Synthesis and evaluation of novel macrocyclic antifungal peptides

Article abstract of DOI:10.1016/j.bmc.2011.08.034

Echinocandins are a novel class of macrocyclic antifungal peptides that act by inhibiting the β-(1,3)-Dglucan synthase complex, which is not present in mammalian cells. Due to the large number of hydroxyl groups present in these complex macrocyclic lipope

Full text of DOI:10.1016/j.bmc.2011.08.034

Bioorganic & Medicinal Chemistry 19 (2011) 6505–6517
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of novel macrocyclic antifungal peptides
Monique P.C. Mulder a, John A.W. Kruijtzer a, Eefjan J. Breukink b, Johan Kemmink a,
Roland J. Pieters a, Rob M.J. Liskamp a,
⇑
a Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, PO Box 80.082, 3508 TB Utrecht, The Netherlands
b Biochemistry of Membranes, Bijvoet Center for Biomolecular Research and Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Echinocandins are a novel class of macrocyclic antifungal peptides that act by inhibiting the b-(1,3)-D-
Received 10 March 2011
Revised 10 August 2011
Accepted 14 August 2011
Available online 22 August 2011
glucan synthase complex, which is not present in mammalian cells. Due to the large number of hydroxyl
groups present in these complex macrocyclic lipopeptides, most structure–activity relationship studies
have relied upon semisynthetic derivatives. In order to probe the influence of the cyclic peptide backbone
on the antifungal activity we developed a successful strategy for the synthesis of novel echinocandins
analogues by on-resin ring closing metathesis or disulfide formation. The specific minimum inhibitory
activity of each mimic was determined against Candida albicans. Our results indicate that ring size is
an important factor for antifungal activity.
Keywords:
Echinocandins
Macrocyclic peptides
Antifungal
Ó 2011 Elsevier Ltd. All rights reserved.
b-(1,3)-D-Glucan synthase inhibitors
On-resin RCM
1. Introduction
To date, three echinocandins have been approved by the Food
and Drug Administration (FDA). The first licensed echinocandin
Invasive fungal infections represent a growing threat and in re-
cent years the incidence and diversity of fungal infections has in-
creased enormously.1,2 Despite advances in antifungal therapies,
systemic fungal infections continue to cause significant morbidity
and mortality in immunocompromised patients. Resistance against
and toxicity of the current antifungal agents underscores the ur-
gent need for development of new antifungal compounds that
act on novel targets.3
was caspofungin (1) in 2001, followed by micafungin (2) in 2005,
and anidulafungin (3) in 2006 (Fig. 1).6 These echinocandins are
all semisynthetic derivatives of the natural fermentation prod-
ucts.7–9
Due to the large number of hydroxyl groups in these lipopep-
tides, total synthesis of the echinocandins is extremely challenging.
The total synthesis of the least complex member of the family, ech-
inocandin D (4), which lacks an ornithine hemiaminal function
(Fig. 1), has been described in the mid 1980s.10,11 However, all re-
ported attempts at the synthesis of echinocandin C (5) have failed
due to difficulties in formation of the hemiaminal moiety.10,12
These synthetic limitations led to isolation and modification of
semi-synthetic derivatives, which were used for most of the struc-
ture–activity relationship (SAR) studies.13 In 1992 the first total
synthesis of simplified echinocandin analogues was described by
Zambias et al.12 These structure–activity relationship data showed
that several of the functional groups, primarily the hydroxyl
groups on positions of R1, R2 and R4 (Fig. 1), were not necessary
for antifungal activity.14 Partly inspired by this work, Klein et al. re-
ported a similar study in 2000.15 Both papers described derivatives
that were obtained by peptide head-to-tail cyclization. Encouraged
by these reports, we became interested in pursuing additional ech-
inocandin analogues by employing alternate peptide cyclization
approaches. We here report the synthesis and activity of novel ech-
inocandin analogues (Fig. 2) obtained by ring closing metathesis
(6) and disulfide formation (7). Using such approaches, the impact
of the size and character of the macrocyclic peptide backbone on
The echinocandins represent the most recent class of antifungal
drugs that reached the market in the last decade.4 They are large
macrocyclic lipopeptide molecules that block fungal cell wall bio-
synthesis by inhibiting the b-(1,3)-
complex is responsible for the production of the major fungal cell
wall biopolymer b-(1,3)- -glucan. Since this glucan is not utilized
D-glucan synthase complex. This
D
in human cells, inhibition of its biosynthesis provides for a selec-
tive approach to generate antifungal agents. By blocking the syn-
thesis of b-(1,3)-D-glucan, the cell wall is weakened, which leads
to lysis of the fungal cells. Different fungi have varying amounts
of chitin, glucans, mannoproteins, and other cell wall constituents.
Therefore, some species are more susceptible to the echinocandins
than others. Since b-(1,3)-D-glucan is a major cell wall component
of Candida and Aspergillus species, these species are more sensitive
to echinocandins.5
⇑
Corresponding author. Tel.: +31 302537396; fax: +31 302536655.
E-mail address: R.M.J.Liskamp@uu.nl (R.M.J. Liskamp).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.08.034

6506
M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
Figure 1. Structures of the FDA approved semisynthetic echinocandins (1–3) and two natural echinocandins (4,5).
the ring of the hexapeptide was opened at the hemiaminal function
and the ornithine residue was replaced to regenerate new cyclic
hexapeptides.17
2. Results and discussion
Ring-closing metathesis (RCM) reactions have been previously
carried out on peptides linked to a solid support.18 However, these
RCM reactions required long reaction times (18–48 h). Recently,
Robinson et al. reported large acceleration of RCM on resin-bound
peptides by microwave heating in the presence of chaotropic re-
agents.19 This procedure was also applied to the preparation of
our mimic 6. As shown in Scheme 1, linear precursors were pre-
pared by SPPS using either the Rink or Wang resin, ultimately pro-
viding the peptide amide (6a) or carboxylic acid (6b), respectively.
Then, RCM reaction of resin-bound peptide 8 was performed using
a 15 mol % solution of Grubbs II in DCM (containing 10 vol % LiCl/
DMA 0.4 M) and microwave irradiation to provide the ring-closed
product 9. Removal of the Fmoc group, coupling of palmitic acid,
and acidolytic cleavage from the resin with concomitant removal
of the side chain protecting groups, gave the cyclic peptide 6 as a
mixture of cis/trans isomers in an overall yield of 11% after purifi-
cation by preparative HPLC. Attempted RCM reactions without
microwave irradiation were less successful. For example, treat-
ment of the linear resin-bound peptide with second generation
Grubbs catalyst (20 mol %) in DCM containing 10 vol % LiCl/DMA
(0.4 M) at 50 °C for 24 h gave only trace amounts of the cyclized
product. After reduction of unsaturated peptides 6a and 6b macro-
cyclic peptides 10a and 10b were obtained.
Synthesis of the disulfide mimic 7 started with SPPS on the Rink
resin as outlined in Scheme 2. Removal of the Fmoc group followed
by introduction of the palmitoyl chain and cleavage from the resin
yielded the deprotected peptide 11, which was then cyclized via
disulfide formation by oxidation with aq DMSO20 yielding peptide
7 in an overall yield of 22% after purification by preparative HPLC.
The antifungal activity of each macrocyclic peptide analogue
was evaluated by broth microdilution using Caspofungin21 as a ref-
erence compound. Minimum inhibitory concentrations (MICs)
were inspected visually and thereby quantified as the lowest con-
centration of compound resulting in inhibition of yeast growth
Figure 2. Structures of the designed synthetic echinocandin analogues.
antifungal activity can be investigated more extensively. In addi-
tion, these new mimics may be attractive alternatives since they
also contain a position for further modification (i.e., the C-termi-
nus; substituent R1).
In comparison to caspofungin 1, mimics 6 and 7 were designed
to contain several alterations (Fig. 2). Consistent with SAR
studies14,15 a number of functional groups (primarily hydroxyl
groups, i.e., R1, R2 and R4) which are not necessary for antifungal
activity, were omitted. Moreover, the lipophilic chain (R3 in com-
pounds 1–5) was replaced by a palmitoyl group (R2 in 6a and
6b), which closely mimics the fatty acid tail of caspofungin. Earlier
SAR studies showed that a C12–C18 fatty acid chain gave an opti-
mal activity.16 In addition, the ornithine derived residue at the top
of the caspofungin structure (1) was replaced by an allyl glycine
residue together with introduction of an allyl glycine residue at
the C-terminus leading to precursor 8. Similarly, replacement by
a cysteine residue at these positions gave precursor 11. Cyclization
of these precursors slightly enlarged the ring to a 23-membered
ring as compared to the original 21-membered ring of caspofungin.
According to the literature17 there was room for alterations at this
site of the molecule. Borromeo et al. reported an approach in which

M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
6507
after overnight incubation at 30 °C (Table 1). Unfortunately, mim-
ics 6, 7 and 10 did not show antifungal activity up to 100 g/mL
(corresponding to 96 M). To more clearly determine the factors
responsible for the observed lack of antifungal activity, further
investigations were performed by varying the lipophilic ‘tail’
(substituent R2, Fig. 3A), introduction of modifications at the C-
terminus (Fig. 3B) and changing the peptide ring size (Fig. 3C).
mild acidolytic cleavage of the protected peptide from the resin
and cyclization gave the protected macrocyclic peptide 15 in an
overall yield of 90%. Removal of the IvDde-group28 from ornithine
with hydrazine and subsequent coupling of the fatty acid chain,
followed by global deprotection and purification, gave cyclic pep-
tides 12 and 13 in overall yields of 17–23%.
Minimum inhibitory concentrations (MICs) were evaluated and
are given in Table 2. These results showed that mimic 13a with a
terphenyl lipophilic tail is 50-fold more active than 12a with a pal-
mitoyl chain. Moreover, mimics containing an ornithine (12b, 13b)
residue were fivefold more active than the mimics containing a
threonine (12a, 13a) residue at this position as is consistent with
previously described results by Klein et al.15
Thus, compounds 12 and 13 having a terphenyl tail gave us a
clue about the high importance of the lipophilic tail in general. This
finding enticed us to synthesize from thereon all compounds a
with a terphenyl chain. For example 6b containing a terphenyl
chain became 16. For the synthesis of RCM mimic 16 with a ter-
phenyl chain, the RCM conditions employed required optimization.
The use of the Hoveyda–Grubbs II catalyst, longer reaction times,
and adding the catalyst in portions did not improve the rate of con-
version or yields of the ring-closed product. We did however find
that changing the sequence of the reactions did allow for success
at the RCM step. Thus, RCM was performed after coupling of the
fatty acid chain instead of with the Fmoc precursor 8 as in Scheme 1
(step vii and viii were switched). Linear precursor 8 was prepared
by SPPS on the Wang resin. Removal of the N-terminal Fmoc group,
coupling of the terphenyl chain followed by RCM reaction of the re-
sin-bound peptide using a 10 mol % solution of Grubbs II in DCM
(containing 10 vol % 0.4 M LiCl in DMA) under microwave irradia-
tion resulted in the ring-closed product. Cleavage from the resin
with concomitant removal of side chain protecting groups gave
cyclic peptide 16 as a mixture of cis/trans isomers in an overall
yield of 15% after purification by preparative HPLC. After Pd/C
assisted reduction of the double bond macrocyclic peptide 17
(Table 1) was obtained.
l
l
2.1. Influence of the lipophilic ‘Tail’
The nature of the fatty acid chain and its influence on the activ-
ity of the echinocandins has been studied extensively. Several re-
ports have described the preparation of semi-synthetic
echinocandin analogues by enzymatic deacylation and chemical
reacylation with alternative fatty acids.16,22–26 It has been shown
that neither the macrocyclic peptide itself, the fatty acid, nor a
mixture of both show significant antifungal activity. These results
clearly demonstrated that the intact echinocandin molecule is re-
quired for inhibition of antifungal growth.23 Moreover, the nature
of the side chain plays a very important role in the biological activ-
ity. In this regard, side chain length, overall lipophilicity, and other
geometric factors such as rigidity, contribute to the SAR of these
analogues.
These previous studies have shown that the lipophilic chain in
the echinocandins is a crucial determinant for antifungal potency.
To determine if the choice of the lipophilic tail for mimics 6, 7 and
10 might be partly responsible for the loss of antifungal activity, a
series of fatty acid derivatives (12a,b and 13a,b; Scheme 3), based
on previous work by Klein et al., were prepared.15 Their work
established that head-to-tail backbone mimics bearing a terphenyl
(‘Ter’) chain showed reasonable biological activity. Therefore,
head-to-tail mimics were prepared with the palmitoyl (12) and
the terphenyl27 (13) fatty acid chain. The synthesis of these mimics
proceeded readily and is outlined in Scheme 3 for mimics 12 and
13. The linear precursor 14 was prepared by SPPS using the trityl
resin. Removal of the e-Fmoc group from ornithine, followed by
Scheme 1. Reagents and conditions: (i) Rink resin: (1) 20% piperidine in NMP; (2) Fmoc-Alg-OH, BOP, DIPEA, NMP; Wang resin: (1) Fmoc-Alg-OH, pyridine, DCBC, DMF; (2)
Ac2O/NMI/DiPEA/DMF (2/1/1/6, v/v/v); (ii–vi) (1) 20% piperidine in NMP; (2) Fmoc-Xxx-OH, BOP, DiPEA, NMP; (vii) Grubbs II (15 mol %), 10 vol % LiCl/DMA (0.4 M), MW,
75 min, 100 °C, DCM; (viii) (1) 20% piperidine in NMP; (2) palmitic acid, HATU, DiPEA, NMP; (ix) TFA/TIS/H2O (95/2.5/2.5, v/v/v); (x) H2, 10% Pd/C, EtOH, rt, 36 h.

6508
M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
Scheme 2. Reagents and conditions: (i) (1) 20% piperidine in NMP; (2) Fmoc-Cys(Trt)-OH, BOP, DiPEA, NMP; (ii–vi) (1) 20% piperidine in NMP; (2) Fmoc-Xxx-OH, BOP, DiPEA,
NMP; (vii) (1) 20% piperidine in NMP; (2) palmitic acid, HATU, DiPEA, NMP; (viii) TFA/TIS/H2O (95/2.5/2.5, v/v/v); (ix) H2O, NH4OAc, DMSO, pH 6.
2.2. Influence of C-terminal modifications
RCM mimic 16 contains a C-terminal moiety allowing for mod-
ification, which is convenient for the preparation of additional ana-
logues. This moiety replaces the site of the hemiaminal group
present in most echinocandins (Fig. 1, R1 = OH), which is known
to be unstable at pH >7.28–30 This has led to several studies on
derivatization of the hemiaminal moiety to improve stability.31–37
In particular, these studies showed that aminoalkylethers (e.g.,
R1 = OCH2CH2NH2) bearing a basic amino group had enhanced
antifungal properties.31 Further optimization led to the closely re-
lated N-alkylamino aminal series37 including clinically used caspo-
fungin 1.
Considering these factors, two C-terminally modified analogues
incorporating basic groups were synthesized (Scheme 4). Synthesis
of these mimics proceeded largely as described for mimic 16, ex-
cept in the last step. The resin-bound ring-closed product was
cleaved from the resin with KCN in MeOH to give the fully tBu/
Boc protected methylester 18. Saponification of 18 gave carboxylic
acid 20, which was coupled to Boc-protected ethylene diamine
Figure 3. Factors studied for antifungal activity.
followed by acidolysis affording 21. Reduction of methylester 18
followed by removal of the protecting groups gave macrocyclic
peptide alcohol 22. Reacting 18 with hydrazine afforded 23
The minimum inhibitory concentrations (MICs) were evaluated
and are shown in Table 1. Although these results showed that
mimics 16 and 17 with a terphenyl chain did not reach their
(Scheme 4).
The minimum inhibitory concentrations (MICs) of mimics 19,
21, 22 and 23 were evaluated and are shown in Table 1. Although
MIC-values at 100
in growth of Candida albicans cells was visible starting at 5
l
g/mL (corresponding to 87
l
M), a difference
g/mL
these results showed that these mimics did not reach their MIC-
values at 100 lg/mL, the same dose-dependent change in the
l
(Fig. S1 in Supplementary data). Further investigation of this obser-
vation by light microscopy has led to a clearer view of the differ-
ence in growth that was observed. A clear-dose-dependent effect
of our compounds in the growth pattern of C. albicans cells was vis-
ible. Higher concentrations of 16 resulted in enlarged rounded cells
that agglutinated, showing large vacuoles and distorted cyto-
plasms. There was a clear reduction in the number of budding cells
and when these appeared more buds were formed simultaneously.
growth pattern of the C. albicans cells was observed for mimics
21, 22 and 23 as for 16.
2.3. The ring size is crucial
The ring size of the RCM compounds (23-membered rings) is
slightly larger than that of the natural echinocandins (21-mem-
bered rings). However, the resulting compounds were completely
devoid of biological activity. This somewhat unexpected finding
enticed us to investigate the influence of ring size on activity,
Although 16 was not fully active at concentrations up to 100 lg/mL
a clear effect on yeast growth was observed (Fig. S2).

M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
6509
Scheme 3. Reagents and conditions: (i) (1) Fmoc-Hyp(tBu)-OH, DIPEA, DCM; (2) DCM/MeOH/DiPEA (17/2/1, v/v/v); (ii–vi) (1) 20% piperidine in NMP; (2) Fmoc-Xxx-OH, BOP,
DiPEA, NMP; (vii) 20% piperidine in NMP; (viii) HFIP/DCM (1/1, v/v); (ix) BOP, DiPEA, DMF; (x) NH2NH2/DMF (5 vol %); (xi) for 12: palmitic acid, DCC, HOBt, DCM; for 13 Ter
chain, DCC, HOBt, DCM; (xii) TFA/TIS/H2O (95/2.5/2.5, v/v/v).
Table 1
Table 2
Antifungal activity of analogues
Antifungal activity of head-to-tail analogues
R1
R2
n
In vitro activity MIC (lg/mL)
Candida albicans CBS 9975
Caspofungin (1)
—
—
—
1
1
1
1
2
0.025
25
4.38
0.47
0.14
>100
12a
12b
13a
13b
26
Threonine
Ornithine
Threonine
Ornithine
Ornithine
Palm
Palm
Ter
Ter
Ter
resin. Removal of the Fmoc group on lysine, coupling of the Ter
chain, removal of the Psoc-protecting38 group and mild acid cleav-
age from the resin gave protected linear peptide 25. Solution-phase
cyclization followed by protecting group removal afforded the
macrocyclic peptide 26 in an overall yield of 21%. The minimum
inhibitory concentration (MIC) of this echinocandin homolog was
evaluated and shown in Table 2.
Even expansion of the peptide macrocycle by just one carbon
atom completely abolished antifungal activity which we had not ex-
pected in view of the limited added flexibility to the peptide
macrocycle.
R1
X
R2
In vitro activity
MIC ( g/mL)
Candida albicans CBS
l
9975
Caspofungin
0.025
(1)
To assure that the low activities of our compounds are not
strain dependent and hence the conclusions regarding the impor-
tance of the ring size are more general, we have tested a represen-
tative selection of the compounds against a panel of Candida
strains. This panel included Candida dubliensis, Candida glabrata,
Candida tropicalis, Candida parapsilosis, Candida krusei and two
strains of C. albicans. The results were in the same order of
magnitude.
6a
6b
7
C(O)NH2
C(O)OH
C(O)NH2
C(O)NH2
C(O)OH
C(O)OH
C(O)OH
C(O)OCH3
CH@CH Palm >100
CH@CH Palm >100
S–S
Palm >100
10a
10b
16
17
19
21
22
23
CH2CH2 Palm >100
CH2CH2 Palm >100
CH@CH Ter
CH2CH2 Ter
CH@CH Ter
>100
>100
>100
>100
>100
>100
C(O)NHCH2CH2NH2 CH@CH Ter
CH2OH
C(O)NHNH2
CH@CH Ter
CH@CH Ter
3. Conclusions
A series of new echinocandin analogues were synthesized by
RCM and disulfide bond formation to explore the influence of the
macrocyclic peptide ring structure of the echinocandins on anti-
fungal activity. Evaluation of the antifungal properties of the result-
ing compounds showed no measurable activity for mimics 6, 7 and
and a 22-membered ring containing analogue 26 of echinocandin
was synthesized by head-to-tail cyclization as is outlined in
Scheme 5. Linear precursor 24 was prepared by SPPS on the trityl

6510
M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
Scheme 4. Reagents and conditions: (i) KCN, MeOH; (ii) LiOH, THF; (iii) N-Boc-ethylene-diamine, HATU, DiPEA, NMP; (iv) TFA/TIS/H2O (95/2.5/2.5, v/v/v); (v) LiBH4, THF; (vi)
N2H4ꢀH2O, DMF; (vii) HCl/Et2O.
Scheme 5. Reagents and conditions: (i) Fmoc-Hyp(tBu)-OH, DIPEA, DCM; (ii–vi) (1) 20% piperidine in NMP; (2) Fmoc-Xxx-OH, BOP, DiPEA, NMP; (vii) (1) 20% piperidine in
NMP; (2) ter tail, HATU, DiPEA, NMP; (viii) TBAFꢀH2O, DCM; (ix) HFIP/DCM (1/1; v/v); (x) BOP, DiPEA, DMF; (xi) TFA/TIS/H2O (95/2.5/2.5, v/v/v).
10. It is also known that the fatty acid chain plays a crucial role in
relation to antifungal potency, therefore analogues 16 and 17, bear-
ing a terphenyl lipophilic chain, were synthesized. These analogues
showed a dose-dependent effect on antifungal growth at concentra-
Therefore, the preparation of other larger derivatives by either RCM
or disulfide formation seems an unattractive avenue.
The mimics described here comprise a novel set of echinocan-
din-based analogues. The influence of ring size on the conforma-
tion(s) of the macrocyclic lipopeptides and the resulting impact
on antifungal activity will be examined in future investigations.
In addition, the double bond resulting from the RCM approach used
here presents an additional site for derivatization. These modifica-
tions and their effects on antifungal activity are presently under
investigation.
tions starting at 5 lg/mL. The C-terminus is another modifiable site
in the echinocandins, as such analogues (21–23) with a modified
C-terminus were prepared and were shown to have the same
dose-dependent effect on fungal growth. Remarkably, a slight
enlargement of the macrocyclic peptide ring from a 21- (13b) to a
22-membered system (26) completely abolishes antifungal activity.

M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
6511
Peptides were characterized using ElectroSpray Ionization Mass
Spectrometry (ESI-MS) on a QP8000 single quadrupole mass spec-
trometer in a positive ionization mode. High resolution mass spec-
trometry (HRMS) analyses were performed using MALDI TOF/TOF
(Applied Biosystems).
4. Experimental section
4.1. Chemistry
4.1.1. General
Unless stated otherwise, all chemicals were obtained from com-
mercial sources and used without further purification. Piperidine,
N,N-diisopropylethylamine (DiPEA), N-methylimidazole (NMI)
peptide grade dichloromethane (DCM), N,N-dimethylformamide
(DMF), 1-Methyl-2-pyrrolidinone (NMP), tert-butyl methylester
(MTBE), trifluoroacetic acid (TFA) and HPLC grade solvents were
purchased 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 hexafl-
urophosphate (BOP), N-hydroxybenzotriazole (HOBt), O-(7-aza-
benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexafluorophos-
phate (HATU) and Na-9-fluorenylmethyloxycarbonyl (Fmoc)
protected amino acids were purchased from GL Biochem Ltd
(Shanghai, China). The tert-butyl side chain protected homotyro-
sine (H-hTyr(tBu)-OH) was purchased from Advanced Chemtech
(Louisville, United States) and subsequently protected with the
Fmoc group. Triisopropylsilane (TIS) was obtained from Merck
(Darmstadt, Germany). 2,6-dichlorobenzoylchloride (DCBC), Sec-
ond generation Grubbs and Hoveyda–Grubbs catalyst were
obtained from Aldrich (Munich, Germany). The 2-chlorotritylchlo-
ride PS resin cross-linked with 1% DVB (200–400 mesh) was pur-
chased from Hecheng Chemicals (Shanghai, China) (theoretical
loading: 1.10 mmol/g). The Wang resin; Tentagel S PHB (theoreti-
cal loading: 0.26 mmol/g) and Rink resin; Tentagel S RAM (theoret-
ical loading: 0.25 mmol/g) were purchased from RAPP Polymere
(Tübingen, Germany).
4.2. General procedures
4.2.1. Solid phase peptide synthesis
Peptides were synthesized manually. Used abbreviations; Palm:
palmitoyl, Ter: terphenyl, Alg: Allylglycine, Hyp: 4-hydroxyproline,
hTyr: homotyrosine, IvDde: isovaleryl substituted dimedone, Psoc:
(2-Phenyl-2-trimethylsilyl)ethoxycarbonyl. Each synthetic cycle
consisted of the following steps.
4.2.1.1. Fmoc removal.
The resin was treated with a 20%
solution of piperidine in NMP (3ꢁ, each 10 min). The solution
was removed by filtration and the resin was washed with NMP
(3ꢁ, each 3 min) and DCM (3ꢁ, each 3 min).
4.2.1.2. Coupling step.
A mixture of Fmoc-Xxx-OH (3 equiv),
BOP (3 equiv) and DiPEA (6 equiv) in NMP (10 mL/mmol)) 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 (3ꢁ, each 3 min) and DCM (3ꢁ, each 3 min). Completion
of the coupling was checked with Kaiser or chloranil test.
4.2.1.3. 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] was added to the resin and N2
was bubbled through the mixture for 20 min The solution was re-
moved by filtration and the resin was washed with NMP (3ꢁ, each
3 min) and DCM (3ꢁ, each 3 min).
All reactions were carried out at room temperature unless sta-
ted otherwise. Solid phase synthesis was performed in plastic
syringes with a polyethylene frit. Microwave reactions were car-
ried out on a Biotage Initiator system. Solution phase reactions
were monitored by TLC on Merck pre-coated Silica 60 plates. Spots
were visualized by UV light, ninhydrin and K2CO3/KMnO4. Solid
phase reactions were monitored with the chloranil test39 in case
of secondary amines or with the Kaiser test40 in case of primary
amines. Column chromatography was performed using Silicycle
4.2.2. TFA cleavage
The resin was shaken in a mixture of TFA/TIS/H2O (95/2.5/2.5,
v/v/v) for 2 h. The peptide was precipitated in MTBE/hexane
(1/1), the supernatant was removed and the crude peptide was
washed twice with MTBE/hexane (1/1) and lyophilized from tert-
BuOH/H2O (1/1, v/v). The isolated peptide was analyzed by HPLC
and characterized by MS.
UltraPure silicagel (40–63 lm).
1H NMR, TOCSY, 1H–13C HSQC and ROESY spectra were recorded
using a Varian INOVA-500 spectrometer (500 MHz); chemical
shifts (d) were obtained in ppm relative to TMS. For measurements
in DMSO, the residual solvent peak was used as a reference.
Analytical HPLC was performed on a Shimadzu automated HPLC
system equipped with an evaporative light scattering detector
(PL-ELS 1000) and a UV/Vis detector operated at 220/254 nm. Pre-
parative HPLC runs were performed on an Applied Biosystems 400
solvent delivery system with an Applied Biosystems 757 UV/VIS
absorbance detector. Two buffer systems were used for HPLC. The
first will be referred to as TFA MeCN/H2O buffer and consists out
of buffer A: 0.1% TFA in MeCN/H2O, 5/95, v/v and buffer B: 0.1%
TFA in MeCN/H2O, 95/5, v/v. For analytical HPLC a flow rate of
1.0 mL/min with a linear gradient of buffer B (100% in 20 min) from
100% buffer A was used. Preparative runs used a flow rate of 12 ml/
min with a linear gradient of buffer B (100% in 40 min) from 100%
buffer A. The second will be referred to as i-PrOH/MeOH/H2O buffer
and consists out of buffer A: 0.1% TFA in i-PrOH/MeOH/H2O, 5/5/90,
v/v/v and buffer B: 0.1% TFA in i-PrOH/MeOH/H2O, 45/50/5, v/v/v.
For analytical HPLC a flow rate of 0.5 mL/min with a linear gradient
of buffer B (100% in 40 min) from 100% buffer A was used. Prepara-
tive runs used a flow rate of 6 ml/min with a linear gradient of buffer
B (100% in 80 min) from 100% buffer A.
4.2.3. Microwave-assisted RCM
A microwave vessel containing a magnetic stirrer bead was
loaded under argon with resin peptide, catalyst and solvent. The
vessel was capped and irradiated at 100 °C. At the end of the reac-
tion period the resin-bound peptide was washed with DMF (3ꢁ
3 mL, each 3 min) DCM (3ꢁ 3 mL, each 3 min) and MeOH (3ꢁ
3 mL, each 3 min).
4.2.4. Hydrogenation
To a solution of the olefinic peptide in tBuOH/H2O (1 mL, 3/1, v/v)
10% Pd/C was added. The reaction mixture was stirred under H2 at
atmospheric pressure overnight. The mixture was then filtered
through a path of Celite and washed extensively with tBuOH. The
mixture was concentrated and lyophilized from tert-BuOH/H2O
(1/1, v/v).
4.2.5. Head-to-tail cyclization
The peptide was dissolved in dry DMF (2 mL/lmol) and BOP
(4 equiv) and DiPEA (8 equiv) were added. The mixture was stirred
overnight followed by evaporation in vacuo. The product was
redissolved in EtOAc and washed with 1 M KHSO4 (2ꢁ), NaHCO3
(2ꢁ) and H2O (2ꢁ). The organic layer was dried over Na2SO4,
filtered and concentrated in vacuo.

6512
M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
4.2.6. Cleavage IvDde group
The resin was drained and washed with DMF (3ꢁ 3 mL, each
2 min) and DCM (3ꢁ 3 mL, each 2 min). Hyp was coupled as the
dipeptide Fmoc-Orn(Boc)-Hyp(tBu)-OH.
The peptide was dissolved in dry DMF (70 mL/mmol) and
N2H4ꢀH2O/DMF (5 v/v%) was added. The mixture was stirred for
2 h. Followed by the addition of brine and extraction with EtOAc
(2ꢁ). The organic layer was washed with H2O, dried over Na2SO4,
filtered and concentrated in vacuo. The crude peptide was purified
by column chromatography (DCM/MeOH 20/1, v/v/v).
ESI-MS calcd for TFA deprotected 8b C54H68N8O14: 1052.49,
found: m/z 1053.10 [M+H]+.
ESI-MS calcd for TFA deprotected 9b C52H64N8O14: 1024.45,
found: m/z 1025.00 [M+H]+.
The crude peptide was purified by column chromatography (i-
PrOH/NH4OH/H2O, 10/1/1, v/v/v). After lyophilization peptide 6b
(12.2 mg; 11%) was obtained as a white solid. Purity was confirmed
by analytical HPLC (Adsorbosphere C8, 40 min run, TFA MeCN/H2O
buffers) and was found to be higher than 95% for a mixture of E/Z
stereoisomers (tR = 22.60 min and 22.81 min). ESI-MS calcd for
C53H84N8O13: 1040.62, found: m/z 1041.15 [M+H]+; HRMS calcd
for C53H84N8O13 [M+H]+ 1041.6236, found 1041.6243; 1H NMR
4.3. cyclo[-(Palm)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-NH2 (6a)
The linear peptide Fmoc-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg-NH2
(8a) was synthesized on Fmoc-Rink-Tentagel resin (1 g;
0.25 mmol) according to the general procedure for solid phase pep-
tide synthesis. ESI-MS calcd for TFA deprotected 8a C54H69N9O13
:
1051.50, found: m/z 1052.20 [M+H]+.
The resulting resin-bound peptide 8a was subjected to the gen-
eral microwave-assisted RCM procedure under the following con-
(CD3OH, 500 MHz): d Alg-1: 7.87 (NH), 4.17 (
(bCH), 5.48 ( CH); Thr-2: 7.95 (NH), 4.70 ( CH), 4.32 (bCH), 1.14
CH); Hyp-3: 4.37 ( CH), 2.18/1.73 (bCH), 4.40 ( CH), 3.68/3.61
(dCH); hTyr-4: 7.64 (NH), 4.02 ( CH), 2.14/1.93 (bCH), 2.48
CH); Orn-5: 7.64 (NH), 4.62 ( CH), 2.00/1.52 (bCH), 1.58 ( CH),
2.86 (dCH); Hyp-6: 4.51 ( CH), 2.20/1.99 (bCH), 4.34 ( CH), 3.68/
3.42 (dCH); Alg-7: 8.23 (NH), 2.47/2.18 (bCH), 5.48 ( CH) ppm.
13C NMR (CD3OH, 125 MHz): d Alg-1: 54.1 (
CH), 35.4 (bCH),
128.9 ( CH); Thr-2: 56.8 ( CH), 67.8 (bCH), 18.7 ( CH); Hyp-3:
61.1 ( CH), 37.2 (bCH), 70.2 ( CH), 55.7 (dCH); hTyr-4: 54.3
CH), 32.3 (bCH), 31.7 ( CH); 115.5 (Ar-H), 129.8 (Ar-H); Orn-5:
50.8 ( CH), 28.5 (bCH), 23.7 ( CH), 39.2 (dCH); Hyp-6: 59.3
CH), 37.9 (bCH), 69.8 ( CH), 55.5 (dCH); Alg-7: 54.7 ( CH),
34.3 (bCH), 128.9 ( CH).
aCH), 2.60/2.13
c
a
ditions: Resin-bound peptide (421 mg; 105.2
(3.8 mL), LiCl/DMA (degassed; 0.4 M; 0.38 mL), Grubbs II
(15 mol %; 13.4 mg; 15.8 mol), 100 °C for 75 min. ESI-MS calcd
52H65N9O13 1023.47, found: m/z
lmol), DCM
(
c
a
c
a
l
(c
a
c
for TFA deprotected 9a
C
:
a
c
c
1024.15 [M+H]+.
The Fmoc group was cleaved and palmitic acid (108 mg;
421 mol) was coupled overnight in the presence of HATU
(160 mg; 421 mol) and DIPEA (147 L; 842 mol) in NMP
a
l
c
a
a
c
l
l
l
c
(3 mL). The resin was washed with NMP (3ꢁ 3 mL, each 3 min)
and DCM (3ꢁ 3 mL, each 3 min) and subjected to the TFA-mediated
cleavage procedure followed by lyophilization. The crude peptide
was purified by column chromatography (i-PrOH/NH4OH/H2O,
15/1/1, v/v/v). After lyophilization peptide 6a (12 mg; 11%) was ob-
tained as a white solid. Purity was confirmed by analytical HPLC
(Adsorbosphere C8, 40 min run, TFA MeCN/H2O buffers) and was
found to be higher than 95% for a mixture of E/Z stereoisomers
(a
c
a
c
(a
c
a
c
4.5. cyclo[-(Palm)-Cys-Thr-Hyp-hTyr-Orn-Hyp-Cys]-NH2 (7)
The linear peptide Fmoc-Cys-Thr-Hyp-hTyr-Orn-Hyp-Cys-NH2
was synthesized on Fmoc-Rink-Tentagel resin (211 mg; 50.6 mol)
(tR = 22.6 min and 22.8 min). ESI-MS calcd for C53H85N9O12
1039.63, found: m/z 1040.61 [M+H]+; HRMS calcd for
53H85N9O12 [M+H]+ 1040.6396, found 1040.6403; 1H NMR
(DMSO, 500 MHz): d Alg-1: 8.05 (NH), 4.16 ( CH), 2.36/2.16
(bCH), 5.74 ( CH); Thr-2: 7.354 (NH), 4.61 ( CH), 4.32 (bCH),
1.09 ( CH); Hyp-3: 4.43 ( CH), 2.13/1.79 (bCH), 4.30 ( CH), 3.64/
3.56 (dCH); hTyr-4: 7.75 (NH), 4.00 ( CH), 2.04/1.90 (bCH), 2.46/
2.36 ( CH); Orn-5: 7.23 (NH), 4.49 ( CH), 1.65/1.53 (bCH), 1.47
CH), 2.75 (dCH); Hyp-6: 4.46 ( CH), 2.02/1.87 (bCH), 4.37
CH), 3.78/3.66 (dCH); Alg-7: 7.93 (NH), 4.093 ( CH), 2.29
CH); Palm: 2.11 (CH2), 1.45 (CH2), 1.26 (CH2), 1.23
(CH2), 0.86 (CH3) ppm. 13C NMR (DMSO, 125 MHz): d Alg-1: 52.2
CH), 34.5 (bCH), 126.2 ( CH); Thr-2: 56.6 ( CH), 66.0 (bCH),
19.4 ( CH); Hyp-3: 58.3 ( CH), 37.3 (bCH), 68.7 ( CH), 55.7
(dCH); hTyr-4: 52.4 ( CH), 32.6 (bCH), 31.1 ( CH); 115.5 (Ar-H),
129.8 (Ar-H); Orn-5: 52.8 ( CH), 28.0 (bCH), 24.0 ( CH), 39.0
(dCH); Hyp-6: 59.3 ( CH), 37.9 (bCH), 69.2 ( CH), 55.7 (dCH);
Alg-7: 52.9 ( CH), 33.8 (bCH), 126.2 ( CH); Palm: 35.2 (CH2),
:
l
according to the general procedure for solid phase peptide synthe-
sis. The Fmoc group was cleaved and palmitic acid (52 mg;
C
a
202.6
lmol) was coupled overnight in the presence of HATU
c
a
(77 mg; 202.6
l
mol) and DiPEA (71 L; 405.2 mol) in NMP
l
l
c
a
c
(2 mL). The resin was washed with NMP (3ꢁ 2 mL, each 3 min)
and DCM (3ꢁ 2 mL, each 3 min) and subjected to the TFA cleavage
procedure and lyophilized. Mass spectral analysis (ESI-MS calcd for
11 C51H85N9O12S2: 1079.58, found: m/z 1079.90 [M+H]+) confirmed
the formation of the linear peptide 11 and the peptide was cyclized
a
c
a
(
(
c
c
a
a
(bCH), 5.74 (
c
by oxidation with DMSO. Peptide 11 (18 mg; 16.7 lmol) was dis-
solved in aq 2.5% AcOH (25 mL). The pH was adjusted to 6 with
aq NH4OH (25%). To this solution DMSO (5.5 mL) was added. After
stirring at rt overnight, the solution was partially concentrated in
vacuo and remaining DMSO was removed by a speedvac apparatus
(42 °C, overnight). After preparative HPLC and lyophilization, pep-
tide 7 (4 mg; 22%) was obtained as a white solid. Purity was con-
firmed by analytical HPLC (Altima C8, 65 min run, i-PrOH/MeOH/
H2O buffers) and was found to be higher than 99% (tR = 40.71 min).
ESI-MS calcd for C51H83N9O12S2: 1077.56, found: m/z 1078.35
[M+H]+; HRMS calcd for C51H83N9O12S2 [M+H]+ 1078.5681, found
1078.5721; 1H NMR (DMSO, 500 MHz): d Cys-1: 8.18 (NH), 4.76
(a
c
a
c
a
c
a
c
a
c
a
c
a
c
28.9 (CH2), 25.4 (CH2), 22.2 (CH2), 14.0 (CH3).
4.4. cyclo[-(Palm)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-OH (6b)
Peptide 6b was obtained analogous to peptide 6a, on Wang-
Tentagel resin (1 g; 0.26 mmol), except for the loading step. For
(
a
CH), 3.29/2.85 (bCH); Thr-2: 7.63 (NH), 4.70 (
1.05 ( CH); Hyp-3: 4.24 ( CH), 2.12/1.80 (bCH), 4.37 (
3.59 (dCH); hTyr-4: 7.34 (NH), 3.90 ( CH), 1.96/1.89 (bCH), 2.52/
2.47 ( CH); Orn-5: 7.25 (NH), 7.24 ( NH), 4.45 ( CH), 1.80/1.61
(bCH), 1.51 ( CH), 1.94 (bCH),
4.28 ( CH), 2.85
(bCH); Palm: 2.11 (CH2), 1.47 (CH2), 1.26 (CH2), 1.23 (CH2), 0.86
(CH3) ppm. 13C NMR (DMSO, 125 MHz): d Cys-1: 51.1 (
CH), 43.4
(bCH); Thr-2: 56.1 ( CH), 66.7 (bCH), 19.3 ( CH); Hyp-3: 61.0
CH), 37.1 (bCH), 69.3 ( CH), 55.9 (dCH); hTyr-4: 53.5 ( CH),
32.4 (bCH), 31.2 ( CH); 115.5 (Ar-H), 129.9 (Ar-H); Orn-5: 49.6
a
CH), 4.29 (bCH),
c
a
cCH), 3.80/
that Tentagel
(364 mg, 1.04 mmol) were dried in vacuo overnight over P2O5.
DMF (5 mL) and pyridine (114 L, 1.42 mmol) were added and
the resin was shaken for 10 min until total dissolution. DCBC
(149 L, 1.04 mmol) was added and the resin was shaken over
S
PHB (1.0 g, 0.26 mmol) and Fmoc-Alg-OH
a
e
c
a
l
c
CH), 2.80 (dCH); Hyp-6: 4.55 (a
CH), 3.56/3.18 (dCH); Cys-7: 8.25 (NH), 4.30 (a
c
l
the weekend. The resin was drained and washed with DMF (5ꢁ
3 mL, each 2 min) and DCM (5ꢁ 3 mL, each 2 min). Subsequently,
unreacted hydroxyl functions of the resin were acetylated by treat-
ment with Ac2O/NMI/DiPEA/DMF (5 mL; 2:1:1:6; v/v/v) for 30 min.
a
a
c
(a
c
a
c

M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
6513
(aCH), 27.6 (bCH), 23.5 (
cCH), 38.5 (dCH); Hyp-6: 59.0 (
aCH), 37.8
(cCH); Hyp-3: 4.42 (
aCH), 2.11/1.82 (bCH), 4.40 (
c
CH), 3.76
(bCH), 68.5 ( CH), 53.5 (dCH); Cys-7: 51.5 (
c
a
CH), 43.4 (bCH); Palm:
(dCH); hTyr-4: 8.46 (NH), 3.62 (
a
CH), 2.19/2.11 (bCH), 2.43/2.34
CH), 6.93/6.65 (Ar-H); Thr-5: 7.45 (NH), 4.12 (bCH), 1.02
CH); Hyp-6: 2.11/1.82 (bCH), 4.27 ( CH), 3.61 (dCH); Palm:
35.3 (CH2), 29.0 (CH2), 25.4 (CH2), 22.1 (CH2), 14.0 (CH3).
4.6. cyclo[-(Palm)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-NH2. (10a)
Hydrogenation of the cyclic unsaturated peptide 6a (3.2 mg;
(
(
c
c
c
0.85 (CH3), 1.26 (CH2), 1.24 (CH2), 1.44 (CH2), 2.07 (CH2) ppm.
13C NMR (DMSO, 125 MHz): d Orn-1: 50.9 (
CH), 28.9 (bCH),
24.0 ( CH), 37.9 (dCH); Thr-2: 58.5 ( CH), 66.5 (bCH), 19.1
CH); Hyp-3: 60.0 ( CH), 37.7 (bCH), 69.1 ( CH), 56.4 (dCH);
hTyr-4: 53.7 ( CH), 31.0 (bCH), 31.2 ( CH), 129.8, 115.5 (Ar-H);
Thr-5: 56.5 ( CH), 67.3 (bCH), 18.9 ( CH); Hyp-6: 59.3 ( CH),
37.7 (bCH), 69.0 ( CH), 56.2 (dCH); Palm: 13.9 (CH3), 22.0 (CH2),
a
c
a
3.07
l
mol) was carried out using the hydrogenation procedure in
(c
a
c
the presence of 3 mg 10% Pd/C. The saturated peptide 10a
(3.1 mg; quant) was obtained as a white solid. Purity was con-
firmed by analytical HPLC (Adsorbosphere C8, 40 min run, TFA
MeCN/H2O buffers) and was found to be higher than 99%
(tR = 22.62 min). ESI-MS calcd for C53H87N9O12: 1041.65, found:
m/z 1042.59 [M+H]+; HRMS calcd for C53H87N9O12 [M+H]+
1042.6552, found 1042.6530.
a
a
c
c
a
c
25.3 (CH2), 29.0 (CH2), 31.4 (CH2), 35.1 (CH2).
4.9. cyclo[-(Palm)-Orn-Thr-Hyp-hTyr-Orn-Hyp] (12b)
Peptide 13a was obtained analogously to peptide 12a except for
the amino acid sequence. The second amino acid coupled was
Fmoc-Orn(Boc)-OH instead of Fmoc-Thr(tBu)-OH. The loading of
the resin was determined after first coupling: 0.543 mmol/g.
After lyophilization, peptide 12b (18.5 mg; 22%) was obtained
as a white solid. Purity was confirmed by analytical HPLC (Altima
C8, 40 min run, TFA MeCN/H2O buffers) and was found to be higher
than 99% (tR = 23.18 min). ESI-MS calcd for C50H82N8O11: 970.61,
found: m/z 971.20 [M+H]+; HRMS calcd for C50H82N8O11 [M+H]+
971.6181, found 971.6187; 1H NMR (DMSO, 500 MHz): d Orn-1:
4.7. cyclo[-(Palm)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-OH (10b)
Hydrogenation of the cyclic unsaturated peptide 6b (4.7 mg;
4.52 lmol) was carried out using the hydrogenation procedure in
the presence of 5 mg 10% Pd/C. The saturated peptide 10b
(4.6 mg; quant.) was obtained as a white solid. Purity was con-
firmed by analytical HPLC (Adsorbosphere C8, 40 min run, TFA
MeCN/H2O buffers) and was found to be higher than 95%
(tR = 22.57 min). ESI-MS calcd for C53H86N8O13: 1042.63, found:
m/z 1043.59 [M+H]+; HRMS calcd for C53H86N8O13 [M+H]+
1043.6393, found 1043.6416.
7.92 (NH), 7.67 (
2.79 (dCH); Thr-2: 8.27 (NH), 4.74 (
Hyp-3: 4.39 ( CH); hTyr-4: 7.59 (NH), 4.03 (
(bCH), 2.43/2.37 ( CH); Orn-5: 7.70 (NH), 7.59 ( NH), 4.52 (
1.89/1.62 (bCH), 1.56 ( CH), 3.40/2.66 (dCH); Hyp-6: 4.40 (
e
NH), 4.14 (
a
CH), 1.64/1.56 (bCH), 1.54 (
CH), 4.36 (bCH), 1.09 (
CH), 1.86/1.75
c
CH);
CH),
a
c
a
a
4.8. cyclo[-(Palm)-Orn-Thr-Hyp-hTyr-Thr-Hyp] (12a)
c
e
a
CH),
c
cCH),
A polystyrene resin functionalized with a 2-chloro Trityl linker
(600 mg; initial loading: 1.1 mmol/g) was loaded with Fmoc-
Hyp(tBu)-OH (810.8 mg; 1.98 mmol) in DCM (5 mL) in the
3.76/3.51 (dCH); Palm: 2.02 (CH2), 1.46 (CH2), 1.32 (CH2), 1.26
(CH2), 0.86 (CH3) ppm. 13C NMR (DMSO, 125 MHz): d Orn-1: 50.6
(
a
CH), 28.3 (bCH), 23.5 (
c
CH), 38.3 (dCH); Thr-2: 56.1 (
a
a
CH),
CH),
presence of DiPEA (690
lL; 3.96 mmol) for 16 h. Subsequently,
66.2 (bCH), 20.4 (
28.1 (bCH), 30.9 (
c
c
CH); Hyp-3: 60.8 ( CH); hTyr-4: 53.2 (
CH); 115.2 (Ar-H), 129.5 (Ar-H); Orn-5: 50.6
a
unreacted tritylchloride moieties were capped with methanol
(DCM/MeOH/DiPEA; 3ꢁ 5 mL, each 2 min; 17/2/1; v/v/v). After
drying in vacuo overnight, the amount of Fmoc-Hyp(tBu)-OH cou-
pled to the resin was determined by a Fmoc determination accord-
ing to Meienhofer41 and was found to be 0.536 mmol/g. The
peptide sequence was synthesized according to the general
procedure for solid phase peptide synthesis.
(a
CH), 28.4 (bCH), 22.2 (
c
CH), 35.2 (dCH); Hyp-6: 69.3 ( CH),
c
55.4 (dCH); Palm: 35.2 (CH2), 27.8 (CH2), 25.9 (CH2), 25.0 (CH2),
13.6 (CH3).
4.10. cyclo[-(Ter)-Orn-Thr-Hyp-hTyr-Thr-Hyp] (13a)
The Fmoc group was cleaved and the linear peptide was re-
leased from the resin by treatment of the resin with HFIP/DCM
(6 mL, 1/1, v/v) for 2 h. This treatment was repeated once for 1 h.
The mixture was concentrated to obtain the linear peptide. A por-
tion of the linear peptide (187 mg; 0.15 mmol) was subjected to
head-to-tail cyclization and the IvDde group was cleaved following
the general procedures. The free amine of the linear peptide
Peptide 13a was obtained analogously to peptide 12a until the
coupling of the tail. Loading of the resin determined after first cou-
pling: 0.536 mmol/g.
The free amine of the linear peptide (34.7 mg; 34.7 lmol) was
coupled with the Ter chain by dissolving the peptide in DCM
(4 mL) and adding 4-(400-pentyloxy-1,10:40,100-terphenyl)-carbox-
ylic acid27 (13.8 mg; 38.2
l
mol), DCC (7.9 mg; 38.2
lmol) and
(54.2 mg; 54.2
lmol) was coupled with palmitic acid by dissolving
HOBt (5.2 mg; 38.2 lmol). The mixture was stirred overnight and
the peptide in dry DCM (4 mL) and adding palmitic acid (15.3 mg;
then the DCU was filtered off over Celite. The crude peptide was
purified by column chromatography (DCM/MeOH 30/1, v/v/v), Fol-
lowed by treatment with a mixture of TFA/TIS/H2O (95/2.5/2.5;
3 mL) for 1 h. The mixture was concentrated in vacuo, redissolved
in a small amount of TFA and precipitated in MTBE/hexane (1/1).
The supernatant was removed and the crude peptide washed twice
with MTBE/hexane (1/1). After lyophilization, peptide 13a
(13.1 mg; 23%) was obtained as a white solid. Purity was confirmed
by analytical HPLC (Altima C8, 65 min run, i-PrOH/MeOH/H2O buf-
fers) and was found to be higher than 99% (tR = 39.72 min). ESI-MS
calcd for C57H71N7O13: 1061.51, found: m/z 1062.65 [M+H]+; HRMS
calcd for C57H71N7O13 [M+H]+ 1062.5188, found 1062.5227; 1H
59.6
59.6
l
mol), DCC (12.3 mg; 59.6
lmol) and HOBt (8.0 mg;
lmol). The mixture was stirred overnight and the DCU filtered
off over Celite. The crude peptide was purified by column chroma-
tography (DCM/MeOH 30/1, v/v/v), Followed by treatment with a
mixture of TFA/TIS/H2O (3 mL, 95/2.5/2.5, v/v/v) for 2 h. The mix-
ture was concentrated in vacuo, redissolved in a small amount of
TFA and precipitated in MTBE/hexane (1/1). The supernatant was
removed and the crude peptide washed twice with MTBE/hexane
(1/1). After lyophilization, peptide 12a (15.6 mg; 19%) was ob-
tained as a white solid. Purity was confirmed by analytical HPLC
(Altima C8, 65 min run, i-PrOH/MeOH/H2O buffers) and was found
to be higher than 99% (tR = 42.18 min). ESI-MS calcd for
NMR (DMSO, 500 MHz): d Orn-1: 8.38 (NH), 7.46 (
CH), 1.91/1.54 (bCH), 1.53 ( CH), 3.09/3.02 (dCH); Thr-2: 7.95
(NH), 4.32 ( CH), 4.08 (bCH), 1.25 ( CH); Hyp-3: 4.43 ( CH),
2.14/1.84 (bCH), 4.40 ( CH), 3.77 (dCH); hTyr-4: 8.40 (NH), 3.68
CH), 2.18/2.13 (bCH), 2.44/2.36 ( CH), 6.95/6.66 (Ar-H); Thr-5:
eNH), 4.53
C
C
49H79N7O12: 957.58, found: m/z 958.95 [M+H]+; HRMS calcd for
(a
c
49H79N7O12 [M+H]+ 958.5865, found 958.5819; 1H NMR (DMSO,
a
c
a
500 MHz): d Orn-1: 7.80 (NH), 4.30 (
a
CH), 1.69/1.38 (bCH), 1.39
CH), 4.01 (bCH), 1.21
c
(cCH), 2.98 (dCH); Thr-2: 7.88 (NH), 4.23 (
a
(a
c

6514
M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
7.48 (NH), 4.80 (
2.11/1.84 (bCH), 4.29 (
a
CH), 4.16 (bCH), 1.06 (
c
CH); Hyp-6: 4.38 (
a
CH),
30 mL, each 3 min). The resin-bound peptide was subjected to
the microwave-assisted RCM procedure under the following condi-
tions: Resin peptide (3.3 g; 0.86 mmol), DCM (27.5 mL), LiCl/DMA
c
CH), 3.64 (dCH); Ter: 0.91 (CH3), 1.37 (CH2),
1.42 (CH2), 1.74 (CH2), 4.01 (OCH2) 7.66/7.04 (Ar-H Ph-3), 7.80/7.74
(Ar-H Ph-2), 7.99/7.81 (Ar-H Ph-1) ppm. 13C NMR (DMSO,
(degassed; 0.4 M; 2.7 mL), Grubbs II (9 mol %; 64 mg; 75.4 lmol),
125 MHz): d Orn-1: 52.1 (
(dCH); Thr-2: 58.4 ( CH), 66.6 (bCH), 19.2 (
CH), 37.6 (bCH), 69.2 ( CH), 56.4 (dCH); hTyr-4: 53.6 (
31.2 (bCH), 31.2 ( CH), 129.8/115.5 (Ar-H); Thr-5: 56.5 (
67.3 (bCH), 19.0 ( CH); Hyp-6: 59.3 (
CH), 56.2 (dCH); Ter: 13.8 (CH3), 21.9 (CH2), 27.7 (CH2), 28.4
a
CH), 28.2 (bCH), 24.1 (
c
CH), 38.0
100 °C for 60 min (performed in three batches due to the size
(10 mL) of the microwave vessel).
a
c
CH); Hyp-3: 60.1
(
a
c
a
a
CH),
CH),
The fully protected peptide 18 was obtained by cleavage from
the resin by treatment with a catalytic amount of KCN in MeOH
(20 mL) for 16 h. The resin was filtered and washed with MeOH
(3ꢁ 15 mL) and the filtrate was concentrated in vacuo to yield
the crude peptide. The peptide was purified by column chromatog-
raphy (DCM/MeOH, 50/1, v/v). After lyophilization peptide 18
(191.5 mg; 15%) was obtained as a white solid. Purity was con-
firmed by analytical HPLC (Altima C8, 65 min run, i-PrOH/MeOH/
H2O buffers) and was found to be higher than 99% (tR = 50.05 min).
ESI-MS calcd for C83H118N8O16: 1482.87, found: m/z 1483.79
[M+H]+; HRMS calcd for C83H118N8O16 [M+H]+ 1483.8744, found
1483.8715; 1H NMR (CDCl3, 500 MHz): d tBu: 1.53–1.37 (CH3),
1.25–1.11 (CH3); hTyr-4: 7.57/7.00 (Ar-H); Me ester: 3.71
(OCH3); Ter: 0.95 (CH3), 1.31 (CH2), 1.41 (CH2), 1.82 (CH2), 4.01
(OCH2) ppm. 13C NMR (CDCl3, 125 MHz): d tBu: 28.6 (CH3), 28.2
(CH3); hTyr-4: 128.7/115.5 (Ar-H); Me ester: 52.7 (OCH3); Ter:
14.1 (CH3), 22.6 (CH2), 28.9 (CH2), 29.2 (CH2), 68.5 (OCH2). Full
NMR characterization of the protected peptide 18 was not possible
due to the high intensity of the tBu/Boc signals and the broadened
amino acid signals. However, full NMR characterization of the
deprotected peptide can be found at the experimental details of
cyclic peptide 19.
c
c
aCH), 37.6 (bCH), 69.1
(c
(CH2), 67.7 (OCH2), 128.2/115.4 (Ar-C Ph-3), 127.8/127.2 (Ar-C
Ph-2), 128.8/126.6 (Ar-C Ph-1).
4.11. cyclo[-(Ter)-Orn-Thr-Hyp-hTyr-Orn-Hyp] (13b)
Peptide 13b was obtained analogously to peptide 13a except for
the amino acid sequence. The second amino acid coupled was
Fmoc-Orn(Boc)-OH instead of Fmoc-Thr(tBu)-OH. Loading of the
resin determined after first coupling: 0.543 mmol/g.
After lyophilization, peptide 13b (9.4 mg; 17%) was obtained as
a white solid. Purity was confirmed by analytical HPLC (Altima C8,
65 min run, i-PrOH/MeOH/H2O buffers) and was found to be 90%
(tR = 38.95 min). ESI-MS calcd for C58H74N8O12: 1074.54, found:
m/z 1075.69 [M+H]+; HRMS calcd for C58H74N8O12 [M+H]+
1075.5504, found 1075.5529; 1H NMR (DMSO, 500 MHz): d Orn-
1: 8.62 (NH), 8.01 (
3.47/2.72 (dCH); Thr-2: 8.34 (NH), 4.78 (
CH); Hyp-3: 4.34 ( CH), 2.00/1.86 (bCH), 4.38 (
(dCH); hTyr-4: 7.60 (NH), 4.07 ( CH), 2.09/1.87 (bCH), 2.46/2.38
CH), 6.95/6.66 (Ar-H); Orn-5: 7.50 (NH), 4.69 (
(bCH), 1.60 ( CH), 2.90/2.84 (dCH); Hyp-6: 4.38 (
(bCH), 4.37 (
e
NH), 4.60 (
a
CH), 1.83/1.65 (bCH), 1.60 (
CH), 4.39 (bCH), 1.13
CH), 3.75/3.69
cCH),
a
(c
a
c
a
4.13. cyclo[-(Ter)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-OH (16)
(c
a
a
CH), 1.83/1.65
CH), 2.16/1.77
c
Peptide 16 was obtained analogous to peptide 18 except for the
last step and obtained by using the TFA cleavage procedure for a
small aliquot of resin. After preparative HPLC and lyophilization,
peptide 16 was obtained as a white solid. Purity was confirmed
by analytical HPLC (Altima C8, 65 min run, i-PrOH/MeOH/H2O buf-
fers) and was found to be higher than 95% for a mixture of E/Z
stereoisomers (tR = 40.25 min and 40.43 min). ESI-MS calcd for
C61H76N8O14: 1144.55, found: m/z 1145.67 [M+H]+; HRMS calcd
for C61H76N8O14 [M+H]+ 1145.5559, found 1145.5547; 1H NMR
c
CH), 3.72/3.49 (dCH); Ter: 0.91 (CH3), 1.37 (CH2),
1.42 (CH2), 1.75 (CH2), 4.02 (OCH2) 7.66/7.04 (Ar-H Ph-3), 7.81/
7.75 (Ar-H Ph-2), 8.03/7.82 (Ar-H Ph-1) ppm. 13C NMR (DMSO,
125 MHz): d Orn-1: 51.8 (
(dCH); Thr-2: 56.5 ( CH), 66.5 (bCH), 19.5 (
CH), 37.7 (bCH), 69.2 ( CH), 55.9 (dCH); hTyr-4: 52.2 (
33.4 (bCH), 31.1 ( CH), 129.9/115.5 (Ar-H); Orn-5: 49.7 (
29.0 (bCH), 22.9 ( CH), 38.9 (dCH); Hyp-6: 59.2 (
(bCH), 69.2 ( CH), 56.1 (dCH); Ter: 13.9 (CH3), 21.9 (CH2), 27.7
aCH), 29.0 (bCH), 22.9 (
cCH), 35.7
a
c
CH); Hyp-3: 60.9
(a
c
a
a
CH),
CH),
c
c
aCH), 37.3
c
(DMSO, 500 MHz): d Alg-1: 8.62 (NH), 4.73 (
(bCH), 5.46 ( CH); Thr-2: 7.66 (NH), 4.68 ( CH), 4.26 (bCH), 1.09
CH); Hyp-3: 4.23 ( CH), 1.79 (bCH), 4.36 ( CH), 3.78/3.58
(dCH); hTyr-4: 7.33 (NH), 3.87 ( CH), 1.95/1.88 (bCH), 2.52/2.47
CH), 6.95/6.65 (Ar-H); Orn-5: 7.62 ( NH), 7.21 (NH), 4.46
CH), 2.83 (dCH); Hyp-6: 4.28
CH), 3.58/3.16 (dCH); Alg-7: 8.15 (NH),
CH), 2.35 (bCH), 5.46 ( CH); Ter: 0.89 (CH3), 1.21 (CH2),
aCH), 2.88/2.55
(CH2), 28.4 (CH2), 67.7 (OCH2), 128.2/115.4 (Ar-C Ph-3), 127.9/
127.2 (Ar-C Ph-2), 128.9/126.6 (Ar-C Ph-1).
c
a
(c
a
c
a
4.12. cyclo[-(Ter)-Alg-Thr(tBu)-Hyp(tBu)-hTyr(tBu)-Orn(Boc)-
Hyp(tBu)-Alg]-OMe (18)
(c
(a
(a
e
CH), 1.83/1.56 (bCH), 1.51 (
CH), 1.91 (bCH), 4.53 (
c
c
Wang-Tentagel S PHB resin (4 g; 1.04 mmol) and Fmoc-Alg-OH
(1.09 g, 4.16 mmol) were dried in vacuo overnight over P2O5. DMF
4.19 (
a
c
1.39 (CH2), 1.73 (CH2), 4.01 (OCH2), 7.65/7.03 (Ar-H Ph-3), 7.79/
7.73 (Ar-H Ph-2), 7.98/7.81 (Ar-H Ph-1) ppm.
(20 mL) and pyridine (555
lL, 6.86 mmol) were added and the re-
sin was shaken for 10 min until total dissolution. DCBC (596
lL,
4.16 mmol) was added and the resin was shaken for two days.
The resin was drained and washed with DMF (5ꢁ 30 mL, each
2 min) and DCM (5ꢁ 30 mL, each 2 min). Subsequently, unreacted
hydroxyl functions of the resin were acetylated by treatment with
Ac2O/NMI/DiPEA/DMF (30 mL; 2/1/1/6; v/v/v) for 30 min. The resin
was drained and washed with DMF (3ꢁ 30 mL, each 2 min) and
DCM (3ꢁ 30 mL, each 2 min). The peptide sequence was synthe-
sized according to the general procedure for solid phase peptide
synthesis. Hyp was coupled as part of the dipeptide Fmoc-Orn
(Boc)-Hyp(tBu)-OH.
4.14. cyclo[-(Ter)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-OH (17)
Hydrogenation of the cyclic unsaturated peptide 16 (3.5 mg;
3.06 lmol) was carried out using the hydrogenation procedure in
the presence of 3 mg of 10% Pd/C. The saturated peptide 17 (quant.)
was obtained as a white solid. Purity was confirmed by analytical
HPLC (Altima C8, 65 min run, i-PrOH/MeOH/H2O buffers) and
was found to be higher than 95% (tR = 41.99 min). ESI-MS calcd
for C61H78N8O14: 1146.56, found: m/z 1147.53 [M+H]+; HRMS calcd
for C61H78N8O14 [M+H]+ 1147.5710, found 1147.5707; 1H NMR
ESI-MS calcd for TFA deprotected 8b C54H68N8O14: 1052.49,
found: m/z 1053.52 [M+H]+.
(DMSO, 500 MHz): d Alg-1: 8.60 (NH), 4.55 (
1.35 ( CH); Thr-2: 7.44 (NH), 4.64 ( CH), 4.30 (bCH), 1.14 (
Hyp-3: 4.37 ( CH), 2.17/1.81 (bCH), 4.32 ( CH), 3.67/3.44 (dCH);
hTyr-4: 7.68 (NH), 3.97 ( CH), 2.11/1.89 (bCH), 2.47/2.38 ( CH),
6.95/6.67 (Ar-H); Orn-5: 7.65 ( NH), 7.64 (NH), 4.64 ( CH), 1.89/
a
CH), 1.76 (bCH),
Removal of the Fmoc group was followed by coupling overnight
of the ter tail (1.44 g; 4 mmol) in the presence of HATU (1.52 g;
4 mmol) and DIPEA (1.39 mL; 8 mmol) in NMP (30 mL). The resin
was washed with NMP (3ꢁ 30 mL, each 3 min) and DCM (3ꢁ
c
a
cCH);
a
c
a
c
e
a

M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
6515
1.62 (bCH), 1.54 (
1.85 (bCH), 4.38 (
1.80/1.60 (bCH), 1.376 (
c
c
CH), 2.57/2.83 (dCH); Hyp-6: 4.47 (
CH), 3.73 (dCH); Alg-7: 8.33 (NH), 4.32 (
CH); Ter: 0.91 (CH3), 1.36 (CH2), 1.42
a
CH), 2.46/
4.17. cyclo[-(Ter)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-
NHCH2CH2NH2. (21)
a
CH),
c
(CH2), 1.75 (CH2), 4.02 (OCH2), 7.67/7.05 (Ar-H Ph-3), 7.81/7.76
To a solution of peptide 20 (12.3 mg; 8.37
ene-diamine (2.68 mg; 16.74 mol) and HATU (3.18 mg;
8.37 mol) in DCM (1 mL) DIPEA (2.92 L; 16.74 mol) was added
and the reaction mixture was stirred for 48 h. As the reaction was
not finished according to TLC (DCM/MeOH/AcOH, 19/1/0.1) an-
lmol), N-boc-ethyl-
(Ar-H Ph-2), 8.04/7.83 (Ar-H Ph-1) ppm. 13C NMR (DMSO,
l
125 MHz): d Alg-1: 54.0 (
56.1 ( CH), 66.3 (bCH), 19.1 (
(bCH), 68.4 ( CH), 55.2 (dCH); hTyr-4: 52.1 (
30.7 ( CH), 129.6/115.2 (Ar-H); Orn-5: 49.3 (
23.2 ( CH), 38.4 (dCH); Hyp-6: 58.0 (
CH), 55.5 (dCH); Alg-7: 49.5 ( CH), 29.6 (bCH), 24.2 (
a
CH), 31.4 (bCH), 24.0 (
c
CH); Thr-2:
l
l
l
a
cCH); Hyp-3: 60.5 (
aCH), 37.1
c
aCH), 32.2 (bCH),
c
c
a
CH), 28.6 (bCH),
other portion of N-boc-ethylene-diamine (2.7 mg; 17
lmol), HATU
aCH), 37.6 (bCH), 68.9
(3.2 mg; 8.4 mol) and DIPEA (2.92 L; 16.74 mol) was added
l
l
l
(c
a
cCH); Ter:
and the mixture was stirred for an additional 24 h. The mixture
was concentrated in vacuo, the residue re-dissolved in EtOAc
(5 mL) and washed with 1 N KHSO4 (3ꢁ 5 mL), 5% NaHCO3 (3ꢁ
5 mL) and brine (5 mL). The organic layer was dried over Na2SO4
and concentrated in vacuo. The crude peptide was purified by col-
umn chromatography (DCM/MeOH/AcOH, 30/1/0.1, v/v/v) yielding
the protected peptide (8.0 mg; 59%) as a white solid. ESI-MS calcd
for C89H130N10O17: 1610.96, found: m/z 1611.53 [M+H]+.
13.6 (CH3), 21.6 (CH2), 27.4 (CH2), 28.1 (CH2), 67.4 (OCH2), 127.9/
115.1 (Ar-H Ph-3), 127.6/126.9 (Ar-H Ph-2), 128.6/126.3 (Ar-H
Ph-1) ppm.
4.15. cyclo[-(Ter)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-OMe (19)
Fully protected peptide 18 (19.9 mg; 13.41 lmol) was treated
The protected peptide (12.7 mg; 7.8 lmol) was treated with a
with a mixture of TFA/TIS/H2O (2 mL, 95/2.5/2.5, v/v/v) for 2 h.
The mixture was concentrated in vacuo, re-dissolved in a small
amount of TFA and precipitated in MTBE/hexane (1/1). The super-
natant was removed and the crude peptide washed twice with
MTBE/hexane (1/1). After preparative HPLC (Altima C8 semiprep,
120 min run, i-PrOH/MeOH/H2O buffers) and lyophilization, pep-
tide 19 (9.3 mg; 60%) was obtained as a white solid. Purity was
confirmed by analytical HPLC (Altima C8, 65 min run, i-PrOH/
MeOH/H2O buffers) and was found to be higher than 99%
(tR = 42.62 min). ESI-MS calcd for C62H78N8O14: 1158.56, found:
m/z 1159.52 [M+H]+; HRMS calcd for C62H78N8O14 [M+H]+
1159.5716, found 1159.5750; 1H NMR (DMSO, 500 MHz): d Alg-
mixture of TFA/TIS/H2O (1 mL, 95/2.5/2.5, v/v/v) for 2 h. The mix-
ture was concentrated in vacuo, the residue re-dissolved in a small
amount of TFA and precipitated in MTBE/hexane (1/1). The super-
natant was removed and the crude peptide washed twice with
MTBE/hexane (1/1). After preparative HPLC (Altima C8 semiprep,
120 min run, i-PrOH/MeOH/H2O buffers) and lyophilization, pep-
tide 21 (7.2 mg; 78%) was obtained as a white solid. Purity was
confirmed by analytical HPLC (Altima C8, 65 min run, i-PrOH/
MeOH/H2O buffers) and was found to be higher than 99% for a mix-
ture of E/Z stereoisomers (tR = 39.20 min and 39.48 min). ESI-MS
calcd for C63H82N10O13: 1186.61, found: m/z 1187.65 [M+H]+;
HRMS calcd for
1187.6152; 1H NMR (DMSO, 500 MHz): d Alg-1: 8.15 (NH), 4.25
CH), 2.40/2.19 (bCH), 5.61 ( CH); Thr-2: 7.67 (NH), 4.65 ( CH),
4.31 (bCH), 1.14 ( CH); Hyp-3: 4.35 ( CH), 2.16/1.83 (bCH), 4.41
CH), 3.77/3.72 (dCH); hTyr-4: 7.74 (NH), 3.96 ( CH), 2.11/1.95
(bCH), 2.48/2.37 ( CH) 6.96/6.67 (Ar-H); Orn-5: 7.71 ( NH), 7.46
(NH), 4.66 ( CH), 1.84/1.68 (bCH), 1.54 ( CH), 2.87 (dCH); Hyp-6:
4.48 ( CH), 2.06/1.89 (bCH), 4.32 ( CH), 3.67/3.44 (dCH); Alg-7:
7.99 (NH), 4.23 ( CH), 2.34 (bCH), 5.47 ( CH); NHCH2CH2NH2
C
61H78N10O13 [M+H]+ 1187.6141, found
1: 8.62 (NH), 4.73 (
7.65 (NH), 4.68 ( CH), 4.26 (bCH), 1.09 (
CH), 2.02/1.83 (bCH), 4.36 ( CH), 3.79/3.59 (dCH); hTyr-4: 7.33
(NH), 3.88 ( CH), 1.94/1.88 (bCH), 2.52/2.46 ( CH), 6.94/6.64 (Ar-
H); Orn-5: 7.63 ( NH), 7.21 (NH), 4.46 ( CH), 1.82/1.57 (bCH),
1.51 ( CH), 2.82 (dCH); Hyp-6: 4.53 ( CH), 1.93/1.88 (bCH), 4.28
CH), 3.58/3.17 (dCH); Alg-7: 8.27 (NH), 4.29 ( CH), 2.35 (bCH),
5.46 ( CH); Me ester: 3.61 (CH3); Ter: 0.89 (CH3), 1.22 (CH2),
aCH), 2.88/2.55 (bCH), 5.46 (cCH); Thr-2:
a
cCH); Hyp-3: 4.22
(a
c
a
(a
c
c
a
a
c
(c
a
e
a
c
e
c
a
a
c
(c
a
a
c
c
a
c
1.38 (CH2), 1.72 (CH2), 4.00 (OCH2), 7.64/7.03 (Ar-H Ph-3), 7.79/
7.73 (Ar-H Ph-2), 7.98/7.81 (Ar-H Ph-1) ppm.
amide: 3.32/3.23 (CH2), 2.86/2.82 (CH2); Ter: 0.92 (CH3), 1.38
(CH2), 1.43 (CH2), 1.75 (CH2), 4.03 (OCH2), 7.67/7.06 (Ar-H Ph-3),
7.82/7.76 (Ar-H Ph-2), 8.01/7.84 (Ar-H Ph-1) ppm.
4.16. cyclo[-(Ter)-Alg-Thr(tBu)-Hyp(tBu)-hTyr(tBu)-Orn(Boc)-
Hyp(tBu)-Alg]-OH (20)
4.18. cyclo[-(Ter)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-[CH2OH] (22)
A solution of 0.2 N LiOH (0.6 mL) was added drop-wise to a
cooled (0 °C) solution of peptide 18 (61.8 mg; 41.7 lmol) in THF
LiBH4 (11.1
18 (18.8 mg; 12.67
l
L; 22.17
lmol) was added to a solution of peptide
lmol) in dry THF (2 mL) and the reaction mix-
(1.5 mL). The reaction mixture was stirred at room temperature
for 16 h followed by evaporation in vacuo. The product was re-dis-
solved in H2O and neutralized with 1 N HCl, followed by lyophili-
zation yielding peptide 20 (67 mg; quant.) as a white solid.
Purity was confirmed by analytical HPLC (Altima C8, 65 min run,
i-PrOH/MeOH/H2O buffers) and was found to be higher than 95%
(tR = 49.63 min). ESI-MS calcd for C82H116N8O16: 1468.85, found:
m/z 1469.75 [M+H]+; HRMS calcd for C82H116N8O16 [M+H]+
1469.8588, found 1469.8579; 1H NMR (CDCl3, 500 MHz): d tBu:
1.48–1.33 (CH3), 1.22–1.08 (CH3); hTyr-4: 7.53/6.96 (Ar-H); Ter:
0.92 (CH3), 1.38 (CH2), 1.28 (CH2), 1.79 (CH2), 3.98 (OCH2) ppm.
13C NMR (CDCl3, 125 MHz): d tBu: 28.1 (CH3), 28.0 (CH3); hTyr-4:
128.3/115.1 (Ar-H); Ter: 13.7 (CH3), 22.2 (CH2), 28.5 (CH2), 28.7
(CH2), 68.0 (OCH2). Full NMR characterization of the protected pep-
tide 20 was not possible due to the high intensity of the tBu/Boc
signals and the broadened amino acid signals. However, full NMR
characterization of the deprotected peptide can be found at the
experimental details of cyclic peptide 16.
ture was stirred for 5 h. The reaction mixture was quenched by
addition of satd aq NaHCO3 and stirred for an additional 10 min.
The aqueous layer was extracted with Et2O (10 mL) and EtOAc
(10 mL). The combined organic layers were dried over Na2SO4, fil-
tered and concentrated in vacuo. The crude peptide was purified by
column chromatography (DCM/MeOH/AcOH, 20/1/0.1, v/v/v)
yielding the protected peptide alcohol (10.2 mg; 55%) as a white
solid. ESI-MS calcd for C82H118N8NaO15: 1477.86, found: m/z
1477.82 [M+Na]+.
The peptide (10 mg; 6.9 lmol) was treated with a mixture of
TFA/TIS/H2O (2 mL; 95/2.5/2.5; v/v/v) for 2 h. The mixture was con-
centrated in vacuo, re-dissolved in a small amount of TFA and pre-
cipitated in MTBE/hexane (1/1). The supernatant was removed and
the crude peptide washed twice with MTBE/hexane (1/1). After
preparative HPLC (Altima C8 semiprep, 120 min run, i-PrOH/
MeOH/H2O buffers) and lyophilization, peptide 20 (3.7 mg; 26%)
was obtained as a white solid. Purity was confirmed by analytical

6516
M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
HPLC (Altima C8, 65 min run, i-PrOH/MeOH/H2O buffers) and was
found to be higher than 99% (tR = 41.83 min). ESI-MS calcd for
4.20. cyclo[-(Ter)-Lys-Thr-Hyp-hTyr-Orn-Hyp] (26)
C
61H78N8O13: 1130.57, found: m/z 1131.57 [M+H]+; HRMS calcd
Linear peptide Fmoc-Lys(Psoc)-Thr(tBu)-Hyp(tBu)-hTyr(tBu)-
Orn(Boc)-Hyp(tBu)-OH (24) was synthesized on trityl resin
for C61H78N8O13 [M+H]+ 1131.5767, found 1131.5795; 1H NMR
(DMSO, 500 MHz): d Alg-1: 8.67 (NH), 4.63 ( CH), 2.55/2.43
(bCH), 5.58 ( CH); Thr-2: 7.70 (NH), 4.64 ( CH), 4.28 (bCH), 1.13
CH); Hyp-3: 4.32 ( CH), 2.13/1.81 (bCH), 4.31 ( CH), 3.66/3.41
(dCH); hTyr-4: 7.75 (NH), 3.91 ( CH), 2.08/1.93 (bCH), 2.46/2.38
CH), 6.95/6.66 (Ar-H); Orn-5: 7.64 ( NH), 7.45 (NH), 4.63
CH), 1.91/1.61 (bCH), 1.55 ( CH), 2.94/2.83 (dCH); Hyp-6: 4.39
CH), 2.00/1.82 (bCH), 4.39 ( CH), 3.74/3.71 (dCH); Alg-7: 7.69
CH), 2.29/1.94 (bCH), 5.54 ( CH); alcohol: 3.28/3.13
a
(200 mg; 93.8
phase peptide synthesis. The Fmoc group was cleaved and the ter
tail (101.4 mg; 281.4 mol) was coupled overnight in the presence
of HATU (107 mg; 281.4 mol) and DiPEA (98 L; 562.8 mol) in
lmol) according to the general procedure for solid
c
a
(c
a
c
l
a
l
l
l
(
(
(
c
a
a
e
NMP (2 mL). The resin was washed with NMP (3ꢁ 3 mL, each
c
c
3 min) and DCM (3ꢁ 3 mL, each 3 min). The Psoc group was
cleaved by treatment with TBAFꢀ3H2O (88.78 mg; 281.4
lmol) for
(NH), 3.71 (
a
c
15 min in DCM (2 mL). Peptide 25 was obtained by treatment of
the resin with HFIP/DCM (3 mL, 1/1, v/v) for 3 h. This was repeated
once for 1 h. The mixture was concentrated to obtain peptide 25
(121.4 mg; 90%) as a foam. Characterization was carried out by
ESI-MS (calcd for C80H118N8O15: 1430.87, found: m/z 1431.89
[M+H]+).
(CH2OH); Ter: 0.91 (CH3), 1.37 (CH2), 1.42 (CH2), 1.75 (CH2), 4.02
(OCH2), 7.67/7.05 (Ar-H Ph-3), 7.82/7.75 (Ar-H Ph-2), 8.01/7.83
(Ar-H Ph-1) ppm. 13C NMR (DMSO, 125 MHz): d Alg-1: 54.0
(
a
CH), 34.0 (bCH), 128.9 (
c
a
CH); Thr-2: 56.2 (
CH), 36.9 (bCH), 68.4 (
CH), 32.0 (bCH), 30.7 ( CH), 129.6/115.2
CH), 28.5 (bCH), 23.3 (
a
CH), 66.3 (bCH),
19.1 ( CH); Hyp-3: 60.5 (
(dCH); hTyr-4: 52.4 (
(Ar-H); Orn-5: 49.5 (
c
cCH), 55.1
a
a
c
Then, the crude linear peptide Ter-Lys-Thr(tBu)-Hyp(tBu)-
hTyr(tBu)-Orn(Boc)-Hyp(tBu)-OH (25) was subjected to the head-
to-tail cyclization according to the general procedure, followed
by treatment with a mixture of TFA/TIS/H2O (10 mL, 95/2.5/2.5,
v/v/v) for 1 h. The mixture was concentrated in vacuo, re-dissolved
in a small amount of TFA and precipitated in MTBE/hexane (1/1).
The supernatant was removed and the crude peptide washed twice
with MTBE/hexane (1/1). After preparative HPLC (Altima C8 semi-
prep, 120 min run, i-PrOH/MeOH/H2O buffers) and lyophilization,
peptide 26 (20.4 mg; 23%) was obtained as a white solid. Purity
was confirmed by analytical HPLC (Altima C8, 65 min run, i-
PrOH/MeOH/H2O buffers) and was found to be higher than 99%
(tR = 41.05 min). ESI-MS calcd for C59H76N8O12: 1088.56, found:
m/z 1089.82 [M+H]+; HRMS calcd for C59H76N8O12 [M+H]+
1089.5661, found 1089.5656; 1H NMR (DMSO, 500 MHz): d Lys-
cCH), 38.4 (dCH);
Hyp-6: 58.2 (
aCH), 38.0 (bCH), 69.0 (cCH), 55.5 (dCH); Alg-7:
50.0 ( CH), 33.0 (bCH), 127.7 (cCH); alcohol: 63.8 (CH2OH); Ter:
a
13.6 (CH3), 21.6 (CH2), 27.4 (CH2), 28.1 (CH2), 67.4 (OCH2), 127.9/
115.1 (Ar-H Ph-3), 127.5/126.9 (Ar-H Ph-2), 128.5/126.3 (Ar-H
Ph-1) ppm.
4.19. cyclo[-(Ter)-Alg-Thr-Hyp-hTyr-Orn-Hyp-Alg]-NHNH2 (23)
To a solution of peptide 18 (9.0 mg; 6.06 lmol) in dry DMF
(1 mL) N2H4ꢀH2O (0.2 mL) was added and the reaction mixture
was stirred for 24 h. The volatiles were removed by evaporation
in vacuo and coevaporation with MeOH (3ꢁ). Lyophilization
yielded the protected peptide hydrazide (8.0 mg; 89%) as a white
solid. ESI-MS calcd for C82H118N10NaO15: 1505.87, found: m/z
1505.80 [M+Na].
1: 8.53 (NH), 7.90 (
e
NH), 4.66 (
a
CH), 2.14/1.72 (bCH), 1.60/1.33
CH); Thr-2: 7.79 (NH), 4.71
CH); Hyp-3: 4.23 ( CH), 2.12/1.78
CH), 3.80/3.59 (dCH); hTyr-4: 7.43 (NH), 3.98
CH), 1.99/1.87 (bCH), 2.52/2.46 ( CH), 6.96/6.66 (Ar-H); Orn-5:
7.69 ( NH), 7.33 (NH), 4.51 ( CH), 1.87/1.63 (bCH), 1.56 ( CH),
2.89 (dCH); Hyp-6: 4.40 ( CH), 1.98/1.77 (bCH), 4.30 ( CH), 3.66/
(cCH), 1.58/1.32 (dCH), 3.56/2.71 (e
(aCH), 1.09 (bCH), 1.09 (
c
a
The protected peptide (8.0 mg; 5.4 lmol) was treated with HCl/
(bCH), 4.37 (
c
Et2O42 for 2 h. The mixture was concentrated in vacuo, re-dissolved
in a small amount of MeOH and precipitated in MTBE/hexane (1/1).
The supernatant was removed and the crude peptide washed twice
with MTBE/hexane (1/1). After preparative HPLC (Altima C8 semi-
prep, 120 min run, i-PrOH/MeOH/H2O buffers) and lyophilization,
peptide according to the general procedure 23 (3 mg; 48%) was ob-
tained as a white solid. Purity was confirmed by analytical HPLC
(Altima C8, 65 min run, i-PrOH/MeOH/H2O buffers) and was found
to be higher than 95% (tR = 39.50 min). ESI-MS calcd for
(a
c
e
a
c
a
c
3.31 (dCH); Ter: 0.91 (CH3), 1.36 (CH2), 1.41 (CH2), 1.74 (CH2),
4.01 (OCH2) 7.65/7.03 (Ar-H Ph-3), 7.80/7.74 (Ar-H Ph-2), 8.03/
7.81 (Ar-H Ph-1) ppm. 13C NMR (DMSO, 125 MHz): d Lys-1: 53.4
(
a
CH), 31.0 (bCH), 23.3 (
CH), 66.4 (bCH), 19.2 (
CH), 55.2 (dCH); hTyr-4: 53.0 (
CH), 129.1/114.6 (Ar-H); Orn-5: 49.4 (
CH), 38.3 (dCH); Hyp-6: 59.2 ( CH), 37.6 (bCH), 68.4 (
c
CH), 26.9 (dCH), 38.4 (
CH); Hyp-3: 60.7 (
CH), 32.3 (bCH), 30.7
CH), 27.7 (bCH), 23.3
CH),
e
CH); Thr-2: 55.7
(a
c
aCH), 36.7 (bCH),
68.9 (
c
a
a
C
61H78N10O13: 1158.57, found: m/z 1159.80 [M+H]+; HRMS calcd
for C61H78N10O13 [M+H]+ 1159.5828, found 1159.5845; 1H NMR
(DMSO, 500 MHz): d Alg-1: 8.72 (NH), 4.62 ( CH), 2.52/2.46
(bCH), 5.62 ( CH); Thr-2: 7.57 (NH), 4.65 ( CH), 4.30 (bCH), 1.13
CH); Hyp-3: 4.46 ( CH), 2.21/2.32 (bCH), 4.40 ( CH), 3.66/3.42
(dCH); hTyr-4: 7.71 (NH), 3.95 ( CH), 2.10/1.92 (bCH), 2.47/2.37
CH), 6.95/6.66 (Ar-H); Orn-5: 7.67 ( NH), 7.45 (NH), 4.65
(
(
c
c
a
c
a
54.5 (dCH); Ter: 13.7 (CH3), 21.6 (CH2), 27.5 (CH2), 28.1 (CH2),
67.2 (OCH2), 127.4/114.7 (Ar-C Ph-3), 127.1/126.4 (Ar-C Ph-2),
128.1/125.8 (Ar-C Ph-1).
c
a
(c
a
c
a
(
(
(
c
a
a
e
4.21. Biological activity
CH), 1.85/1.65 (bCH), 1.52 (
CH), 2.03/1.83 (bCH), 4.30 (
CH), 2.15 (bCH), 5.63 (cCH); Ter: 0.91 (CH3), 1.37 (CH2),
cCH), 2.92/2.83 (dCH); Hyp-6: 4.34
cCH), 3.74 (dCH); Alg-7: 8.30 (NH),
Antifungal activity was evaluated by broth microdilution. The
media used in this assay was Yeast Extract Peptone Dextrose
(YPD) containing 1% yeast extract, 2% peptone, 1% dextrose in dis-
tilled water. Test compounds were dissolved in distilled water or
10% DMSO, depending on the solubility characteristics of the com-
pounds, to a concentration of 1 mg/mL. After solubilization each
compound was diluted five times in YPD medium rendering a stock
4.31 (
a
1.42 (CH2), 1.74 (CH2), 4.02 (OCH2), 7.76/7.05 (Ar-H Ph-3), 7.84/
7.67 (Ar-H Ph-2), 8.02/7.82 (Ar-H Ph-1) ppm. 13C NMR (DMSO,
125 MHz): d Alg-1: 54.0 (
56.2 ( CH), 66.2 (bCH), 19.1 (
(bCH), 69.0 ( CH), 55.1 (dCH); hTyr-4: 52.2 (
30.7 ( CH), 129.6/115.2 (Ar-H); Orn-5: 49.3 (
23.2 ( CH), 38.4 (dCH); Hyp-6: 60.6 (
CH), 55.5 (dCH); Alg-7: 50.5 ( CH), 37.0 (bCH), 129.3 (
a
CH), 33.9 (bCH), 127.3 (
c
CH); Thr-2:
a
cCH); Hyp-3: 58.0 (
aCH), 34.1
c
a
CH), 32.2 (bCH),
c
c
a
CH), 28.6 (bCH),
solution of 200
cant (Merck Sharp & Dohme B.V., Haarlem, Netherlands), was in-
cluded as control. Serial twofold dilutions of the test
l
g/mL. Caspofungin,20 purchased from the fabri-
aCH), 37.6 (bCH), 68.4
(c
a
c
CH);
a
Ter: 13.5 (CH3), 21.6 (CH2), 27.4 (CH2), 28.0 (CH2), 67.4 (OCH2),
126.9/115.1 (Ar-H Ph-3), 126.4/127.9 (Ar-H Ph-2), 128.5/127.5
(Ar-H Ph-1) ppm.
compounds in YPD medium were prepared as followed. To each
well of a sterile Greiner bio-one Cellstar 96 well, U bottomed
microtiter plate 100 ll of YPD was dispensed. Manually, 100 ll of

M. P. C. Mulder et al. / Bioorg. Med. Chem. 19 (2011) 6505–6517
6517
9. Norris, T.; VanAlsten, J.; Hubbs, S.; Ewing, M.; Cai, W.; Jorgensen, M. L.; Bordner,
J.; Jensen, G. O. Org. Process Res. Dev. 2008, 12, 447.
10. Kurokawa, N.; Ohfune, Y. J. Am. Chem. Soc. 1986, 108, 6041.
11. Evans, D. A.; Weber, A. E. J. Am. Chem. Soc. 1987, 109, 7151.
12. Kurokawa, N.; Ohfune, Y. Tetrahedron 1993, 49, 6195.
stock compounds was delivered to each well in colomn 1. Then
using a 8-channel pipet, compounds in column 1 were serially di-
luted twofold.
The plates containing the diluted compounds were inoculated
with 100 ll of the appropriate microorganism. The yeast strains
13. For an excellent review of the total and semisynthetic approaches, see: Renslo,
A. R. Anti-Infect. Agents Med. Chem. 2007, 6, 201.
used were isolates obtained from the CBS-KNAW Fungal Biodiver-
sity Centre (Utrecht, The Netherlands). The collection included C.
dubliensis, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei and
two strains of C. albicans. Stock cultures of the yeast strains in li-
quid media (YPD + 15% glycerol) were maintained at ꢂ80 °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 media
and aerated for 24 h at 30 °C on a shaker set at 300 rpm. The broth
cultures were diluted 10 times with media and the optical density
of this suspension was measured at a wavelength of 600 nm. The
suspension was further diluted to an OD600 of 0.01 and this suspen-
sion was further diluted 1:100 in YPD media. This final dilution
was used for inoculating the plates. Thus, the final number of cells
per well is approximately 1.5 ꢁ 103 cells/ml.43 The final volume/
14. Zambias, R. A.; Hammond, M. L.; Heck, J. V.; Bartizal, K.; Trainor, C.; Abruzzo,
G.; Schmatz, D. M.; Nollstadt, K. M. J. Med. Chem. 1992, 35, 2843.
15. Klein, L. L.; Li, L.; Chen, H.; Curty, C. B.; DeGoey, D. A.; Grampovnik, D. J.; Leone,
C. L.; Thomas, S. A.; Yeung, C. M.; Funk, K. W.; Kishore, V.; Lundell, E. O.; Wodka,
D.; Meulbroek, J. A.; Alder, J. D.; Nilius, A. M.; Lartey, P. A.; Plattner, J. J. Bioorg.
Med. Chem. Lett. 2000, 8, 1677.
16. Debono, M.; Abbott, B. J.; Turner, J. R.; Howard, L. C.; Gordee, R. S.; Hunt, A. S.;
Barnhart, M.; Molloy, R. M.; Willard, K. E.; Fukuda, D.; Butler, T. F.; Zeckner, D. J.
Ann. N.Y. Acad. Sci. 1988, 544, 152.
17. Borromeo, P. S.; Cohen, J. D.; Gregory, G. S.; Henle, S. K.; Hitchcock, S. A.;
Jungheim, L. N.; Mayhugh, D. R.; Shepherd, T. A.; Turner, W. W., Jr. (Eli Lilly and
Company), US patent 6,653,281 B1, 2003.
18. Examples of on-resin RCM: (a) Stymiest, J. L.; Mitchell, B. F.; Wong, S.; Vederas,
J. C. Org. Lett. 2003, 5, 47; (b) Stymiest, J. L.; Mitchell, B. F.; Wong, S.; Vederas, J.
C. J. Org. Chem. 2005, 70, 7799; (c) Elaridi, J.; Patel, J.; Jackson, W. R.; Robinson,
A. J. J. Org. Chem. 2006, 71, 7538; (d) Jiang, S.; Li, P.; Lee, S. L.; Lin, C. Y.; Long, Y.;
Johnson, M. D.; Dickson, R. B.; Roller, P. P. Org. Lett. 2007, 9, 9; (e) Addona, D. D.;
Carotenuto, A.; Novellino, E.; Piccand, V.; Reubi, J. C.; Cianni, A. D.; Gori, F.;
Papini, A. M.; Ginanneschi, M. J. Med. Chem. 2008, 51, 512.
19. Robinson, A. J.; Elaridi, J.; van Lierop, B. J.; Mujcinovic, S.; Jackson, W. R. J. Pept.
Sci. 2007, 13, 280.
well, including organism and compound was 200 ll. The last row
of the plate contained drug-free wells dedicated for growth and
sterility controls for each organism tested.
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 inhibi-
tion of yeast growth.
20. Tam, J. P.; Wu, C.-R.; Liu, W.; Zhang, J.-W. J. Am. Chem. Soc. 1991, 113, 6657.
21. Caspofungin is present in and commercially available as ‘Cancidas’ from Merck.
22. Debono, M.; Abbott, B. J.; Fukuda, D. S.; Barnhart, M.; Willard, K. E.; Molloy, R.
M.; Michel, K. H.; Turner, J. R.; Butler, T. F.; Hunt, A. H. J. Antibiot. 1989, XLII,
389.
23. Taft, C. S.; Selitrennikoff, C. P. J. Antibiot. 1990, XLIII, 433.
24. Debono, M.; Turner, W. W.; LaGrandeur, L.; Burkhardt, F. J.; Nissen, J. S.;
Nichols, K. K.; Rodriguez, M. J.; Zweifel, M. J.; Zeckner, D. J.; Gordee, R. S.; Tang,
J.; Parr, T. R. J. Med. Chem. 1995, 38, 3271.
25. Jamison, J. A.; Rodriguez, M. J.; Vasudevan, V. US Patent 6,670,324, 2003.
26. Jamison, J. A.; Zeckner, D. J.; Rodriguez, M. J. J. Antibiot. 1997, 50, 562.
27. The terphenyl tail was first synthesized according to the procedure of Debono
et al.24 However, low yields and solubility problems led to the use of the
procedure described by: Scherer, S.; Haber, S. (Clariant GMBH), Patent WO 00/
50375, 2000.
28. Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.; Chan, W. C.
Tetrahedron Lett. 1998, 39, 1603.
29. Balkovec, J. M.; Black, R. M.; Hammond, M. L.; Hock, J. V.; Zambias, R. A.;
Abruzzo, G.; Bartizal, K.; Kropp, H.; Trainor, C.; Schwartz, R. E.; McFadden, D. C.;
Nollstadt, K. H.; Pittarelli, L. A.; Powles, M. A.; Schmatz, D. M. J. Med. Chem.
1992, 35, 194.
30. Bouffard, F. A.; Hammond, M. L.; Arison, B. H. Tetrahedron Lett. 1995, 36, 1405.
31. Bouffard, F. A.; Zambias, R. A.; Dropinski, J. F.; Balkovec, J. M.; Hammond, M. L.;
Abruzzo, G. K.; Bartizal, K. F.; Marrinan, J. A.; Kurtz, M. B.; McFadden, D. C.;
Nollstadt, K. H.; Powles, M. A.; Schmatz, D. M. J. Med. Chem. 1994, 37, 222.
32. Jamison, J. A.; LaGrandeur, L. M.; Rodriguez, M. J.; Turner, W. W.; Zeckner, D. J. J.
Antibiot. 1997, 51, 239.
33. Balkovec, J. M.; Dropinski, J. F.; Bouffard, F. A. US Patent 5,741,775, 1998.
34. Rodriguez, J. M.; Nesler, M. J.; Zweifel, M. J. US Patent 60/112,433, 2000.
35. Lal, B.; Gund, V. G.; Gangopadhyay, A. K.; Nadkarni, S. R.; Dikshit, V.; Chatterjee,
D. K.; Shirvaikar, R. Bioorg. Med. Chem. 2003, 11, 5189.
Acknowledgments
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 recording
HRMS spectra. From the CBS-KNAW Fungal Biodiversity Centre
(Utrecht, The Netherlands) we would like to thank Dr. T. Boekhout
for providing us with the yeast strains and Dr. J. Dijksterhuis for his
help with the visualization of yeast by light microscopy. And finally
we thank Dr. D. T. S. Rijkers and Dr. N. I. Martin for critically read-
ing the manuscript.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.08.034.
References and notes
36. Lal, B.; Gund, V. G. Bioorg. Med. Chem. Lett. 2004, 14, 1123.
37. Balkovec, J. M.; Dropinski, J. F.; Bouffard, F. A. US Patent 6,268,338, 1996.
38. Wagner, M.; Heiner, S.; Kunz, H. Synlett 2000, 1753.
39. Vojkovsky, T. Pept. Res. 1995, 8, 236.
40. Kaiser, E.; Colescott, R. L.; Bossinger, L. D.; Cook, P. I. Anal. Biochem. 1970, 34,
595.
41. Meienhofer, J.; Waki, M.; Heimer, E. P.; Lambros, T. J.; Makofske, R. C.; Chang,
C.-D. Int. J. Pept. Protein Res. 1979, 13, 35.
42. When the peptide was cleaved using the standard TFA procedure, the TFA
adduct of the hydrazine amide was formed. Characterization of the TFA adduct
was carried out by ESI-MS (calcd for C63H77F3N10O14: 1254.56, found: m/z
1255.64 [M+H]+).
1. Gullo, A. Drugs 2009, 69, 65.
2. Lai, C.-C.; Tan, C.-K.; Huang, Y.-T.; Shao, P.-L.; Hsueh, P.-R. J. Infect. Chemother.
2008, 14, 77.
3. Kanafani, Z. A.; Perfect, J. R. Clin. Infect.Dis. 2008, 46, 120.
4. Denning, D. W. J. Antimicrob. Chemother. 2002, 49, 889.
5. Examples of reviews discussing the b-(1,3)-D-glucan synthase complex: (a)
Douglas, C. M. Med. Mycol. 2001, 39, 55; (b) Liu, J.; Balasubramanian, M. K. Curr.
Drug Targets Infect. Disord. 2001, 1, 159; (c) Bowman, S. M.; Free, S. J. BioEssays
2006, 28, 799; (d) Latgé, J. P. Mol. Microbiol. 2007, 66, 279.
6. Sucher, A. J.; Chahine, E. B.; Balcer, H. E. Ann. Pharmacother. 2009, 43, 1647.
7. Leonard, W. R.; Belyk, K. M.; Conlon, D. A.; Bender, D. R.; DiMichele, L. M.; Liu, J.;
Hughes, D. L. J. Org. Chem. 2007, 72, 2335.
43. Treco, D. A.; Winston, F. Curr. Protoc. Mol. Biol. 2008, 82, 13.2.1.
8. Tomishima, M.; Ohki, H.; Yamada, A.; Maki, K.; Ikedia, F. Bioorg. Med. Chem. Lett.
2008, 18, 2886.