Solid-Phase Synthesis of the Peptaibol Alamethicin U-22324 by Using a Double-Linker Strategy

Article abstract of DOI:10.1002/ejoc.201601102

The facile Fmoc (Fluorenylmethoxycarbonyl) solid-phase synthesis of the α-helical-channel-forming peptaibol alamethicin U-22324 is described. A late-stage reduction by using a new double-linker method was used to introduce the C-terminal alcohol. Furthermore, we also report the synthesis of an alamethicin analogue with a C-terminal carboxylic acid.

Full text of DOI:10.1002/ejoc.201601102

A Journal of
Accepted Article
Title: Solid-Phase Synthesis of the Peptaibol Alamethicin U-22324 Using a Double
Linker Strategy
Authors: Margaret Anne Brimble; Andrew Siow; Paul Harris; Kuo-Yuan Hung
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601102
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Solid-Phase Synthesis of the Peptaibol Alamethicin U-22324
Using a Double Linker Strategy
Andrew Siow[a,b], Kuo-yuan Hung[a,b], Paul W. R. Harris[a,b], Margaret A. Brimble*[a,b]
Abstract: The facile Fmoc solid-phase synthesis of the α-helical channel-forming peptaibol alamethicin U-22324 is described. A late-stage
reduction using a novel double linker method was employed to introduce the C-terminal alcohol. Furthermore, we also report the synthesis of
an alamethicin analogue with a C-terminal carboxylic acid.
Introduction
Peptaibols are a class of nonribosomally synthesised peptides
derived from a variety of fungi.[1] Naturally occurring peptaibols
usually contain 4-20 residues and are structurally identified by
the presence of multiple α,α-dialkyl α-amino acids such as the
non-proteinogenic α-aminoisobutyric acid (Aib, B), which is
ubiquitous in peptaibols. They often also contain an acetylated
N-terminus and a β-amino alcohol C-terminus. One such
Figure 1. Structure of alamethicin U-22324 (1).
peptaibol
is
alamethicin
U-22324
(1)
(Ac-
BPBABAQBVBGLBPVBBEQ-Pheol) (Figure 1). This peptaibol 1
was first extracted from the culture broth of the fungus
Trichoderma viride in 1967 and was shown to be active against
Gram-positive bacteria and fungi.[2] Alamethicin U-22324 is a
member of the alamethicin family of peptaibols, which have
been extensively studied due to their vast range of biological
activities, including antibacterial and antifungal effects.[3] Various
model studies have been conducted to determine the mode of
action of the alamethicin class of peptaibols and it has been
postulated that these peptaibols interact with cell membranes by
Skrydstrup et al. reported the synthesis of 1 using a 2-
chlorotrityl chloride resin preloaded with phenylalaninol.[10]
Cyanuric fluoride was used to convert Fmoc-Aib-OH to its acid
fluoride reactive intermediate in situ and couplings were carried
out using microwave irradiation to afford 1 in 24% yield with >
90% purity. Kiso et al. utilised a solution phase synthesis
approach to prepare 1, using CIP with 1-hydroxy-7-
azabenzotriazole (HOAt) to execute the difficult Aib couplings.[11]
The full synthesis was then performed via a convergent
approach attaching four different fragments of the peptaibol in
solution to afford 1 in 16% yield. Given our group’s ongoing
interest in antimicrobial peptaibols (AMP)[12,13] we endeavoured
to undertake a total synthesis of alamethicin 1, using this as a
platform to develop a new method that enables facile formation
of the C-terminal alcohol.
forming
a voltage-gated ion-channel, due in part to the
hydrophobicity and the helical structure of the peptaibol,
resulting in the formation of transmembrane pores that leads to
cell lysis.[4-6]
The synthesis of this class of peptaibols has been challenging,
owing to the difficult couplings of the sterically hindered Aib
residues contained in these peptaibols.[7-9] Current methods to
synthesise alamethicin peptaibols use expensive coupling
Peptaibols containing C-terminal alcohols are ubiquitous in
nature and constitute an important class of biologically active
compounds.[14] Examples of these peptaibols include atroviridin,
trichokonin, polysporin or the alamethicin family that contain a
phenylalaninol C-terminus.[1] Current methods to form C-terminal
alcohols using SPPS are limited. One common method to form
C-terminal alcohols uses less readily available C-terminal amino
alcohols, which are preloaded to 2-chlorotrityl resin.[10] Due to
the sensitive nature of 2-chlorotrityl resin, microwave SPPS
conditions can result in premature cleavage of the peptide from
resin.[15] Although the synthesis of alamethicin analogue F50/5
reagents,
including
2-chloro-1,3-dimethylimidazolidinium
hexafluorophosphate (CIP), tetramethylfluoroformamidinium
hexafluorophosphate or Fmoc-amino acid fluorides.[10,11]
[a]
School of Biological Sciences, The University of Auckland,
3A Symonds St, Auckland 1142, New Zealand
E-mail: m.brimble@auckland.ac.nz
has been reported using
a 2-chlorotrityl resin, utilising
microwave irradiation at 70 °C throughout the synthesis.[16]
Furthermore, this method is compromised by the reported
difficulty to attach alcohols to 2-chlorotrityl resin resulting in low
yields.[17] Other methods to form C-terminal alcohols rely on O-N
acyl transfers, aminolysis of peptaibols on resin using amino
alcohol cleavage agents and reduction of peptides on resin.[17,18]
http://www.auckland.ac.nz/
[b]
Maurice Wilkins Centre for Molecular Biodiscovery, School of
Biological Sciences, The University of Auckland
Auckland 1142, New Zealand
Supporting information for this article is given via a link at the end of
the document.
We attempted to investigate a facile method to form the
corresponding C-terminal alcohol in situ by incorporating a late
stage reduction step using standard SPPS conditions. Herein,
we describe a successful synthetic approach to alamethicin 1
1
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European Journal of Organic Chemistry
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using a double linker reduction method as a general method to
alamethicin analogue
5
upon acidic cleavage using
form the β-amino alcohol moiety at the C-terminus.
TFA/DODT/H2O/TIPS (94:2.5:2.5:1, v/v/v/v) affording 5 in 21%
yield after HPLC purification with >98% purity (see Supporting
Information).
Results and Discussion
Our first synthetic route (Scheme 1) incorporated 4-
hydroxymethylbenzoic acid (HMBA) as a linker for peptide
synthesis, which can be cleaved by basic hydrolysis using
NaOH to form a C-terminal acid or reduced at the ester bond
between HMBA and the peptide to form the corresponding C-
terminal alcohol. We therefore envisaged that reduction of this
linker with sodium borohydride (NaBH4) could provide the C-
terminal alcohol for the synthesis of 1. Disappointingly, the on
resin reduction was found to be low yielding for a six residue
peptaibol (see Supporting Information). Further difficulties were
encountered using this method as attempts to optimise coupling
conditions could not be monitored easily by LCMS, due to the
difficulty of cleaving the peptide from the HMBA linker.
Scheme 2. Use of acid cleavable Fmoc-Phe-HMPP linker to afford analogue 5.
In an attempt to develop an alternative approach to effect a C-
terminal reduction accessing the key phenylalaninol residue, we
turned to work previously performed by our group. We have
utilised a Rink amide linker resin to form Fmoc-Arg-HMBA-
(Arg)6-NH2 in which the electron withdrawing HMBA was still
attached to the sequence.[21] We chose to adapt this model for
our work using the Rink amide linker which could be cleaved
under acidic conditions to enable monitoring of the reaction
progress (Scheme 3). Given the successful use of NaBH4 for the
reduction of N-protected α-amino acid carboxylic-carbonic
anhydrides to form the corresponding alcohol,[20] we envisioned
that use of NaBH4 on our proposed peptide sequence in which
the HMBA electron deficient linker was still attached, would
result in reduction of the ester bond between the peptide
sequence and the HMBA linker to form the corresponding
alcohol at the C-terminal phenylalanine residue.
Scheme 1. Use of HMBA linker for on-resin reduction.
In an alternative synthetic approach, we envisioned that the
use
of
the
commercially
available
Fmoc-Phe-3-(4-
Upon adoption of our final synthetic route (Scheme 3), initial
attempts to prepare the HMBA-Rink amide linker were carried
out by first attaching Rink amide linker 7 to aminomethyl resin
using DIC/6-Cl-HOBt in DMF, followed by Fmoc deprotection to
form 6. The next step required forming the double linker by
coupling the electron deficient HMBA directly to the Rink amide
resin 6. This was achieved using DIC/6-Cl-HOBt in DMF to
afford 8. With the double linker resin 8 in hand, Fmoc-Phe-OH
was anchored to 8 using the aforementioned coupling conditions
for 2. The resin was then treated with acetic anhydride/DMF
(20:80, v/v) to cap any remaining free amino groups. With the
first Fmoc-protected amino acid attached to our double linker
complex 9, analysis was feasible due to facile acidic cleavage of
the Rink amide resin from HMBA that enables LCMS analysis.
(hydroxymethyl)phenoxy)propanoic acid (HMPP) linker 3 would
provide an acid labile moiety that would enable us to monitor the
peptide coupling reactions by LCMS, enabling optimization of
the coupling conditions for the synthesis of 1 (Scheme 2). To
design our acid cleavable peptide, HMPP linker 3 was attached
to the aminomethyl resin using N,N’-diisopropylcarbodiimide
(DIC) followed by Fmoc deprotection to afford 4. Preliminary
tests showed that coupling the subsequent Fmoc-Gln(Trt)-OH
using benzotriazole derivative O-(7-azabenzotriazol-1-yl)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU) to 4
was feasible. The next step encountered a difficult coupling
between Fmoc-Glu(OtBu)-OH and resin bound NH2-Gln(Trt).
Extensive trials demonstrated that the coupling reagent
(benzotriazol-1yloxy)tris(dimethylamino)phosphonium
hexafluorophosphate (BOP) effectively coupled the amino acids
with no uncoupled dipeptide observed. The next challenge was
the coupling of the repeating sterically demanding Aib units;
previous attempts to couple consecutive Aib residues using
HATU resulted in deletions of Aib (see Supporting Information).
A more efficient reagent system was screened using a mixture
Given the successful installation of the double linker on resin,
the synthesis of the peptaibol sequence using the same Fmoc
SPPS coupling conditions used for the synthesis of alamethicin
analogue 5 was carried out (see Supporting Information). The
full sequence of amino acids was attached with no noticeable
side products affording crude 10 in reasonable purity (Figure 2).
With our peptide sequence fully assembled our next focus was
the key reductive cleavage of HMBA from peptaibol 10 to afford
the C-terminal alcohol of alamethicin 1. Initial attempts to
perform the reduction of HMBA complex 10 using NaBH4
resulted in poor yields as determined by LCMS of the crude
sample, due in part to the poor solubility of 1. i-PrOH was
determined to be the solvent of choice and the reduction of 10
was successfully achieved using a protocol adapted from that
used to effect reduction of mixed anhydrides of N-protected α-
amino acids.[20] A sample of 10 was dissolved in i-PrOH and a
solution of NaBH4 in H2O was added dropwise at 0 °C. This
reduction method successfully formed the C-terminal alcohol
affording alamethicin 1 with the remaining peak attributed to
ester hydrolysis product 5, which can be easily separated by
RP-HPLC (Figure 3A).
of
(1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-
morpholino-carbenium hexafluorophosphate (COMU)/ ethyl 2-
cyano-2-(hydroxyimino)acetate (Oxyma). This combination was
trialled due to its reported success to facilitate amide bond
formation between sterically demanding Aib residues.[12,19] Using
a COMU/Oxyma mixture proved sufficient in facilitating the
coupling between Aib residues and was utilised for ensuing Aib
couplings, instead of relying on the other known documented
method of using a DIC/Oxyma mixture.[16]
Using our optimized coupling conditions the remaining
residues of 1 were attached to the growing peptide chain (see
Supporting Information). However attempts to cleave the final
sequence from the HMPP-linker resin using NaBH4 to form
phenylalaninol failed,[20] in part due to the electron rich nature of
the HMPP linker. This method however, did allow us to form
2
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European Journal of Organic Chemistry
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Scheme 3. Synthesis of 1 using the double linker method.
Figure 2. LCMS profile of crude 10. (Ion polarity positive); (m/z [M+2H]2+ calcd:
1056.1; found 1056.6).
U-22324 was isolated in 18% yield after purification by HPLC
with >98% purity, which is comparable to the most recent
reported yield for 1.[10] Compound 1 was identified by LCMS (m/z
[M-H]- calculated: 1962.1; observed: 1962.8) (Figure 3B).
A sample of synthetic 1 was co-injected with a commercially
available sample of alamethicin 1 obtained from fermentation of
Trichoderma viride and showed no significant deviation in
retention time on HPLC (Figure 4). Samples were analysed by
LRMS and showed similar mass signals (see Supporting
Information).
Both the LRMS and HRMS for 1 (in positive mode) displayed
fragment peaks at m/z 1189.6942 and m/z 775.4361 (see
Supporting Information), that corresponded to cleavage of the
tertiary amide bond between Aib and Pro (Figure 5).[22-24] This
cleavage leads to the predicted diprotonated N-terminal ion y7
fragment and C-terminal acylium ion b13.[24]
Figure 3. LCMS profile of (A) crude after reduction, 5 was identified as side
product due to hydrolysis of 10, (Ion polarity negative); (m/z [M-2H]2- calcd:
987.5; found 987.7). (B) Purified 1. (Ion polarity negative); (m/z [M-H]- calcd:
1962.1; found: 1962.8).
3
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European Journal of Organic Chemistry
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In summary, we have successfully synthesised alamethicin 1 via
late-stage reduction of a new double linker system, that enabled
the formation of the C-terminal phenylalaninol residue which is
ubiquitous in many bioactive peptaibols.[1,3] We also document
the synthesis of alamethicin analogue
5 using an acidic
cleavage method. The work reported herein provides a useful a
new method to prepare peptides that contain C-terminal alcohol
residues.
Experimental Section
Procedure for attachemnt of HMBA to 6: To Fmoc-deprotected Rink
amide resin 6 (0.1 mmol) pre-swollen in DMF (5 mL, 5 min), was added
4-hydroxymethylbenzoic acid (HMBA) (61 mg, 0.4 mmol) and 6-Cl-HOBt
(132 mg, 0.8 mmol) dissolved in DMF (1.5 mL), followed by addition of
DIC (77.5 µL, 0.5 mmol). The reaction mixture was gently agitated at
room temperature for 2 h. The resin was filtered and washed with DMF (3
x 3 mL) to afford 8.
Figure 4. HPLC profile of co-injection mixture of synthetic 1 and commercial
alamethicin 1.
Procedure for the attachment of Fmoc-Phe-OH to the 8: To HMBA
resin 8 (0.1 mmol) pre-swollen in DMF (5 mL, 5 min), was added a
solution of Fmoc-Phe-OH (78 mg, 0.2 mmol) in DMF (1 mL). To the
reaction mixture was added DMAP (1.22 mg, 0.009 mmol) dissolved in
DMF (100 µL), followed by addition of DIC (31 µL, 0.2 mmol). The
reaction mixture was gently agitated at room temperature for 2 h, filtered
and repeated once for a further 2 h with fresh reagents. The resin was
filtered and washed with DMF (3 x 3 mL) and CH2Cl2 (3 x 3 mL) to afford
9.
Surprisingly, the other Aib-Pro bond of alamethicin displayed no
such fragmentation as determined by LCMS. The commercial
sample also displayed fragment peaks under LRMS (in positive
mode) at m/z 1189.6 and m/z 774.4 (see Supporting
Information). This fragmentation also affected the Aib-Pro bond
of alamethicin 5 displaying fragment peaks b13 m/z 1189.7 and
y7 m/z 789.4 (see Supporting Information).
General Procedure for Fmoc-SPPS: Please refer to Supporting
Information for synthesis of 10.
Procedure for reduction of HMBA 10 complex: Alamethicin-HMBA-
NH2 10 (316 mg, 0.2 mmol) was dissolved in isopropanol (16 mL) and a
solution of NaBH4 (0.45 g, 12 mmol) in H2O (10 mL) was added dropwise
at 0 °C and stirred for 20 min. The resulting reaction solution is quenched
with 12 M HCl (800 µL) to pH 2 and the aqueous layer was extracted with
EtOAc (2 x 5 mL) and concentrated in vacuo. The resulting residue
(557.8 mg) was diluted with H2O/CH3CN (1:1, 10 mL) and lyophilised.
Purification by RP-HPLC using a semipreperative Gemini C-18 column
(Phenomenex, 5 µm, 10.0 x 250 mm) gave peptide 1 (50 mg, 18% based
on a 0.1 mmol scale); tR = 24.25 min. ESI-MS(m/z [M-H]- calcd: 1962.1;
found: 1962.8).
Figure 5. Fragmentation of U-22324 to form b13 and y 7 fragment ions.
Acknowledgments
We acknowledge financial support from the the Maurice Wilkins
Centre for Molecular Biodiscovery. The authors would also like
to thank Professor Gregory M. Cook and Ayana Menorca for
antimicrobial compound testing.
The antimicrobial (MIC) values of synthetic alamethicin 1 and
analogue 5 against Gram-positive S. aureus and Gram-negative
E. coli were determined (Table 1). The antimicrobial activity for
synthetic peptaibol 1 were less potent than those reported in the
literature.[2] The antimicrobial assay results showed that
Keywords: Alamethicin • C-terminal alcohol • Reduction •
Double linker • Phenylalaninol
replacing the C-terminal alcohol in
1 for the C-terminal
carboxylic acid in 5 had no significant effect on the activity.
Interestingly recent studies have documented that substitution of
native amino acids in alamethicin with β-silicon-β3-amino acids
provides a 20-fold increase in membrane permeability,[25] and
incorporation of α,α-substituted disilylated amino acids modified
alamethicin analogues physicochemical properties.[26] These
method could be applied to future alamethicin studies.
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