Solid-Phase Total Synthesis of Oscillamide Y and Analogues

Article abstract of DOI:10.1021/jo970671o

We report an efficient solid phase synthesis of oscillamide Y and three analogues. The cyclic peptide was prepared using a combination of Fmoc and allyl chemistries and an acid labile Wang type linker. The urea functionality was smoothly incorporated usin

Full text of DOI:10.1021/jo970671o

J . Org. Chem. 1997, 62, 6199-6203
6199
Solid -P h a se Tota l Syn th esis of Oscilla m id e Y a n d An a logu es
Ian R. Marsh and Mark Bradley*
Department of Chemistry, University of Southampton, Highfield, Southampton, SO17 1BJ , UK
Simon J . Teague
Department of Medicinal Chemistry, Astra Charnwood, Bakewell Road, Loughborough,
Leicestershire, LE11 0RH, UK
Received April 15, 1997X
We report an efficient solid phase synthesis of oscillamide Y and three analogues. The cyclic peptide
was prepared using a combination of Fmoc and allyl chemistries and an acid labile Wang type
linker. The urea functionality was smoothly incorporated using NR-(4-nitrophenyloxycarbonyl)-
Nꢀ-(9-fluorenylmethoxycarbonyl)-D-lysine allyl ester. Coupling to the N-methyl amino acid was
readily achieved using HATU, monitoring the reaction using bromophenol blue. Allyl deprotection
was accomplished using Pd(PPh3)4 and dimedone, and cyclization was smoothly accomplished using
PyBroP. All reactions were monitored using mass spectrometry methodology. The cyclized
materials were cleaved by acidolysis and purified by RP HPLC. In all cases the material isolated
was the major product and gave the expected molecular ion information. HPLC comparison with
an authentic sample of oscillamide Y showed that the isomer containing L-N-methylalanine and
1
L-homotyrosine was the natural product. 1H NMR and H-1H COSY NMR experiments further
confirmed this identification. The four compounds were tested as competitive and slow-tight binding
inhibitors of chymotrypsin but showed, contrary to literature expectations, no inhibitory activity.
In tr od u ction
Oscillamide Y (Figure 1) is a cyclic ureido-containing
hexapeptide produced by a “toxic” strain of the freshwater
cyanobacterium Oscillatoria agardhii and whose struc-
ture was first reported in 1995 as a potent inhibitor of
chymotrypsin.1 It contains several unusual functional-
ities including a urea, N-methylalanine, and homoty-
rosine as well as a bridging D-lysine residuesa bridge
found in other cyclic peptides such as bacitracin A2 and
glidobaktin3 but usually with L-stereochemistry. It also
bears a striking resemblance to the reported structure
of konbamide,4 a calmodulin antagonist isolated from the
sea sponge Theonella. The stereochemistries of the
F igu r e 1. Oscillamide Y (* unknown stereochemistry).
homotyrosine and N-methylalaine residues were un-
known1 so we decided to synthesize four possible ana-
logues of oscillamide Y to firstly determine the stereo-
chemistry of the natural product and secondly, to
determine the relative chymotrypsin inhibitory properties
of the four compounds. The development of solid phase
methodology to oscillamide Y was investigated because
of our desire to use oscillamide Y as a template for library
generation and the design of serine protease inhibitors.
Thus (hydroxymethyl)phenoxyacetic acid was coupled
to aminomethyl polystyrene resin using diisopropylcar-
bodiimide (DIC)/hydroxybenzotriazole (HOBt) in dichlo-
romethane/DMF. This, in our hands, provides a much
more reliable resin-linker system than the traditional
methods of Wang linker synthesis. Coupling of the first
residue was carried out using FmocTyr(OBut)OH in THF/
pyridine with DIC activation followed by capping with
acetic anhydride and gave an initial loading of 0.4 mmol/g
as determined by quantitative Fmoc and ninhydrin tests.
The Fmoc group was removed with piperidine and the
free amine treated with 4 equiv of NR-(4-nitrophenyloxy-
carbonyl)-Nꢀ-(9-fluorenylmethoxycarbonyl)-D-lysine allyl
ester (2) in DMF/diisopropylethylamine (DIPEA). Bases
such as N-methylmorpholine and pyridine were found to
be inapplicable in this step as was the use of dichlo-
romethane. The NR-activated lysine compound (2) was
prepared as shown in Scheme 2 in good yield (59%) from
D-Lys(Cbz)OH.
Resu lts a n d Discu ssion
Our synthetic approach is outlined in Scheme 1 and
utilized Fmoc/But/allyl chemistries as have been reported
previously by several other groups including our own.2,5
The urea functionality was incorporated using a nitro-
phenyl carbamate activated lysine residue.
X Abstract published in Advance ACS Abstracts, August 1, 1997.
(1) Sano, T.; Kaya, K. Tetrahedron Lett. 1995, 36, 5933.
(2) Lee, J . H.; Griffin, J . H.; Nicas, T. I. J . Org. Chem. 1996, 61,
3983.
(3) Schmidt, U.; Kleefeldt, A.; Mangold, R. J . Chem. Soc., Chem.
Commun. 1992, 1687.
(4) Schmidt, U.; Weinbrenner, S. Angew. Chem., Int. Ed. Engl. 1996,
35, 1336.
(5) Egner, B. J .; Langley, G. J .; Bradley, M. J . Org. Chem. 1995,
60, 2652.
Successful urea formation was monitored by removal
of a small quantity of resin (ca. 5 mg) followed by
trifluoroacetic acid (TFA) treatment and subsequent
HPLC and ES (electrospray) MS analysis.6 Fmoc peptide
synthesis continued on the side chain of the lysine
residue using Fmoc-L-Phe and then either Fmoc-L-N-
S0022-3263(97)00671-3 CCC: $14.00 © 1997 American Chemical Society

6200 J . Org. Chem., Vol. 62, No. 18, 1997
Marsh et al.
Sch em e 1
using Pd(0) and dimedone as the scavenger.10 The resin
bound linear peptide was deprotected with piperidine and
washed with pyridinium hydrochloride in DMF/dichlo-
romethane (1:1) to ensure all traces of piperidine were
removed. The success of this final allyl deprotection was
monitored by mass spectrometry5,6 and in all cases the
full length, but deprotected peptide, was the only mate-
rial observed. Cyclization proceeded without incident
using PyBroP in DMF/dichloromethane with DIPEA and
was again monitored by mass spectrometry. The com-
pounds were cleaved from the support using TFA/H2O
(95:5) for 2 h, the solvent was evaporated, and the crude
products were dissolved in water/acetonitrile and lyoph-
ilized before purification by reverse phase HPLC. The
desired compound was the major component in all cases
showing the high efficiency of the synthesis. HPLC
comparison of the four synthetic materials and compari-
son with an authentic sample of oscillamide Y (kindly
provided by Tomoharu Sano) showed that the isomer
containing L-N-methylalanine and L-homotyrosine cor-
responded to the natural material (Figure 2).
Sch em e 2
MeAla or Fmoc-D-N-MeAla. Coupling to the N-methyl-
amino acid was attempted with three coupling mixtures.
(i) DIC/HOBt, (ii) PyBroP,7 (iii) HATU8/HOBt, monitoring
the reaction in each case by the use of bromophenol blue.9
HATU (O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate) proved to be the most
successful coupling agent giving complete coupling in 2-3
h.
The four oscillamide Y isomers were assayed as
competitive inhibitors of chymotrypsin using the sub-
strate succinyl-AlaAlaProPhe-p-nitroanilide,11 monitoring
the release of p-nitrophenol at 410 nm. At concentrations
up to 100 µM, none of the four isomers showed any
competitive inhibitory behavior with chymotrypsin. In
a similar assay authentic oscillamide Y was also found
not to inhibit chymotrypsin. The four isomers of oscil-
The remaining residue (Fmoc-L-Ile) coupled without
incident using DIC/HOBt followed by allyl ester removal
(6) Typical ESMS monitoring procedure: A 5-10 mg sample of resin
is treated with 20% piperidine/DMF for 10 min and washed with DMF
and dichloromethane before cleavage with TFA/H2O (95:5, 1 mL) for
1 h. After filtration and evaporation of the solvents, the product is
analyzed by ES MS.
(7) Frerot, E.; Coste, J .; Pantaloni, A.; Dufour, M.-N.; J ouin, P.
Tetrahedron 1991, 47, 259.
(8) Carpino, L. A.; El-Faham, A.; Minor, C. A., Albericio, F. J . Chem.
Soc., Chem. Commun. 1994, 201.
(9) Krchnak, V.; Vagner, J .; Safar, P.; Lebl, M. Coll. Czech. Chem.
Commun. 1988, 53, 2542.
(10) Kunz, H.; Unverzagt, C. Angew. Chem., Int. Ed. Engl. 1984,
23, 436.
(11) DelMar, E. G.; Largman, C.; Brodrick, J . W.; Geokas, M. C.
Anal. Biochem. 1979, 99, 316.

Solid-Phase Total Synthesis of Oscillamide Y
J . Org. Chem., Vol. 62, No. 18, 1997 6201
water (1:1, 80 mL) and sodium hydroxide (2 M, 9 mL) at 4 °C
and the mixture stirred for 1.5 h. The reaction mixture was
concentrated under reduced pressure, poured into water (200
mL) and acidified with 2 M KHSO4. The aqueous phase was
extracted with ethyl acetate (60 mL, 5 × 30 mL), the extracts
were combined, washed (brine, 60 mL), dried (MgSO4), and
filtered, and the solvent was removed in vacuo to give a sticky
oil (6.55 g, 98%): 1H NMR (300 MHz, d6-DMSO) δ 1.26-1.44
(s + m, 14 H), 1.50-1.64 (m, 1 H), 2.97 (dt, J ) 5.9 and 6.6
Hz, 2 H), 3.82 (ddd, J ) 8.1, 8.8 and 5.1 Hz, 1 H), 5.00 (s, 2
H), 7.02 (d, J ) 8.1 Hz, 1 H), 7.24 (t, J ) 5.9 Hz, 1 H), 7.29-
7.38 (m, 5 H), 12.41 (broad s, 1H); 13C NMR (75 MHz,
d6-DMSO) δ 22.8, 28.1, 28.9, 30.3, 39.9, 53.4, 65.0, 77.9, 127.6,
128.3, 137.2, 155.5, 156.0, 174.2.
Nr-(ter t-Bu t yloxyca r b on yl)-NE-(9-flu or en ylm et h oxy-
ca r bon yl)-D-lysin e.13 A solution of NR-(tert-butyloxycarbon-
yl)-Nꢀ-(benzyloxycarbonyl)-D-lysine (6.55 g, 17.2 mmol) in
ethanol (250 mL) was purged with nitrogen. Palladium on
activated carbon (10%, 0.65 g) was added, the solution de-
gassed, the apparatus filled with hydrogen (×3), and the
reaction mixture stirred overnight. The reaction was filtered
through Celite and the solvent removed under reduced pres-
sure. The product was redissolved in dioxane (100 mL) and
NaHCO3 (5% aq, 50 mL), the solution cooled in an ice bath, a
solution of Fmoc-succinimide (5.50 g, 16.3 mmol) added
dropwise over 40 min, and the mixture stirred for another 30
min. The reaction mixture was concentrated under reduced
pressure, diluted to 250 mL by the addition of water, and
acidified to pH 3 with 2 M KHSO4. The aqueous phase was
extracted with ethyl acetate (75 mL, 3 × 50 mL). The extracts
were combined, washed (brine, 50 mL), dried (MgSO4), and
filtered, and the solvent was removed in vacuo to give the
product as a colorless foam (7.57 g, 99%): 1H NMR (300 MHz,
d6-DMSO) δ 1.25-1.45 (s + m, 14 H), 1.45-1.70 (m, 1 H), 2.97
(m, 2 H), 3.84 (ddd, J ) 4.4, 5.2 and 7.4 Hz, 1 H), 4.20 (t, J )
6.6 Hz, 1H), 4.29 (d, J ) 6.6 Hz, 2 H), 7.03 (d, J ) 7.4 Hz, 1
H), 7.28 (t, J ) 5.2 Hz, 1 H), 7.32 (t, J ) 7.4 Hz, 2 H), 7.40 (t,
J ) 7.4 Hz, 2 H), 7.68 (d, J ) 7.4 Hz, 2 H), 7.88 (d, J ) 7.4 Hz,
2 H), 12.41 (broad s, 1 H); 13C NMR (75 MHz, d6-DMSO) δ
22.9, 27.9, 28.9, 30.4, 39.9, 46.7, 53.4, 66.3, 77.9, 120.0, 125.1,
127.0, 127.5, 140.7, 143.9, 155.6, 156.0, 174.2.
F igu r e 2. HPLC chromatograms showing coinjections of (A)
oscillamide Y + synthetic oscillamide Y (L-MeAla, L-Htyr); (B)
oscillamide Y + synthetic oscillamide Y (L-MeAla, D-Htyr); (C)
oscillamide Y + synthetic oscillamide Y (D-MeAla, D-Htyr); (D)
oscillamide Y + synthetic oscillamide Y (D-MeAla, L-Htyr)
[gradient conditions: t ) 0, 75% A, 25% B; t ) 10, 75% A,
25% B; t ) 30, 25% A, 75% B; t ) 35, 100% B; A ) water/0.1%
TFA, B ) acetonitrile/0.1% TFA]. Approximately equal amounts
of synthetic and authentic oscillamide Y were coinjected in
each run (as determined by UV absorbance). (* ) Oscillamide
Y).
Nr-(ter t-Bu t yloxyca r b on yl)-NE-(9-flu or en ylm et h oxy-
ca r bon yl)-D-lysin e Allyl Ester . NR-(tert-Butyloxycarbonyl)-
Nꢀ-(9-fluorenylmethoxycarbonyl)-D-lysine (7.57 g, 16.2 mmol)
was dissolved in DMF (40 mL) and cesium carbonate (3.98 g,
12.2 mmol) added. The mixture was stirred for 1 h. Allyl
bromide (13.8 g, 9.9 mL, 0.114 mol) was added to the mixture
and stirring continued for a further 1 h resulting in a milky
solution. The mixture was diluted to 250 mL with water and
acidified with 2 M KHSO4. The aqueous phase was extracted
with dichloromethane (80 mL, 3 × 40 mL). The extracts were
combined, washed (brine, 80 mL), dried (MgSO4), and filtered,
and the solvent was removed in vacuo to give a white solid
which was recrystallized from ethyl acetate/hexane to give
white crystals (6.89 g, 83%): mp 119-121 °C; IR (Nujol) 3345,
lamide Y were tested for slow, tight-binding inhibitory
activity of chymotrypsin, but again, no inhibition was
observed, clearly at variance with other published re-
sults.1
We have therefore produced a very efficient solid phase
synthesis of a urea-containing cyclic peptide and have
set the stage for the use oscillamide Y as a template for
solid phase chemistry. Unfortunately we could not
repeat the work of previous workers who claimed that
oscillamide Y was a potent inhibitor of chymotrypsin.
1
1749, 1690, 1522; H NMR (300 MHz, CDCl3) δ 1.40-1.50 (s
+ m, 11 H), 1.50-1.58 (m, 2 H), 1.62-1.71 (m, 1 H), 1.75-
1.90 (m, 1 H), 3.19 (m, 2 H), 4.21 (t, J ) 6.6 Hz, 1 H), 4.33 (m,
1 H), 4.40 (d, J ) 6.6 Hz, 2 H), 4.63 (dt, J ) 5.9 and 1.5 Hz, 2
H), 4.95 (broad s, 1 H), 5.15 (d, J ) 7.4 Hz, 1 H), 5.25 (dd, J
) 10.3 and 1.5 Hz, 1 H), 5.33 (dd, J ) 16.9 and 1.5 Hz, 1 H),
5.84-6.00 (ddt, J ) 16.9, 10.3 and 5.9 Hz, 1 H), 7.32 (dt, J )
1.5 and 7.4 Hz, 2 H), 7.41 (t, J ) 7.4 Hz, 2 H), 7.60 (d, J ) 7.4
Hz, 2 H), 7.77 (d, J ) 7.4 Hz, 2 H); 13C NMR (75 MHz, CDCl3)
δ 22.4, 28.2, 29.3, 32.3, 40.5, 47.2, 53.2, 65.8, 66.4, 79.8, 118.8,
119.9, 125.0, 126.9, 127.6, 131.5, 141.2, 143.9, 155.4, 156.4,
172.4. HRMS: C29H37O6N2 requires 509.2539; found 509.2617.
Nr-(4-Nitr oph en yloxycar bon yl)-NE-(9-flu or en ylm eth oxy-
ca r bon yl)-D-lysin e Allyl Ester (2). A solution of NR-(tert-
butyloxycarbonyl)-Nꢀ-(9-fluorenylmethoxycarbonyl)-D-lysine al-
lyl ester (0.97 g, 1.9 mmol) and p-toluenesulfonic acid (0.33 g,
Exp er im en ta l Section
Gen er a l. An authentic sample of oscillamide Y was gener-
ously provided by Tomoharu Sano. Specific sources of reagents
and solvents were as follows: Peptide synthesis grade N,N-
dimethylformamide (DMF) and HPLC grade acetonitrile,
Rathburn Chemical Co.; Fmoc N-Me-Ala (D and L), Htyr (D
and L), HOBt and PyBroP, Advanced ChemTech; HATU,
PerSeptive Biosystems; other Fmoc-protected amino acids,
NovaBiochem.
Nr-(ter t-Bu tyloxyca r bon yl)-NE-(ben zyloxyca r bon yl)-D-
lysin e.12 A solution of di-tert-butyl dicarbonate (3.82 g, 17.5
mmol) in dioxane (30 mL) was added to a solution of Nꢀ-
(benzyloxycarbonyl)-D-lysine (5.03 g, 18.0 mmol) in dioxane/
(12) Acosta, C. K.; Bahr, M. L.; Burdett, J . E.; Cessac, J . W.;
Martinez, R. A.; Rao, P. N. J . Chem. Res. (M) 1994, 914.
(13) Schiller, P. W.; Weltrowska, G.; Nguyen, T. M.-D.; Wilkes, B.
C.; Chung, N. N.; Lemieux, C. J . Med. Chem. 1992, 35, 3956.

6202 J . Org. Chem., Vol. 62, No. 18, 1997
Marsh et al.
Ta ble 1. 1H NMR (360 MHz, d 7-DMF ) Da ta for
1.9 mmol) in dichloromethane/ethanol (1:1, 20 mL) was
evaporated under reduced pressure at 45 °C and the evapora-
tion repeated six times until no starting material remained.
A solution of this product in dichloromethane (15 mL) was
added to a suspension of 4-nitrophenyl chloroformate in
dichloromethane (10 mL) and pyridine (1.2 mL, 15.2 mmol)
with cooling in an ice/salt bath. After stirring and allowing
the temperature to increase to 5 °C over 3 h, the reaction
mixture was acidified by addition of 1 M KHSO4 (50 mL). The
organic layer was separated and the aqueous phase extracted
with dichloromethane (3 × 15 mL). The extracts were
combined, dried (MgSO4), and filtered, and the solvent was
removed in vacuo. The crude product was purified by column
chromatography (silica gel eluting with 2:1 hexane/ethyl
acetate to remove 4-nitrophenol and 2:3 hexane/ethyl acetate
to elute the product) and gave the title compound as pale
yellow crystals (0.78 g, 72%): mp 126-128 °C; IR (Nujol) 3327,
1724, 1683, 1530; 1H NMR (300 MHz, CDCl3) δ 1.35-1.70 (m,
4 H), 1.70-2.05 (m, 2 H), 3.24 (m, 2 H), 4.21 (t, J ) 6.6 Hz, 1
H), 4.41 (m, 3 H), 4.68 (d, J ) 5.9 Hz, 2 H), 4.93 (t, J ) 5.2 Hz,
1 H), 5.29 (d, J ) 10.3 Hz, 1 H), 5.36 (d, J ) 16.2 Hz, 1 H),
5.86-5.95 (ddt, J ) 10.3, 16.2 and 5.9 Hz, 1 H), 6.02 (d, J )
8.1 Hz, 1 H), 7.22-7.32 (m, 4 H), 7.40 (t, J ) 7.4 Hz, 2 H),
7.60 (d, J ) 7.4 Hz, 2 H), 7.75 (d, J ) 7.4 Hz, 2 H), 8.18 (d, J
) 8.8 Hz, 2 H); 13C NMR (75 MHz, CDCl3) δ 22.2, 29.6, 31.6,
40.2, 47.3, 54.2, 66.3, 66.9, 119.4, 120.1, 122.0, 125.1, 125.2,
127.1, 127.9, 131.5, 141.4, 144.0, 144.9, 153.0, 155.8, 157.0,
171.7. HRMS: C31H32O8N3 requires 574.2189; found 574.2153.
(Hyd r oxym eth yl)p h en oxya ceta m id om eth yl P olysty-
r en e Resin . A solution of diisopropylcarbodiimide (1.67 g,
2.0 mL, 13.2 mmol), HOBt (1.78 g, 13.2 mmol), and 4-(hy-
droxymethyl)phenoxyacetic acid (2.40 g, 13.2 mmol) in CH2Cl2/
DMF (1:1, 30 mL) was added to aminomethyl polystyrene resin
(8.26 g, 6.6 mmol NH2) suspended in dichloromethane (130
mL) and the mixture shaken for 2.5 h. At this time a
ninhydrin test was negative. The resin was filtered, washed
with DMF (4 × 80 mL), dichloromethane (4 × 100 mL), and
ether (2 × 80 mL) and dried in vacuo.
Oscilla m id e Y
position
CHR
δ (ppm)
(m, Hz)
Phe
4.67
3.52
2.98
7.37
7.30
9.11
5.09
1.28
2.05
5.04
2.05
1.98
2.85
2.62
7.18
6.91
9.17
4.30
2.05
1.82
1.37
1.26
1.37
7.54
4.26
2.01
1.85
1.60
1.40
3.72
2.90
7.60
6.97
4.63
3.14
3.00
7.18
6.87
6.40
(m)
â
CH2
aryl
NH
(dd, J ) 3.1, 13.3 Hz)
(d, J ) 13.3 Hz)
(m)
(m)
(d, J ) 9.1 Hz)
(q, 6.8 Hz)
(d, J ) 6.8 Hz)
(s)
(m)
(m)
(m)
MeAla
Htyr
CHR
â
CH3
NMe
CHR
â
CH2
CH2
aryl
NH
γ
(obscured by solvent)
(m)
(overlapping with Tyr)
(d, J ) 8.5 Hz)
(d, J ) 4.3 Hz)
(t, J ) 6.4 Hz)
(m)
Ile
CHR
CHâ
γ
CH2
(m)
(m)
γ
δ
CH3
CH3
NH
(d, J ) 6.9 Hz)
(t, J ) 7.4 Hz)
(d, J ) 6.4 Hz)
(dd, J ) 6.9, 9.0 Hz)
(m)
(m)
(m)
(m)
(m)
(obscured by solvent)
(broad)
(d, J ) 7.4 Hz)
(m)
(dd, J ) 5.1, 14.0 Hz)
(obscured by solvent)
(overlapping with Htyr)
(d, J ) 8.5 Hz)
(d, J ) 7.9 Hz)
Lys
CHR
â
CH2
CH2γ + CH2
δ
δ
CH2
CH2
ꢀ
NHR
NHꢀ
CHR
Tyr
â
CH2
Nr-(F lu or en ylm et h oxyca r b on yl)-O-ter t-b u t yl-L-t yr o-
sin yl(h yd r oxym et h yl)p h en oxya cet a m id om et h yl P oly-
styr en e Resin . Diisopropylcarbodiimide (1.13 g, 1.39 mL, 8.0
mmol) was added to an ice cold solution of FmocTyr(tBu)OH
(3.68 g, 8.0 mmol) and HOBt (1.19 g, 8.0 mmol) in THF (30
mL). The preactivated amino acid solution was added to
(hydroxymethyl)phenoxyacetamidomethyl polystyrene resin
(2.5 g, 2.0 mmol) suspended in pyridine (10 mL) and the
mixture shaken for 3.5 h. The resin was filtered and washed
with THF (2 × 30 mL), DMF (4 × 30 mL), and dichlo-
romethane (4 × 30 mL) and dried in vacuo. Resin substitution
was determined by quantitative Fmoc and ninhydrin analysis
and found to be 0.4 mmol/g. Residual resin sites were capped
by treatment with acetic anhydride (0.36 g, 0.34 mL, 3.6 mmol)
and pyridine (0.28 g, 0.29 mL, 3.6 mmol) in dichloromethane
(30 mL) for 1.25 h. The resin was filtered, washed with
dichloromethane (3 × 30 mL), DMF (4 × 30 mL), and
dichloromethane (5 × 30 mL), and dried in vacuo.
aryl
NH
Me-AlaOH (130 mg, 0.4 mmol), HOBt (54 mg, 0.4 mmol), and
DIC (63 µL, 0.4 mmol) in dichloromethane/DMF (4 mL:10
drops), 1.5 h. (c) Fmoc-L-HtyrOH (168 mg, 0.4 mmol), HOBt
(28 mg, 0.2 mmol), HATU (0.15 g, 0.4 mmol), and DIPEA (138
µL, 0.8 mmol) in dichloromethane/DMF (1:1), 2 h. A bro-
mophenol blue test indicated complete coupling. (d) Fmoc-
IleOH (141 mg, 0.4 mmol), HOBt (54 mg, 0.4 mmol), and DIC
(63 µL, 0.4 mmol) in dichloromethane/DMF (4 mL:10 drops),
2 h. Following all couplings the resin was filtered and washed
with DMF (6 × 4 mL) and dichloromethane (4 × 4 mL). All
couplings (except c) were analyzed using the ninhydrin test.
A solution of palladium tetrakis (triphenylphosphine) (116
mg, 0.1 mmol) and dimedone (140 mg, 1.0 mmol) in dichlo-
romethane/THF (1:1, 4 mL, sparged with nitrogen) was added
to the resin and the mixture shaken overnight. The resin was
filtered, washed with dichloromethane (2 × 4 mL), DMF (2 ×
4 mL), 0.5% DIPEA + 0.5% diethyldithiocarbamic acid sodium
salt in DMF (4 × 4 mL), DMF (3 × 4 mL), and dichloromethane
(4 × 4 mL). The resin was treated with 20% piperidine in
DMF (4 mL) for 30 min, filtered, and washed with DMF (4 ×
4 mL), 10% pyridinium hydrochloride in dichloromethane/DMF
(1:1, 3 × 4 mL), and dichloromethane (4 × 4 mL). A solution
of PyBroP (280 mg, 0.6 mmol) and DIPEA (103 µL, 0.6 mmol)
in dichloromethane/DMF (1:1, 4 mL) was added to the resin
and the mixture shaken for 2.5 h. The resin was filtered and
washed with dichloromethane (2 × 4 mL), DMF (4 × 4 mL),
and dichloromethane (4 × 4 mL). A ninhydrin test was
negative. The product was cleaved from the solid support by
treatment with TFA/H2O (95:5, 5 mL) for 2.5 h. The resin was
filtered and washed with TFA (2 × 2 mL) and dichlo-
rormethane (3 × 4 mL), and the filtrate and washes were
combined. Removal of the solvent under reduced pressure
gave a white solid which was dissolved in water/acetonitrile
and lyophilized. Purification was achieved using semiprepara-
tive RP HPLC on a Phenomenex Prodigy ODS 5 µm 250 × 10
Oscilla m id e Y (L-MeAla , L-Htyr ) (1a ). NR-(Fluorenyl-
methoxycarbonyl)-O-tert-butyl-L-tyrosinyl(hydroxymethyl)phe-
noxyacetamidomethyl polystyrene resin (0.27 g, 0.1 mmol) was
treated with 20% piperidine in DMF (4 mL) for 30 min, filtered,
and washed with DMF (4 × 4 mL) and dichloromethane (4 ×
4 mL). The resin was suspended in DMF (2 mL), a solution
of NR-(4-nitrophenyloxycarbonyl)-Nꢀ-(9-fluorenylmethoxycar-
bonyl)-D-lysine allyl ester (229 mg, 0.4 mmol) in DMF (2 mL)
and DIPEA (69 µL, 0.4 mmol) were added, and the mixture
was shaken for 25 min. The resin was filtered and washed
with DMF (6 × 4 mL) and dichloromethane (4 × 4 mL).
A
ninhydrin test showed complete coupling.
The resin was subjected to Fmoc-peptide synthesis using
the following conditions:
(i) Fmoc deprotection: 20% piperidine in DMF (4 mL) for
30 min, followed by washing with DMF (4 × 4 mL) and
dichloromethane (4 × 4 mL).
(ii) Coupling conditions: (a) Fmoc-PheOH (155 mg, 0.4
mmol), HOBt (54 mg, 0.4 mmol), and DIC (63 µL, 0.4 mmol)
in dichloromethane/DMF (4 mL:10 drops), 1.5 h. (b) FmocN-

Solid-Phase Total Synthesis of Oscillamide Y
J . Org. Chem., Vol. 62, No. 18, 1997 6203
mm column, eluting at 2.0 mL/min. The following gradient
was used: t ) 0-10 min, 75% A, 25% B; t ) 30 min, 25% A,
75% B, t ) 35 min, 100% B. (A ) water, 0.1% TFA, B )
MeCN, 0.1% TFA). Under these conditions (L-MeAla, L-Htyr)
oscillamide Y eluted after 24.8 min. Yield 8 mg. λmax
(MeOH): 278 nm, ꢀ ) 2595. [lit.1 λmax (H2O): 278 nm, ꢀ )
2595]. ESMS: 858.3 (M + H)+, 880.4 (M + Na)+; HRMS: M
HCl/0.01 M CaCl2 buffer at a concentration of 20 µg/ mL.
Kinetic analysis using the substrate Suc-Ala-Ala-Pro-Phe-pNA
at pH 8 in 0.1 M Tris-HCl/0.01 M CaCl2 and monitoring the
release of 4-nitrophenol at 410 nm gave Km and kcat values of
48 µM and 350 s-1, respectively, essentially identical to the
literature.11 Addition of any of the four oscillamide Y ana-
logues, up to a concentration of 100 µM (using the substrate
Suc-Ala-Ala-Pro-Phe-pNA at a concentration of 0.67 Km), gave
no observable inhibition. Preincubation of the oscillamide Y
analogues (25 µM) with chymotrypsin likewise gave no observ-
able inhibition.
1
+ H C45H60O10N7 requires 858.4402, Found 858.4385. For H
NMR data see Table 1 and Supporting Information.
Oscilla m id e Y (L-MeAla , D-Htyr ; D-MeAla , L-Htyr ; a n d
D-MeAla , D-Htyr Isom er s) (1b, 1c, 1d ). The above procedure
was repeated using 0.88 g (0.4 mmol) of NR-(fluorenylmethoxy-
carbonyl)-O-tert-butyl-L-tyrosinyl(hydroxymethyl)phenoxyacet-
amidomethyl polystyrene resin. After coupling of FmocPheOH,
the resin was split into three equal portions (A, B, C). Fmoc-
L-MeAlaOH was coupled to A, and Fmoc-D-MeAlaOH to B and
C. At the next synthetic stage, Fmoc-D-HtyrOH was coupled
to A, and B and C were treated with Fmoc-D-HtyrOH and
Fmoc-L-HtyrOH, respectively. Subsequent synthetic steps
were identical. Purification by semipreparative RP HPLC
using the conditions above gave oscillamide Y (L-MeAla,
D-Htyr), 25.5 min, 9 mg; oscillamide Y (D-MeAla, L-Htyr), 25.8
min, 3 mg; oscillamide Y (D-MeAla, D-Htyr), 25.3 min. ESMS:
Oscillamide Y (L-MeAla, D-Htyr) 858.3 (M + H)+, 880.4 (M +
Na)+. Oscillamide Y (D-MeAla, D-Htyr) 858.4 (M + H)+, 880.4
(M + Na)+. Oscillamide Y (D-MeAla, L-Htyr) 858.3 (M + H)+,
880.2 (M + Na)+.
Ack n ow led gm en t. The authors would like to thank
Astra Charnwood for financial support, Mrs. J . Street
for high field NMR experiments, and Dr. G. J . Langley
for MS analyses. M.B. thanks the Royal Society for a
University Research Fellowship.
Su p p or tin g In for m a tion Ava ila ble: 1H and 1H-1H
COSY NMR spectra for oscillamide Y (L-MeAla, L-Htyr) (4
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.
Ch ym otr yp sin In h ibition Assa ys. Bovine pancreatic
R-chymotrypsin (from Calbiochem) was dissolved in 0.1 M Tris-
J O970671O