Anthelmintic PF1022A: Stepwise solid-phase synthesis of a cyclodepsipeptide containing N-methyl amino acids

Article abstract of DOI:10.1016/j.tet.2011.12.026

Cyclodepsipeptides of the enniation-, PF1022-, and verticilide-family represent a diverse class of highly interesting natural products with respect to their manifold biological activities. However, until now no stepwise solid-phase synthesis has been accomplished due to the difficult combination of N-methyl amino acids and hydroxycarboxylic acids. We report here the first stepwise solid-phase synthesis of the anthelmintic cyclooctadepsipeptide PF1022A based on an Fmoc/THP-ether protecting group strategy on Wang-resin. The standard conditions of our synthesis allow an unproblematic adaption to an automated peptide synthesizer.

Full text of DOI:10.1016/j.tet.2011.12.026

Tetrahedron 68 (2012) 2068e2073
Contents lists available at SciVerse ScienceDirect
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journal homepage: www.elsevier.com/locate/tet
Anthelmintic PF1022A: stepwise solid-phase synthesis of a cyclodepsipeptide
containing N-methyl amino acids
*
€
€
Sebastian Luttenberg, Frank Sondermann, Jurgen Scherkenbeck
€
Bergische Universitat Wuppertal, Fachgruppe Chemie, Gaußstraße 20, D-42119 Wuppertal, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Cyclodepsipeptides of the enniation-, PF1022-, and verticilide-family represent a diverse class of highly
interesting natural products with respect to their manifold biological activities. However, until now no
stepwise solid-phase synthesis has been accomplished due to the difficult combination of N-methyl
amino acids and hydroxycarboxylic acids. We report here the first stepwise solid-phase synthesis of the
anthelmintic cyclooctadepsipeptide PF1022A based on an Fmoc/THP-ether protecting group strategy on
Wang-resin. The standard conditions of our synthesis allow an unproblematic adaption to an automated
peptide synthesizer.
Received 28 September 2011
Received in revised form 4 December 2011
Accepted 10 December 2011
Available online 16 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
enniatins (6e9), a large family of cyclohexadepsipeptides, display
diverse biological activities, including antibiotic, antifungal, in-
Depsipeptides are defined as heterodetic peptides with at least
one ester bond. While most depsipeptides contain only few, usually
irregular ester groups, depsipeptides with alternating ester bonds
are less common. Examples (Fig. 1) of these highly symmetric
natural products comprise PF1022A (1), verticilide (3), bassianolide
(4), valinomycin (5), and the enniatins (6e9).1 These regular
cyclodepsipeptides share several characteristic structural features,
among those the occurrence of (R)-hydroxycarboxylic acids and N-
methyl amino acids.
Natural depsipeptides, in particular cyclodepsipeptides, are of
increasing interest for pharmaceutical research because of their
wide range of biological activities (Fig. 1).2 For instance, valino-
mycin (5) a well known potassium-selective ionophore was re-
cently reported to be the most potent agent against severe acute
respiratory-syndrome coronavirus (SARS-CoV).3 Verticilide (3),
a cyclooctadepsipeptide isolated from a Verticilium sp. shows
a strong and selective inhibiting activity on ryanodine binding in
insects.4 Bassianolide (4), another cyclooctadepsipeptide, and the
secticidal, antiproliferative, and cell migration inhibitory activities.5
The 24-membered cyclooctadepsipeptide PF1022A (1), a metabo-
lite of Mycelia sterilia (Rosselinia sp.), originally isolated from leaves
of Camellia japonica has been established as a resistance-breaking
anthelmintic with low toxicity in animals (Fig. 1).6,7 Other cyclo-
depsipeptides act by selective ion transport through cellular
membranes.
Despite the high potential of cyclodepsipeptides for drug dis-
covery, only very limited screening and structureeactivity data
have been reported until now, probably due to the difficult access to
these compounds. The combination of amino acids and bulky
hydroxycarboxylic acids represents a formidable challenge for
a solid-phase synthesis, which is on the other hand a prerequisite
for the preparation of larger compound collections needed for bi-
ological screening. The stepwise assembly of such compounds is
expected to be particularly difficult when N-methyl amino acids are
involved.8 Due to the increased steric hindrance couplings need
more time with the consequence that dioxomorpholine formation
and epimerization can become the predominant side-reactions.9e11
The difficulties, resulting from the stepwise assemblage of
N-methyl amino acids have been impressively demonstrated in
several cyclosporine syntheses.12,13
Abbreviations: ACN, acetonitrile; Boc, tert-butyloxycarbonyl; BOPCl, N,N0-bis(2-
oxo-3-oxazolidinyl)phosphinic chloride; DCM, dichloromethane; DEAD, dieth-
ylazodicarboxylate; DHP, 3,4-dihydro-2H-pyrane; DIC, N,N0-diisopropylcarbodii-
mide; DIEA, diisopropylethylamine; DMAP, 4-dimethylaminopyridine; DMF,
dimethylformamide; DMSO, dimethylsulfoxide; EDCI, 1-(3-Dimethylaminopropyl)-
3-ethylcarbodiimide methiodide; Fmoc, 9-Fluorenyl-methoxycarbonyl; HATU,
N,N,N0,N0-tetramethyl-O-(7-azabenzo-triazol-1-yl)uroniumhexa-fluorophosphate;
HOAt, 1-hydroxy-7-azabenzotriazole; HOBt, 1-hydroxy-benzotriazole; MeOH,
methanol; TEA, triethylamine; TFA, trifluoro acetic acid; THF, tetrahydrofuran; THP,
tetrahydropyranyl; TPP, triphenylphosphane; p-TsOH, para-toluenesulfonic acid.
* Corresponding author. Tel.: þ49 202 439 2654; fax: þ49 202 439 3464; e-mail
address: scherkenbeck@uni-wuppertal.de (J. Scherkenbeck).
Although advanced reagents, such as BMTB and DFET have been
developed and applied for the coupling of sterically hindered
N-methyl amino acids, high-yielding ester bond assemblage on
solid support remains a synthetic problem.14,15 Besides standard
reagents, such as DIC/DMAP alsotriphosgene and hexa-
fluoroacetonides of
a-hydroxycarboxylic acids have been employed
for the construction of depsipeptides.16e18
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.12.026

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S. Luttenberg et al. / Tetrahedron 68 (2012) 2068e2073
2069
H
C
5
R
11
N
O
O
N
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
N
C H
5 11
O
O
O
O
N
O
O
O
N
O
O
O
O
O
O
O
H
C
5
N
N
11
N
O
N
O
O
O
N
N
C H
5
11
R
PF1022A (1):
Verticilide (3)
Bassianolide (4)
R = H
emodepside (2):
N
R =
O
O
R\S1
O
O
O
O
O
N
O
O
O
N
N
O
N
R\S2
O
O
O
O
N
N
O
O
O
R\S3
N
O
O
O
O
O
O
N
N
O
O
O
O
Valinomycine (5)
Fig. 1. Naturally occurring cyclodepsipeptides.
Usually, the ester bonds are either assembled beforehand in
solution and only the peptide bonds are formed on the resin or the
hydroxycarboxylic acids are introduced without any protection.19,20
Only a few procedures have been published for stepwise solid-
phase depsipeptide syntheses, the most impressive one comprises
a twenty-four step total synthesis of the cyclododecadepsipeptide
valinomycine (5), including the on-resin formation of six ester
bonds.21e23 However, no stepwise solid-phase syntheses of dep-
sipeptides containing N-methyl amino acids have been accom-
plished until now. In this paper we wish to report a solid-phase total
synthesis of the cyclooctadepsipeptide PF1022A in which all ester-
and amide-bonds are formed on the support.
Silyl ethers and acetyl groups have been employed in solid-
phase syntheses as protecting groups for the OH-function of hy-
droxy acids.24 While acetyl groups are cleaved under strong basic
conditions, bearing again the risk of racemization, silyl ethers ap-
pear generally unsuited for the coupling of N-methyl amino acids
and hydroxy acids due to the sterical shielding of the bulky silyl
residue. For these reasons we chose the THP-protecting group,
which is cleaved under mild acidic conditions, compatible even
with the Boc-protecting group.25 Fmoc protected N-methyl leucine
and THP protected hydroxy acids were prepared according to
standard literature procedures (Scheme 1).26e30
2.2. Solid-phase synthesis of PF1022A
2. Results and discussion
First we used the Kaiser-oxime resin, which we had already
applied successfully in the course of a didepsipeptide segment
synthesis of PF1022A (1) and emodepside (2). However, in the
considerably longer stepwise synthesis of PF1022A, deletion- and
failure-sequences accumulated, accompanied by premature cleav-
ages from the resin, which resulted in an inseparable mixture of
products after six or seven couplings.
In a second attempt we chose the Wang-resin, being well aware
of the potential problems arising from the basic cleavage conditions
of the Fmoc-group. Since the strongly solvating DMF has been
shown to be an unfavorable solvent in critical couplings with re-
spect to yield, diketopiperazine formation and degree of racemi-
zation we used the less solvating THF, in which all reagents were
soluble and the Wang-resin still showed good swelling proper-
ties.14,31 After each coupling step a small portion of the resin was
cleaved and the product analyzed by HPLC-MS. The results of our
optimization experiments are summarized in Table 1.
2.1. Protecting group strategy and preparation of building
blocks
In principle, the most frequently used Fmoc- and Boc-protecting
group strategies should be applicable also for the solid-phase
synthesis of depsipeptides. However, both of them have their
own problems when ester bonds are involved. The basic conditions
needed for the removal of the Fmoc-group may cause a partial ester
cleavage, diketopiperazine formation and enhanced racemization
of the chiral hydroxy acids. On the other hand, Fmoc-protecting
schemes are well established for standard supports, such as the
Wang-resin and can be easily adapted on a peptide synthesizer.
Diketopiperazine formation is drastically reduced or even sup-
pressed under the strong acidic cleavage conditions used for the
cleavage of Boc protecting groups. However, activated ester bonds
as those from PhLac for instance, may be affected, too. Additionally,
the resins compatible with Boc-protecting, mainly the Kaiser-
oxime and the PAM-resin are significantly more expensive and
have lower loadings compared to Wang-resin.
As expected, the coupling-yields were strongly dependent on
the residue used as the first building block, on the coupling re-
agents and conditions. THP-PhLac 18, which was used as the
anchoring-residue due to its lower tendency to form

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2070
(a)
O
b
a
c
OH
FmocN
11
FmocN
Me
FmocHN
COOH
O
O
10
12
O
O
O
d
f
(b)
O
OH
OTHP
O
O
OTHP
OH
13
14
15
O
O
e
OH
OH
OTHP
OH
(c)
NH2
16
OH
17
18
Scheme 1. Preparation of Fmoc-NMe-leucine (a), THP-Lac (b) and THP-PhLac (c). Reagents and conditions: (a) paraformaldehyde, p-TsOH, toluene, 112 ꢀC, 24 h, 100%; (b) AlCl3,
Et3SiH, DCM, rt, 1 h, 63%; (c) DHP, p-TsOH, toluene, 50 ꢀC, 3 h, 93%; (d) 1 M THF/LiOH (5:3, aq), 0 ꢀC, 5 h, 85%; (e) 40% NaNO2 (aq), H2SO4, 2 h 0 ꢀC, 16 h rt, then add 40% NaNO2 in
H2SO4, 6 h 0 ꢀC, then 16 h rt, 62%; (f) DHP, p-TsOH, pyridine, DCM, rt, 16 h, 100%.
Table 1
PF1022A solid phase-synthesisdoptimization of coupling-steps 1e4
Step (entry)
Building block (3.0 equiv)
THP-PhLac
Product (bond formed)
No. of couplings
Conditions (equiv)
Yield (%)
1a
1b
1c
1d
2a
2b
2c
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
19 (ester)
3
2
2
3
3
2
2
2
2
2
2
1
2
2
1
1
2
DIC (3.0), DMAP (0.1)
DIC (3.0), DMAP (3.0), HOBt (3.0)
EDCI (3.0), DMAP (0.1)
HATU (2.0), DIEA (3.0)
DIC (3.0), DMAP (0.1)
DIC (3.0), DMAP (3.0), HOBt (3.0)
EDCI (3.0), DMAP (0.1)
HATU (2.0), DIEA (3.0)
DIC (3.0), DMAP (0.1)
DIC (3.0), DMAP (3.0), HOBt (3.0)
EDCI (3.0), DMAP (0.1)
15 acid chloride (3.0), DMAP (1.0)
DIC (3.0), DMAP (0.1)
DIC (3.0), DMAP (3.0), HOBt (3.0)
Acid chloride (3.0), DMAP (1.0)
(Fmoc-NMe-Leu)2O (3.0 equiv), DMAP (3.0 equiv)
TPP (3.0 equiv), DEAD (3.0 equiv)
62a
92a
17a
36a
91b
93b
44b
77a
54a
48a
19a
34a
51b
77b
5b
Fmoc-NMe-Leu
20 (ester)
THP-Lac
21 (amide)
Fmoc-NMe-Leu
22 (ester)
38b
51b
a
Gravimetric determination of the product after deprotection and cleavage from the resin.
UV-determination after Fmoc removal. Yields calculated on a loading of 1.07 mmol/g resin. THF was used as solvent in all experiments.
b
dioxomorpholines compared to THP-Lac 15, could be coupled to the
resin in over 90% yield with the reagent mixture DIC/DMAP/HOBt
(entry 1b, Table 1) in two coupling cycles. Without HOBt the re-
activity of the reagent system was distinctly reduced and even after
three couplings the yield was only 62%. Remarkably, EDCI failed
completely and even HATU, which has been used quite frequently
as reagent for ester formation gave only a moderate coupling yield
in the anchoring step.32,33 In the first chain-extension reaction an
ester bond between the anchored PhLac and Fmoc-NMe-Leu had to
be established. Best yields (93%) were obtained again with DIC/
DMAP/HOBt after two coupling cycles.
for this step to a content lower than 10e15% in the cleavage solution.
Noteworthy, in all following Fmoc-group removals dixomorpholine
formation was found between only 1% and 5% at maximum.
Several methods were tested for the coupling of THP-Lac 15 to
resin-bound NMe-Leu (20/21), including THP-Lac acid chloride
(entry 3e, Table 1 and Scheme 2). While this method gave only low
yields largely due to insufficient coupling, epimerization, and resin-
cleavages, good results for the amide bond formation were ob-
tained with HATU and the DIC/DMAP/HOBt reagent. Since the
carbodiimides are known for their risk of racemization, HATU was
used for all subsequent amide forming couplings.
The next step, removal of the Fmoc-group from the didepsipep-
tide segment 20, is in particular prone to the formation of dioxo-
morpholines. Extensive studies in the literature demonstrate the
critical role of the first and second amino acid as well as the coupling
conditions. Sterically demanding amino acids (Val, Leu, Phe) in the
first position and N-methyl amino acids in the second position,
which exactly complies with the PF1022A situation, generally fa-
cilitate piperazinedione formation.34e36 Even after careful optimi-
zations, we were not able to reduce the dioxomorpholine formation
The reaction between resin-bound Lac and NMeLeu (21/22)
turned out to be more difficult than the previous ester formations
between the Wang-resin and PhLac (18/19) on the one side and
PhLac/NMeLeu (19/20) on the other side (Scheme 2). Thus, in
addition to DIC, further procedures frequently used for the forma-
tion of difficult ester bonds, such as the highly reactive Fmoc-NMe-
Leu acid chloride, the Yamaguchi dichlorobenzoate active ester
(entry 4d, Table 1) and the Mitsunobo (entry 4e, Table 1) coupling
with (S)-lactic acid were studied.37,38 Both the amino acid chloride

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2071
Scheme 2. Solid-phase synthesis of PF1022A. Reagents and conditions: (a) DIC, HOBt, DMAP, THF, rt, 16 h; (b) p-TsOH, DCM/MeOH 97:3, rt, 2ꢁ1 h; (c) Fmoc-NMe-Leu 12, DIC, HOBt,
DMAP, THF, rt, 16 h; (d) 25% piperidine/THF, rt, 30 min; (e) THP-Lac 15, HATU, DIEA, THF, rt, 16 h; (f) THP-PhLac 18, HATU, DIEA, THF, rt, 16 h; (g) 50% TFA/DCM, rt, 1.5 h; (h) BOPCl,
DIEA, DCM, rt, 48 h.
and the Yamaguchi method afforded insufficient yields while the
Mitsunobo reaction gave somewhat higher yields. However, the
DIC/DMAP/HOBt reagent system remained superior also for this
coupling step.
respect to biological and spectroscopical data with a natural ref-
erence sample.
3. Summary and conclusion
Standard conditions were used for the removal of the Fmoc-
protecting group (THF, 25% piperidine, 30 min). The THP-
protecting groups were removed quantitatively with diluted p-
TsOH (5 mg/mL in DCM/MeOH 97:3, 2ꢁ1 h) according to the
procedure published by Kuisle.23 Due to the symmetry of
PF1022A the remaining four chain-elongation reactions are sim-
ple repeats of the first couplings. Thus, no more optimizations
were conducted. The yield of the linear octadepsipeptide 25 after
removal of the N-terminal Fmoc-group and cleavage from the
resin (50% TFA in DCM, 1.5 h, quantitative) was 16%, which cor-
responds to an average yield of 89% for each of the sixteen steps
on solid support (Scheme 2).
We have developed a methodology for the stepwise coupling of
N-methyl amino acids and hydroxycarboxylic acids on solid sup-
port, which allows the preparation of the anthelmintic cyclo-
octadepsipeptide PF1022A (1) in an overall yield in a range of
13e16% (89% average yield for each step). The synthesis comprises
seventeen steps, sixteen of which are performed on solid support
followed by a solution macrocyclization. Albeit there is still room
for improvements in particular in the amide-bond forming steps,
our standard conditions allow an easy adaption to an automated
synthesizer as
a prerequisite for the preparation of cyclo-
depsipeptide screening-libraries.
The macrocyclization reaction, performed under high-dilution
conditions in DCM with BOPCl as coupling reagent, afforded
PF1022A in almost quantitative yield, regardless of whether the
purified or the crude precursor 25 was employed. We found it very
convenient to work with the crude octadepsipeptide because this
avoided a costly and time-consuming preparative HPLC purifica-
tion. Furthermore, PF1022A can be easily separated from non-
cyclized by-products by flash-chromatography with silica gel due
to its considerably higher lipophilicity. The unusual high macro-
cyclization yield can be attributed to a cis-amide bond between
a leucine and lactic acid, which brings the N- and C-terminus in
close proximity and thus facilitates the cylization reaction.39
PF1022A (1) obtained by synthesis was completely identical with
4. Experimental
4.1. General
The starting material (D)-phenyllactic acid, and was either pur-
chased or prepared by standard literature procedures.28 All re-
actions except the saponification were performed in dried solvents.
Dichloromethane and the reagents DIEA and piperidine were
refluxed for 1 h over calcium hydride and distilled. THF was refluxed
for several hours over LiAlH4 and then distilled and toluene was
dried by filtration through basic alumina. The instrumentation used
was as follows: 1H NMR and 13C NMR spectra were recorded at 25 ꢀC

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S. Luttenberg et al. / Tetrahedron 68 (2012) 2068e2073
2072
on a Bruker Avance 400 (400.13 MHz for 1H NMR, 100.62 MHz for
13C NMR) or Bruker Avance III 600 (600.13 MHz for 1H NMR,
150.90 MHz for 13C NMR). IR-spectra were measured on a Nicolet
PROTEGE 460 Spectrometer E.S.P. HPLC-MS was performed on
a Varian 500 Ion-Trap LC-ESI-MS system or on a Bruker MicroTOF
(column: 5 mm RP18) with ACN/H2O gradient 10/90 to 95/5. High-
resolution MS were measured on a Bruker MicroTOF. Preparative
low pressure chromatography (LPLC) was performed using silica gel
THPeether 14 (16.84 g, 89.47 mmol, 93%) as a colorless liquid. IR
(film): 1754 cmꢂ1; 1H NMR (400 MHz, CDCl3):
d
¼1.34 (d, J¼7.0 Hz,
1H, CbH3), 1.39 (d, J¼7.0 Hz, 2H, CbH3), 1.49 (m, 2H, CH2, THPeH3e5),
1.64 (m, 2H, CH2, THPeH3e5), 1.79 (m, 2H, CH2, THPeH3e5), 3.39 and
3.77 (2m, 2H, CH2, THPeH6), 3.68 (s, 3H, OCH3), 4.15 and 4.37 (2 q,
J¼7.0 Hz, 1H, CaH), 4.64 (m, 1H, CH, THPeH2) ppm; 13C NMR
ꢀ
ꢀ
(100 MHz, CDCl3):
d
¼18.7, 19.1, 25.3, 30.4, 51.7, 62.3, 69.9, 97.5,
173.5 ppm; MS (ESI): m/z (%)¼206 (4), 211 ([MþNa]þ, 100); HRMS
(ESI): MþþNa, found 211.0941; C9H16NaO4 requires 211.0946.
€
60
m
m (230e400 mesh, MachereyeNagel), Buchi pump. TLC: Silica
gel 60 F254 (Merck). Melting points (mp) were determined with
a Buechi 535 melting point apparatus and are uncorrected. The
progress of the solid phase reactions was detected either by the
bromophenolblue color test, determination of resin loading by UV
determination of the Fmoc-group cleavage product or by test
cleavages and HPLC-MS analysis.40
4.2.4. (2R)-2-(Tetrahydro-2H-pyran-2-yloxy)-propanoic acid (15). A
solution of methyl ester 14 (2.07 g, 11.00 mmol) was stirred in 1 M
THF/LiOH (5:3 aq, 50 mL) for 5 h at 0 ꢀC. Then the reaction mixture
was acidified to pH 3 with 10% hydrochloric acid and extracted four
times with ethyl acetate. The combined extracts were washed with
a saturated NaCl solution (1ꢁ), dried with MgSO4, and filtered. A
clear liquid (1.64 g, 9.40 mmol, 85%) of THP protected lactic acid 15
was obtained after evaporation of the solvent and drying in high
vacuum. Further purification was not necessary. IR (film): 1735,
4.2. Synthesis of the building blocks in solution
4.2.1. (S)-4-Isobutyl-5-oxo-oxazolidine-3-carboxylic acid 9H-fluo-
ren-9-yl methyl ester (11). A mixture containing paraformaldehyde
3103 cmꢂ1
;
1H NMR (400 MHz, CDCl3):
d
¼1.45 (d, J¼7.0 Hz, 1H,
(45.28 g, 1.49 mol), Fmoc-(L)-leucine (80.00 g, 0.22 mol), and p-
CbH3), 1.50 (d, J¼7.0 Hz, 2H, CbH3), 1.55 (m, 2H, CH2, THPeH3e5), 1.70
(m, 2H, CH2, THPeH3e5),1.83 (m, 2H, CH2, THPeH3e5), 3.52 and 3.86
(2m, 2H, CH2, THPeH6), 4.25 and 4.43 (2q, J¼7.0 Hz, 1H, CaH), 4.65
and 4.73 (2m, 1H, CH, THPeH2), 9.76 (s, 1H, CO2H) ppm; 13C NMR
TsOH (4.52 g, 23.53 mmol) was refluxed in toluene (400 mL) for
24 h in a dean stark apparatus until water separation was complete.
The reaction was cooled to room temperature, washed with satu-
rated NaHCO3 solution, dried with Na2SO4, and concentrated in
vacuo to yield oxazolidine 11 (81.89 g, 0.22 mol, 100%) as a white
solid, which was used without further purification. Mp 68e70 ꢀC;
(100 MHz, CDCl3):
d
¼19.1, 19.6, 25.3, 30.3, 62.8, 70.1, 98.0,
178.1 ppm; MS (ESI): m/z (%)¼89 (15), 101 (11), 173 ([MꢂH]ꢂ, 100),
174 (11); HRMS (ESI): MþþNa, found 197.0785; C8H14NaO4 requires
197.0790.
IR (KBr): 1721, 1799 cmꢂ1
6H, CdH3-Leu), 1.33e1.87 (m, 3H, CbH2-Leu, CgH-Leu), 4.25 (m, 2H,
CH2eFmoc), 4.70 (m, 2H, CHeFmoc, CaH-Leu), 5.08 (m, 2H,
CH2eoxazolidinone), 7.21e7.79 (m, 8H, AreH) ppm; 13C NMR
;
1H NMR (400 MHz, CDCl3):
d
¼0.84 (m,
4.2.5. (R)-3-Phenyl-2-(Tetrahydro-2H-pyran-2-yloxy)-propanoic
acid (18). To a solution of ( )-PhLac 17 (6.71 g, 40.38 mmol) in DCM
D
(100 MHz, CDCl3):
d
¼6.4, 21.0, 23.1, 24.7, 37.2, 47.2, 56.4, 67.6, 120.0,
(200 mL), 1 equiv of 3,4-dihydro-2H-pyrane (3.43 g, 40.38 mmol)
and a solution of p-TsOH (605.16 mg, 3.15 mmol) and pyridine
(251.65 mg, 3.15 mmol) in DCM (10 mL) were added. The mixture
was stirred for 16 h at ambient temperature. After that the mixture
was evaporated under reduced pressure and the residue was dis-
solved in diethylether (100 mL). The ether solution was washed
with brine, dried over Na2SO4, filtered, and evaporated. The crude
product was dried in vacuo and was used in solid phase-synthesis
without further purification. The reaction provides the product 18
as white solid with a yield of 100% (10.11 g, 40.21 mmol). IR (film):
124.7, 124.8, 125.0, 125.1, 127.1, 127.1, 127.7, 141.4, 143.8, 143.9, 156.5,
157.1, 177.6 ppm; MS (ESI): m/z¼144 (7), 179 (100), 366 ([MþH]þ,
21), 388 (26); HRMS (ESI): MþþNa, found 388.1519; C22H23NNaO4
requires 388.1519; Anal. Calcd for C22H23NO4: C, 72.31; H, 6.34; N,
3.83; found: C, 72.36; H, 6.47; N, 3.86.
4.2.2. (S)-2-[(9H-Fluoren-9-yl-methoxycarbonyl)-methyl-amino]-4-
methyl-pentanoic acid (12). Triethylsilane (36.61 g, 0.31 mol) was
added to a suspension of compound 11 (56.94 g, 0.156 mol) and
AlCl3 (41.98 g, 0.31 mol) in DCM (1000 mL). After stirring for 60 min
at room temperature the mixture was washed with 1 M HCl (3ꢁ,
400 mL each), dried with Na2SO4, concentrated in vacuo, and
crystallized (cyclohexane/DCM 8:2) to provide N-Fmoc-N-methyl
leucine 12 (35.94 g, 0.098 mol, 63%) as a white crystalline solid. Mp
1739, 3437 cmꢂ1
;
1H NMR (400 MHz, CDCl3):
d
¼1.48 (m, 2H, CH2,
THPeH3e5), 1.60 (m, 2H, CH2, THPeH3e5), 1.67 (m, 2H, CH2,
THPeH3e5), 3.00 (m, 1H, CbH2), 3.06, 3.27, 3.44, and 3.95 (4m, 2H,
CH2, THPeH6), 3.19 (dd, J¼4.1, 9.7 Hz, 1H, CbH2), 4.19 and 4.79 (2m,
1H, CH, THPeH2), 4.27 and 4.52 (2 dd, J¼3.6, 5.6 Hz, 1H, CaH),
7.22e7.31 (m, 5H, AreH), 10.00 (s, 1H, CO2H) ppm; 13C NMR
112e114 ꢀC (lit.30 113e116 ꢀC); ½a 2D5
ꢃ
ꢂ19.5 (c 1.0, DMF); IR (KBr):
1655, 1754, 3427 cmꢂ1
;
1H NMR (400 MHz, CDCl3):
d
¼0.94e1.03
(100 MHz, CDCl3):
d
¼20.4, 24.8, 32.0, 38.5, 61.0, 74.2, 96.5, 126.7,
(m, 6H, CH3-Leu), 1.47e1.79 (m, 3H, CbH2-Leu, CgH-Leu), 2.91 (s, 3H,
NCH3), 4.24 (m, 1H, CHeFmoc), 4.46 (m, 2H, CH2eFmoc), 4.62 and
4.98 (2m, 1H, CaH-Leu), 7.28e7.44 (m, 4H, AreH), 7.57 (m, 2H,
128.2, 128.3, 129.5, 129.8, 137.0, 176.5 ppm; MS (ESI): m/z (%)¼184
(17), 251 ([MþH]þ, 4), 268 ([MþNH4]þ, 100); HRMS (ESI): MþþNa,
found 273.1097; C14H18NaO4 requires: 273.1103.
AreH), 7.79 (m, 2H, AreH) ppm; 13C NMR (100 MHz, CDCl3):
d
¼6.8,
21.1, 37.2, 24.6, 30.2, 47.2, 56.3, 67.6, 119.9, 124.7, 124.8, 125.0, 125.1,
127.1, 127.1, 127.6, 141.3, 143.8, 143.9, 156.4, 157.1, 177.1 ppm; MS
(ESI): m/z¼146 (7), 179 (7), 368 ([MþH]þ, 100), 385 (16); HRMS
(ESI): MþþNa, found 390.1681; C22H25NNaO4 requires 390.1676;
Anal. Calcd for C22H25NO4: C, 71.91; H, 6.86; N, 3.81; found: C, 71.61;
H, 6.96; N, 3.82.
4.3. Solid-phase synthesis
4.3.1. Coupling of the first residue. A solution of PhLaceOTHP 18
(3 equiv), DIC (3 equiv), HOBt (3 equiv), and DMAP (3 equiv) in THF
(1.0 mL/100 mg resin) was added to Wang-resin (500 mg,
1.07 mmol/g loading level), swollen in THF (1.0 mL/100 mg resin,
30 min, then solvent is removed), and stirred for 16 h at room
temperature. The resin was washed three times (3 min each) with
DCM (10 mL), acetone (10 mL), DCM (10 mL), and dried in high
vacuum until constant mass was reached. The whole procedure was
repeated once.
4.2.3. (2R)-2-(Tetrahydro-2H-pyran-2-yloxy)-propanoic acid methyl
ester (14). Dihydropyran (8.98 g,105.66 mmol) was dropped within
1 h at 50 ꢀC to a solution of methyl (
D)-lactate (10.00 g, 96.06 mmol)
and p-TsOH (165.41 mg, 0.96 mmol) in toluene (100 mL). After
stirring for 2 h at this temperature the solution was washed with
saturated NaHCO3 solution, dried with Na2SO4, filtrated, and
evaporated. Kugelrohr distillation (8.9ꢁ10ꢂ2 mbar, 70 ꢀC) provided
4.3.2. General procedure for the cleavage of the THP-protecting
group. A suspension of the resin (500 mg) and 10 mL of p-TsOH

€
S. Luttenberg et al. / Tetrahedron 68 (2012) 2068e2073
2073
in DCM/MeOH 97:3 (5 mg/mL) was agitated for 1 h at room tem-
perature. After that time the solvent was removed and the resin
was washed with the cleavage solution one time (3 min). The whole
procedure was repeated once. The resin was filtered off, washed
three times (3 min each) with DCM (10 mL), acetone (10 mL), THF
(10 mL), and dried in high vacuum.
20.8, 20.8, 20.9, 21.0, 23.0, 23.1, 23.2, 23.2, 23.4, 23.9, 24.2, 24.3,
24.3, 24.4, 28.9, 30.1, 30.1, 30.2, 30.3, 30.6, 35.7, 36.3, 36.5, 36.6, 36.7,
36.7, 36.8, 37.1, 37.3, 53.0, 53.2, 53.3, 56.4, 66.7, 67.5, 67.7, 70.1, 70.9,
71.0, 126.7, 126.7, 126.8, 128.1, 128.2, 128.2, 129.5, 135.1, 135.2, 135.9,
169.0, 169.0, 169.3, 169.5, 169.7, 170.2, 170.3, 170.7, 170.9 ppm; MS
(ESI): m/z¼475 (4), 950 ([MþH]þ, 1), 966 ([MþNH4]þ, 100), 971
(23); HRMS (ESI): MþþNa, found 971.5351; C52H76N4NaO12 re-
quires 971.5352.
4.3.3. General procedure for ester couplings. A solution of N-Fmoc-
N-methyl-leucine 12 (3 equiv), DIC (3 equiv), HOBt (3 equiv), and
DMAP (3 equiv) in THF (1 mL/100 mg resin) was dropped at room
temperature to the swollen resin (in THF, 1.0 mL/100 mg resin,
30 min, then the solvent was removed). The reaction was stirred for
16 h at room temperature. Then the resin was filtered off and
washed three times (3 min each) with DCM (10 mL), acetone
(10 mL), DCM (10 mL), and dried in high vacuum until constant
mass was reached. The whole coupling and washing procedures
were repeated once.
Acknowledgements
Generous financial support by Bayer Animal Health GmbH is
gratefully acknowledged.
References and notes
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2. Lemmens-Gruber, R.; Kamyar, M. R.; Dornetshuber, R. Curr. Med. Chem. 2009, 16,
1122e1137.
4.3.4. General procedure for the cleavage of the Fmoc-group. A
suspension of the swollen resin (500 mg) and piperidine in THF
(25%, 10.0 mL) was shaken for 30 min at room temperature. Then
the resin was filtered off and washed three times (3 min each) with
DCM (10 mL), acetone (10 mL), DCM (10 mL), and dried in high
vacuum.
3. Cheng, Y.-Q. ChemBioChem 2006, 7, 471e477.
4. Monma, S.; Sunazuka, T.; Nagai, K.; Arai, T.; Shiomi, K.; Matsui, R.; Omura, S.
Org. Lett. 2006, 8, 5601e5604.
ꢀ
5. Xu, Y.; Wijeratne, E. M. K.; Espinosa-Artiles, P.; Gunatilaka, A. A. L.; Molnar, I.
ChemBioChem 2009, 10, 345e354.
6. Scherkenbeck, J.; Jeschke, P.; Harder, A. Curr. Top. Med. Chem. 2002, 2, 759e777.
7. Jeschke, P.; Iinuma, K.; Harder, A.; Schindler, M.; Murakami, T. Parasitol. Res.
2005, 97, 11e16.
8. Coin, I. J. Pept. Sci. 2010, 16, 223e230.
4.3.5. General procedure for amide couplings. A solution of the
hydroxycarboxylic acid (15 or 18) (3 equiv), HATU (2 equiv), and
DIEA (3 equiv) in THF (1.0 mL/100 mg resin) was added to the
swollen resin (in THF, 1.0 mL/100 mg resin, 30 min, then the solvent
was removed) and shaken for 16 h at room temperature. The resin
was filtered off and washed three times (3 min each) with DCM
(10 mL), acetone (10 mL), DCM (10 mL), and dried in high vacuum.
The whole coupling and washing procedures were repeated once.
9. Teixido, M.; Albericio, F.; Giralt, E. J. Pept. Res. 2005, 65, 153e166.
10. Koshla, M. C.; Smeby, R. R.; Bumpus, F. M. J. Am. Chem. Soc. 1972, 94, 4721e4724.
11. Coin, I.; Beerbaum, M.; Schmieder, P.; Bienert, M.; Beyermann, M. Org. Lett.
2008, 10, 3857e3860.
12. Koo, S. Y.; Wenger, R. M. Helv. Chim. Acta 1997, 80, 695e705.
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1998, 63, 5734e5735.
14. Jastrzabek, K. G.; Subiros-Funosas, R.; Albericio, F.; Kolesinska, B.; Kaminski, Z. J.
J. Org. Chem. 2011, 76, 4506e4513.
15. Wischnat, R.; Rudolph, J.; Hanke, R.; Kaese, R.; May, A.; Theis, H.; Zuther, U.
Tetrahedron Lett. 2003, 44, 4393e4394.
16. Thern, B.; Rudolph, J.; Jung, G. Angew. Chem., Int. Ed. 2002, 41, 2307e2309.
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19. Lee, B. H. Tetrahedron Lett. 1997, 38, 757e760.
4.3.6. General procedure for the cleavage from the resin. The resin
(500 mg) was suspended in a solution (10 mL) consisting of TFA/
DCM 1:1 and agitated for 1.5 h at room temperature. The resin was
filtered of and washed with DCM (10ꢁ, 10 mL each). The combined
DCM rinses were washed with brine, dried with Na2SO4, and con-
centrated in vacuo. Usually, the crude product was immediately
used for the macrocyclization reaction without further purification.
20. Miyashita, M.; Nakamori, T.; Akamatsu, M.; Murai, T.; Miyagawa, H.; Ueno, T.
Pept. Chem. 1996, 34, 137e140.
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16762e16771.
~
ꢀ
23. Kuisle, O.; Quinoa, E.; Riguera, R. J. Org. Chem. 1999, 64, 8063e8075.
24. Davies, J. S.; Howe, J.; Jayatilake, J.; Riley, T. Lett. Pept. Sci. 1997, 441e445.
4.4. Macrocyclization in solution
~
ꢀ
25. Kuisle, O.; Quinoa, E.; Riguera, R. Tetrahedron Lett. 1999, 40, 1203e1206.
€
26. Zhang, S.; Govender, T.; Norstrom, T.; Arvidsson, P. I. J. Org. Chem. 2005, 70,
Cyclooctadepsipeptide PF1022A (1). BOPCl (62.0 mg, 0.24 mmol)
was added to a solution of the crude linear octadepsipeptide 25
6918e6920.
27. Donners, M. P. J.; Hersmis, M. C.; Custers, J. P. A.; Meuldijk, J.; Vekemans, J. A. J.
M.; Hulshof, L. A. Org. Process Res. Dev. 2002, 6, 606e610.
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259e264.
29. Zubia, A.; Mendoza, L.; Vivanco, S.; Aldaba, E.; Carrascal, T.; Lecea, B.; Arrieta,
A.; Zimmerman, T.; Vidal-Vanaclocha, F.; Cossío, F. P. Angew. Chem., Int. Ed.
2005, 44, 2903e2907.
30. Kim, H. O.; Olsen, R. K.; Choi, O. S. J. Org. Chem. 1987, 52, 4531e4536.
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32. Pon, R. T.; Yu, S.; Sanghvi, Y. S. Bioconjugate Chem. 1999, 10, 1051e1057.
33. Ingram, A.; Stokes, R. J.; Redden, J.; Gibson, K.; Moore, B.; Faulds, K.; Graham, D.
Anal. Chem. 2007, 79, 8578e8583.
34. Nidhino, N.; Xu, M.; Mihara, H.; Fujimoto, T. Bull. Chem. Soc. Jpn. 1992, 65,
991e994.
35. Smith, R. A.; Bobko, M. A.; Lee, W. Bioorg. Med. Chem. Lett. 1998, 8, 2369e2374.
36. Fischer, P. M. J. Pept. Sci. 2003, 9, 9e35.
37. Lengweiler, U. D.; Fritz, M. G.; Seebach, D. Helv. Chim. Acta 1996, 79, 670e701.
38. Kobayashi, M.; Nanba, T.; Toyama, T.; Saito, A. Annu. Rep. Sankyo Res. Lab. 1994,
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8459e8470.
(196.80 mg content: 16%, 31.49 mg, 32.56 mmol) and DIEA (87 ml,
0.51 mmol) in DCM (200 mL) at 0 ꢀC. The reaction was allowed to
warm up to room temperature and stirred for 24 h. Then the same
amounts of BOPCl (62.0 mg, 0.24 mmol) and DIEA (87
ml,
0.51 mmol) were added and stirring was continued for another
24 h. After that time the mixture was washed twice with saturated
NaHCO3 solution, dried with Na2SO4, and concentrated in vacuo.
The crude product was purified by flash-chromatography (toluene/
isopropanol 20:1) to afford PF1022A 1 (25.0 mg, 26.34 mmol, 81%,
based on the content of the raw linear octadepsipeptide 25) as
a white solid. IR (KBr): 1660, 1739 cmꢂ1; 1H NMR (400 MHz, DMSO-
d6):
d
¼0.68e0.99 (m, 21H, CdH3-Leu, CbH3-Lac), 1.06e1.78 (m, 14H,
CbH2-Leu, CgH-Leu),1.28 (d, J¼6.7 Hz, 3H, CbH3-Lac), 2.68, 2.77, 2.80,
2.85, and 2.91 (5s, 12H, NCH3), 3.08 (m, 2H, CbH2-PhLac), 4.41, 5.11,
and 5.22 (3m, 4H, CaH-Leu), 5.01, 5.32, and 5.42 (3m, 2H, CaH-Lac),
5.52, 5.68, and 5.71 (3m, 2H, CaH-PhLac), 7.24e7.31 (m, 10H, AreH)
40. Winter, M.; Warrass, R. In Combinatorial Chemistry; Fenniri, H., Ed.; Oxford
University: Oxford, 2000; pp 117e138.
ppm; 13C NMR (100 MHz, DMSO-d6):
d¼15.5, 16.3, 16.8, 20.4, 20.6,