A combined solid- and solution-phase approach provides convenient access to analogues of the calcium-dependent lipopeptide antibiotics

Article abstract of DOI:10.1039/c3ob42238k

The calcium-dependent lipopeptide antibiotics represent a promising new class of antimicrobials for use in combating drug-resistant bacteria. At present, daptomycin is the only such lipopeptide used clinically and displays potent antimicrobial activity against a number of pathogenic Gram-positive bacteria. Given the increasing need for new antibiotics, practical synthetic access to unnatural analogues of daptomycin and related antimicrobial lipopeptides is of value. We here report an efficient synthetic route combining solid- and solution-phase techniques that allows for the rapid preparation of daptomycin analogues. Using this approach, four such analogues, including two enantiomeric variants, were synthesized and their antimicrobial activities and hydrolytic stabilities evaluated.

Full text of DOI:10.1039/c3ob42238k

Organic &
Biomolecular Chemistry
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A combined solid- and solution-phase approach
provides convenient access to analogues of the
calcium-dependent lipopeptide antibiotics†
Cite this: Org. Biomol. Chem., 2014,
12, 913
Peter ’t Hart,‡a Laurens H. J. Kleijn,‡a Gerjan de Bruin,b Sabine F. Oppedijk,a
Johan Kemminka and Nathaniel I. Martin*a
The calcium-dependent lipopeptide antibiotics represent a promising new class of antimicrobials for use
in combating drug-resistant bacteria. At present, daptomycin is the only such lipopeptide used clinically
and displays potent antimicrobial activity against a number of pathogenic Gram-positive bacteria. Given
the increasing need for new antibiotics, practical synthetic access to unnatural analogues of daptomycin
and related antimicrobial lipopeptides is of value. We here report an efficient synthetic route combining
solid- and solution-phase techniques that allows for the rapid preparation of daptomycin analogues.
Using this approach, four such analogues, including two enantiomeric variants, were synthesized and
their antimicrobial activities and hydrolytic stabilities evaluated.
Received 11th November 2013,
Accepted 10th December 2013
DOI: 10.1039/c3ob42238k
www.rsc.org/obc
Introduction
The accelerated appearance of antibiotic resistance presents a
serious and growing global health risk. Despite the increasing
need for new antibacterial agents, only two mechanistically
and structurally new antibiotics have reached the clinic in the
past 40 years: linezolid and daptomycin.1,2 While linezolid is
of synthetic origin, daptomycin (Fig. 1) is a natural product
isolated from fermentations of Streptomyces roseosporus.
Daptomycin is rapidly bactericidal against Staphylococcus
aureus, including methicillin-resistant S. aureus (MRSA), vanco-
mycin-intermediate S. aureus (VISA) and vancomycin-resistant
S. aureus (VRSA) strains.3–5 Marketed under the trade name
Cubicin, daptomycin is the first lipopeptide antibiotic of its
kind to be approved for clinical use. Structurally unique, dap-
tomycin is a cyclic depsipeptide composed of 13 amino acids
(including non-proteinogenic and D-amino acids) and bears an
N-terminal 10-carbon lipophilic tail.
Fig. 1 Structure of daptomycin (1) with non-proteinogenic and
D-amino acids indicated.
model for daptomycin’s mode of action involves interaction
with the bacterial membrane leading to rapid depolarization
and a loss of membrane potential resulting in bacterial cell
death without rupturing the cell.7 Daptomycin’s activity is
calcium-dependent with serum levels of free calcium (45–55 μg
mL−1) sufficient to induce full antimicrobial activity.8 Owing
to the presence of four carboxylate side chains in the peptide,
daptomycin is negatively charged at physiological pH and as
such interacts with Ca2+ ions.9 Upon binding to Ca2+ daptomy-
cin is believed to oligomerize after which it is able to effectively
disrupt the cell membranes of sensitive bacteria leading to
defective cell division and cell wall synthesis.6,7
While the precise mechanistic details of daptomycin’s anti-
bacterial activity are unclear, it is known to disrupt aspects of
bacterial cell membrane function.6,7 In this regard, the current
aMedicinal Chemistry & Chemical Biology Group, Utrecht Institute for
Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG Utrecht,
The Netherlands. E-mail: n.i.martin@uu.nl
bBio-organic Synthesis Group, Leiden Institute of Chemistry, Einsteinweg 55,
2333 CC Leiden, The Netherlands
†Electronic supplementary information (ESI) available: Preparation of Fmoc-
(Dmb)Gly-OH and analytical RP-HPLC traces, CD spectra, and 2D-NMR spectra
for peptides 2, 3, ent-2 and ent-3. See DOI: 10.1039/c3ob42238k
‡Denotes equal contribution by P. ’t Hart and L. H. J. Kleijn.
Given its role as the prototypical calcium-dependent lipo-
peptide antibiotic, daptomycin has garnered much attention
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from the synthetic community. Modified daptomycin ana-
logues have been prepared via semi-synthesis10–13 and the syn-
thesis of daptomycin itself has been achieved using both
chemo-enzymatic approaches or total synthesis. The Marahiel
group demonstrated that linear precursor peptide thioesters
could be converted to their corresponding daptomycin macro-
cycles by action of appropriate recombinant cyclase enzyme.14
In addition, the two total syntheses of daptomycin have
also been reported to date.15,16 In both cases the synthetic
routes described are labor intensive and make use of multi-
step synthetic strategies. For these reasons we were drawn
towards developing a convenient synthetic route for the prepa-
ration of daptomycin analogues so as to provide more rapid
access to structurally diverse derivatives of this important
antibiotic.
Results and discussion
In designing the daptomycin analogues to be prepared
(Scheme 1) we opted to replace the L-threonine residue at posi-
tion 4 with L-diaminopropionic acid. In doing so we were able
to circumvent incorporation of the synthetically challen-
ging15,16 ester linkage between Thr4 and Kyn13 found in the
natural daptomycin macrocycle by replacing it with the more
accessible amide linkage. In this regard, we also speculated
that the corresponding macrocyclic amide analogue of dapto-
mycin might show improved hydrolytic stability (vide infra).
Aside from the amide for ester modification, we also incorpor-
ated L-glutamic acid at position 12 in place of the (2S,3R)-
3-methyl glutamate normally found in daptomycin. While
published preparations of (2S,3R)-3-methyl glutamate are
available,15–17 they are rather lengthy (>10 steps) and time con-
suming. In addition, Marahiel and coworkers previously inves-
tigated the same Glu for 3-MeGlu substitution in their chemo-
enzymatic approach to daptomycin analogues and found it to
have a relatively small effect on antimicrobial activity (MIC
values increased by ca. 7-fold).14
A variety of synthetic approaches were considered and
explored in developing the route towards the daptomycin ana-
logue illustrated in Scheme 1. Initial attempts at performing
the entire synthesis on the solid phase by attachment to the
resin via either of the aspartate side chains present in the
macrocycle were unsuccessful and led us to explore the com-
bined solid- and solution-phase approach indicated. To avoid
any racemization in the later cyclization step, the peptide syn-
thesis began at the glycine residue corresponding to position 5
in daptomycin. Employing an acid sensitive resin (2-chlorotri-
tyl) and using standard SPPS techniques, the N-terminus of
the peptide was first installed, including the C10 fatty acid tail.
At this point, the diaminopropionic acid side chain was depro-
tected providing an attachment point for the kynurenine
residue (position 13) after which the remainder of the peptide
was assembled without incident. Of particular note was the
need to incorporate a rare Fmoc-Kyn building block which was
obtained via a recently described methodology for the gram-
Scheme 1 Synthetic route developed for the preparation of daptomy-
cin analogue 2. Reagents and conditions: (a) Fmoc-Xaa-OH (or decanoic
acid), BOP, DIPEA, DMF; (b) Pd(PPh3)4, PhSiH3, CH2Cl2; (c) Fmoc-Xaa-
OH, BOP, DIPEA, DMF; (d) HFIP–CH2Cl2; (e) BOP, DIPEA, CH2Cl2, 40 h;
(f) TFA–TIS–H2O. Highlighted in blue are the structural differences
between compound 2 and daptomycin.
scale production of kynurenine in either L- or D-stereo-
chemistry.18 In addition, DMB-protected glycine was intro-
duced at position 10 to avoid aspartamide formation with the
neighboring Asp residue. Upon completion, the intermediate
protected peptide was cleaved from the resin using mild acid
conditions and dissolved in CH2Cl2 at high dilution (0.5 mM)
followed by treatment with BOP/DIPEA which led to clean for-
mation of the desired macrocycle (Scheme 1). Following depro-
tection and purification by RP-HPLC, daptomycin analogue 2
was obtained.
Based upon the concise route developed for the preparation
of daptomycin analogue 2, a second analogue, compound 3,
was also prepared wherein L-kynurenine at position 13 was
replaced by the structurally similar L-tryptophan. The synthesis
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Table 1 Antimicrobial activity of daptomycin analogues against
S. aureus (ATCC 29123)
Compound
MICa (μM)
1 (Daptomycin)
2
1.23
201.2
3
100.8
ent-2
ent-3
Not activeb
Not activeb
a Culture broth supplemented with Ca2+ (50 mg L−1). b Not active at the
highest concentration tested = 804 μM.
of 3 proceeded without incident after which the antimicrobial
activity of analogues 2 and 3 was compared with that of auth-
entic daptomycin (Table 1). Using a standard broth dilution
assay employing S. aureus (ATCC 29213) as an indicator strain,
analogues 2 and 3 were both found to exhibit calcium-depen-
dent antimicrobial activity albeit at a significantly reduced
level relative to that of daptomycin. The approximate 100-fold
decrease in activity was somewhat expected given the structural
modifications introduced in analogues 2 and 3. Marahiel and
coworkers reported similarly attenuated activities for daptomy-
cin analogues obtained via their chemo-enzymatic approach.14
It is clear that replacement of the ester linkage in daptomy-
cin with an amide dramatically alters antimicrobial activity.
This effect may be due to conformational changes/restrictions
in the macrocycle that result from incorporation of the amide.
The amide for ester substitution is also expected to impart
greater hydrolytic stability. Thus, the serum stability of ana-
logues 2 and 3 was also evaluated and compared with that of
daptomycin. Each peptide was incubated with human plasma
serum at 37 °C and sampled at specific time points. Under
these conditions, daptomycin itself underwent significant
degradation with an approximate 50% loss in the first
24 hours (Fig. 2). This degradation is presumably due to hydro-
lytic opening of the macrocyclic lactone as evidenced by the
appearance of a new M + H2O species. By comparison, amide
analogues 2 and 3 were much more stable under the same con-
Fig. 2 Serum stability of authentic daptomycin 1 (A) compared with
ditions with only minimal degradation detected over extended analogues 2 (B) and 3 (C). Significant degradation of daptomycin is
observed upon incubation at 37 °C while amide analogues 2 and 3
display increased stability.
time periods of up to 48 hours.
The relative ease with which analogues 2 and 3 where
assembled prompted us to also prepare the corresponding
enantiomeric analogues ent-2 and ent-3. The preparation of
enantiomeric analogues were shown to have identical reten-
tion times by analytical RP-HPLC (Fig. 3) and exhibited optical
rotations of equal magnitude but opposite sign. In addition,
the circular dichroism spectra obtained for the daptomycin
analogues further supported their enantiomeric nature
(ESI Fig. S3†).
The antibacterial activities of ent-2 and ent-3 were next eval-
uated using the same assay as described above and revealed
no detectable activity for both enantiomeric analogues
(Table 1). These results indicate that a specific chiral inter-
action(s) is required for the activity of those analogues bearing
the “native” daptomycin stereochemistry and may also support
a similarly stereospecific mode of action for daptomycin itself.
biologically active peptides in both enantiomeric forms is an
established approach used in establishing the role of chiral
biomolecular targets.19–22 If as some have proposed, daptomy-
cin kills bacteria via membrane disruption without invoking a
specific chiral bacterial target biomolecule, one would expect
the “mirror-image” enantiomeric form of daptomycin to show
an equal antibiotic activity. Conversely, should the enantio-
meric form of daptomycin not show antibacterial activity, it
can be taken as an indication that a chiral interaction with a
specific biomolecular target is integral to daptomycin’s mode
of action. To this end the enantiomeric analogues ent-2 and
ent-3 were assembled using the appropriate stereochemically-
inverted amino acid building blocks. As expected, the
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Fig. 3 Overlays of analytical RP-HPLC traces obtained for enantiomeric daptomycin analogues (A) 2 and ent-2 and (B) 3 and ent-3.
Plausible candidates for the requisite chiral target could biomolecule(s) in bacterial strains that are sensitive to
include any number of membrane proteins or chiral phospho- daptomycin.
lipids. In this regard, recent reports suggest that phosphatidyl-
glycerol may play a role. Specifically, in daptomycin-resistant
strains of enterococci and S. aureus levels of phosphatidylgly-
cerol are significantly reduced.23
Experimental
Reagents and general methods
All reagents employed were of American Chemical Society
(ACS) grade or finer and were used without further purification
unless otherwise stated. Both L-and D-kynurenine were syn-
Conclusions
In summary, we have developed a convenient approach thesized according to a previously described procedure18 and
employing both solid- and solution phase techniques for the converted into the requisite Fmoc-building blocks based on
preparation of analogues of the calcium-dependent lipopep- established protocols.14 A large scale preparation of Fmoc-
tide family of antibiotics. While the antibacterial activity of the (Dmb)Gly-OH was also developed based on existing literature
synthetic analogues was significantly reduced relative to that procedures (see ESI† for details).24,25 All known compounds
of daptomycin, their hydrolytic stability was greatly increased. prepared had NMR spectra, mass spectra, and optical rotation
The synthetic route here described should also be readily values consistent with the assigned structures. Orthogonally
amenable to the production of new lipopeptide analogues with protected Fmoc-L-Dap(Aloc)-OH and Fmoc-D-Dap(Aloc)-OH
enhanced properties. Future work will be aimed at evaluating were obtained from Iris Biotech GmbH. All reactions and
the effect of incorporating different amino acids in an attempt fractions from column chromatography were monitored by
to increase antibacterial activity while maintaining the hydro- thin layer chromatography (TLC) using plates with a UV
lytic stability of these analogues. Support for this approach is fluorescent indicator (normal SiO2, Merck 60 F254). One or
evidenced by the observation that a tryptophan-for-kynurenine more of the following methods were used for visualization:
substitution as in compound 3 results to a more active ana- UV absorption by fluorescence quenching; iodine staining;
logue. In addition, our findings with the enantiomeric ana- phosphomolybdic acid : ceric sulfate : sulfuric acid : H2O (10 g :
logues implicate the involvement of a specific chiral target 1.25 g : 12 mL : 238 mL) spray; and ninhydrin staining. Flash
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Table 2 Analytical data for compounds 1 (daptomycin), 2, ent-2, 3, and ent-3
Exact mass
(calc.)
Exact mass
(found)
Compound
Rt (min)
[α]2D5
1 (Daptomycin)
2
ent-2
3
ent-3
30.2
29.3
29.3
29.6
29.6
N.D.a
1620.7182 [M + H]+
1591.7029 [M + H]+
1591.7029 [M + H]+
1587.7080 [M + H]+
1587.7080 [M + H]+
1620.7167 [M + H]+
1591.6997 [M + H]+
1591.6995 [M + H]+
1587.7069 [M + H]+
1587.7063 [M + H]+
−7.98 (0.28, H2O)
+8.70 (0.28, H2O)
−9.45 (0.65, H2O)
+9.24 (0.49, H2O)
a N.D. = not determined.
chromatography was performed using Merck type 60, DIPEA (0.17 mL, 1.0 mmol, 4.0 equiv.) were added. The cycliza-
230–400 mesh silica gel.
tion reaction was monitored by TLC (5% MeOH in CH2Cl2)
and was typically complete within 24 to 40 h. The reaction
mixture was then concentrated, dissolved in EtOAc (250 mL)
and washed with 1 M KHSO4 (2 × 200 mL) followed by satu-
rated NaHCO3 (2 × 200 mL). The organic layer was dried over
Na2SO4, filtered and concentrated under vacuum. The pro-
tected cyclic peptide was treated with a solution of TFA–TIS–
H2O (95 : 2.5 : 2.5, 10 mL) for 1.5 hours. The mixture was then
added to cold ether and the precipitated peptide collected
by centrifugation and further washed with cold ether (2×).
Crude peptides were purified by RP-HPLC using a Maisch
ReproSil-Pur C18-AQ column (250 × 22 mm, 10 µm) and
employing a gradient of 30% to 70% Buffer B with a flow of
12 mL min−1 (Buffer A: 95% H2O, 5% MeCN, 0.1% TFA; Buffer
B: 5% H2O, 95% MeCN, 0.1% TFA). Product containing frac-
tions were pooled and lyophilized to yield between 5–10 mg of
pure peptide (1.2–2.4% overall yield). Table 2 summarizes the
analytical data obtained for the four daptomycin analogues
prepared as well as for authentic daptomycin.
Instrumentation for compound characterization
NMR spectra were recorded at 300 or 500 MHz with chemical
shifts reported in parts per million (ppm) downfield relative to
tetramethylsilane (TMS). H NMR data are reported in the fol-
1
lowing order: multiplicity (s, singlet; d, doublet; t, triplet; q,
quartet; qn, quintet and m, multiplet), number of protons,
coupling constant (J) in hertz (Hz) and assignment. When
appropriate, the multiplicity is preceded by br, indicating that
the signal was broad. 13C NMR spectra were recorded at
75.5 MHz with chemical shifts reported relative to CDCl3
δ 77.0. 2D NMR experiments (TOCSY and HSQC) 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 using a 2 mm cuvet.
Peptide synthesis
Lipopeptide analogue 2 was synthesized beginning with Fmoc-
Gly loaded 2-chlorotrityl resin (410 mg, 0.25 mmol). Fmoc
groups were removed with 20% piperidine in DMF (10 mL,
1 × 1 min., 1 × 30 min.). Coupling reactions were done in DMF
(10 mL) with 4 equivalents of Fmoc-amino acid (or capric
acid), 4 equivalents of BOP and 8 equivalents of DIPEA
(1 hour). For removal of the Aloc protecting group, the resin
was first washed with CH2Cl2 (2 × 10 mL) under argon after
which PhSiH3 (0.74 mL, 6.0 mmol) in CH2Cl2 (4 mL) and Pd-
(PPh3)4 (74 mg, 0.06 mmol) in CH2Cl2 (12 mL) were added and
the mixture swirled under argon for 1 hour. The reaction
mixture was drained and the procedure was repeated. To
remove residual palladium catalyst, the resin was then washed
with CH2Cl2 (5 × 10 mL), a 0.5% solution of diethyldithiocar-
bamic acid trihydrate sodium salt in DMF (5 × 10 mL), and
Biological activity assays
The daptomycin analogues were tested against S. aureus (ATCC
29213) as an indicator strain. Two-fold serial dilutions of each
compound were made in microtiter plates using Mueller–
Hinton broth. Each well was inoculated at 1 × 106 CFU
and incubated at 37 °C for 16 h followed by visual determi-
nation of MIC values. In these assays calcium- and mag-
nesium-free Mueller–Hinton broth (Fluka) was supplemented
with CaCl2 (final Ca2+ concentration 50 mg L−1) and with
MgCl2 (final Mg2+ concentration 10 mg L−1) or alternatively
with MgCl2 alone to assess the effect of calcium on anti-
bacterial activity.
Serum stability assays
DMF (5 × 10 mL). After Aloc removal, the remainder of the Daptomycin and analogues 2 and 3 were added to human
peptide was assembled on resin using standard SPPS plasma serum to a final concentration of 150 µg mL−1, fol-
approaches. The protected peptide was cleaved from the resin lowed by incubation at 37 °C. At 0, 24 and 48 hours a 100 μL
by addition of hexafluoroisopropanol (HFIP) in CH2Cl2 (1 : 4, aliquot was taken and added to a 200 μL volume of methanol
6 mL, 1 hour) and the filtrate collected. The resin was rinsed to precipitate plasma proteins (methanol also contained
with additional HFIP–CH2Cl2 (2 × 3 mL) and the combined fil- 0.075 mg mL−1 ethylparaben as an internal standard). The
trates concentrated under vacuum. The crude linear peptide mixture was vortexed for 5 seconds and allowed to stand at
was dissolved in CH2Cl2 (500 mL, approximate peptide concen- room temperature for 15 minutes. After centrifugation at
tration of 0.5 mM) and BOP (0.22 g, 0.50 mmol, 2.0 equiv.) and 13 000 rpm for 5 minutes, the supernatant was removed and
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used for HPLC analysis. Analysis of the samples was per-
formed by analytical RP-HPLC with a Maisch ReproSil-Pur 120
C18-AQ column (250 × 4.6 mm, 5 μm) and a linear gradient of
0–100% buffer B over 48 minutes at a flow rate of 1 mL min−1
(Buffer A: 95% H2O, 5% MeCN, 0.1% TFA; Buffer B: 5% H2O,
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intact peptide to be calculated based upon the ratio of peak
areas corresponding to the internal standard and the intact
peptides.
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Circular dichroism spectroscopy
A 60 μM solution of each peptide was prepared by dissolving
in 20 mM HEPES buffer (pH 7.4). Samples were measured at
room temperature from 210–250 nm at a scan rate of 20 nm
min−1 and a bandwidth of 0.1 nm. The spectra were converted
to molar ellipticities in units of deg × cm2 dmol−1
.
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Acknowledgements
We thank Eefjan Breukink for providing authentic daptomycin
and Frederike Müskens for preparing L- and D-kynurenine.
Javier Sastre Toraño is acknowledged for providing HRMS ana-
lysis and Jonas Dörr for assistance in acquiring the CD
measurements. Linda Quarles van Ufford is kindly thanked for
assisting with antibacterial assays. Financial support provided
by The Netherlands Organization for Scientific Research (VIDI
grant to NIM).
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