A practical solid-phase synthesis of Glu7-phalloidin and entry into fluorescent F-actin-binding reagents

Article abstract of DOI:10.1002/anie.200700017

Simple synthesis of a potent probe: An efficient solid-phase synthesis of Glu⁷-phalloidin (the resin-bound cyclic peptide shown) features the one-pot I₂-mediated deprotection of S-tritylcysteine and formation of the Cys-Trp thioether bridge. A fluorescent conjugate of the resulting phalloidin analogue shows specific staining of F-actin (filamentous actin) in fixed mammalian epithelial cells (see fluorescence micrograph). Fmoc = 9-fluorenylmethoxycarbonyl. (Chemical Equation Presented).

Full text of DOI:10.1002/anie.200700017

Angewandte
Chemie
DOI: 10.1002/anie.200700017
Cyclic Peptides
A Practical Solid-Phase Synthesis of Glu7-Phalloidin and Entry into
Fluorescent F-Actin-Binding Reagents**
Laura A. Schuresko and R. Scott Lokey*
Phalloidin (1) is a potent actin-binding toxin whose chemistry
and bioactivity have been studied since the early 1900s.[1]
Phalloidin binds with high affinity to filamentous actin (F-
actin) and lowers the critical concentration ofactin polymer-
ization in solution.[2] It has been used extensively to study
actin dynamics in vitro, and fluorescent analogues of phallo-
idin serve as highly specific reagents for microscopic visual-
[3]
ization ofthe actin cytoskeleton.
The natural source of
phalloidin, Amanita phalloides, is a mushroom that lives in a
complex ecological relationship with associated flora and is
widely considered to be uncultivable.[1] Thus, an efficient
synthetic route to phalloidin would be highly desirable as an
alternative source for this reagent and as an entry into related
cyclic peptides. Although there have been a number of
syntheses ofphalloidin analogues both in solution [4–7] and on a
solid phase,[8] no synthetic route has been published with
yields significant enough to provide this reagent in practical
quantities. The yields reported for these syntheses, which
relied on the preparation ofrelatively complex building
blocks in solution, ranged from 0.5[4] to 1.3%.[6,8] Herein we
report an efficient and practical solid-phase synthesis of Glu7-
phalloidin (2) in 50% overall yield from simple starting
Central to any total synthesis ofphalloidin and its
derivatives is the formation of the transannular thioether
bridge between the Cys and Trp residues. This transformation
has been effected in previous syntheses by the treatment of a
cysteine-derived sulfenyl chloride with a suitably protected
tryptophan derivative,[8,11,12] or through the attack ofa
cysteine sulfhydryl group on an oxidatively activated trypto-
phan indole.[13,14] Although these syntheses provided efficient
access to the Trp–Cys thioether adduct in solution, there has
been no report ofthe direct thionation ofa Trp indole on the
solid phase.
Our approach to the formation of the critical thioether
bridge was inspired by a side reaction reported to occur
during solution-phase I2-mediated deprotection of S-tritylcys-
teine (Cys(Trt)) in peptides containing tryptophan.[15] The
minor product resulted from thioether formation between the
Cys and Trp residues, which occurred presumably through the
attack ofthe tryptophan indole on the suelfnyl iodide
intermediate that forms upon trityl deprotection.[16] By
using peptides based on the sequence Cys(Trt)-Glyn-Trp, the
same authors showed that I2 treatment in dilute solution led to
thioether formation that was more efficient than disulfide
dimerization for n > 3.
7
materials. Derivatization ofthe Glu side chain yielded a
fluorescent analogue that stains F-actin in fixed cells at a
concentration comparable to that ofcommercial phalloidin-
based probes.
Phalloidin is a bicyclic heptapeptide which contains an
unusual bridging thioether linkage between the Cys and Trp
residues. The natural product contains four common l-amino
acid residues, a d-threonine residue, an unusual g,d-dihy-
droxy-l-leucine residue, and the rare cis epimer of4-hydroxy-
l-proline. Structure–activity studies have shown that the g,d-
dihydroxy-l-leucine side chain is not essential for actin
binding.[7,9] We therefore replaced this residue with glutamic
acid to provide the derivative 2 with both a handle for linkage
to the solid phase and a site for fluorophore attachment. The
cis-4-hydroxy-l-proline residue was prepared according to
published methods.[8,10]
When we applied similar conditions to the solid-phase
synthesis ofmodel peptides based on the thioether-containing
sequence ofphalloidin, the only observed products were the
desired thioether and the dimer resulting from on-resin
[*] L. A. Schuresko, Dr. R. S. Lokey
Department of Chemistry and Biochemistry
University of California Santa Cruz
Santa Cruz, CA, 95064 (USA)
intermolecular disulfide formation. For the sequence H2N-
À1
Cys-Pro-Ala-Trp-OH, at a loading value of0.1 mmolg
,
cyclization was twice as efficient as dimerization (Scheme 1).
When the l-proline residue was replaced with TIPS-protected
cis-4-hydroxy-l-proline, the ratio ofthe thioether to the
disulfide product increased to 6.6:1. These results encouraged
us to undertake a solid-phase synthesis ofGlu 7-phalloidin
with an I2-mediated cyclization as the key step to form the
thioether bridge.
Fax: 831-459-2935
E-mail: lokey@chemistry.ucsc.edu
[**] The support of the NIH (1RO1 CA104569-01) for this research is
gratefully acknowledged. We also thank Craig Tamble for assistance
in cell culture and actin staining.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2007, 46, 3547 –3549
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
Scheme 1. Assessmentof solid-phase I 2-mediated cyclization as a
strategy for the construction of the thioether bridge in phalloidin
derivatives by using tetrapeptide model systems. The thioether/disul-
fide productratios are 2:1 (for Fmoc-NH-Cys-Pro-Ala-Trp-OH) and
6.6:1 (for Fmoc-NH-Cys-Hyp(OTIPS)-Ala-Trp-OH). DMF=N,N-dime-
thylformamide, Fmoc=9-fluorenylmethoxycarbonyl, TFA=trifluoroace-
tic acid, TIPS=triisopropylsilyl.
To generate the appropriate peptide precursor, the Glu7
residue was protected at its C terminus as an allyl ester and
linked through its side chain to 2-chlorotrityl polystyrene
resin. Elongation to give the heptapeptide was carried out by
using standard Fmoc chemistry (Scheme 2), and after removal
[17]
ofthe N-terminal Fmoc group
and C-terminal allyl ester,
the peptide backbone was cyclized by treatment with
diphenylphosphorylazide (DPPA). Following cleavage of
the product from the resin, HPLC–MS analysis showed that
the macrolactamization had proceeded efficiently. No cyclo-
dimer or higher oligomers were observed.
When the resin-bound cyclic peptide 4 was treated with I2
in DMF, complete conversion into the thioether was
observed, with no intermolecular disulfide dimer detected.
Upon cleavage from the resin with 1% trifluoroacetic acid/
CH2Cl2, the crude, fully protected cyclic product was found to
be more than 90% pure by LC–MS; no higher oligomers were
present. Removal ofthe protecting groups on the d-Thr and
4-hydroxy-l-proline side chains by sequential treatment with
TFA/CH2Cl2 (1:1) and 50% HF–pyridine in THF, and
purification by reversed-phase HPLC, yielded a single
Scheme 2. Solid-phase peptide synthesis (SPPS) of Glu7-phalloidin.
DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, DIPEA=N,N’-diisopropyl-
ethylamine, NMM=N-methylmaleimide.
1
major product whose circular dichroism (CD) and H NMR
spectra[8] were consistent with Glu7-phalloidin (see the
Supporting Information for complete characterization). The
overall yield ofthe purified material was 50% on the basis of
the initial resin loading.
fluorescent adduct 5 (Scheme 3). Cultured mammalian epi-
thelial (BS-C-1) cells were fixed and treated with 5 at
1 mgmLÀ1 by following standard protocols (see the Support-
ing Information). Fluorescence microscopy revealed the F-
actin staining pattern typical ofcommercial phalloidin con-
jugates (Figure 1). Actin-filament staining was completely
abolished when the fixed cells were pretreated with natural
phalloidin, thus demonstrating the specificity of 5 for F-actin.
In summary, we have developed a simple synthesis of
Glu7-phalloidin and its derivatives, including a fluorescent
bioactive probe that is as effective as natural phalloidin
conjugates in staining F-actin in fixed cells. This route will
Synthetic phallotoxins can exist as two isolatable atrop-
isomers.[1] The synthetic route reported herein provides
exclusive access to the natural atropisomer, as determined
by comparison ofthe CD spectrum ofGlu 7-phalloidin with
that ofthe authentic natural product. Furthermore, the
distinctive upfield chemical shift of the Ala5 methyl group is
diagnostic ofthe natural atropisomer because ofthe prox-
imity ofthis group to the anisotropy ifeld ofthe tryptophan
indole ring.[8]
Tetramethylrhodamine cadaverine was conjugated to
compound 2 by using PyBOP/HOBt in DMF to yield the
3548
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Angewandte
Chemie
Keywords: cyclization · fluorescent probes · natural products ·
.
peptides · solid-phase synthesis
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[10] C. A. Weir, C. M. Taylor, J. Org. Chem. 1999, 64, 1554.
[11] T. Wieland, C. Jochum, H. Faulstich, Justus Liebigs Ann. Chem.
1969, 727, 138.
Scheme 3. Conversion of Glu7-phalloidin (2) into a rhodamine deriva-
tive. HOBt=1-hydroxy-1H-benzotriazole, PyBOP=benzotriazol-1-yloxy-
tripyrrolidinophosphonium hexafluorophosphate.
[12] T. Wieland, O. Weiberg, E. Fischer, G. Horlein, Liebigs Ann.
Chem. 1954, 587, 146.
[13] W. E. Saviage, A. Fontana, Int. J. Pept. Protein Res. 1980, 15, 102.
[14] W. E. Saviage, A. Fontana, J. Chem. Soc. Chem. Commun. 1976,
600.
[15] P. Sieber, B. Kamber, B. Riniker, W. Rittel, Helv. Chim. Acta
1980, 63, 2358.
[16] Alternatively, thionation may occur by iodination ofthe indole
to form a 3-iodoindolenine intermediate, which undergoes
nucleophilic attack at C2 by the sulfur atom followed by
dehydrohalogenation.
Figure 1. a) Fluorescence micrographs of a) BS-C-1 cells stained with
the tetramethylrhodamine–phalloidin derivative 5 and b) cells pre-
treated with natural phalloidin (1) prior to the addition of 5; inset
shows a phase-contrast image of the same field.
[17] Initial attempts to remove the final Fmoc group by using 20%
piperidine in DMF resulted in the formation of unidentified side
products and a low overall yield ofGlu 7-phalloidin. Treatment of
the linear peptide with 1% DBU in DMF, however, led to clean
deprotection ofthe N terminus and a dramatic increase in the
yield ofthe final product.
generate sufficient amounts of fluorescently labeled phalloi-
din to perform high-throughput image-based screens for
compounds that affect actin morphology, and will allow us to
make extensive modifications to the phalloidin scaffold for
future structure–activity studies.
Received: January 3, 2007
Published online: March 27, 2007
Angew. Chem. Int. Ed. 2007, 46, 3547 –3549
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3549