Mutual influence of backbone proline substitution and lipophilic tail character on the biological activity of simplified analogues of caspofungin

Article abstract of DOI:10.1039/c2ob25951f

The echinocandins represent the most recent class of antifungal drugs. Previous structure-activity relationship studies on these lipopeptides have relied mainly upon semisynthetic derivatives due to their complex chemical structures. A successful strategy for the rapid enantioselective synthesis of the branched fatty acid chain of caspofungin and analogues was developed to synthesize several simplified analogues of caspofungin. The specific minimum inhibitory activity of each mimic was determined against a panel of Candida strains. This approach gave access to new fully synthetic derived caspofungin mimics with high and selective antifungal activities against Candida strains. In addition, the data suggested an important role of the hydroxy proline residue in the bioactive conformation of the macrocyclic peptide ring structure.

Full text of DOI:10.1039/c2ob25951f

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PAPER
Mutual influence of backbone proline substitution and lipophilic tail
character on the biological activity of simplified analogues of caspofungin†
Monique P. C. Mulder,‡a Peter Fodran,‡b Johan Kemmink,a Eefjan J. Breukink,c John A. W. Kruijtzer,a
Adriaan J. Minnaard*b and Rob M. J. Liskamp*a
Received 17th May 2012, Accepted 18th July 2012
DOI: 10.1039/c2ob25951f
The echinocandins represent the most recent class of antifungal drugs. Previous structure–activity
relationship studies on these lipopeptides have relied mainly upon semisynthetic derivatives due to their
complex chemical structures. A successful strategy for the rapid enantioselective synthesis of the
branched fatty acid chain of caspofungin and analogues was developed to synthesize several simplified
analogues of caspofungin. The specific minimum inhibitory activity of each mimic was determined
against a panel of Candida strains. This approach gave access to new fully synthetic derived caspofungin
mimics with high and selective antifungal activities against Candida strains. In addition, the data
suggested an important role of the hydroxy proline residue in the bioactive conformation of the
macrocyclic peptide ring structure.
fungal cell wall, by inhibiting the β-(1,3)-glucan synthase
Introduction
enzyme.7 In view of their mode of action as well as their
Over the past decades, the incidence and diversity of life-
threatening fungal infections have increased dramatically. In part
this is due to an increase of the immunosuppressed patient popu-
lation.1,2 In addition, the toxicity and growing antifungal resist-
ance of the current antifungal agents underscore the urgent need
for development of new antifungal compounds that act on un-
related and novel targets.3,4
pharmacological and toxicological profiles, the echinocandin
compounds exhibit the most promising target selectivity, as 1,3-
β-glucan is only found in fungi and not in mammalian cells.7
This resulted in less toxic effects, as compared to the other
classes of antifungal agents. Moreover, so far the clinically
approved agents of this class of compound have rarely met
fungal resistance.8
Amphotericin B used for treatment of severe mycoses is ham-
pered by its nephrotoxicity and many other side effects. Azoles
are the most widely used antifungal agents because of their high
therapeutic index. However, their widespread use has resulted in
severe drug resistance.5 Development of echinocandin like
cyclolipohexapeptides for the treatment of invasive fungal infec-
tions represented a breakthrough in antifungal chemotherapy.6
This family of antifungal drugs interferes with the synthesis of
β-(1,3)-glucan, a major component of the framework of the
The echinocandins are a broad group of lipopeptide antifungal
agents that typically consists of a cyclic hexapeptide acylated
with a lipophilic tail on its N-terminus. The first licensed echino-
candin was caspofungin (1, Fig. 1), a semi-synthetic compound
based on pneumocandin B0, in 2001. Since then, two other
semi-synthetic echinocandins, micafungin (2005) and anidula-
fungin (2006), have been approved by the Food and Drug
Administration (FDA).9 These clinically relevant echinocandins
are all semisynthetic derivatives of the natural fermentation
products.10–12
The complex chemical structures of the echinocandins have
led to extensive structure–activity relationship (SAR) studies by
semisynthetic modification of the natural product.13 This
approach relies on the availability of the lipopeptides in large
quantities from fermentation and of course these semisynthetic
modifications cannot access a complete SAR of the echino-
candins. With respect to this, synthetic approaches offer an
opportunity to obtain valuable additional SAR information.
In 1992 the first total synthesis of simplified echinocandin
analogues was described by Zambias et al.14 Structure–activity
relationship data of these analogues showed that several of the
functional groups, primarily the hydroxyl groups, were not
aMedicinal Chemistry and Chemical Biology, Utrecht Institute for
Pharmaceutical Sciences, Utrecht University, PO Box 80082, 3508 TB
Utrecht, The Netherlands. E-mail: R.M.J.Liskamp@uu.nl;
Fax: +31 30 2536655
bDepartment of Bio-organic Chemistry, Stratingh Institute for Chemistry,
University of Groningen, Nijenborgh 7, 9747 AG Groningen, The
Netherlands. E-mail: A.J.Minnaard@rug.nl; Fax: +31 50 3634296
cBiochemistry of Membranes, Bijvoet Center for Biomolecular Research
and Institute of Biomembranes, Utrecht University Padualaan 8, 3584
CH Utrecht, The Netherlands
†Electronic supplementary information (ESI) available: 1H spectra for
1
caspofungin analogues 4–7, 19–21; H and 13C spectra for compounds
8–16. See DOI: 10.1039/c2ob25951f
‡These authors contributed equally to this work.
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Fig. 1 Structures of caspofungin (1), previously described mimics (2 and 3)16 and the designed synthetic caspofungin analogues (4–7).
essential for antifungal activity.14 Partly inspired by this work,
Klein et al. reported a similar study in 2000.15 The total syn-
thesis approach has provided valuable echinocandin SAR data
that would be difficult or impossible to obtain via modification
of the natural products themselves.16
antifungal activity. Also analogues were included with either one
(5) or no (6, 7) methyl groups (Fig. 1).
Results and discussion
These reports and our own findings17 showed that the role of
the fatty acid tail was very dominant. Our previous analogues
(e.g. 3, Fig. 1) were designed to contain a palmitoyl chain,17
instead of the dimethylmyristoyl chain, which is consistent with
SAR studies showing that a regular C12–C18 fatty acid side
chain gives optimal activity.19 Unfortunately, mimics equipped
with this chain showed low to no activity. Therefore, based on
work by Klein et al.,15 simplified head-to-tail analogues were
synthesized equipped with a terphenyl fatty acid side chain (e.g.
2 Fig. 1). Terphenyl lipophilic tail containing analogue 2 was
30-fold more active than mimic 3 with a palmitoyl chain.17
Other reports in the literature discussed the importance of both
the stereochemistry and methyl branching patterns of fatty acids
in biological recognition.20,21 This raised the interest in an
investigation of the influence of the presence and stereochemistry
of methyl groups in the dimethylmyristoyl chain on the anti-
fungal activity.
Although extensive structure–activity studies have been per-
formed in the past on the nature of the fatty acid side chain and
its influence on the activity of the echinocandins,18,22–26 the role
of the stereochemistry and number of methyl groups in the
natural dimethylmyristoyl chain of caspofungin (1) has not been
investigated so far.
Here, we report the synthesis and antifungal activity of a sim-
plified caspofungin analogue (4) bearing its natural (10R,12S)-
dimethylmyristoyl chain.27 In addition, the influence of the
methyl groups in this chain on antifungal activity was deter-
mined by synthesis of several analogues and evaluation of their
Chemistry
Both the relative and absolute configuration of the fatty acid side
chain of caspofungin (1) have been elucidated by Leonard
et al.27 Their reported stereoselective synthesis of the (R,S)-
enantiomer was conducted in 8 steps and provided (10R,12S)-
dimethylmyristic acid 8 in a 90 : 10 (syn/anti) ratio of diastereo-
mers. Based on this, we designed an efficient asymmetric syn-
thesis using the copper/Josiphos28a,b catalysed addition of
methylmagnesium bromide for the construction of the stereo-
genic centers (Scheme 1).27
Commercially available (E)-pent-2-enoic acid 9 was converted
into thioester 10 with Steglich conditions.29 This thioester under-
went copper/Josiphos catalyzed enantioselective conjugate
addition of MeMgBr smoothly, affording the desired compound
11 in 92% yield with a high er (24 : 1).28b Subsequent DIBAL-H
reduction followed by the Horner–Wadsworth–Emmons28b reac-
tion gave the elongated thioester 12 in good yield (80%) with an
excellent E : Z ratio (20 : 1). After separation of the unwanted
Z-stereoisomer with column chromatography, the α,β-unsaturated
thioester 12 was subjected to a second conjugate addition. This
reaction afforded a 20 : 1 mixture of C3 epimers which was
separable again by column chromatography. The desired dia-
stereomer 13 was isolated in 80% yield. After DIBAL-H
reduction and Wittig reaction with a carboxylate functionalized
ylide, a mixture of double bond isomers 14 in an approximate
3 : 1 ratio was obtained in 47% combined yield. In order to avoid
any epimerization of the nearby stereocenter, the double bond of
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and is outlined in Scheme 3 for mimic 4. Linear precursor 17
was prepared by SPPS using the trityl resin as follows. After
removal of the ε-Fmoc group from ornithine dimethylmyristic
acid 8 was coupled. Mild acidolytic cleavage of the Mtt group
using trifluoroethanol and acetic acid also liberated the peptide
chain from the resin and gave the linear fully protected peptide
precursor 18. Cyclization and subsequent deprotection afforded
the macrocyclic peptide 4 in an overall yield of 51% after purifi-
cation by preparative reverse phase HPLC. Synthesis of macro-
cyclic peptides 5–7 was performed analogously to the synthesis
of mimic 4 in Scheme 3. For the preparation of peptides 5–7
other fatty acids, i.e. methylmyristic acid, palmitic acid and
myristic acid, were included in step (h) instead of dimethyl-
myristic acid 8 (Scheme 3). Peptides 5–7 were obtained in
overall yields of 48–88% after purification by preparative reverse
phase HPLC.
Scheme 1 Synthesis of (10R,12S)-dimethylmyristic acid 8. (a) EtSH,
DCC, DMAP, DCM, 0–21 °C, 3 h, 95%; (b) MeMgBr, CuBr–(S,RFe)-
Josiphos, MTBE, −78 °C, 16 h, 92%; (c) DIBAL-H, DCM, −50 °C,
1 h; (d) (MeO)2P(O)CH2COSEt, nBuLi, THF, 0–21 °C, 16 h, 70%; (e)
MeMgBr, CuBr–(S,RFe)-Josiphos, MTBE, −78 °C, 16 h, 80%; (f)
DIBAL-H, DCM, −50 °C, 1 h; (g) Br−Ph3P+(CH2)6CO2H, LiHMDS,
THF, 21 °C, 20 min, 47%; (h) NH2NH2·H2O, O2, Cat, EtOH, 21 °C,
70%.
Structure–activity relationships
The antifungal activity of each analogue (4–7) was evaluated
using a broth microdilution microtiter assay against a panel of
common Candida species.31 This panel included C. dubliniensis,
C. glabrata, C. tropicalis, C. parapsilosis, C. krusei and two
strains of C. albicans. Caspofungin 132 was used as a reference
compound and our previously described compounds (2 and 3)17
were also included in the test for comparison. The results of
these tests are expressed as the minimum inhibitory concen-
tration (MIC) value, the minimum concentration of compound
which completely inhibits visible fungal growth, and are shown
in Table 1.
The simplified caspofungin analogue 4, bearing the natural
side chain, showed substantial antifungal activity against the
Candida strains (0.07–0.19 μg mL−1) with a somewhat higher
MIC value for C. krusei (2.25 μg mL−1) and proved to be in-
active against C. parapsilosis. Only a 5-fold decrease in the
inhibitory activity of 4, compared with that of caspofungin (1),
was observed against C. albicans and the closely related
C. dubliniensis.33 In addition, analogue 4 proved to have a more
selective antifungal spectrum than caspofungin (1).
Scheme 2 Synthesis of (12S)-methylmyristic acid 16. (a) DIBAL-H,
DCM, −50 °C, 1 h; (b) Br−Ph3P+(CH2)8CO2H, LiHMDS, THF, 21 °C,
20 min, 60% over two steps; (c) NH2NH2·H2O, O2, Cat, EtOH, 21 °C,
80%.
14 was subsequently reduced by diimide, in situ formed by
catalytic oxidation of hydrazine,30 affording 8 in 70% yield.
Overall, the side chain 8 of caspofungin was synthesized in a
linear 8 step synthesis with an overall yield of 16%. The catalytic
enantioselective conjugate addition in combination with a Wittig
reaction using a carboxylate functionalized ylide represents an
efficient approach towards methyl-branched fatty acids. It is
important to mention that several of the intermediates in the
synthesis were rather volatile, which complicated isolation.
The synthesis of the mono methyl analogue 16 was performed
in a similar manner (Scheme 2). Thioester (11) was reduced by
DIBAL-H and after a Wittig reaction, a mixture of E and Z
isomers (15) was isolated in 60% yield. Reduction of the result-
ing double bond was achieved by in situ generated diimide30 and
gave 16 in 80% yield.
In order to assess the contribution of the methyl groups in the
dimethylmyristoyl chain 8 of caspofungin (1), the mono methyl
derivative methylmyristic acid 16, as well as non-methylated
palmitic and myristic fatty acid chains were incorporated as a
replacement for 8 in analogue 4 to give 5, 6 and 7, respectively.
Remarkably, all of the analogues in the series showed substan-
tial antifungal activity against Candida. Similar to analogue 4,
these analogues were more selective than caspofungin, having
higher MIC values for C. krusei and showing no activity against
C. parapsilosis. These results also showed that there is no
obvious relationship between the presence of the methyl groups
in the dimethylmyristoyl chain 8 of caspofungin (1) and anti-
fungal activity.
Most surprisingly analogue 6 with a palmitoyl chain, as is
also present in analogue 3, showed a 10–35-fold increase in anti-
fungal activity against Candida. The only difference between
these analogues is the position of the hydroxyl group in the left-
upper hydroxyproline (residue Hyp-6) of the cyclic hexapeptide
(R2 and R3 in Fig. 1).
Now that we had both the (10R,12S)-dimethylmyristic acid 8
and its mono methyl analogue (12S)-methylmyristic acid 16 in
hand, attention was directed toward the synthesis of the head-to-
tail mimics. The synthesis of these mimics proceeded readily
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Scheme 3 Reagents and conditions: (a) (1) Fmoc-3-Hyp(tBu)-OH, DIPEA, DCM; (2) DCM/MeOH/DiPEA (17/2/1, v/v/v); (b)–(f) (1) 20%
piperidine in NMP; (2) Fmoc-Xxx-OH, BOP, DiPEA, NMP; (g) 20% piperidine in NMP; (h) dimethylmyristic acid 8, HATU, DIPEA, NMP; (i)
TFE/AcOH/DCM (2/1/7, v/v/v); ( j) BOP, DiPEA, DMF; (k) TFA/TIS/H2O (95/2.5/2.5, v/v/v).
Table 1 In vitro antifungal activity of caspofungin and analogues against common Candida species displayed as MIC values in μg mL−1
Candida
albicans
CBS9975
Candida
albicans
CBS8758
Candida
dubliniensis
CBS7987
Candida
glabrata
CBS138
Candida
krusei
CBS573
Candida
parapsilosis
CBS604
Candida
tropicalis
CBS94
Peptide
Caspofungin 1
0.023
0.140
>4.25
0.117
0.188
0.313
0.203
0.039
0.156
>4.25
0.188
0.281
0.406
0.25
0.014
0.063
1.75
0.070
0.047
0.102
0.047
0.027
0.188
3.8
0.469
0.625
0.234
0.438
0.006
0.078
>4.25
2.25
1.875
0.375
0.813
0.281
>4.25
>4.25
>4.25
>4.25
3.5
0.006
0.063
3.8
0.094
0.188
0.109
0.063
2
3
4
5
6
7
>4.25
This somewhat surprising influence of the hydroxyl position
in the proline residue warranted the synthesis of three additional
analogues. The synthesis of these macrocyclic peptides 19–21
was performed analogously to the synthesis of mimic 4 in
Scheme 3 and their antifungal activity was evaluated and
expressed as MIC values as is shown in Table 2.
These analogues confirm the finding that the position of the
hydroxyl group in the top-left proline residue (R2 and R3 in
Hyp-6) is crucial for antifungal activity. Analogues 3, 20 and 21
(R2 = OH) bearing the same lipophilic tail as analogues 6, 4 and
7 (R3 = OH), respectively, were practically inactive or showed a
tremendous decrease in antifungal activity against Candida.
Clearly R2 = OH is only allowed in caspofungin derivative 2
bearing a terphenyl fatty acid side chain. Here analogue 19 with
R3 = OH and analogue 2 with R2 = OH both showed substantial
antifungal activity against Candida. Analogue 19 was even more
active than analogue 2 and had activities comparable to those of
caspofungin (1). In addition the selectivity of inhibition of
Candida parapsilosis by 19 was remarkable and was not shown
by any of the other analogues (see Tables 1 and 2).
These results suggest that the position of the hydroxyl group
in the top-left hydroxyproline of the cyclic hexapeptide
backbone (R2 and R3 in Hyp-6, Fig. 1) in combination with the
character of the lipophilic fatty acid chain was a crucial factor for
antifungal activity.
In attempts to interpret this finding we evaluated the analogues
by circular dichroism (CD) in order to probe their conformation-
al properties.
Circular dichroism spectroscopy
In addition to secondary structure prediction, circular dichroism
spectroscopy can give valuable insights into peptide structure.34
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Table 2 In vitro antifungal activity of caspofungin analogues against common Candida species displayed as MIC values in μg mL−1
Candida
albicans
CBS9975
Candida
albicans
CBS8758
Candida
dubliniensis
CBS7987
Candida
glabrata
CBS138
Candida
krusei
CBS573
Candida
parapsilosis
CBS604
Candida
tropicalis
CBS94
Peptide
19
20
21
0.051
1.63
>4.25
0.039
3.0
>4.25
0.031
1.5
3.0
0.027
2.5
>4.25
0.027
>4.25
>4.25
0.75
>4.25
>4.25
0.141
2.5
>4.25
Fig. 2 CD spectra of caspofungin and analogues (2–7, 19–21). (A) + (B) Measured in MeCN/H2O (1/1, v/v); (C) + (D) measured in TFE/H2O (1/1, v/v).
All peptides were measured at 0.1 mM concentration.
As was described by Ovchinnikov and Ivanov,35 CD spectra can
provide evidence for the perturbation or alteration of the confor-
mational state in a series of related peptides.34 Therefore, CD
may provide an attractive technique to evaluate our analogues
and determine whether the observed antifungal activity can be
correlated with the conformational state.
Thus, the secondary structure of caspofungin (1) and
analogues (2–7, 19–21) was investigated by circular dichroism
as presented in Fig. 2. In addition to measurements in MeCN/
H2O, CD spectra were measured in TFE/H2O, since TFE is
known to enhance secondary structure formation of peptides.36
As is shown in Fig. 2, spectra of the cyclic peptides 4–7 in
both MeCN/H2O and TFE/H2O are similar. Peptides 4–7 are
characterized by a negative band at ca. 220 nm and a positive
band at 205 nm. In general, these peptides (4–7) showed CD
spectra characteristic for a type II β-turn.37–40 However, their
pattern below 215 nm is clearly different from caspofungin (1).
This difference may be explained by the deletion of several
hydroxyl groups in our analogues. Moreover, analogue 19
showed a distinctly different pattern at wavelengths below
210 nm and higher ellipticities of the band compared to peptides
4–7. This observation may be explained by a large contribution
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of the aromatic residues present in the side chain of analogue 19
as is supported by CD spectra of other analogues (data in ESI†).
Remarkably, cyclic peptides 3, 20 and 21 (R2 = OH; Fig. 2B
and D) exhibit different spectra than series 4–7 (R3 = OH;
Fig. 2A and C). Differences are apparent in the ellipticities of
the bands, which could indicate that these peptides have a more
disordered structure with less β-turn character. The only struc-
tural difference between these analogues is the position of the
hydroxyl group in the left-upper hydroxyproline of the hexa-
peptide ring (R2 and R3 in Hyp-6, Fig. 1). Apparently, merely
the position of the OH-function was responsible for the largely
different CD-spectra and possibly conformational behaviour of
the peptides.
Extensive NMR studies showed that in solution these peptides
comprise many rapidly exchanging conformations, which result
in averaged NMR spectra, and we have not been able to detect
any conformational preferences of the peptides with either the
hydroxyl group at the R2 or R3 position. So far, crystallization
attempts of these peptides were also unsuccessful. We assume
that the inherent flexibility of the cyclic peptides is the main
reason for this. CD measurements showed subtle but distinctive
changes indicative of structural changes in the conformation of
these cyclic peptides. The results of this investigation suggest
that the top-left proline residue somehow plays an important role
in the bio-active conformation of the macrocyclic peptide ring
structure.
For further insights into the influence of the OH-function on
the conformation of our analogues, models were built and super-
imposed with the reported crystal structure of Echinocandin
B (ECBN)12 (data in ESI, Fig. S3†). Overall, the more active
analogue 6 appeared to have a better superimposition with the
crystal structure. Analogue 3 had a more puckered structure with
the Hyp-6 residue situated more in the back of the molecule as
compared to analogue 6. In addition, the Orn-5 residue was
pointing out more to the front of the macrocyclic peptide ring
structure in 3. These models support the finding that the position
of the OH-function in Hyp-6 induces subtle changes in the con-
formation of the peptides. Apparently, these add up and were
sufficient for a large effect on the biological activity.
Finally, we have observed that the choice of the fatty acid
derivative (rigid as in 2 or flexible as in 3) in combination with
the conformational character of the ring may be a determining
factor for antifungal activity.
Experimental section
Reagents, materials and analysis methods
Unless stated otherwise, all chemicals were obtained from com-
mercial suppliers and used without further purification. Piper-
idine, N,N-diisopropylethylamine (DiPEA), peptide grade
dichloromethane (DCM), N,N-dimethylformamide (DMF),
N-methylpyrrolidone (NMP), tert-butyl methyl ether (MTBE),
trifluoroacetic acid (TFA) and HPLC grade solvents were pur-
chased from Biosolve B.V. (Valkenswaard, The Netherlands) and
used directly, with the exception of DMF, NMP and DCM,
which were dried on molecular sieves (4 Å). The coupling
reagents benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium
hexafluorophosphate (BOP), O-(7-azabenzotriazol-1-yl)-N,N,N′,
N′-tetramethyluronium hexafluorophosphate (HATU) and
Nα-9-fluorenylmethyloxycarbonyl (Fmoc) protected amino acids
were purchased from GL Biochem Ltd (Shanghai, China). Pro-
tected homotyrosine (H-hTyr(tBu)-OH) was purchased from
Advanced Chemtech (Louisville, United States) and furnished
with the Fmoc group. Fmoc-Orn(Mtt)-OH was obtained from
Nova Biochem and Fmoc-3-Hyp(tBu)-OH was synthesized
according to a literature procedure.41 Triisopropylsilane (TIS)
was obtained from Merck (Darmstadt, Germany). The 2-chloro-
tritylchloride PS resin cross-linked with 1% DVB (200–
400 mesh) was purchased from Hecheng Chemicals (Shanghai,
China) (theoretical loading: 1.10 mmol g−1).
Conclusion
A versatile approach for the rapid enantioselective synthesis of
the dimethylmyristoyl chain of caspofungin and a one methyl
group containing analogue is described. These fatty acids were
coupled to simplified analogues of the caspofungin skeleton. In
this way access was obtained to new fully synthetic derived
caspofungin mimics with high and selective antifungal activities
against Candida. Unexpectedly, no obvious relationship between
the presence of the methyl groups in the dimethylmyristoyl tail 8
of caspofungin (1) and antifungal activity was observed. The
dimethyl containing lipophilic tail derivative 4 did not lead to
significant improvement of its bioactivity as compared to that of
the non-methylated lipophilic chain. In fact the activities of non-
methylated derivatives 6 and 7 and the methylated derivatives 4
and 5 were quite similar in most species. The position of the
hydroxyl group in the top-left proline residue (R2 and R3 in
Hyp-6) was apparently more crucial: when this group is moved
from the beta (R3) to the gamma (R2) position, activity is lost
when the fatty acid side chain is an acyl side chain. Clearly
R2 = OH is only allowed in caspofungin derivative 2 bearing a
terphenyl fatty acid side chain. Thus, the position of the hydroxy
group in the top-left hydroxyproline of the cyclic hexapeptide
backbone (R2 and R3 in Hyp-6, Fig. 1) in combination with the
character of the lipophilic fatty acid chain was a crucial factor for
antifungal activity. The position of the hydroxy group turned out
to be very important for the general activity against Candida
species as was shown by the higher activity of analogue 19
compared to analogue 2, but also for selectivity of inhibition.
Compound 19 was capable of a much more selective inhibition
of Candida parapsilosis than analogue 2.
All reactions were carried out at room temperature unless
stated otherwise. Solid phase synthesis was performed in plastic
syringes with a polyethylene frit. Synthesis in solution was moni-
tored by TLC on Merck pre-coated silica 60 plates. Spots were
visualized by UV light, K2CO3/KMnO4 or Seebach stain. Solid
phase reactions were monitored with the chloranil test42 in the
case of secondary amines or with the Kaiser test43 in the case
of primary amines. Column chromatography was performed
using Silicycle UltraPure silica gel (40–63 μm).
1H NMR spectra were recorded on a Varian AMX 400 MHz
spectrometer and chemical shifts are given in ppm relative to
TMS (0.00 ppm). 13C NMR spectra were recorded using the
attached proton test (APT) sequence on a Varian AMX 400
(101 MHz) spectrometer and chemical shifts are given in ppm
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relative to CDCl3 (77.0 ppm). For measurements in DMSO, the
residual solvent peak was used as a reference. For the peptides,
1H NMR, TOCSY, 1H–13C HSQC and ROESY spectra were
recorded using a Varian INOVA-500 spectrometer (500 MHz).
Purity of the peptides was confirmed by analytical HPLC
using an Alltima C8 column (4.6 × 250 mm; 5 μm) at a flow rate
of 0.5 mL min−1 using a linear gradient of buffer B (100% in
40 min) from 100% buffer A. Analytical HPLC was performed
on a Shimadzu automated HPLC system equipped with an eva-
porative light scattering detector (PL-ELS 1000) and a UV/Vis
detector operated at 220/254 nm. Preparative HPLC runs were
performed using an Alltima C8 column (22 × 250 mm; 10 μm)
at a flow rate of 6 mL min−1 with a linear gradient of buffer
B (100% in 80 min) from 100% buffer A on an Applied Bio-
systems 400 solvent delivery system with an Applied Biosystems
757 UV/VIS absorbance detector. The buffer system used con-
sists of buffer A: 0.1% TFA in MeCN/H2O, 20/80, v/v and
buffer B: 0.1% TFA in i-PrOH/MeCN/H2O, 45/50/5, v/v/v.
ESI-MS spectra were obtained in the positive ion mode on a
Shimadzu QP8000 single quadrupole mass spectrometer. HRMS
analyses were performed on a MALDI TOF/TOF (Applied
Biosystems).
0.520 mmol g−1. Subsequently, unreacted trityl chloride moieties
were capped with methanol (DCM/MeOH/DiPEA; 3 × 5 mL,
each 2 min; 17/2/1; v/v/v). The peptide sequence was syn-
thesized according to the general procedure for solid phase
peptide synthesis. The resin-bound peptide 17 was divided in
portions for the synthesis of the analogues 4–7.
The Fmoc group was cleaved and the fatty acid side chain was
coupled by adding the tail (3 equiv.), HATU (3 equiv.) and
DiPEA (6 equiv.) in NMP (3 mL) to the resin. The mixture was
shaken overnight, subsequently the coupling solution was
removed by filtration and the resin washed with NMP
(3 × 4 mL, each 3 min) and DCM (3 × 4 mL, each 3 min). Com-
pletion of the coupling was checked with the Kaiser test. Then,
mild acidolytic cleavage, by treatment with TFE/AcOH/DCM
(2/1/7, v/v/v, 5 mL) for 2 h, of the Mtt group as well as cleavage
from the resin gave protected peptide precursor 18. The mixture
was concentrated in vacuo and the peptide was precipitated in
MTBE/hexanes (3×, 1/1, v/v).
The linear peptide was then subjected to head-to-tail cycliza-
tion by dissolving it in dry DMF (2 mL μmol−1) followed by the
addition of BOP (4 equiv.) and DiPEA (8 equiv.). The mixture
was stirred for 48 h and then evaporated in vacuo. The product
was redissolved in EtOAc and washed with 1 M KHSO4 (2×),
NaHCO3 (2×) and H2O (2×). The organic layer was dried over
Na2SO4, filtered and concentrated in vacuo. Final protecting
group removal by treatment with a mixture of TFA/TIS/H2O
(2 mL, 95/2.5/2.5, v/v/v) for 2 h and precipitation in MTBE/
hexanes (3×, 1/1, v/v) afforded the crude peptide. After lyophili-
zation from tert-BuOH/H2O (1/1, v/v) the peptide was purified
by preparative HPLC.
Chemistry
General procedures for solid phase synthesis. Peptides were
synthesized manually. Used abbreviations: DMT-Myr:
(10R,12S)-dimethylmyristoyl; Me-Myr: (12S)-methylmyristoyl;
Myr: myristoyl; Palm: palmitoyl; Ter: terphenyl; 3-Hyp:
3-hydroxyproline; hTyr: homotyrosine; Mtt: 4-methyltrityl. Each
synthetic cycle consisted of the following steps.
Cyclo[-(DMT-Myr)-Orn-Thr-Hyp-hTyr-Orn-3-Hyp] (4). Peptide
4 was obtained according to the general procedure for the prep-
aration of the caspofungin analogues using 149.4 μmol of resin
bound peptide 17. After lyophilization, peptide 4 (74.5 mg;
51%) was obtained as a colourless solid.
Fmoc-group removal: The resin was treated with a 20% solu-
tion of piperidine in NMP (3×, each 10 min). The solution was
removed by filtration and the resin was washed with NMP
(3×, each 3 min) and DCM (3×, each 3 min).
Coupling step: A mixture of Fmoc-Xxx-OH (3 equiv.), BOP
(3 equiv.) and DiPEA (6 equiv.) in NMP (10 mL mmol−1) was
added to the resin and N2 was bubbled through the mixture for
2 h. The solution was removed by filtration and the resin washed
with NMP (3×, each 3 min) and DCM (3×, each 3 min). Com-
pletion of the coupling was checked with the Kaiser or chloranil
test.
Capping of the remaining free amines: Capping solution
[Ac2O (50 mmol, 4.7 mL), HOBt (1.9 mmol, 220 mg), DiPEA
(12.5 mmol, 2.2 mL) in 100 mL NMP] (10 mL mmol−1) was
added to the resin and N2 was bubbled through the mixture for
20 min. The solution was removed by filtration and the resin was
washed with NMP (3×, each 3 min) and DCM (3×, each 3 min).
Purity was confirmed by analytical HPLC and was found to
be higher than 99% (Rt = 41.28 min). ESI-MS calcd for
C50H82N8O11: 970.61, found: m/z 971.90 [M + H]+; HRMS
calcd for C50H82N8O11 [M + H]+ 971.6181, found 971.6124;
1H-NMR (CD3OH, 500 MHz): δ Orn-1: 8.34 (NH), 4.48 (αCH),
2.31/1.79 (βCH), 1.68 (γCH), 3.60/2.85 (δCH); Thr-2: 8.73
(NH), 4.90 (αCH), 4.53 (βCH), 1.22 (γCH); Hyp-3: 4.49 (αCH),
2.27/1.97 (βCH), 4.50 (γCH), 3.78/3.70 (δCH); hTyr-4: 7.62
(NH), 4.81 (αCH), 1.96/1.67 (βCH), 2.98/2.92 (γCH), 6.92/6.69
(Ar-H); Orn-5: 7.50 (NH), 4.30 (αCH), 2.21/1.92 (βCH), 2.17
(γCH), 2.57 (δCH); Hyp-6: 4.28 (αCH), 4.08 (βCH), 2.27/1.97
(γCH), 3.78/3.71 (δCH); Tail: 2.25 (C(O)CH2), 1.81/1.75 (CH2),
1.67/1.61 (CH2), 1.49, 1.42, 1.35, 1.33/1.27, 1.31/1.06, 1.28
(CH2), 1.24/0.92 (CHCH2CH), 1.09, 0.87 (CH3), 0.85 (CH3)
ppm. 13C-NMR (CD3OH, 125 MHz): δ Orn-1: 53.1 (αCH), 38.1
(βCH), 24.0 (γCH), 37.6 (δCH); Thr-2: 57.9 (αCH), 68.9 (βCH),
19.7 (γCH); Hyp-3: 62.4 (αCH), 34.2 (βCH), 71.4 (γCH), 56.9
(δCH); hTyr-4: 50.8 (αCH), 30.0 (βCH), 40.6 (γCH), 130.7/
116.5 (Ar-H); Orn-5: 54.7 (αCH), 34.4 (βCH), 27.0 (γCH), 32.6
(δCH); Hyp-6: 74.5 (αCH), 69.4 (βCH), 34.2 (γCH), 46.5
(δCH); Tail: 45.7 (CHCH2CH), 37.8, 36.7 (C(O)CH2), 32.6,
30.9, 30.0, 27.7, 26.9 (CH2), 23.9 (CH2), 30.8 (CH2), 30.1,
20.05 (CH3), 11.2 (CH3).
General procedure for the preparation of the caspofungin
analogues (4–7). A polystyrene resin functionalized with a 2-
chloro trityl chloride linker (550 mg; initial loading: 1.1 mmol g−1
)
was loaded with Fmoc-3-Hyp(tBu)-OH (999.4 mg;
2.44 mmol) in DCM (5 mL) in the presence of DiPEA (850 μL;
4.88 mmol) for 16 h. The solution was removed by filtration and
the resin washed with DCM (6 × 5 mL, each 3 min). After
drying in vacuo overnight, the amount of Fmoc-3-Hyp(tBu)-OH
coupled to the resin was determined by an Fmoc determination
according to Meienhofer et al.44 and was found to be
This journal is © The Royal Society of Chemistry 2012
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Cyclo[-(Me-Myr)-Orn-Thr-Hyp-hTyr-Orn-3-Hyp] (5). Peptide
5 was obtained according to the general procedure for the prep-
aration of the caspofungin analogues using 39 μmol of resin
bound peptide 17. After lyophilization, peptide 5 (17.9 mg;
48%) was obtained as a colourless solid.
preparation of the caspofungin analogues using 39 μmol of resin
bound peptide 17. After lyophilization, peptide 7 (32.4 mg;
88%) was obtained as a colourless solid.
Purity was confirmed by analytical HPLC and was found to
be higher than 99% (Rt = 39.33 min). ESI-MS calcd for
C48H78N8O11: 942.58, found: m/z 944.00 [M + H]+; HRMS
calcd for C48H78N8O11 [M + H]+ 943.5868, found 943.5867;
1H-NMR (CD3OH, 500 MHz): δ Orn-1: 8.16 (NH), 8.34 (εNH),
4.48 (αCH), 2.31/1.78 (βCH), 1.67 (γCH), 3.59/2.85 (δCH);
Thr-2: 8.72 (NH), 4.89 (αCH), 4.53 (βCH), 1.22 (γCH); Hyp-3:
4.49 (αCH), 2.27/1.97 (βCH), 4.50 (γCH), 3.78/3.70 (δCH);
hTyr-4: 7.62 (NH), 4.81 (αCH), 1.96/1.66 (βCH), 2.97/2.92
(γCH), 6.99/6.69 (Ar-H); Orn-5: 7.53 (NH), 4.30 (αCH), 2.21/
1.92 (βCH), 2.17 (γCH), 2.57 (δCH); Hyp-6: 4.28 (αCH), 4.08
(βCH), 2.27/1.96 (γCH), 3.78/3.71 (δCH); Tail: 2.25 (C(O)CH2),
1.81/1.75 (CH2), 1.66/1.60 (CH2), 1.32 (CH2), 1.31 (CH2), 1.29
(CH2), 1.27 (CH2), 0.90 (CH3) ppm. 13C-NMR (CD3OH,
125 MHz): δ Orn-1: 53.1 (αCH), 38.1 (βCH), 24.0 (γCH), 37.6
(δCH); Thr-2: 58.0 (αCH), 69.0 (βCH), 19.7 (γCH); Hyp-3: 62.3
(αCH), 34.2 (βCH), 71.3 (γCH), 56.9 (δCH); hTyr-4: 50.8
(αCH), 29.9 (βCH), 40.6 (γCH), 130.7/116.5 (Ar-H); Orn-5:
54.7 (αCH), 34.4 (βCH), 27.0 (γCH), 32.7 (δCH); Hyp-6: 74.5
(αCH), 69.4 (βCH), 34.2 (γCH), 46.5 (δCH); Tail: 36.7 (C(O)-
CH2), 32.9 (CH2), 30.4 (CH2), 30.0 (CH2), 27.0 (CH2), 23.9
(CH2), 23.4 (CH2), 14.2 (CH3).
Purity was confirmed by analytical HPLC and was found to
be higher than 95% (Rt = 40.48 min). ESI-MS calcd for
C49H80N8NaO11: 979.58, found: m/z 979.85 [M + Na]+; HRMS
calcd for C49H80N8O11: [M + H]+ 957.6025, found 957.6016;
1H-NMR (CD3OH, 500 MHz): δ Orn-1: 8.15 (NH), 8.33 (εNH),
4.48 (αCH), 2.30/1.79 (βCH), 1.67 (γCH), 3.59/2.84 (δCH);
Thr-2: 8.72 (NH), 4.89 (αCH), 4.53 (βCH), 1.22 (γCH); Hyp-3:
4.48 (αCH), 2.27/1.96 (βCH), 4.49 (γCH), 3.77/3.70 (δCH);
hTyr-4: 7.72 (NH), 4.80 (αCH), 1.95/1.66 (βCH), 2.98/2.91
(γCH), 6.99/6.68 (Ar-H); Orn-5: 7.52 (NH), 4.30 (αCH), 2.20/
1.91 (βCH), 2.17 (γCH), 2.56 (δCH); Hyp-6: 4.28 (αCH), 4.07
(βCH), 2.27/1.96 (γCH), 3.77/3.70 (δCH); Tail: 2.24 (C(O)CH2),
1.80/1.75 (CH2), 1.66/1.59 (CH2), 1.32 (CH2), 1.30, 1.30/1.27,
1.30/1.09, 1.29 (CH2), 1.27 (CH2), 1.13, 0.87 (CH3), 0.85(CH3)
ppm. 13C-NMR (CD3OH, 125 MHz): δ Orn-1: 53.1 (αCH), 38.1
(βCH), 24.0 (γCH), 37.6 (δCH); Thr-2: 57.9 (αCH), 68.9 (βCH),
19.8 (γCH); Hyp-3: 62.3 (αCH), 34.2 (βCH), 71.3 (γCH), 56.8
(δCH); hTyr-4: 50.8 (αCH), 30.0 (βCH), 40.6 (γCH), 130.7/
116.5 (Ar-H); Orn-5: 54.6 (αCH), 34.4 (βCH), 26.9 (γCH) 32.6
(δCH); Hyp-6: 74.5 (αCH), 69.4 (βCH), 34.2 (γCH), 46.5
(δCH); Tail: 37.6, 36.6 (C(O)CH2), 35.4, 30.7 (CH2), 30.3, 30.1
(CH2), 27.9, 26.9 (CH2), 23.9 (CH2), 23.8 (CH2), 19.3 (CH3),
11.4 (CH3).
(E)-S-Ethyl pent-2-enethioate (10). To a cooled solution
(0 °C) of (E)-pentenoic acid 9 (5.01 g; 50 mmol), DCC
(11.35 g; 55 mmol) and DMAP (611 mg; 5 mmol, 10 mol%) in
pentane (500 mL), neat EtSH (7.22 mL; 100 mmol) was added
dropwise. The mixture was allowed to slowly reach room temp-
erature and stirred for 16 h. After filtration through a short silica
pad, volatiles were evaporated. The crude residue was distilled
(Kugelrohr) and afforded product 10 as a colorless liquid
(6.85 g; 95%).
Cyclo[-(Palm)-Orn-Thr-Hyp-hTyr-Orn-3-Hyp] (6). Peptide 6
was obtained according to the general procedure for the prep-
aration of the caspofungin analogues using 39 μmol of resin
bound peptide 17. After lyophilization, peptide 6 (24.5 mg;
65%) was obtained as a colourless solid.
Purity was confirmed by analytical HPLC and was found to
be higher than 99% (Rt = 42.28 min). ESI-MS calcd for
C50H82N8O11: 971.23, found: m/z 972.45 [M + H]+; HRMS
calcd for C50H82N8O11 [M + H]+ 971.6181, found 971.6197;
1H-NMR (CD3OH, 500 MHz): δ Orn-1: 8.16 (NH), 4.48 (αCH),
2.30/1.78 (βCH), 1.67 (γCH), 3.59/2.84 (δCH); Thr-2: 8.72
(NH), 4.88 (αCH), 4.52 (βCH), 1.21 (γCH); Hyp-3: 4.49 (αCH),
2.26/1.96 (βCH), 4.49 (γCH), 3.77/3.70 (δCH); hTyr-4: 7.72
(NH), 4.81 (αCH), 1.96/1.66 (βCH), 2.97/2.91 (γCH), 6.99/6.68
(Ar-H); Orn-5: 7.52 (NH), 4.30 (αCH), 2.20/1.91 (βCH), 2.17
(γCH), 2.57 (δCH); Hyp-6: 4.28 (αCH), 4.07 (βCH), 2.26/1.96
(γCH), 3.77/3.71 (δCH); Tail: 2.24 (C(O)CH2), 1.78/1.75 (CH2),
1.66/1.60 (CH2), 1.31 (CH2), 1.30 (CH2), 1.28 (CH2), 1.27
(CH2), 0.89 (CH3) ppm. 13C-NMR (CD3OH, 125 MHz): δ Orn-
1: 53.1 (αCH), 38.1 (βCH), 24.0 (γCH), 37.6 (δCH); Thr-2: 58.0
(αCH), 68.9 (βCH), 19.7 (γCH); Hyp-3: 62.3 (αCH), 34.2
(βCH), 71.3 (γCH), 56.9 (δCH); hTyr-4: 50.8 (αCH), 30.0
(βCH), 40.6 (γCH), 130.7/ 116.5 (Ar-H); Orn-5: 54.6 (αCH),
34.4 (βCH), 27.0 (γCH), 32.7 (δCH); Hyp-6: 74.5 (αCH), 69.4
(βCH), 34.2 (γCH), 46.5 (δCH); Tail: 36.7 (C(O)CH2), 32.8
(CH2), 30.5 (CH2), 30.0 (CH2), 27.0 (CH2), 23.9 (CH2), 23.4
(CH2), 14.1 (CH3).
1H NMR (400 MHz, CDCl3): δ 6.96–6.84 (m, 1H), 6.06 (dd,
J = 15.5, 1.5 Hz, 1H), 2.95–2.84 (m, 2H), 2.18 (qd, J = 7.2,
3.7 Hz, 2H), 1.28–1.01 (m, 3H), 0.99 (s, 3H) ppm. 13C NMR
(101 MHz, CDCl3): δ 190.03 (Cq), 146.46 (CH), 127.79 (CH),
25.18 (CH2), 22.95 (CH2), 14.78 (CH3), 12.05 (CH3). Spectral
data correspond to literature.28a
(S)-S-Ethyl 3-methylpentanethioate (11). (S,RFe)-Josiphos·
EtOH adduct (43.2 mg; 67 μmol) and CuBr·Me2S (12.8 mg;
62 μmol) were stirred in freshly distilled MTBE (56 mL) until
the mixture remained homogeneous (typically 10–30 min). Then
the mixture was cooled to −78 °C and after 10 min a solution of
MeMgBr in Et2O (2.7 mL; 8.1 mmol) was added dropwise
during 10 min. After 15 min of stirring a solution of thioester 10
(721 mg; 5 mmol) in MTBE (6.2 mL) was added over 3 h by a
syringe pump. The reaction mixture was stirred for an additional
16 h at −78 °C, quenched with EtOH (5 mL) and allowed to
reach ambient temperature. Then a solution of NH4Cl (1 M;
50 mL) was added. The organic layer was separated and the
aqueous layer extracted with Et2O (3 × 20 mL). The combined
organic layers were dried over MgSO4 and carefully evaporated
(the product is volatile). The residual yellow liquid was purified
by flash chromatography (Et2O : pentane; 1 : 4) to afford
Cyclo[-(Myr)-Orn-Thr-Hyp-hTyr-Orn-3-Hyp] (7). Peptide 7
was obtained according to the general procedure for the
7498 | Org. Biomol. Chem., 2012, 10, 7491–7502
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(3S,5S)-S-Ethyl 3,5-dimethylheptanethioate (13). (S,RFe)-Josi-
phos–CuBr complex (29.1 mg; 39 μmol) was dissolved in
freshly distilled MTBE (23.7 mL) until the mixture remained
homogeneous (typically 10–30 min). Then the mixture was
cooled to −78 °C and after 10 min a solution of MeMgBr in
Et2O (3 M in Et2O; 1.1 mL; 3.42 mmol) was added dropwise.46
After 15 min of stirring, a solution of thioester 12 (490 mg;
2.63 mmol) in MTBE (2.6 mL) was added over 3 h by a syringe
pump. The mixture was stirred for an additional 16 h at −78 °C.
The reaction was quenched by addition of EtOH (2 mL) and the
mixture was allowed to reach ambient temperature. Then an
aqueous solution of NH4Cl (1 M, 30 mL) was added, the
organic layer separated and the aqueous layer extracted with
Et2O (3 × 20 mL). The combined organic layers were dried over
MgSO4 and carefully evaporated (the product is volatile). The
residual yellow liquid was purified by flash chromatography
(MTBE : pentane; 1 : 250) to afford (3S,5S)-S-ethyl 3,5-
dimethylheptanethioate 13 (417.7 mg; 78%) as a colorless
liquid.
(S)-S-ethyl 3-methylpentanethioate 11 (800 mg; 80%) as a col-
ourless liquid.
HRMS: calcd for C8H16OS [M + H]+ 161.0994, found
1
161.0996; H NMR (400 MHz, CDCl3): δ 2.87 (q, J = 7.4 Hz,
2H), 2.53 (dd, J = 14.4, 6.1 Hz, 1H), 2.34 (dd, J = 14.4, 8.1 Hz,
1H), 2.17–1.72 (m, 1H), 1.45–1.30 (m, 1H), 1.24 (t, J = 7.4 Hz,
4H), 1.08–0.72 (m, 6H) ppm. 13C NMR (101 MHz, CDCl3)
δ 199.36, 51.01, 32.63, 29.23, 23.24, 19.02, 14.78, 11.21. NMR
spectra contain traces of solvents (≈5%). Spectral data corres-
pond to literature.28a
The enantiomeric ratio was determined on the corresponding
methyl ester by chiral stationary phase gas chromatography on a
Chiraldex G-TA column (30 m × 0.25 mm), 60 °C, retention
times: 5.94 (R)/6.05 (S) min: 85 : 1 (er), 97% ee (as reported in
the literature28a) [α]D = +8.4 (c = 1.0 in CHCl3).
The absolute configuration was determined on the alcohol:28a
HRMS: calcd for C11H22OS [M + H]+ 203.147, found
1
203.110; [α]D = +4.2 (c = 1.4 in CHCl3); H NMR (400 MHz,
[α]D = +7.4 (c = 0.95 in CHCl3).
Literature45 reports the opposite enantiomer with αD = −8.5
(c = 1 in CHCl3).
CDCl3): δ 2.86 (q, J = 7.4 Hz, 2H), 2.52 (dd, J = 14.4, 5.4 Hz,
1H), 2.28 (dd, J = 14.4, 8.5 Hz, 1H), 1.43–1.27 (m, 2H), 1.24
(t, J = 7.4 Hz, 4H), 1.14–0.94 (m, 3H), 0.92 (d, J = 6.6 Hz, 3H),
0.85 (d, J = 6.5 Hz, 6H) ppm. 13C NMR (101 MHz, CDCl3):
δ 199.33, 51.29, 44.07, 31.53 (CH1), 29.00, 28.66 (CH1),
23.23, 20.15 (CH3), 19.48 (CH3), 14.79 (CH3), 11.07 (CH3).
(S,E)-S-Ethyl 5-methylhept-2-enethioate (12). A solution of
(S)-S-ethyl 3-methylpentanethioate 11 (801 mg; 5 mmol) in
CH2Cl2 (50 mL) was cooled to −55 °C and then a solution of
DIBAL (1 M in CH2Cl2; 6 mL; 6 mmol) was added. The
mixture was stirred until complete conversion of the starting
material (ca. 1.5 h). Subsequently, the mixture was poured into a
saturated Rochelle’s salt (potassium sodium tartrate) solution
and stirred until the phases separated (mostly within 2 h). Layers
were separated and the aqueous layer was extracted with CH2Cl2
(3 × 15 mL). The combined layers were dried and carefully
evaporated (the product is volatile) until the weight corresponded
to a quantitative yield (501 mg).
(10S,12S)-Dimethyltetradec-7-enoic acid (14). A solution of
(S)-S-ethyl 3-methylpentanethioate (417 mg; 2.06 mmol) in
CH2Cl2 (20 mL) was cooled to −55 °C and a solution of
DIBAL (1 M in CH2Cl2; 2.5 mL; 2.5 mmol) was added. The
mixture was stirred until complete conversion of the starting
material (ca. 1.5 h), then poured into saturated aqueous
Rochelle’s salt and stirred until phases separated (mostly within
2 h). Layers were separated and the aqueous layer was extracted
with CH2Cl2 (3 × 15 mL). The combined layers were dried and
carefully evaporated (the product is volatile) to a weight corres-
ponding to a quantitative yield (294 mg).
To a cooled solution (0 °C) of HWE reagent (1.8 g;
7.5 mmol) in THF (25 mL) a solution of n-BuLi (1.6 M in
hexanes; 3.44 mL; 5.55 mmol) was added dropwise. The reac-
tion mixture was stirred for 20 min at 0 °C. Then (S)-3-methyl-
pentanal (501 mg; 5 mmol) in a small amount of THF (0.3 mL)
was added and the reaction mixture was stirred overnight (16 h).
The reaction was quenched with water (10 mL). Layers
were separated and the aqueous layer extracted with Et2O
(3 × 15 mL). The combined organic layers were dried over
MgSO4 and carefully evaporated (the product is volatile). The
residue was purified by flash chromatography on SiO2 (MTBE :
pentane; 250 : 1) and afforded (S,E)-S-ethyl 5-methylhept-
2-enethioate 12 (510 mg; 55%) as a colorless liquid.
To a stirred suspension of 7-(bromotriphenylphosphoranyl)-
heptanoic acid (1.65 g; 3.5 mmol) in THF (2 mL) at ambient
temperature a solution of LiHMDS (1 M in THF; 6.2 mL;
6.18 mmol) was added dropwise. The mixture was stirred until
the suspension turned into a deep red solution. Then a solution
of (3S,5S)-3,5-dimethylheptanal (293 mg; 2 mmol) in a small
amount of THF (300 μL) was added and the reaction mixture
was stirred until complete consumption of the starting material
(2 h). The mixture was acidified to pH = 1 by dilute aq. HCl and
extracted with Et2O (3 × 20 mL). The combined organic layers
were dried and evaporated. The resulting thick liquid was
purified by column chromatography (Et2O : pentane; 1 : 4) and
afforded 14 (331 mg; 47%) as a colorless liquid (a mixture of
E and Z isomers).
HRMS calcd for C10H18OS [M + H]+ 187.1151, found
1
187.1152; [α]D = +8.2 (c = 1.8 in CHCl3); H NMR (400 MHz,
CDCl3): δ 6.85 (dtd, J = 8.6, 7.6, 1.2 Hz, 1H), 6.08 (dd, J =
15.5, 1.3 Hz, 1H), 3.05–2.83 (m, 2H), 2.17 (ddd, J = 7.2, 6.5,
3.5 Hz, 1H), 2.09–1.91 (m, 1H), 1.53 (dq, J = 13.3, 6.9 Hz, 1H),
1.36 (dt, J = 14.5, 6.8 Hz, 1H), 1.26 (ddd, J = 7.4, 4.9, 1.2 Hz,
3H), 0.86 (td, J = 7.0, 3.2 Hz, 6H) ppm. 13C NMR (101 MHz,
CDCl3): δ 189.95, 144.27, 129.69, 39.21, 34.19, 29.18, 22.99,
19.14, 14.79, 11.34 + 2 peaks at 65.81 and 15.24 as Et2O
residues.
HRMS: calcd for C16H30O2 [M + H]+ 255.232, found
1
255.235; [α]D = +12.2 (c = 1.8 in CHCl3); H NMR (400 MHz,
CDCl3): δ 5.51–5.22 (m, 2H), 2.35 (t, J = 7.5 Hz, 2H),
2.13–0.55 (m, 25H) ppm. 13C NMR (101 MHz, CDCl3):
δ 131.22, 130.17, 129.03, 128.67, 44.23, 44.00, 39.77, 34.26,
34.03, 32.35, 31.61, 31.55, 30.70, 30.42, 29.29, 29.16, 28.72,
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28.49, 27.08, 24.57, 24.51, 20.16, 20.06, 19.68, 11.14 –
additional peaks observed due to the inseparable E/Z mixture.
Spectral data correspond to the literature.27
by dilute aq. HCl and extracted with Et2O (3 × 20 mL). The
combined organic layers were dried and evaporated. The residual
red liquid was purified by column chromatography (Et2O :
pentane; 1 : 4) and afforded 16 (50 mg, 83%) as a colorless thick
liquid.
(10R,12S)-Dimethyltetradecanoic acid (8). To a vigorously
stirred solution of (10S,12S)-dimethyltetradec-7-enoic acid 14
(310.1 mg; 1.2 mmol) and flavine catalyst (49.6 mg; 0.12 mmol,
10 mol%) in EtOH (1 mL) under an oxygen atmosphere, hydra-
zine hydrate (1.6 mL; 31.7 mmol) was added in one portion.
Vigorous stirring continued for 16 h. Then the reaction mixture
was acidified to pH = 1 by dilute aq. HCl and extracted with
Et2O (3 × 20 mL). The combined organic layers were dried and
evaporated. The residual red liquid was purified by column
chromatography (Et2O : pentane; 1 : 4) and afforded 8 (524 mg;
81%) as a colorless thick liquid.
HRMS: calcd for C15H31O2 [M + H]+ 243.231, found
1
243.233; [α]D = +0.3 (c = 0.5 in CHCl3); H NMR (400 MHz,
CDCl3): δ 2.34 (t, J = 7.5 Hz, 2H), 1.65–1.60 (m, 2H),
1.32–1.23 (m, 20H), 0.87–0.81 (m, 6H) ppm. 13C NMR
(101 MHz, CDCl3): δ 220.66, 36.62, 34.38, 34.07, 29.99, 29.66,
29.58, 29.48, 29.42, 29.23, 29.05, 27.09, 24.66, 19.20, 11.39.
Cyclo[-(Ter)-Orn-Thr-Hyp-hTyr-Orn-3-Hyp] (19). Peptide 19
was obtained according to the general procedure for the prep-
aration of the caspofungin analogues using 39 μmol of resin
bound peptide 17. After lyophilization, peptide 19 (18 mg; 43%)
was obtained as a colourless solid.
HRMS: calcd for C16H32O2 [M + H]− 255.232, found
1
255.216; [α]D = +14.1 (c = 1.3 in CHCl3); H NMR (400 MHz,
CDCl3): δ 11.20 (bs, 1H), 2.34 (t, J = 7.5 Hz, 2H), 1.95–1.51
(m, 1H), 1.57–0.32 (m, 31H) ppm. 13C NMR (101 MHz,
CDCl3): δ 180.15 (Cq), 44.70, 36.85, 34.21, 31.56 (CH), 30.00
(CH), 29.93, 29.46, 29.24, 29.20, 29.07, 26.86, 24.73, 20.26
(CH3), 19.72 (CH3), 11.17(CH3).
Purity was confirmed by analytical HPLC and was found to
be higher than 95% (Rt = 21.55 min). ESI-MS calcd for
C58H74N8O12: 1074.54, found: m/z 1075.85 [M + H]+; HRMS
calcd for C58H74N8O12 [M + H]+ 1075.5504, found 1075.5529;
1H-NMR (DMSO, 500 MHz): δ Orn-1: 8.59 (NH), 7.98 (εNH),
4.53 (αCH), 2.15/1.68 (βCH), 1.77 (γCH), 3.45/2.73 (δCH);
Thr-2: 8.22 (NH), 4.76 (αCH), 4.39 (βCH), 1.13 (γCH); Hyp-3:
4.33 (αCH), 2.16/1.76 (βCH), 4.38 (γCH), 3.74/3.69 (δCH);
hTyr-4: 7.55 (NH), 4.04 (αCH), 2.08/1.87 (βCH), 2.46/2.37
(γCH), 6.95/6.66 (Ar-H); Orn-5: 7.59 (NH), 4.70 (αCH), 1.84/
1.69 (βCH), 1.59 (γCH), 2.88/2.83 (δCH); Hyp-6: 4.13 (αCH),
4.02 (βCH), 2.12/1.87 (γCH), 3.65 (δCH); Ter: 0.92 (CH3), 1.37
(CH2), 1.41 (CH2), 1.75 (CH2), 4.00 (OCH2), 7.67/7.05 (Ar-H
Ph-3), 7.83/7.75 (Ar-H Ph-2), 8.03/7.81 (Ar-H Ph-1) ppm.
13C-NMR (DMSO, 125 MHz): δ Orn-1: 51.1 (αCH), 25.2
(βCH), 22.7 (γCH), 35.1 (δCH); Thr-2: 55.5 (αCH), 65.6 (βCH),
18.7 (γCH); Hyp-3: 59.9 (αCH), 36.4 (βCH), 68.4 (γCH), 54.8
(δCH); hTyr-4: 51.4 (αCH), 32.5 (βCH), 30.2 (γCH), 128.5/
114.1 (Ar-H); Orn-5: 48.2 (αCH), 28.0 (βCH), 22.1 (γCH), 37.9
(δCH); Hyp-6: 71.5 (αCH), 66.7 (βCH), 32.5 (γCH), 44.1
(δCH); Ter: 13.2 (CH3), 21.1 (CH2), 26.9 (CH2), 27.6 (CH2),
67.2 (OCH2), 126.8/114.1 (Ar-H Ph-3), 125.3/125.8 (Ar-H
Ph-2), 127.6/126.6 (Ar-H Ph-1) ppm.
Spectral data correspond to the literature.27
(S)-12-Methyltetradec-9-enoic acid (15). To a stirred solution
of (S)-S-ethyl 3-methylpentanethioate 11 (80 mg; 0.5 mmol) in
CH2Cl2 (0.75 mL) at −50 °C, DIBAL-H was added. After com-
plete conversion of the thioester (2 h), the reaction mixture was
poured into a saturated solution of Rochelle’s salt. After clear
layer separation, the organic layer was separated and the aqueous
layer extracted with Et2O (3 × 10 mL). The combined organic
layers were dried and carefully evaporated until the weight corres-
ponded to a quantitative yield (43 mg).
Then, to a stirred suspension of 9-(bromotriphenyl phospho-
ranyl)nonanoic acid (425 mg; 0.85 mmol) in THF (0.75 mL) a
solution of LiHMDS (1 M in THF; 1.5 mL; 1.5 mmol) was
added until the solution stayed deep red. To this solution,
3-methyl-pentanal in a small amount of THF (300 μL) was
added. The reaction mixture was stirred until complete conver-
sion of the starting material (2 h) and acidified to pH = 1 by
dilute aq. HCl. The resulting solution was extracted with Et2O
(3 × 20 mL) and the combined organic layers were dried and
evaporated. The resulting thick liquid was purified by column
chromatography (Et2O : pentane; 1 : 4) and afforded 15
(65.9 mg; 55%) as a colorless liquid (as a mixture of E and Z
isomers).
Cyclo[-(DMT-Myr)-Orn-Thr-Hyp-hTyr-Orn-Hyp] (20). Peptide
20 was obtained analogously to peptide 4, except for the amino
acid sequence, using 31 μmol of resin bound peptide 17. The
first amino acid coupled was Fmoc-Hyp(tBu)-OH instead of
Fmoc-3-Hyp(tBu)-OH. After lyophilization, peptide 20 (12 mg;
40%) was obtained as a colourless solid.
HRMS: calcd for C15H29O2 [M + H]+ 241.216, found
1
241.217; [α]D = +0.3 (c = 0.5 in CHCl3); H NMR (400 MHz,
Purity was confirmed by analytical HPLC and was found to
be higher than 99% (Rt = 23.25 min). ESI-MS calcd for
C50H82N8O11: 970.61, found: m/z 971.75 [M + H]+; HRMS
calcd for C50H82N8O11 [M + H]+ 971.6181, found 971.6123;
1H-NMR (DMSO, 500 MHz): δ Orn-1: 8.07 (NH), 7.91 (εNH),
4.35 (αCH), 2.02/1.43 (βCH), 1.65 (γCH), 3.40/2.66 (δCH);
Thr-2: 8.21 (NH), 4.74 (αCH), 4.38 (βCH), 1.07 (γCH); Hyp-3:
4.31 (αCH), 2.15/1.75 (βCH), 4.34 (γCH), 3.72 (δCH); hTyr-4:
7.53 (NH), 4.03 (αCH), 2.06/1.84 (βCH), 2.45/2.36 (γCH), 6.94/
6.65 (Ar-H); Orn-5: 7.61 (εNH), 7.39 (NH), 4.66 (αCH), 1.79/
1.59 (βCH), 1.55 (γCH), 2.79 (δCH); Hyp-6: 4.34 (αCH), 1.99/
1.82 (βCH), 4.36 (γCH), 3.68/3.47 (δCH); Tail: 2.12 (C(O)CH2),
CDCl3): δ 5.37 (dt, J = 6.0, 4.6 Hz, 2H), 2.35 (t, J = 7.5 Hz,
2H), 2.01 (d, J = 5.4 Hz, 2H), 1.68–1.59 (m, 2H), 1.40–1.21 (m,
14H), 0.96–0.80 (m, 6H) ppm. 13C NMR (101 MHz, CDCl3):
δ 177.89, 130.32, 128.66, 35.11, 34.13, 33.63, 29.69, 29.46,
29.19, 28.94, 28.88, 27.19, 24.64, 19.11, 11.55.
(S)-12-methyltetradecanoic acid (16). To a vigorously stirred
solution of (S)-12-methyltetradec-9-enoic acid 15 (60 mg;
0.25 mmol) and flavine catalyst (10 mg; 25 μmol, 10 mol%) in
EtOH (1 mL) under oxygen atmosphere, hydrazine hydrate
(375 μl; 7.5 mmol) was added in one portion. Vigorous stirring
was continued for 16 h and the mixture was acidified to pH = 1
7500 | Org. Biomol. Chem., 2012, 10, 7491–7502
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1.47, 1.45/1.22, 1.39, 1.31/1.06, 1.26/1.21, 1.25/1.02, 1.19/0.89,
0.83 (CH3), 0.82 (CH3) ppm. 13C-NMR (DMSO, 125 MHz):
δ Orn-1: 49.4 (αCH), 25.1 (βCH), 22.2 (γCH), 34.6 (δCH); Thr-
2: 55.4 (αCH), 68.4 (βCH), 18.6 (γCH); Hyp-3: 59.9 (αCH),
36.4 (βCH), 68.0 (γCH), 54.7 (δCH); hTyr-4: 51.2 (αCH), 32.5
(βCH), 30.2 (γCH), 128.5/114.1 (Ar-H); Orn-5: 48.7 (αCH),
27.9 (βCH), 21.8 (γCH), 37.9 (δCH); Hyp-6: 58.1 (αCH), 36.8
(βCH), 65.5 (γCH), 55.0 (δCH); Tail: 43.3, 35.4, 34.3
(CHCH2CH), 30.2, 28.6, 27.8, 27.6, 25.5, 24.6, 19.1 (CH3),
10.2 (CH3).
free wells dedicated for growth and sterility controls for each
organism tested.
The plates containing the diluted compounds were inoculated
with 100 μl of the appropriate microorganism. The yeast strains
used were isolates obtained from the CBS-KNAW Fungal Bio-
diversity Centre (Utrecht, The Netherlands). The collection
included C. dubliensis, C. glabrata, C. tropicalis, C. parapsilo-
sis, C. krusei and two strains of C. albicans. Stock cultures of
the yeast strains in liquid medium (YPD + 15% glycerol) were
maintained at −80 °C. For use in this assay, yeast cultures were
streaked on YPD agar plates and incubated for 24 h at 30 °C.
Then, using a sterile disposable loop, cells from a colony were
suspended in 5 mL of YPD medium and aerated for 24 h at
30 °C on a shaker set at 300 rpm. The broth cultures were
diluted 10 times with the medium and the optical density of this
suspension was measured at a wavelength of 600 nm. The sus-
pension was further diluted to an OD600 of 0.01 resulting in a
concentration of (1–5) × 106 cfu mL−1. This suspension
was further diluted 1 : 100 in YPD medium to yield (1–5) ×
104 cfu mL−1. This final dilution was used for inoculating the
plates. Plates containing the diluted compounds were inoculated
with 100 μL per well of the appropriate microorganism using an
8-channel pipet. The final volume per well, including organism
and compound, was 200 μL. Thus, the final number of cells per
Cyclo[-(Myr)-Orn-Thr-Hyp-hTyr-Orn-Hyp] (21). Peptide 21
was obtained analogously to peptide 7, except for the amino acid
sequence, using 31 μmol of resin bound peptide 17. The first
amino acid coupled was Fmoc-Hyp(tBu)-OH instead of Fmoc-
3-Hyp(tBu)-OH. After lyophilization, peptide 21 (6 mg; 20%)
was obtained as a colourless solid.
Purity was confirmed by analytical HPLC and was found to
be higher than 95% (Rt = 22.25 min). ESI-MS calcd for
C48H78N8O11: 942.58, found: m/z 943.65 [M + H]+; HRMS
calcd for C48H78N8O11 [M + H]+ 943.5868, found 943.5870;
1H-NMR (DMSO, 500 MHz): δ Orn-1: 8.07 (NH), 7.91 (εNH),
4.29 (αCH), 1.96/1.37 (βCH), 1.58 (γCH), 3.34/2.60 (δCH);
Thr-2: 8.21 (NH), 4.68 (αCH), 4.31 (βCH), 1.01 (γCH); Hyp-3:
4.24 (αCH), 2.09/1. 69 (βCH), 4.27 (γCH), 3.66 (δCH); hTyr-4:
7.57 (NH), 3.97 (αCH), 2.39/2.30 (βCH), 2.00/1.78 (γCH), 6.88/
6.69 (Ar-H); Orn-5: 7.65 (εNH), 7.39 (NH), 4.59 (αCH), 1.72/
1.52 (βCH), 1.49 (γCH), 2.73 (δCH); Hyp-6: 4.27 (αCH), 1.93/
1.76 (βCH), 4.30 (γCH), 3.62/3.41 (δCH); Tail: 2.06 (C(O)CH2),
1.41, 1.20, 1.17/1.01, 1.14, 0.87 (CH3), 0.80 (CH3) ppm.
13C-NMR (DMSO, 125 MHz): δ Orn-1: 49.4 (αCH), 25.1
(βCH), 22.1 (γCH), 34.6 (δCH); Thr-2: 55.4 (αCH), 68.4 (βCH),
18.6 (γCH); Hyp-3: 59.9 (αCH), 36.4 (βCH), 67.9 (γCH), 54.7
(δCH); hTyr-4: 51.2 (αCH), 32.5 (βCH), 30.2 (γCH), 128.5/
114.1 (Ar-H); Orn-5: 48.6 (αCH), 27.9 (βCH), 21.9 (γCH), 37.9
(δCH); Hyp-6: 58.0 (αCH), 36.8 (βCH), 65.5 (γCH), 55.1
(δCH); Tail: 34.3 (C(O)CH2), 28.1, 27.5, 24.7, 21.3, 18.0 (CH3),
13.2 (CH3).
well was approximately 5–25 × 103 cfu mL−1
.
Tests were incubated overnight at 30 °C prior to recording
MICs. The in vitro activity was determined visually at 24 h of
incubation as the lowest concentration of compound resulting in
full inhibition of yeast growth.
CD spectroscopy. CD spectra were recorded at 298 K on a
JASCO J-810 spectrometer using 0.1 cm path length quartz
cells. The CD spectra are an average of four scans, collected at
0.2 nm intervals between 190 and 250 nm. The peptides were
prepared at concentrations of 0.1 mM in MeCN/H2O (1/1, v/v)
or 0.1 mM in TFE/H2O (1/1, v/v). Ellipticity is reported as the
mean residue ellipticity [θ] in degrees cm2 dmol−1
.
Acknowledgements
We wish to thank Mr H.W. Hilbers for recording ESI-MS mass
spectra and Mr C. Versluis and Mevr. J.M.A. Damen for record-
ing HRMS spectra. We thank Prof A. Killian for helpful discus-
sions regarding the interpretation of CD spectra and the use of
the CD equipment. In addition, we thank Dr T. Boekhout from
the CBS-KNAW Fungal Biodiversity Centre (Utrecht, The Neth-
erlands) for generously providing us with the Candida strains.
Biology
Candida MIC assay. Antifungal activity was evaluated by
broth microdilution. The medium used in this assay was yeast
extract peptone dextrose (YPD) containing 1% yeast extract, 2%
peptone, 1% dextrose in distilled water. Test compounds were
dissolved in distilled water or 10% DMSO, depending on the
solubility of the compounds, to a concentration of 1 mg mL−1
.
After dissolving each compound was diluted 20 times in YPD
medium rendering a stock solution of 50 μg mL−1. Caspofun-
gin,32 purchased from Merck Sharp & Dohme B.V. (Haarlem,
Netherlands), was included as a reference. Serial 2-fold dilutions
of the test compounds in YPD medium were prepared as
follows. To each well of a sterile Greiner bio-one Cellstar 96
well, U bottomed microtiter plate 100 μl of YPD was dispensed.
Manually, 100 μl of stock compounds was delivered to each well
in column 1. Then using an 8-channel pipet, compounds in
column 1 were serially diluted 2-fold over the microtiter plate
until column 11. The last column of the plate contained drug-
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7502 | Org. Biomol. Chem., 2012, 10, 7491–7502
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