Synthesis of phakellistatin 11: A Micronesia (Chuuk) marine sponge cyclooctapeptide

Article abstract of DOI:10.1021/np0100441

The cyclic octapeptide phakellistatin 11 (1), a constituent of The Federated States of Micronesia (Chuuk) marine sponge Phakellia sp., was synthesized using solid-phase techniques. An initial solution-phase synthesis proved to be inadequate owing to spontaneous deprotection of the Fmoc group at the heptapeptide stage. Using the PAL resin attachment and proceeding from Fmoc-Glu-α-allyl ester, linear elongation of the octapeptide was performed until the final unit Pro was added. The allyl ester was removed using Pd⁰[P(C₆H₅)₃]₄. Cleavage of the final Fmoc group and cyclization with PyAOP provided phakellistatin 11 (1) in 17% overall yield. The synthetic specimen of phakellistatin 11 (1) was found to be chemically but not biologically (cancer cell lines) identical to the natural product. The result suggested a conformational difference or more likely the presence of a trace amount of a highly active antineoplastic agent that binds noncovalently to the natural cyclic octapeptide 1.

Full text of DOI:10.1021/np0100441

J . Nat. Prod. 2001, 64, 883-891
883
Syn th esis of P h a k ellista tin 11: A Micr on esia (Ch u u k ) Ma r in e Sp on ge
Cycloocta p ep tid e1
George R. Pettit,* J ohn W. Lippert III, Stuart R. Taylor, Rui Tan, and Michael D. Williams
Cancer Research Institute and Department of Biochemistry, Arizona State University, Tempe, Arizona 85287-2404
Received J anuary 31, 2001
The cyclic octapeptide phakellistatin 11 (1), a constituent of The Federated States of Micronesia (Chuuk)
marine sponge Phakellia sp., was synthesized using solid-phase techniques. An initial solution-phase
synthesis proved to be inadequate owing to spontaneous deprotection of the Fmoc group at the heptapeptide
stage. Using the PAL resin attachment and proceeding from Fmoc-Glu-R-allyl ester, linear elongation of
the octapeptide was performed until the final unit Pro was added. The allyl ester was removed using
Pd0[P(C6H5)3]4. Cleavage of the final Fmoc group and cyclization with PyAOP provided phakellistatin 11
(1) in 17% overall yield. The synthetic specimen of phakellistatin 11 (1) was found to be chemically but
not biologically (cancer cell lines) identical to the natural product. The result suggested a conformational
difference or more likely the presence of a trace amount of a highly active antineoplastic agent that binds
noncovalently to the natural cyclic octapeptide 1.
From 1965 to 1966 onward2 our group has pursued the
isolation, structural elucidation, and synthesis of an array
of marine animal constituents where certain members have
proven to be remarkably active and clinically promising
anticancer drugs.3 The marine invertebrate phylum Porifera
has been of continuous interest to us as a source of
structurally unique and potentially important anticancer
agents such as the spongistatins4 and the halichondrins/
halistatins.5 Over fifty years ago the pioneering research
of Bergmann6 with marine sponge nucleoside constituents
had already provided the scientific foundation for the now
well-known anticancer and antiviral drugs ARA-C and
ARA-A. Recent advances in this very productive area
directed at sponge peptide constituents include the discov-
ery by Boyd and colleagues7 of a new and promising
hemiasterlin (C) with very potent human cancer cell line
inhibitory activity and the murine P388 lymphocytic
leukemia active cyclodepsipeptides thiomycalolides A and
B by the Fusetani group.8 Other new marine sponge
cyclodepsipeptides with biological activity (cytotoxic and/
or antifungal or under study) include cyclolithistide A,9a
keramides K and L,9b microsclerodermins C-E,9c geodi-
amolides H and I,9d and theopalanamide.9e
Over the past 15 years we have been isolating and
elucidating the structures of marine sponge cyclic peptides.
In the course of the research we have been examining the
cancer cell line activity of these peptides versus the later
synthetic specimens with focus on those from Federated
States of Micronesia (Chuuk) marine sponge Phakellia sp.
(phakellistatins 1-12). The present investigation was
directed at phakellistatin 11 (1). Previously the natural
cycloheptapeptides phakellistatin 2 (2)10 and 5 (3)11 exhib-
ited anticancer cell line (P388 leukemia) activity with ED50
0.34 and 0.23 µg/mL, respectively. Subsequently the syn-
thetic products were found to be inactive, exhibiting ED50
values of 24 and >10 µg/mL.12,13 Similar results were
obtained with three of the axinastatins (4a -c),14 isolated
from the marine sponge Axinella sp., stylostatin 1 (5),15
isolated from the marine sponge Stylotella aurantium, and
stylopeptide 1 (6), isolated from both Phakellia costata and
Stylotella aurantium.16 Interestingly, both natural speci-
mens of stylopeptide 1 (6) were found to be identical
chemically (NMR, TLC, HPLC, and mp), but their ability
* To whom correspondence should be addressed. Tel: 480-965-3351.
Fax: 480 965-8558.
10.1021/np0100441 CCC: $20.00
© 2001 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/16/2001

884 J ournal of Natural Products, 2001, Vol. 64, No. 7
Pettit et al.
sp., the total synthesis of phakellistatin 11 (1) was under-
taken.
Resu lts a n d Discu ssion
An initial investigation of a synthetic approach to
phakellistatin 11 (1) involved the solution-phase sequential
amino acid addition and N-Fmoc/tert-butyl protection
strategy20 similar to one we used to obtain both axinast-
atins 4a and 4b and stylopeptide 6.14,16a The tert-butyl
ester21 for C-terminal protection was selected because they
do not readily undergo nucleophilic attack and are there-
fore useful to minimize diketopiperzine (DKP) formation.21d,22
For N-terminal blocking the 9-fluorenylmethoxycarbonyl
(Fmoc) group21d,23 was chosen, as cleavage is easily ac-
complished with diethylamine22a,b or tris(2-aminoethyl)-
amine (TAEA).24 Formation of peptide bonds was per-
formed in either dichloromethane (DCM) or dimethylform-
amide (DMF) utilizing diethylphosphorocyanidate (DEPC)25
(8) with diisopropylethylamine (DIEA) as the base. Solu-
to inhibit growth of the P388 lymphocytic leukemia cell line
differed by more than 10-fold (ED50 ∼10 vs 0.1 µg/mL,
respectively).16 That result suggested the cell growth
inhibitory specimens of stylopeptide 1 from P. costata might
be transporting (by complex or other means), or are simply
contaminated by, one or more of the extraordinarily active
halistatin-type (cf., 7)5a antineoplastic agents in a trace
amount detectable only by biological means.16 Furthermore,
during the isolation of halichondrin B (7a )5a one of the
major difficulties encountered was the separation of trace
(∼10-6%) cyclic peptide constituents such as the heptapep-
tides axinastatins 4a ,b.14
tion-phase synthesis of phakellistatin 11 (1) proceeded
without problem until the heptapeptide stage (18). While
most of the N-Fmoc peptide tert-butyl esters seem to lose
the Fmoc group over time, heptapeptide 18 seemed par-
ticularly fragile since dibenzylfulvene (DBF) was observed
by TLC after a few days and the insoluble DBF polymer
formed (Scheme 1).
As a result of the unstable nature of heptapeptide 18,
isolated from solution-phase coupling, a change in the
synthetic strategy was adopted in which a three-dimen-
sional orthogonal (Fmoc/tert-butyl/allyl) solid-phase ap-
proach was attempted.26-28 The mild conditions used to
remove the allyl groups are compatible with classical Fmoc/
tBu methods for solid-phase peptide synthesis (SPPS).26,28
The R-allyl ester serves as the third dimension of protection
and is easily cleaved without disrupting the other protect-
ing groups by using Pd0[P(C6H5)3]4 under neutral condi-
tions.26,28 Allyl-based protecting groups have been used
extensively in organic synthesis and have recently been
applied to nucleotide, carbohydrate, and peptide synthe-
ses.27 The use of allyl protection for the R-carboxyl group
of glutamic acid in combination with classical Fmoc and
tBu strategy allowed for synthesis of the cyclic octapeptide
directly on the resin. In addition, by choosing the correct
solid-phase resin, in this case the PAL polymer, cleavage
of the bound peptide with trifluoroacetic acid (TFA) re-
sulted in formation of the corresponding amide of glutamic
acid (glutamine) required for phakellistatin 11 (1). Once
the glutamic acid was successfully anchored to the resin
with the C-terminus protected with the allyl group and the
amino portion protected with the Fmoc group, the carbam-
ate was removed and stepwise elongation performed until
finally both allyl and terminal Fmoc groups were removed
and cyclization effected (Scheme 1).
In 1986-1987 the yellow-orange sponge Phakellia sp.
(class Demospongiae, order Axinellida) was collected by
scuba (500 kg wet wt, at depths of 25 to 40 m), and we
isolated 34 mg of phakellistatin 11 (1) (6.8 × 10-6%)17 along
with phakellistatin 10 and the already known, from the
closely related P. costata, phakellistatins 7-9.18 Cyclooc-
tapeptide 1 was found to significantly inhibit growth of the
murine P388 lymphocytic leukemia (ED50 0.20 µg/mL).17
Furthermore phakellistatin 11 (1) was tested (10-5 M high
test concentration; log10 dilutions) in the NCI’s 60-cell line
human tumor in vitro screen,19a-c and a variety of data
analyses19b-d were performed.17 Phakellistatin 11 (1) gave
overall panel-averaged GI50 concentrations of (1.32 ( 0.49)
× 10-7 M.17 TGI-COMPARE correlation analyses19d of the
differential cytotoxicity of cyclic octapeptide 1 showed
Pearson correlation coefficients of 0.83, 0.96, and 0.87 with
profiles of phakellistatin 4, phakellistatin 10, and vinblas-
tine, respectively.17 In a continuing effort to explore the
biological activity of cyclic peptides isolated from Phakellia
Initially, N-Fmoc-L-Glu-R-O-allyl was anchored to the
deprotected amino group of the 5-(4-Fmoc-aminomethyl-

Synthesis of Phakellistatin 11
J ournal of Natural Products, 2001, Vol. 64, No. 7 885
3,5-dimethoxyphenoxy)valeric acid-bonded (PAL) resin
through the glutamic acid â-carboxyl group (11). Solid-
F igu r e 1. HPLC chromatogram of the crude peptide mixture: (A)
MeOH; (B) phakellistatin 11 (1); (C) undesired products; see Experi-
mental Section for solvent gradient system.
phase peptide synthesis of phakellistatin 11 (1) employed
Fmoc deprotection with 20% piperidine29 followed by pep-
tide-bond formation using HATU (9).30 After addition of
the Pro unit, the Fmoc amine protecting group was
removed, and the allyl ester was cleaved by means of the
Pd0 reagent. The cyclization reaction on the resin was
performed with PyAOP (10) in order to minimize possible
guanidinium formation that may occur in the potentially
slow cyclization coupling step.30 Phakellistatin 11 (1) bound
to the resin was then cleaved from the polymer attachment
with 90% TFA in the presence of radical scavengers.
Isolation of the resulting product was performed using
HPLC (Figure 1).
F igu r e 2. HPLC chromatogram of phakellistatin 11 (1): (A) synthetic;
(B) natural; (C) mixture; see experimental for solvent system. Arrow
points to a conformer or possible impurity in the natural product.
rotation value of the synthetic sample was significantly less
25
negative ([R]D -130° vs -163°), which points to the
possibility of the natural product being associated with a
chemically undetected contaminant or complex or repre-
senting a different conformer during cancer cell line
evaluation. HPLC analyses of the synthetic, natural, and
mixture samples are presented in Figure 2. Each specimen
was shown to have the same retention time.
Comparison of the synthetic and natural specimens of
phakellistatin 11 (1) illustrated that the two compounds
were identical chemically but not biologically. The synthetic
sample of phakellistatin 11 (1) was shown to have a P388
leukemia ED50 value of >10 µg/mL, whereas the natural
product ED50 was 0.2 µg/mL. In addition, the optical
Sch em e 1. Solid-Phase Synthesis and Partial Solution-Phase Synthesis of Phakellistatin 11 (1)

886 J ournal of Natural Products, 2001, Vol. 64, No. 7
Pettit et al.
F igu r e 3. 1H NMR overlay of phakellistatin 11 (1) at 25 °C.
F igu r e 4. 1H NMR overlay comparisons of phakellistatin 11 (1) at 70 °C.
Interestingly, the HPLC of the natural product exhibited
a small shoulder peak (possible conformer or impurity) not
displayed by the synthetic sample. In addition, phakel-
listatin 11 (1) was found to exist as two conformers in
solution. The ratio of conformers is dependent on the
solvent, temperature, and concentration of sample. Three
proline amide bonds are present in phakellistatin 11 (1),
which have an ability to give rise to conformational changes
owing to the low rotational energy barrier that needs to
upon a rise in temperature, the cyclic octapeptide adopts
almost exclusively one spatial orientation. Specifically
diagnostic of this fact is the H NMR spectrum of the Ile
1
side-chain methyl groups for the mixture specimen (Figure
5). Over the course of the temperature experiment one
conformer obviously is more stable relative to the other.
As a final check of the correct 1H NMR spectrum of the
synthetic versus the natural specimens of phakellistatin
11 (1), spectra were taken in deuterated DMSO and the
peaks were shown to be identical with those reported in
our earlier report.17
To study the contamination of cyclic peptides isolated
from marine natural products with biologically potent
compounds, some detailed experiments were undertaken.
Synthetic stylopeptide 1 (6) was mixed with diminishing
small quantities of homohalichondrin (7c). The 1H NMR
and biological data (P388 cell line) are illustrated in Figure
1
be overcome. Initial H NMR studies performed in deuter-
ated acetonitrile (Figure 3) showed the presence of two
conformers, which prompted us to conduct an NMR experi-
ment in which the temperature was raised from 25 °C to
70 °C in an effort to force the conformational equilibrium
1
to one conformer. Figure 4 illustrates the H NMR spectra
collected at 70 °C for the synthetic, natural, and mixture
specimens of phakellistatin 11 (1). The data suggest that,

Synthesis of Phakellistatin 11
J ournal of Natural Products, 2001, Vol. 64, No. 7 887
F igu r e 5. Temperature-dependent 1H NMR spectra for the isoleucine side chain of phakellistatin 11 (1).
F igu r e 6. 1H NMR biological activity study of stylopeptide contaminated with homohalichondrin (see Table 1 and text for explanation of data).
Ta ble 1. NMR Biological Activity Study of Stylopeptide (1 mg)
Contaminated with Homohalichondrin (see Figure 6 for NMR
data)
specimen becomes extremely potent (P388 cell line ED50
0.005 µg/mL; Table 1). The remaining data illustrate the
results of chemically undetectable amounts of the antican-
cer drug that are readily detected biologically using the
murine (P388) leukemia cell line.
homohalichondrin
P388 cell line
experiment NMR
(µg)
wt % (ED50 µg/mL)
1
2
3
4
5
6
A
B
C
100
50
10
1.0
0.1
10
5
1
0.1
0.01
0.005
0.032
0.041
0.51
4.5
The results of this and our previous research in this area
illustrate that certain natural cyclic peptides are able to
complex with or otherwise carry trace (too small for usual
NMR and chromatographic detection) amounts of excep-
tionally potent antineoplastic compounds such as the
sponge halichondrins/halistatins.14 Alternatively, albeit
much less likely, there may be a conformational explana-
tion under conditions of the biological experiments. We are
now currently engaged in attempting to isolate these trace
contaminants. Clearly, it is important to confirm the
D
6.0
6 and Table 1, respectively. The results show that when
100 µg of homohalichondrin (7c) is added to a 1 mg sample
of synthetic stylopeptide (6), the impurity is almost non-
detectable by 1H NMR (Figure 6, A) yet biologically the

888 J ournal of Natural Products, 2001, Vol. 64, No. 7
Pettit et al.
small portions of DCM, so the final concentration with respect
to the amino tert-butyl ester was ∼0.1-0.2 M. The reaction
mixture was usually stirred under argon for 3-4 h between
-10 and 0 °C. TLC monitoring showed dibenzofulvene (DBF)
byproduct(s) and the desired N-Fmoc tert-butyl ester.
biological activity of such cyclic peptides by means of total
syntheses.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. All solvents were
redistilled except for acetonitrile (HPLC grade, EM Science)
and DMF (anhydrous, Aldrich). All coupling reactions were
conducted under argon (solution-phase) or nitrogen (solid-
phase). The L-proline tert-butyl ester, diethyl phosphorocya-
nidate (DEPC, 93%), diisopropylethylamine (DIEA), diethy-
lamine (Et2NH), and trifluoroacetic acid (TFA) were used as
received from Sigma-Aldrich Co. The peptide-bond-forming
reagents used in the solid-phase synthesis, N-[(dimethy-
lamino)-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-me-
thylmethanaminium hexafluorophosphate N-oxide (HATU)
and 7-azabenzotriazol-1-yloxy)tris(pyrrolidino)phosphonium
hexafluorophosphate (PyAOP), were purchased from PerSep-
tive Biosystems. The N-Fmoc-L-amino acids were supplied by
NovaBiochem or the Sigma-Aldrich Co. and used as received
except for the N-Fmoc-L-Glu-R-allyl ester, which was pur-
chased from PerSeptive Biosystems. Flash column chroma-
tography employed 230-400 mesh, 0.040-0.063 mm silica gel
60 (Merck). The solid-phase syntheses were performed with a
9050 Plus PepSynthesizer following procedures recommended
by PerSeptive Biosystems Bioresearch Products. The PAL
resin was also obtained from PerSeptive BioSystems. Organic
extracts of aqueous solutions were dried over sodium sulfate.
Solvents were removed using a rotary evaporator.
Melting points were determined using an Electrothermal
9100 unit and are uncorrected. Thin-layer chromatography
was performed using silica gel GHLF Uniplates (Analtech),
and the plates were visualized by UV light and/or ceric
sulfate-sulfuric acid (by heating 2-3 min). All compounds
were visible under short-wave UV light. Optical rotation data
were collected using a Perkin-Elmer 241 polarimeter (1 mL,
1 dm cell) at the sodium D line (589 nm at indicated
temperatures). IR spectra were obtained on a Nicolet FT-IR
MX-1 using a thin film of compound evaporated from DCM
on a NaC1 plate. 1H NMR and 13C NMR spectra were recorded
employing Varian Gemini 300, Varian Unity 400, or Varian
VXR-500S instruments using a deuterated solvent and were
referenced to either TMS or the solvent. Analytical samples
were dried in vacuo (Abderhalden over P2O5 at CH3OH or
water reflux temperatures for several hours). Elemental
analyses were done by Galbraith Laboratories, Inc. (Knoxville,
TN). Atmospheric pressure chemical ionization high-resolution
mass spectrometry (ACPIHRMS) was recorded using a LC-
mate J EOL LCMS system. Fast-atom bombardment high-
resolution mass spectroscopy (FABHRMS) was done at the
Midwest Center for Mass Spectrometry, Department of Chem-
istry, University of Nebraska (Lincoln, NE 68588-0362).
Gen er a l Dep r otection P r oced u r e. A solution of the
N-Fmoc tert-butyl ester in 1:1 CH3CN-Et2NH (∼0.2 M) was
stirred at room temperature for 2 h. We avoided the use of
DCM as solvent in the deprotection step because it was found
to be a variable that adversely affected the yield of final
product. Deprotection was observed to be complete by TLC
after several minutes, but longer times were used to ensure
maximum deprotection. Reaction of ninhydrin by mild heating
of the TLC plate indicated the free amino tert-butyl ester near
the origin. Solvents were removed by rotary evaporation for
several minutes followed by evaporation of CH3CN in portions
(2 × 5 mL) to yield viscous amber syrups. Further removal of
trace solvent was also done in vacuo (typically ∼0.3 Torr, 30
min). The free amino tert-butyl ester thus obtained was used
immediately in couplings without further purification.
N-F m oc-P r o-O-Bu t (12). A 500 mL pressure bottle was
charged with a magnetic stirrer and N-Fmoc-Pro (23.8 g, 70.6
mmol, and 150 mL of DCM) and then sealed with a rubber
septum secured tightly with wire. The bottle was cooled in an
i-PrOH/dry ice bath while condensed isobutylene (70 mL) was
transferred via cannula into the bottle held in the cold bath.
After about 20 mL of isobutylene was added, it was followed
by H2SO4 (0.7 mL), and the addition of isobutylene was
continued. The cold reaction mixture was stirred for 2 h. After
stirring for an additional 16 h at room temperature, the bottle
was cooled in an i-PrOH/dry ice bath and opened carefully.
The reaction mixture was allowed to degas fully by being
stirred for several minutes open to the air at room tempera-
ture. Saturated aqueous NaHCO3 (100 mL) was carefully
added to the reaction mixture, and it was stirred for 2 h at
room temperature. The pH of the aqueous layer was about 8.
(Addition of several milliliters of water removed an emulsion
that sometimes formed in the neutralization.) The aqueous
layer was washed with DCM (50 mL). The combined DCM
extract was washed with saturated aqueous NaHCO3 (3 × 20
mL), water (3 × 20 mL), and saturated aqueous NaCl (20 mL).
The organic layer was dried (MgSO4) and filtered. Evaporation
of the solvent gave 29.1 g of syrup, from which 23.9 g of a crude
powder was precipitated using hexane/minimal EtOAc (86%
yield after one crop). TLC (1:1 EtOAc-hexane) of this powder
showed two spots: N-Fmoc-Pro-O-But, Rf 0.6, and an unidenti-
fied minor impurity, Rf 0.45. (Crystallization was precluded
in the presence of this impurity, which was always present
from the acid-catalyzed tert-butyl esterification of N-Fmoc-Pro,
but was obtained in larger amounts when excess TsOH was
used instead of catalytic H2SO4.) Silica gel chromatography
(1:1:1 hexane-EtOAc-DCM eluent) of the crude powder gave
fractions that recrystallized readily (from hexane-minimal
EtOAc). This sequence of precipitation/chromatography/re-
crystallization gave colorless crystals in 84% overall isolated
yield from several reactions (38.33 g of N-Fmoc-Pro was
converted to 37.74 g of N-Fmoc-Pro-O-But): mp 108-109 °C;
Rf 0.63 (EtOAc); [R]25 -80.6° (c 1.0, CHCl3); UV λmax nm, (log
D
ꢀ) 235 (3.8); 265 (4.3); 289 (3.7); 300 (3.8); IR (NaCl thin film)
ν
max cm-1 3070, 2976, 2882, 1740, 1707, 1451, 1416, 1350, 1154,
1119, 1090, 741; 1H NMR (500 MHz, CDCl3) conformers δ 1.44,
1.47 (2S, 9H total), 1.88, 2.10 (2m, 3H), 2.24 (m, 1H), 3.53 (m,
1H), 3.66 (m, 1H), 4.28 (m, 1H), 4.23, 4.32 (2m, 2H), 4.44 (m,
1H), 7.32 (t, J ) 6.9, 2H), 7.40 (t, J ) 6.9, 2H), 7.64 (m, 2H),
7.77 (d, J ) 7.7, 2H); 13C NMR (APT, 100.6 MHz, CDCl3)
conformers δ 23.2, 24.2, 27.9, 29.8, 31.0, 46.4, 46.9, 47.2, 47.3,
59.5, 59.8, 67.3, 67.5, 81.2, 81.3, 119.8, 125.1, 125,3, 127.0,
127.6, 141.1, 141.2, 143.6, 143.9, 144.1, 144.3, 154.7, 155.4,
171.7, 171.8; EIMS m/z 393 [M]+; anal. C 73.73%, H 6.93%, N
3.56%, calcd for C24H27NO4 C 73.22%, H 6.92%, N 3.56%.
N-F m oc-P h e-P r o-O-Bu t (13). Using the above general
procedures, N-Fmoc-Pro-O-But (5.00 g, 12.71 mmol) was
N-deprotected in 2:2:1 CH3CN-Et2NH-DCM and coupled
with N-Fmoc-Phe. After coupling, the mixture was kept at 0
°C for 16 h and then subjected to chromatography (2:1
hexane-EtOAc), which gave crystals (4.5 g, 66%) from hot
EtOAc-hexane or hexane-DCM by slow evaporation.
Alternatively, a solution of pure Pro-O-But (5.0 g, 29.20
mmol) and DIEA in DCM at room temperature was trans-
ferred via cannula to a mixture of N-Fmoc-Phe-DEPC in DCM
and DMF (10 mL, added first to N-Fmoc-Phe for better
solubility) at -10 °C under argon. After coupling, chromatog-
raphy (2:1 hexane-EtOAc) yielded 15.4 g (98%) of colorless
crystals (from hexane-EtOAc). This method was repeated on
the same scale to yield an additional 14.6 g (93%) of pure
dipeptide: mp 142.5-143.5 °C; Rf 0.24-0.28 (2:1 hexane-
EtOAc); [R]26D -40.2° (c 1.41, CHCl3); IR (NaCl thin film) νmax
cm-1: 3279, 3063, 3028, 3005, 2978, 2936, 2884, 1734, 1717,
1653, 1645, 1636, 1541, 1506, 1449, 1368, 1248, 1152, 1040,
Am in o Acid Cou p lin g P r oced u r e. A solution of the crude
amino tert-butyl ester in DCM (∼0.5 M) containing DIEA (1
equiv) at room temperature was transferred slowly under
positive argon pressure via cannula to a solution of N-Fmoc-
amino acid (1 equiv) and DEPC (1 equiv) in DCM (∼0.3 M) at
-10 °C. (The calculated volume of 93% pure DEPC was added
to give 1 equiv). Transfer was completed via cannula with
1
758, 741, 700; H NMR (300 MHz, CDCl3) δ minor 1.37, 1.49

Synthesis of Phakellistatin 11
J ournal of Natural Products, 2001, Vol. 64, No. 7 889
(2s, C(CH3)3, 9H); minor 1.47, 1.66, 1.97 (3 m, Pro γ-CH2, 2H);
1.85, 2.15 (2m, Pro â-CH2, 2H); 2.95 (dd, J ) 13.7, 6.0, Phe
â-CH2, 1H), 3.17 (dd, J ) 13.7, 6.0, Phe â-CH2, 1H); minor
3.30, 3.37, minor 3.54, 3.66 (4 m, Pro δ-CH2, 2H); 4.16, 4.23
(2m, Fmoc â-CH2, 2H); 4.32 (m, Fmoc R-CH, 1H); 4.41 (m, Pro
R-CH, 1H); Phe R-CH, 1H, minor 4.45 (m), 4.74 (q, J ) 7.3);
Phe NH, 1 H: 5.62 (d, J ) 8.2), 5.69 (d, J ) 9.3), 5.80 (d, J )
8.2); Phe Ar H: 7.20-7.33 (m, 5H); Fmoc ArH: 7.20-7.33 (2H),
7.39 (2H), 7.55 (2H), 7.75 (2H); 13C NMR (APT, 75.5 MHz,
CDCl3) δ minor 22.1, 24.8 (Pro γ-CH2); 27.7 (Pro C(CH3)3); 28.0,
minor 29.0, minor 30.5 (Pro â-CH2); 38.6, minor 40.4 (Phe
â-CH2); minor 45.9, 46.9 (Pro δ-CH2); 47.0 (Fmoc R-CH); 53.5,
minor 54.5 (Phe R-CH); 59.7 (Pro R-CH); 67.0 (Fmoc â-CH2);
81.3 (Pro C(CH3)3); 119.9, 125.1, 127.0, 127.6 (Fmoc Ar CH);
128.3, minor 128.5, minor 129.4, 129.8 (Phe ArCH); 136.0,
minor 136.3 (Phe Ar Cq); 141.2, 143.8 (Fmoc Ar Cq); 155.7
(urethane CO); 170.0 (Phe CO); 171.0 (Pro CO); FABHRMS
m/z 541.2697 [M + H]+ (calcd for C33H37N2O5, 541.2702); anal.
C 73.79%, H 6.96%, N 5.08%, calcd for C33H36N2O5 C 73.31%,
H 6.71%, N 5.18%.
N-F m oc-Ile-P h e-P r o-O-Bu t (14). By application of the
general methods N-Fmoc-Phe-Pro-O-But (19 g, 35.07 mmol)
was N-deprotected and coupled with N-Fmoc-Ile in DCM and
DMF (12 mL, added first). Chromatography (5:4 hexane-
EtOAc) yielded a foam in 90% yield (21.3 g): mp 72-74 °C
(foam); Rf 0.19-0.22 (2:1 hexane-EtOAc); [R]27D -41° (c 1.01,
CHCl3); IR (NaCl thin film) νmax cm-1 3295, 3061, 2969, 2936,
2878, 1724, 1640, 1537, 1451, 1368, 1240, 1154, 1034, 760, 740,
700; 13C NMR (APT, 75.5 MHz, CDCl3) δ 11.3 (Ile δ-CH3); 15.4
(Ile γ-CH3); minor 22.0, 24.4 (Pro γ-CH2); 24.7 (Ile γ-CH2); 27.9
(Pro C(CH3)3); 28.9, minor 30.6 (Pro â-CH2); 37.4 (Ile â-CH),
38.2, minor 40.8 (Phe â-CH2); minor 46.0, 46.9 (Pro δ-CH2);
47.1 (Fmoc R-CH); 51.7 (Ile R-CH); 52.9 (Phe R-CH); 59.6,
minor 60.3 (Pro R-CH); 66.9 (Fmoc â-CH2); 81.3, minor 82.3
(Pro C(CH3)3); 119.9, 125.1, minor 126.8, 127.0, 127.6 (Fmoc
Ar CH); 128.2, minor 128.4, minor 129.3, 129.7 (Phe Ar CH);
135.8, minor 136.1 (Phe Ar Cq); 141.2, 143.8 (Fmoc Ar Cq);
156.0 (urethane CO); 169.4, minor 169.8 (Phe CO); 170.5 (Ile
CO); 170.9 (Pro CO); FABHRMS m/z 654.3542 [M + H]+ (calcd
for C39H48N3O6 654.3543); anal. C 71.75%, H 7.57%, N 6.38%,
calcd for C39H47N3O6 C 71.65%, H 7.25%, N 6.43%.
134.5 °C; Rf 0.23 (1:4 hexane-EtOAc), 0.46 (EtOAc); [R]26
D
-77.6° (c 0.87, CHCl3); 13C NMR (APT, 75.5 MHz, CDCl3) δ
11.3 (Ile δ-CH3); 15.3 (Ile γ-CH3); 22.0, 24.5, 24.7 (1,5Pro γ-CH2,
Ile γ-CH2); 27.9 (Pro C(CH3)3); 28.7, 29.0, minor 30.6 (1,5Pro
â-CH2); 36.8 (Ile â-CH), 38.1, minor 40.9 (2,4Phe â-CH2); minor
46.0, 46.9 (1,5Pro δ-CH2); 47.1 (Fmoc R-CH); 51.7, 52.9, 54.2,
57.7,58.2, 59.4, 59.7, 60.8 (1,5Pro R-CH, 2,4Phe R-CH, Ile R-CH);
67.8 (Fmoc â-CH2); 81.1, minor 82.4 (Pro C(CH3)3); 120.0,
125.0, 126.6, 126.9, 127.1, 127.8 (Fmoc Ar CH); 128.3, 128.5,
128.6, 129.1, 129.4, 129.7 (2,4Phe Ar CH); 136.5 (2,4Phe Ar Cq);
141.3, 143.6 (Fmoc Ar Cq); 156.0 (urethane CO); 169.4, 169.8,
170.1, 170.4, 171.1, 171.8 (1,5Pro CO, 2,4Phe CO, Ile CO);
FABHRMS m/z 898.4781 [M + H]+ (calcd for C53H64N5O8
898.4755); anal. C 70.86%, H 7.08%, N 7.89%, calcd for
C
53H63N5O8 C 70.88%, H 7.07%, N 7.80%.
N-F m oc-6P h e-5P r o-4P h e-3Ile-2P h e-1P r o-O-Bu t (17). Us-
ing the general procedures summarized above, N-Fmoc-Pro-
Phe-Ile-Phe-Pro-O-But (29.0 g, 32.29 mmol) was N-deprotected
and coupled with N-Fmoc-Phe in DCM and DMF (12 mL,
added first for better solubility). For chromatography, EtOAc
as eluent was too polar and required another chromatographic
pass (1:4 hexane-EtOAc) due to poor resolution. Precipitation
occurred with some difficulty from hexane-minimal EtOAc
to yield 28.6 g (85%) of the hexapeptide: mp 118-121 °C; Rf
0.37 (4:1 EtOAc-hexane); [R]25 -50.6° (c 0.16, CHCl3); 13C
D
NMR (BB, 75.5 MHz, CDCl3) δ 11.3 (Ile δ-CH3); 15.3 (Ile
γ-CH3); 22.6, 24.7 (1,5Pro γ-CH2, Ile γ-CH2); 27.9 (Pro C(CH3)3);
29.0 (1,5Pro â-CH2); 37.1 (Ile â-CH); 38.8 (2,4,6Phe â-CH2); 46.9
(
1,5Pro δ-CH2); 47.0 (Fmoc R-CH); 51.8, 54.9, 58.0, 59.4, 59.6,
60.3 (1,5Pro R-CH, 2,4,6Phe R-CH, Ile R-CH); 67.0 (Fmoc â-CH2);
81.3 (Pro C(CH3)3); 119.9, 125.2, 126.8, 127.0, 127.7 (Fmoc Ar
CH); 128.4, 128.7, 128.9, 129.4, 129.7 (2,4,6Phe Ar CH); 136.3
(
2,4,6Phe Ar Cq); 141.2, 143.8 (Fmoc Ar Cq); 155.9 (urethane
CO); 169.7, 170.2, 170.5, 171.0, 171.2 (1,5Pro CO, 2,4,6Phe CO,
Ile CO); FABHRMS m/z 1045.5453 [M + H]+ (calcd for
C26H73N6O9 1045.5439); anal. C 70.84%, H 7.30%, N 7.77%,
calcd for C62H72N6O9 C 71.24%, H 6.94%, N 8.04%.
N-F m oc-7P r o-6P h e-5P r o-4P h e-3Ile-2P h e-1P r o-O-Bu t (18).
By means of the above general procedures, N-Fmoc-Phe-Pro-
Phe-Ile-Phe-Pro-O-But (27.8 g, 26.58 mmol) was N-deprotected
and coupled with N-Fmoc-Pro. After coupling, the mixture was
stored 2 days at -25 °C. Chromatography (20:1 EtOAc-i-
PrOH) yielded 28 g (92%) of crude heptapeptide, which was
subjected to another chromatographic separation owing to
inadequate resolution and purity: the pure heptapeptide
N-F m oc-4P h e-3Ile-2P h e-1P r o-O-Bu t (15). By application
of the general procedures, N-Fmoc-Ile-Phe-Pro-O-But (23.7 g,
36.29 mmol) was N-deprotected and coupled with N-Fmoc-Phe
in DCM and DMF (12 mL, added first for better solubility).
After coupling, the mixture was stored 16 h at -25 °C.
Chromatography (1:1 hexane-EtOAc) yielded a slightly im-
pure foam, which required additional chromatography. Yield:
28.7 g (99%); Rf 0.27-0.30 (1:1 hexane-EtOAc); [R]25D -39.6°
(c 0.72, CHCl3); 13C NMR (APT, 75.5 MHz, CDCl3) δ 11.4 (Ile
δ-CH3); minor 15.0, 15.2 (Ile γ-CH3); minor 22.0, 24.7, minor
24.8 (Pro γ-CH2, Ile γ-CH2); 27.9 (Pro C(CH3)3); 29.0, minor
30.6 (Pro â-CH2); 37.5 (Ile â-CH), minor 38.1, 38.3, minor 41.0
exhibited Rf 0.34 (20:1 EtOAc-i-PrOH), 0.21 (EtOAc); [R]25
D
-71° (c 0.10, CHCl3); 13C NMR (BB, 75.5 MHz, CDCl3) δ 11.4
(Ile δ-CH3); 15.4, minor 15.5 (Ile γ-CH3); minor 21.1, minor
22.0, minor 24.2, 24.4, 24.7 (1,5,7Pro γ-CH2, Ile γ-CH2); 27.9 (Pro
C(CH3)3); 29.0, 30.1, 30.6 (1,5,7Pro â-CH2); 36.8 (Ile â-CH), 38.2,
41.5 (2,4,6Phe â-CH2); 46.9 (1,5,7Pro δ-CH2); 47.1 (Fmoc R-CH);
51.9, 53.8, 54.4, 56.0, 57.6, 58.2, 59.4, 59.7, 60.6 (1,5,7Pro R-CH,
2,4,6Phe R-CH, Ile R-CH); 67.8, 68.5 (Fmoc â-CH2); 81.1 (Pro
C(CH3)3); 120.1, 120.4, 124.5, 124.9, 126.7, 127.0, 127.3 (Fmoc
(
2,4Phe â-CH2); minor 46.0, 46.9 (Pro δ-CH2); 47.1 (Fmoc R-CH);
51.8 (Ile R-CH); minor 53.0, 56.1, minor 57.5, 57.7 (2,4Phe
R-CH); minor 59.4, 59.6 (Pro R-CH); 67.0 (Fmoc â-CH2); 81.3,
minor 82.3 (Pro C(CH3)3); 119.9, 125.1, minor 126.8, minor
126.9, 127.0, 127.6 (Fmoc Ar CH); 128.3, minor 128.5, 128.7,
129.3, 129.8 (2,4Phe Ar CH); 136.0, minor 136.2, 136.5 (2,4Phe
Ar Cq); 141.2, 143.8 (Fmoc Ar Cq); 156.0 (urethane CO); 169.5,
minor 169.8, 170.1, minor 170.4, minor 170.6, 170.7 (2,4Phe CO,
Ile CO); 171.0 (Pro CO); FABHRMS m/z 801.4204 [M + H]+
(calcd for C48H57N4O7 801.4227); anal. C 71.72%, H 7.36%, N
7.02, calcd for C48H56N4C7 C 71.98%, H 7.05%, N 6.99%.
Ar CH); 128.3, 128.4, 128.8, 129.0, 129.2, 129.4, 129.8 (2,4,6
-
Phe Ar CH); 136.1, 136.4 (2,4,6Phe Ar Cq); 141.0, 141.5, 145.1,
145.9 (Fmoc Ar Cq); 158.0 (urethane CO); 169.3, 169.4, 169.9,
170.2, 170.4, 170.6, 170.8, 171.2, 171.4 (1,5,7Pro CO, 2,4,6Phe CO,
Ile CO); anal. C 68.86%, H 7.28%, N 9.04, calcd for C67H79N7O10
C 70.44%, H 6.94%, N 8.58%.
Cyclo-(P r o-P h e-P r o-P h e-Ile-P h e-P r o-Gln ) [ph akellista-
t in 11] (1). Gen er a l P r oced u r e for t h e Solid -P h a se
Syn th esis. The Fmoc-protected PAL polymer (1.1 g, 0.26
mmol scale, 0.24 mmol/g), swelled in DMF, was added to a
glass column (15 × 1 cm). Eight of 10 vials were prepared to
contain the necessary amino acids (3 equiv) and HATU (2.9
equiv) in the following order: Fmoc-Glu-R-O-allyl, Fmoc-Pro,
Fmoc-Phe, Fmoc-Ile, Fmoc-Phe, Fmoc-Pro, Fmoc-Phe, Fmoc-
Pro. To the ninth and tenth vials were added Pd0[P(C6H5)3]4
(0.48 g) and PyAOP (3 equiv), respectively. Both the resin
amino group and the amino acid units were deprotected with
20% piperidine in DMF (15 min). Peptide bond formation was
N-F m oc-5P r o-4P h e-3Ile-2P h e-1P r o-O-Bu t (16). With the
general procedure (see above) N-Fmoc-Phe-Ile-Phe-Pro-O-But
(28.8 g, 35.91 mmol) was N-deprotected and coupled with
N-Fmoc-Pro. The reaction was stored for 5 days at -25 °C.
Chromatography (4:1 hexane-EtOAc) yielded fractions from
which a 29.2 g precipitate (90%) was obtained. The precipitate
was very difficult to redissolve in EtOAc (used as eluent), but
easily dissolved in DCM. Deprotection occurred slowly and
unpredictably in some fractions, evidenced by the forma-
tion of insoluble material and verified by TLC: mp 132.5-

890 J ournal of Natural Products, 2001, Vol. 64, No. 7
Pettit et al.
D. L.; Boyd, M. R.; Hamel, E.; Bai, R.; Schmidt, J . M.; Tackett, L. P.;
Ru¨tzler, K. Gazz. Chim. It. 1993, 123, 371-377. (d) Pettit, G. R.;
Ichihara, Y.; Wurzel, G.; Williams, M. D.; Schmidt, J . M.; Chapuis,
J .-C. J . Chem. Soc., Chem. Commun. 1995, 383-385.
allowed to proceed for 30 min using DIEA (3.0 equiv) in DMF.
Allyl deprotection using palladium(0) complex was recycled
through the column for 2 h, and cyclization of the polypeptide
was accomplished in a 1 h period. After the final cycle
(dichloromethane wash), the resin was removed and dried
under high vacuum for 14 h (0.01 mmHg). A 20 mL (10 mL/
0.5 g resin) deprotecting solution of TFA (90%), thioanisole
(5%), 1,2-ethanedithiol (3%), and anisole (2%) was freshly
prepared and added to the peptide-bound resin. The slurry was
stirred for 2 h under argon, followed by filtration of the solution
and concentration to an oil. The oil was dissolved in DCM and
product precipitated with hexane. Separation of the impurities
and isolation of the desired product were accomplished through
the use of reversed-phase HPLC on a C8 column in acetoni-
trile-water HPLC gradient: at t ) 0-40 min, 45% A, 55% B;
at t ) 50 min, 80% A, 20% B; at t ) 60 min, 45% A, 55% B,
where solvent A is HPLC grade acetonitrile and solvent B is
distilled water (flow rate 6 mL/min), to afford 42 mg (17%) of
(6) Bergmann, W.; Feeney, R. J . J . Am. Chem. Soc. 1950, 72, 2809.
(7) Gamble, W. R.; Durso, N. A.; Fuller, R. W.; Westergaard, C. K.;
J ohnson, T. R.; Sackett, D. L.; Hamel, E.; Cardellina, J . H., II; Boyd,
M. R. Bioorg. Med. Chem. 1999, 7, 1611-1615.
(8) Matsunaga, S.; Nogata, Y.; Fusetani, N. J . Nat. Prod. 1998, 61, 663-
666.
(9) (a) Clark, D. P.; Carroll, J .; Naylor, S.; Crews, P. J . Org. Chem. 1998,
63, 8757-8764. (b) Uemoto, H.; Yahiro, Y.; Shigemori, H.; Tsuda, M.;
Takao, T.; Shimonishi, Y.; Kobayashi, J . Tetrahedron 1998, 54, 6719-
6724. (c) Schmidt, E. W.; Faulkner, D. J . Tetrahedron 1998, 54, 3043-
3056. (d) Tinto, W. F.; Lough, A. J .; McLean, S.; Reynolds, W. F.; Yu,
M.; Chan, W. R. Tetrahedron 1998, 54, 4451-4458. (e) Schmidt, E.
W.; Bewley, C. A.; Faulkner, D. J . J . Org. Chem. 1998, 63, 1254-
1258.
(10) (a) Pettit, G. R.; Gao, F.; Cerny, R. L.; Doubek, D. L.; Tackett, L. P.;
Schmidt, J . M.; Chapuis, J .-C. J . Med. Chem. 1994, 37, 1165-1168.
(b) Pettit, G. R.; Tan, R.; Williams, M. D.; Tackett, L.; Schmidt, J . M.
Bioorg. Med. Chem. Lett. 1993, 3, 2869-2874.
(11) Pettit, G. R.; Xu, J .-P.; Cichacz, Z. A.; Williams, M. D.; Dorsaz, A.-C.;
Brune, D. C.; Boyd, M. R.; Cerny, R. L. Bioorg. Med. Chem. Lett. 1994,
4, 2091-2096.
(12) Pettit, G. R.; Rhodes, M. R.; Tan, R. J . Nat. Prod. 1999, 62, 409-
414.
(13) Pettit, G. R.; Toki, B. E.; Xu, J .-P.; Brune, D. C. J . Nat. Prod. 2000,
63, 22-28.
(14) (a) Pettit, G. R.; Holman, J . W.; Boland, G. M. J . Chem. Soc., Perkin
Trans. 1 1996, 2411-2416. (b) Bates, R. B.; Caldera, S.; Ruane, M.
D. J . Nat. Prod. 1998, 61, 405. (c) Mechnich, O.; Hessler, G.; Kessler,
H.; Bernd, M.; Kutscher, B. Helv. Chim. Acta 1997, 80, 1338-1354.
(15) (a) Pettit, G. R.; Srirangam, J . K.; Herald, D. L.; Erickson, K. L.;
Doubek, D. L.; Schmidt, J . M.; Tackett, L. P.; Bakus, G. J . J . Org.
Chem. 1992, 57, 7217-7220. (b) Bourne, G. T.; Meutermans, W. D.
F.; Alewood, P. F.; McGeary, R. P.; Scanlon, M.; Watson, A. A.;
Smythe, M. L. J . Org. Chem. 1999, 64, 3095-3101. ASU-CRI cell
line laboratories; natural stylostatin (4): ED50 0.8 µg/mL; synthetic
stylostatin (4): ED50 >10 µg/mL.
a colorless solid (1): mp 188-189 °C (lit. 194-196 °C); [R]25
D
-130° (c 1.00, CH3OH) (lit. [R]25 -163°, c 0.08, CH3OH);
D
APCIHRMS m/z 974.5140 (calcd for C53H68N9O9, 974.5132);
anal. C 60.29%, H 7.14%, N 11.80%, calcd for C53H68N9O9‚41/
2H2O.
Comparison of the natural and synthetic specimens of
phakellistatin 11 (1) by 1H and 13C NMR in DMSO gave
identical NMR signals.
HP LC An a lysis of th e Syn th etic, Na tu r a l, a n d Mixtu r e
Sp ecim en s of P h a k ellista tin 11 (1). HPLC analysis of the
synthetic, natural, and mixed specimens of phakellistatin 11
(1) was carried out on a C18 column using a 35% acetonitrile-
65% water solvent system and a flow rate of 1 mL/min.
(16) (a) Pettit, G. R.; Taylor, S. R. J . Org. Chem. 1996, 61, 2322-2325.
(b) Pettit, G. R.; Srirangam, J . K.; Herald, D. L.; Xu, J .-P.; Boyd, M.
R.; Cichacz, Z.; Kamano, Y.; Schmidt, J . M.; Erikson, K. L. J . Org.
Chem. 1995, 60, 8257-8261.
Ack n ow led gm en t. We gratefully acknowledge for support
of this research Outstanding Investigator Grant CA 44344-6-
12 from the Division of Cancer Treatment and Diagnosis, NCI,
DHHS; the Arizona Disease Control Research Commission;
Diane Cummings Halle; Gary L., Diane R. Tooker, and Polly
Trautman; the Caitlin Robb Foundation; and the Robert B.
Dalton Endowment. We are pleased to thank for other as-
(17) Pettit, G. R.; Tan, R.; Ichihara, Y.; Williams, M. D.; Doubek, D. L.;
Tackett, L. P.; Schmidt, J . M. J . Nat. Prod. 1995, 58, 961-965.
(18) Pettit, G. R.; Xu, J .-P.; Dorsaz, A.-C.; Williams, M. D.; Boyd, M. R.;
Cerny, R. L. Bioorg. Med. Chem. Lett. 1995, 5, 1339-1344.
(19) (a) Boyd, M. R. In Cancer: Principles and Practices of Oncology
Update; DeVita, V. T., J r., Hellman, S., Rosenberg, S. A., Eds.;
Lippincott: Philadelphia, 1989; Vol. 3, No. 10, pp 1-12. (b) Boyd, M.
R.; Paull, K. D.; Rubinstein, L. R. In Cytotoxic Anticancer Drugs:
Models and Concepts for Drug Discovery and Development; Valeriote,
F. A., Corbett, T., Baker, L., Eds.; Kluwer Academic Publishers:
Amsterdam, 1992; pp 11-34. (c) Boyd, M. R. In Current Therapy in
Oncology; Niederhuber, J . R., Ed.; Decker, Inc.: Philadelphia, 1993;
pp 11-22. (d) Boyd, M. R.; Paull, K. D. Drug Dev. Res. 1995, 34, 91-
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Tetrahedron 1993, 49, 9307-9320.
(21) (a) Adamson, J . G.; Blaskovich, M. A.; Groenevelt, H.; Lajoie, G. A.
J . Org. Chem. 1991, 56, 3447-3449. (b) Costopanagiotis, A. A.;
Preston, J .; Weinstein, B. J . Org. Chem. 1966, 31, 3398-3400. (c)
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J . The Chemical Synthesis of Peptides; Oxford University Press: New
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(22) (a) Fischer, P. M.; Solbakken, M.; Undheim, K. Tetrahedron 1994,
50, 2277-2288. (b) Tung, R. D.; Dhaon, M. K.; Rich, D. H. J . Org.
Chem. 1986, 51, 3350-3354. (c) Hoeg-J ensen, T.; J akobsen, M. H.;
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Adermann, K. Liebigs Ann. Chem. 1989, 751-769 and 771-780.
(23) For a review see: (a) Fields, G. B.; Noble, R. L. Int. J . Rept. Protein
Res. 1990, 35, 161. (b) Chang, C.; Waki, M.; Ahmad, M.; Meinenhofer,
J .; Lundell, E. O.; Haaug, J . D. Int. J . Rept. Protein Res. 1980, 15,
59-66. (c) Bodanszky, M.; Deshmane, S. S.; Martinez, J . J . Org.
Chem. 1979, 44, 1622-1625.
(24) Carpino, L. A.; Sadat-Aalaee, D.; Beyermann, M. J . Org. Chem. 1990,
55, 1673-1675.
(25) Hamada, Y.; Rishi, S.; Shioiri, T.; Yamada, S.-I. Chem. Pharm. Bull.
1977, 25, 224-230. DEPC is also called diethylphosphoryl cyanide
or diethyl cyanophosphonate.
sistance Drs. Gregory T. Bourne (for
a generous gift of
synthetic stylostatin 1), Cherry L. Herald, Fiona Hogan, J ean
M. Schmidt, and J . Charles Chapuis, as well as Laura Crews
and Lee Williams.
Refer en ces a n d Notes
(1) Contribution 476 of the Series Antineoplastic Agents. For Part 475
see: Pettit, G. R.; Grealish, M. P. J . Org. Chem. in preparation.
(2) Pettit, G. R.; Day, J . F.; Hartwell, J . L.; Wood, H. B. Nature 1970,
227, 962-963.
(3) (a) Pettit, G. R. The Dolastatins. In Progress in the Chemistry of
Organic Natural Products; Herz, W., Kirby, G. W., Steglich, W.,
Tamm, C., Eds.; Springer-Verlag: New York, 1997; Vol. 70, pp 1-79.
(b) Pettit, G. R.; Herald, C. L.; Hogan, F. Biosynthetic Products for
Anticancer Drug Design and Treatment. In Anti-Cancer Drug Design;
Baguley, C., Ed.; Academic Press: in preparation. (c) Pettit, G. R.
Evolutionary Biosynthesis of Anticancer Drugs. In New Prospects in
Anticancer Agents for the 21st Century; in preparation. (d) Pettit, G.
R. J . Nat. Prod. 1996, 59, 812-821. (e) Pettit, G. R. The Bryostatins.
In Progress in the Chemistry of Organic Natural Products; Herz, W.,
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York, 1991; Vol. 57, pp 153-195.
(4) (a) Ovechkina, Y. Y.; Pettit, R. K.; Cichacz, Z. A.; Pettit, G. R.; Oakley,
B. R. Antimicrob. Agents Chemother. 1999, 1993-1999. (b) Smith,
A. B., III; Lin, Q.; Pettit, G. R.; Chapuis, J .-C.; Schmidt, J . M. Bioorg.
Med. Chem. Lett. 1998, 8, 567-568. (c) Pettit, R. K.; McAllister, S.
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Antimicrob. Agents 1998, 9, 147-152. (d) Pettit, G. R.; Cichacz, Z.
A.; Herald, C. L.; Gao, F.; Boyd, M. R.; Schmidt, J . M.; Hamel, E.;
Bai, R. J . Chem. Soc., Chem. Commun. 1994, 1605-1606. (e) Pettit,
G. R.; Cichacz, Z. A.; Gao, F.; Herald, C. L.; Boyd, M. R.; Schmidt, J .
M.; Hooper, J . N. A. J . Org. Chem. 1993, 58, 1302-1304.
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