Synthesis of the marine sponge cycloheptapeptide stylopeptide 1

Article abstract of DOI:10.1021/jo951986b

The solution-phase synthesis of the marine sponge stylopeptide 1 (2), cyclo-(Leu-Ile-Phe-Ser-Pro-Ile-Pro), was conveniently accomplished using N-terminal Fmoc- and C-terminal tert-butyl ester protection with tert-butyl ether side-chain blocking for serine. Peptide bond formation for each step except for the final cyclization was effected with diethyl phosphorocyanidate to give the linear heptapeptide in 19% yield. Both TBTU (4) or BOP-Cl (5) were used to cyclize the heptapeptide and resulted in 67% and 13% yields of stylopeptide 1 (2), respectively. Peptide 2 was obtained in 11% overall yield based on the TBTU cyclization procedure. The general approach represents a useful improvement in the synthesis of such cyclic peptides. The synthetic stylopeptide 1 (2) proved to be identical with the natural product.

Full text of DOI:10.1021/jo951986b

2322
J . Org. Chem. 1996, 61, 2322-2325
Syn th esis of th e Ma r in e Sp on ge Cycloh ep ta p ep tid e Stylop ep tid e 11
George R. Pettit* and Stuart R. Taylor
Cancer Research Institute and Department of Chemistry, Arizona State University,
Box 871604, Tempe, Arizona 85287-1604
Received November 8, 1995X
The solution-phase synthesis of the marine sponge stylopeptide 1 (2), cyclo-(Leu-Ile-Phe-Ser-Pro-
Ile-Pro), was conveniently accomplished using N-terminal Fmoc- and C-terminal tert-butyl ester
protection with tert-butyl ether side-chain blocking for serine. Peptide bond formation for each
step except for the final cyclization was effected with diethyl phosphorocyanidate to give the linear
heptapeptide in 19% yield. Both TBTU (4) or BOP-Cl (5) were used to cyclize the heptapeptide
and resulted in 67% and 13% yields of stylopeptide 1 (2), respectively. Peptide 2 was obtained in
11% overall yield based on the TBTU cyclization procedure. The general approach represents a
useful improvement in the synthesis of such cyclic peptides. The synthetic stylopeptide 1 (2) proved
to be identical with the natural product.
In 1983, we located the orange Indo-Pacific sponge
recently to obtain axinastatins 2 and 3.5 The tert-butyl
esters6 for C-terminal protection were selected because
they do not readily undergo nucleophilic attack and are
therefore useful to minimize diketopiperazine forma-
tion.4b-d For N-terminal blocking the 9-fluorenylmeth-
oxycarbonyl (Fmoc) group7abc was chosen as cleavage4b,c
is easily accomplished with diethylamine. Peptide bond
formation was conducted in dichloromethane (DCM)
employing diethyl phosphorocyanidate (DEPC)8 with
diisopropylethylamine (DIEA) as base. The solvent
selection was based on studies that indicated less epimer-
ization9 than with DMF.10 When DEPC was employed
in the final peptide cyclization step, the yield of stylopep-
tide 1 (2) was barely detectable. Interestingly, the
related azide DPPA has been used in peptide cyclization
reactions with varying degrees of success.11,12a When
TBTU12ab,13 (4)/DIEA in DMF and BOP-Cl14 (5)/DIEA in
DCM were employed, moderate yields (21% and 13%,
respectively) of stylopeptide 1 (2) were realized. Maxi-
mum yield (67%) of the cyclic peptide 2 was obtained
using TBTU/DIEA in DCM. The initial low yields of
stylopeptide 1 employing DEPC for the cyclization step
Stylotella aurantium northwest of New Ireland (PNG)
in the Bismark Archipelago. Subsequently, we isolated
the cycloheptapeptides designated stylostatin 1 (1)2a and
stylopeptide 1 (2)2b and confirmed our initial structural
assignments by X-ray crystal structure determinations.
Stylopeptide 1 was also isolated from the Federated
States of Micronesia (Chuuk) marine sponge Phakellia
costata.2c In both sponges stylopeptide 1 was essentially
a trace (∼10-5% yields) constituent. While the specimens
of stylopeptide 1 from the two different sponge sources
appeared quite pure by TLC, HPLC, mp, and high field
(400 MHz) 1H-NMR, their ability to inhibit growth of the
P388 lymphocytic leukemia cell line differed by more
than ten fold (ED50 ∼10 vs 0.1 µg/mL, respectively) Such
observations suggested the more cell growth inhibitory
specimens of stylopeptide 1 from P. costata might be
transporting (by complex or other means), or simply
contaminated by one or more of the extraordinarily active
halistatin-type3ab (cf., 3) antineoplastic agents in a trace
amount only detectable by biological means. To resolve
this cell growth inhibitory dilemma and to provide a
larger supply of stylopeptide 1 (2) for additional biological
evaluation, its total synthesis was achieved as described
in the sequel.
(5) Pettit, G. R.; Holman, J . W.; Boland, G. M. J . Chem. Soc., Perkin
Trans. 1, submitted.
A sequential amino acid addition and N-Fmoc/tert-
butyl protection strategy4 (Scheme 1) was employed to
obtain stylopeptide 1 (2) that was similar to one we used
(6) Adamson, J . G.; Blaskovich, M. A.; Groenevelt, H.; Lajoie, G. A.
J . Org. Chem. 1991, 56, 3447. We obtained lower yields with this
procedure using excess p-toluenesulfonic acid compared to a catalytic
amount of sulfuric acid. Costopanagiotis, A. A.; Preston, J .; Weinstein,
B. J . Org. Chem. 1966, 31, 3398. Roeske, R. J . Org. Chem. 1963, 28,
1251. Anderson, G. W.; Callahan, F. M. J . Am. Chem. Soc. 1960, 82,
3359.
(7) (a) For a review see: Fields, G. B.; Noble, R. L. Int. J . Pept.
Protein Res. 1990, 35, 161. (b) Chang, C.; Waki, M.; Ahmad, M.;
Meienhofer, J .; Lundell, E. O.; Haug, J . D. Int. J . Pept. Protein Res.
1980, 15, 59. (c) Bodanszky, M.; Deshmane, S. S.; Martinez, J . J . Org.
Chem. 1979, 44, 1622.
(8) Hamada, Y.; Rishi, S.; Shioiri, T.; Yamada, S.-I. Chem. Pharm.
Bull. 1977, 25, 224. DEPC is also called diethylphosphoryl cyanide or
diethyl cyanophosphonate.
(9) Benoiton, N. L. Int. J . Pept. Protein Res. 1994, 44, 399.
(10) Haver, A. C.; Smith, D. D. Tetrahedron Lett. 1993, 34, 2239.
Miyazawa, T.; Otomatsu, T.; Yamada, T.; Kuwata, S. Int. J . Pept.
Protein Res. 1992, 39, 229. Benoiton, N. L.; Kuroda, K. Int. J . Pept.
Protein Res. 1981, 17, 197.
(11) Schmidt, U.; Langner, J . J . Chem. Soc., Chem. Commun. 1994,
2381.
(12) (a) Zimmer, S.; Hoffmann, E.; J ung, G.; Kessler, H. Liebigs Ann.
Chem. 1993, 497. (b) Trzeciak, A.; Bannwarth, W. Tetrahedron Lett.
1992, 33, 4557.
(13) Ehrlich, A.; Rothemund, S.; Brudel, M.; Beyermann, M.; Car-
pino, L. A.; Bienert, M. Tetrahedron Lett. 1993, 34, 4781.
(14) Suggested by Prof. R. B. Bates, University of Arizona. Also
designated N,N-bis(2-oxo-3-oxazolidinyl)phosphinic chloride.
* Author to whom correspondence should be addressed.
X Abstract published in Advance ACS Abstracts, March 1, 1996.
(1) Unit 346 in the series “Antineoplastic Agents” and for the
preceding report see: J ayson, G. C.; Crowther, D.; Prendiville, J .;
McGown, A. T.; Scheid, C.; Stern, P.; Young, R.; Brenchley, P. Chang,
J .; Owens, S.; Pettit, G. R. Br. J . Cancer 1995, 72, 461.
(2) (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. (b) Pettit, G. R.; Srirangam, J . K.; Herald, D.
L.; Xu, J .-P.; Boyd, M. R.; Cichacz, Z.; Kamano, Y.; Schmidt, J . M.;
Erickson, K. L. J . Org. Chem. 1995, 60, 8257. (c) Unpublished results.
(3) (a) Pettit, G. R.; Tan, R.; Gao, F.; Williams, M. D.; Doubek, D.
L.; Boyd, M. R.; Schmidt, J . M.; Chapuis, J .-C.; Hamel, E.; Bai, R.;
Hooper, J . N. A.; Tackett, L. P. J . Org. Chem. 1993, 58, 2538. (b) Pettit,
G. R.; Gao, F.; Doubek, D. L.; Boyd, M. R.; Hamel, E.; Bai, R.; Schmidt,
J . M.; Tackett, L. P.; Ru¨tzler, K. Gazz. Chim. Ital. 1993, 123, 371.
(4) (a) Perich, J . W.; J ohns, R. B. Aust. J . Chem. 1990, 43, 1623;
Riniker, B.; Flo¨rsheimer, A.; Fretz, H.; Sieber, P.; Kamber, B.
Tetrahedron 1993, 49, 9307. (b) For an exception see Fischer, P. M.;
Solbakken, M.; Undheim, K. Tetrahedron, 1994, 50, 2277. (c) Tung,
R. D.; Dhaon, M. K.; Rich, D. H. J . Org. Chem. 1986, 51, 3350. (d)
Høeg-J ensen, T.; J akobsen, M. H.; Holm, A. Tetrahedron Lett. 1991,
32, 6387. (e) Paulsen, H.; Adermann, K. Liebigs Ann. Chem., 1989,
751 and 771.
0022-3263/96/1961-2322$12.00/0 © 1996 American Chemical Society

Synthesis of Cycloheptapeptide Stylopeptide 1
J . Org. Chem., Vol. 61, No. 7, 1996 2323
Ch a r t 1
Sch em e 1
may have been due in part to the unblocked serine
hydroxyl group, though it is usually not necessary to
protect this hydroxyl group.15 Fortunately, the problem
with DEPC was nicely avoided using TBTU (4).
identical with the natural product by comparing mixture
mp, TLC, [R]D, IR, and high field (400 MHz) H NMR
1
data. Our earlier concern about the possibility of the
natural stylopeptide 1 serving as a carrier for an unde-
tected (by physical and chemical means) but powerful
halistatin-like (cf., 3) component was amply realized by
results of careful evaluation of the synthetic cyclic peptide
against the murine P388 lymphocytic leukemia cell line
and a selection of human cancer cell lines. In each case
(15) Burke, T. R., J r.; Knight, M.; Chandrasekhar, B.; Ferretti, J .
A. Tetrahedron Lett. 1989, 30, 519. Fischer, P. M.; Retson, K. V.; Tyler,
M. I.; Howden, M. E. H. Int. J . Pept. Protein Res. 1991, 38, 491.
Barbato, G.; D’Auria, G.; Paolillo, L.; Zanotti, G. Int. J . Pept. Protein
Res. 1991, 37, 388.
The synthetic stylopeptide 1 (2) was found to be

2324 J . Org. Chem., Vol. 61, No. 7, 1996
Pettit and Taylor
peptide was subjected to medium pressure silica gel chroma-
tography.16 To avoid clogging,4c filtration was sometimes
required to remove insoluble byproducts (formed by allowing
the oily residue to stand for several h) prior to column
chromatography. Multiple precipitations/crystallizations of
the product gave colorless powders (except for the di-, tri, and
tetrapeptides which were foams) which were sufficiently pure
for the next coupling reaction. During the process of multiple
precipitations/crystallizations from hot solutions, filtration was
sometimes required to remove insoluble material (polymer?)
which probably formed from DBF.
the synthetic peptide (2) proved to be inactive while the
natural specimens from P. costata maintained their
activity suggesting the presence of this unknown active
constituent. Since we have recently discovered an analo-
gous situation among some of our other marine Porifera
cyclic peptides,5 it is now clear that such cyclic peptides
are capable of associating with certain strongly active
polyether-type antineoplastic agents that are only detect-
able by biological methods. In short, the results of these
experiments should serve as a useful caution signal when
evaluating such sponge peptides for cancer cell growth
inhibitory properties.
N-F m oc-Ile-P r o-OBu t (7). Using the above general pro-
cedures N-Fmoc-Pro-OBut (6, 3.00 g, 7.62 mmol)5,6 was N-
deprotected and coupled with N-Fmoc-Ile. The concentrated
residue was subjected to silica gel chromatography (2:1 hexane/
EtOAc, column: 50 mm x 6.5 in silica depth, flow: ∼1 in./90
s) to give 2.76 g (72% yield) of a foamy solid. Analytical purity
was achieved by additional chromatography: Rf 0.53 (1:1
Exp er im en ta l Section
Gen er a l. Except for acetonitrile (HPLC grade, EM Science)
and DMF (anhyd, Aldrich), all solvents were redistilled. All
the amino acids corresponded to the L-configuration, and
coupling reactions were conducted under argon. The N-Fmoc-
L-amino acids (Sigma-Aldrich), N,N-bis(2-oxo-3-oxazolidinyl)-
phosphorodiamidic choride (BOP-Cl, 5, TCI America Co.),14
and p-toluenesulfonic acid monohydrate (TsOH‚H2O, Mathe-
son, Coleman & Bell) were used as received. Diethyl phos-
phorocyanidate (DEPC, 93%),8 diisopropylethylamine (DIEA),
diethylamine (Et2NH), and O-(1H-benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium tetrafluoroborate (TBTU, 4),12,13 and tri-
fluoroacetic acid (TFA) were used as received from Sigma-
Aldrich Co. 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 for 2-3 min). Column chromatography employed
230-400 mesh, 0.040-0.063 mm silica gel 60 (Merck). Melt-
ing points are uncorrected. Optical rotation data were col-
lected using a 1 mL 1-dm cell at the sodium D line (589 nm
hexane/EtOAc); [R]28 -57.5° (c 1.275, CHC13); UV (nm, log
D
ꢀ): 235, 3.8; 267, 4.3; 289, 3.7; 300, 3.8; EIMS m/ z calcd for
C30H38N2O5 506, found 506. Anal. Calcd for C30H38N2O5
(506.64): 71.12 C, 7.56 H, 5.53 N. Found: 71.26 C, 7.39 H,
5.20 N.
N-F m oc-P r o-Ile-P r o-OBu t (8). Using the above general
procedures N-Fmoc-Ile-Pro-OBut (7, 6.90 g, 13.62 mmol) was
N-deprotected and coupled with N-Fmoc-Pro. The concen-
trated oil was purified by silica gel chromatography (4:1
EtOAc/hexane, column: 90 mm x 7.5 in silica depth, flow; ∼1
in./6 min ) 23 mL/min to yield 7.6 g of a foam. Rechromatog-
raphy gave 6.28 g (76% yield) of a colorless foam; mp 85-89
°C; Rf 0.45 (EtOAc); [R]23D -105° (c 0.11, CHC13); UV (nm, log
ꢀ) 235, 3.8; 267, 4.2; 289, 3.7; 300, 3.8; FABHRMS m/ z calcd
for C35H46N3O6 604.3386, found 604.3372 [M + H]+. Anal.
Calcd for C35H45N3O6‚H2O (621.77): 67.61 C, 7.62 H, 6.76 N.
Found: 67.72 C, 7.72 H, 6.43 N.
N-F m oc-Ser (Bu t)-P r o-Ile-P r o-OBu t (9). Using the above
general procedures N-Fmoc-Pro-Ile-Pro-OBut (8, 6.66 g, 11.03
mmol) was N-deprotected and coupled with N-Fmoc-Ser(But).
After concentration, silica gel chromatography (4:1 EtOAc/
hexane, column: 90 mm x 8 in silica depth, flow: 23 mL/70 s)
was used to afford 6.77 g (80% yield) of a foam. Analytical
purity was achieved by additional chromatography: Rf 0.22-
1
and temperatures noted). The H NMR δ values are relative
to TMS in CDCl3 (except for stylopeptide 1 which are relative
to DMSO-d6 at δ 2.49) and relative to CDCl3 (δ 77.0) for 13C
NMR spectra. J values are in Hz. Analytical samples were
dried in vacuo (Abderhalden over P2O5 at methanol or water
reflux temperatures for several h). Elemental analyses were
completed by Galbraith Laboratories, Inc. (Knoxville, TN).
Gen er a l Dep r otection P r oced u r e.4b,c,7b A solution of the
N-Fmoc tert-butyl ester in 1:1 CH3CN/Et2NH (∼0.2 M) was
stirred at rt for 2 h. After several minutes, deprotection was
observed to be complete (by TLC), but longer times were used
to insure maximum deprotection. Reaction with ninhydrin by
mild heating of the TLC plate indicated the free amino tert-
butyl ester near the origin. Solvents were removed by rotary
evaporation to yield viscous amber syrups. Further removal
of trace solvent sometimes was done in vacuo (typically ∼0.3
torr, 30 min). The crude free amino tert-butyl ester was used
immediately without further purification.
0.30 (4:1 EtOAc/hexane); [R]24.5 -72° (c 3.24, CHC13); FAB-
D
HRMS m/ z calcd for C42H59N4O8 747.4333, found 747.4314 [M
+ H]+. Anal. Calcd for C42H58N4O8‚H2O (764.96): 65.95 C,
7.91 H, 7.32 N. Found: 66.10 C, 7.90 H, 7.29 N.
N-F m oc-P h e-Ser (Bu t)-P r o-Ile-P r o-OBu t (10). Using the
above general procedures N-Fmoc-Ser(But)-Pro-Ile-Pro-OBut
(9, 6.67 g, 8.93 mmol) was N-deprotected and coupled with
N-Fmoc-Phe. The reaction became homogeneous as the reac-
tion progressed. Silica gel chromatography (4:1 EtOAc/hexane,
column: 90 mm x 6.5 in silica depth, flow: 23 mL/90 s)
afforded 7.18 g (90% yield) of a powder. A pure sample was
obtained by additional chromatography and precipitation: mp
Am in o Acid Cou p lin g P r oced u r e.7b,8 A solution of the
crude amino tert-butyl ester in DCM (∼0.5 M) containing DIEA
(1 equiv) at rt was transferred slowly under positive argon
pressure via cannula to a solution of the 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 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 stirred under argon for 3-4 h between -10 and
-5 °C. TLC monitoring showed dibenzofulvene (DBF) byprod-
uct(s) and the desired N-Fmoc tert-butyl ester. If the order of
addition was reversed (Fmoc-amino acid/DEPC added to the
amino tert-butyl ester/DIEA mixture), the yields decreased.
When the DEPC coupling (in DCM or DMF/DCM) was
performed with anhydrous sodium carbonate5 as base, the
reverse order of addition was used. In general, the DEPC/
DIEA method was judged to be more convenient.
114-119 °C; Rf 0.21-0.30 (4:1 EtOAc/hexane); [R]25 -78° (c
D
0.54, CHC13); FABHRMS m/ z calcd for C51H68N5O9 894.5017,
found 894.5017 [M + H]+. Anal. Calcd for C51H67N5O9
(894.12); 68.51 C, 7.55 H, 7.83 N. Found: 68.48 C, 7.68 H,
7.60 N.
N-F m oc-Ile-P h e-Ser (Bu t)-P r o-Ile-P r o-OBu t (11). Using
the above general procedures N-Fmoc-Phe-Ser-(But)-Pro-Ile-
Pro-OBut (10, 7.25 g, 8.11 mmol) was N deprotected and
coupled with N-Fmoc-Ile. Homogeneity of the mixture was
observed as the reaction progressed. Silica gel chromatogra-
phy (EtOAc, column: 90 mm x 6 in silica depth, flow: 23 mL/
105 s) gave 5.34 g (65% yield) of a powder from EtOAc/hexane.
Additional chromatography and precipitation provided an
analytical sample: mp 154-159 °C; Rf 0.22 (10:1 EtOAc/
hexane, 0.15 (4:1 EtOAc/hexane); [R]26D -68° (c 0.26, CHC13);
IR (NaC1 thin film) νmax (cm-1): 3293 (s), 3065 (w), 2969 (s),
2936 (m), 2878 (m), 1724 (m), 1628 (s), 1535 (s), 1449 (s), 1391
(w), 1366 (m), 1234 (m), 1196 (w), 1155 (m), 1097 (w), 1034
(w), 758 (m), 743 (m); FABHRMS m/ z calcd for C57H79N6O10
P u r ifica tion of th e P ep tid es 7-12. After concentrating
the reaction mixture, the crude product could be directly
chromatographed quite effectively without an aqueous wash-
ing procedure.8 The crude N-Fmoc tert-butyl ester-protected
(16) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923.

Synthesis of Cycloheptapeptide Stylopeptide 1
J . Org. Chem., Vol. 61, No. 7, 1996 2325
1007.5857, found 1007.5871 [M + H]+. Anal. Calcd for
C57H78N6O10 (1007.28): 67.97 C, 7.81 H, 8.34 N. Found: 67.51
C, 7.83 H, 8.24 N.
Calcd for C40H61N7O8‚2H2O (803.99): 59.76 C, 8.15 H, 12.19
N. Found: 59.54 C, 7.80 H, 12.23 N. Complete NMR (1H,
13C, HMBC, NOESY, ROESY in DMSO-d6) and X-ray data for
stylopeptide 1 have been previously reported.2b
N-Fm oc-Leu -Ile-P h e-Ser (Bu t)-P r o-Ile-P r o-OBu t (12). Us-
ing the above general procedures N-Fmoc-Ile-Phe-Ser(But)-Pro-
Ile-Pro-OBut (11, 5.47 g, 5.43 mmol) was N-deprotected and
coupled with N-Fmoc-Leu. Silica gel column chromatography
(EtOAc, column: 90 mm x 6.5 in silica depth, flow: 23 mL/75
s) provided 4.59 g (76% yield) of a colorless powder. Additional
chromatography and precipitation afforded a pure sample: Rf
0.32 (EtOAc); [R]26D -76° (c 0.55, CHC13); UV (nm, log ꢀ): 236,
3.8; 266, 4.2; 289, 3.7; 300, 3.8; FABHRMS m/ z calcd for
C63H90N7O11 1120.6698, found 1120.6721 [M + H]+. Anal.
Calcd for C63H89N7O11 (1120.44): 67.54 C, 8.01 H, 8.75 N.
Found: 67.94 C, 8.36 H, 8.61 N.
Meth od B. BOP -C1 Cou p lin g: The preceding Fmoc and
tert-butyl deprotection reactions (method A) were repeated
using 12 (54 mg, 0.0483 mmol). A solution of the TFA salt of
Leu-Ile-Phe-Ser-Pro-Ile-Pro and DIEA (160 equiv) in DCM (35
mL) was transferred slowly via cannula (over 1 h) under argon
to a stirred solution of BOP-C1 (22 equiv) in DCM (400 mL)
at 0 °C. The reaction mixture was stirred at 0 °C for 30 min
and at rt for 3 weeks. The solvent was evaporated and the
residue triturated and then dissolved in EtOAc (50 mL). The
ethyl acetate solution was washed with water (3 × 2 mL), and
the aqueous layer was back-extracted with EtOAc (3 × 2 mL).
The combined EtOAc extract was washed with saturated
aqueous NaHCO3 (3 × 2 mL) and brine (2 mL), dried (MgSO4),
and filtered. Solvent was removed to yield an oil (0.1 g). The
oil was subjected to silica gel chromatography using 2:1
i-PrOH/hexane as eluent. Stylopeptide 1 (2) was obtained as
colorless needles from CH3OH (4.9 mg, 13% yield). The
synthetic peptide was identical by TLC with authentic natural
stylopeptide 1.
Stylop ep tid e 1 (2). Meth od A. TBTU Cou p lin g: N-
Fmoc-Leu-Ile-Phe-Ser-(But)-Pro-Ile-Pro-OBut (12, 1.00 g, 0.897
mmol) was N-deprotected using the above general procedure
in a 1 L round-bottom flask. After thorough removal of
solvents, neat TFA (30 mL) was added to the crude Leu-Ile-
Phe-Ser(But)-Pro-Ile-Pro-OBut at rt (precipitation was ob-
served immediately), and the mixture was stirred for 2.5 h.
Excess TFA was removed in vacuo, followed by evaporation
of 2 × 5 mL portions of DCM. To the TFA salt in DCM (600
mL) was added TBTU (1.44 g, 4.48 mmol) dissolved in CH3-
CN (15 mL) at 0 °C under argon. DIEA (6 mL, 1% v/v) was
added slowly (∼15 min) at rt while stirring. Dilution of the
heptapeptide corresponded to 1.4 mM. The reaction mixture
was stirred for 2 h at 0 °C and continued for 14 d at rt. After
removal of solvent in vacuo, a solution of the crude product in
DCM (200 mL) was washed with 10% aqueous citric acid (3 ×
10 mL), saturated aqueous NaHCO3 (3 × 10 mL), and water
(10 mL). The aqueous layers were back-extracted with EtOAc
(10 mL). The combined organic layers were dried (MgSO4) and
filtered. Solvents were removed in vacuo to yield 1.67 g of an
oil which was chromatographed on silica gel using a 25 mm x
10 in (silica depth) column and elution with 1:1 hexane/i-PrOH
under slight pressure to allow ∼23 mL/4 min. Stylopeptide 1
was followed by ceric sulfate-sulfuric acid-developed TLC (Rf
0.40, 4:1 EtOAc/CH3OH). (Stylopeptide 1 was not visualized
by UV or I2). Straw-colored crystals were obtained from
saturated solutions in CH3OH. Recrystallization was contin-
ued until the mother liquor was completely colorless (at least
three recrystallizations) to give 0.46 g of colorless needles (67%
final step yield and 11% overall).
Ack n ow led gm en t . The financial support for this
investigation was provided by Outstanding Investigator
Grant CA44344-01-07 awarded by the Division of Can-
cer Treatment, NCI, DHHS, the Arizona Disease Con-
trol Research Commission, the Fannie E. Rippel Foun-
dation, Virginia Piper, the Robert B. Dalton Endowment
Fund, Eleanor W. Libby, Gary L. Tooker, Diane M.
Cummings, J ohn and Edith Reyno, Lottie Flugel, Polly
Trautman, the Fraternal Order of Eagles Art Ehrmann
Cancer Fund, and the Ladies Auxiliary to the Veterans
of Foreign Wars. For other very useful assistance we
are pleased to thank Drs. Michael R. Boyd, J ean-
Charles Chapuis, Fiona Hogan, Robin K. Pettit, Michael
D. Williams, Mr. David M. Carnell, and Mr. Lee
Williams, the U. S. National Science Foundation (Grants
BBS 88-04992, CHE-92-14799, CHE-88-13109, CHE-
8409644) and the NSF Regional Instrumentation Facil-
ity in Nebraska (Grant CHE-8620177).
Comparison of the natural and synthetic specimens of
stylopeptide 1 (2) by mixed mp, TLC, [R]D, IR, and 1H NMR
(400 MHz, CD3OD and DMSO-d6) showed that both were iden-
tical: mp 228.0-229.5 °C (synthetic), mp 228.0-229.0 °C
(natural),2b mixed mp 227.0-228.0 °C; Rf 0.51 (i-PrOH); 0.40
(4:1 EtOAc/CH3OH); 0.27-0.31 (2:1 i-PrOH/hexane; 0.18 (1:1
1
Su p p or tin g In for m a tion Ava ila ble: List of 1H (from H-
1H COSY) and 13C (APT and BB) NMR assignments for Fmoc
tert-butyl ester intermediates, 7-12 and H NMR spectra and
IR spectra of stylopeptide 1 (2) (8 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal and can be
ordered from the ACS; see any current masthead page for
ordering information.
1
hexane/i-PrOH); [R]27D -129° (c 0.21, CH3OH, synthetic) [R]25
D
-128° (c 0.20, CH3OH, natural),2b synthetic FABHRMS m/ z
calcd for C40H62N7O8 768.4660, found 768.4653 [M + H]+;
natural FABHRMS m/ z found 768.4647 [M + H]+.2b Anal.
J O951986B