A one-pot chemoenzymatic synthesis of (2S, 4R)-4-methylproline enables the first total synthesis of antiviral lipopeptide cavinafungin B

Article abstract of DOI:10.1016/j.tet.2018.09.046

We report an efficient ten-step (longest linear sequence) synthesis of antiviral natural product cavinafungin B in 37% overall yield. By leveraging a one-pot chemoenzymatic synthesis of (2S,4R)-4-methylproline and oxazolidine-tethered (AT (Boc)G-Rink resin) SPPS methodology, the assembly of our molecular target could be conducted in an efficient manner. This general strategy could prove amenable to the construction of other natural and unnatural linear lipopeptides. The value of incorporating biocatalytic steps in complex molecule synthesis is highlighted by this work.

Full text of DOI:10.1016/j.tet.2018.09.046

Accepted Manuscript
A one-pot chemoenzymatic synthesis of (2S, 4R)-4-methylproline enables the first
total synthesis of antiviral lipopeptide cavinafungin B
Christian R. Zwick, III, Hans Renata
PII:
S0040-4020(18)31142-6
10.1016/j.tet.2018.09.046
TET 29818
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 10 June 2018
Revised Date: 21 September 2018
Accepted Date: 24 September 2018
Please cite this article as: Zwick III CR, Renata H, A one-pot chemoenzymatic synthesis of (2S, 4R)-4-
methylproline enables the first total synthesis of antiviral lipopeptide cavinafungin B, Tetrahedron (2018),
doi: https://doi.org/10.1016/j.tet.2018.09.046.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
A One-Pot Chemoenzymatic Synthesis of (2S,4R)-4-Methylproline Enables the First Total
Synthesis of Antiviral Lipopeptide Cavinafungin B
Christian R. Zwick III, Hans Renata*
Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, Jupiter, FL 33458
hrenata@scripps.edu
Abstract
We report an efficient ten-step (longest linear sequence) synthesis of antiviral natural
product cavinafungin B in 37% overall yield. By leveraging a one-pot chemoenzymatic synthesis
of (2S,4R)-4-methylproline and oxazolidine-tethered (AT(Boc)G-Rink resin) SPPS
methodology, the assembly of our molecular target could be conducted in an efficient manner.
This general strategy could prove amenable to the construction of other natural and unnatural
linear lipopeptides. The value of incorporating biocatalytic steps in complex molecule synthesis
is highlighted by this work.
Keywords: biocatalysis, lipopeptide, chemoenzymatic synthesis, hydroxylation
1
. Introduction
The aldehyde lipopeptides cavinafungin A (1) and B (2) were originally isolated in 2015
as minor constituents of crude extracts from Colispora cavincola, collected during a bioactivity
1
guided screen for potentiation of antifungal agent caspofungin. Although the sought-after
potentiation activity was eventually attributed to the depsipeptide colisporifungin, the
cavinafungins demonstrated broad-spectrum antifungal properties (MIC of 0.4–4 µg/mL against
Candida species and 8 µg/mL against A. fumigatus). Cavinafungin A and B are nearly identical
lipopentapeptide aldehydes, differing only by acetylation at
L
-homoserine (Figure 1). The
-homoserine, and (2S,4R)-4-
cavinafungins contain three noncanonical amino acids ( -alaninal,
L
L
methylproline [(2S,4R)-4-MePro]), as well as an N-terminal oleamide lipid tail. A related family
of C-terminal peptide aldehydes called the fellutamides A–D (3–6) (Figure 1) display a range of
2
activities, including proteasome inhibition and nerve growth factor synthesis induction.
1

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Figure 1. The cavinafungins and related aldehyde lipopeptides.
More recently, cavinafungin A (1) was identified to be potently active against all four
dengue virus serotypes (IC =1–5 nM, >100-fold selectivity over uninfected cells) and the Zika
5
0
3
virus (IC =150 nM, ca. 30-fold selectivity over uninfected cells). Aldehyde reduction to
5
0
alcohol (cavinafungol) neutralized both antifungal and antiviral properties, suggesting the
1
,3
aldehyde is structurally important for biological activity. A novel CRISPR/Cas9 protocol for
genome-wide profiling in HCT116 cells identified the endoplasmic-reticulum-associated signal
4
peptidase (ER-associated SPase) as the efficacy target of 1. SPase is an essential membrane-
bound serine protease involved in cleaving signal peptides of secretory and membrane proteins
in the ER. Mechanistically, 1 is proposed to anchor to the ER membrane through its lipophilic
tail, followed by subsequent binding of the oligopeptide motif to the SEC11A peptide binding
cleft of SPase. A proximal catalytic serine residue likely attacks the C-terminal aldehyde of
cavinafungin A, resulting in the formation of a hemiacetal intermediate and covalent inhibition
3
of SPase activity. This proposed mechanism draws parallels to the classical serine protease
5
inhibition mechanism. Cavinafungin A was subsequently used to confirm that cleavage by
SPase is essential for dengue virus protein processing, indicating that cavinafungin A would
3
likely inhibit other homologous flavivirus possessing similar infection mechanism. As
cavinafungin A (1) and B (2) share identical antifungal activity, it is plausible that they also
display identical antiviral properties. Hypothetically, cavinafungin A may give rise to B in vivo
upon acetyl hydrolysis, explaining their identical antifungal activities. Dengue and Zika viruses
2

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have no FDA-approved vaccination or specific antiviral treatment and cavinafungin is the only
known selective eukaryotic SPase inhibitor. Thus, obtaining ample quantities of material for
3
,6
further study and application is of significant value.
The establishment of a robust synthetic
strategy would also enable “Hit-to-Lead” optimization starting from cavinafungin B.
Recently, peptide-based drugs (~2–50 amino acids) have experienced a renaissance in the
pharmaceutical industry due to their ability to bridge the beneficial properties of biological and
7
small molecule therapeutics. Advances in high-throughput sequencing technologies,
bioinformatics-based genome mining, and combinatorial biosynthesis have catalyzed their re-
7
a,8
entry into drug discovery programs. Although peptides modulate a suite of useful biological
activities, they routinely display poor pharmacokinetic/dynamic properties, such as membrane
7
impermeability and lability to peptidases, resulting in biological instability. In contrast, natural
7
,9
and artificial lipopeptides can remedy some of these undesired properties. Examples of FDA-
approved lipopeptides for medical use include the antifungal micafungin and the antibiotics
7
b,9,10
polymyxin B and daptomycin.
To attenuate the current supply demand for flaviviral probes and drug candidates, we
targeted synthetic access to the cavinafungins. This communication documents our synthesis of
cavinafungin B in 10 steps from known intermediates. Of note, we developed a one-pot
1
1
chemoenzymatic synthesis of (2S,4R)-4-MePro which expedites the assembly of 2.
Furthermore, our strategy delineates a protocol for constructing linear lipopeptides using
fluorenylmethyloxycarbonyl (Fmoc) based solid-phase peptide synthesis (SPPS), opening the
door towards analogs derived from cavinafungin’s core structure.
2
. Results and discussion
At the outset of our synthetic planning, we identified the C-terminal aldehyde, the
(2S,4R)-4-MePro residue, and the oleamide motif as key synthetic challenges. Peptide aldehydes
have diverse applications in chemical ligation, high-throughput protein profiling, peptide
12
macrocyclization, and protease inhibition. Several methods are known for introducing
aldehydes into a peptide of interest, including alcohol oxidation, reduction of activated
amides/esters/semicarbazones, and aldehyde protection via oxazolidine/thiazolidine
1
2a
2
heterocycles.
Unfortunately, peptide aldehydes are prone to epimerization on isolation,
though this feature appears substrate dependent. Our synthetic approach was inspired by a recent
report published by Baran and co-workers on a general solid-phase peptide synthesis (SPPS)
strategy towards peptide aldehyde synthesis and macrocyclization, which resulted in the
1
2b
synthesis of 28 natural and unnatural peptide aldehydes (Figure 2).
Briefly, the strategy
involved a tert-butoxycarbonyl (Boc) protected oxazolidine tether constructed from a suitable
1
2d
Fmoc-amino aldehyde and threonine–glycine (TG)-functionalized Rink amide resin.
3
After

ACCEPTED MANUSCRIPT
iterative amide couplings and a final Fmoc deprotection, the resulting peptide could be easily
cleaved from the TG-Rink amide resin, affording the desired peptide aldehyde after HPLC
purification. Subsequently, suitable nucleophiles were used to capture the transient iminium
species to yield the corresponding macrocyclic peptides.
Figure 2. Previous application of XT(Boc)G-Rink resin SPPS in peptide aldehyde synthesis and
subsequent macrocyclization.
This protocol proved applicable to cavinafungin B (2), starting from known Boc–
alaninal–oxazolidine–threonine–glycine Rink amide resin (AT(Boc)G-Rink resin) (Figure 3).
Taking notes from the previously reported protocol, PyAOP and NMM were selected for amide
coupling steps, and a solution of 20% piperidine/DMF was used for Fmoc deprotection steps
1
2b
(
Figure 3). Although fragments 11, 14, and 16 are commercially available, 8 and 9 required
preparation. The first fragment, 9, was initially targeted as the O-trityl ether, but due to synthetic
difficulties, the primary alcohol was instead protected as the tert-butyldimethylsilyl (TBS) ether.
Although (2S,4R)-4-MePro is present in a suite of biologically relevant natural peptides, it has
1
3
proven nontrivial to construct in the literature. Previous reports require the use of
dangerous/expensive reagents (MeOTf/LiHMDS or Crabtree’s Catalyst), directing groups,
several protecting groups/functional group interconversions, and do not display complete
1
3c,d
stereocontrol.
chemoenzymatic synthesis of (2S,4R)-4-MePro from
iron- and α-ketoglutarate dependent dioxygenase (Fe/αKG) GriE to perform C–H-oxygenation at
Recently, we reported a step efficient and high yielding one-pot
1
1
L
-leucine (7). Our protocol leverages the
1
4
the δ-position of 7 (Figure 3). The resulting aminoaldehyde spontaneously cyclizes to an imine,
and can be efficiently reduced to the secondary amine upon addition of NH •BH , resulting in
3
3
1
5
yields as high as 88%. Fmoc protection of (2S,4R)-4-MePro yielded 8 in 57% over two steps.
Here, this sequence was carried out on 100 mg scale to provide adequate supply of material for
SPPS. According to previous reports, peptide lipidation via SPPS is commonly performed,
1
6
although a general procedure for coupling oleic acid via SPPS could not be found. In turn we
4

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adjusted known conditions (EDC/HOBt/DIPEA) reported by Zeng et al. for solution phase
1
6b
oleamide synthesis. Following global deprotection and cleavage from the resin, cavinafungin
B was isolated in 37% overall yield after HPLC purification.
A. One-Pot Chemoenzymatic Synthesis of 8
a. GriE,
Fe2+, αKG;
δ
Me
β
Me
γ
α
NH •BH3
Me
CO H
3
2
b. Fmoc-OSu
δ
CO H
2
Me NH2
CO H
2
N
N
5
7% over
steps
Fmoc
L-leucine (7)
2
[
δ-oxidation & condensation]
8
B. SPPS Synthesis of Cavinafungin B (2)
Me
Me
O
O
NH2
ai. 9, PyAOP,
O
bi. 11, PyAOP,
NH2
Me
N
NMM
Me
NMM
H
NBoc
NBoc
aii. 20% pip./DMF
bii. 20% pip./DMF
AT(Boc)G-
Rink resin
OTBS
1
0
Me
HN
NH2
O
NH
O
Me
O
Me
Me
O
Me
O
ci. 8, PyAOP,
NH Me
O
NMM
N
NH Me
O
Me
N
H
Me
H
NBoc
cii. 20% pip./DMF
NBoc
OTBS
1
2
OTBS
1
3
Me
NH2
Me
N
di. 14,
PyAOP,
NMM
tBuO
O
O
O
NH
dii. 20%
pip./DMF
Me
Me
O
ei. 16, EDC,
HOBt, DIPEA
NH Me
O
cavinafungin B (2)
N
H
Me
eii. 95:5 TFA:H O
2
NBoc
3
7% yield over
0 steps
1
OTBS
1
5
Me
Me
t
O Bu
CO H
CO H
2
2
CO H
2
CO H
2
Me
Me
CO H
C
17
H
32
2
N
TBSO
NHFmoc
9
NHFmoc
11
Fmoc
NHFmoc
8
14
16
Figure 3. Chemoenzymatic synthesis of 8 and cavinafungin B (2).
5

ACCEPTED MANUSCRIPT
3
. Conclusions
This work describes the first synthesis of antiviral lipopetide cavinafungin B, completed
in 10 steps from known intermediates. Of note, we utilized a one-pot chemoenzymatic synthesis
of (2S,4R)-4-MePro to expedite the assembly of cavinafungin B. Furthermore, our strategy
delineates a method for linear lipopeptide construction using Fmoc-based SPPS. With an
established synthesis of cavinafungin B, this work opens the door towards further studies
involving this useful family of lipopeptides. Our synthetic strategy highlights the value of
1
7
incorporating chemoenzymatic steps in complex molecule synthesis.
4
. Experimental
4
.1. General materials and methods
Unless otherwise noted, all chemicals and reagents for chemical reactions were purchased at the
highest commercial quality and used without further purification. SPPS reaction vessels were
purchased from Torviq. SPPS was performed using 100-200 mesh Rink Amide-Am Resin (0.55
TM
mmol/g) purchased from Creosalus (SA6061). A KJ-201BD orbital shaker or New Brunswick
®
Innova 42/42R incubator shaker was used for the general mixing and agitation of solid-phase
reactions. Reactions were monitored by thin layer chromatography (TLC) and liquid
chromatography/mass spectrometry (LC/MS). TLC was performed with 0.25 mm E. Merck silica
4
plates (60F-254) using short-wave UV light as the visualizing agent, and ninhydrin, KMnO , or
phosphomolybdic acid and heat as developing agents. LC/MS was performed with Agilent 1260
Infinity System equipped with Poroshell 120 EC-C18 column (3.0 x 50 mm, 2.7 micron). Flash
®
column chromatography was performed using an Biotage Isolera One automated purification
®
system loaded with Zip KP-Sil cartridges filled with SilicaFlash P60 silica gel (230-400 mesh).
Preparative HPLC was performed on Shimadzu LC-8A system equipped with SunFire C18 OBD
column (30 x 250 mm, 10 micron). NMR spectra were recorded on a Bruker spetrometer and
calibrated using residual undeuterated solvent. Optical rotations were measured on Autopol IV
polarimeter (Rudolph Research Analytical). Enzymes (DpnI, Q5 polymerase) were purchased
from New England Biolabs (NEB, Ipswich, MA). Expression and purification of GriE were
1
1a
performed by following previously reported protocols.
6

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4
.2. Synthesis of N-(((9H-fluoren-9-yl)methoxy)carbonyl)-O-(tert-butyldimethylsilyl)- -
L
homoserine (9)
Fmoc- -homoserine (1.00 g, 2.93 mmol, 1.0 eq) and imidazole (399 mg, 5.86 mmol, 2.0
eq) were dissolved in anhydrous DMF (3.0 mL, 1.0M). tert-Butyldimethylsilyl chloride (883 mg,
.86 mmol, 2.0 eq) was then added portionwise at 23 °C. The reaction was stirred for 9 hours,
then diluted with H O (~20 mL) and EtOAc (~20 mL). The aqueous layer was separated and
extracted with EtOAc (3 x 20 mL). The combined organic layers were washed with sat. aq. NaCl
30 mL), dried over MgSO , and concentrated in vacuo. Purification by silica gel
L
5
2
(
4
chromatography (1:9 EtOAc:hexanes to EtOAc via gradient elution) afforded TBS ether 9 (0.609
1
g, 46% yield) as a slightly tan foam. H NMR spectra of 9 matches those reported in the
1
8
literature.
4
.3. Synthesis of (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-methylpyrrolidine-2-
carboxylic acid (8)
A 100-mL beaker was charged with
final concentration), -ascorbic acid (70.4 mg, 0.400 mmol, 0.5 equiv), α-ketoglutaric acid
disodium salt dihydrate, 542 mg, 2.40 mmol, 3.0 equiv), followed by 40.0 mL of 50 mM kPi
L
-leucine (105 mg, 0.800 mmol, 1.0 equiv, 20 mM
L
(
buffer (pH 7.0). After addition of 200 µL of 200 mM FeSO solution (0.0400 mmol, 0.05 equiv),
4
the reaction was started by the addition of GriE stock solution (final concentration = 0.030 mM,
0
2
.0015 equiv). The mixture was shaken at 20 ºC, 150 rpm. After 2 h, solid NH •BH (61.5 mg,
3 3
.00 mmol, 2.5 equiv) was added and the reaction was shaken for 4 h. The reaction was acidifed
with 1 M HCl (1.0 mL), and centrifuged at 15,000 rpm for 5 min. The supernatant was collected,
lyophilized, and used for the next step without further purification.
3
The crude material from the previous step was suspended in 8.0 mL of sat. aq. NaHCO .
A solution of Fmoc-OSu (675 mg, 2.00 mmol, 2.5 equiv) in MeCN (4.0 mL) was added, and the
reaction was stirred at rt overnight. The reaction was acidified with 1 M HCl until pH = 2–3. The
aqueous layer was extracted with EtOAc (3 x 10 mL), and the combined organic layers were
washed with sat. aq. NaCl, dried over MgSO , and concentrated in vacuo. Purification by silica
4
gel chromatography (1:2 EtOAc:hexanes to 1:0.05 EtOAc:AcOH via gradient elution) afforded 8
1
as a white foam (161 mg, 57% yield over 2 steps). H NMR spectra of 8 match those reported in
2
0
literature.
7

ACCEPTED MANUSCRIPT
4
.4. Synthesis of cavinafungin B (2)
AT(Boc)G-Rink resin was prepared as previously reported and stored at 0 °C after
1
2b,21
lyophilization.
AT(Boc)G-Rink resin (100 mg, 0.0282 mmol, substitution = 0.282 mmol/g,
1
.0 eq) was placed in a 2-mL reaction vessel and swollen with anhydrous DMF (3.0 mL) for 0.5
h. A solution of Fmoc-O-TBS- -homoserine (9) (51.5 mg, 0.113 mmol, 4.0 eq), PyAOP (58.9
L
mg, 0.113 mmol, 4.0 eq), and N-methylmorpholine (NMM) (24.8 µL, 0.226 mmol, 8.0 eq) in
anhydrous DMF (0.30 mL, ~ 0.1M) was pre-mixed for 10 min in a scintillation vial. The reaction
vessel was purged of DMF, filled with the pre-mixed solution, and agitated on an orbital shaker
at 23 °C for 5 hours. The resin was washed with DMF (3 x 3.0 mL), DCM (3 x 3.0 mL), and
DMF (3 x 3.0 mL) and capped with a solution of acetic anhydride/pyridine (1.0 mL, 1:9 v/v) for
1
0 min. The resin was washed with DMF (3 x 3.0 mL), DCM (3 x 3.0 mL), and DMF (3 x 3.0
mL), then treated with 20% piperidine/DMF (3.0 mL, 2 x 10 min) to effect Fmoc deprotection
and washed with DMF (3 x 3.0 mL), DCM (3 x 3.0 mL), and DMF (3 x 3.0 mL). This procedure
was repeated for three subsequent amide couplings and Fmoc-deprotections with Fmoc-
L-valine
(11) (38.3 mg, 0.113 mmol, 4.0 eq – agitated 24 h), Fmoc-(2S,4R)-Me-L-Pro (8) (39.7 mg, 0.113
t
mmol, 4.0 eq – agitated 24 h), and Fmoc-O- Bu-
L
-threonine (14) (44.9 mg, 0.113 mmol, 4.0 eq –
agitated 24 h). A solution of oleic acid (16) (0.0370 mL, 0.116 mmol, 3.9 eq), EDC (21.9 mg,
0
5
.141 mmol, 5.0 eq), HOBt (21.6 mg, 0.141 mmol, 5.0 eq), and DIPEA (24.6 µL, 0.141 mmol,
.0 eq) in anhydrous DMF (0.30 mL, 0.1 M) was pre-mixed for 10 min in a scintillation vial. The
reaction vessel was filled with the pre-mixed solution, and agitated on an orbital shaker at 23 °C
for 24 h. The resin was washed with DMF (3 x 3.0 mL), DCM (3 x 3.0 mL), and DMF (3 x 3.0
mL), and capped with a solution of acetic anhydride/pyridine (1.0 mL, 1:9 v/v) for 10 min. The
resin was washed with DMF (3 x 3.0 mL), DCM (3 x 3.0 mL), and DMF (3 x 3.0 mL). A
mixture of TFA/H O (95:5 v/v) was added to the resin and agitated for 2 h (take caution, as the
2
solution experiences a strong exotherm). The cleavage solution was collected in a scintillation
vial and the resin washed with TFA (3 x 2.0 mL) and DCM (3 x 2.0 mL). The resulting solution
was dried under stream of air. The resulting film was dissolved in H O:MeCN (~1:1 v/v, 4.0 mL)
2
and purified by HPLC (5–100% H
2
O:MeCN for 35 min then 100% MeCN for 5 min, flow rate =
5
0 mL/min) yielding cavinafungin B (2) (7.7 mg, 37% yield) as a white powder (t = 32.1 min).
R
1
1
D
NMR spectra of 2 match those reported in literature. [α] = – 39.0 (c 0.4, MeOH); H NMR
8

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(700 MHz, DMSO-d
6
) δ 9.35 (d, J = 3.2, 1H), 8.26 (d, J = 6.6 Hz, 1H), 7.90 – 7.78 (m, 3H), 5.32
(
m, 2H), 4.70 – 4.65 (m, 1H), 4.49 – 4.44 (m, 1H), 4.44 – 4.39 (m, 1H), 4.38 – 4.27 (m, 1H),
.20 – 4.03 (m, 2H), 3.83 – 3.76 (m, 2H), 3.44 – 3.35 (m, 1H), 3.29 – 3.24 (m, 1H), 2.41 – 2.29
m, 1H), 2.17 – 2.07 (m, 2H), 2.00 – 1.95 (m, 4H), 1.84 – 1.73 (m, 1H), 1.73 – 1.61 (m, 2H),
.48 – 1.41 (m, 2H), 1.30 – 1.20 (m, 20H), 1.15 (dd, J = 7.2, 5.2 Hz, 1H), 1.08 (d, J = 6.3 Hz,
4
(
1
1
1
3
1
1
3
6
H), 0.98 – 0.94 (m, 3H), 0.89 – 0.79 (m, 9H); C NMR (176 MHz, DMSO-d ) δ 201.1, 172.2,
71.9, 171.5, 170.8, 169.4, 129.6, 69.8, 66.9, 59.2, 57.8, 57.5, 56.2, 53.9, 53.8, 49.8, 36.5, 34.8,
2.0, 31.2, 30.3, 29.1, 29.1, 29.0, 28.8, 28.7, 28.6, 26.6, 25.2, 22.1, 19.3, 19.1, 18.0, 17.1, 13.9,
72 5 8
3.5; HRMS (ESI): calc for C40H N O : 750.5381; found 750.5146.
Acknowledgement
We thank Justine deGruyter and Alex Nanna for technical assistance with solid phase
peptide synthesis, Paige Dickson and Dr. Patrick McEnaney for assistance with MALDI, and Dr.
Kevin Pels for helpful discussion. We thank the Roush, Shen, and Kodadek labs for access to
their instrumentations. Financial support for this work is generously provided by The Scripps
Research Institute (TSRI) and the Farris Foundation.
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