Total Synthesis of the Highly N-Methylated Acetylene-Containing Anticancer Peptide Jahanyne

Article abstract of DOI:10.1021/acs.orglett.7b03925

The first total synthesis of the highly N-methylated acetylene-containing lipopeptide jahanyne, an apoptosis-inducing natural product from marine cyanobacteria, is reported. A late-stage solution-phase coupling enabled introduction of the C-terminal keton

Full text of DOI:10.1021/acs.orglett.7b03925

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Total Synthesis of the Highly N-Methylated Acetylene-Containing
Anticancer Peptide Jahanyne
†
‡
†,§
,†,§,‡
‡
§,‡
Andrew Siow, George Opiyo, Iman Kavianinia, Freda F. Li, Daniel P. Furkert,
Paul W. R. Harris,† and Margaret A. Brimble*
,§
†
School of Biological Sciences, The University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
School of Chemical Sciences, The University of Auckland, 23 Symonds Street, Auckland 1010, New Zealand
Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, 3A Symonds Street, Auckland 1010, New Zealand
‡
§
*
S Supporting Information
ABSTRACT: The first total synthesis of the highly N-
methylated acetylene-containing lipopeptide jahanyne, an
apoptosis-inducing natural product from marine cyanobacteria,
is reported. A late-stage solution-phase coupling enabled
introduction of the C-terminal ketone pyrrolidine moiety. A
modified Fmoc solid-phase synthesis strategy was adopted to
effectively couple multiple sterically hindered N-methylated
amino acids while suppressing epimerization. The total
synthesis has enabled confirmation of the proposed absolute
configuration of natural jahanyne.
arine cyanobacteria are a well-known source of
structurally unique bioactive molecules. The biological
Scheme 1. Retrosynthetic Analysis of Jahanyne (1a)
1
,2
M
activity of these intriguing natural products ranges from
anticancer to antiviral activity, which makes these secondary
metabolites attractive leads for biomedical research and drug
2
discovery. Several of these compounds have been isolated,
such as dolastatin-10. Synthetic analogues of dolastatin-10 are
clinically used as payloads in antibody−drug conjugates for
3
cancer therapy. A small subset of these secondary metabolites
share an unusual terminal alkynyl fatty acid motif, including
dragonamide, kurahyne, viridamides, carmabin, almiramides,
4
−9
and jahanyne.
These heavily N-methylated acetylene-
containing lipopeptides exhibit a range of antimicrobial,
antifungal, and antitumor activities; hence, further studies
enabled by the chemical synthesis of this subfamily of
lipopeptides may afford new leads for drug discovery.
9
In early 2015, Suenaga et al. reported the isolation and
characterization of a novel acetylene-containing lipopeptide
jahanyne (1a) from the marine cyanobacterium Lyngbya sp.
(Scheme 1). Jahanyne (1a) contains the rare N-terminal
acetylene-containing fatty acid (2R,4S)-2,4-dimethyldec-9-ynoic
acid (2), a C-terminal (S)-1-(pyrrolidin-2-yl)ethan-1-one
(
Oep) (3), and multiple N-methylated amino acids. The
secondary metabolites. To the best of our knowledge, no
member of this family of acetylene-containing N-methylated
lipopeptides has been synthesized using a 9-fluorenylmethoxy-
carbonyl (Fmoc)-solid-phase approach. Currently, the only
methodology used to synthesize this class of lipopeptides is a
substitution of canonical amino acids for N-methylated amino
acids has been shown to alter biological functions of
polypeptides such as improved stability, binding, and oral
1
0−12
bioavailability.
This makes efficient incorporation of N-
methyl amino acids of paramount importance for peptide-based
drug discovery programs. However, the lack of efficient
methods to synthesize highly N-methylated and acetylene-
containing lipopeptides hampers detailed investigation of the
properties and the biomedical potential of this family of
4
,5
solution-based approach.
Received: December 17, 2017
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.7b03925
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
Herein, we describe the first total synthesis of jahanyne (1a)
using a combination of solid-phase peptide synthesis and a late
stage solution-phase coupling. It was envisaged that the total
synthesis of 1a would be achieved by a late stage convergent
solution-phase coupling of Oep 3 with the C-terminal fragment
the isolated natural product sample, was also synthesized (see
Supporting Information). The spectral data of both samples
were in excellent agreement, confirming that the fatty acid
moiety of the natural product possessed the 2,4-anti, (2R,4S)
9
configuration.
4
a (Scheme 1). Conceivably, intermediate 4a would be
The requisite C-terminal Oep 3 was synthesized following a
15
synthesized by the solid-phase coupling of 5 with the N-
terminal building block 2, followed by mild linker cleavage.
Assembly of the peptide chain 5 would be accomplished by a
linear Fmoc SPPS approach on resin.
literature protocol.
With the necessary building blocks in hand, we then focused
on elaboration of the N-methylated peptide sequence. The
particular challenge of synthesizing N-methylated peptides is
their poor reactivity in SPPS due to steric hindrance imposed
We envisaged that the required fatty acid 2 would be
prepared in six steps from commercially available 5-hexyn-1-ol
11,16
by the additional methyl group.
Yields are additionally
(6) (Scheme 2). Hence, the synthesis began by converting
compromised by diketopiperazine (DKP) formation which is
prevalent in both the N-methyl amino acid coupling and Fmoc-
16,17
Scheme 2. Synthesis of Fatty Acid Fragment 2
removal steps.
Furthermore, epimerization at the α-C
position of activated N-methyl amino acids during the coupling
11,16
step is also problematic.
We avoided using the tert-butyloxycarbonyl (Boc) protecting
group strategy for SPPS, due to the documented acid lability of
N-methylated amino acids, which are prone to fragment as well
17,18
as undergo DKP formation.
We therefore examined linkers
suitable for Fmoc SPPS that can be cleaved under mild
conditions and have sufficient steric hindrance on the solid
support to help suppress DKP formation.
Our first synthetic approach focused on using the 2-
chlorotrityl-functionalized polystyrene resin (2-CTC) pre-
loaded with Pro (0.62 mmol/g) at elevated temperatures to
promote coupling of hindered amino acids. Initial results
indicated that 2-CTC was unstable to microwave (MW)
conditions and the high temperature used in MW assisted
couplings led to hydrolysis of the chlorotrityl ester bond
resulting in premature cleavage of the peptide from the C-
1
3
alcohol 6 to iodide 7 via an Appel reaction. Asymmetric
alkylation of the enolate generated from 8 with iodide 7, under
Myers conditions, proceeded smoothly to afford amide 9.
Reductive cleavage of the chiral auxiliary in 9 afforded alcohol
1
4
19
terminus (see pp S16−S19 in Supporting Information).
Given our findings using 2-CTC, we next investigated the use
of the mild acid labile 4-(4-hydroxymethyl-3-methoxy-
phenoxy)butyric acid (HMPB) linker (Scheme 3). We attached
Fmoc-Pro-OH to preformed HMPB-aminomethyl polystyrene
resin 13 and obtained an acceptable loading of 0.4 mmol/g
(66%) based on the Fmoc-release assay (see p S8 in the
Supporting Information). The resulting HMPB handle of 14 is
cleavable under mild conditions using hexafluoroisopropanol
(HFIP) facilitating the ability to monitor the reaction by
1
0 which was then converted to 11. Iodide 11 was employed in
asymmetric alkylation of the enolate generated from ent-8 to
afford 12 thus installing the required 2,4-anti-dimethyl
stereochemistry in the alkyl chain of amide 12. Finally, basic
hydrolysis of pseudoephedrine amide 12 afforded the target
carboxylic acid 2 (dr = ca. 15:1). To confirm the absolute
stereochemistry, the (S)-phenylglycine amide of 2, prepared in
the original isolation paper from 2 obtained by degradation of
Scheme 3. Total Synthesis of Jahanyne (1a) and Analogue 1b
B
DOI: 10.1021/acs.orglett.7b03925
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
HPLC/MS analysis. Removal of the Fmoc group from the
Fmoc-N-methylated amino acids was carried out using the
hindered Fmoc-deblocking reagent 2-methylpiperidine (2-
using COMU/Oxyma and NMM, with a prolonged reaction
time of 3 h at room temperature, followed by HFIP cleavage to
afford 4b. We then successfully appended synthetic fatty acid 2
to peptidyl resin 5 using similar coupling conditions with that
of 4b. Treatment of the fully assembled resin-bound peptide
with 20 vol % HFIP in CH Cl , and analysis by HRMS,
2
0−22
MP).
prevent the risk of DKP formation or epimerization of sensitive
This was used as a precautionary measure to
2
3,24
amino acids.
Pleasingly, 2-MP quantitatively removed the
2
2
Fmoc-protecting group, in an acceptable reaction time (1 × 5
min, 1 × 7 min) with no detectable side reactions, and was used
for all subsequent Fmoc-removals.
confirmed the presence of the desired peptide 4a as the major
product (ca. 66% as judged by peak area integration of RP-
HPLC, λ = 210 nm, t = 59.6 min) (Figure 1). Fragmentation
R
Attention next turned to the optimal choice of coupling
reagents to efficiently couple sequential sterically hindered
Fmoc-protected N-methylated amino acids. For the majority of
the jahanyne sequence, a mixture of (1-cyano-2-ethoxy-2-
oxoethylidenaminooxy) dimethylaminomorpholinocarbenium
hexafluorophosphate (COMU) and ethyl 2-cyano-2-
(
hydroxyimino)acetate (Oxyma) was used as previously
25,26
reported to facilitate hindered amide bond formation.
Epimerization of 15 at the Phe residue from the COMU/
Oxyma mediated coupling between Fmoc-NMe-Phe-OH and
resin bound 14 could be negated by using 2,4,6-trimethylpyr-
idine (sym-collidine) as a weaker base (see Figure S7 in the
Supporting Information). However, use of MW irradiation was
critical to expedite the amidation of 14 to 15 and was used for
the following coupling steps involving Fmoc-protected N-
27
methylated amino acids. Fmoc-removal of 15 resulted in a
mixture of HPLC separable peaks with identical mass, which we
Figure 1. RP-HPLC analysis of crude 4a using a Gemini C-18 column
17,27,28
+
(
5 μm, 10 mm × 250 mm). HRMS (EI): m/z [M + H] calculated for
attributed to cis/trans amide conformers.
This isomerism
+
+
C H N O : 976.6481, observed: 976.6472; [M + Na] calculated
for C H N O Na : 998.6301, observed: 998.6317.
could be controlled by heating the HPLC column to 77 °C
resulting in a single peak due to accelerated cis/trans
interconversion, thereby enabling accurate monitoring of
54 86
7
9
+
54
85
7
9
27
reactions (see Figures S8−S9 in the Supporting Information).
of peptide 4a was observed during HRMS, as a consequence of
the labile NMe-Phe-Pro and NMe-Val-NMe-Phe amide bonds
(see p S30 in the Supporting Information). The resulting crude
peptide 4a was then directly subjected to the final convergent
solution-phase installation of the C-terminal Oep moiety.
We anticipated poor reactivity for the final solution-phase
coupling of Oep 3 to linear peptide 4a/4b, due to the
hydrophobic properties of our heavily N-methylated peptide
fragment and the need to effect formation of a tertiary amide
bond. We therefore conducted model studies using Fmoc-Pro-
OH to identify suitable coupling conditions. Initial studies
using N,N′-dicyclohexylcarbodiimide (DCC) or N-(3-
(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride
(EDC·HCl) to effect the coupling of Oep 3 to Fmoc-Pro-
OH were disappointing. However, condensation using N,N′-
diisopropylcarbodiimide (DIC) with 6-Cl-HOBt as an additive
using DIPEA as a base was successful.
Unfortunately, extension of these model conditions to effect
the desired coupling of Oep with 4a/4b were unsuccessful. It
was eventually determined that use of a modified cocktail of
DIC/6-Cl-HOBt/DIPEA and COMU/Oxyma with DIPEA
activation and MW irradiation successfully effected the
hindered condensation of 4a/4b with 3 (see Figures S26−
S27 in the Supporting Information). Subsequent purification of
the crude reaction mixture by RP-HPLC afforded jahanyne (1a,
Next, we focused on the synthesis of the adjacent sterically
demanding NMe-Val sequence. Attempts to couple the first
Fmoc-NMe-Val-OH to deprotected 15 using COMU/Oxyma
with sym-collidine under MW irradiation resulted in low
coupling efficiency. Use of N,N-diisopropylethylamine
(DIPEA) not only quantitatively coupled Fmoc-NMe-Val-OH
but also promoted epimerization. Employing N-methylmorpho-
line (NMM) and COMU/Oxyma cleanly yielded the desired
Fmoc-protected peptide 16 (see Figures S11−S13 in the
Supporting Information).
Disappointingly, despite extensive screening of optimal
coupling reagents, coupling of the second and third bulky
Fmoc-NMe-Val-OH to peptidyl resin 16 was low yielding (see
Table S1 in the Supporting Information). Pleasingly, the
HNMe-Val coupling steps were solved by generating the Fmoc-
amino acyl chlorides in situ using bis(trichloromethyl)carbonate
29
(
BTC) as reported by Falb et al. Fmoc-NMe-Val-OH in dry
THF was activated with BTC, and sym-collidine was then
added to neutralize the liberated HCl. DIPEA was also added to
resin bound 16 to prevent premature acidic cleavage from the
resin as well as promote the ensuing reaction. The activated
Fmoc-NMe-Val-Cl was added to the peptidyl resin pretreated
with DIPEA and stirred at room temperature for 4 h, resulting
in quantitative conversion to 17 with no detectable
1
6,29
epimerization.
The remaining amino acid couplings to
47%, > 98% purity, t = 54.2 min) (Figure 2). Pleasingly, the
R
afford resin bound 5 were effected using COMU/Oxyma and
appropriate base under MW irradiation.
Having established a reliable synthetic method to form the
linear backbone of jahanyne, we next investigated coupling of
the acetylene-containing fatty acid moiety 2 to the N-terminus
of 5. For model studies, we utilized commercially available 10-
undecynoic acid (10-UDYA) to couple with peptidyl resin 5
spectral data for synthetic 1a were observed to correspond well
with those originally reported for the natural product 1a (see
9
pp S43−S62 in the Supporting Information).
In summary, we have developed the first synthesis of
enantiopure jahanyne (1a) and an analogue 1b containing an
achiral alkynoic acid, via Fmoc SPPS. The synthesis described
herein not only confirmed the correct structure for the natural
C
DOI: 10.1021/acs.orglett.7b03925
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
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4
−8
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ASSOCIATED CONTENT
Supporting Information
■
*
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.7b03925.
(22) Ali, A. M.; Taylor, S. D. J. Pept. Sci. 2010, 16, 190−199.
(23) Brieke, C.; Cryle, M. J. Org. Lett. 2014, 16, 2454−2457.
(24) Szantai-Kis, M.; Walters, C. R.; Barrett, T. M.; Hoang, E. M.;
Petersson, E. J. Synlett 2017, 28, 1789−1794.
Experimental procedures and spectral data (PDF)
(25) Siow, A.; Hung, K.-y.; Harris, P. W.; Brimble, M. A. Eur. J. Org.
Chem. 2017, 2017, 350−354.
AUTHOR INFORMATION
Corresponding Author
(26) Kavianinia, I.; Kunalingam, L.; Harris, P. W.; Cook, G. M.;
Brimble, M. A. Org. Lett. 2016, 18, 3878−3881.
■
*
(27) Rodríguez, H.; Suarez, M.; Albericio, F. J. Pept. Sci. 2010, 16,
E-mail: m.brimble@auckland.ac.nz.
ORCID
1
36−140.
(28) Schnitzer, T.; Wennemers, H. J. Am. Chem. Soc. 2017, 139,
5356−15362.
1
(
Daniel P. Furkert: 0000-0001-6286-9105
Margaret A. Brimble: 0000-0002-7086-4096
Notes
29) Falb, E.; Yechezkel, T.; Salitra, Y.; Gilon, C. J. Pept. Res. 1999,
5
3, 507−517.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We acknowledge financial support from the Maurice Wilkins
Centre for Molecular Biodiscovery and The University of
Auckland for the award of a Doctoral Scholarship to G.O. The
̈
authors would also like to thank Chloe Marcourt (The
University of Auckland) for the synthesis of Oep.
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DOI: 10.1021/acs.orglett.7b03925
Org. Lett. XXXX, XXX, XXX−XXX