A solid-phase approach to the phallotoxins: Total synthesis of [Ala 7]-phalloidin

Article abstract of DOI:10.1021/jo0503153

Herein we report a solid-phase synthetic approach to [Ala ⁷]-phalloidin (1). Prior syntheses of phallotoxins were carried out using solution-phase routes that required large scale and preclude library production. The route presented here consis

Full text of DOI:10.1021/jo0503153

A Solid-Phase Approach to the Phallotoxins: Total Synthesis of
[Ala7]-Phalloidin
Marc O. Anderson,† Anang A. Shelat,‡ and R. Kiplin Guy*,†,§
Department of Pharmaceutical Chemistry, Chemistry and Chemical Biology Program, and
Department of Cellular and Molecular Pharmacology, University of California at San Francisco,
San Francisco, California 94143-2280
rguy@cgl.ucsf.edu
Received February 18, 2005
Herein we report a solid-phase synthetic approach to [Ala7]-phalloidin (1). Prior syntheses of
phallotoxins were carried out using solution-phase routes that required large scale and preclude
library production. The route presented here consists of solution-phase preparation of key
orthogonally protected amino acid building blocks, followed by a solid-phase peptide synthesis
sequence, featuring two resin-bound macro-cyclization reactions. The final product mixture was
composed of two atropisomeric compounds, one designated “natural” (1) and the other designated
“non-natural” (1′). The structures of these species were modeled using restrained energy minimiza-
tion with NMR-derived restraints.
Introduction
metastasis.7 Actin has been suggested as a target for
cancer chemotherapy.7b
Phalloidin (2) is a cyclic heptapeptide fungal toxin
produced by Amanita phalloides (the Death Cap mush-
room).1 This compound is a potent stabilizer of filamen-
tous actin, and fluorescently labeled phallotoxins have
been used in cellular biology to study the biochemistry
of the actin system.1a,2 The actin polymerization equilib-
rium plays an important role in cellular processes such
as signaling,3 motility,4 endocytosis,5 tumorigenesis,6 and
Although the biochemistry and toxicology of phalloidin
(2) have been explored for nearly 200 years, a total
synthesis of this compound remains to be reported. A
notable amount of synthetic work on phalloidin as well
as natural and non-natural analogues of this compound
(phallotoxins) was carried out by Wieland et al.1a,8 More
recently, Paolillo et al. published a synthesis of [Ala7]-
phalloidin (1)9 and several other analogues.10
† Department of Pharmaceutical Chemistry.
(4) (a) Pantaloni, D.; Le Clainche, C.; Carlier, M.-F. Science 2001,
292 (5521), 1502-1506. (b) Carlier, M.-F.; Le Clainche, C.; Wiesner,
S.; Pantaloni, D. BioEssays 2003, 25(4), 336-345. (c) Pollard, T. D.
Nature 2003, 422 (6933), 741-745.
‡ Chemistry and Chemical Biology Program.
§ Department of Cellular and Molecular Pharmacology.
(1) See reviews: (a) Wieland, T. Peptides of Poisonous Amanita
Mushrooms; Springer-Verlag: New York, 1986. (d) Cooper, J. A. J.
Cell. Bio. 1987, 105 (4), 1473-1478. (b) Wieland, T.; Jochum, C.; et al.
Liebigs Ann. Chem. 1969, 727, 138-142. (c) Wieland, T.; Faulstich,
H. CRC Crit. Rev. Biochem. 1978, 5(3), 185-260. (e) Vetter, J. Toxicon
1998, 36, 13-24.
(2) Adams, A. E.; Pringle, J. R. Methods Enzymol. 1991, 194, 729-
731.
(3) (a) DeMali, K. A.; Wennerberg, K.; Burridge, K. Curr. Opin. Cell
Biol. 2003, 15, 572-582. (b) Stammes, M. Curr. Opin. Cell Biol. 2002,
14, 428-433. (c) Meyer, G.; Feldman, E. L. J. Neurochem. 2002, 83,
490-503. (e) Zigmond, S. H. Curr. Opin. Cell Biol. 1996, 8, 66-73.
(5) (a) Schott, D.; Huffaker, T.; Bretscher, A. Curr. Opin. Mol. Biol.
Genet. 2002, 5, 564-574. (b) Qualmann, B.; Kessels, M. M. Intl. Rev.
Cytol. 2002, 220, 93-144.
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11, 41-47. (b) Boschek, C. B. Organizational Changes of Cytoskeletal
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Klein, G., Ed.; Raven Press: New York, 1982; Vol. 1, pp 173-187.
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7, 111-117. (b) Jordan, M. A.; Wilson, L. Curr. Opin. Cell Biol. 1998,
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Gastroenterol. 2003, 19, 23-30.
10.1021/jo0503153 CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/16/2005
4578
J. Org. Chem. 2005, 70, 4578-4584

Total Synthesis of [Ala7]-Phalloidin
FIGURE 1. Retrosynthetic analysis of [Ala7]-phalloidin (1). The route is designed to allow for solid-supported synthesis of libraries
with maximal diversity.
The previous synthetic routes to the phallotoxins relied
upon large-scale solution-phase peptide synthesis tech-
niques. Our own retrosynthetic analysis suggested that
a solid-phase approach might be advantageous for the
rapid and efficient construction of the phallotoxins. In
particular, such an approach would allow access to a
diverse library of compounds. Herein we report a novel
solid-phase approach to the phallotoxins, namely, the
total synthesis of [Ala7]-phalloidin (1).
The Thr2 side chain was protected as an acid-labile tert-
butyl ether group, allowing simultaneous resin cleavage
and side chain deprotection in the forward direction. Next
in the retrosynthesis, the B ring was opened between
residues Ala7 and Trp6, leading to precursor 4. Strategi-
cally, our decision for the order of cyclizing the A and B
rings, as well as the specific residues to cyclize, was based
on precedent in the solution-phase phallotoxin synthesis
literature.8c,9,10 Disconnection of precursor 4 using stan-
dard peptide synthesis transformations gives rise to
species 5-7, the free solid support 8, and other com-
mercially available protected amino acids.
Results and Discussion
A retrosynthesis of [Ala7]-phalloidin (1) is presented
in Figure 1. The molecule is a bicyclic heptapeptide with
an unusual Trp6-Cys3 thioether unit bridging the mol-
ecule. Two interesting features are the Hyp4 residue,
which has the rare cis relative configuration, and Thr2,
which has the less common D-stereochemistry at the
R-carbon. We envisaged the synthesis of [Ala7]-phalloidin
(1) proceeding with the compound anchored to a solid
support through the cis-Hyp4 side chain using the acid-
labile tetrahydropyranyl polystyrene linker (THPP).11
The A ring was opened early in the retrosynthesis,
between residues Trp6 and Ala5, leading to resin-bound
intermediate 3. In intermediate 3, the Ala5 carboxy group
was protected as a fluoride-labile 2-trimethylsilylethyl
(Tmse) ester12 and the Trp6 amino group was protected
as the thiolate-labile o-nitrobenzenesulfonamide (Ns).13
Compound 5 was prepared through Mitsunobu reaction
of a corresponding inexpensive trans-hydroxyproline
derivative, using conditions previously developed for the
inversion of this class of compounds14 (see Figure S1,
Supporting Information). Compound 6 was available in
two steps from commercially available Cbz-Ala-OH.15
The synthesis of the central Trp6-Cys3 unit (7) is
depicted in Figure 2. Commercially available protected
cystine 9 was transformed to di-tert-butoxycarbamate
10.16 Next, disulfide 10 was oxidatively cleaved with
sulfuryl chloride17 to generate 2 equiv of the correspond-
ing sulfenyl chloride 11. This species was then im-
(12) Sieber, P. Helv. Chim. Acta 1977, 60, 2711. Gerlach, H. Helv.
Chim. Acta 1977, 60, 9039.
(13) (a) Fukuyama, T.; Jow, C.-K.; Cheung, M. Tetrahedron Lett.
1995, 36, 6373-6374. (b) Fukuyama, T.; Cheung, M.; Jow, C.-K.; Hidai,
Y.; Kan, T. Tetrahedron Lett. 1997, 38, 5831-5834. (c) Nihei, K.; Kato,
M. J.; Yamane, T.; Palma, M. S.; Konno, K. Synlett 2001, 1167-1169.
(d) Miller, S. C.; Scanlan, T. J. Am. Chem. Soc. 1997, 119, 2301-2302.
(e) Miller, S. C.; Scanlan, T. J. Am. Chem. Soc. 1998, 120, 2690-2691.
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1977, 99, 6151-6153. (b) Heber, H.; Faulstich, Wieland, T. Int. J. Pept.
Protein Res. 1974, 6, 381-389. (c) Wieland, T.; Miura, T.; Seeliger, A.
Int. J. Pept. Protein Res. 1983, 21, 3-10.
(9) Zanotti, G.; Falcigno, L.; Saviano, M.; D’Auria, G.; Bruno, B. M.;
Campanile, T.; Paolillo, L. Chem. Eur. J. 2001, 7, 1479-1485.
(10) Falcigno, L.; Costantini, D’Auria, G.; Bruno, B. M.; Zobeley, S.;
Zanotti, G.; Paolillo, L. Chem. Eur. J. 2001, 7, 4665-4673.
(11) (a) Thompson, L. A.; Ellman, J. A. Tetrahedron Lett. 1994, 35,
9333-9336. (b) Liu, G.; Ellman, J. A. J. Org. Chem. 1995, 60, 7712-
7713. (c) Bianco, A.; Sonksen, C. P.; Roepstorff, P.; Briand, J.-P. J.
Org. Chem. 2000, 65, 2179-2187.
(15) Godfrey, J. D. Jr.; Gordon, E. M.; Langen, D. V.; Engebrecht,
J.; Pluscec, J. J. Org. Chem. 1986, 51, 3073-3075.
(16) Bergeron, R. J.; Wollenweber, M.; Wiegand, J. J. Med. Chem.
1994, 37, 2889-2895.
(17) (a) Harpp, D. N.; Friedlander, B. T.; Larsen, C.; Steliou, K.;
Stockton, A. J. Org. Chem. 1978, 43, 3481-3485. (b) Liu, L.; Tanke,
R. S.; Miller, M. J. J. Org. Chem. 1986, 51, 5333-5337.
J. Org. Chem, Vol. 70, No. 12, 2005 4579

Anderson et al.
FIGURE 3. Synthesis of tetrapeptide 16. Reagents and
conditions: (a) Fmoc-cis-Hyp-O-Allyl (5), pyridinium p-tolu-
enesulfonate, 1,2-dichloroethane, 18 h, 80 °C; (b) Pd(PPh3)4,
N,N-dimethylbarbituric acid, CH2Cl2, 2 h, room temperature;
(c) H-Ala-O-Tmse (6), (benzotriazole-1-yloxy) tripyrrolidino-
phosphonium hexafluorophosphate (PyBOP), 1-hydroxy-ben-
zotriazole (HOBt), (i-Pr)2EtN, DMF/CH2Cl2 (1:1), 2 × 30 min,
room temperature; (d) piperidine (20% in DMF), 10 min, room
temperature; (e) Fmoc-Cys-[S-(2-((o-NO2Ph)SO2-Trp-O-Allyl))]-
OH (7), (7-azabenzotriazole-1-yloxy) tripyrrolidinophospho-
nium hexafluorophosphate (PyAOP), 1-hydroxy-7-azabenzo-
triazole (HOAt), 2,4,6-collidine, DMF/CH2Cl2 (1:1), 30 min,
room temperature.
FIGURE 2. Synthesis of intermediate 7. Reagents and
conditions: (a) di-tert-butyl dicarbonate, MeOH, Et3N, 10 min,
60 °C, 95%; (b) SO2Cl2, CHCl3, under air, 45 min, room
temperature; (c) (o-NO2Ph)SO2-Trp-O-Allyl (12), NaHCO3,
CHCl3, under argon, 15 min, room temperature (71% two
steps); (d) CF3CO2H, (i-Pr)3SiH, 24 h, room temperature; (e)
9-fluorenylmethyl chloroformate, Na2CO3 (1 M aq), dioxane,
2 h, 0 °C (66% two steps).
mediately coupled8b,18 with orthogonally protected tryp-
tophan fragment 12 to yield thioether 13. In our
exploration of this two-step process, it was found that
the generation of sulfenyl chloride 11 must be carried
out under an atmosphere of air and that the reaction does
not proceed under inert atmosphere (e.g., argon). We
speculate that the transformation of a disulfide to a
sulfenyl chloride using sulfuryl chloride may occur by a
radical mechanism requiring an initiator (in this case,
oxygen diradical). Finally, removal of the acid-labile Cys3
protecting groups, followed by reprotection of the amino
group, was carried out to arrive at target fragment 7.
The initial steps of the solid-phase synthesis sequence
are illustrated in Figure 3. First, dihydropyranyl poly-
styrene resin 8 was loaded with protected cis-Hyp4 5 to
yield tetrahydropyranyl polystyrene-bound intermediate
14 (53% loading by mass of the resulting resin). Pd0-
catalyzed deprotection of the allyl ester gave the free acid.
Coupling with protected Ala5 6 in the presence of (ben-
zotriazole-1-yloxy) tripyrrolidinophosphonium hexafluo-
rophosphate (PyBOP),19 1-hydroxybenzotriazole (HOBt),
and (i-Pr)2EtN, generated dipeptide 15. The Hyp4 Fmoc
group was removed to generate the free secondary amine.
To limit the recognized problem of racemization when
coupling to carbamate-protected cysteines,20 the reagents
(7-azabenzotriazole-1-yloxy) tripyrrolidino-phosphonium
hexafluorophosphate (PyAOP), 1-hydroxy-7-azabenzot-
riazole (HOAt), and the weak base 2,4,6-collidine were
then used to effect coupling of 15 with acid 7 to generate
tetrapeptide 16. Progress of the reactions was generally
confirmed by qualitative bead-analysis tests, such as the
standard Kaiser and chloranil tests, as well as microscale
cleavage followed by RP-HPLC and either MALDI-TOF
or ESI-MS analysis.
The next sequence of steps is shown in Figure 4.
Iterative Fmoc peptide synthesis was carried out on
intermediate 16 to attach the D-Thr2, Ala1, and Ala7
residues, yielding compound 4. The allyl ester and Fmoc
groups were removed by Pd0-catalyzed trans-allylation
and by treatment with base, respectively. Two sets of
conditions were examined for the subsequent cycliza-
tion: (1) PyAOP,21 HOAt, (i-Pr)2EtN, DMF/CH2Cl2, 24
h; and (2) diphenylphosphoryl azide,22 (i-Pr)2EtN, DMF,
24 h. After microscale cleavage and analysis by RP-HPLC
and MALDI-TOF-MS, both sets of conditions showed a
single homogeneous species without evidence of epimer-
ization or cyclodimerization. The less toxic reagent
PyAOP was chosen to effect the cyclization and generate
3.
The final steps to be carried out were deprotection of
the Ala5-Tmse and Trp6-Ns groups, cyclization of the A
ring, resin-cleavage, and deprotection to generate the
product 1. Removal of the Ala5-Tmse group was affected
smoothly using tetrabutylammonium fluoride (TBAF).
Deprotection of the Trp6-Ns group was surprisingly
difficult. Typical reported solid-phase conditions are
(21) (a) Ehrlich, A.; Heyne, H.-U.; Winter, R.; Beyermann, M.;
Haber, H.; Carpino, L.; Bienert, M. J. Org. Chem. 1996, 61, 8831-
8838. (b) Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.; Carpino, L.
A.; Kates, S. A. Tetrahedron Lett. 1997, 38, 4853-4856. (c) Albericio,
F.; Bofill, J. M.; El-Faham, A.; Kates, S. A. J. Org. Chem. 1998, 63,
9678-9683.
(22) (a) Shioiri, T.; Yamada, S.-I. Chem. Pharm. Bull. 1974, 22, 859.
(b) Evans, D. A.; Weber, A. E. J. Am. Chem. Soc. 1987, 109, 7151-
7157.
(18) (a) Crich, D.; Davies, J. W. Tetrahedron Lett. 1989, 30, 4307-
4308. (b) Tupper, D. E.; Pullar, I. A.; Clemens, J. A.; Fairhurst, J.;
Risius, F. C.; Timms, G. H.; Wedley, S. J. Med. Chem. 1993, 36, 912-
918.
(19) Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 1990,
31, 205-208.
(20) Han, Y.; Albericio, F.; Barany, G. J. Org. Chem. 1997, 62, 4307-
4312.
4580 J. Org. Chem., Vol. 70, No. 12, 2005

Total Synthesis of [Ala7]-Phalloidin
FIGURE 4. Synthesis of [Ala7]-phalloidin (1). Reagents and conditions: (a) piperidine (20% in DMF), 20 min, room temperature;
(b) Fmoc-D-Thr(O-t-Bu)-OH, PyBOP, HOBT, (i-Pr)2EtN, DMF/CH2Cl2 (1:1), 30 min, room temperature; (c) Fmoc-Ala-OH, PyBOP,
HOBT, (i-Pr)2EtN, DMF/CH2Cl2 (1:1), 30 min, room temperature; (d) Pd(PPh3)4, N,N-dimethylbarbituric acid, CH2Cl2, 2 h, room
temperature; (e) PyAOP, HOAt, (i-Pr)2EtN, DMF/CH2Cl2 (1:1), 24 h, room temperature; (f) tetrabutylammonium fluoride (1 M in
THF) 2 × 24 h, room temperature; (g) â-mercaptoethanol, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), DMF under argon, 6 × 30
min, room temperature; (h) diphenylphosphoryl azide, (i-Pr)2EtN, DMF, 48 h, room temperature. (i) CF3CO2H/H2O/Et3SiH
(8:2:10), 30 min, room temperature.
treatment with â-mercaptoethanol and 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU) under argon.13d,e Achieving
complete deprotection using these conditions required six
successive iterations, presumably because of difficulty in
removing residual oxygen from the resin, thus resulting
in the oxidation of the thiol reagent.
same molecular weight as the desired product and
seemed likely to be atropisomers of [Ala7]-phalloidin,
generated in the final cyclization. Species 1 and 1′ were
characterized by 1H NMR, HMQC, DQF-COSY, and
ROESY. All 1H spin systems were assigned by compari-
son of DQF-COSY and ROESY spectra (Tables S1 and
S3), and all protonated carbons were assigned by cor-
relation with the HMQC spectra (Tables S2 and S4).
Species 1 possessed spectroscopic data consistent with
[Ala7]-phalloidin as previously characterized,9 whereas
1′ had noticeably different data. On the basis of these
observations, species 1 was tentatively designated the
“natural” atropisomer of [Ala7]-phalloidin, whereas spe-
cies 1′ was designated the “non-natural” atropisomer.
Atropisomeric products have been reported before in
cyclizations to form phallotoxins. For example, in the
solution-phase synthesis of [L-Thr2, Ala7]-phalloidin,10 two
species were isolated and identified as atropisomers by
spectroscopic techniques and computational modeling. By
circular dichroism (CD) analysis, one species was found
to be consistent with the natural bioactive phallotoxins,1a
whereas the other had a spectrum that was generally
inverted. Thus, CD analysis was performed on isolated
species 1 and 1′, as well as on authentic phalloidin (2)
(Figure 5). Phalloidin (2) and species 1 both showed
positive Cotton effects at 250 and 305 nm. This contrasts
with the CD spectrum of species 1′, which showed
negative Cotton effects at the same wavelengths. That
the CD spectra of 1 and 1′ are not perfectly symmetric is
expected, as the species are not enantiomeric. The
similarity in CD of 1 with phalloidin (2) suggests that
this species possesses the positive helicity of the natural
bioactive phallotoxins, whereas 1′ possesses non-natural
Conditions were then examined for the cyclization of
the A ring. The closure of this ring was more difficult
than for the B ring. Three sets of conditions were
examined: (1) PyAOP,21 HOAt, (i-Pr)2EtN, DMF/CH2Cl2,
48 h; (2) (benzotriazol-1-yloxy)-dipyrrolidinocarbenium
hexafluoro-phosphate (HbPyU),8,9,23 (i-Pr)2EtN, DMF:
CH2Cl2, 48 h; and (3) diphenylphosphoryl azide,22
(i-Pr)2EtN, DMF, 48 h. After cleavage and analysis by
RP-HPLC, it was found that PyAOP and HbPyU were
both unsuccessful, showing neither consumption of start-
ing material nor formation of product. Interestingly,
HbPyU has been used successfully for solution-phase
cyclization of phallotoxins to form the same amide
bond.9,10 Fortunately, diphenylphosphoryl azide effected
complete conversion of starting material to cyclized
product, although a degree of side-product formation was
observed, including suspected oligomers.
Finally, concomitant cleavage from the resin and
deprotection of the D-Thr2 side chain tert-butyl ether
group was accomplished with CF3CO2H/H2O/Et3SiH
(8:2:10). Careful analysis of the crude material by RP-
HPLC showed that the product was composed of two
species with very similar retention times, in a ∼1:2 ratio.
RP-HPLC afforded the two species 1 and 1′. Both
compounds were shown by MALDI-TOF-MS to have the
(23) (a) Chen, S. Q.; Xu, J. C. Chin. Chem. Lett. 1993, 4(10), 846-
850. (b) Li, P.; Xu, J.-C. Tetrahedron 2000, 56, 4437-4445.
J. Org. Chem, Vol. 70, No. 12, 2005 4581

Anderson et al.
FIGURE 5. CD spectra of [Ala7]-phalloidin atropisomers (1
and 1′) and authentic phalloidin (2) (in CH3OH at 20 °C). The
spectra were recorded at 0.2, 0.6, and 0.6 mmolar, respectively,
and normalized to determine molar elipticity [∆ ꢀ]M.
FIGURE 6. Stereo drawing of the [Ala7]-phalloidin “natural”
atropisomer 1 derived from computational method I.
negative helicity. It should be noted that biosynthesized
phallotoxins are produced exclusively in positive helical
form and that the negative helicity of the latter species
is not observed in nature.
The purified yields of the two atropisomeric species 1
and 1′ were 1.3% and 3.2%, respectively, based on 53%
initial loading of hydroxyproline 5 onto DHPP resin.
While low, these yields can partially be accounted for by
the difficulty that was faced in separating the two
isomeric products of very similar polarity. The purity of
the intermediates throughout the synthesis did seem
quite high, implying that most couplings and deprotec-
tions proceeded with good conversion (see RP-HPLC
traces in Supporting Information). We suggest that the
two sequential cyclization reactions (uncommon in pep-
tide synthesis), and particularly the more problematic
second cyclization (Figure S20 in Supporting Information)
also help to account for the low yield. Furthermore, it
should be considered that for a solid-phase synthesis
consisting of 18 steps, an overall yield of 1.3% corre-
sponds to an average of 79% yield per step, which is not
unusual in natural product synthesis. Last, it is notable
that the overall yield we report is comparable to the
previous solution-phase synthesis of [Ala7]-phalloidin9
(eight linear steps, 1.3% overall).
Restrained energy minimizations were performed to
gain insight into the structures of 1 and 1′. NH-HR
torsional angle and qualitative distance restraints were
derived from DQF-COSY J-value and ROESY NMR data,
respectively. To model species 1, the computational
starting point was the solution structure of [Ala7]-
phalloidin established by Paolillo et al.9 (method I). This
structure was minimized over 10 000 steps using our own
NMR-derived restraints, yielding a model consistent with
the previously reported structure9 (Figure 6, Tables S5
and S6). By convention, the atropisomerism of bicyclic
peptides is designated “U” or “D” on the basis of the
orientation of the bridge (up or down) when the peptide
is observed in a clockwise manner. The model had a
U-type conformation with a positive indole-thioether
angle, consistent with the bioactive phallotoxins.1a In
addition, the anisotropy field of the Trp6 indole ring is
located in the vicinity of the Ala5 methyl, which accounts
for the upfield shift of this group (Table S1). This
phenomenon is generally associated with the bioactive
phallotoxins.1a
FIGURE 7. Stereo drawing of the [Ala7]-phalloidin “non-
natural” atropisomer 1′ derived from computational method
II.
indole-thioether angle. However, the validity of this
model was questioned because of the relatively high total
energy and the significant violation of torsional angle and
distance restraints (Table S7). Furthermore, the “in-
verted” CD spectrum observed for 1′ is inconsistent with
a positive indole-thioether angle, based on X-ray analysis
of early crystalline phalloidin analogues.1a,24
The first method was then modified to assume an
initial inverted D-type structure (method II). This simu-
lation produced a reasonable model of species 1′ (Figure
7 and Tables S8 and S9) with significantly less violation
and lower total energy than the previous model. Notably,
the model had a negative indole-thioether angle, as
expected on the basis of the inverted CD spectra of this
species.1a,24 Furthermore, this model places the aniso-
tropy field of the Trp6 indole residue near Hyp4-HR and
Cys3-HR, accounting for the relative upfield shifts of these
protons (Table S3). Overall, this model of 1′ is fairly
consistent with a model proposed for a “non-natural”
atropisomer of [L-Thr2, Ala7]-phalloidin,10 a species also
showing upfield shifts of Hyp4-HR and Cys3-HR. An
interesting mechanistic possibility that may have led to
the inverted structure of 1′ is that, during activation
preceding the final cyclization, Ala5 may have epimerized
(24) (a) Wieland, T.; Beijer, B.; Seeliger, A.; Dabrowski, K.; Zanotti,
G.; Tonelli, A. E.; Gieren, A.; Dederer, B.; Lamm, V.; Hadicke, E.
Liebigs Ann. Chem. 1981, 2318-2334. (b) Zanotti, G.; Beijer, B.;
Wieland, T. Int. J. Pept. Protein Res. 1987, 30, 323-329.
When method I was applied to model 1′, the resulting
structure had a U-type conformation and a positive
4582 J. Org. Chem., Vol. 70, No. 12, 2005

Total Synthesis of [Ala7]-Phalloidin
to D-Ala5, and that cyclization of the resulting peptide
could have led to 1′. In this case, 1′ would be formally
described as a conformationally inverted diastereomer of
1. Indeed, the use of DPPA has been shown to cause
racemization in slower cyclizations.25 As the NMR-RMD
approach utilized in these studies cannot conclusively
establish stereochemistry, this explanation also seems
reasonable and is worth consideration.
Hz, CDCl3): δ 28.1, 28.5, 29.1, 41.3, 54.3, 57.6, 66.4, 81.0, 84.1,
111.6, 114.45, 118.6, 119.1, 120.0, 123.2, 125.6, 126.8, 127.3,
130.3, 131.4, 132.8, 133.3, 134.1, 136.8, 147.3, 157.8, 170.1,
170.9. HRMS calcd for C32H40N4O10S2 [M] 704.2186, found [M]
704.2183.
Fmoc-Cys-[S-(2-((o-NO2Ph)SO2-Trp-O-Allyl))]-OH (7).
To a solution of Boc-Cys-[S-(2-((o-NO2Ph)SO2-Trp-O-Allyl))]-
O-t-Bu (13) (1.137 g, 1.62 mmol) in a minimal amount of CH2-
Cl2 (∼5 mL) was added triisopropylsilane (1 mL, 4.84 mmol,
3 equiv) and CF3CO2/H2O (8:2 v/v) (21 mL). The heterogeneous
solution was vigorously stirred for 24 h at room temperature.
Low resolution ESI-MS of the crude reaction mixture showed
the presence of the desired deprotected material. The solution
was concentrated in vacuo and resuspended in a mixture of
1,4-dioxane (16 mL), Na2CO3 (1 M aq, 16 mL), and H2O (10
mL). The solution was then stirred in an ice bath for 5 min,
and then a solution of 9-fluorenylmethyl chloroformate (0.554
g, 1.2 equiv) in 1,4-dioxane (2 × 5 mL) was added over 5 min.
The solution was stirred vigorously for 1 h at 0 °C, with
frequent addition of NaOH (1 M aq) to maintain the pH
between 9 and 10 (tested with pH paper). To the solution was
added brine (100 mL) followed by HCl (1 M aq) to adjust the
pH to 2. The crude product was then extracted with CH2Cl2
(3 × 50 mL), washed with brine (50 mL), dried over MgSO4,
and concentrated in vacuo on silica. The product was isolated
by dry-loaded silica flash column chromatography (1:1 hexane/
ethyl acetate to 1:2 hexanes/ethyl acetate (1% acetic acid)). The
product was typically contaminated with acetic acid, which was
removed azeotropically by the addition and evaporation of
toluene (10 mL × 3), yielding the purified product as a yellow
Conclusion
A convergent solid-phase synthesis of [Ala7]-phalloidin
(1) has been reported. These studies demonstrate the
rapid solid-phase construction of an orthogonally pro-
tected linear peptide sequence followed by two sequential
on-resin macrocyclization reactions. The final cyclization
was shown to generate two species (1 and 1′) believed to
be atropisomers. The “natural” atropisomer (1) was
minimized to a reasonable structure consistent with the
previously reported structure of [Ala7]-phalloidin. This
contrasts with the “non-natural” atropisomer (1′), which
required an initial starting structure with an overall
inverted shape relative to species 1. In future studies,
we plan to reexamine cyclization of the A ring in order
to optimize for formation of the natural atropisomer. The
extension of this strategy to target phalloidin (2), as well
as other phallotoxins, will be reported in due course.
foam (0.826 g, 66%): [R]25 ) -90.6 (c CH2Cl2). IR (CDCl3):
D
1
3154, 2984, 2901, 1793 cm-1. H NMR (400 MHz, CDCl3): δ
Experimental Section
2.88-2.94 (mult, 1H), 3.30-3.38 (mult, 3H), 4.17 (t, 1H, J )
7 Hz), 4.35-4.40 (mult, 3H), 4.53-4.56 (mult, 3H), 4.65 (bs,
1H), 5.09-5.16 (mult, 2H), 5.62-5.70 (mult, 1H), 5.75 (d, 1H,
J ) 8 Hz), 6.17 (d, 1H, J ) 8 Hz), 6.93 (t, 1H, J ) 8), 7.08 (t,
1H, J ) 8 Hz), 7.18-7.46 (mult, 8H), 7.56-7.61 (mult, 3H),
7.72-7.74 (mult, 3H), 9.43 (bs, 1H). 13C NMR (100 Hz,
CDCl3): δ 27.1, 39.8, 47.2, 54.1, 57.4, 66.6, 67.9, 111.6, 115.1,
118.7, 119.3, 120.2, 120.2, 123.5, 125.4, 125.5, 125.9, 127.3,
127.4, 128.0, 130.2, 131.2, 132.9, 133.4, 133.9, 136.8, 141.5,
143.8, 147.0, 171.1, 177.2. HRMS calcd for C38H34N4O10S2 [M
+ H] 771.1794, found [M + H] 771.1792.
For general experimental information, see Supporting In-
formation.
Boc-Cys-[S-(2-((o-NO2Ph)SO2-Trp-O-Allyl))]-O-t-Bu
Ester (13). A solution of (Boc-Cys-O-t-Bu)2 (10)26 (1.017 g, 1.84
mmol) in anhydrous chloroform (18 mL) was vigorously stirred
under air and then treated dropwise with sulfuryl chloride
(0.44 mL, 5.5 mmol, 3.0 equiv). The solution was stirred for
45 min at room temperature, under air, becoming progressively
more yellow over this period of time. TLC indicated that the
generation of the sulfenyl chloride was mostly complete (5:2
hexanes/ethyl acetate, UV, I2, product Rf ) 0.4). The solution
was quickly concentrated in vacuo to remove excess sulfuryl
chloride, yielding the sulfenyl chloride (11) as a viscous yellow
oil.
General Procedures for Solid-Phase Synthesis. (Based
on estimated initial loading of 0.217 mmol).
General Procedure A: Washing of Solid-Phase Resin.
The reaction vessel containing the resin-bound peptide was
washed with DMF (4 × 4 mL), CH2Cl2 (4 × 4 mL), CH3CN (2
× 4 mL), diethyl ether (2 × 4 mL), and CH2Cl2 (2 × 4 mL).
For each individual wash, solvent was added, the vessel was
capped and inverted twice, and then the solvent was removed
by vacuum filtration.
General Procedure B: Deprotection of an Allyl Ester
Group. The reaction vessel containing the resin-bound peptide
was treated with CH2Cl2 (4 mL) and degassed by bubbling with
argon for 5 min. N,N-Dimethylbarbituric acid (5 equiv) and
tetrakis(triphenylphosphine)-palladium(0) (0.2 equiv) were
added. The vessel was capped, agitated for 2 h, washed with
diethyldithiocarbamic acid sodium salt (2% in DMF) (2 × 4
mL), and then washed according to General Procedure A.
General Procedure C: Deprotection of an Fmoc Group.
To the reaction vessel containing the resin-bound peptide was
added piperidine (20% in DMF) (4 mL). The vessel was capped,
agitated for 20 min, and then washed according to General
Procedure A.
General Procedure D: Deprotection of a Trimethyl-
silylethyl (Tmse) Ester. The reaction vessel containing the
resin-bound peptide was treated with DMF (4 mL) and
tetrabutylammonium fluoride (1 M in THF) (1.2 equiv). The
vessel was capped, agitated for 24 h, and then washed
according to General Procedure A. By microscale cleavage and
The sulfenyl chloride was immediately dissolved in anhy-
drous CHCl3 (18 mL) and stirred for 5 min in an ice bath,
under argon. The solution was quickly treated with NaHCO3
(0.309 g, 3.68 mmol, 2.0 equiv), followed immediately by a
solution of (o-NO2Ph)SO2-Trp-O-Allyl ester (12) (0.869 g, 2.02
mmol, 1.1 equiv) in anhydrous CHCl3 (18 mL). The solution
was stirred in an ice bath for 15 min and then warmed to room
temperature. TLC (5:2 hexanes/ethyl acetate, UV) showed the
product (Rf ) 0.3), as well as a small amount of Ns-Trp-O-
Allyl ester (Rf ) 0.1). The solution was concentrated in vacuo
on silica. The product was isolated by dry-loaded silica flash
column chromatography (5:2 hexanes/ethyl acetate) as a yellow
oil (0.916 g, 71%): [R]25 91.4 (c CHCl3). IR (CDCl3): 3154,
D
2983, 1733, 1696 cm-1. 1H NMR (400 MHz, CDCl3): δ 1.40 (s,
9H), 1.46 (s, 9H), 2.68 (t, 1H, J ) 14 Hz), 3.12-3.35 (mult,
2H), 4.37-4.53 (mult, 4H), 5.12-5.18 (mult, 2H), 5.33 (d, 1H,
J ) 8 Hz), 5.66-5.67 (mult, 1H), 6.04 (d, 1H), 6.97 (t, 1H, J )
8 Hz), 7.12 (t, 1H, J ) 8 Hz), 7.27 (d, 1H, J ) 8 Hz), 7.36 (d,
1H, J ) 8 Hz), 7.52-7.54 (mult, 2H), 7.63 (dd, 1H, J ) 2, 8
Hz), 7.81 (dd, 1H, J ) 2, 8 Hz), 10.2 (bs, 1H). 13C NMR (100
(25) Katoh, T.; Ueki, M. Int. J. Pept. Protein Res. 1993, 42 (3), 264-
269.
(26) Bergeron, R. J.; Wollenweber, M.; Wiegand, J. J. Med. Chem.
1994, 37, 2889-2895.
J. Org. Chem, Vol. 70, No. 12, 2005 4583

Anderson et al.
RP-HPLC analysis, it was found that two iterations of this
procedure were required to affect complete deprotection.
General Procedure E: Deprotection of an o-Nitroben-
zenesulfonyl (Ns) Group. A container of DMF was degassed
for at least 30 min by bubbling argon through it. The reaction
vessel containing the resin-bound peptide was treated with
degassed DMF (4 mL), â-mercaptoethanol (20 equiv), and 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) (10 equiv). The vessel
was capped, agitated for 30 min, and then quickly washed with
DMF (2 × 4 mL). By microscale cleavage and RP-HPLC
analysis, it was found that six iterations of this procedure were
required to affect complete deprotection. The beads were finally
washed according to General Procedure A.
General Procedure F: Coupling of Amino Acids using
PyBOP. To the reaction vessel containing the resin-bound
peptide was added DMF/CH2Cl2 (1:1) (4 mL), the amino acid
(3 equiv), 1-hydroxybenzotriazole (HOBt) hydrate (3 equiv),
(i-Pr)2EtN (6 equiv), and benzotriazole-1-yloxy-tripyrrolidino-
phosphonium hexafluorophosphate (PyBOP) (3 equiv). The
vessel was capped, agitated for 15 min, and then washed
according to General Procedure A.
Resin-Bound Intermediate 3. The Trp6 allyl ester and
Ala7 Fmoc groups were removed (General Procedures B and
C). The Kaiser test was positive, confirming the presence of
free primary amine. To the reaction vessel containing the
resin-bound peptide were added DMF/CH2Cl2 (1:1) (4 mL),
1-hydroxy-7-azabenzotriazole (HOAt) (3 equiv), (i-Pr)2EtN (6
equiv), and (7-azabenzotriazole-1-yloxy) tripyrrolidinophos-
phonium hexafluorophosphate (PyAOP) (3 equiv). The vessel
was capped, agitated for 24 h, and then washed (General
Procedure A). The Kaiser test was negative, confirming that
the cyclization was complete. Microscale cleavage, followed by
separation using RP-HPLC, and analysis by MALDI-TOF-MS
showed the presence of intermediate 3 (Figures S6-S8; note
that partial deprotection of Tmse was observed under the
microscale cleavage conditions).
[Ala7]-Phalloidin (1). The Ala5 trimethylsilylethyl (Tmse)
and Trp6 o-nitrobenzenesulfonyl (Ns) groups were removed
(General Procedures D and E). The Kaiser test was performed,
confirming the presence of free primary amine. Microscale
cleavage followed by separation using RP-HPLC and analysis
by ESI-MS showed the presence of the deprotected peptide
(Figures S9 and S10).
Solid-Phase Loading Sequence: Resin-Bound Inter-
mediate 16. To a solution of Fmoc-cis-Hyp-O-Allyl ester (5)
(0.483 g, 1.23 mmol, 3 equiv) in 1,2-dichloroethane (5 mL) was
added pyridinium p-toluenesulfonate (0.205 g, 0.817 mmol, 2
equiv) and 3 Å molecular sieves. The solution was briefly
agitated to dry, decanted into a 25-mL round-bottom flask, and
treated with 3,4-dihydro-2H-pyran-2-yl-methoxy methyl (“di-
hydropyranyl”) polystyrene resin (8) (NovaBiochem part no.
01-64-0192; loading ) 0.950 mmol/g) (0.430 g, 0.409 mmol, 1
equiv). The suspension was heated at 80 °C for 20 h, and then
the resin was vacuum filtered through a Buchner funnel. The
resin was then transferred into a Bio-rad Biospin 10-mL
chromatography column (“reaction vessel”) attached to a Bio-
Rad 3-way stopcock, and the resin was washed (General
Procedure A). The mass of the resulting dry resin was 0.515 g
(53% loading); thus for stoichiometry calculations in the
remainder of the resin-bound steps, loading was assumed to
be 0.217 mmol.
To the reaction vessel containing the resin-bound peptide
were added DMF (4 mL), (i-Pr)2EtN (5 equiv), and diphen-
ylphosphoryl azide (5 equiv). The vessel was capped, agitated
for 48 h, and then washed according to General Procedure A.
Microscale cleavage followed by crude analysis with MALDI-
TOF-MS were performed, showing molecular weight consistent
with [Ala7]-phalloidin (1).
The Bio-Rad 3-way stopcock was removed from the reaction
vessel and replaced with a CF3CO2H-resistant polypropylene
plug. A heterogeneous mixture of CF3CO2H/H2O/Et3SiH
(8:2:10 v/v/v) (6 mL) was prepared and added to the reaction
vessel. The vessel was capped, agitated for 30 min, and then
filtered into 100-mL recovery flask. The beads were then
sequentially washed and filtered into the flask with CH2Cl2
(4 mL), CH3CN (4 mL), CH2Cl2 (4 mL), and CH3OH (2 × 4
mL). The resulting solution was concentrated in vacuo. Crude
analysis by RP-HPLC (solvent gradient B) showed a mixture
of compounds, including a ∼1:2 mixture of [Ala7]-phalloidin
atropisomers, 1 (11.8 min) and 1′ (12.0 min) (Figure S10). RP-
HPLC separation of 1 and 1′ was performed (solvent gradient
B) to yield [Ala7]-phalloidin natural atropisomer 1 (5.7 min,
0.002 g, 1.3% yield based on initial loading 8) and non-natural
atropisomer 1′ (6.0 min, 0.005 g, 3.2% yield).
The Hyp4 allyl ester group was removed (General Procedure
B), followed by the coupling of H-Ala-O-Tmse (6)15 (General
Procedure D). The coupling procedure was carried out twice
to ensure complete reaction.
The Hyp4 Fmoc group was removed by treatment with
piperidine (General Procedure C), although for no more than
10 min because of concern of diketopiperazine formation. The
chloranil test was performed to confirm the presence of free
secondary amine.
1
[Ala7]-Phalloidin Natural Atropisomer (1). H and 13C
NMR (800 MHz, DMSO-d6, 298 K): summarized in Tables S1
and S2. UV (CH3CN/H2O): λmax 291. CD (CH3OH, 20 °C):
positive Cotton effects 250, 305 nm. HRMS calcd for C32H42-
N8O9S [M + Na] 737.2693, found [M + Na] 737.2681. (See
Figures S12-S14.)
Coupling conditions were adapted from Barany et al.,20 to
minimize racemization of cysteine. Thus, a solution of Trp6-
Cys3 fragment (7) (1.9 equiv) in DMF/CH2Cl2 (1 mL) was
treated with 1-hydroxy-7-azabenzotriazole (HOAt) (2 equiv),
2,4,6-collidine (4 equiv), and (7-azabenzotriazole-1-yloxy) tri-
pyrrolidinophosphonium hexafluorophosphate (PyAOP) (2
equiv). The mixture was agitated by pipet for no more than
3-5 s and then quickly washed into the reaction vessel with
DMF/CH2Cl2 (3 × 1 mL). The vessel was capped, agitated for
30 min, and then washed (General Procedure A). The chloranil
test was negative, confirming absence of secondary amine and
indicating complete coupling. Microscale cleavage and crude
analysis by MALDI-TOF-MS were performed, revealing the
presence of intermediate 16.
[Ala7]-Phalloidin Non-Natural Atropisomer (1′). 1H
NMR (800 MHz, DMSO-d6, 298 K): summarized in Tables S3
and S4. UV (CH3CN/H2O): λmax 295. CD (CH3OH, 20 °C):
negative Cotton effects 250, 305 nm. HRMS calcd for C32H42-
N8O9S [M + Na] 737.2693, found [M + Na] 737.2708. (See
Figures S15-S17.)
Acknowledgment. We thank Jamie Moser for op-
timization work in the synthesis of 7 and Mark Kelly
for assistance in high-field NMR experiments. This work
has been supported by grants from the U.S. National
Science Foundation (CHE-9984277), and the UCSF-NIH
Biochemistry Training Grant (T-32 CA 09270-27).
Resin-Bound Intermediate 4. The Cys3 Fmoc group was
removed (General Procedure C, carried out twice for this one
sluggish deprotection), and the resulting free amine was
coupled with Fmoc-D-Thr(O-t-Bu)-OH (General Procedure F).
This deprotection-coupling procedure was then repeated to
attach Ala1 and then Ala7. Microscale cleavage followed by
separation using RP-HPLC and analysis by MALDI-TOF-MS
showed the presence of intermediate 4 (Figures S3-S5; note
that partial deprotection of tBu was observed under the
cleavage conditions).
Supporting Information Available: Supporting syn-
thetic procedures for 5 and 12; NMR spectra for 5, 7, 13, 1, 1′;
RP-HPLC chromatograms and MALDI-TOF/ESI-MS spectra
for 15, 16, 4, 3, 1; tabulated 1H and 13C NMR data; and
computationally derived parameters for species 1 and 1′. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JO0503153
4584 J. Org. Chem., Vol. 70, No. 12, 2005