Improved Strategy for the Synthesis of the Anticancer Agent Culicinin D

Article abstract of DOI:10.1002/ejoc.201500872

The anticancer peptaibol culicinin D was synthesised via a newly developed pathway. This route included an improved attachment of a C-terminal amino acid alcohol building block to 2-chlorotrityl chloride resin. A model system utilising readily available Fmoc-alaninol as the substitute for the unusual APAE building block was developed to investigate the resin-loading by N-anchoring of the first C-terminal residue and an intramolecular O-N acyl shift. The use of both Fmoc SPPS and the crucial O-N acyl transfer afforded a C-terminal alcohol that enabled the synthesis of a library of five related peptaibols. This model system was then applied to the synthesis of culicinin D. The C-terminal APAE building block was anchored to 2-chlorotrityl chloride resin in 67 % loading yield using the optimised conditions, and culicinin D (6.31 mg, 4 %) was prepared by SPPS prior to peptide cleavage and O-N acyl shift. This synthetic strategy was also used to prepare a diastereomer of culicinin D containing the unnatural (S)-AHMOD amino acid. The anticancer agent Culicinin D, active against breast tumor cells, was synthesised on the solid phase. The resin loading was improved by attachment of first residue to solid-phase via the amine rather than the alcohol. After SPPS and cleavage, the peptide alcohol was formed via an intramolecular O-N transfer reaction. Successful synthesis of several culicinin D analogues demonstrated the feasibility of this new synthetic strategy.

Full text of DOI:10.1002/ejoc.201500872

FULL PAPER
DOI: 10.1002/ejoc.201500872
Improved Strategy for the Synthesis of the Anticancer Agent Culicinin D
Michaela Stach,[a] Andreas J. Weidkamp,[a] Sung-Hyun Yang,[a] Kuo-yuan Hung,[b]
Daniel P. Furkert,[a] Paul W. R. Harris,[a,b] Jeff B. Smaill,[c,d] Adam V. Patterson,[c,d] and
Margaret A. Brimble*[a,b,c]
Keywords: Solid-phase synthesis / Peptides / Medicinal chemistry / Antitumor agents / Acyl transfer reaction
The anticancer peptaibol culicinin D was synthesised via a
newly developed pathway. This route included an improved
attachment of a C-terminal amino acid alcohol building block
to 2-chlorotrityl chloride resin. A model system utilising read-
ily available Fmoc-alaninol as the substitute for the unusual
APAE building block was developed to investigate the resin-
loading by N-anchoring of the first C-terminal residue and
an intramolecular O–N acyl shift. The use of both Fmoc SPPS
and the crucial O–N acyl transfer afforded a C-terminal
alcohol that enabled the synthesis of a library of five related
peptaibols. This model system was then applied to the syn-
thesis of culicinin D. The C-terminal APAE building block
was anchored to 2-chlorotrityl chloride resin in 67% loading
yield using the optimised conditions, and culicinin D
(6.31 mg, 4%) was prepared by SPPS prior to peptide cleav-
age and O–N acyl shift. This synthetic strategy was also used
to prepare a diastereomer of culicinin D containing the un-
natural (S)-AHMOD amino acid.
Culicinin D
1 (Figure 1) is a 10-membered linear
Introduction
peptaibol[4] that was first isolated from the fungus Culicino-
myce clavisporus and exhibits selective inhibitory activity
against PTEN-negative MDA468 breast tumor cells with
an IC50 value of less than 6 nM.[4] Culicinin D (1) not only
consists of the unnatural amino acids Aib and AHMOD
but also contains the non-proteinogenic residues 2-
(2-aminopropylamino)ethanol (APAE), and 2-amino-4-
methyldecanoic acid (AMD) (Figure 1).[4]
Peptaibols are short non-ribosomal peptides containing
five to twenty amino acid residues and are mostly isolated
from fungi in soil.[1] As indicated by the name, peptaibols
usually consist of the non-proteinogenic amino acid amino-
isobutyric acid (Aib) and contain a C-terminal alcohol and
an acylated N-terminus. The presence of non-proteinogenic
amino acids such as the helix promoting, conformationally
constrained Aib, isovaline (Iva) and hydroxyproline (Hyp)
are an important feature of peptaibols.[1a,1b] The more com-
plex unnatural amino acids etnorvaline (α-amino-α-ethyl-n-
pentanoic acid) and 2-amino-6-hydroxy-4-methyl-8-oxo-
decanoic acid (AHMOD) have also been found, although
less frequently.[1a] Notably, peptaibols lack charged and po-
lar amino acids,[1b] and nearly all structures are primarily
helical which has a significant effect on biological ac-
tivity.[1b] Most peptaibols exhibit antibacterial or antifungal
activity due to their ability to permeate lipid bilayers and
form transmembrane pores.[1a,1b,2] In addition, some
peptaibols exhibit cytotoxicity against cancer cells.[3]
The synthesis of culicinin D (1) and its epimer at the C-
6 position of the AHMOD residue was recently reported
by our group.[4b] Upon completion of the synthesis, the
stereochemistry of the hydroxyl group at the C-6 position
of the AHMOD residue was established to be (R) by NMR
analysis. The syntheses of the unnatural Fmoc-protected
building blocks AHMOD, AMD and APAE were reported,
and 1 was synthesised by Fmoc solid phase peptide synthe-
sis (SPPS).[4b] The synthesis of 1 involved some difficult
coupling steps, and the final product was obtained in low
yield mainly due to the insufficient attachment of Fmoc-
protected APAE (Figure 1) to 2-chlorotrityl-functionalised
aminomethyl polystyrene resin via the less reactive hydroxyl
group (23% loading yield).[4b] Furthermore, the coupling
between the two sterically hindered Aib residues was diffi-
cult, and elimination of the β-hydroxyl ketone moiety of
AHMOD was observed upon TFA-mediated peptide cleav-
age, resulting in an overall recovery of less than 1 mg of
culicinin D 1 (Ͻ 0.62% yield).[4b]
[a] School of Chemical Sciences, The University of Auckland,
23 Symonds Street, Auckland 1010, New Zealand
E-mail: m.brimble@auckland.ac.nz
http://brimble.chem.auckland.ac.nz/
[b] School of Biological Sciences, The University of Auckland,
3A Symonds Street, Auckland 1010, New Zealand
[c] Maurice Wilkins Centre for Molecular Biodiscovery,
The University of Auckland,
Private Bag 92019, Auckland 1010, New Zealand
[d] Auckland Cancer Society Centre, School of Medical Sciences,
The University of Auckland,
Multi-milligram amounts of 1 are needed to further vali-
date its biological functions and mandates a substantial im-
provement to the initial chemical synthesis reported. Ad-
ditionally, the synthesis of structurally related analogues of
Private Bag 92019, Auckland 1010, New Zealand
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500872.
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M. A. Brimble et al.
FULL PAPER
Figure 1. Structure of culicinin D and the unnatural amino acid building blocks (R)-AHMOD, AMD and APAE.
1 containing diverse amino acid substitutes of the AHMOD Herein we report the use of O–N acyl transfer for the chal-
building block will pay a way for structure-activity relation- lenging synthesis of the anticancer agent 1 and its ana-
ship studies, thus leading towards the development of more logues.
potent anticancer agents.
The reported synthesis of peptide alcohols based on O–
N acyl transfer showed a substantial increase in the loading
Results and Discussion
of hydroxyl-functionalised amines to trityl resins.[5] Using
this method a fragment of gramicidin A, a fragment of the
Due to the previously discussed challenges associated
antimicrobial peptide trichogin GA IV and octreotide[5] with Fmoc SPPS and the poorly nucleophilic hydroxyl
were synthesised. Intramolecular O–N acyl migration was group,[5,11] a new strategy was implemented based on an-
also used to prepare the following compounds: the pro- choring of the more nucleophilic amine to 2-chlorotrityl
drugs of paclitaxel and other taxoids, the prodrugs of HIV chloride resin followed by O–N acyl transfer in solution to
protease,[6] the 42 amino acid containing β-amyloid peptide generate the desired C-terminal hydroxyl group
(Aβ1–42),[7] β-secretase inhibitors,[8] the prolyl endopeptid- (Scheme 1).[5,11] This strategy involves anchoring of an
ase inhibitor eurystatin A[9] and several cyclic peptides.[10] Fmoc-β-amino alcohol to 2-chlorotrityl chloride resin via
Scheme 1. Strategy for feasibility study using Fmoc-alaninol to improve the loading yield. a) Fmoc-alaninol (3 equiv.), 2% DBU in
anhydr. DMF, room temp., 6–12 h, repeat once; b) Fmoc-βala-OH (6 equiv.), DIC/DMAP (3 equiv./0.3 equiv.), anhydr. DMF/CH2Cl2 (v/
v, 1:1), r.t., 6–12 h, repeat once; c) Fmoc SPPS: i) 40% piperidine in DMF (v/v) (3ϫ 10 min); ii) Fmoc-AA-OH (5 equiv.), HATU
(4.8 equiv.), iPr2NEt (10 equiv.), DMF, 45 min; then Fmoc-AA-OH (5 equiv.), Oxyma (5 equiv.), DIC (10 equiv.), DMF, 45 min; Fmoc-
Aib-OH coupling: Fmoc-Aib-OH (5 equiv.), TFFH (5 equiv.), iPr2NEt (10 equiv.), DMF, 45 min; butyric acid coupling: butyric acid
(5 equiv.), HATU (5 equiv.), iPr2NEt (10 equiv.), DMF/CH2Cl2 (v/v, 1:1), 45 min; then: butyric acid (5 equiv.), Oxyma (5 equiv.), DIC
(10 equiv.), DMF/CH2Cl2 (v/v, 1:1), 45 min; d) CH2Cl2/HFIP (v/v, 4:1), r.t., 30 min; e) 20% piperidine in DMF (v/v); C4 = butanoyl.
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Improved Strategy for the Synthesis of Culicinin D
the amino group upon simultaneous removal of the Fmoc allows the challenging coupling between two Aib amino ac-
group, followed by formation of an ester bond with the ids to be carried out with less difficulty.[4b,15] The coupling
carboxylic acid of the next Fmoc-protected amino acid.
between the two Aib residues in 5c–e proceeded in almost
Elongation of the peptide using Fmoc SPPS and cleavage quantitative yield with the use of TFFH. For the release of
of the isopeptide using 1% TFA enables the intramolecular isopeptides from the solid support a mixture of (1,1,1,3,3,3-
O–N acyl transfer of the isopeptide to give the desired pept- hexafluoropropan-2-ol) (HFIP)/CH2Cl2 (1:4, v/v) was em-
ide alcohol at the C-terminus.[12] In order to demonstrate ployed in place of 1% TFA/CH2Cl2 mixture.
the feasibility of this strategy for the synthesis of 1 and ana-
HFIP is a mild and acid-free cleavage reagent that can
logues, N-Fmoc alaninol 2 was used as a simplified substi- prevent the undesired elimination of the β-hydroxyl ketone
tute of the more complex N-Fmoc-protected APAE build- moiety of the AHMOD amino acid, an issue which was
ing block. N-Fmoc-Alaninol (2) was synthesised by NaBH4 previously observed during TFA-mediated peptide cleav-
reduction of the activated N-Fmoc alanine in an organic/ age.[4b] After RP-HPLC purification isopeptides 6a–e were
aqueous medium (Scheme 1).[13] In a simultaneous fashion, subjected to the intramolecular O–N acyl shift reaction
the Fmoc group on 2 was removed and the free amino using 20% piperidine in DMF (v/v). The reaction was con-
group loaded onto the resin upon treatment with a solution veniently monitored by RP-HPLC as isopeptide 6a eluted
of 2% DBU in anhydrous DMF. Fmoc-β-Ala-OH was next earlier at 17.93 min, while the O–N acyl shift product 7a
coupled to the free hydroxyl group on resin 3 in the pres- eluted later at 20.13 min. The reaction proceeded within a
ence of DIC/DMAP to generate functionalised resin 4. couple of minutes, after which all of isopeptide 6a were con-
Pleasingly, the resin loading was determined[14] to be 54– verted into the desired peptide alcohol 7a (Figure 2 and
67% (Table 1) which is 2–3 times higher than that achieved Figure S25 in the Supporting Information). Peptide
using our previously reported method.[4b]
Isopeptides 5a–e were then assembled from resin 4 under purification (Table 1).
alcohols 7a–e were recovered in good yields after RP-HPLC
standard Fmoc SPPS conditions using 5 equiv. of Fmoc-
In contrast to 6a, which was obtained with satisfactory
protected amino acid, 4.8 equiv. of O-(7-azabenzotriazol-1- purity prior to purification, RP-HPLC analysis of crude
yl)-N,N,NЈ,NЈ-tetramethyluronium
hexafluorophosphate isopeptides 6b–e revealed two peaks with different retention
(HATU) and 10 equiv. of iPr2NEt. Fluoro-N,N,NЈ,NЈ- times but identical mass values. Based on our previous ob-
tetramethylformamidinium hexafluoro phosphate, (TFFH) servation we assumed that the peaks with earlier eluting
is a coupling agent which generates acid fluoride in situ and retention time correspond to isopeptide 6b–e, whereas the
Table 1. Loading yields and final yields of compounds synthesised via O–N acyl shift method.
Sequence[a]
Loading of resin 4%[b]
Yield [%], (mg)
HRMS calcd./found[c]
7a
7b
7c
7d
7e
LeuAlaLeuβalaAla-ol
67
65
54
63
63
36, (0.59)
33, (1.04)
43, (2.26)
17, (16.91)
11, (11.35)
444.3186/444.3175
979.6556/979.6569
1021.7025/1021.7042
937.6086/937.6063
989.6399/989.6430
C4ProLeuAlaAlaLeuLeuAlaLeuβalaAla-ol
C4ProLeuAibAibLeuLeuAibLeuβalaAla-ol
C4ProAlaAibAibAlaLeuAibLeuβalaAla-ol
C4ProProAibAibProLeuAibLeuβalaAla-ol
[a] C4 = butanoyl, -ol indicates an alcohol at the C-terminus. [b] Loading yield was determined according to reference 14 and is show as
median value of two experiments. [c] Mass calcd./found for [M + H]+, observed by HRMS positive-ion mode ESI.
Figure 2. RP-HPLC chromatograms of the intramolecular O–N acyl shift in (S)-2-aminopropyl-Leu-Ala-Leu-βalanoate (6a). a) Isopeptide
(S)-2-aminopropyl-Leu-Ala-Leuβ-alanoate (6a) before rearrangement; b) peptide alcohol Leu-Ala-βala-Ala-ol (7a) after rearrangement;
c) co-elution of isopeptide Leu-Ala-βala-Ala-NH2 (6a) and peptide alcohol Leu-Ala-βala-Ala-ol (7a).
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M. A. Brimble et al.
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peaks with later retention time were the desired peptide (v/v), and measurement of the absorbance of the Fmoc-
alcohols 7b–e. The two peaks were separated by RP-HPLC piperidine adduct[14] showed an acceptable loading of 65%,
purification and subjected to the Kaiser test which con- which is a substantial improvement to that obtained in our
firmed the presence of primary amine as expected for the previous synthesis of culicinin D (1) (23% loading).[4b]
early eluting isopeptides 6b–e. In contrast, no free amine
To assemble the culicinin D framework, Fmoc-Leu-OH,
was present as predicted for the later eluting peptaibols 7b– Fmoc-Aib-OH and Fmoc-Leu-OH were incorporated using
e. We assume that in the case of isopeptides 6b–e some a double coupling strategy employing HATU/iPr2NEt fol-
premature rearrangement to peptaibol 7b–e had taken place lowed by Oxyma/DIC as the coupling reagents. Further-
during workup and handling procedures. This was not more, to ensure complete Fmoc removal, a solution of 40%
deemed to be a concern as the mixture could be converted piperidine in DMF was employed and deprotection re-
exclusively to peptaibols 7b–e upon treatment with 20% peated three times. The valuable AMD building block[4b]
piperidine/DMF.
(Figure 1) was singly coupled to the resin using only
To adapt this O–N acyl shift strategy for the synthesis of 2 equiv. of Fmoc-AMD-OH instead of the typical 5 molar
culicinin D, the Nα-Fmoc-N-Ns-protected APAE building excess amount used for readily available N-Fmoc-amino
block 15 was required (Ns = nosyl, Scheme 2). The nosyl acids. After Fmoc deprotection on resin 19 using 20%
protecting group was chosen for the secondary amine in- piperidine/DMF, Fmoc-Aib-OH was coupled to the resin
stead of the previously used benzyl group due to its facile using a single coupling strategy with HATU/iPr2NEt for
removal either on-resin or in solution using a mixture of 2- 20 min to give resin 20. It was initially found that coupling
mercaptoethanol and DBU. Thus, ethanolamine 8 was N- of Fmoc-Aib-OH using HATU/iPr2NEt for 45 min af-
protected with 2-nitrobenzenesulfonyl chloride (NsCl) in forded several unknown by-products. By reducing the reac-
78% yield, and alcohol 9 was quantitatively protected as tion time from 45 min to 20 min the formation of these by-
tert-butyldimethylsilyl (TBS) ether 10 with TBS-Cl. Alkyl products was significantly reduced. Similarly, the difficult
iodide 12 was obtained in 89% yield from alcohol 11 upon Aib-Aib condensation was accomplished by single coupling
reaction with imidazole, triphenylphosphine and iodine. using TFFH/iPr2NEt for 20 min. Longer reaction times of
Protected ethanolamine 10 was then alkylated with iodide up to 45 min led to more complex product HPLC profiles.
12 using Cs2CO3 in DMF at 80 °C to afford the N-Boc- The coupling of (R)-AHMOD (Figure 1) was performed
and O-TBS-protected APAE 13 in 64% yield. Cleavage of once for 45 min using only 2 equiv. of Fmoc-(R)-AHMOD-
both TBS and Boc protecting groups from compound 13 OH and HATU/iPr2NEt as the coupling reagents. Follow-
was carried out simultaneously using neat TFA for 4 hours, ing removal of the Fmoc group on 22a–b, Fmoc-Pro-OH
yielding amino alcohol 14 which was directly N-Fmoc pro- was coupled to the resin using HATU/iPr2NEt and sub-
tected using Fmoc-OSu in dioxane/H2O to afford 15 in 58% sequently N-acylated with butyric acid using HATU/
yield over two steps.
iPr2NEt in DMF/CH2Cl2 (1:1, v/v). With resin-bound
With the AHMOD, APAE and AMD building blocks in culicinin D (23a) in hand, we decided to effect nosyl depro-
hand, we proceeded with the revised SPPS of culicinin D tection and intramolecular O–N acyl shift in a one-pot re-
(Scheme 3). The amine of Fmoc-APAE(Ns)-OH (15) was action. The peptide was cleaved from the resin using a solu-
anchored to 2-chlorotrityl chloride resin by treatment with tion of HFIP in CH2Cl2 (1:4, v/v) for 30 min, and crude
2% DBU/DMF to effect Fmoc removal and attachment of peptide 24a was recovered after solvent evaporation and
the amino group in a simultaneous fashion. The resultant lyophilisation. Peptide 24a was next dissolved in DMF at a
resin 16 was esterified with Fmoc-β-Ala-OH using DIC/ concentration of 20 mgmL–1 and treated with 20 equiv. of
DMAP as the coupling agents in DMF/CH2Cl2 (1:1, v/v) 2-mercaptoethanol and 10 equiv. of DBU. The reaction was
to give the modified resin 17. The loading was determined monitored by LC-MS (Figure S28, Supporting Infor-
by removal of the Fmoc group in 20% piperidine in DMF mation), which showed initial deprotection of the nosyl
Scheme 2. Synthesis of the Nα-Fmoc-N-Ns-protected APAE building block 15 (Ns = nosyl). a) Na2CO3 (1.3 equiv.), NsCl (1 equiv.),
EtOAc, 10 h; b) imidazole (2.6 equiv.), TBS-Cl (1.25 equiv.) in anhydr. DMF, 15 h; c) imidazole (1.2 equiv.), PPh3 (1.2 equiv.), iodine
(1.3 equiv.), anhydr. THF, –15 °C for 4 h, then room temp. for 6 h; d) CsCO3 (2 equiv.), iodide 12 (1.5 equiv.), anhydr. DMF, 80 °C, 60 h;
e) (1 equiv.) TFA, 4 h; f) Na2CO3 (10%), Fmoc-OSu (1.2 equiv.), dioxane/H2O, 0 °C, 1 h.
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Improved Strategy for the Synthesis of Culicinin D
Scheme 3. Solid-phase peptide synthesis of culicinin D and its analogues. a) Fmoc-APAE(Ns)-OH (15) (1.5 equiv.), 2% DBU in anhydr.
DMF, room temp., 6–12 h, repeat once; b) Fmoc-βAla-OH (6 equiv.), DIC (3 equiv.), DMAP (0.3 equiv.), anhydr. DMF/CH2Cl2 (v/v,
1:1), r.t., 6–12 h, repeat once; c) i) 40% piperidine in DMF (v/v) (3ϫ 10 min); ii) Fmoc-AA-OH (5 equiv.), HATU (4.8 equiv.), iPr2NEt
(10 equiv.), DMF, 45 min; iii) Fmoc-AA-OH (5 equiv.), Oxyma (5 equiv.), DIC (10 equiv.), DMF, 45 min; d) i): 40% piperidine in DMF
(v/v) (3ϫ 10 min); ii) Fmoc-AMD-OH (2 equiv.), HATU (1.8 equiv.), iPr2NEt (4 equiv.), DMF, 45 min; e) i) 20% piperidine in DMF (v/
v) (2ϫ 10 min); ii) Fmoc-Aib-OH (5 equiv.), HATU (4.8 equiv.), iPr2NEt (10 equiv.), DMF, 20 min; f) i) 20% piperidine in DMF (v/v)
(2ϫ 10 min); ii) Fmoc-Aib-OH (5 equiv.), TFFH (5 equiv.), iPr2NEt (10 equiv.), DMF, 20 min; g) i) 20% piperidine in DMF (v/v) (2ϫ
10 min); ii) Fmoc-AA-OH (2 equiv.), HATU (1.8 equiv.), iPr2NEt (4 equiv.), DMF, 45 min; h) i) 20% piperidine in DMF (v/v) (2ϫ
10 min); ii) Fmoc-Pro-OH (5 equiv.), HATU (4.8 equiv.), iPr2NEt (10 equiv.); iii) 20% piperidine in DMF (v/v) (2ϫ 10 min); vi) butyric
acid (5 equiv.), HATU (5 equiv.), iPr2NEt (10 equiv.), DMF/CH2Cl2 (1:1); i) CH2Cl2/HFIP (v/v, 4:1), r.t., 30 min; j) 2-mercaptoethanol/
DBU (20 equiv./10 equiv.) in DMF (20 mg/mL).
Table 2. Resin loading yields and final purified yield of culicinin D 25a and analogue 25b.
Loading of resin
Yield [%],
(mg)
Sequence[a]
HRMS calcd./found[c]
17%[b]
25a C4Pro[(R)-AHMOD]AibAib(AMD)LeuAlaLeuβala(APAE)OH
25b C4Pro[(S)-AHMOD]AibAib(AMD)LeuAlaLeuβala(APAE)OH
65
61
4, (6.31)
1, (1.02)
1234.8754/1234.8751
1234.8754/1234.8732
[a] C4 = butanoyl. [b] Loading yield was determined according to reference 14 and is shown as median value of two experiments. [c]
Mass calcd./found for [M + H]+, observed by HRMS positive-ion mode ESI.
group over 2–3 h followed by the intramolecular O–N acyl (S)-AHMOD was synthesised successfully to assess the bio-
shift. Following completion of nosyl deprotection a further logical importance of the stereochemistry of the C-6
portion of DBU (10 equiv.) was added to ensure the O–N hydroxyl group in the AHMOD amino acid. Thus 25b was
acyl shift proceeded quantitatively. Immediate dilution with synthesised from resin 17 (loading 61%) using the analo-
H2O/CH3CN and purification by RP-HPLC afforded multi gous conditions in an adequate yield (based on a 0.06 mmol
milligram quantities of culicinin D 25a (6.31 mg) in 4% scale) (Table 2).
overall yield (based on resin loading). To confirm that the
culicinin D isopeptide 24a had undergone the desired re-
arrangement reaction to give peptide 25a, the Kaiser test
was performed and confirmed that no free primary amine
Conclusions
was present in peptide 25a.
In the pursuit for an effective, robust synthesis of the
To assess the robustness of the synthetic protocols estab- potent anticancer agent culicinin D 1, both an improved
lished herein, the diastereoisomeric culicinin D analogue synthesis of the C-terminal APAE building block 15 was
25b whereby the (R)-AHMOD residue is substituted by developed as well as its high yielding attachment to 2-
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determined on an Electrothermal® melting point apparatus and are
uncorrected. Optical rotations were determined at 20 °C with a Per-
kin–Elmer 341 polarimeter and are given in units of 10–1
degcm2 g–1. Nuclear magnetic resonance (NMR) spectra were re-
corded as indicated on either the Bruker AVANCE DRX300 spec-
trometer (1H, 300 MHz, 13C, 75 MHz), or the Bruker DRX400
spectrometer (1H, 400 MHz, 13C, 100 MHz). Chemical shifts are
reported in parts per million (ppm) relative to the tetramethylsilane
signal recorded at δH = 0.00 ppm in CDCl3/SiMe4 solvent or were
referenced to the residual DMSO signal at δH = 2.50 ppm in [D6]-
chlorotrityl chloride resin. These improvements allowed for
rapid assembly of culicinin D 25a in milligram quantities.
Key strategies employed in this revised synthesis include N-
anchoring of the APAE residue in Ͼ 60% yield, shortened
coupling times for the Aib amino acid and one-pot nosyl
deprotection/intramolecular O–N acyl shift which simpli-
fied the recovery of the peptaibol by RP-HPLC purifica-
tion. Given that other peptaibols such as microbacterins A
and B,[16] cephaibols[17] and alamethicins[1c,18] contain sim-
ilar C-terminal alcohol residues comparable to our model DMSO solvent or the residual benzene signal at δH = 7.16 ppm in
C6D6 or the residual MeOH signal at δH = 3.31 ppm in MeOD.
The 13C values were referenced to the residual chloroform signal
at δC = 77.0 ppm in CDCl3/SiMe4 solvent or were referenced to the
residual DMSO signal at δC = 39.5 ppm in [D6]DMSO solvent or
were referenced to residual benzene signal at δC = 128.0 ppm in
C6D6 solvent or were referenced to the residual MeOH signal at δC
peptide alcohols 7a–e, we envisage that this improved pro-
cedure will enable access to these related antibacterial
peptaibols. Additionally, several naturally occurring peptai-
bols are known to be antimicrobial and anticancer agents,[3]
peptaibols 7a–e may also be active against cancer cells.
Therefore, biological testing of 7a–e against the growth of
PTEN-negative breast tumor cells would give insight into
the structure-activity relationship of culicinin D 1.
1
= 49.00 ppm. H NMR shift values are reported as chemical shift
(δH), relative integral, multiplicity (s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; dd, doublet of doublets; dq, doublet of
quartets; ddd, doublet of doublet of doublets), coupling constant
Coupling of Fmoc-Aib-OH to the AMD residue on-resin
requires further refinement due to the formation of un- (J in Hz) and assignments. 13C values are reported as chemical
shift (δC) and assignment. Assignments are made with the aid of
DEPT135, COSY, NOESY and HSQC experiments. Accurate high
resolution mass spectra were recorded on a Bruker micrOTOFQ
mass spectrometer using electrospray ionization (ESI) in the posi-
tive mode. LCMS was performed using an Agilent (Santa Clara,
USA) 1100 Compact HPLC equipped with a single wavelength UV
detector at 214 nm with an in-line Hewlett–Packard (Palo Alto,
CA) 1100MSD mass spectrometer using electrospray ionization
(ESI) in the positive mode. An Agilent Zorbax 300SB-C3 (3.5 μ;
3.0ϫ150 mm) column was employed using a linear gradient of 1–
99% B over 33 min at 0.3 mL/min. The solvent system used was A
(0.1% formic acid in H2O) and B (0.1% formic acid in CH3CN).
Analytical RP-HPLC was performed using a Dionex P680 System
using either an XTerra® C18, (300 Å, 5 μm, 4.6ϫ150 mm),
Phenomenex Gemini C18 (110 Å, 5 μm, 4.6ϫ250 mm) or a Phe-
nomenex Gemini C18 (110 Å, 5 μm, 4.6ϫ150 mm), using a linear
gradient of 0.1% TFA/H2O (buffer A) and 0.1% TFA/CH3CN
(buffer B) with detection at 210 nm. Peptides were purified on a
Waters 600 Systems using an XTerra® C18 (300 Å, 10 μm,
19ϫ300 mm) at a flow rate of 10 mLmin–1, using gradients with
0.1% TFA/H2O (buffer A) and 0.1% TFA/CH3CN (buffer B) with
detection at 210 nm or on Dionex Ultimate 3000 system using a
Phenomenex Gemini C18 (110 Å, 5 μm, 10ϫ250 mm) at a flow
rate of 5 mLmin–1, using gradients with 0.1% TFA/H2O (buffer A)
and 0.1% TFA/CH3CN (buffer B) with detection at 210 nm.
known by-products as observed by LC-MS following pept-
ide cleavage. However, this improved revised strategy
allowed culicinin D 25a and its isomer containing the un-
natural (S)-AHMOD, 25b to be successfully synthesised in
ample quantities. Biological evaluation of these peptaibols
prepared herein against human breast tumor cells of vary-
ing PTEN status is currently underway.
Experimental Section
General Information: All the reagents were purchased as reagent
grade and used without further purification. Solvents for reactions
were dried according to standard procedures. Solvents for RP-
HPLC were purchased as HPLC grade and used without further
purification. O-(7-Azabenzotriazol-1-yl)-N,N,NЈ,NЈ-tetramethyl-
uronium hexafluorophosphate (HATU), were purchased from GL
Biochem (Shanghai, China). Dimethylformamide (DMF, AR
grade) and acetonitrile (CH3CN, HPLC grade) were purchased
from Scharlau (Barcelona, Spain). N,NЈ-Diisopropylethylamine
(iPr2NEt), piperidine, N,NЈ-diisopropylcarbodiimide (DIC) and
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were purchased from
Sigma–Aldrich (St. Louis, USA). N-Methylpyrrolidine was pur-
chased from Fluka (Buchs, Switzerland). 4-(Dimethylamino)pyr-
idine (DMAP), 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP) and
N,N,NЈ,NЈ-tetramethylfluoroformamidinium hexafluorophosphate
(TFFH) were obtained from AK Scientific (Union City, USA). Tri-
fluoroacetic acid (TFA) was purchased from Oakwood Chemical
(River Edge, USA). 2-Chlorotrityl chloride resin and ethyl 2-cyano-
2-(hydroxyimino)acetate (Oxyma) were purchased from Novabi-
ochem (Merck, Germany). Fmoc-amino acids (Fmoc-Aib-OH,
(9H-Fluoren-9-yl)methyl N-[(2S)-1-Hydroxypropan-2-yl]carbamate
(Fmoc-alaninol) (2):[19] A solution of Fmoc-protected alanine (1.0 g,
3.21 mmol) in DME (1,2-dimethoxyethane) (6.42 mL, 0.5 mol/L)
was cooled to –15 °C (acetone/dry ice) under nitrogen atmosphere.
N-Methylmorpholine (299 μL, 3.21 mmol) and isobutyl chloro-
formate (458 μL, 3.21 mmol) were added dropwise successively. Af-
Fmoc-Ala-OH, Fmoc-βAla-OH, Fmoc-Leu-OH, Fmoc-Pro-OH) ter 1 h stirring, the reaction mixture was filtered and washed 3
were purchased from GL Biochem. Thin-layer chromatography times with DME (1 mL). The filtrates were cooled to –15 °C (acet-
(TLC) was performed using 0.2 mm plates of silica gel F254
(Merck) and compounds were visualized by ultraviolet fluorescence
or by staining with potassium permanganate, ninhydrin or vanillin
stain, followed by heating the plate as appropriate. Flash column
chromatography was performed using silica gel S 0.063–0.1 mm
(Riedel de Hahn) silica gel with indicated solvents. Infrared spectra
were recorded on a Perkin–Elmer Spectrum 100 infrared spectrom-
eter and reported in wavenumbers (ν [cm–1]). Melting points were
one/dry ice), and a solution of NaBH4 (243 mg, 6.42 mmol) in H2O
(3 mL) was added in one portion, followed by H2O (80 mL). The
suspension was filtered, and the residue was washed with H2O (3ϫ
80 mL) and hexane (3ϫ 80 mL) and dried under high vacuum to
give 2 (0.73 g, 76%) as a white solid, m.p. 122–124 °C (ref.[19]
120 °C); δH (400 MHz, CDCl3): δ = 1.18 (d, J = 5.6 Hz, 3 H, CH3),
1.98 (br. s, 1 H, OH), 3.60 (d, J = 46.19 Hz, 2 H, CH2OH), 3.83
(br. s, 1 H, CHNH), 4.22 (t, J = 6.6 Hz, 1 H, Fmoc-CH), 4.43 (s,
6346
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Eur. J. Org. Chem. 2015, 6341–6350

Improved Strategy for the Synthesis of Culicinin D
2 H, Fmoc-CH2), 4.84 (s, 1 H, NH), 7.32 (t, J = 7.5, J = 1.0 Hz, 2
tert-Butyl (S)-[1-(N-{2-[(tert-butyldimethylsilyl)oxy]ethyl}-2-
H, Fmoc-Ar-CH), 7.41 (t, J = 7.4 Hz, 2 H, Fmoc-Ar-CH), 7.60 (t, nitrophenylsulfonamido)propan-2-yl]carbamate (13): Sulfonamide 10
J = 7.5 Hz, 2 H, Fmoc-Ar-CH), 7.77 (t, J = 7.5 Hz, 2 H, Fmoc- (300 mg, 0.832 mmol) was dissolved in dry DMF (2.5 mL) at room
Ar-CH) ppm; δC (100 MHz, CDCl3) 17.4 (CH3), 47.4 (Fmoc-CH), temperature under a nitrogen atmosphere. Cs2CO3 (543 mg,
49.1 (CHNH), 66.8 (CH2OH, Fmoc-CH2), 67.0 (CH2OH, Fmoc- 1.67 mmol) was added, and the reaction mixture was stirred for
CH2), 120.1 (Fmoc-Ar-CH), 125.1 (Fmoc-Ar-CH), 127.2 (Fmoc-
Ar-CH), 127.8 (Fmoc-Ar-CH), 141.5 (Fmoc-Ar-quatC), 144.0
(Fmoc-Ar-quatC) ppm. MS m/z (ESI+) [M + H]+ 298, calcd. for
C18H19NO3 298.
30 min. Iodide 12 (357 mg, 1.25 mmol) was added and the reaction
mixture was heated to 80 °C and stirred for 60 h. After cooling to
room temperature, saturated aqueous NH4Cl (10 mL) was added,
and the mixture was extracted with diethyl ether (3ϫ 10 mL). The
combined organic extracts were washed with brine (20 mL), dried
with anhydrous Na2SO4, filtered and evaporated under reduced
pressure. Purification of the crude product by flash chromatog-
raphy (hexane/EtOAc (4:1) buffered with 0.5% Et3N) afforded 13
(276 mg, 0.533 mmol, 64 %) as a yellow oil. Rf = 0.65 (hexane/
N-(2-Hydroxyethyl)-2-nitrobenzenesulfonamide (9):[20] To a solution
of ethanolamine (500 mg, 8.19 mmol) in EtOAc (3 mL) at room
temperature was added a solution of Na2CO3 (1.13 g, 10.6 mmol)
in H2O (5 mL), followed by dropwise addition of a solution of
NsCl (1.74 g, 7.84 mmol) in EtOAc (5 mL). After stirring for 10 h
the organic layer was washed with saturated aqueous NH4Cl
(10 mL), brine (10 mL), dried with Na2SO4, filtered and evaporated
to yield 9 (1.57 g, 6.40 mmol, 78%) as a white crystal. Rf = 0.65
(EtOAc/MeOH-aq. NH3, 10:1:0.01); m.p. 79.9–83.3 °C (ref.[20] m.p.
not determined); δH (400 MHz, CD3OD): δ = 8.03 (m, 1 H, H-
ArC), 7.80 (m, 1 H, H-ArC), 7.74 (m, 2 H, H-ArC), 3.52 (t, J =
6.0 Hz, 2 H, 2-H) and 3.08 (t, J = 5.8 Hz, 2 H, 1-H) ppm; δC
(100 MHz, CD3OD) 149.8 (ArC), 135.1 (ArC), 134.9 (ArC-SO2R)
133.8 (ArC), 131.8 (ArC), 126.2 (ArC), 61.9 (C2), 46.7 (C1) ppm.
EtOAc, 3:1); IR (neat): ν
= 3388, 2928, 1698, 1541, 1158,
˜
max
736 cm–1. [α]2D0 = –44.0 (c = 0.60 in CHCl3); δH (400 MHz, CDCl3):
δ = 8.01 (m, 1 H, H-ArC), 7.66 (m, 2 H, H-ArC), 7.61 (m, 1 H,
H-ArC), 4.78 [s, 1 H, 4-NHCO2C(CH3)3], 4.00 (s, 1 H, 4-H), 3.73
(t, J = 6.4 Hz, 2 H, 1-H), 3.41–3.55 (m, 3 H, 2-H, 3-Ha), 3.35 (dd,
J1 = 6.2, J2 = 14.9 Hz, 1 H, 3-Hb), 1.39 [s, 9 H, 4-NHCO2C(CH3)3],
1.12 (d, J = 7.1 Hz, 3 H, CH3), 0.83 [s, 9 H, 1-OSi(CH3)2C(CH3)3]
and –0.01 [s, 6 H, 1-OSi(CH3)2C(CH3)3] ppm. δC (100 MHz,
CDCl3) 155.6 [4-NHCO2C(CH3)3], 148.3 (ArC), 131.9 (ArC), 130.8
(ArC), 134.2 (ArC), 133.6 (ArC), 124.4 (ArC), 79.4 [4-
NHCO2C(CH3)3], 61.8 (C1), 53.2 (C2), 49.6 (C3), 44.3 (C4), 30.5
[1-OSi(CH3)2C(CH3)3] 28.5 [4-NHCO2C(CH3)3],26.0 [1-OSi(CH3)2-
N-{2-[(tert-Butyldimethylsilyl)oxy]ethyl}-2-nitrobenzenesulfon-
amide (10): To a solution of alcohol 9 (1.0 g, 4.06 mmol) in dry
DMF (3.6 mL) at room temperature under nitrogen atmosphere
was added imidazole (719 mg, 10.6 mmol) followed by TBS-Cl
(766 mg, 5.08 mmol). After stirring for 15 h the solution was
poured into H2O (50 mL). The aqueous phase was extracted with
diethyl ether (2ϫ 30 mL), and the combined organic phases were
washed with water (50 mL), brine (50 mL), dried with anhydrous
Na2SO4 and evaporated in vacuo. The crude product was purified
by flash column chromatography (hexane/EtOAc, 4:1 to 2:1) to af-
ford 10 (1.46 g, quant.) as a white crystal. Rf = 0.57 (hexane/
C(CH3)3], 18.9 (CH3) and 2ϫ –5.4 [1-OSi(CH3)2C(CH3)3] cm–1
.
HRMS (ESI+) [M + H]+ 518.2359 calcd. for C22H40N3O7SSi
518.2351.
(S)-N-(2-Aminopropyl)-N-(2-hydroxyethyl)-2-nitrobenzenesulfon-
amide (14): Amine 13 (250 mg, 0.48 mmol) was dissolved in TFA
(10 mL) at room temperature and stirred for 3 h. The solution was
removed first by rotary evaporator under reduced pressure and
then by azeotropic distillation with hexane. Compound 14 was used
in the next step without further purification.
EtOAc, 2:1); m.p. 62.2–63.9 °C; IR (neat): νmax = 2956, 2928, 2856,
˜
1534, 1164 cm–1. δH (400 MHz, CDCl3): δ = 8.13 (m, 1 H, H-ArC),
7.87 (m, 1 H, H-ArC), 7.74 (m, 2 H, H-ArC), 5.77 (s, 1 H,
-NHSO2Ar), 3.69 (t, 2 H, 2-H), 3.20 (dt, J1 = 5.5, J2 = 5.5 Hz, 2
H, 1-H), 0.83 [s, 9 H, C(CH3)3] and –0.01 (s, 6 H, Si-CH3) ppm;
δC (100 MHz, CDCl3) 148.4 (ArC), 133.9 (ArC), 133.7 (ArC), 132.9
(ArC), 61.3 (C2), 46.1 (C1), 25.9 [C(CH3)3], 18.3 [3ϫ C(CH3)3] and
–5.4 [Si(CH3)2] ppm. HRMS (ESI+) [M + H]+ 361.1251 calcd. for
C14H25N2O5SSi 361.1248.
(S)-(9H-Fluoren-9-yl)methyl{1-[N-(2-hydroxyethyl)-2-nitrophenyl
Sulfonamido]propan-2-yl}carbamate (15): Compound 14 was dis-
solved in a solution of aqueous Na2CO3 (2.5 mL, 10%), and the
solution was cooled to 0 °C. A solution of Fmoc-OSu (195 mg,
0.578 mmol) in dioxane (2.5 mL) was added dropwise, and the reac-
tion was warmed to room temperature and stirred for 1 h. The
reaction mixture was extracted with EtOAc (3ϫ 5 mL), and the
combined organic extracts were washed with brine (5 mL), dried
with anhydrous Na2SO4, filtered and evaporated under reduced
pressure. The crude product was purified by flash column
chromatography (EtOAc/hexane-Et3N, 5:1:0.2) to give 15 (147 mg,
0.280 mmol, 58% over 2 steps) as a yellow oil. Rf = 0.34 (hexane/
tert-Butyl (S)-(1-Iodopropan-2-yl)carbamate (12):[21] Imidazole
(467 mg, 6.85 mm) and PPh3 (1.80 g, 6.85 mmol) were dissolved in
THF (20 mL) under a nitrogen atmosphere and cooled to 0 °C.
Iodine (1.88 g, 7.42 mmol) was added in portions and the mixture
was stirred for 30 min. A solution of alcohol 11 (1.0 g, 5.71 mmol)
in THF (5 mL) was added dropwise at 0 °C. After stirring for 4 h
at this temperature the reaction mixture was warmed to ambient
temperature and stirred for further 6 h. Diethyl ether (30 mL) was
added and the mixture was filtered through celite®, washed with
saturated aqueous Na2S2O3, brine, dried with anhydrous Na2SO4
and purified by flash column chromatography (hexane then hexane/
EtOAc, 1:1); IR (neat): ν
= 1694, 1541, 1253, 1119, 871 cm–1.
˜
max
[α]2D0 = –53.2 (c = 0.25 in CHCl3); δH (400 MHz, CDCl3): δ = 7.98
(m, 1 H, ArC), 7.74 (d, J = 7.5 Hz, 2 H, 10-H, 11-H), 7.65–7.55
(m, 5 H, 7-H, 14-H, ArC), 7.38 (dt, J1 = 7.5, J2 = 1.0 Hz, 2 H, 9-
H, 12-H), 7.30 (m, 2 H, 8-H, 13-H), 5.20 (d, J = 9.2 Hz, 1 H,
FmocNHR), 4.42 (m, 1 H, 6-H), 4.24 (m, 2 H, 5-H), 4.13 (m, 1 H,
EtOAc (10:1) buffered with 3% Et3N). The resulting product was 4-H), 3.75 (m, 2 H, 1-H), 3.57 (m, 2 H, 2-H), 3.29 (m, 1 H, 3a-H),
washed with 10 % aqueous citric acid to yield 12 (1.46 g, 3.17 (dd, J1 = 15.1, J2 = 4.5 Hz, 1 H, 3b-H) and 1.19 (d, J = 6.5 Hz,
5.10 mmol, 89%) a white crystal. Rf = 0.25 (hexane/EtOAc, 10:1);
3 H, CH3) ppm; δC (100 MHz, CDCl3) 155.9 (R1OCONHR2),
147.2 (ArC), 143.1 (C6a, C14a), 140.4 (C10a, C10b), 132.9 (ArC),
m.p. 58.6–61.0 °C (ref.[21] 58–60 °C). [α]2D0 = –21.5 (c = 0.26 in
CHCl3) [ref.[22] –19.8 (c = 5.80)]; δH (400 MHz, CDCl3): δ = 4.58 131.9 (ArC), 130.9 (ArC), 130.3 (ArC), 126.8 (9-H, 12-H), 126.2
(s, 1 H, -NHBoc), 3.52 (s, 1 H, 2-H), 3.41 (s, 1 H, 1a-H), 3.30 (dd,
J1 = 10.4, J2 = 4.6 Hz, 1 H, 1b-H), 1.45 [s, 9 H, (CH3)3] and 1.20
(10-H, 11-H), 124.5 (8-H, 13-H), 123.3 (ArC), 119.0 (C7, C14),
66.2 (C5), 59.8 (C1), 54.0 (C3), 51.3 (C2), 46.3 (C6), 44.9 (C4)
(d, J = 7.3 Hz, 3 H, CH3) ppm; δC (100 MHz, CDCl3) 28.6 and 17.8 (CH3) ppm. HRMS (ESI+) [M + H]+ 526.1641 calcd. for
(CH3)3 and 21.4 (CH3) ppm.
C26H28N3O7S 526.1642.
Eur. J. Org. Chem. 2015, 6341–6350
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6347

M. A. Brimble et al.
FULL PAPER
General Procedures for SPPS of Culicinin D Analogues 7a–e, Culici-
nin D (25a), Culicinin D Analogue 25b
and MeOH (3 ϫ 5 mL). The same Fmoc-protected amino acid
(5 equiv.) was dissolved in 0.23 m Oxyma (5 equiv.) solution in
DMF, and the solution was added to the resin. After the addition
of DIC (10 equiv.) the resin was agitated at room temperature for
45 min, and the resin was washed with DMF (3 ϫ 5 mL) and
MeOH (3ϫ 5 mL). The Fmoc protecting group was removed by
agitating the resin in a solution of 40% piperidine in DMF (v/v,
5 mL, 3ϫ 10 min) and followed by washing with DMF (3ϫ 5 mL)
and MeOH (3ϫ 5 mL).
General Procedure for the Attachment of Fmoc-β-Amino Alcohol 2
to Resin via the Amino Group: Fmoc-alaninol 2 (3 equiv.) was dis-
solved in a solution of 2% DBU (6 equiv.) in anhydrous DMF, and
the mixture was added to the 2-chlorotrityl chloride resin (1 equiv.,
loading: 1.51 mmol/g). The resin was stirred at room temperature
for approx. 14 h, and the reaction mixture was removed by fil-
tration and washed with DMF (3ϫ 5 mL) and CH2Cl2 (3ϫ 5 mL).
After drying under high vacuum the same procedure was repeated
for 6 h. The reaction mixture was removed by filtration and the
resin was washed with DMF (3ϫ 5 mL), 10% iPr2Net/MeOH (3ϫ
5 mL) and CH2Cl2 (3ϫ 5 mL). The unreacted resin was capped by
reaction with MeOH (3ϫ 10 mL, 3ϫ 10 min), and the resin was
dried for 15 min to afford resin 3.[5]
General Procedure for Coupling of Fmoc-Aib-OH to Resin Bound
Aib in Culicinin D Analogues 7a–e: Fmoc-Aib-OH (5 equiv.) and
TFFH (5 equiv.) was dissolved in DMF (3 mL), and the solution
was added to the resin. A solution of 2 m iPr2NEt (10 equiv.) in
NMP was added to the resin, and the reaction was agitated at room
temperature for 45 min. The resin was washed with DMF (3ϫ
5 mL) and MeOH (3ϫ 5 mL) and the coupling was repeated. A
solution of 40% piperidine in DMF (v/v, 5 mL) was then added to
resin for Fmoc deprotection (3ϫ 10 min.) followed by washing with
DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL).
General Procedure for the Attachment of Fmoc-Protected Alcohol
15 to Resin via the Amino Group: Compound 15 (3 equiv.) was dis-
solved in a solution of 2% DBU (6 equiv.) in anhydrous DMF, and
the mixture was added to the 2-chlorotrityl chloride resin (1 equiv.,
loading: 1.51 mmol/g). After agitation at room temperature for ap-
prox. 14 h the reaction mixture was removed by filtration, and the
resin was washed with DMF (3ϫ 5 mL) and CH2Cl2 (3ϫ 5 mL).
After drying the resin under high vacuum the procedure was re-
peated at room temperature for 6 h. The reaction mixture was re-
moved by filtration, and the resin was washed with DMF (3ϫ
5 mL), 10% iPr2NEt/MeOH (3ϫ 5 mL) and CH2Cl2 (3ϫ 5 mL).
Unreacted resin was capped by reaction with MeOH (3ϫ 10 mL,
3ϫ 10 min), and the resin was dried for 15 min to afford resin 16.[5]
General Procedure for the Attachment of Fmoc-AMD-OH and
Fmoc-AHMOD-OH in Culicinin D (25a) and Analogue 25b: Fmoc-
protected amino acid (2 equiv.) was dissolved in a 0.23 m HATU
(1.8 equiv.) solution in DMF and the solution was added to the
resin, followed by addition of a solution of 2 m iPr2NEt in NMP
(4 equiv.). After agitating at room temperature for 45 min, the resin
was washed with DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL). The
Fmoc group was removed by agitating the resin in a solution of
20% piperidine/DMF (v/v, 5 mL, 2ϫ 10 min), followed by washing
with DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL).
General Procedure for the Attachment of Fmoc-β-Alanine-OH to
Functionalized 2-Chlorotrityl Chloride Resin 3: Fmoc-β-alanine-OH
(6 equiv.) and DMAP (0.3 equiv.) were dissolved in a mixture of
anhydrous. CH2Cl2 and anhydrous. DMF (1:1, v/v). The resulting
solution was added to the resin 3 (1 equiv., loading: 1.51 mmol/g),
DIC (3 equiv.) was added, and the reaction was agitated at room
temperature for approx. 14 h. After washing with DMF (3ϫ 5 mL),
MeOH (3ϫ 5 mL) and CH2Cl2 (3ϫ 5 mL) the resin was dired un-
der high vacuum. The same procedure was repeated for 6 h.[5] The
loading was determined by spectrophotometric analysis[14] and the
results are shown below; culicinin D analogue 7a: 0.64 mmol/g
(67 %), 0.64 mmol/g (67 %), 0.59 mmol/g (64 %), 0.59 mmol/g
(64%); culicinin D analogue 7b: 0.61 mmol/g (66%), 0.59 mmol/g
(64%); culicinin D analogue 7c: 0.58 mmol/g (63%), 0.41 mmol/g
(45%); culicinin D analogue 7d–e: 0.57 mmol/g (62%), 0.58 mmol/
g (63%).
General Procedure for Coupling of Fmoc-Aib-oH to Resin Bound
AMD in Culicinin D (25a) and Analogue 25b: Fmoc-Aib-OH
(5 equiv.) and HATU (4.8 equiv.) were dissolved in DMF (3 mL),
and the solution was added to the resin. A solution of 2 m iPr2NEt
(10 equiv.) in NMP was added to the resin, and the reaction was
agitated at room temperature for 20 min. The resin was washed
with DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL) and the coupling
was repeated. A solution of 20% piperidine in DMF (v/v, 5 mL)
was then added to resin for Fmoc deprotection (2ϫ 10 min) fol-
lowed by washing with DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL).
General Procedure for Coupling of Fmoc-Aib-OH to Resin Bound
Aib in Culicinin D (25a) and Analogue 25b: Fmoc-Aib-OH
(5 equiv.) and TFFH (5 equiv.) were dissolved in DMF (3 mL), and
the solution was added to the resin. A solution of 2 m iPr2NEt
(10 equiv.) in NMP was added to the resin, and the reaction was
agitated at room temperature for 20 min. The resin was washed
with DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL) and the coupling
was repeated. A solution of 20% piperidine in DMF (v/v, 5 mL)
was then added to resin for Fmoc deprotection (2ϫ 10 min.) fol-
lowed by washing with DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL).
General Procedure for the Attachment of Fmoc-β-alanine-OH to
Functionalised 2-Chlorotrityl Chloride Resin 16: Fmoc-β-alanine-
OH (6 equiv.) and DMAP (0.3 equiv.) were dissolved in a mixture
of anhydrous. CH2Cl2 and anhydrous. DMF (1:1, v/v). The re-
sulting solution was added to resin 16 (1 equiv., loading:
1.51 mmol/g), DIC (3 equiv.) was added, and the reaction was agi-
tated at room temperature for approx. 14 h. After washing with
DMF (3ϫ 5 mL), MeOH (3ϫ 5 mL), CH2Cl2 (3ϫ 5 mL) and dry-
ing under high vacuum, the same procedure was repeated for 6 h.[5]
The loading was determined by spectrophotometric analysis and
the results are shown below;[14] culicinin D (25a) and culicinin D
analogue 25b: 0.46 mmol/g (56%), 0.53 mmol/g (65%).
General Procedure After the Last Aib Residue in Culicinin D (25a)
and Analogue 25b: The appropriate Fmoc-protected amino acid
(5 equiv.) was dissolved in 0.23 m HATU (4.8 equiv.) solution in
DMF and the solution was added to the resin. A solution of 2 m
iPr2NEt (10 equiv.) in NMP was added to the resin, and the reac-
tion was agitated at room temperature for 45 min. The resin was
washed with DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL). The Fmoc
protecting group was removed by agitating the resin in a solution
of 20% piperidine in DMF (v/v, 5 mL, 2ϫ 10 min) and followed
by washing with DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL).
General Procedure for Solid-Phase Peptide Synthesis (SPPS): The
appropriate Fmoc-protected amino acid (5 equiv.) was dissolved in
0.23 m HATU (4.8 equiv.) solution in DMF and the solution was
added to the resin. A solution of 2 m iPr2NEt (10 equiv.) in NMP
was added to the resin, and the reaction was agitated at room tem-
perature for 45 min. The resin was washed with DMF (3ϫ 5 mL)
General Procedure for the Attachment of N-Terminal Butyric Acid:
Butyric acid (5 equiv.) and HATU (5 equiv.) were dissolved in mix-
6348
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Eur. J. Org. Chem. 2015, 6341–6350

Improved Strategy for the Synthesis of Culicinin D
ture of CH2Cl2 and DMF (v/v, 1:1, 2 mL). iPr2NEt (10 equiv.) was
added to the solution, and the solution was added to the resin.
Butanoyl-ProLeuAlaAlaLeuLeuAlaLeuβalaAlaol (7b): From 6b
(3.14 mg, 0.031 mmol), culicinin D analogue 7b was obtained as
After agitation at room temperature for 45 min, the resin was a white foamy solid after semi-preparative RP-HPLC purification
washed with DMF (3ϫ 5 mL) and MeOH (3ϫ 5 mL). Butyric
acid (5 equiv.) and Oxyma (5 equiv.) were dissolved in a mixture of
CH2Cl2 and DMF (v/v, 1:1, 2 mL). DIC (10 equiv.) was added to
the solution, and the mixture was added to the resin. After agitat-
ing at room temperature for 45 min, the resin was washed with
DMF (3ϫ 5 mL), MeOH (3ϫ 5 mL) and dried for 15 min under
vacuum.
(1.04 mg, 3.21 μmol, 33%). Anal. RP-HPLC: tR = 28.09 min (Phe-
nomenex Gemini C18, 4.6ϫ250 mm, 5u, 1% B to 46% B at 3% B
per min, 46% B to 65% B in 1% B per min, flow rate 1 mL/min).
HRMS (ESI+): C48H87N10O11 [M + H]+ calcd./found 979.6556/
979.6569, C48H86N10O11Na [M + Na]+ calcd./found 1001.6375/
1001.6393.
(S)-2-Aminopropyl-butanoyl-ProLeuAibAibLeuLeuAibLeuβalano-
ate (6c): From 2-chlorotrityl chloride resin (79.9 mg, 0.0396 mmol,
loading: 0.495 mmol/g), culicinin D analogue 6c was obtained as
a white foamy solid after semi-preparative RP-HPLC purification
(8.86 mg, 8.67 μmol, 22%). Anal. RP-HPLC: tR = 24.13 min (Phe-
nomenex Gemini C18, 4.6ϫ250 mm, 5u, 1% B to 46% B at 3% B
per min then 46% B to 65% B at 1% B per min, flow rate 1 mL/
min). MS (ESI+): C51H92N10O11 [M]+ calcd./found 1021.3/1021.6.
General Procedure for HFIP-Mediated Resin Cleavage and Purifica-
tion: Resin-bound culicinin D and culicinin D analogues were
cleaved from the resin by gentle agitation in a mixture of CH2Cl2/
HFIP (4:1, v/v, 5 mL) for 30 min. The solvent was evaporated on
a rotary evaporator at ambient temperature, and the residue was
dissolved in a solution of H2O/acetonitrile (1:1, v/v, 10 mL) and
lyophilised. The white powder was then dissolved in H2O/aceto-
nitrile (1:1, v/v) and purified by semi-preparative RP-HPLC.
Butanoyl-ProLeuAibAibLeuLeuAibLeuβalaAlaol (7c): From 6c
(5.27 mg, 5.20 μmol), culicinin D analogue 7c was obtained as a
white foamy solid after semi-preparative RP-HPLC purification
(2.26 mg, 2.21 μmol, 43%). Anal. RP-HPLC: tR = 40.98 min (Phe-
nomenex Gemini C18, 4.6ϫ250 mm, 5u, 1% B to 71% B at 1% B
per min, flow rate 1 mL/min). HRMS (ESI+): C51H93N10O11 [M +
H]+ calcd./found 1021.7025/1021.7042, C51H92N10O11Na [M +
Na]+ calcd./found 1043.6845/1043.6881.
General Procedure for the Solution Phase O–N Acyl Shift in Culici-
nin D Analogues 7a–e: Purified culicinin D analogues were dis-
solved in 20% piperidine/DMF (v/v, peptide concentration 5 mg/
mL), and the reaction was stirred for 3–5 h. For incomplete reac-
tions the reaction mixture was stirred under microwave irradiation
at 50 °C and 50 W until completion of reaction as determined by
analytical RP-HPLC. After evaporation of the solvent the crude
product was dissolved in a solution of H2O/acetonitrile (1:1, v/v,
10 mL), lyophilised and purified by semi-preparative RP-HPLC.
Butanoyl-ProAlaAibAibAlaLeuAibLeuβalaAlaol (7d): From 2-
chlorotrityl chloride resin (187.2 mg, 0.1079 mmol, loading:
0.58 mmol/g), culicinin D analogue 7d was obtained as a white
foamy solid after semi-preparative RP-HPLC purification
(16.91 mg, 18.0 μmol, 17%). Anal. RP-HPLC: tR = 44.07 min (Phe-
nomenex Gemini C18, 4.6ϫ150 mm, 5u, 1% B to 65% B at 1% B
per min, flow rate 1 mL/min). HRMS (ESI+): C45H81N10O11 [M +
H]+ calcd./found 937.6086/937.6063, C45H80N10O11Na [M + Na]+
calcd./found 959.5906/959.5874.
General Procedure for Simultaneous Deprotection of Nosyl-Protect-
ing Group and O–N Acyl Shift in Culicinin D (25a) and Analogue
25b: To a solution of culicinin D or culicinin D analogues (1 equiv.)
in DMF (peptide concentration 20 mg/mL) was added 2-mercapto-
ethanol (20 equiv.) and DBU (10 equiv.), and the reaction mixture
was stirred at room temperature for 2–3 h until completion of nosyl
deprotection followed by LC-MS. DBU (10 equiv.) was added and
the reaction was stirred for 1 h until the completion of the O–N
acyl shift as monitored by LC-MS. The reaction mixture was di-
luted with H2O + 0.1% TFA (20 mL) and purified by semi-prepar-
ative RP-HPLC.
(S)-2-Aminopropyl-butanoyl-ProProAibAibProLeuAibLeuβalanoate
(6e): From 2-chlorotrityl chloride resin (175.5 mg, 0.1012 mmol,
loading: 0.58 mmol/g), culicinin D analogue 6e was obtained as a
white foamy solid after semi-preparative RP-HPLC purification
(4.72 mg, 4.77 μmol, 5%). Anal. RP-HPLC: tR = 37.57 min (Phe-
nomenex Gemini C18, 4.6ϫ150 mm, 5u, 1% B to 65% B at 1% B
per min, flow rate 1 mL/min). MS (ESI+): C49H84N10O11 [M]+
calcd./found 989.3/989.6.
(S)-2-Aminopropyl-LeuAlaLeuβalanoate (6a): From 2-chlorotrityl
chloride resin (50.0 mg, 0.031 mmol, loading: 0.62 mmol/g), culici-
nin D analogue 6a was obtained as a white foamy solid after semi-
preparative RP-HPLC purification (3.34 mg, 5.02 μmol, 16%).
Anal. RP-HPLC: tR = 18.04 min (Phenomenex Gemini C18,
4.6 ϫ 250 mm, 5 µ, 1 % B to 65 % B at 1 % B per min, flow rate
1 mL/min). MS (ESI+) C21H42N5O5 [M + H]+ calcd./found 444.6/
444.4.
Butanoyl-ProProAibAibProLeuAibLeuβalaAlaol (7e): From 2-
chlorotrityl chloride resin (175.5 mg, 0.1012 mmol, loading:
0.58 mmol/g), culicinin D analogue 7e was obtained as a white
foamy solid after semi-preparative RP-HPLC purification
(11.35 mg, 11.47 μmol, 11%). Anal. RP-HPLC: tR = 41.60 min
(Phenomenex Gemini C18, 4.6ϫ150 mm, 5u, 1% B to 65% B at
1% B per min, flow rate 1 mL/min). HRMS (ESI+): C49H85N10O11
[M + H]+ calcd./found 989.6399/989.6430, C49H84N10O11Na [M +
Na]+ calcd./found 1011.6219/1011.6278.
LeuAlaLeuβalaAlaol (7a): From 6a (1.64 mg, 3.70 μmol), culici-
nin D analogue 7a was obtained as a white foamy solid after semi-
preparative RP-HPLC purification (0.59 mg, 1.33 μmol, 36%).
Anal. RP-HPLC: tR = 20.13 min (Phenomenex Gemini C18,
4.6 ϫ 250 mm, 5 µ, 1 % B to 41 % B at 1 % B per min, flow rate
1 mL/min). HRMS (ESI+): C21H42N5O5 [M + H]+ calcd./found
444.3186/444.3175, C21H41N5O5Na [M + Na]+ calcd./found
466.3005/466.2990.
Butanoyl-Pro[(R)-AHMOD]AibAib(AMD)LeuAibLeuβala(APAE)
OH (25a): From functionalized 2-chlorotrityl chloride resin 17
(267 mg, 0.1423 mmol, loading: 0.53 mmol/g) culicinin D 25a was
obtained as a white foamy solid after semi-preparative RP-HPLC
(S)-2-Aminopropyl-butanoyl-ProLeuAlaAlaLeuLeuAlaLeuβalano-
ate (6b): From 2-chlorotrityl chloride resin (98.34 mg, 0.059 mmol,
loading: 0.60 mmol/g), culicinin D analogue 6b was obtained as a
white foamy solid after semi-preparative RP-HPLC purification
(4.17 mg, 4.25 μmol, 7%). Anal. RP-HPLC: tR = 50.27 min (Phe-
nomenex Gemini C18, 4.6ϫ250 mm, 5u, 1% B to 65% B at 1% B
purification (6.31 mg, 5.10 μmol, 4 %). Anal. RP-HPLC: tR
=
54.01 min (Phenomenex Gemini C18, 4.6ϫ150 mm, 5u, 1% B to
65 % B at 1 % B per min, flow rate 1 mL/min). HRMS (ESI+):
C63H116N11O13 [M + H]+ calcd./found 1234.8754/1234.8751,
C63H116N11O13Na [M + Na]+ calcd./found 1256.8574/1256.8564.
per min, flow rate 1 mL/min). MS (ESI+): C48H86N10O11 [M]+ Butanoyl-Pro[(S)-AHMOD]AibAib(AMD)LeuAibLeuβala(APAE)-
calcd./found 979.2/979.5.
OH (25b): From functionalized 2-chlorotrityl chloride resin 17
Eur. J. Org. Chem. 2015, 6341–6350
© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6349

M. A. Brimble et al.
FULL PAPER
Hayashi, T. Kimura, Y. Kiso, Bioorg. Med. Chem. 2005, 13,
6167–6174.
[8] a) H. Tamamura, T. Kato, A. Otaka, N. Fujii, Org. Biomol.
Chem. 2003, 1, 2468–2473; b) H. Tamamura, T. Hori, A.
Otaka, N. Fujii, J. Chem. Soc. Perkin Trans. 1 2002, 577–580.
[9] T. D. Owens, G.-L. Araldi, R. F. Nutt, J. E. Semple, Tetrahe-
dron Lett. 2001, 42, 6271–6274.
(127 mg, 0.0632 mmol, loading: 0.50 mmol/g) culicinin D analogue
25b was obtained as a white foamy solid after semi-preparative RP-
HPLC purification (1.02 mg, 0.83 μmol, 1.31%). Anal. RP-HPLC:
tR = 53.24 min (Phenomenex Gemini C18, 4.6ϫ150 mm, 5 µ, 1% B
to 75% B at 1% B per min, flow rate 1 mL/min). HRMS (ESI+):
C63H116N11O13 [M
+
H]+ calcd./found 1234.8754/1234.8732,
C63H116N11O13Na [M + Na]+ calcd./found 1256.8574/1256.8549.
[10] J. Lécaillon, P. Gilles, G. Subra, J. Martinez, M. Amblard, Tet-
rahedron Lett. 2008, 49, 4674–4676.
[11] J. Tailhades, N. A. Patil, M. A. Hossain, J. D. Wade, J. Pept.
Sci. 2015, 21, 139–147.
[1] a) T. Degenkolb, H. Brückner, Chem. Biodiversity 2008, 5,
1817–1843; b) C. d. l. Fuente-Núñez, L. Whitmore, B. A. Wal-
lace, in: Handbook of Biologically Active Peptides, 2nd edition
(Ed.: A. J. Kastin), Academic Press, Boston, USA, 2013, p.
150–156; c) B. Leitgeb, A. Szekeres, L. Manczinger, C.
Vágvölgyi, L. Kredics, Chem. Biodiversity 2007, 4, 1027–1051;
d) C. R. Röhrich, W. M. Jaklitsch, H. Voglmayr, A. Iversen,
A. Vilcinskas, K. F. Nielsen, U. Thrane, H. von Döhren, H.
Brückner, T. Degenkolb, Fungal Divers. 2014, 69, 117–146.
[2] G. Bills, Y. Li, L. Chen, Q. Yue, X.-M. Niu, Z. An, Nat. Prod.
Rep. 2014, 31, 1348–1375.
[3] a) M. Shi, H.-N. Wang, S.-T. Xie, Y. Luo, C.-Y. Sun, X.-L.
Chen, Y.-Z. Zhang, Mol. Cancer 2010, 9, 26; b) S. Ayers, B. M.
Ehrmann, A. F. Adcock, D. J. Kroll, E. J. Carcache de Blanco,
Q. Shen, S. M. Swanson, J. O. Falkinham, M. C. Wani, S. M.
Mitchell, C. J. Pearce, N. H. Oberlies, J. Pept. Sci. 2012, 18,
500–510.
[12] a) M. El Khatib, M. Elagawany, F. Jabeen, E. Todadze, O.
Bol’shakov, A. Oliferenko, L. Khelashvili, S. A. El-Feky, A.
Asiri, A. R. Katritzky, Org. Biomol. Chem. 2012, 10, 4836–
4838; b) Y. Sohma, T. Yoshiya, A. Taniguchi, T. Kimura, Y.
Hayashi, Y. Kiso, Biopolymers 2007, 88, 253–262; c) L. Mouls,
G. Subra, C. Enjalbal, J. Martinez, J.-L. Aubagnac, Tetrahe-
dron Lett. 2004, 45, 1173–1178.
[13] a) M. Rodriguez, M. Llinares, S. Doulut, A. Heitz, J. Martinez,
Tetrahedron Lett. 1991, 32, 923–926; b) A. Boeijen, J. van Ame-
ijde, R. M. J. Liskamp, J. Org. Chem. 2001, 66, 8454–8462.
[14] W. C. Chan, P. D. White, Fmoc solid phase peptide Synthesis,
Oxford University Press, New York, USA, 2000.
[15] K. Ben Haj Salah, N. Inguimbert, Org. Lett. 2014, 16, 1783–
1785.
[16] D. Liu, H. Lin, P. Proksch, X. Tang, Z. Shao, W. Lin, Org.
Lett. 2015, 17, 1220–1223.
[4] a) H. He, J. E. Janso, H. Y. Yang, V. S. Bernan, S. L. Lin, K.
Yu, J. Nat. Prod. 2006, 69, 736–741; b) K.-y. Hung, P. W. R.
Harris, M. A. Brimble, Org. Lett. 2012, 14, 5784–5787.
[5] J. Tailhades, M.-A. Gidel, B. Grossi, J. Lécaillon, L. Brunel, G.
[17] a) U. Orcel, M. De Poli, M. De Zotti, J. Clayden, Chem. Eur.
J. 2013, 19, 16357–16365; b) M. Schiell, J. Hofmann, M. Kurz,
F. R. Schmidt, L. Vertesy, M. Vogel, J. Wink, G. Seibert, J.
Antibiot. 2001, 54, 220–233.
Subra, J. Martinez, M. Amblard, Angew. Chem. Int. Ed. 2010, [18] L. Kredics, A. Szekeres, D. Czifra, C. Vágvölgyi, B. Leitgeb,
49, 117–120; Angew. Chem. 2010, 122, 121.
Chem. Biodiversity 2013, 10, 744–771.
[6] a) M. Skwarczynski, Y. Hayashi, Y. Kiso, J. Med. Chem. 2006,
49, 7253–7269; b) Y. Hamada, J. Ohtake, Y. Sohma, T. Kimura,
Y. Hayashi, Y. Kiso, Bioorg. Med. Chem. 2002, 10, 4155–4167.
[7] a) A. Taniguchi, Y. Sohma, M. Kimura, T. Okada, K. Ikeda,
Y. Hayashi, T. Kimura, S. Hirota, K. Matsuzaki, Y. Kiso, J.
Am. Chem. Soc. 2006, 128, 696–697; b) L. A. Carpino, E.
Krause, C. D. Sferdean, M. Schümann, H. Fabian, M. Bienert,
M. Beyermann, Tetrahedron Lett. 2004, 45, 7519–7523; c) Y.
Sohma, Y. Chiyomori, M. Kimura, F. Fukao, A. Taniguchi, Y.
[19] a) A. Boeijen, J. van Ameijde, R. M. J. Liskamp, J. Org. Chem.
2001, 66, 8454–8462; b) M. Rodriguez, M. Llinares, S. Doulut,
A. Heitz, J. Martinez, Tetrahedron Lett. 1991, 32, 923–926.
[20] T. James, P. MacLellan, G. M. Burslem, I. Simpson, J. A.
Grant, S. Warriner, V. Sridharana, A. Nelson, Org. Biomol.
Chem. 2014, 12, 2584–2591.
[21] C. S. Söderman, L. A. Schwan, Org. Lett. 2011, 13, 4192–4195.
Received: July 2, 2015
Published Online: August 18, 2015
6350
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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2015, 6341–6350