycin, and the active synthetic analogues
were antagonized by various bacterial cell wall precursors
including: lipid I, lipid II, UDP-MurNAc-pentapeptide, UDP-
1
Figure 2. Structures of analogues 6-11 with variations at positions X
2
and X . Compound 12 is a hybrid comprised of the peptide macrocycle
of laspartomycin C and the exocyclic tripeptide unit and C10 lipid of
daptomycin.
GlcNAc,
undecaprenyl-pyrophosphate
(C55-PP),
and
undecaprenyl-phosphate (C55-P). The antibiotics (as well as
vancomycin as a positive control) were pre-incubated with the
various bacterial cell wall precursors and administered to S.
simulans 22, which was used as an indicator strain (Supplemental
Table S2). As expected, the activity of vancomycin was fully
antagonized by lipid I, lipid II, and UDP-MurNAc-pentapeptide,
each of which contains the D-Ala-D-Ala motif recognized by
vancomycin. In contrast, daptomycin and analogues 6-12 were not
antagonized by any of the bacterial cell wall precursors. For
laspartomycin C, however, both C55-P and C55-PP gave an
indication of antagonism. Upon repeating the assay with the water
soluble C15-P and C15-PP it became clear that only the
monophosphate species is capable of antagonizing the activity of
laspartomycin C.
MIC or complete loss of antibacterial activity (see Supplemental
Table S1). Of particular note is the observation that compound 12,
a daptomycin-laspartomycin C hybrid, is completely inactive. The
decreased activity of these analogues does not, however, appear to
be due to an inability to bind to calcium. The circular dichroism
spectra obtained (Supplemental Figure S1) indicate that all
analogues undergo conformational changes upon mixing with
calcium, as was previously observed for daptomycin and the
amphomycin-derived MX-2401. Particularly intriguing are D-Pip
and Pro bearing analogues 11 and 12, which, although devoid of
activity, exhibit calcium-induced conformational changes very
similar to that of laspartomycin C. These findings prompted us to
further investigate the antibacterial mechanism of laspartomycin C.
9
,18
Next, a TLC-based binding assay was employed to assess the
binding of laspartomycin C to C55-P. Mixing of C55-P with
We began by examining the effect of laspartomycin C on
bacterial cell wall biosynthesis by specifically looking for
accumulation of the cytoplasmic lipid II precursor UDP-MurNAc-
pentapeptide in response to administration of antibiotic. As is
clearly seen in Figure 3, when S. aureus cells are treated with
laspartomycin C there is a significant accumulation of UDP-
MurNAc-pentapeptide, an effect not seen with daptomycin. These
findings indicate that the target of laspartomycin C lies
downstream of UDP-MurNAc-pentapeptide, implicating one of
2
+
laspartomycin C in Ca -containing buffer led to formation of a
surprisingly stable complex that could be clearly visualized by TLC
(Figure 4). By comparison, there was no indication of C55-P
binding by daptomycin. When the same TLC experiment was
performed with lipid II, no complex formation was observed
(Supplemental Figure S4) further indicating that laspartomycin C
1
2
selectively targets
C55-P. We next investigated whether
laspartomycin C is capable of inhibiting lipid II synthesis by means
of an in vitro assay. To do so, C55-P was pre-treated with
laspartomycin C at a variety of concentrations, followed by addition
of UDP-GlcNAc, UDP-MurNAc-pentapeptide, and M. flavus
membrane vesicles known to contain the lipid II-producing
enzymes MraY and MurG. Under these conditions laspartomycin
C blocked lipid II synthesis in a dose-dependent manner while
incubation with daptomycin had no effect lipid II synthesis
(Supplemental Figure S5). These findings support a mode of action
for laspartomycin C wherein the sequestration of C55-P leads to
a
-1
Table 1. MICs (µg mL ) measured against indicator strains S. simulans 22
and S. aureus 29213 supplemented with 5.0 and 10 mM Ca
2+
Compound
S. aureus 29213
S. simulans 22
2
+
2+
2+
2+
5 mM Ca
10 mM Ca
5 mM Ca
10 mM Ca
Laspartomycin C (1)
Daptomycin (2)
4
2
4
≤1
0.25
0.125
0.031
≤0.031
a
MIC = minimum inhibitory concentration
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CONCLUSION
In summary, we here describe the total synthesis of
laspartomycin C by means of a flexible route that also allows for the
preparation of structural analogues. While the synthesis of
daptomycin, the preeminent depsipeptide CDA, has been
previously described,
represents the first of its kind among the macrolactam sub-family of
CDAs. Hybrid structures combining aspects of laspartomycin C
and daptomycin were also prepared and evaluated for antibacterial
activity. In all cases these variants were less active than either parent
compound, suggesting a significant difference in the modes of
action of laspartomycin C and daptomycin. Following up on these
findings we established that unlike daptomycin, laspartomycin C
exerts its antibiotic effect by tightly complexing C55-P. In further
assessing this interaction, we also report the first ITC-based
characterization of a C55-P-targeting CDA and determined the
thermodynamic parameters governing the binding of
laspartomycin C to C55-P. Of particular note is the low nanomolar
13,14
our synthesis of laspartomycin C
Figure 4. TLC analysis of C55-P incubation with 0.5-2 eq. of
laspartomycin C and daptomycin. Laspartomycin C forms a stable
complex with C55-P (red box) contrary to daptomycin. MS analysis of
the laspartomycin C:C55-P complex recovered from the TLC plate
indicated the presence of intact laspartomycin C. The brightness and
contrast of the figure has been adjusted to enhance visibility of the
laspartomycin reference band (the original unadjusted figure is
included in the supplemental information, see Figure S3).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
blocked formation of lipid II and prevention of bacterial cell wall
biosynthesis.
To obtain a more quantitative understanding of the binding of
K
d
value associated with laspartomycin C’s binding to its
structurally simple phospholipid target.
C
55-P by laspartomycin C we turned to isothermal titration
At present, daptomycin is the only clinically approved CDA and
our findings show it to be a generally more potent antibiotic than
laspartomycin and the structural analogue here investigated. That
said, laspartomycin C’s ability to kill a range of Gram-positive
calorimetry (ITC). ITC was used to study the interaction of
laspartomycin C with large unilamellar vesicles (LUVs) comprised
of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1.5
mol% C55-P, a system mimicking a simple membrane environment.
Titration of the C55-P containing LUVs into a solution of
laspartomycin C results in an isotherm that appears to be the
combination of two distinct binding events (Figure 5). The initial
stage of the isotherm (left side) shows an endothermic event
resulting from the interaction of laspartomycin C with the DOPC
membrane vesicles. Similarly, titration of “empty” DOPC vesicles,
not containing C55-P, into laspartomycin C solution resulted in an
isotherm displaying an identical interaction (Supplemental Figure
S6). However, as the quantity of C55-P injected increases, a second
binding event is apparent (Figure 5, enlarged). This is ascribed to
the binding of C55-P by laspartomycin C and indicates a remarkably
high affinity interaction with a dissociation constant in the low
2-3
pathogens via a mode of action different than that of daptomycin
indicates that it may have potential for development. At present, no
clinically used antibiotic acts via a C55-P targeting mode of action.
In this regard our synthetic route provides the means for future
structure-activity relationship studies with this interesting class of
CDAs. While the therapeutic potential of C55-P targeting peptides
requires further validation, such compounds may be of value in
addressing the growing threat posed by antibiotic-resistant bacteria.
EXPERIMENTAL SECTION
General procedures
All reagents employed were of American Chemical Society (ACS)
grade or finer and were used without further purification unless
otherwise stated. Fmoc-(Dmb)Gly-OH and Fmoc-Kyn-OH were
nanomolar range (K
employed further reveals that enthalpic (ΔH = -9.8 ± 0.6 kJ mol )
d
= 7.3 ± 3.8 nM). The ITC approach
-1
-1
-1
and entropic (ΔS = 122.9 ± 4.7 J mol K ) factors both contribute
to the tight binding of C55-P by laspartomycin C.
15,19
obtained via previously published procedures.
Fmoc-L-Dap(Aloc)-
OH, Fmoc-D-allo-Thr and 2-chlorotrityl resin were obtained from Iris
Biotech GmbH and the latter was used without protection of the side
chain hydroxyl moiety. All known compounds prepared had NMR
spectra, mass spectra, and optical rotation values consistent with the
assigned structures.
1
0
5
0
0
5
-
Instrumentation for compound characterization
-
5
NMR spectra were recorded at 400 or 500 MHz with chemical shifts
reported in parts per million (ppm) downfield relative to
tetramethylsilane (TMS). 2D NMR experiments (TOCSY, HSQC and
NOESY) were performed on a 500 MHz instrument. High-resolution
mass spectrometry (HRMS) analysis was performed using an ESI
instrument. Circular Dichroism spectra were recorded on a Jasco J-810
CD-spectrometer and ITC experiments were carried out using a
MicroCal Auto-ITC200. Automated peptide synthesis was performed
on a CS Bio CS336x peptide synthesizer.
-10
0
-
10
0.50
.0
0.5
molar ratio
1.0
0.75
1.00
1.25
molar ratio
Figure 5. Isothermal titration calorimetry: DOPC vesicles
containing 1.5 mol% C55-P were titrated into a solution of
laspartomycin C in HEPES buffer containing CaCl
2
. Fitting of the
second binding curve (red) provides a K value of 7.3 ± 3.8 nM. For a
d
full summary of all associated thermodynamic parameters see
Supplemental Table 3. Each point is the average of three independent
experiments.
Preparation of Laspartomycin C and analogues
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Journal of Medicinal Chemistry
Chlorotrityl resin (5.0 g, 1.60 mmol/g) was loaded with DMB-
Fmoc-Gly-OH. Resin loading was determined after coupling of the
This material is available free of charge via the Internet at
http://pubs.acs.org.
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second amino acid because complete Fmoc deprotection of resin
bound DMB-Fmoc-Gly required nonstandard conditions: DMB-
Fmoc-Gly 2-chlorotrityl resin (6.0 g) was thus treated with
ethanolamine:DMF (1:4 v:v, 1x30 min, 1x90 min) followed by washing
AUTHOR INFORMATION
t
Corresponding Author
Phone: 011 +31 (0)6 1878 5274. E-mail: n.i.martin@uu.nl.
with DMF. Overnight coupling of Fmoc-Asp( Bu)-OH (3.7 g, 9.0
mmol), BOP (4.0 g, 9.0 mmol) and DiPEA (3.1 mL, 18.0 mmol) in
DMF followed by end capping with Ac
2
O:DiPEA:DMF (0.5:0.5:9
Funding Sources
v:v:v, 20 mL) yielded Fmoc-Asp(tBu)-(DMB)-Gly 2-chlorotrityl resin
-
1
No competing financial interests have been declared. Financial
support provided by The Natural Sciences and Engineering
Research Council of Canada (NSERC discovery grant to L.A.M.)
and The Netherlands Organization for Scientific Research (NWO
PhD scholarship to L.H.J.K. and NWO-VIDI grant to N.I.M.).
(
0.52 mmol.g as determined spectrophotometrically).
Linear precursor peptides encompassing Gly
0
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3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
to Asp
1
were
assembled via standard Fmoc solid-phase peptide synthesis (SPPS)
either via manual synthesis (resin bound AA:Fmoc-AA:BOP:DiPEA,
1:4:4:8 molar eq.) or automated synthesis (resin bound AA:Fmoc-
AA:HBTU:HOBt:DiPEA, 1:4:3.75:3.75:8 molar eq.) typically on 0.25
mmol scale. NMP or DMF was used as solvent and Fmoc
deprotections were carried out with piperidine:DMF or
piperidine:NMP (1:4 v:v). Amino acid side chains were protected as
ACKNOWLEDGMENT
t
Javier Sastre Toraño is kindly acknowledged for measuring HR-
MS. We thank Jonas Dörr for assistance with CD measurements
and Linda Quarles van Ufford and Heather Wassink for technical
assistance.
follows: Boc for Orn and Trp, Trt for D-Asn, Aloc for DAP, Bu for Asp,
Glu and D-Ser, DMB for Gly in Asp-Gly sequences. Kyn and D-allo-
Thr were introduced without side chain protection. Following coupling
and Fmoc deprotection of Asp , N-terminal acylation was achieved by
1
coupling (E)-13-methyltetradec-2-enoic acid using the same coupling
conditions used for the SPPS.
The resin-bound, Alloc protected intermediate was next washed
ABBREVIATIONS USED
with CH
PhSiH (0.74 mL, 6.0 mmol) in CH
hour. The resin was subsequently washed with CH
followed by a solution of diethyldithiocarbamic acid trihydrate sodium
2
Cl
2
and treated with Pd(PPh
3
)
4
(74 mg, 0.06 mmol) and
(ca. 10 mL) under argon for 1
Cl (5x10 mL),
Alloc, allyloxycarbonyl; CD, circular dichroism; CDA, calcium-
dependent antibiotic; C15-P, farnesyl phosphate; C15-PP, farnesyl
3
2
Cl
2
2
2
pyrophosphate;
undecaprenyl pyrophosphate; DBU, 1,8-diazabicyclo[5.4.0]undec-
-ene; Dmb, 3,4-dimethoxybenzyl; DMF, dimethylformamide;
DOPC, l-α-phosphatidylcholine; d-Pip, d-pipecholic acid; FDA,
Food and Drug Administration; Fmoc,
fluorenylmethyloxycarbonyl; HPLC, high performance liquid
chromatography; GlcNAc, N-acetylglucosamine; K , dissociation
C
55-P, undecaprenyl phosphate;
C55-PP,
-
1
salt (5 mg mL in DMF, 5x10 mL), and DMF (5x10 mL). The
remaining three amino acids where added via standard Fmoc SPPS
with removal of the final Fmoc protecting group to yield the complete
linear resin-bound peptide with a free N-terminal amine. The resin was
7
treated with (CF₃)₂CHOH:CH
with additional (CF₃)₂CHOH:CH
washings were then evaporated to yield the linear protected peptide
with free C- and N-termini. The residue was dissolved in CH Cl (250
mL) and treated with BOP (0.22 g, 0.5 mmol) and DiPEA (0.17 mL,
.0 mmol) and the solution was stirred overnight after which TLC
indicated complete cyclization. The reaction mixture was concentrated
and directly treated with TFA:TIS:H O (95:2.5:2.5, 10 mL) for 60-90
minutes. The reaction mixture was added to Et O:hexanes (1:1) and
the resulting precipitate washed once more with Et O:hexanes (1:1).
The crude cyclic peptide was lyophilized from BuOH:H O (1:1) and
purified with reverse phase HPLC by applying a gradient of 25% to
5% buffer B (buffer A: H O:MeCN:TFA, 95:5:0.1 v:v:v; buffer B:
O:MeCN:TFA, 5:95:0.1 v:v:v) over 1 hour with a flow rate of 12
2
Cl
2
(1:4, 10 mL) for 1 hour and rinsed
2
Cl and CH Cl . The combined
2
2
2
d
constant; l-2,3-Dap, l-2,3-diaminopropionic acid; LUV, large
unilamellar vesicle; MIC, minimum inhibitory concentration;
MRSA, methicillin-resistant S. aureus; MS, mass spectrometry;
MurNAc, N-acetylmuramic acid; NMR, nuclear magnetic
resonance; SPPS, solid-phase peptide synthesis; TLC, thin-layer
chromatography; UDP, uridine diphosphate glucose; VISA,
vancomycin-intermediate S. aureus; VRSA, vancomycin-resistant S.
aureus.
2
2
1
2
2
2
t
2
6
2
REFERENCES
H
2
-
1
mL min on a C18 Maisch 250x22 mm column. Pure fractions were
pooled and lyophilized to yield the desired cyclic lipopeptide products
in >95% purity (based on analytical HPLC analysis) as white powders,
typically in 10-20 mg quantity (4.2-9.3 % yield based on resin loading).
1
.
D. B. Borders, R. A. Leese, H. Jarolmen, N. D. Francis, A. A.
Fantini, T. Falla, J. C. Fiddes, A. Aumelas. Laspartomycin, an acidic
lipopeptide antibiotic with a unique peptide core. J. Nat. Prod.
2
007, 70, 443-446.
2
3
.
.
H. Naganawa, M. Hamada, K. Maeda, Y. Okami, T. Takeuchi, H.
Umezawa. Laspartomycin a new anti-staphylococcal peptide. J.
Antibiot. 1968, 21, 55-62.
W. V. Curran, R. A. Leese, H. Jarolmen, D. B. Borders, D.
Dugourd, Y. C. Chen, D. R. Cameron. Semisynthetic approaches
to laspartomycin analogues. J. Nat. Prod. 2007, 70, 447-450.
ASSOCIATED CONTENT
Supporting Information
Synthetic procedures and analytical data for all new compounds
including characterization data and MIC determinations.
Supplemental figures and tables for: CD spectra, bacterial cell wall
synthesis antagonization assays, lipid II binding assays, ITC
binding studies, 2D-NMR spectra, and analytical RP-HPLC traces.
4. F. M. Kong, G. T. Carter. Structure determination of glycinocins A
to D, further evidence for the cyclic structure of the amphomycin
antibiotics. J. Antibiot. 2003, 56, 557-564.
5
.
H. S. Sader, R. K. Flamm, R. N. Jones. Antimicrobial activity of
daptomycin tested against Gram-positive pathogens collected in
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Europe, Latin America, and selected countries in the Asia-Pacific
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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Region (2011). Diagn. Micr. Infec. Dis. 2013, 75, 417-422.
6. N. Yin, J. Li, Y. He, P. Herradura, A. Pearson, M. F. Mesleh, C. T.
Mascio, K. Howland, J. Steenbergen, G. M. Thorne, D. Citron, A.
D. Van Praagh, L. I. Mortin, D. Keith, J. Silverman, C. Metcalf.
Structure-activity relationship studies of a series of semisynthetic
lipopeptides leading to the discovery of surotomycin, a novel cyclic
lipopeptide being developed for the treatment of clostridium
difficile-associated diarrhea. J. Med. Chem. 2015, 58, 5137-5142.
7
.
M. Strieker, M. A. Marahiel. The structural diversity of acidic
lipopeptide antibiotics. ChemBioChem 2009, 10, 607-616.
L. Robbel, M. A. Marahiel. Daptomycin, a bacterial lipopeptide
synthesized by a nonribosomal machinery. J. Biol. Chem. 2010,
0
1
2
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0
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2
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0
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.
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85, 27501-27508.
9. D. Jung, A. Rozek, M. Okon, R. E. Hancock. Structural transitions
as determinants of the action of the calcium-dependent antibiotic
daptomycin. Chem. Biol. 2004, 11, 949-957.
10. J. Pogliano, N. Pogliano, J. A. Silverman. Daptomycin-mediated
reorganization of membrane architecture causes mislocalization of
essential cell division proteins. J. Bacteriol. 2012, 194, 4494-4504.
1
1. E. Rubinchik, T. Schneider, M. Elliott, W. R. P. Scott, J. Pan, C.
Anklin, H. Yang, D. Dugourd, A. Muller, K. Gries, S. K. Straus, H.
G. Sahl, R. E. W. Hancock. Mechanism of action and limited cross-
resistance of new lipopeptide MX-2401. Antimicrob. Agents
Chemother. 2011, 55, 2743-2754.
1
2. T. Schneider, K. Gries, M. Josten, I. Wiedemann, S. Pelzer, H.
Labischinski, H. G. Sahl. The lipopeptide antibiotic Friulimicin B
inhibits cell wall biosynthesis through complex formation with
bactoprenol phosphate. Antimicrob. Agents Chemother. 2009,
53, 1610-1618.
13. H. Y. Lam, Y. F. Zhang, H. Liu, J. C. Xu, C. T. T. Wong, C. Xu, X.
C. Li. Total synthesis of daptomycin by cyclization via a
chemoselective serine ligation. J. Am. Chem. Soc. 2013, 135,
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272-6279.
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4. C. R. Lohani, R. Taylor, M. Palmer, S. D. Taylor. Solid-phase total
synthesis of daptomycin and analogs. Org. Lett. 2015, 17, 748-
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5. P. ’t Hart, L. H. J. Kleijn, G. de Bruin, S. F. Oppedijk, J. Kemmink,
N. I. Martin. A combined solid- and solution-phase approach
provides convenient access to analogues of the calcium-dependent
lipopeptide antibiotics. Org. Biomol. Chem. 2014, 12, 913-918.
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8. D. Dugourd, H. Y. Yang, M. Elliott, R. Siu, J. J. Clement, S. K.
Straus, R. E. W. Hancock, E. Rubinchik. Antimicrobial properties
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presence of lung surfactant. Antimicrob. Agents Chemother.
2
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TABLE OF CONTENTS GRAPHIC
O
O
OH
O
P
O
D
9
N
O
N
H
O
O
O
HN
HN
O
Ca2+
O
+
Laspartomycin C:C55-P
d
(tight complex, low nM K )
N
H
HN
N
H
O
7
3
O
OH
O
O
O
N
D
OH
NH
NH
O
O
H
N
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
N
H
O
C
55-P
(unique target)
Laspartomycin C
(via synthesis)
O
OH
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