Solid-phase synthesis of a novel phalloidin analog with on-bead and off-bead actin-binding activity

Article abstract of DOI:10.1039/C8CC08379G

Specific effectors of actin polymerization have found use as dynamic probes of cellular morphology that may be used to gauge cellular response to stimuli and drugs. Of various natural products that target actin, phalloidin is one of the most potent and selective inhibitors of actin depolymerization. Phalloidin and related members of the phallotoxin family are macrocyclic heptapeptides bearing a characteristic and rigidifying transannular tryptathionine bridge. Here we describe a solid-phase synthesis of a new phalloidin analog as a prototype for library development with the potential for on- and off-bead screening. To validate our method, we labelled the phalloidin derivative with a fluorescent dye which stained actin in CHO cells. Furthermore, a bioassay was developed allowing actin polymerization on beads carrying a phalloidin derivative.

Full text of DOI:10.1039/C8CC08379G

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Solid-phase synthesis of a novel phalloidin analog
with on-bead and off-bead actin-binding activity†
Cite this: Chem. Commun., 2019,
5, 385
5
Antoine Blanc,
Mihajlo Todorovic
and David M. Perrin
*
Received 19th October 2018,
Accepted 28th November 2018
DOI: 10.1039/c8cc08379g
rsc.li/chemcomm
Specific effectors of actin polymerization have found use as
dynamic probes of cellular morphology that may be used to gauge
cellular response to stimuli and drugs. Of various natural products
that target actin, phalloidin is one of the most potent and selective
inhibitors of actin depolymerization. Phalloidin and related members
of the phallotoxin family are macrocyclic heptapeptides bearing a
characteristic and rigidifying transannular tryptathionine bridge. Here
we describe a solid-phase synthesis of a new phalloidin analog as
a prototype for library development with the potential for on- and
off-bead screening. To validate our method, we labelled the phalloidin Fig. 1 Structure of phalloidin. Amino acid positions are numbered for
derivative with a fluorescent dye which stained actin in CHO cells. reference.
Furthermore, a bioassay was developed allowing actin polymerization
on beads carrying a phalloidin derivative.
targeted cells dramatically increases thus highlighting an oppor-
3b
The best-known and most specific ligand for F-actin is the tunity to use PHD for precision therapeutics against cancer.
natural product phalloidin (PHD), a bicyclic heptapeptide Yet, the rational design of PHD analogs has proved challenging
that belongs to a small class of structurally related peptides due to the absence of a high-resolution co-crystal structure of PHD
composing the phallotoxin family (Fig. 1).
bound to F-actin. To date, XPD studies along with biochemical
Phallotoxins are biosynthesized from a ribosomally trans- studies, supported by a limited number of synthetic PHD
lated leader peptide that is cyclized and then further oxidized derivatives, have implicated key structure–activity relationships
1
4
to yield the characteristic tryptathionine crosslink inter alia.
PHD binds non-covalently, selectively, and with high affinity
o 40 nM) at the subunit interface of two actin monomers in crosslink that provides essential rigidity while bisecting PHD
(SARs).
A key feature of PHD is the transannular tryptathionine
(K
d
3
4
5
6
F-actin to inhibit depolymerization, even in the presence of into turns I and II. Turn I, (Cys -cis-Hyp -Ala -Trp ), forms
ions and proteins known to trigger F-actin depolymerization.
2
a rigid b-turn that is largely responsible for the binding inter-
Given actin’s central role in cellular homeostasis, it is often actions contributing to PHD toxicity. By contrast, turn II
6
7
1
hijacked by numerous pathogens and is dysregulated in neo- (Trp -[g, d-di or g-mono-hydroxy-Leu] -Ala -[D-Thr/D-b-hydroxy-
2
3
plastic tissue. Such events have inspired the design of actin- aspartic acid (D-Hya)] -Cys ) is relatively plastic and can accom-
modate various modifications as turn II is believed to extrude
As such, fluorescently labelled PHD bioconjugates are widely from the binding site.
3
specific ligands as research tools and candidate therapeutics.
4
f,5
Wieland and co-workers pioneered
used as specific probes of F-actin. With the exception of hepato- the synthesis of phallotoxins; by exploiting the work of Savige
cytes, the well-known poor cellular uptake of PHD explains its and Fontana, they reacted N-a-protected 3a-hydroxyhexa-
3b
6
relatively low apparent cytotoxicity (IC50 410 mM). Yet when hydro-pyrrolo[2,3-b]indole-(2-carboxamide) (HPI), with the
PHD is conjugated to a targeting agent, toxicity against the S-Tr-cysteine in TFA to introduce the characteristic tryptathionine
bridge. Alternate routes to PHD have been reported by Lokey and
7
Guy.
Chemistry Department, UBC, 2036 Main Mall, Vancouver, V6T-1Z1, Canada.
Despite these reports, PHD and its derivatives remain rela-
E-mail: dperrin@chem.ubc.ca
Electronic supplementary information (ESI) available. See DOI: 10.1039/c8cc08379g tively unexplored, particularly in terms of combinatorial based
†
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approaches. Hence, a long-term goal would be the discovery of
new PHD analogues with specificity for certain pathognomonic
actin isoforms using a One-Bead-One-Compound (OBOC)
library, which in turn would expose new SARs of this venerated
8
toxin. More generally still, as there is interest in developing
libraries of bicyclic peptide macrocycles, PHD could be a
privileged scaffold for generating novel peptidic ligands.
Towards these ends, here we report the Solid Phase Peptide
Synthesis (SPPS) of an entirely synthetic PHD analogue to
highlight the potential for on-bead and off-bead validation
and inform the design of bicyclic heptapeptide libraries that
may be used to target filamentous proteins as well as others.
To consider a synthesis of PHD with the potential for
on-bead screening and off-bead validation, the PHD derivative
would need to remain anchored to the bead throughout the
synthesis yet be cleaved following. For this purpose, we sought
a linker arm that would survive (i) the base treatment used in
Fmoc deprotection and (ii) the relatively long (3–5 h) trifluoro-
acetic acid (TFA) treatment used to install the tryptathionine
crosslink yet would be cleavable under mild conditions follow-
ing synthesis. Hence, tartrate-based linker 6 was synthesized
9
on TentaGel Macrobead (MB). To assess whether this linker, Scheme 1 Solution phase CuAAC with linker (24) and tripeptide (23),
2
7
followed by synthesis of [D-Thr (PEG-tartrate-TentaGel-MB), Leu ]-PHD
once linked to PHD, would permit on-bead screening, an azide-
linker was coupled with commercially available [Lys ]-PHD and
7
(30) on the solid phase.
conjugated to 6 by a Copper(I)-catalyzed Cycloaddition (CuACC)
1
0
as shown in Fig. 2 (see ESI† for details). The beads linked to route to provide a PHD analogue on the same beads. Although
cpd 12 were then exposed to a solution of fluorescent G-actin. the success with 12 might have suggested grafting at position-7,
2
Following washing, the bead-bound F-actin was detected as a we chose position-2 instead. Our choice to graft onto Thr was
2
vividly fluorescent halo (Fig. 2) which was not observed in the primarily informed by a report that D-Thr substitution by
presence of beads bearing only linker 6 (see ESI† for structure D-aminobutyric acid (D-Abu) did not significantly diminish
4
c,e,11
and full details).
PHD toxicity.
In order to couple to a primary amine on
Having validated the F-actin-stabilizing activity of a bead- the growing chain, we induced the tryptathionine crosslink
bound PHD-bioconjugate, we considered a convergent SPPS immediately following HPI introduction thereby unmasking
the N-terminal amine. This necessarily imposed directionality
on an otherwise circularly permutable synthetic route that may
1
2
be worthy of future consideration. To minimize the potential
for epimerization upon ring closure, we opted to macrocyclize
4
5
between Hyp and Ala (Scheme 1), which necessitated the
synthesis of a tripeptide with the sequence D-Thr-Cys-cis-Hyp.
With these considerations in mind, we approached the
a
b
synthesis of the said PHD. The tripeptide N -Fmoc-O -propargyl-
b
g
D-threonyl-S -trityl-cysteinyl-(1-carbamoylmethyl)-O -(tert-butyl-
diphenylsilyl)-cis-hydroxyprolinate ester (23) was first synthesized
(
ESI†). Tripeptide 23 containing the Thr-O-propargyl ether was then
13
conjugated to the azido tartrate-linker 24 via CuACC (Scheme 1).
We also added a Br-Phe into 24 to aid in eventual MS-identification
of the final product, a feature found to be unnecessary for 12 that
used linker 6. Furthermore, we obtained a better yield by appending
the alkyne functionality to the Thr of the tripeptide 23; hence, an
azide was introduced into the tartrate-based linker 24.
Peptide-linker 25 was then loaded onto the TentaGel MB
(
ESI†) (Scheme 1) that afforded bidirectional synthetic
Fig. 2 Confocal image of beads (280–320 mm) with only linker
6
14
capability. The carboxamidomethyl ester (CAM) was chosen
owing to its stability to both organic bases and to TFA. The
linear [D-Thr (PEG-tartrate-TentaGel-MB), Leu ]-PHD heptapeptide
precursor was synthesized by standard Fmoc/tert-Bu SPPS starting
(
[
negative control, for the structure of 6, see ESI†) (Panel A) or linked to
Lys ]-PHD (12) (Panel B). White rings correspond to 668 nm emission
1
5
7
2
7
of F-actin polymerization, doped with actin that was labeled with Alexa
Fluor 647 (545 nm excitation).
3
86 | Chem. Commun., 2019, 55, 385--388
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with tripeptide-linker resin 26 and terminating with the incorpora-
a
tion of N -Boc-HPI to yield hepta-peptide 27 on the solid support
a
(
Scheme 1 and ESI†). The N -Boc-3a-hydroxyhexahydro-pyrrolo-
[2,3-b]indole-2-carboxylate (HPI) (10) was prepared as previously
described and coupled to the resin-bound peptide. The resulting
bead-bound hexapeptide (27) was then treated with TFA for 3–5 h
6d
a
2
to install the tryptathionine bridge. This unmasked the H N of
the tryptathionine, which was then coupled to Fmoc-protected
alanine. Following deprotection, a PyBOP-promoted macrocycliza-
2
7
tion gave the [D-Thr (PEG-tartrate-TentaGel-MB), Leu ]-PHD (30)
Scheme 1). Each coupling step was effected with COMU in
the presence of DMF-ethyl acetate-acetonitrile, followed by mild
(
16
capping reaction.
Subsequently, the TentaGel Macrobeads, linked to (30)
were exposed to a solution allowing the polymerization of
fluorescent G-actin to F-actin (ESI†). F-actin bound to bead-
linked cpd 30, was detected as a fluorescent ring (Fig. 3), albeit
with diminished intensity compared to the case where the
native PHD was directly linked to the beads. Such would be
anticipated since the overall yield of PHD in this case is low,
thus reducing the effective concentration of PHD on the bead
Scheme 2 Oxidative cleavage of the phalloidin derivative 30 and oximation
with a fluorescent dye 32 to yield a fluorescent phalloidin conjugate 34.
(but not that of the linker arm or truncated/monocyclic) products.
Fig. 4 Confocal microscopy (Obj 10Â) of CHO cells exposed to
fluorescent phalloidin (PHD) derivatives. Fixed and permeabilized CHO
2
7
cells were incubated with [D-Thr (EDANS), Leu ]-PHD (34) (Panel A,
7
2
7
405 nm excitation) or commercially available [HyLeu (TRITC)]-PHD
Fig. 3 Confocal image of beads (280–320 mm) linked to [D-Thr , Leu ]-PHD
30) and probed using fluorescent actin as demonstrated in Fig. 2.
(
Panel B, 545 nm excitation).
(
To verify that the PHD on the bead was specific for actin, we that staining by 34 is target-specific (for blocking experiment
cleaved the peptide from the bead and conjugated it to a results, see ESI†).
fluorescent dye for a solution-phase actin staining assay. To do
Herein we prepared a synthetic PHD that can be assayed
this, IO4 treatment cleaved the diol, which had resulted from on- and off-beads by fluorescence. To demonstrate on-bead
isopropylidene hydrolysis during the acid-catalyzed tryptathiony- screening, we linked a commercially available PHD analog to the
lation reaction, to afford a glyoxamide handle. In a one-pot beads to demonstrate that a bead-bound PHD can elicit spatially
reaction, Boc-Aoa-EDANS (31) was deprotected and directly mixed defined F-actin polymerization. A major difference between the
under neutral conditions (pH 6–7) with the PHD glyoxamide natural product and the synthetic PHD derivative 30 lies in the
derivative (33) to afford
a
new, fluorescently labelled amino acid composition at position-2; in the bioactive natural
2
7
[
D-Thr (PEG-EDANS), Leu ]-phalloidin (34) that was isolated product a non-alkylated D-Thr occupies this position. While the
1
7
with an unoptimized yield of 2–3% (Scheme 2 and ESI†).
role that D-Thr plays in stabilizing F-actin remains ambiguous, our
2
Following HPLC purification, the resulting [D-Thr (PEG-EDANS), derivative corroborates past reports regarding a tolerance for
7
b
Leu ]-phalloidin (34) effectively stained Chinese hamster ovary D-Thr O -alkylation. As actin can be polymerized in a dynamic
18
(CHO) cells (Fig. 4) that had been permeabilized and fixed. chain-reaction by various ligands, it would be important to
In addition, no fluorescent signal was detected by fluorescent differentiate specific polymerization from non-specific aggrega-
confocal microscopy when the CHO cells were pre-blocked with tion that might be induced by intermediate products, the linker,
7
commercially available [HyLeu (TRITC)]-PHD thus demonstrating or even the bead itself. Notably, no polymerization was observed
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This work was supported by funds from NSERC.
We thank Dr E. Polishchuk and Ms J. Chen for cell-culture
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Conflicts of interest
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3
88 | Chem. Commun., 2019, 55, 385--388
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