Convergent synthesis of dolastatin 15 by solid phase coupling of an N- methylamino acid

Article abstract of DOI:10.1021/jo981055a

Convergent synthesis of dolastatin 15 (1), a cytostatic depsipeptide isolated from the Indian Ocean sea hare, has been described. For construction of the backbone, a single-step condensation of peptide fragment 2 and pyrrolidone fragment 3 was successfull

Full text of DOI:10.1021/jo981055a

J . Org. Chem. 1999, 64, 405-411
405
Con ver gen t Syn th esis of Dola sta tin 15 by Solid P h a se Cou p lin g of
a n N-Meth yla m in o Acid
Kenichi Akaji,*,† Yuzo Hayashi,† Yoshiaki Kiso,† and Naohiro Kuriyama‡
Department of Medicinal Chemistry, Kyoto Pharmaceutical University,
Yamashina-ku, Kyoto 607-8414, J apan, and YMC Company, Ltd, Kumiyama-cho, Kyoto 613-0024, J apan
Received J une 2, 1998
Convergent synthesis of dolastatin 15 (1), a cytostatic depsipeptide isolated from the Indian Ocean
sea hare, has been described. For construction of the backbone, a single-step condensation of peptide
fragment 2 and pyrrolidone fragment 3 was successfully performed using 2-chloro-1,3-dimethyl-
2-imidazolinium hexafluorophosphate (CIP) developed by us as an efficient coupling reagent.
Coupling of an N-methylamino acid on solid support was first achieved using CIP for the efficient
synthesis of peptide fragment 2. The effectiveness of CIP for the coupling of N-methylamino acids
in solution and on solid support were clarified by the syntheses of model di- and tripeptides.
Dolastatin 15 is a cytostatic depsipeptide isolated from
the marine mollusk Dolabella auricularia by Pettit et al.
in 1989.1 Among the dolastatins, an unprecedented series
of linear and cyclic antineoplastic and/or cytostatic pep-
tide isolated from the Indian Ocean sea hare,2 dolastatin
15 as well as dolastatin 10 represent the two most
important members because of the strong and selective
activities. Dolastatin 15 inhibits growth of the P388
lymphocytic leukemia cell line with an ED50 value of 2.4
ng/mL.
segment synthetic strategy and obtained a final com-
pound exhibiting essentially the same physical properties
as those obtained by Poncet et al. In those syntheses,
various coupling procedures including mixed anhydride,
chlorocarbonate, carbodiimide, and diethyl cyanophos-
phonate (DEPC),7 were selected case by case at each
activation step. For the construction of the backbone of
dolastatin 15, however, a two-step procedure involving
separate incorporation of N,N-dimethylvaline or bism-
ethylation of valine was necessary because of the low
efficiency of these activating reagents in a single-step
condensation.6
Recently, we have developed a new coupling reagent,
2-chloro-1,3-dimethyl-2-imidazolinium hexafluorophos-
phate (CIP) and found that CIP in the presence of the
additive 1-hydroxy-7-azabenzotriazole (HOAt)8 effectively
couples sterically hindered R,R-dialkylamino acids.9 In
these couplings, the initially formed oxazolone intermedi-
ate is transformed to a highly active ester by the catalytic
additive to produce the desired condensation product
without detectable racemization. Here, we describe a
method of convergent synthesis of dolastatin 15 by single-
step fragment condensation, in which all couplings
including those of N-methylamino acids on solid support
are conducted by CIP-mediated activation.
Structurally, dolastatin 15 is a lipophilic pentapeptide
esterified by an N-acylpyrrolidone and contains seven
chiral carbons all having S-stereochemistry. In the pep-
tide part, two N-methylated amino acids, N-methylvaline
and N,N-dimethylvaline,3 are included. The first synthe-
sis of dolastatin 15 was communicated in 1991 by Pettit
and co-workers.4 In 1992, Poncet et al.5 reported total
synthesis of the proposed structure and described that
the synthetic compound exhibited physical properties
different from those reported by Pettit and co-workers
for the natural dolastatin 15 and their synthesis product.
The absolute configuration and physical properties of
dalastatin 15 was confirmed by the synthesis by Pettit
and co-workers in 1994,6 in which they employed a
Resu lts a n d Discu ssion
Scheme 1 shows our retrosynthetic route of dolastatin
15. For completing the synthesis of dolastatin 15, single-
step segment condensation between peptide fragment 2
and nonpeptide fragment 3 by the CIP/HOAt method was
used, since activation at the Pro residue is resistant to
isomerization. In contrast to the previous syntheses, an
† Kyoto Pharmaceutical University.
‡ YMC Co.
(1) Pettit, G. R.; Kamano, Y.; Dufresne, C.; Cerny, R. L.; Herald, C.
L.; Schmidt, J . M. J . Org. Chem. 1989, 54, 6005.
(2) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Fujii, Y.; Kizu, H.; Boyd,
M. R.; Boettner, F. E.; Doubek, D. L.; Schmidt, J . M.; Chapuis, J . C.;
Michel, C. Tetrahedron 1993, 49, 9151.
(3) Bowman, R. E.; Stroud, H. H. J . Chem. Soc. 1950, 1342.
(4) Pettit, G. R.; Herald, D. L.; Singh, S. B.; Thornton, T. J .;
Mullaney, J . T. J . Am. Chem. Soc. 1991, 113, 6692.
(5) Patino, N.; Fre´rot, E.; Galeotti, N.; Poncet, J .; Coste, J .; Dufour,
M.-N.; J ouin, P. Tetrahedron 1992, 48, 4115.
(6) Pettit, G. R.; Thornton, T. J .; Mullaney, J . T.; Boyd, M. R.;
Herald, D. L.; Singh, S- B.; Flahiva, E. J . Tetrahedron 1994, 50, 12097.
(7) Shioiri, T.; Yokoyama, Y.; Kasai, Y.; Yamada, S. Tetrahedron
1976, 32, 2211.
(8) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397.
(9) (a) Akaji, K.; Kuriyama, N.; Kimura, T.; Fujiwara, Y.; Kiso, Y.
Tetrahedron Lett. 1992, 33, 3177. (b) Akaji, K.; Kuriyama, N.; Kiso, Y.
Tetrahedron Lett. 1994, 35, 3315.
10.1021/jo981055a CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/05/1999

406 J . Org. Chem., Vol. 64, No. 2, 1999
Akaji et al.
Sch em e 1
Ta ble 1. Mod el P ep tid es by Solu tion P h a se Syn th esis
N,N-dimethylamino acid was incorporated in peptide
fragment 2 since CIP-mediated activation is expected to
be efficient enough for hindered couplings in solution, as
shown from our previous syntheses of mirabazoles.10 Pro,
2-hydroxyisovaleric acid (Hiva), and Phe were employed
for the preparation of fragment 3. The pyrrolidone ring
of 3 was constructed by the CIP-mediated coupling of Phe
and Meldrum’s ester employed as C2 unit.11 For the
preparation of peptide fragment 2, CIP-mediated activa-
tion for the coupling of an N-methylamino acid on solid
support was selected as a preferable scheme to facilitate
the practical synthesis of dolastatin 15. Thus, prior to
the synthesis of dolastatin 15 according to the scheme,
we evaluated CIP-mediated activation for its efficiency
in preparing a peptide sequence containing N-methy-
lamino acids on solid support.
Cou p lin g of N-Meth yla m in o Acid on Solid Su p -
p or t. N-Methylamino acid is difficult to incorporate by
standard peptide coupling methods even in solution
phase reactions because of its steric hindrance and
marked tendency to racemize.12 To overcome these dif-
ficulties, several halogenophosphonium reagents have
been evaluated for the coupling of N-methylamino acid.13
In the practical synthesis of destruxin B containing two
consecutive N-methylamino acid residues, however, pro-
longed reaction times (more than 18 h) were necessary
to obtain moderate to good isolation yields even with
newly developed coupling reagents including BOP-Cl and
PyBroP.14 For the coupling of an N-methylamino acid on
solid support, few syntheses employing HATU as an
effective coupling reagent have been reported15 and no
practical solid-phase syntheses of natural products have
time
(h)
yield
(%)a
D-isomer
content (%)
Z-MeLeu-Val-OMe 4
Z-MeVal-Val-OMe 5
Z-Val-MeVal-OMe 6
Z-MeLeu-MeLeu-OMe 7
Z-MeLeu-MeVal-OMe 8
1
1
3
3
3
95
91
92
97
91
<0.5b
<0.5b
<1.0b
<0.5c
<1.5d
a
b
Isolation yield after silica gel column chromatography. De-
termined by reverse phase HPLC. c Determined by reverse phase
HPLC and 1H NMR. Determined by 1H NMR.
d
been achieved. These reports have prompted us to
examine the application of CIP-mediated activation for
coupling in solution followed by coupling on solid support.
To evaluate the coupling efficiency in solution, dipep-
tides 4 to 8 containing an N-methylamino acid were
synthesized using CIP-HOAt (Table 1). Each coupling
reaction was conducted at 25 °C in CH2Cl2, and the
desired product was isolated by silica gel column chro-
matography. Coupling of N-methylamino acids proceeded
within 1 h to give dipeptides 4 and 5 with quantitative
yields. The more difficult coupling to N-methylamino
acids gave dipeptides 6 to 8 quantitatively after a 3 h
reaction using CIP-HOAt.
Epimerization during the CIP-mediated activation was
measured by the determination of D-isomer content in
the crude isolated products. For dipeptides 4 to 7, reverse
phase HPLC was employed to analyze the D-isomer
contents. For dipeptide 8, however, comparison of peaks
1
of methy ester by H NMR was employed since baseline
separation of the D-isomer by HPLC was difficult. In the
comparison, signals could be found with chemical shifts
that differ in the isomers.16 In each analysis, the D-isomer
dipeptide was identified by comparison with standard
dipeptide separately synthesized starting from the cor-
responding D-amino acid.
Table 1 shows that most couplings resulted in a
D-isomer content less than 0.5%. Even in the most
sterically hindered dipeptide 8, the D-isomer content was
less than 1.5%. These results indicate that CIP-mediated
(10) (a) Akaji, K.; Kuriyama, N.; Kiso, Y. J . Org. Chem. 1996, 61,
3350. (b) Kuriyama, N.; Akaji, K.; Kiso, Y. Tetrahedron 1997, 53, 8323.
(11) J ouin, P.; Castro, B.; Nisato, D. J . Chem. Soc., Perkin Trans. 1
1987, 1177.
(12) (a) Fre´rot, E.; Coste, J .; Poncet, J .; J ouin, P.; Castro, B.
Tetrahedron Lett. 1992, 33, 2815. (b) Calmes, M.; Cavelier-Frontin,
F.; J acquier, R.; Mercadier, J .-L.; Sabil, S.; Verducci, J .; Quiot, J .-M.;
Vey, A. Int. J . Pept. Protein Res. 1993, 41, 528.
(13) Coste, J .; Fre´rot, E.; J ouin, P. J . Org. Chem. 1994, 59, 2437.
(14) Ward, D. E.; Lazny, R.; Pedras, S. C. Tetrahedron Lett. 1997,
38, 339.
(15) (a) Angell, Y. M.; Garc´ıa-Echeverr´ıa, C.; Rich, D. H. Tetrahedron
Lett. 1994, 33, 5981. (b) Carpino, L. A.; El-Faham, C.; Minor, A.;
Albericio, F. J . Chem. Soc., Chem. Commun. 1994, 201.
(16) Coste, J .; Dufour, M.-N.; Pantaloni, A.; Castro, B. Tetrahedron
Lett. 1990, 31, 669.

Convergent Synthesis of Dolastatin 15
J . Org. Chem., Vol. 64, No. 2, 1999 407
Ta ble 2. Mod el P ep tid es by Solid P h a se Syn th esis
yield
(%)
D-isomer
time
content (%)a
Z-MeVal-Val-OMe 9
Z-Val-MeVal-Gly-OMe
1 h × 1 ≈100b
<0.5
<1.0
<2.0
<2.0
<2.0
2 h × 2
59c
74c
53d
56d
10 12 h × 1
Fmoc-MeLeu-MeVal-Gly-OMe
3 h × 1
11 12 h × 1
a
b
Determined by reverse phase HPLC. Determined by Kaiser
ninhydrin test. c Determined by amino acid analysis of peptide-
d
resin. Determined by UV analysis of the Fmoc group.
activation is an efficient method for coupling in solution,
with practically no racemization within 1 to 3 h at 25
°C. In contrast, it had been reported in solution synthesis
of destruxin B that DCC/DMAP activation, a highly
efficient conventional peptide coupling procedure, re-
sulted in a 72% isolation yield with 37% isomerization
after a 24 h reaction.14
Coupling efficiency on solid support was then examined
using three model peptides: Z-MeVal-Val-OMe 9, Z-Val-
MeVal-Gly-OMe 10, and Fmoc-MeLeu-MeVal-Gly-OMe
11. As a solid support, 2-chlorotrityl chloride (ClTrt)
polystyrene resin,17 from which the desired model pep-
tides could be cleaved by treatment with weak acid such
as AcOH, was selected. Incorporation of the N-methy-
lamino acid was carried out by use of a double coupling
(2 h × 2) or a 12 h single coupling employing 5 equiv of
the N-methylamino acid. The extent of the reaction was
monitored by the Kaiser ninhydrin test for dipeptide 9,
by the amino acid ratio of Val/Gly in acid hydrolysate of
the product resin for tripeptide 10, and by UV analysis
of the Fmoc deprotection step18 for tripeptide 11. Each
product obtained by cleavage from the resin followed by
methylation with (trimethysilyl)diazomethane19 was char-
acterized by the same methods described by solution
phase reaction. D-Isomer content of each crude product
was analyzed using HPLC or methyl ester-derived signal
comparison by 1H NMR. Each corresponding D-isomer
was identified by separate syntheses using D-amino acid.
Table 2 summarizes the coupling yields and D-isomer
contents in model peptide syntheses on solid support.
Quantitative to moderate coupling yields were obtained
by single coupling reactions, although somewhat longer
reaction times than that in solution phase synthesis were
necessary. Slightly lower yields were obtained by a double
coupling protocol for each model peptide. As a reaction
solvent, DMF gave better results than CH2Cl2. D-Isomer
contents were less than 2%, indicating practically no
racemization occurred during the CIP-mediated activa-
tion despite prolonged reaction times. The efficiency of
CIP-mediated coupling might be mediated by the forma-
tion of the HOAt active ester intermediate, which is
believed to have enhanced reactivity because of its
neighboring nitrogen effect, as described in our previous
synthesis of mirabazole C.10
F igu r e 1. Synthetic route for peptide fragment 2.
synthesized dolastatin 15 by a convergent route shown
in Scheme 1 employing CIP-mediated coupling of N-
methylamino acids on solid support.
Peptide fragment 2 was synthesized by chain elonga-
tion on solid support according to the route shown in
Figure 1. As a solid support, 2-chlorotrityl chloride (ClTrt)
resin was selected as in the above model experiment. It
is particularly useful in the synthesis of prolyl peptide
as the bulk of the trityl linker helps prevent diketopip-
erazine formation.17 The initial Fmoc-Pro-O-ClTrt resin
was prepared by the reaction of ClTrt resin with Fmoc-
Pro-OH in the presence of DIEA. After cleavage of the
Fmoc group of the resin by piperidine treatment, Fmoc-
MeVal-OH was coupled by a 1.5 h reaction using CIP-
HOAt. Fmoc deprotection by piperidine and CIP-medi-
ated double coupling reactions were repeated for the
incorporation of Fmoc-Val-OH and DOV (dolavaline,
N,N′-dimethylvaline) to give tetrapeptide resin 12.
The peptide chain was then cleaved from the resin by
treatment with AcOH/TFE/CH2Cl2 (1:1:8).20 The crude
product obtained was purified by flash chromatography
to give the desired peptide fragment 2 with a 50%
isolation yield calculated from the starting Fmoc-Pro-O-
ClTrt resin. The purified fragment 2 showed a single
peak on an analytical HPLC and was shown to be a
monomer by FAB-MS spectrometry.
The nonpeptide fragment 20 was synthesized according
to the route shown in Figure 2. L-Hydroxyisovalelic acid
(Hiva) 13 and Phe-OBzl 14 were condensed by a 1 h
reaction using CIP-HOBt to give 15 without difficulty.
The hydroxy group of 15 was protected using tert-
butyldimethylsilyl chloride in the presence of imidazole,21
and then the Bzl group was cleaved by hydrogenolysis
to yield 17 quantitatively. Condensation of 17 and
Meldrum’s ester was then carried out by a 4 h reaction
at 25 °C using CIP in the presence of DMAP.22 In this
particular coupling step, addition of DMAP gave better
results than HOAt probably because of its basicity. The
Syn th esis of Dola sta tin 15. The model studies
described above suggest that the coupling of an N-
methylamino acid is feasible using CIP-HOAt. We then
(17) (a) Barlos, K.; Gatos, D.; Kallitsis, J .; Papaphotiu, G.; Sotiriu,
P.; Wenqing, Y.; Scha¨fer, W. Tetrahedron Lett. 1989, 30, 3943. (b)
Barlos, K.; Gatos, D.; Kapolos, S.; Papaphotiu, G.; Scha¨fer, W.;
Wenqing, Y. Tetrahedron Lett. 1989, 30, 3947.
(20) Barlos, K.; Chatzi, O.; Gatos, D.; Stavropoulos, G. Int. J . Pept.
Protein Res. 1991, 37, 513.
(18) Meienhofer, J .; Waki, M.; Heimer, E.; Lambros, T. J .; Makofske,
R.; Chang, C.-D. Int. J . Pept. Protein Res. 1979, 13, 35.
(19) Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull.
1981, 29, 1475.
(21) Corey, E. J .; Venkateswarlu, A. J . Am. Chem. Soc. 1972, 94,
6190.
(22) Hassner, A.; Krepski, R.; Alexanian, V. Tetrahedron 1978, 34,
2069.

408 J . Org. Chem., Vol. 64, No. 2, 1999
Akaji et al.
Con clu sion
We have achieved an efficient synthesis of dolastatin
15, a cytostatic depsipeptide containing a pyrrolidone
amino acid and N-methylamino acids, in which all
condensation reactions were carried out by CIP-mediated
activation. In our synthesis, a convergent route utilizing
single-step condensation of peptide fragment 2 and
pyrrolidone fragment 3 using CIP-HOAt as an efficient
coupling reagent was employed. The CIP-mediated reac-
tion was also successfully applied for coupling of N-
methylamino acid on solid support, demonstrating the
first practical application of the solid-phase procedure for
natural product synthesis.
Exp er im en ta l Section
Gen er a l. Solvents were reagent grade and dried prior to
use. All flash chromatographic separations were carried out
on Wakogel FC-40 obtained from WAKO Pure Chemical Ind.
Ltd. Fmoc amino acid derivatives and 2-chlorotrityl chloride
resin were obtained from Calbiochem Novabiochem and used
without further purification. For quantification of Fmoc amino
acid on the resin, absorbance at 301 nm after cleavage of the
Fmoc group with piperidine was measured according to the
procedure described by Meienhofer et al.18
F igu r e 2. Synthetic route for nonpeptide fragment 20.
Melting points were uncorrected. The 1H and 13C NMR
spectra were recorded on a 270 MHz spectrometer with TMS
as an internal standard. Optical rotations were measured at
ambient temperature using a 1 mL cell. Analytical HPLC was
carried out on a reverse phase column (4.6 × 150 mm), which
was eluted with CH3CN in 0.1% aqueous TFA: Rt1, YMC Pack
C8 AM202, detected at OD 214 nm; Rt2, YMC Pack ODS
AM302, flow rate 0.9 mL/min, detected at OD230 nm.
Syn th eses of Mod el P ep tid es 4-8 in Solu tion (Ta ble
1). Z-Val-MeVal-OMe 6: To a solution of Z-MeVal-OMe (0.5
mmol) in CHCl3 (0.5 mL) was added 25% HBr/AcOH (1.5 mL)
at an ice-bath temperature, and the mixture was stirred for
60 min at 25 °C. The solvent and AcOH were removed by
azeotroping with heptane (3 times), yielding a light brown oil.
To the solution of Z-Val-OH (2 equiv) in CH2Cl2 (3 mL) were
added DIEA (6 equiv) and HOAt (1 equiv). CIP (2 equiv) and
the above HBr‚H-MeVal-OMe in CH2Cl2 (2 mL) were added
to the mixture. The reaction mixture was stirred for 3 h at 25
°C. The solvent was removed in vacuo, and the residue was
extracted with AcOEt. The organic layer was washed with 5%
citric acid, 5% NaHCO3, and H2O and dried (MgSO4). The
crude product was isolated by silica gel column chromatogra-
phy using hexane/AcOEt (7:3) to give the desired dipeptide as
an oil. Model peptides 4, 5, 7, and 8 were similarly synthesized,
and the isolation yield and reaction time are shown in Table
1. Z-MeLeu-Val-OMe 4: [R]25D -41.63 (c ) 1.10, MeOH), single
peak on HPLC Rt1 23.06 min (CH3CN 45%, 1.0 mL/min), FAB-
MS, m/z 393.2403 for [M + H]+ (calcd 393.2389 for C21H33N2O5).
F igu r e 3. Synthetic route for dolastatin 15.
resulting Meldrum’s ester adduct, without purification,
was heated in refluxing AcOEt to afford cyclized product.
Methylation of the enolic function of the cyclized product
was conducted under Mitsunobu conditions at 25 °C for
2 h to give methyl vinyl ether.23 The product was a
mixture of diastereoisomers due to partial epimerization
caused by the activation of an N-acylamino acid. After
purification by flash chromatography, the desired TB-
DMS-(S)-Hiva-(S)Dpy 18 was easily separated and ob-
tained at a 46% overall yield calculated from 17 accom-
panied with 9% formation of (S,R) isomer. The silyl group
of 18 was quantitatively cleaved by TFA treatment to
afford the hydroxy product 19. Esterification of 19 with
Boc-Pro-OH was performed by a 10 h reaction at 25 °C
using CIP-DMAP. After purification by flash column
chromatography, the desired nonpeptide fragment 20 was
obtained at a 92% yield.
Z-MeVal-Val-OMe 5: [R]25 -88.22 (c ) 0.37, MeOH), single
D
peak on HPLC Rt1 15.06 min (CH3CN 45%, 1.0 mL/min), FAB-
MS, m/z 379.2217 for [M + H]+ (calcd 379.2233 for C20H31N2O5).
Z-Val-MeVal-OMe 6: [R]25D -102.22 (c ) 0.32, MeOH), single
peak on HPLC Rt1 23.42 min (CH3CN 35%, 2.0 mL/min), FAB-
MS, m/z 379.2243 for [M + H]+ (calcd 379.2233 for C20H31N2O5).
The final coupling for dolastatin 15 was carried out as
shown in Figure 3. The Boc group of 20 was cleaved by
a 30 min treatment with TFA at 25 °C, and the product,
without purification, was condensed with peptide frag-
ment 2 by a 2 h reaction using CIP-HOAt. The crude
product was purified by flash chromatography to yield
dolastatin 15 with an 89% isolation yield. The synthetic
material had the same spectroscopic and physical proper-
ties as those reported for natural and synthetic dolastatin
Z-MeLeu-MeLeu-OMe 7: [R]26 -121.08 (c ) 0.37, MeOH),
D
single peak on HPLC Rt1 24.38 min (CH3CN 45%, 2.0 mL/
min), FAB-MS, m/z 421.2714 for [M + H]+ (calcd 421.2702 for
C23H37N2O5). Z-MeLeu-MeVal-OMe 8: [R]25D -135.49 (c ) 0.36,
MeOH), single peak on HPLC Rt1 26.59 min (CH3CN 42%, 2.0
mL/min), FAB-MS, m/z 407.2556 for [M + H]+ (calcd 407.2546
for C22H35N2O5).
Corresponding dipeptides containing D-amino acid were
synthesized by the same procedure. Z-(D)-MeVal-Val-OMe:
[R]25D 78.13 (c ) 0.32, MeOH), single peak on HPLC Rt1 16.92
min (CH3CN 45%, 1.0 mL/min), FAB-MS, m/z 379.2225 for [M
+ H]+ (calcd 379.2233 for C20H31N2O5). Z-(D)-Val-MeVal-
1
15.5,6 In addition, the H NMR spectrum of our synthetic
dolastatin 15 was identical to the synthetic dolastatin
15 obtained by Poncet and co-workers.
(23) Bajwa, J . S.; Anderson, R. C. Tetrahedron Lett. 1990, 48, 6973.
OMe: [R]21 -47.73 (c ) 0.44, MeOH), single peak on HPLC
D

Convergent Synthesis of Dolastatin 15
J . Org. Chem., Vol. 64, No. 2, 1999 409
Rt1 25.16 min (CH3CN 35%, 2.0 mL/min), FAB-MS, m/z
379.2229 for [M + H]+ (calcd 379.2233 for C20H31N2O5). Z-(D)-
1H NMR signals derived form methyl ester: Z-MeLeu-
MeLeu-OMe 7; 3.49, 3.68, and 3.69 ppm. Z-(D)-MeLeu-MeLeu-
OMe; 3.62, 3.67, and 3.74 ppm. Z-MeLeu-MeVal-OMe 8; 3.43,
3.65, 3.67, and 3.69 ppm. Z-(D)-MeLeu-MeVal-OMe; 3.61, 3.67,
3.70, and 3.71 ppm.
DOV-Va l-MeVa l-P r o-OH (2). Fmoc-Pro-O-ClTrt resin was
prepared from ClTrt resin (3.0 g, 3.90 mmol) and Fmoc-Pro-
OH (0.34 g, 1.0 mmol) in CH2Cl2 (20 mL) in the presence of
DIEA (2.7 mL, 15.6 mmol) according to the procedure described
for the synthesis of model peptide on solid support to yield
3.10 g of desired starting resin with substitution 0.297 mmol/
g. Piperidine (20%) in DMF was added to the resin swelled
with DMF, and the mixture was agitated for 20 min at 25 °C.
The NR-deprotected resin was filtered and washed with DMF.
To the solution of Fmoc-MeVal-OH (0.97 g, 2.75 mmol) in
DMF (20 mL) were added HOAt (0.188 g, 1.38 mmol), DIEA
(1.44 mL, 8.26 mmol), and CIP (0.767 g, 2.75 mmol). The
mixture was then added to the above resin, and the resin-
mixture was agitated for 1.5 h. After washing the resin with
DMF, the NR-Fmoc group was removed by treatment with 20%
piperidine/DMF as described above. Fmoc-Val-OH was then
introduced by a double coupling procedure (3 h × 2) using
Fmoc-Val-OH (1.56 g, 4.6 mmol), HOAt (0.31 g, 2.3 mmol),
DIEA (2.24 mL, 12.85 mmol), and CIP (1.28 g, 4.59 mmol) in
DMF (20 mL). The Fmoc group of the tripeptide resin was
removed with 20% piperidine, and DOV was introduced by a
double coupling procedure (1.5 h × 2) using DOV (0.40 g, 2.75
mmol), HOAt (0.28 g, 2.06 mmol), DIEA (1.76 mL, 10.1 mmol),
and CIP (1.15 g, 4.13 mmol) in DMF (20 mL). The resin
obtained was filtered, washed with DMF and MeOH, and dried
to yield 3.10 g of desired tetrapeptide-O-ClTrt resin 12.
MeLeu-MeLeu-OMe: [R]24 37.38 (c ) 0.31, MeOH), single
D
peak on HPLC Rt1 27.03 min (CH3CN 45%, 2.0 mL/min), FAB-
MS, m/z 421.2704 for [M + H]+ (calcd 421.2702 for C23H37N2O5).
Z-(D)-MeLeu-MeVal-OMe: [R]26 -0.548 (c ) 0.37, MeOH),
D
single peak on HPLC Rt1 27.54 min (CH3CN 42%, 2.0 mL/
min), FAB-MS, m/z 407.2542 for [M + H]+ (calcd 407.2546 for
C22H35N2O5).
Syn th eses of Mod el P ep tid es 9-11 on Solid Su p p or t
(Ta ble 2). Z-MeVal-Val-OMe 9: To 1 g (1.05 mmol) of
2-chlorotrityl chloride (ClTrt) resin were added Fmoc-Val-OH
(1.07 g, 3.15 mmol) in CH2Cl2 (12 mL) and DIEA (0.82 mL,
4.73 mmol), and the mixture was agitated for 1 h at 25 °C.
MeOH/DIEA (9:1) (4 mL) was added, and the mixture was
further agitated for 30 min. The resin was filtered, washed
with DMF and MeOH, and then dried in vacuo to yield 1.34 g
of Fmoc-Val-O-ClTrt resin with substitution 0.687 mmol/g.
To the solution of Z-MeVal-OH (0.77 g, 2.76 mmol) in DMF
(12 mL) were added HOAt (188 mg, 1.38 mmol), DIEA (1.1
mL, 6.44 mmol), and CIP (0.77 g, 2.76 mmol). The mixture
was then added to the above resin swelled with DMF, and the
resin solution was agitated at 25 °C. After 60 min, the resin
was ninhydrin test negative. The mixture was filtered, and
the resin was washed with MeOH and dried to yield 1.14 g of
desired dipeptide resin.
To the Z-MeVal-Val-ClTrt resin (400 mg) obtained above
was added TFA/anisole (3.8 mL/0.2 mL), and the mixture was
stirred at 0 °C for 1 h. After filtration, the filtrate was
concentrated in vacuo, and the residue was extracted with
AcOEt. The organic layer was washed with 10% citric acid and
H2O and dried (MgSO4). To the oily product (85 mg, 0.233
mmol) in MeOH (2 mL) was added 10% (trimethylsilyl)-
diazomethane in n-hexane (2.2 mL, 1.4 mmol), and the mixture
was stirred at 25 °C for 1 h under Ar atomosphere. AcOH was
added to the mixture, and the solvent was removed in vacuo.
The residue was extracted with AcOEt, and the organic layer
was washed with 5% citric acid, 5% NaHCO3, and H2O and
dried (MgSO4). The crude product was purified by silica gel
column chromatography using hexane/AcOEt (7:3) to yield 82
AcOH/TFE/CH2Cl2 (1:1:8, 45 mL) was added to the DOV-
Val-MeVal-Pro-O-ClTrt resin 12 (2.97 g), and the resin-mixture
was stirred at 25 °C for 1.5 h. The mixture was filtered and
the solvent of the filtrate was removed in vacuo. The resulting
oily residue was purified by flash chromatography using
CHCl3/MeOH (5:1) followed by recrystallization with CHCl3/
hexane to yield 0.2 g (50%, calculated from the starting Fmoc-
Pro-O-ClTrt resin) of the desired tetrapeptide 2 as a solid:
[R]25 -160.36 (c ) 0.28, MeOH), mp 155-157 °C, Rt2 12.34
D
min [CH3CN (10-60%/30 min)], 1H NMR (270 MHz, CDCl3) δ
0.76 (d, J ) 6.6 Hz, 3H), 0.85-0.99 (m, 12H), 1.19 (d, J ) 6.3
Hz, 3H), 1.76-1.89 (m) 1.93-2.25 (overlapping m) 2.23-2.40
(overlapping m) total 8H, 2.45 (br s, 6H), 3.08 (s, 3H), 3.62-
3.81 (m, 2H), 4.37-4.44 (m, 1H), 4.77-4.83 (m, 1H), 5.17 (d,
J ) 10.9 Hz, 1H), 7.34 (br d, J ) 8.6 Hz, 1H). 13C NMR (67.8
MHz, CDCl3) δ 17.92, 18.26, 18.40, 19.16, 19.68, 24.83, 26.99,
27.82, 29.16, 30.64, 31.02, 41.76, 43.31, 47.26, 53.89, 59.59,
60.07, 74.23, 74.37, 168.89, 170.55, 172.58, 176.26. FAB-MS,
m/z 455.3236 for [M + H]+ (Calcd 455.3233 for C23H43N4O5).
mg (67%) of the dipeptide 9 as an oil: [R]18 -84.44 (c ) 0.36,
D
MeOH), single peak on HPLC Rt1 14.70 min (CH3CN 45%, 1.0
mL/min), FAB-MS, m/z 379.2280 for [M + H]+ (calcd 379.2233
for C20H31N2O5).
Model Peptides 10 and 11 were similarly synthesized. For
the estimation of coupling yield, amino acid analysis of the
corresponding tripeptide resin was used for model peptide 10,
and UV absorption of the Fmoc group for model peptide 11.
Z-Val-MeVal-Gly-OMe 10: [R]25 -143.78 (c ) 0.37, MeOH),
D
single peak on HPLC Rt1 10.22 min (CH3CN 35%, 2.0 mL/
Hiva -P h e-OBzl 15. To the solution of Hiva (0.36 g, 3.04
mmol) in THF (15 mL) were added Phe-OBzl tosylate (1.04 g,
2.43 mmol), DIEA (2.11 mL, 12.1 mmol), HOBt (2.10 g, 13.7
mmol), and CIP (1.27 g, 4.56 mmol), and the mixture was
stirred at 25 °C for 3 h. The solvent was removed in vacuo
and the residue was extracted with AcOEt. The organic layer
was washed with 5% citric acid, 5% NaHCO3, and H2O, dried
(MgSO4), and rotary evaporated. The crude product was
purified by silica gel column chromatography using hexane/
AcOEt (2:1) followed by precipitation with AcOEt/hexane to
min), FAB-MS, m/z 458.2292 for [M + Na]+ (calcd 458.2267
for C22H33N3O6Na). Fmoc-MeLeu-MeVal-Gly-OMe 11: [R]20
D
-96.00 (c ) 0.30, MeOH), single peak on HPLC Rt1 23.48 min
(CH3CN 44%, 2.0 mL/min), FAB-MS, m/z 552.3085 for [M +
H]+ (calcd 552.3074 for C31H42N3O6).
Corresponding tripeptides containing a D-amino acid were
synthesized by the same procedure. Z-(D)-Val-MeVal-Gly-
OMe: [R]19 -63.93 (c ) 0.31, MeOH), single peak on HPLC
D
Rt1 12.62 min (CH3CN 44%, 2.0 mL/min), FAB-MS, m/z
458.2313 for [M + Na]+ (calcd 458.2267 for C22H33N3O6Na).
yield 0.75 g (87%) of 15 as a solid: [R]26 -38.5 (c ) 0.5,
D
Fmoc-(D)-MeLeu-MeVal-Gly-OMe: [R]20 -19.41 (c ) 0.34,
D
MeOH), mp 85-86 °C, Rt2 11.78 min [CH3CN (40-80%/
30min)], IR (KBr) 3265, 1739, 1704, 1649, 1529, 1261, 1176
cm-1 1H NMR (270 MHz, CDCl3) δ 0.72 (d, J ) 6.9 Hz, 3H),
0.92 (d, J ) 6.9 Hz, 3H), 2.02-2.12 (m, 1H), 3.05 (d, J ) 5.3
Hz, 1H), 3.11 (dd, J ) 6.9 Hz, J ) 6.9 Hz, 2H), 3.92 (dd, J )
5.3 Hz, J ) 3.3 Hz, 1H), 4.96 (dd, J ) 6.9 Hz, J ) 8.2 Hz, 1H),
5.09 (d, J ) 12.2 Hz, 1H), 5.15 (d, J ) 12.2 Hz, 1H), 6.96 (br
MeOH), single peak on HPLC Rt1 27.96 min (CH3CN 44%, 2.0
mL/min), FAB-MS, m/z 552.3066 for [M + H]+ (calcd 552.3074
for C31H42N3O6).
Estim a tion of D-Isom er Con ten t. HPLC separation of
D-isomer was carried out using YMC-Pack C8 AM-202 column
(4.6 × 150 mm), which was eluted with a isocratic condition
of CH3CN in 0.1% aqueous TFA. The eluant was monitored
by measuring UV absorption at 214 nm. The elution conditions
for model peptides were as follows. CH3CN (35%) with a flow
rate of 1 mL/min; model peptide 5 and 9: 2 mL of 35% CH3-
CN/min; model peptide 6 and 10: 2 mL of 44% CH3CN/min;
model peptide 11: 2 mL of 45% CH3CN/min; model peptide 7.
d, J ) 8.2 Hz, 1H), 7.03-7.07 (m, 2H), 7.10-7.38 (m, 8H). 13
C
NMR (67.8 MHz, CDCl3) δ 15.49, 19.03, 31.79, 38.08, 52.70,
67.35, 76.24, 127.11, 128.53, 128.62, 129.20, 135.06, 135.65,
171.61, 173.20. Anal. Calcd for C21H25NO4: C, 70.96; H, 7.09;
N, 3.94. Found: C, 71.03; H, 7.26; N. 4.13. FAB-MS, m/z
356.1868 for [M + H]+ (Calcd 356.1862 for C21H26NO4).

410 J . Org. Chem., Vol. 64, No. 2, 1999
Akaji et al.
TBDMS-Hiva -P h e-OBzl 16. To the stirred solution of 15
(1.3 g, 3.66 mmol) in DMF (8 mL) were added tert-butyldim-
ethylsilyl chloride (1.1 g, 7.32 mmol) and imidazole (1.0 g, 14.63
mmol), and the mixture was stirred at 25 °C for 1 h. AcOEt/
H2O (1:1, 40 mL) was added to the mixture, and the organic
layer was washed with H2O and dried (MgSO4). The solvent
was removed in vacuo, and the product was purified by silica
gel column chromatography using hexane/AcOEt (4:1) to yield
27.86 min [CH3CN 55%, 2.0 mL/min], IR (KBr) 1720, 1691,
1
1633, 1460, 1384, 1245, 1145, 1072 cm-1, H NMR (270 MHz,
CDCl3) δ 0.01 (s, 3H), 0.03 (s, 3H), 0.94 (s, 9H), 0.98 (d, J )
6.9 Hz, 3H), 1.05 (d, J ) 6.9 Hz, 3H), 1.92-2.18 (m, 1H), 3.17
(dd, J ) 3.0 Hz, J ) 14.0 Hz, 1H), 3.45 (dd, J ) 5.3 Hz, J )
14.2 Hz, 1H), 3.82 (s, 3H), 4.82 (s, 1H), 4.91 (dd, J ) 3.0 Hz,
J ) 5.3 Hz, 1H), 5.14 (d, J ) 2.0 Hz, 1H), 6.96-7.01 (m, 2H),
7.17-7.22 (m, 3H). 13C NMR (67.8 MHz, CDCl3) δ -5.32,
-4.83, 15.47, 18.42, 20.56, 25.86, 32.02, 34.86, 58.35, 59.69,
76.35, 94.66, 127.06, 128.07, 129.59, 134.34, 169.43, 173.84,
178.18. Anal. Calcd for C23H35NO4Si: C, 66.15; H, 8.45; N, 3.35.
Found: C, 65.91; H, 8.29; N. 3.52. FAB-MS, m/z 418.2408 for
[M + H]+ (Calcd 418.2414 for C23H36NO4Si).
(S)-Hiva -(S)-Dp y 19. To the stirred solution of 18 (145 mg,
0.347 mmol) in CH2Cl2 (4 mL) in an ice bath was added TFA
(0.4 mL). The mixture was stirred at 25 °C for 3 h, and the
solvent was removed in vacuo. The oily residue was purified
by flash chromatography to yield 102 mg (97%) of 19 as an
oil: [R]26D 259.0 (c ) 0.40, MeOH), Rt2 7.26 min [CH3CN (40-
80%/30 min)], IR (KBr) 3500, 1730, 1635, 1305 cm-1, 1H NMR
(270 MHz, CDCl3) δ 0.87 (d, J ) 6.9 Hz, 3H), 1.08 (d, J ) 6.9
Hz, 3H), 2.06-2.18 (m, 1H), 3.12 (dd, J ) 3.0 Hz, J ) 13.9
Hz, 1H), 3.60 (d, J ) 8.3 Hz, 1H), 3.65 (dd, J ) 5.0 Hz, J )
13.9 Hz, 1H), 3.84 (s, 3H), 4.77 (dd, J ) 3.3 Hz, J ) 8.3 Hz,
1H), 4.82 (s, 1H), 4.83 (dd, J ) 3.0 Hz, J ) 5.0 Hz, 1H), 6.92-
7.00 (m, 2H), 7.18-7.25 (m, 3H). 13C NMR (67.8 MHz, CDCl3)
δ 15.35, 19.95, 30.44, 34.66, 58.53, 60.05, 75.61, 94.82, 127.24,
128.26, 129.61, 133.71, 169.81, 174.25, 178.45. FAB-MS, m/z
304.1553 for [M + H]+ (Calcd 304.1549 for C17H22NO4).
1.76 g (96%) of 16 as an oil: [R]26 -46.9 (c ) 0.74, MeOH),
D
Rt2 22.10 min [CH3CN (60-80%/30min)], IR (KBr) 1743, 1681,
1506, 1253, 1178, 1051 cm-1 1H NMR (270 MHz, CDCl3) δ
,
-0.05 (s, 3H), 0.03 (s, 3H), 0.75 (d, J ) 6.9 Hz, 3H), 0.85 (s,
9H), 0.89 (d, J ) 6.9 Hz, 3H), 1.98-2.12 (m, 1H), 3.04 (dd, J
) 5.9 Hz, J ) 13.9 Hz, 1H), 3.14 (dd, J ) 5.9 Hz, J ) 13.9 Hz,
1H), 3.93 (d, J ) 3.3 Hz, 1H), 4.99 (ddd, J ) 5.9 Hz, J ) 5.9
Hz, J ) 8.9 Hz, 1H), 5.09 (d, J ) 12.2 Hz, 1H), 5.15 (d, J )
12.2 Hz, 1H), 6.94 (br d, J ) 8.9 Hz, 1H), 7.04-7.10 (m, 2H),
7.18-7.38 (m, 8H). 13C NMR (67.8 MHz, CDCl3) δ -5.25,
-5.03, 16.12, 17.90, 19.26, 25.73, 32.72, 38.53, 52.29, 67.10,
76.58, 127.11, 128.46, 128.59, 128.66, 129.22, 135.16, 135.63,
171.12, 172.97. FAB-MS, m/z 470.2740 for [M + H]+ (Calcd
470.2727 for C27H40NO4Si).
TBDMS-Hiva -P h e-OH 17. To the stirred solution of 16
(1.346 g, 2.87 mmol) in MeOH (15 mL) was added catalytic
amount of 10% Pd-C, and the mixture was stirred at 25 °C
for 1 h under H2 atomosphere. Pd-C (10%) was removed by
filtration, and the filtrate was rotary evaporated. The product
was recrystallized from hexane to yield 1.0 g (91%) of 17 as a
solid: [R]25 -21.0 (c ) 0.38, MeOH), mp 60-61 °C, Rt2 21.70
D
min [CH3CN (40-80%/30 min)], IR (KBr) 1730, 1646, 1625,
Boc-P r o-Hiva -Dp y 20. To the stirred solution of Boc-Pro-
OH (83 mg, 0.386 mmol) in CH2Cl2 (6 mL) in an ice bath were
added DIEA (0.224 mL, 1.29 mmol), CIP (0.215 g, 0.771 mmol),
19 (78 mg, 0.257 mmol), and DMAP (16 mg, 0.129 mmol). The
mixture was stirred at 25 °C for 10 h, and the solvent was
removed in vacuo. The residue was extracted with AcOEt, and
the organic layer was washed with 5% citric acid, 5% NaHCO3,
and H2O, dried (MgSO4), and rotary evaporated. The product
was purified by flash chromatography using CHCl3/AcOEt (10:
1541, 1253, 1049 cm-1 1H NMR (270 MHz, CDCl3) δ -0.02
,
(s, 3H), 0.04 (s, 3H), 0.71 (d, J ) 6.9 Hz, 3H), 0.84 (s, 9H),
0.86 (d, J ) 6.9 Hz, 3H), 1.95-2.08 (m, 1H), 3.16 (d, J ) 6.3
Hz, 2H), 3.98 (d, J ) 3.3 Hz, 1H), 4.96 (dt, J ) 6.3 Hz, J ) 8.6
Hz, 1H), 6.97 (d, J ) 8.6 Hz, 1H), 7.15-7.32 (m, 5H), 9.10 (br
s, 1H). 13C NMR (67.8 MHz, CDCl3) δ -5.32, -5.00, 16.01,
17.89, 19.25, 25.71, 32.65, 37.93, 52.22, 76.57, 127.26, 128.77,
129.25, 135.54, 174.01, 175.07. Anal. Calcd for C20H33NO4Si:
C, 63.29; H, 8.76; N, 3.69. Found: C, 63.17; H, 8.71; N. 3.91.
FAB-MS, m/z 380.2266 for [M + H]+ (Calcd 380.2257 for
1) to yield 0.12 g (92%) of 20 as a solid: [R]27 117.8 (c ) 0.51,
D
MeOH), mp 152-154 °C, Rt2 21.26 min [CH3CN (40-80%/30
C
20H34NO4Si).
min)], IR (KBr) 1751, 1732, 1705, 1681, 1631, 1456, 1394, 1305
1
cm-1, H NMR (270 MHz, CDCl3) two conformers in the ratio
TBDMS-(S)-Hiva -(S)-Dp y 18. To the stirred solution of 17
of 3:1, δ 0.95 (d, J ) 6.9 Hz, 3H), 1.08 (d, J ) 6.9 Hz, 3H),
1.46, 1.48 (s, 9H), 1.83-2.05 (m, 2H), 2.18-2.35 (overlapping
m, 3H), 3.06 (dd, J ) 3.3 Hz, J ) 13.9 Hz, 1H), 3.25-3.65
(overlapping m, 3H), 3.75, 3.78 (s, 3H), 4.40 (t, J ) 6.1 Hz)
4.53 (dd, J ) 2.6 Hz, J ) 8.3 Hz, total 1H), 4.73-4.83
(overlapping m, 2H), 5.87, 5.93 (each d, J ) 2.6 Hz, 1H), 7.09-
7.29 (m, 5H). 13C NMR (67.8 MHz, CDCl3) δ 15.83, 19.83, 23.47,
28.32, 28.50, 30.75, 34.93, 46.34, 58.35, 58.72, 59.98, 79.60,
79.89, 94.73, 126.97, 128.25, 129.92, 133.98, 154.03, 169.14,
169.58, 172.67, 178.42. Anal. Calcd for C27H36N2O7: C, 64.78;
H, 7.25; N, 5.60. Found: C, 64.60; H, 7.29; N. 5.92. FAB-MS,
m/z 501.2616 for [M + H]+ (Calcd 501.2601 for C27H37N2O7).
Dola st a t in 15 (1). To the stirred solution of 20 (85 mg,
0.170 mmol) in CH2Cl2 (4 mL) in an ice bath was added TFA/
anisole (0.85 mL-85 µL), and the mixture was stirred at 25 °C
for 1 h. The TFA was removed by azeotroping with hexane
(three times) to yield 3 as a light yellow oil.
(0.50 g, 1.31 mmol) in THF (5 mL) in an ice bath was added
DIEA (0.91 mL, 5.22 mmol), Meldrum’s ester (0.21 g, 1.44
mmol), CIP (0.54 g, 1.94 mmol), and DMAP (0.08 g, 0.65
mmol). The mixture was stirred at 25 °C for 4 h, and the
solvent was removed in vacuo. The residue was extracted with
AcOEt, and the organic layer was washed with 10% citric acid,
H2O, dried (MgSO4), and rotary evaporated. The product was
dissolved in AcOEt (80 mL), and the mixture was refluxed for
1 h. The solvent was removed in vacuo, and the residue was
dissolved in THF (5 mL). To this solution in an ice bath were
added MeOH (0.08 mL, 2.00 mmol), Ph3P (0.52 g, 2.00 mmol),
and diethyl diazocarboxylate (0.31 mL, 2.00 mmol). The
mixture was stirred at 25 °C for 2 h, and the solvent was
removed in vacuo. The product was purified by flash chroma-
tography using CHCl3 to yield 0.25 g (46%) of 18 as a solid:
[R]24 189.5 (c ) 0.51, MeOH), mp 104-106 °C, Rt1 26.54 min
D
[CH3CN 55%, 2.0 mL/min], IR (KBr) 1720, 1689, 1631, 1460,
1384, 1251, 1147, 1070 cm-1 1H NMR (270 MHz, CDCl3) δ
,
To the stirred solution of 2 (116 mg, 0.255 mmol) in CH2Cl2
(2.5 mL) in an ice bath were added DIEA (0.136 mL, 0.78
mmol), HOAt (26 mg, 0.19 mmol), CIP (53 mg, 0.19 mmol),
and the above obtained H-Pro-Hiva-Dpy 3 in CH2Cl2/DIEA (2.5
mL/72 µL). The mixture was stirred at 25 °C for 2 h, and the
solvent was removed in vacuo. The residue was extracted with
AcOEt, and the organic layer was washed with 5% citric acid,
5% NaHCO3, and H2O, dried (MgSO4), and rotary evaporated.
The product was purified by flash chromatography using
hexane/acetone (3:2) followed by reprecipitation with AcOEt/
hexane to yield 119 mg (84%) of 1 as a solid: [R]26D -80.7 (c )
0.56, MeOH), mp 177-179 °C, Rt2 9.08 min [CH3CN (40-70%/
0.10 (s, 3H), 0.14 (s, 3H), 0.85 (d, J ) 6.6 Hz, 3H), 0.98 (s,
9H), 1.00 (d, J ) 6.6 Hz, 3H), 1.88-2.05 (m, 1H), 3.18 (dd, J
) 3.0 Hz, J ) 13.8 Hz, 1H), 3.62 (dd, J ) 5.3 Hz, J ) 13.8 Hz,
1H), 3.80 (s, 3H), 4.76 (dd, J ) 3.0 Hz, J ) 5.3 Hz, 1H), 4.80
(s, 1H), 5.27 (d, J ) 3.0 Hz, 1H), 6.99-7.07 (m, 2H), 7.18-
7.23 (m, 3H). 13C NMR (67.8 MHz, CDCl3) δ -4.91, -4.44,
15.62, 18.40, 20.05, 25.91, 32.02, 35.58, 58.31, 60.41, 75.97,
94.88, 127.02, 128.16, 129.67, 134.55, 169.93, 173.85, 178.29.
Anal. Calcd for C23H35NO4Si: C, 66.15; H, 8.45; N, 3.35.
Found: C, 65.85; H, 8.42; N. 3.67. FAB-MS, m/z 418.2421 for
[M + H]+ (Calcd 418.2414 for C23H36NO4Si).
A small amount of diastereoisomer, TBDMS-(S)-Hiva-(R)-
Dpy, was recovered from the flash chromatography: yield 50
30 min)], IR (KBr) 3423, 1735, 1629, 1448, 1309, 1191 cm-1
,
1H NMR (270 MHz, CDCl3) δ 0.78 (d, J ) 6.6 Hz, 3H), 0.92-
1.09 (overlapping m, 21H), 1.80-2.43 (overlapping m, 12H),
mg (9%), [R]22 -265.5 (c ) 0.60, MeOH), mp 99-101 °C, Rt1
D

Convergent Synthesis of Dolastatin 15
J . Org. Chem., Vol. 64, No. 2, 1999 411
2.26 (s, 6H), 2.47 (d, J ) 6.3 Hz, 1H), 3.05 (dd, J ) 3.3 Hz,
13.8 Hz, 1H), 3.18 (s, 3H), 3.54 (dd, J ) 4.3 Hz, J ) 13.8 Hz,
1H), 3.57-3.65 (m, 1H), 3.75 (s, 3H), 3.72-3.94 (m, 3H), 4.62-
4.67 (m, 1H), 4.73 (s, 1H), 4.76-4.87 (m, 3H), 5.14 (d, J ) 10.8
Hz, 1H), 5.90 (d, J ) 2.3 Hz, 1H), 6.90 (br d, J ) 9.2 Hz, 1H),
7.13-7.22 (m, 5H). 13C NMR (67.8 MHz, CDCl3) δ 15.81, 17.70,
18.15, 18.54, 19.16, 19.57, 19.86, 20.14, 24.64, 24.73, 27.31,
27.69, 28.39, 28.57, 28.88, 30.73, 31.09, 34.88, 42.87, 46.41,
47.85, 53.71, 58.09, 58.29, 58.35, 59.23, 59.89, 76.37, 77.86,
94.75, 127.01, 128.16, 130.01, 134.16, 169.14, 169.34, 169.49,
170.26, 171.50, 171.68, 173.01, 178.22. Anal. Calcd for
C45H69N6O9: C, 64.57; H, 8.19; N, 10.04. Found: C, 64.47; H,
7.98; N. 9.89. FAB-MS, m/z 837.5132 for [M + H]+ (Calcd
837.5126 for C45H69N6O9).
Ack n ow led gm en t. We thank Professor J oe¨l Poncet
for sending the 1H NMR spectrum of his synthetic
dolastatin 15.
1
Su p p or tin g In for m a tion Ava ila ble: HPLC and H NMR
analysis for the estimation of D-isomer content and 1H and
13C NMR spectra for compound 1, 2, and 15-20 (21 pages).
This material is contained in libraries on microfiche, im-
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J O981055A