Total synthesis of biseokeaniamides A-C and late-stage electrochemically-enabled peptide analogue synthesis

Article abstract of DOI:10.1039/d0sc03701j

The first total synthesis of cytotoxic cyanobacterial peptide natural products biseokeaniamides A-C is reported employing a robust solid-phase approach to peptide backbone construction followed by coupling of a key thiazole building block. To rapidly access natural product analogues, we have optimized an operationally simple electrochemical oxidative decarboxylation-nucleophilic addition pathway which exploits the reactivity of native C-terminal peptide carboxylates and abrogates the need for building block syntheses. Electrochemically-generated N,O-acetal intermediates are engaged with electron-rich aromatics and organometallic reagents to forge modified amino acids and peptides. The value of this late-stage modification method is highlighted by the expedient and divergent production of bioactive peptide analogues, including compounds which exhibit enhanced cytotoxicity relative to the biseokeaniamide natural products. This journal is

Full text of DOI:10.1039/d0sc03701j

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DOI: 10.1039/D0SC03701J
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Total Synthesis of Biseokeaniamides A-C and Late-Stage
Electrochemically-Enabled Peptide Analogue Synthesis
Yutong Lina and Lara R. Malins*a
Received 00th January 20xx,
Accepted 00th January 20xx
The first total synthesis of cytotoxic cyanobacterial peptide natural products biseokeaniamides A-C is reported employing a
robust solid-phase approach to peptide backbone construction followed by coupling of a key thiazole building block. To
rapidly access natural product analogues, we have optimized an operationally simple electrochemical oxidative
decarboxylation-nucleophilic addition pathway which exploits the reactivity of native C-terminal peptide carboxylates and
abrogates the need for building block syntheses. Electrochemically-generated N,O-acetal intermediates are engaged with
electron-rich aromatics and organometallic reagents to forge modified amino acids and peptides. The value of this late-stage
modification method is highlighted by the expedient and divergent production of bioactive peptide analogues, including
compounds which exhibit enhanced cytotoxicity relative to the biseokeaniamide natural products.
DOI: 10.1039/x0xx00000x
Introduction
The rich structural diversity of peptide natural products extends
well beyond the canonical amino acids to the incorporation of
modified residues and rigidifying elements—motifs which are
often crucial to bioactivity. Replicating and expanding on
Nature’s chemical arsenal is an aspirational goal of synthetic
chemistry, and an endeavor further fuelled by the growing
importance of peptide natural products as lead structures for
therapeutic development.1 Nevertheless, the optimization of
strategies for the direct, late-stage chemical modification of
complex peptides is challenging, particularly methods which
exploit proteinogenic functionalities for the mild and selective
diversification of native peptide substrates.2
In our efforts to develop new strategies for the late-stage
modification of peptides, we have become particularly
interested in C-terminal modifications owing to the
fundamental importance of C-terminal composition to peptide
and protein bioactivity.3 The prospect of exploiting the
ubiquitous C-terminal peptide carboxylate motif for direct
access to differentially functionalized peptide products provides
additional impetus for the development of new synthetic tools.4
In the course of these endeavors, we identified marine
Scheme 1. A) The biseokeanamide natural products; B) An electrochemical
approach to late-stage peptide modification for the rapid synthesis of natural
product analogues.
cyanobacterial natural products biseokeaniamides A-C (1a-c,
pattern of N-methylation, thus offering additional opportunities
for probing the effects of N-methylation on peptide
conformation and hydrophobicity, important features in
overcoming the conventional liabilities of peptide drugs (e.g.
poor bioavailability and membrane permeability).6 Importantly,
Scheme 1A) as promising targets for methodology
development. Isolated from Okeania sp. cyanobacterium in
2017,5 these lipopeptides feature an intriguing C-terminal
thiazole motif as well as an N-terminal lipid chain and a heavily
N-methylated backbone. The analogues vary only in their
compounds 1a-c also exhibit moderate cytotoxicity and were
shown to inhibit sterol O-acyltransferase (SOAT), an enzyme
which mediates the esterification of cholesterol and is
implicated in hypercholesterolemia and atherosclerosis—
disease states which involve the accumulation of cholesterol
a.Research School of Chemistry, Australian National University, Canberra, ACT
2601, Australia. Email: lara.malins@anu.edu.au
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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Journal Name
NHMe
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S
N
O
3
DOI: 10.1039/D0SC03701J
O
3:7 (v/v)
HFIP/DCM
Fmoc-N(R1)-Val-OH
DIEA, DMF
O
N
nButyric acid
(3 steps to prepare)
N
O
SPPS
Cl
R2
N
O
microwave
amide-bond
formation
or pre-activation
Me HN
OH
Oxyma/DIC
DMF
N
peptide
cleavage
iterative
peptide
assembly
2-chlorotrityl
chloride
resin
resin
loading
lipidation
R1
O
O
See SI for
optimization
5 oC, 16 h
Yields from resin loading:
2a: 18%; 2b: 28%; 2c: 18%
Iterative Fmoc-SPPS Conditions
ꢀ = 230 nm
1b
Biseokeaniamides A-C
O
Deprotection: 1:9 (v/v) piperidine/DMF, rt, 2 x 3 min
Coupling: Fmoc-AA-OH, Oxyma pure®, DIC, DMF, rt, 16 h
Capping: 1:9 (v/v) Ac2O/pyridine, rt, 3 min
O
N
N
O
R2
N
O
Me
S
Lipidation: A) nButyric acid-NHS ester, DIEA, DMF, rt, 16 h
B) nButyric acid, Oxyma pure®, DIC, 50 oC, 200 W, ꢀ -wave, 3 h
Me HN
N
N
N
Resin cleavage: 3:7 (v/v) HFIP/DCM, rt, 2 h
2
2
4
6
6
8
8
1a: R1 = Me, R2 = Me, 67%
1b: R1 = H, R2 = Me, 53%
1c: R1 = Me, R2 = H, 69%
4
Retentiontime(min)
R1
O
Scheme 2. Total synthesis of biseokeaniamides A-C employing iterative Fmoc-SPPS followed by coupling of thiazole building block 3.
esters. The novel structural features together with generation approach to the biseokeaniamides could be
opportunities to probe structure-activity relationships of accomplished through the synthesis of truncated acid
natural product analogues prompted the undertaking of a derivatives 2a
synthesis campaign. Our goals were two-fold: 1) Complete the building block
first total synthesis of biseokeaniamides A-C; 2) Explore new
-
3
2c followed by coupling of a preformed thiazole
(Scheme 2).
methods for late-stage, C-terminal analogue synthesis which Accordingly, Fmoc-Val-OH and Fmoc-Me-Val-OH were first
directly exploit the reactivity of C-terminal carboxylic acids. loaded onto the highly acid-labile 2-chlorotrityl chloride resin to
Herein, we disclose the realization of these endeavors through account for the differential backbone methylation patterns of
an efficient solid-phase approach to biseokeaniamides A-C and biseokeaniamides A and C (R1 = Me) and biseokeaniamide B (R1
the development of
a strategy for C-terminal peptide = H). The resins were elongated using manual Fmoc-SPPS. Due
modification which leverages a key electrochemical oxidative to the heavily N-methylated backbone structures of the
decarboxylation step to generate reactive N,O-acetals capable biseokeaniamides and the preponderance of bulky hydrophobic
of engaging a diverse array of nucleophiles (Scheme 1B). This residues (e.g. Val, Leu), each coupling was carried out for 16 h
direct strategy for the diversification of C-terminal peptide acids using the highly activating coupling combination of Oxyma
is applied to peptide substrates and biseokeaniamide Pure® and DIC.16 Lower yields and incomplete couplings were
analogues, leading to a collection of natural product derivatives, observed with alternative reagents (e.g. PyBOP). The N-terminal
including those which exhibit enhanced cytotoxic activity nbutyric acid lipid tail was coupled on-resin using Oxyma/DIC
relative to the natural products.
under microwave irradiation (when R2 = Me) or through the
coupling of a preactivated nbutyric acid-NHS ester (when R2 =
H) (see supporting information for details). Liberation of
Results and discussion
peptides 2a-2c from the resin was accomplished by treatment
with HFIP in DCM, affording the truncated natural products in
18-28% isolated yield (based on the original resin loadings)
following reverse-phase HPLC purification.
First total synthesis of biseokeaniamides A-C
In designing a synthetic approach to the biseokeaniamides, we
envisioned that the robust nature of solid-phase peptide
synthesis (SPPS) could be exploited to rapidly construct the
peptide backbone structure. A handful of other C-terminal
thiazole-containing peptide natural products have been
isolated from marine organisms (e.g. cytotoxic peptides
dolastatin 107 and symplostatin 1,8 barbamide,9 lyngbyapeptins
A,10 B and C11, the highly backbone N-methylated apramides A-
G,12 micromide,13 and virenamides A-E14) and several have been
the subject of recent total synthesis campaigns.13, 15 However,
to the best of our knowledge, none have exploited a solid-phase
approach to peptide backbone construction. Since conventional
solid-phase methods require immobilization of the C-terminal
peptide acid and elongation in the C- to N- direction,
incorporation of the terminal thiazole motif must necessarily
occur after resin cleavage. As such, we envisioned a rapid, first-
Thiazole
3 was prepared in three steps according to literature
methods,13 and a variety of coupling conditions and reactant
stoichiometries were screened with truncated biseokeaniamide
C peptide 2c in order to reduce epimerization—a notable
drawback of peptide elongation in the N- to C-direction.
Standard protocols involving preactivation of the C-terminal Val
residue in 2c with Oxyma/DIC and subsequent treatment with
excess thiazole
integrity at the Val
However, optimization of reaction temperature (5 oC) and
elimination of the preactivation step preferentially afforded the
3
led to complete loss of stereochemical
-position (see supporting information).

natural product over the corresponding
85:15). With optimal conditions in hand, thiazole
to precursor peptides 2a 2c, to afford the natural products 1a
D
-Val epimer (d.r. =
3
was coupled
-
-
2 | J. Name., 2012, 00, 1-3
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1c in 53-69% yield following HPLC purification, which enabled acetals—as common intermediates for the divergVeienwtAsryticnlethOenlsinies
facile removal of the minor diastereomer. Importantly, spectral of natural product analogues—would be DaOvIa: l1u0a.1b0l3e9/aDd0dSCit0io3n70t1oJ
data for the three compounds is fully consistent with that the existing toolbox of late-stage peptide modifications.
reported for the original isolates (see supporting information).5
Electrochemical decarboxylation of a sarcosine model
Devising a strategy for C-terminal modification
To probe the feasibility of the proposed electrochemical
Although our optimized methods enabled robust access to the oxidative decarboxylation and evaluate the scope of suitable
natural products, the inability to fully suppress epimerization in nucleophiles, we first examined the reactivity of Boc-sarcosine
the coupling of the terminal thiazole was a powerful motivation
(5) as a model substrate. Compound 5 was dissolved in
for further exploration of late-stage installation protocols which methanol and treated with triethylamine (15 mol%) for in situ
would circumvent the need for peptide extension in the N- to C- generation of the electrolyte.21b Electrolysis was performed at
direction. Existing syntheses of C-terminal thiazole-containing constant current (8 mA) in an undivided electrochemical cell
natural products have largely exploited building block equipped with carbon electrodes (see supporting information).
approaches, particularly in cases where the thiazole component Mechanistically, this non-Kolbe electrolysis likely proceeds
bears defined
sarcosine (N-methyl glycine)-derived terminal thiazole, the loss of CO2 to afford a stabilized
preservation of -chirality upon incorporation of the aryl motif oxidation affords the N-acyliminium, which is trapped by the

-chirality.15 As the biseokeaniamides feature a through an initial oxidation of the carboxylate with concomitant
-nitrogen radical. A second


is not a prerequisite, thus opening additional opportunities for solvent methanol (Scheme 3).21b Accordingly, concentration of
late-stage installation. Such an approach would avoid the the crude electrolysis reaction afforded the methanol N,O-
preparation of a discrete thiazole building block and open new acetal 6a. As attempts to purify 6a on silica gel led to
vistas for the divergent functionalization of a common peptide decomposition, the material was used crude in subsequent
precursor, facilitating rapid access to natural product transformations.
analogues. This attractive late-stage functionalization logic has
been a prominent feature of several recent approaches to
modified peptides.2
To this end, our attention first turned to the feasibility of direct
Scheme 3. Proposed mechanism of the electrochemical oxidative
thiazole incorporation using decarboxylative cross-coupling
chemistry—a robust approach to C–C bond formation,17
including in the diversification of peptides.18 Preparation of a
biseokeaniamide precursor (bearing a carboxylic acid in place of
decarboxylation to forge N,O-acetal intermediates.
Table 1. Probing the electrochemical generation and reactivity of N,O-acetals.
the C-terminal thiazole motif, see
4 Scheme 5, vide infra) and
activation as the corresponding redox-active ester19 were
followed by treatment with various nickel catalysts and
organothiazole derivatives. Unfortunately, in our hands,
attempts at decarboxylative arylation were unsuccessful, likely
owing to the thermal instability of the thiazole-derived
organozinc reagent.20 Nevertheless determined to exploit the C-
terminal carboxylate functionality, we were encouraged by
initial reports from Seebach and coworkers disclosed in the late
1980s describing the electrochemical oxidative decarboxylation
of amino acids and small peptides in the presence of methanol
to forge N,O-acetals (e.g. Boc-Ala-N(Me)-methoxymethyl
acetal, derived from Boc-Ala-Sar-OH and MeOH).21 Acetal
intermediates could be engaged (via in situ formation of the
corresponding N-acyliminium) with various nucleophiles,
including phosphites,22 allylsilanes, and simple Grignard
reagents.21 Notably, in recent years electrochemical
transformations have attracted considerable attention as mild,
tunable, and sustainable complements to conventional
synthetic chemistry.23 However, there remains remarkably few
examples of the exploitation of anodic oxidation on peptide
Entry
R =
pKa (R–OH)27, 28
N,O-acetal
Yield of 7a
1
2
3
4
5
6
7
8
9
Meb
15.5
12.5
9.3
6a
6b
6c
6d
6e
6f
n.d. (46%c)
53%
d
CF3CH2
(CF3)2CHd
Acd
69%
4.76
4.2
65%
Bz
71%
CHO
3.77
2.86
1.29
0.85
47%
ClCH2CO
Cl2CHCO
Cl3CCO
6g
6h
6i
73%
66%
11%
aYield determined by 1H NMR using dibromomethane as an internal standard,
0.1 mmol scale; n.d. = not determined; bneat methanol used as solvent; Friedel-
c
Crafts reaction under -wave irradiation (50 oC), isolated yield (see supporting
information for details); d0.05 mmol scale.
The reactivity of the methanol-derived N,O-acetal 6a was
examined using a Friedel-Crafts-type reaction with thiophene in
the presence of TFA (2.0 equiv.), which serves to regenerate the
N-acyliminium for nucleophilic attack. Interestingly, under
these reaction conditions, no arylated product
(Table 1). We hypothesized that N,O-acetal 6a was not
substrates aside from electrolyses of cyclic dipeptide24 and
lactam derivatives25 and work from Moeller leveraging Shono-
-
type
oxidations
of
peptide
analogues
bearing
7 was observed
electroauxiliaries.26 We therefore envisioned that broader
application of electrochemically-generated peptide N,O-
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DOI: 10.1039/D0SC03701J
Scheme 4. Electrolysis of Boc-Sar-OH to generate N,O-acetal intermediates followed by diversification with various classes of nucleophiles.
sufficiently reactive at room temperature to generate the we next probed direct organometallic addition to the acetal
requisite N-acyliminium under the conditions employed, or that intermediates. Although Grignard additions to amino acid-
methanol was outcompeting thiophene as a nucleophile leading derived N,O-acetals have been explored,21a the scope of
to reversible generation of the starting N,O-acetal. However, organometallic reagents (e.g. methylmagnesium chloride,
o
repeating the reaction under microwave irradiation (50 C) led cyclohexylmagnesium bromide) is limited. Inspired by the
to productive formation of thiophene
7. As an alternative structure of the biseokeaniamides, we focused our attention on
approach to enhancing reactivity at room temperature, we the addition of thiazole-derived organometallic nucleophiles so
reasoned that electrolysis in the presence of alcohols with lower as to devise an alternative approach to the late-stage
pKa values27 (e.g. better leaving groups) might afford N,O- installation of C-terminal thiazoles. After extensive
acetals with superior reactivity in the arylation reaction (see optimization, including screening of various N,O-acetals, we
Table 1) owing to more facile formation of the key N- were able to obtain thiazole derivative 12 in a modest 28% yield
acyliminium intermediate (see Scheme 3). Pleasingly, the novel over the two steps. The optimal method employed BF3·OEt2 for
trifluoroethanol (TFE) (pKa = 12.5)28-derived acetal 6b reacted the generation of the reactive N-acyliminium at –78 oC followed
with thiophene at ambient temperature to afford 7 in 53% yield by the addition of thiazole-2-cuprate, generated in situ from the
over the two steps (entry 2). Electrolysis in the presence of corresponding organo-lithium reagent. Notably, treatment of
hexafluoroisopropanol (HFIP) (pKa = 9.3)28 afforded N,O-acetal 12 with TFA led to facile deprotection of the Boc group,
6c, which proceeded to product
7 in 69% yield over the two providing an alternative route to key building block 3 (see
steps (entry 3). To further probe the correlation between supporting information). Sulfonylation of the original methanol
enhanced yield and leaving group ability of intermediate N,O- N,O-acetal 6a likewise afforded 13 in 71% yield and provided an
acetals, various carboxylic acids (e.g. acetic acid, entry 4; indirect approach to organometallic addition.29 It is intriguing
benzoic acid, entry 5; formic acid, entry 6; and chloroacetic acid that room temperature activation of 6a was possible under the
derivatives, entries 7-9) were also screened. While pKa and yield sulfonylation conditions (PhSO2H, CaCl2) given the recalcitrance
were not correlated in all cases, owing to the volatility of certain of 6a to TFA-promoted Friedel-Crafts reaction with thiophene—
intermediate N,O-acetals (e.g. 6f, R = CHO) and the subtle an observation which suggests that, in addition to pKa, the
interplay between enhanced reactivity and a tendency toward method of activation is an important determinant of acetal
competitive hydrolysis (e.g. 6i, R = Cl3CCO), it is notable that the reactivity. Engaging intermediate sulfone 13 with arylzinc
highest overall yield (73%) was obtained with chloroacetic acid reagents readily forged the target arylated products, including
(entry 7, pKa = 2.8627). Considering both operational simplicity anisole derivative 11 (obtained as a single regioisomer and in
and ease of handling, MeOH, TFE, HFIP, and acetic acid-derived higher yield than the corresponding Friedel-Crafts approach) as
acetals 6a-6d were identified as the most viable N,O-acetal well as N-methylindole 14. The instability of the thiazole zinc
intermediates in subsequent chemical transformations.
reagent at room temperature, however, precluded the
application of this method to the synthesis of thiazole derivative
.
Encouraged by initial results with thiophene, we explored 12
several additional electron-rich aromatic nucleophiles in
Friedel-Crafts reactions. Using acetic acid-derived N,O-acetal
Late-stage modification of a model tetrapeptide
6d
,
dimethylaniline
(
8
), 2,6-dimethoxytoluene
(9
)
and
The utility of model N,O-acetals as divergent precursors to
thioanisole
(
10)-derived carbamates were accessible in
arylated products
7-14 next prompted extension of the
moderate yields (Scheme 4). Anisole 11 afforded a mixture of
ortho/para-substituted products in 45% yield via the TFE-
derived N,O-acetal 6b. In the interest of expanding the scope of
arylation chemistry beyond electron-rich aromatic substrates,
chemistry to model peptide substrates. N-acyl tetrapeptide 15
,
featuring a C-terminal sarcosine residue, bears structural
similarity to the biseokeaniamides and served as a practical
model for the screening of conditions (Scheme 5A). Remarkably,
4 | J. Name., 2012, 00, 1-3
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Electrochemically-enabled late-stage peptide modifications
View Article Online
DOI: 10.1039/D0SC03701J
A) Model tetrapeptides:
[non-Kolbe
electrolysis]
Thiophene
DCM
(9:1 v:v)
TFA
MeOH, Et3N
4 mA, rt, 5 h
O
O
O
AcHN
O
Me
AcHN
O
Me
AcHN
O
Me
O
S
50 oC, MW
5 h
HN
N
HN
N
OMe
HN
N
N
N
N
OH
H
H
H
O
O
O
17: 66% (over 2 steps)
15
[Friedel-Crafts]
16: N,O-acetal - (stable to isolation)
TMS
BF3•Et2O/THF
O
PhSO2H, CaCl2
O
AcHN
O
Me
AcHN
O
Me
O
O
DCM
HN
N
S
0 oC → rt
20 h
HN
N
rt, 20 h
N
H
N
H
O
O
18: 56% (over 2 steps)
19: 27% (over 2 steps)
[sulfonylation]
[allylation]
B) Natural product analogues:
[non-Kolbe
electrolysis]
O
MeOH, Et3N
2 mA, rt, 8 h
O
hydrolysis
pathway
O
O
O
N
O
N
N
O
Me
N
O
O
Me
N
O
Me
N
Me
O
Me HN
N
OMe
Me HN
N
N
N
OH
H
H
H
O
N
O
20: N,O-acetal
(stable to isolation)
4
Me
O
23
C-terminal amide
O
O
O
O
O
O
N
N
Thiophene/DCM
(9:1 v:v)
N
O
N
O
Me
Me
PhSO2H, CaCl2
DCM
N
O
Me
N
O
Me
S
O
O
TFA
Me HN
N
Me HN
N
S
50 oC, MW
2 h
rt, 20 h
N
N
H
H
O
O
22: 21% (over 2 steps)
21: 18% (over 2 steps)
[Friedel-Crafts]
[sulfonylation]
Scheme 5. Late-stage peptide modifications employing: A) model tetrapeptide 15 and B) biseokeaniamide peptide carboxylic acid derivative 4.
electrolysis in the presence of methanol under the conditions would likely lead be plagued by epimerization. Tetrapeptide 17
previously described led to an N,O-acetal intermediate 16 which is also the first example of a C-terminal thiophene peptide,
was stable to isolation following HPLC purification; accordingly, rendering this chemistry a valuable addition to the menu of late-
the structure of 16 was unambiguously characterized, stage modifications.
confirming selective oxidation of the C-terminal carboxylate. As
competitive hydrolysis of more reactive N,O-acetals (derived N,O-acetal 16 was further amenable to sulfonylation29 under
from electrolysis in solvents other than methanol) occurred, conditions similar to those employed for the model amino acid
crude peptide N,O-acetals were used in the screening of substrate, leading to arylsulfone 18 in 56% yield over two steps.
subsequent reactions for late-stage modification. Intriguingly, Unfortunately, replication of the thiazole cuprate addition (see
thiophene addition was most successful with the methanol- 12, Scheme 3) was unsuccessful on the peptide substrate.
derived acetal 16 under microwave irradiation conditions However, reminiscent of work by Seebach on the allylation of
(50 oC) in the presence of excess TFA, affording 17 in 66% yield small peptide N,O-acetals,21 we were able to access terminal
over the two steps from acid 15 (Scheme 5A). Despite the alkene 19 upon treatment with allylsilane and BF3·OEt2 (27%
favorable reactivity of more activated N,O-acetals in the model over two steps). In each of these transformations, hydrolysis of
sarcosine study (see Table 1), employing acetic acid and the intermediate N,O-acetal to afford the C-terminal N-methyl
chloroacetic acid-derived tetrapeptide acetals did not lead to amide was a frequently observed byproduct (see supporting
improvements in yield. Notably, direct access to thiophene information), particularly in the presence of Lewis acid
product 17 is not otherwise possible using existing literature activators. This side pathway was enhanced with more reactive
methods, and a hypothetical building block approach to the N,O-acetals (e.g. acetic acid and chloroacetic acid derivatives),
incorporation of a C-terminal N-methylamino thiophene unit even when employing anhydrous reaction conditions, thus
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providing key rationale for the preferential use of the methanol- tetrapeptide 19 both exhibited enhanced cyVtioewtoAxrtiicclietyOnlinine
DOI: 10.1039/D0SC03701J
derived N,O-acetal in peptide-based systems.
relation to the natural products (Figure 1A), suggesting that C-
terminal modification may play an important role in optimizing
bioactivity. Compound 19 in particular had observed IC50 values
in the low micromolar range against both cancer cell lines
(Figure 1B). Further structural modifications may deliver
additional analogues with enhanced therapeutic capacity.
Given the relative hydrophobicity of the peptides, future work
will focus in particular on the effect of N-methylation and C-
terminal modifications on peptide conformation and
membrane permeability. Likewise, in light of the observed
activity of 1a-1c as SOAT inhibitors, evaluation of the peptide
analogues as anti-atherosclerotic agents will be the subject of
subsequent studies.
Preparation of natural product analogues
The late-stage decarboxylative modifications were finally
applied to the biseokeaniamide scaffold to generate analogues
of the natural products (Scheme 5B). Peptide acid 4 bearing a C-
terminal sarcosine residue, and derived directly from solid-
phase peptide synthesis on 2-chlorotrityl chloride resin (see
supporting information), was electrolysed in the presence of
methanol to afford N,O-acetal 20, which was once again stable
to isolation. Taken crude, treatment of 20 under the optimized
conditions devised for the diversification of the model
tetrapeptide afforded thiophene 21 and sulfone 22 in
synthetically useful yields (ca. 20%) over the two steps.
Although competitive hydrolysis (see 23, Scheme 5B)30 was
observed as a common byproduct, the protocols nevertheless
abrogate the need for the synthesis of various pre-
functionalized building blocks and avoid all complications with
epimerization. These electrochemically-enabled approaches
therefore expedite the production of diverse natural product
analogues by exploiting the inherent reactivity of accessible C-
terminal peptide carboxylates.
Conclusions
Herein we have disclosed the first total synthesis of
cyanobacterial natural products biesokeaniamides A-C,
structurally unique lipopeptides bearing a C-terminal thiazole
motif. A solid-phase approach to the construction of the highly
N-methylated peptide backbone was employed followed by
installation of a preformed N-methyl amino thiazole building
block to enable a rapid approach to the peptides. Motivated by
the resilience of the solid-phase approach and a desire to
overcome challenges with epimerization upon building block
incorporation, we examined non-Kolbe electrolysis as a means
of direct decarboxylative C-terminal peptide modification.
Harnessing the reactivity of key N,O-acetal intermediates
forged via electrochemical decarboxylation, we accomplished
the divergent synthesis of various model sarcosine derivatives
using Friedel-Crafts reactions, direct organometallic addition
and sulfonylation chemistry. These methods were readily
extendable to model peptides and natural product precursors
to deliver C-terminally modified peptides from native,
unactivated peptide substrates in an epimerization-free
manner and without the need for lengthy building block
syntheses. In some cases, peptide analogues exhibited
improved cytotoxicity relative to the natural products,
showcasing the value of these late-stage modification strategies
for the efficient syntheses of bioactive peptides. Given that C-
terminal peptide carboxylic acids are directly accessible using
solid-phase peptide synthesis, we envisage that this proof-of-
principle study will find widespread application in the late-stage
modification of peptides.
Evaluation of cytotoxicity
With several natural product analogues in hand, we ultimately
proceeded to probe the cytotoxicity of our compounds relative
to the biseokeaniamide natural products. Tetrapeptides (15
-
19), natural product analogues (21 22) and their precursors
,
(e.g. acid derivatives and N,O-acetal intermediates), as well as
peptide hydrolysis byproducts were screened in cell viability
assays against HeLa and A549 cancer cell lines (see supporting
information for details) and compared to the natural products
1a-1c. Notably, thiophene natural product derivative 21 and
Conflicts of interest
The authors declare no competing financial interests.
Acknowledgements
This work was supported by the Australian Research Council
(DE180100092; fellowship to L.R.M.). We would like to thank Dr.
Amee George, Dr. Jinshu He, and Ms. Kate Parsons at the ANU
Centre for Therapeutic Discovery for performing the biological
assays. We would also like to acknowledge Ms. Anitha
Figure 1. A) Comparative HeLa cell viability assays employing 1a-1c and
analogues 21 and 19; B) Dose-response curves for compounds 21 and 19
versus HeLa and A549 cells.
6 | J. Name., 2012, 00, 1-3
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Chemical Science
Page 8 of 8
View Article Online
DOI: 10.1039/D0SC03701J
A late-stage electrochemical decarboxylation enables rapid access to structural analogues of
biseokeaniamides A-C, cytotoxic lipopeptide natural products.