Total synthesis of motuporin and 5-[L-Ala]-motuporin

Article abstract of DOI:10.1021/jo982145i

Total synthesis of the cyclic peptide hepatotoxin motuporin is described, including an efficient synthesis of the constituent amino acid Adda. Three strategies to motuporin are outlined with their relative strengths and weaknesses. Cyclization of the linear peptide precursor was found to proceed moderately well for peptides containing the N- methyldehydrobutyrine residue masked as a threonine, but significant C- terminal epimerization occurred in the presence of the dehydroamino acid. Replacement of the N-methyldehydrobutyrine residue by L-alanine was explored to assess the contribution of this dehydroamino acid to the biochemical activity of motuporin. Some epimerization also was observed during cyclization of the alanine-containing peptide. Synthetic motuporin and both isomers of 5-[L-Ala]-motuporin inhibit the activity of protein phosphatase-1 (PP1) in rat adipocyte lysates with comparable IC₅₀ values. These results indicate that the N-methyldehydrobutyrine residue is not essential for PP1 inhibition.

Full text of DOI:10.1021/jo982145i

J . Org. Chem. 1999, 64, 2711-2728
2711
Tota l Syn th esis of Motu p or in a n d 5-[L-Ala ]-Motu p or in
Raghu Samy,† Hong Yong Kim,† Matthew Brady,‡ and Peter L. Toogood*,†
Willard H. Dow Laboratory, Department of Chemistry, University of Michigan, and Department of
Physiology, School of Medicine, University of Michigan, Ann Arbor, Michigan 48105
Received October 26, 1998
Total synthesis of the cyclic peptide hepatotoxin motuporin is described, including an efficient
synthesis of the constituent amino acid Adda. Three strategies to motuporin are outlined with
their relative strengths and weaknesses. Cyclization of the linear peptide precursor was found to
proceed moderately well for peptides containing the N-methyldehydrobutyrine residue masked as
a threonine, but significant C-terminal epimerization occurred in the presence of the dehydroamino
acid. Replacement of the N-methyldehydrobutyrine residue by L-alanine was explored to assess
the contribution of this dehydroamino acid to the biochemical activity of motuporin. Some
epimerization also was observed during cyclization of the alanine-containing peptide. Synthetic
motuporin and both isomers of 5-[L-Ala]-motuporin inhibit the activity of protein phosphatase-1
(PP1) in rat adipocyte lysates with comparable IC50 values. These results indicate that the
N-methyldehydrobutyrine residue is not essential for PP1 inhibition.
In tr od u ction
they are hepatotoxic as a consequence of their accumula-
tion in the liver, possibly through bile acid transporters.11
This activity is believed to account for the death of
livestock that drink from water contaminated with cy-
anobacterial blooms,12 and recently the use of contami-
nated water in dialysis led to the death of 50 human
patients.13 Moroever, increased incidence of cancer in
regions where cyanobacteria proliferate may be related
to the presence of these toxins, since microcystin-LR has
been shown to promote tumor formation in mice.14 The
antineoplastic activity of motuporin1 and the tumor-
promoting activity of microcystin-LR15,16 and nodularin17
represent an interesting paradox in the biochemical
profiles of these peptides and suggest that the biological
outcome of phosphatase inhibition is critically dependent
upon cellular localization and the specific phosphatase
that is targeted. The structural differences between
microcystin-LR and motuporin may contribute to a
difference in localization of these compounds in tissues
or cells. Thus, interest in these natural products centers
on their use as biochemical tools for studying the func-
tions of phosphatases in cells and their role as lead
Motuporin (1, Figure 1) is a cyclic pentapeptide isolated
from the marine sponge Theonella swinhoei (Gray).1 It
is cytotoxic toward a variety of human cancer cells in
vitro and inhibits protein phosphatase type 1 (PP1) with
Kd < 1 nM. The structure of motuporin was determined
using NMR spectroscopy and mass spectrometry. It bears
remarkable resemblance to the cyanobacterial natural
products known as nodularins2,3 and is related to the
heptapeptide microcystins (e.g., compounds 2-4, Figure
1).4-7 Specifically, these cyclic peptides all contain the
unusual amino acid (2S,3S,8S,9S,4E,6E)-3-amino-9-
methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid
(Adda),2 an R,â-unsaturated amino acid such as N-
methyldehydroalanine (mdha) or N-methyldehydro-
butyrine (mdhb), an iso-linked D-glutamate, and an iso-
linked D-aspartate or â-methyl D-aspartate residue.
Nodularins and microcystins are potent, competitive
inhibitors of protein phosphatases PP1 and PP2A, dis-
playing a slight selectivity for the latter.8-10 In animals,
* Corresponding author. Present address: Parke-Davis Pharma-
ceutical Research, 2800 Plymouth Road, Ann Arbor, MI 48105. Tel:
(734) 622-1335. Fax: (734) 622-7879. Email: peter.toogood@wl.com.
† Willard H. Dow Laboratory.
(8) Honkanen, R. E.; Zwiller, J .; Moore, R. E.; Daily, S. L.; Khatra,
B. S.; Dukelow, M.; Boynton, A. L. J . Biol. Chem. 1990, 265, 19401-
19404.
‡ Dept. of Physiology.
(9) Mackintosh, C.; Beattie, K. A.; Klump, S.; Cohen, P.; Codd, G.
A. FEBS Lett. 1990, 264, 187-192.
(1) Dilip de Silva, E.; Williams, D. E.; Anderson, R. J .; Klix, H.;
Holmes, C. F. B.; Allen, T. A. Tetrahedron Lett. 1992, 12, 1561-1564.
(2) Rinehart, K. L.; Harada, K.-I.; Namikoshi, M.; Chen, C.; Harvis,
C. A.; Munro, M. H.; Blunt, J . W.; Mulligan, P. E.; Beasley, V. R.;
Dahlem, A. M.; Carmicheal, W. W. J . Am. Chem. Soc. 1988, 110, 8557-
8558.
(3) Namikoshi, M.; Choi, B. W.; Sakai, R.; Sun, F.; Rinehart, K. L.;
Carmicheal, W. W.; Evans, W. R.; Cruz, P.; Munro, M, H. G.; Blunt, J .
W. J . Org. Chem. 1994, 59, 2349-2357.
(4) Botes, P. B.; Wessels, P. L.; Kruger, H.; Runnegar, M. T. C.;
Santikaarn, S.; Smith, R. J .; Barna, J . C. J .; Williams, D. H. J . Chem.
Soc., Perkin Trans. 1 1985, 2747-2748.
(5) Carmichael, W. W.; Beasley, V. R.; Bunner, D. L.; Eloff, J . N.;
Falconer, I.; Gorham, P. R.; Harada, K. I.; Yu, M.-J .; Krishnamurthy,
T.; Moore, R. E.; Rinehart, K. L.; Runnegar, M.; Skulberg, O. M.;
Watanabe, M. Toxicon 1988, 26, 971-973.
(6) Namikoshi, M.; Rinehart, K. L.; Sakai, R.; Sivonen, K.; Car-
micheal, W. W. J . Org. Chem. 1990, 55, 6135-6139.
(7) Namikoshi, M.; Rinehart, K. L. Sakai, R.; Stotts, R. R.; Dahlem,
A. M.; Beasley, V. R.; Carmicheal, W. W.; Evans, W. R. J . Org. Chem.
1992, 57, 866-872 and references therein.
(10) Honkanen, R. E.; Codispoti, B. A.; Tse, K.; Boynton, A. L.
Toxicon 1994, 32, 229-350.
(11) Runnegar, M. T.; Berndt, N.; Kaplowitz, N. Toxicol. Appl.
Pharmacol. 1995, 134, 264-272.
(12) Carmicheal, W. W. In Handbook of Natural Toxins: Charac-
terization, Pharmacology and Therapeutics; Ownby, C. L., Odell, G.
V., Eds.; Pergamon: London, 1988; pp 3-16.
(13) Pouria, S.; de Andrade, A.; Barbosa, J .; Cavalcanti, R. L.;
Barreto, V. T. S.; Ward, C. J .; Preiser, W.; Poon, G. K.; Neild, G. H.;
Codd, G. A. Lancet 1998, 352, 21-36.
(14) Falconer, I. R.; Buckley, T. H. Med. J . Aust. 1989, 150, 351.
(15) Yoshizawa, S.; Matsushima, R.; Watanabe, M. F.; Harada, K.-
I.; Ichihara, A.; Carmicheal, W. W.; Fujiki, H. J . Cancer Res. Clin.
Oncol. 1990, 116, 709-614.
(16) Nishiwaki-Matsushima, R.; Ohtam T.; Nishiwaki, S.; Sug-
anuma, M.; Kohyama, K.; Ishikawa, T.; Carmicheal, W. W.; Fujiki, H.
J . Cancer Res. Clin. Oncol. 1992, 118, 420-424.
(17) Fujiki, H.; Matsushima, R.; Yoshizawa, S.; Suganuma, M.;
Nishiwaki, S.; Ishikawa, T.; Carmicheal, W. W. Proc. Am. Assoc. Cancer
Res. 1991, 32, 157.
10.1021/jo982145i CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/26/1999

2712 J . Org. Chem., Vol. 64, No. 8, 1999
Samy et al.
F igu r e 1.
compounds for the development of subclass specific
phosphatase inhibitors.
first total synthesis of motuporin,31 and a total synthesis
of microcystin-LA.32 We have published a synthetic route
to N-Boc-Adda methyl ester33 and the structurally related
â-methyl-D-aspartate.23 Herein, we describe the optimiza-
tion of this chemistry and elaboration of these fragments
to provide synthetic motuporin and a related analogue,
5-[L-Ala]-motuporin.
A crystal structure of microcystin-LR bound to PP1 was
published in 1995.18 The Adda residue lodges in a
hydrophobic groove on the surface of the protein, and the
glutamate R-carboxyl group and the adjacent amide
carbonyl make hydrogen bonds to two metal-coordinated
water molecules. A covalent bond is formed between the
inhibitor and the protein through conjugate addition of
the thiol from cysteine-273 to the dehydroalanine in the
cyclic peptide. Microcystin undergoes very little confor-
mational adjustment upon binding to PP1, suggesting
that the peptide is preorganized for binding to this
enzyme. Motuporin contains the essential features of
microcystin that appear to be required for binding to PP1,
and it adopts a similar three-dimensional structure.19-22
Molecular modeling studies suggest that motuporin binds
to PP1 in a manner similar to microcystin-LR, although
the mdhb â-carbon in PP1-bound motuporin is predicted
to rest approximately 7 Å from the cysteine-273 thiol,
accounting for the observation that motuporin does not
covalently modify the protein. Structural differences such
as this may be significant with respect to binding affinity
and phosphatase selectivity, and synthetic approaches
to the microcystin and nodularin peptides are expected
to facilitate further investigations of their structural
requirements for phosphatase inhibition and selectivity.
We initiated a program to synthesize motuporin with
the intention of developing a convergent route that could
be adapted to the preparation of other nodularin and
microcystin natural products, as well as related ana-
logues. Our general strategy has been described previ-
ously.23 Prior to, and during the course of our work,
several other groups have published the results of their
studies in this area, including syntheses of Adda,24-30 the
Syn th etic Str a tegy. At the outset of this work, we
proposed to prepare motuporin in a convergent manner
through the assembly and cyclization of linear pentapep-
tide dimethyl ester 5. Installation of the dehydrobutyrine
double bond was anticipated to be performed late in the
synthesis, although we recognized that the synthetic
route could be adapted to incorporate this double bond
earlier. We chose to cyclize the linear precursor at the
C-terminus of the valine residue to achieve maximum
convergency. This approach would allow incorporation of
the Adda residue as the last residue of the pentapeptide
to achieve the shortest linear synthesis. Moreover, we
reasoned that none of the peptide bonds in motuporin or
its precursors displayed any special advantage over the
others with regard to the chances of R-epimerization
during cyclization and that closure at an unsaturated
acid, if we chose to introduce the dehydrobutyrine double
bond early, could present a significant hurdle. Our
retrosynthetic analysis is summarized in Figure 2.
Syn th esis of N-Boc-Ad d a Meth yl Ester (9). The
Adda residue is both unique to microcystins and nodu-
larins and characteristic of these natural products. As
such, the synthesis of this residue constitutes the founda-
tion of any total synthesis of molecules in this class. For
convergency, we chose to synthesize N-Boc-Adda methyl
ester (9) via a Wittig or related olefination reaction
between a nucleophile derived from bromide 10 and
aldehyde 11 as shown in Figure 3. Ample literature
precedent lends support to this approach and indicates
(18) Goldberg, J .; Huang, H.-B.; Kwon, Y.-G.; Greengard, P.; Nairn,
A. C.; Kuriyan, J . Nature 1995, 376, 745-753.
(19) Rudolph-Bo¨hner, S.; Mierke, D. F.; Moroder, L. FEBS Lett.
1994, 349, 319-323.
(20) Bagu, J .; So¨nnichsen, F. D.; Williams, D.; Anderson, R. J .;
Sykes, B. D.; Holmes, C. F. B. Nat. Struct. Biol. 1995, 2, 114-116.
(21) Trogen, G.-B.; Eriksson, J .; Kontteli, M.; Meriluoto, J .; Sethson,
I.; Zdunek, J .; Edlund, U. Biochemistry 1996, 35, 3197-3205.
(22) Annila, A.; Lehtima¨k, J .; Mattila, K.; Eriksson, J . E.; Sivonen,
K.; Rantala, T. T.; Drakenberg, T. J . Biol. Chem. 1996, 271, 16695-
16702.
(23) Kim, H. Y.; Stein, K.; Toogood, P. L. J . Chem. Soc., Chem.
Commun. 1996, 1683-1684.
(24) Namikoshi, M.; Rinehart, K. L.; Dahlem, A.; Beasley, V. R.;
Carmicheal, W. W. Tetrahedron Lett. 1989, 30, 4349-4352.
(25) Chakraborty, T. K.; J oshi, S. P. Tetrahedron Lett. 1990, 31,
2143-2146.
(26) Beatty, M. F.; J ennings-White, C.; Avery, M. A. J . Chem. Soc.,
Perkin Trans. 1 1992, 1637-1641.
(27) D’aniello, F.; Mann, A.; Taddei, M. J . Org. Chem. 1996, 61,
4870-4871.
(28) Sin, N.; Kallmerten, J . Tetrahedron Lett. 1996, 37, 5645-5648.
(29) Panek, J .; Hu, T. J . Org. Chem. 1997, 62, 4914-4915.
(30) Cundy, D. J .; Donohue, A. C.; McCarthy, T. D. Tetrahedron Lett.
1998, 39, 5125-5128.
(31) Valentkovich, R. J .; Schreiber, S. L. J . Am. Chem. Soc. 1995,
117, 9069-9070.
(32) Humphrey, J . M.; Aggen, J . B.; Chamberlin, A. R. J . Am. Chem.
Soc. 1996, 118, 11759-11770.
(33) Kim, H. Y.; Toogood, P. L. Tetrahedron Lett. 1996, 37, 2349-
2352.

Total Synthesis of Motuporin and 5-[L-Ala]-Motuporin
J . Org. Chem., Vol. 64, No. 8, 1999 2713
F igu r e 2.
we investigated ways of improving existing chemistry
from our group and others to produce an efficient and
convergent route to 9 that retained many of the strongest
aspects of the previously published chemistry but avoided
some of the pitfalls.
Upon reinvestigation of work by Rinehart24 and Be-
atty,26 we decided to retain the Evans aldol chemistry
employed by these workers for the synthesis of the C5-
C10 fragment of Adda. Thus, aldol 15 was prepared in
69% yield and 95% de from phenylacetaldehyde, then
transaminated with N,O-dimethylhydroxylamine in the
presence of Al(CH3)3 (78% yield; Scheme 2), and methy-
lated (NaH, MeI, 95%) to give compound 16. Reduction
of the Weinreb amide with LiAlH4 gave an aldehyde that
was homologated under standard Wittig conditions with
isopropylidenetriphenylphosphorane to provide the alk-
ene 17 in 80% yield for the two steps. As reported
previously,33 we then were able to employ the completely
regioselective oxidation mediated by SeO2 to convert this
alkene to the E-allylic alcohol 18. Some overoxidation
occurs in the SeO2 reaction, and consequently the crude
reaction mixture was treated with CeCl3‚7H2O and
NaBH4 to reduce the enal back to the allylic alcohol prior
to workup. Alcohol 18 is a known compound26 and may
be converted to the corresponding bromide 10 by treat-
ment with triphenylphosphine and carbon tetrabromide.
F igu r e 3.
that we might be able to improve upon the yields and
selectivities observed by previous workers.24-26
In a previously published approach to Adda,33 we made
extensive use of Ireland-Claisen rearrangement chem-
istry to produce both fragments of Adda from a common
precursor, namely, (R)-3-pentyn-2-ol (Scheme 1). This
approach contains several attractive elements, such as
the derivation of both components of Adda from a
common precursor and a high level of efficiency in the
introduction of all four asymmetric carbons. However,
overall this route was considered to be too long for large
scale synthesis. Also, we encountered some difficulties
in reproducing the hydrolysis of lactone 14 without
epimerization of the adjacent C2 chiral center. Therefore,
The C1-C4 fragment of Adda was prepared from (R)-
3-pentyn-2-ol as reported previously,23 with some modi-
Sch em e 1

2714 J . Org. Chem., Vol. 64, No. 8, 1999
Samy et al.
Sch em e 2
Sch em e 4
these conditions cleanly provided the corresponding
alcohol in a pleasing 84% yield, with no apparent lactone
formation. Immediate oxidation of the seco-ester to the
desired aldehyde 11 proceeded quantitatively under
standard Swern conditions. No attempt was made to
purify this sensitive aldehyde, which was used im-
mediately in olefination reactions to form N-Boc-Adda
methyl ester.
Sch em e 3
At the outset of our work, all previous syntheses of
Adda had employed Wittig chemistry to install the
disubstituted 4,5-carbon-carbon double bond.24-26
A
shortcoming of this approach is the relatively poor
selectivity for E-double bond geometry that is obtained
using standard Wittig reagents. Subsequently, several
groups have employed palladium-mediated cross-coupling
reactions to introduce the 4,5-double bond with excellent
stereocontrol.27,29,32 However, we were motivated to at-
tempt improving upon the conventional olefination reac-
tion by use of modified ylid chemistry. A survey of the
literature indicated that we might achieve better E-
selectivity by replacing the standard triphenylphospho-
rane with either a phosphine oxide or a trialkylphospho-
rane. The use of tributylphosporane looked particularly
promising on the basis of previous work by Vedejs.38-40
Bromide 10 was transformed to phosphonate 22,
phosphine oxide 23, and tributylphosphorane 24 (Scheme
4), and each of these reagents was explored as a potential
coupling partner for aldehyde 11 with the following
results. Phosphonate 22 could be deprotonated with
nBuLi to give a characteristic red anion but was insuf-
ficiently reactive to couple with aldehyde 11. The anion
formed from phosphine oxide 23 and either nBuLi or
tBuLi reacted with aldehyde 11 to provide a product,
which was tentatively identified as the result of anion
acylation by the ester function in compound 11. Finally,
we were disappointed to find that tributylphosphorane
24 failed to react with aldehyde 11, even upon warming,
fications. Acylation of the alkynol with N-Boc-L-glycine
under standard dicyclohexylcarbodiimide conditions, fol-
lowed by hydrogenation over Lindlar’s catalyst, provided
cis-olefin 12 in 88% overall yield (Scheme 1). Then,
following the precedents established by Kazmaier,34
compound 12 was treated with LDA at -78 °C, in the
presence of zinc chloride, to promote a highly stereo-
selective Claisen rearrangement to acid 13, which was
isolated in 75% yield. This acid was converted to its
corresponding methyl ester 19 in 95% yield using sodium
bicarbonate and methyl iodide in DMF (Scheme 3).35
Reduction of ester 19 with LiAlH4, followed by protection
of the product alcohol with TBDMSCl, gave silyl ether
20 (86% over two steps). Then, the alkene was converted
directly to a methyl ester under the conditions described
by Marshall36 to provide compound 21. Careful monitor-
ing of this reaction was essential to avoid epimerization
at the center adjacent to the newly formed ester carbonyl,
and workup was performed immediately upon observa-
tion of the characteristic blue color of the ozone-saturated
solvent without allowing the reaction mixture to warm
to room temperature (Scheme 3).
t
following deprotonation with nBuLi, or BuOK and 18-
Removal of the silyl group at this juncture was prone
to butyrolactone formation under either acidic or basic
conditions, and prior experience had taught us that
saponification of this lactone was difficult to achieve
without epimerization. Thus, extremely mild deprotection
conditions were essential. The method of Taylor,37 em-
ploying NBS in aqueous DMSO, proved eminently suit-
able for this task, and treatment of silyl ether 21 under
crown-6. Under most of these reaction conditions, alde-
hyde 11 was destroyed during the reaction, and the
instability of this compound may be responsible for the
failure of the attempted olefination reactions.
Faced with these results, we eventually resorted to
attempting to optimize the known Wittig reaction be-
tween triphenylphosphorane 25 and aldehyde 11. Un-
(34) Kazmaier, U. Angew. Chem., Int. Ed. Engl. 1994, 33, 998.
(35) Bocchi, V.; Casnati, G.; Dossena, A.; Marchelli, R. Synthesis
1979, 961.
(36) Marshall, J . A.; Garofalo, A. W. J . Org. Chem. 1993, 58, 3675.
(37) Batten, R. J .; Dixon, A. J .; Taylor, R. J . K. Synthesis 1980, 234.
(38) Tamura, R.; Saegusa, K.; Kakihana, M.; Oda, D. J . Org. Chem.
1988, 53, 2723.
(39) Tamura, R.; Kato, M.; Saegusa, K.; Kakihana, M.; Oda, D. J .
Org. Chem. 1987, 52, 4121.
(40) Vedejs, E.; Fang, H. W. J . Org. Chem. 1984, 49, 210.

Total Synthesis of Motuporin and 5-[L-Ala]-Motuporin
J . Org. Chem., Vol. 64, No. 8, 1999 2715
Sch em e 5
Sch em e 7
υ
Sch em e 6
introduction of this double bond would avoid any poten-
tial complications due to conjugate addition to this center.
A similar approach was initiated by Valentekovich and
Schreiber, who derived this residue from D-threonine,
although these workers eventually achieved dehydration
concomitantly with ester saponification to provide the
product in one step following cyclization of the pep-
tide.31,43 In the event, we found that dehydration of the
threonine residue late in the synthesis presented a
number of problems, and consequently we simultaneously
examined incorporation of the mdhb residue early in the
synthesis as a component of the glutamyl-containing
fragment used for our assembly of the macrocycle (see
below).
Commercially available N-Boc-O-benzyl-L-threonine
was converted to N-Boc-N-Me-O-benzyl-L-threonine in
75% yield under the conditions developed by Benoiton,44
and the carboxyl group was protected as its trimethylsilyl
ethyl (Tse) ester 27 (Scheme 7). Following removal of the
Boc group using HCl-saturated ether, this residue was
condensed with N-Boc-glutamate R-methyl ester45 in the
presence of the BOP reagent to provide dipeptide 28 in
86% yield. The benzyl group in this dipeptide was
removed by hydrogenolysis over 10% Pd/C in ethanol to
give the desired alcohol 7 in 86% yield. This fragment
was either deprotected at the C-terminus by treatment
with TBAF and used in the synthesis as a glutamyl-N-
methylthreonine dipeptide or dehydrated prior to re-
moval of the Tse group and incorporated as a glutamyl-
N-methyldehydrobutyrine dipeptide. Initial attempts to
dehydrate compound 28 using Burgess salt,46 or Ph3P and
DEAD,47 gave no reaction, even upon warming; however,
dehydration was finally achieved using the Martin sul-
furane.48,49 Thus, on treating dipeptide 28 with Martin
fortunately, no significant improvement in E/Z-selectivity
could be achieved either by using “salt-free” conditions
or by performing the reaction at higher temperatures,
suggesting that the product mixture may represent the
result of kinetic control. Under our best conditions, a 2:1
mixture of E and Z isomers of N-Boc-Adda methyl ester
was obtained and after careful chromatographic separa-
tion, the desired E-isomer (E,E-9) was isolated in a
relatively poor 41% yield (Scheme 5).
Unwilling to accept this low yield at such an early stage
in our synthesis we determined to explore conditions for
isomerizing the side product Z,E-isomer of N-Boc-Adda
methyl ester (Z,E-9) to the corresponding E,E-isomer.41
These investigations proved fruitful when it was found
that tungsten lamp irradiation of a solution of the diene
in CH2Cl2 containing a catalytic amount of iodine pro-
moted a clean isomerization of the Z,E-diene to the
desired E,E-diene (Scheme 5). This reaction appears to
reach an equilibrium in which the E,E-diene is preferred,
and on a preparative scale the E,E-diene was isolated in
an acceptable 51% yield, increasing the overall conversion
of alcohol 10 to N-Boc-Adda methyl ester (9) to 50%.
Syn th esis of th e iso-D-â-Meth yla sp a r tyl-L-va lin e
Dip ep tid e F r a gm en t. As reported previously, the â-me-
thylaspartate residue that occurs in microcystins and
nodularins, including motuporin, may be obtained from
an intermediate that is common to our synthesis of Adda.
Thus, alkene 19 was transformed to carboxylic acid 26
by treatment with NaIO4 and RuCl3,42 then converted to
its pentafluorophenyl ester, and coupled with L-valine
allyl ester to provide dipeptide 8 in 80% yield (Scheme
6).
Syn th esis of iso-D-Glu ta m yl-Con ta in in g Dip ep -
tid es. In our original synthetic plans, we envisaged
incorporating the mdhb residue required for motuporin
in the form of an L-threonine residue that could be
dehydrated in the penultimate step of the synthesis. Late
(43) Valentkovich, R. J . Ph.D. Thesis, Harvard University, 1995.
(44) Cheung, S. T.; Benoiton, N. L. Can. J . Chem. 1977, 55, 906.
(45) Sawayama, T.; Tsukamoto, M.; Nishimura, K.; Yamamoto, R.;
Deguchi, T.; Takeyama, K.; Hosoki, K. Chem. Pharm. Bull. 1989, 37,
2417. This compound is commercially available from Bachem.
(46) Wipf, P.; Miller, C. P. Tetrahedron Lett. 1992, 33, 907.
(47) Wojciechowska, H.; Pawlowicz, R.; Andruszkiewicz, R.; Grzy-
bowska, J . Tetrahedron Lett. 1978, 19, 4063.
(41) Saltiel, J .; D’Agostino, J .; Megarity, E. D.; Metts, L.; Neuberger,
K. R.; Wrighton, M.; Zafiriou, O. C. Org. Photochem. 1973, 3, 1-113.
(42) Carlsen, Per H. J .; Katsuki, T.; Martin, V. S.; Sharpless, K. B.
J . Org. Chem. 1981, 46, 3936.
(48) Martin, J . C.; Arhart, R. J . J . Am. Chem. Soc. 1971, 93, 4327.

2716 J . Org. Chem., Vol. 64, No. 8, 1999
Samy et al.
Sch em e 8
sulfurane in DMF at 120 °C, clean formation of a
dehydrobutyrine-containing peptide was observed, giving
29 in 70% yield. Martin sulfurane is known to display a
strong preference for a trans-coplanar disposition of the
leaving groups in the dehydration of secondary alcohols,
and therefore we anticipated that the product of this
dehydration should contain the desired (Z)-methyldehy-
drobutyrine. That this was indeed the case was estab-
lished by comparison of the chemical shift of the vinylic
proton with similar examples in the literature and by
HCl-saturated ether were unsuccessful, possibly as a
result of the operation of an intramolecular N,O-transa-
cylation reaction under these conditions. The coupling
of compounds 6 and 30 was achieved using HATU53,54 to
provide tripeptide 31 in 79% yield, whereas the use of
diphenylphosphinic chloride55 in this step resulted in a
mixture of similar products that was presumed to arise
from epimerization of the chiral center at C-2 in the Adda
residue. Subsequent removal of the trimethylsilylethyl
group using TBAF and condensation with fragment 33a
using BOP56 gave a 41% yield of the target pentapeptide
5a . HATU proved to be slightly superior in this coupling
step, providing 50% of the desired product. In contrast,
reaction of the C-terminal pentafluorophenyl ester was
slow and did not give a clean product.57
Although the above route was generally satisfactory,
to attain a higher level of convergency in our synthesis,
we returned to the strategy of assembling the pentapep-
tide from a tetrapeptide plus N-Boc-Adda as originally
intended. Thus, the glutamyl-N-methylthreonine dipep-
tide ester 7 was deprotected with TBAF and condensed
with 33 using HATU to form tetrapeptide 34 in 59%
overall yield as shown in Scheme 9. Following removal
of the Boc group using TFA,58 N-Boc-Adda pentafluo-
rophenyl ester (35) was attached in the presence of
Hu¨nig’s base to produce pentapeptide 5a in 69% yield
(Scheme 10). Although the yields via this route are
slightly lower, the opportunity to introduce the complex
Adda residue later in the synthesis represents a signifi-
cant advantage in terms of material throughput.
1
analysis of the H and 13C NMR spectra of compound 29
in comparison with those of motuporin1 and other known
N-acyl crotonates.50 Compound 29 exhibits a vinylic
proton chemical shift of 7.00 ppm (quartet) and a vinylic
methyl chemical shift of 1.81 ppm. These values are
consistent with Z-olefin geometry and closely match the
correponding chemical shifts for motuporin (7.02 and 1.72
ppm, respectively). As further evidence for the Z-double
bond geometry in 29, the 1H-13C coupling constant
between the dehydrobutyrine carbonyl and vinylic proton
is 2.7 Hz. A coupling constant of ∼10 Hz would be
expected for the E-isomer.51,52
Assem bly of th e Lin ea r P en ta p ep tid e. We have
explored several strategies for the assembly of linear
pentapeptide precursors to the motuporin macrocycle.
These strategies differ in the order of fragment assembly
and in the timing of the dehydration step to install the
mdhb double bond. Because we initially experienced some
difficulty in forming an amide bond between the C-
terminus of N-Boc-Adda and an appropriate tetrapeptide,
we were led to explore the coupling of N-Boc-Adda with
dipeptide fragment 7 (Scheme 8). Saponification of N-Boc-
Adda methyl ester (9) using aqueous lithium hydroxide
provided the necessary carboxylic acid 6. Meanwhile,
treatment of dipeptide 7 with TFA in CH2Cl2 gave the
complementary coupling partner 30. It is worth noting
that attempts to deprotect the amino terminus of 7 using
A third strategy for synthesis of the pentapeptide was
developed to simplify the final steps of the synthesis
when it was found that dehydration of the threonine
(53) Carpino, L. A.; El-Faham, A. J . Org. Chem. 1994, 59, 695.
(54) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397.
(55) Ramage, R.; Hopton, D.; Parrott, M. J .; Richardson, R. S.;
Kenner, G. W.; Moore, G. N. J . Chem. Soc., Perkin Trans. 1 1985, 461-
470.
(49) Arhart, R. J .; Martin, C. J . J . Am. Chem. Soc. 1972, 94, 5003;
Tetrahedron Lett. 1992, 33, 1561.
(50) Srinivasan, A.; Stephenson, R. W.; Olsen, R. K. J . Org. Chem.
1977, 42, 2256.
(51) Prokof’ev, E. P.; Karpeiskaya, E. I. Tetrahedron Lett. 1979, 20,
737.
(52) Braun, S. Org. Magn. Reson. 1978, 11, 197.
(56) Castra, B.; Dormoy, J . R.; Evin, G.; Selve, C. Tetrahedron Lett.
1975, 1219-1222.
(57) Dory, Y. L.; Mellor, J . M.; McAleer, J . F. Tetrahedron 1996,
52, 1343-1360.
(58) The use of HCl/ether in this deprotection resulted in problems
similar to the ones observed for compound 7, most likely as a result of
N,O-transacylation.

Total Synthesis of Motuporin and 5-[L-Ala]-Motuporin
J . Org. Chem., Vol. 64, No. 8, 1999 2717
Sch em e 9
Sch em e 11
Sch em e 12
Sch em e 10
the macrocycle in a pleasing 79% yield. In contrast, use
of diphenylphosphoryl azide62 instead of FDPP only
proceeded to a meager 26% yield. Alternatively, treat-
ment of 5b with pentafluorophenol and DCC converted
the C-terminus to its pentafluorophenyl ester. When this
ester was treated with TFA/CH2Cl2 to remove the Boc
group, then dissolved in CH2Cl2, and added slowly to a
solution of NMM in the same solvent, macrocycle 39 was
obtained in approximately 55% yield over the three steps.
This approach provided a sample of the macrocycle that
was slightly cleaner than the one obtained using FDPP
on a single occasion, but unfortunately this result was
not reproducible.
Macrocyclization of the mdhb-containing pentapeptide
38 was anticipated to proceed more readily than cycliza-
tion of the threonine-containing peptide because of the
additional conformational constraint imposed by the
presence of the mdhb double bond. In the event, cycliza-
tion was achieved under high dilution conditions (1 mM)
via the pentafluorophenyl ester as shown in Scheme 13,
but a 4:1 mixture of two similar products was obtained.
Chromatographic separation of this mixture, followed by
NMR and MS analysis, resulted in assignment of the
minor component of this mixture as motuporin dimethyl
ester (40) on the basis of its spectral similarity to material
residue late in the synthesis presented some difficulties
and appeared to contribute to a loss of purity due to side
reactions (see below). Thus, deprotection of the C-
terminus of dipeptide 29 using TBAF, followed by con-
densation with fragment 33 using HATU, provided
tetrapeptide 37 in a modest 47% yield. The low yield for
this step is not surprising given the known difficulty of
coupling to R,â-unsaturated acids.59 Following removal
of the Boc group in TFA/CH2Cl2, the tetrapeptide was
coupled with N-Boc-Adda pentafluorophenyl ester 35 in
the presence of diisopropylethylamine to form the linear
pentapeptide 38 in 53% yield (Scheme 11).
Ma cr ocycliza tion Stu d ies. Cyclization of pentapep-
tide 5 was investigated using a variety of reagents to
ascertain the best conditions for formation of the mac-
rocycle (Scheme 12). Cleavage of the allyl ester in 5a was
achieved in 93% yield using Pd(PPh3)4 and dimedone to
give 5b, and acidolytic removal of the Boc group followed
using TFA/CH2Cl2 to provide 5c. Cyclization in the
presence of pentafluorophenyl diphenylphosphinate (FD-
PP)60,61 proceeded smoothly and consistently to provide
(59) Bernasconi, S.; Comini, A.; Corbella, A.; Gariboldi, P.; Sisti, M.
Synthesis 1980, 385-387.
(60) Chen, S.; Xu, J . Tetrahedron Lett. 1991, 32, 6711.
(61) Dudash, J ., J r.; J iang, J .; Mayer, S. C.; J oullie, M. M. Synth.
Commun. 1993, 23, 349.
(62) Shioiri, T.; Ninomiya, K.; Yamada, S.-I. J . Am. Chem. Soc. 1972,
94, 6203-6205

2718 J . Org. Chem., Vol. 64, No. 8, 1999
Samy et al.
Sch em e 13
to the production of side products, lowering the overall
yield of motuporin and making the final purification
difficult. Therefore, we decided to adopt an alternative
strategy for the synthesis of motuporin in which the
mdhb double bond was introduced prior to the macro-
cylization step. We anticipated that the shorter reaction
time that would be necessary for the final step in this
synthesis would lead to a cleaner final product.
These considerations led to the preparation of mdhb-
containing macrocycle 40. Unfortunately, saponification
of this compound gave rise to even more side products
than before, making purification by RP-HPLC, prepara-
tive TLC, or ion-exchange chromatography especially
difficult. Deprotection of macrocycle 40 by SN2-type
dealkylation65,66 of the two methyl esters using either NaI
or KOTMS67 gave a crude mixture that appeared to
contain a product of the correct mass but was unlikely
to readily yield pure motuporin.
prepared by Anderson from the natural product.1 The
major component was tentatively assigned as a diaste-
reomer of 40 resulting from epimerization at the valine
R-carbon.
Some comments on the purification of motuporin are
warranted. As a diacid, this cyclic peptide is particularly
difficult to purify using standard methods. Reverse-phase
HPLC using methanol-aqeuous TFA mixtures provided
samples that eluted as a single peak, but invariably these
samples were found to contain multiple components when
analyzed under different solvent conditions. We have
found that the most reliable and informative conditions
for analyzing the purity of motuporin samples are 60:40
MeOH/0.7% Na2SO4(aq). Unfortunately, the large amount
of Na2SO4 that is obtained following evaporation of
product-containing fractions when these conditions are
used for preparative purposes makes extraction of the
pure peptide cumbersome. Other techniques such as ion-
exchange chromatography and preparative TLC were
found not to possess sufficient resolution to separate the
similar components (probably diastereomers) of our
product mixture.
In summary, several routes to motuporin have been
explored, but none of these routes is entirely satisfactory.
The dimethyl ester of motuporin was prepared cleanly
and in good overall yield, but the final ester cleavage step
gave complex product mixtures from which we were
unable to extract pure motuporin. The best sample of
motuporin that was obtained displays general spectro-
scopic similarity to authentic motuporin by NMR, but it
is clearly not homogeneous and exhibits an optical
rotation that is significantly lower than that observed for
the natural product.
In summary, both FDPP and the pentafluorophenyl
ester route provide good yields of the threonine-contain-
ing macrocycle 39, with the latter approach giving a
cleaner product. In contrast, cyclization of the dehy-
drobutyrine-containing peptide 38 proceeds primarily
with epimerization and gives the desired macrocycle 40
only as a minor product.
Sa p on ifica tion of th e Meth yl Ester s: Syn th esis
of Motu p or in . To prepare motuporin from macrocycle
39, it was necessary to dehydrate the threonine residue
and saponify the two methyl esters. Initially, we proposed
to perform the dehydration in a separate step prior to
saponification, because we expected that this route would
permit milder conditions and a shorter reaction time to
be employed in the final step, possibly minimizing the
formation of side products. However, model studies with
compound 28 indicated that this dehydration would be
difficult to achieve, and an attempt to dehydrate macro-
cycle 39 using the Martin sulfurane was unsuccessful.63
Consequently, because we only had small quantities of
material available, we elected to exploit the chemistry
of Valentekovich and Schreiber and perform the dehy-
dration concomitantly with the saponification.31 Thus,
dimethyl ester 39 was treated LiOH in aqueous metha-
nol,32,64 and the product was purified by HPLC using a
mixture of methanol and 0.7% Na2SO4(aq). These condi-
tions provided a sample of motuporin disodium salt that
was ∼95% pure by HPLC and that coeluted with an
authentic sample of motuporin provided by Prof. Ray-
mond Anderson. The FABMS of this sample matches that
Syn th esis of 5-[L-Ala ]-Motu p or in . As part of our
studies of motuporin, we were interested in developing
a convergent approach that would be amenable to the
preparation of nodularin and microcystin analogues68-73
for studying the structure-function relationships for
inhibition of protein phosphatases by these molecules.
1
expected for motuporin disodium salt, and the H NMR
spectrum is consistent with previously published data but
is contaminated by small amounts of aliphatic impurities.
The final step in this synthesis is difficult to follow.
Saponification of the two methyl esters appears to
proceed rapidly; however, the dehydration reaction is
slower, and previous workers have indicated that the
double bond equilibrates under the reaction conditions,
requiring a longer reaction time to obtain a single double
bond isomer. We reasoned that leaving the saponified
cyclic peptide under basic conditions for longer than
necessary to cleave the ester functionalities was leading
(65) McMurry, J . E.; Wong, G. B. Synth. Commun. 1972, 2, 389.
(66) McMurry, J . Org. React. 1976, 24, 187.
(67) Laganis, E. D.; Chenard, B. L. Tetrahedron Lett. 1984, 25, 5831.
(68) Taylor, C.; Quinn, R. J . Bioorg. Med. Chem. Lett. 1996, 6, 2107-
2112.
(69) Mehrota, A. P.; Gani, D. Tetrahedron Lett. 1996, 37, 6915-
6918.
(70) Webster, K. L.; Rutherford, T. J .; Gani, D. Tetrahedron Lett.
1997, 38, 5713-5716.
(71) Taylor, C.; Quinn, R. J . Bioorg. Med. Chem. Lett. 1996, 6, 2113-
2116.
(72) Mehrota, A. P.; Webster, K. L.; Gani, D. J . Chem. Soc., Perkin
Trans. 1 1997, 2495-2511.
(73) Maude, A. B.; Mehrota, A. P.; Gani, D. J . Chem. Soc., Perkin.
Trans. 1 1997, 2513-2526.
(63) Previous workers reported that they failed to achieve the
selective dehydration of a D-threonine residue at the same position,
see ref 43.
(64) Kaestle, K. L.; Anwer, M. K.; Audhya, T. K.; Goldstein, G.
Tetrahedron Lett. 1991, 32, 327-330.

Total Synthesis of Motuporin and 5-[L-Ala]-Motuporin
J . Org. Chem., Vol. 64, No. 8, 1999 2719
Sch em e 14
F igu r e 4.
It has been shown that, in microcystins, Adda and the
acidic residues â-methylaspartate and glutamate par-
ticipate in noncovalent bonding interactions with PP1,18,74
and that the mdha residue is required for secondary
covalent attachment to the protein.75 In contrast, the
mdhb residue in motuporin does not bind covalently to
PP1.20,76 Structural studies have indicated that the mdhb
side chain in motuporin is turned downward and shifted
away from the critical cysteine-273 residue, whereas the
mdha side chain in microcystins binds in close proximity
to the thiol and undergoes covalent attachment via a
conjugate addition reaction.20 As may be expected by
comparison to motuporin and nodularin, the ability to
covalently modify PP1 is not essential for inhibition of
phosphatase activity by microcystins or for microcystin
toxicity.73,77 Sodium borohydride mediated reduction of
the mdha residue in microcystin-LR gave a mixture of
diastereomeric dihydromicrocystins, each of which is a
nanomolar inhibitor of PP1.73 These reports suggest that
modified nodularins that lack the dehydrobutyrine double
bond also should be potent PP1 inhibitors. Further
comparison of the mdha-containing microcystins and
mdhb-containing nodularins indicates that the vinylic
methyl group in nodularins also may be unnecessary,
although the location of this carbon in the complex with
PP1 is known to differ between the two ring systems. To
investigate these ideas, we decided to prepare 5-[L-Ala]-
motuporin (41, Figure 4), in which the mdhb residue is
replaced by L-alanine. This particular analogue was
selected because L-alanine is readily available and in-
corporates the two modifications to the nodularin struc-
ture that were of interest, namely reduction of the double
bond and removal of the methyl group that differs
between the nodularin and microcystin ring systems.
5-[L-Ala]-Motuporin is only available by total synthesis.
Our approach to the total synthesis of 5-[L-Ala]-
motuporin followed that for motuporin, differing only in
the replacement of glutamyl-N-methylthreonine frag-
ment 7 by a glutamyl-N-methylalanine-containing dipep-
tide. Thus, commercially available N-Boc-L-alanine was
converted to N-Boc-N-Me-L-alanine in 90% yield under
the conditions of Benoiton.44 The carboxyl group was
protected as a trimethylsilyl ethyl ester to give N-Boc-
N-Me-L-alanine trimethylsilyl ethyl ester 42 in 89% yield
(Scheme 14). Removal of the Boc group with TFA gave
the corresponding amine TFA salt, which was coupled
Sch em e 15
with N-Boc-D-glutamic acid-R-methyl ester using HATU
to give dipeptide 43 in 72% yield. Removal of the Tse
ester using TBAF and condensation of the resulting acid
with fragment 33 in the presence of HATU gave tet-
rapeptide 44 in 86% yield (Scheme 15). Then, removal of
the Boc group with TFA followed by treatment with
N-Boc-Adda pentafluorophenyl ester (35) in the presence
of diisopropyl ethylamine provided the pentapeptide 45
in 81% yield (Scheme 16). As for motuporin, deprotection
of the linear peptide and macrocyclization was achieved
via the C-terminal pentafluorophenyl ester under high
dilution conditions to form the macrocycle 46 in a
combined 27% yield for the four steps (Scheme 17). A
minor cyclization product (18%) also was isolated and
tentatively assigned as the valine R-carbon epimer (47).
The structural assignments for these two molecules were
made on the basis of their relative yields and spectral
similarities between compound 46 and motuporin dim-
ethyl ester 39. Saponification of 46, followed by HLPC
purification, gave a product that displays NMR and mass
spectral data consistent with 5-[L-Ala]-motuporin (41).
For comparison, saponification of compound 47 was
performed in a similar manner, to provide a product
tentatively assigned as structure 48.
(74) Bagu, J . R.; Sykes, B. D.; Craig, M. M.; Holmes, C. F. B. J .
Biol. Chem. 1997, 272, 5087.
En zym e Assa ys. Synthetic motuporin (1) and the two
5-[L-Ala]-motuporin isomers (41 and 48) were evaluated
as inhibitors of protein phosphatase-1 (PP1) activity in
rat adipocyte lysates78 using microcystin-LR as a refer-
ence. Under the assay conditions employed, microcystin-
(75) Runnegar, M.; Berndt, N.; Kong, S.-M.; Lee, E. Y. C.; Zhang,
L. Biochem. Biophys. Res. Commun. 1995, 216, 162-169.
(76) Annila, A.; Lehtimaki, J .; Mattila, K.; Eriksson, J . E.; Sivonen,
K.; Rantala, T. T.; Drakenberg, T. J . Biol. Chem. 1996, 271, 16695.
(77) Namikoshi, M.; Wook, B.; Sun, F.; Rinehart, K. L.; Evans, W.
R.; Carmicheal, W. W. Chem. Res. Toxicol. 1993, 6, 151-158.

2720 J . Org. Chem., Vol. 64, No. 8, 1999
Samy et al.
Sch em e 16
PP1 activity than motuporin itself, with IC50 ) 18.4 nM;
nonetheless, the high level of activity shown by 5-[L-Ala]-
motuporin indicates that the N-methyldehydrobutyrine
residue in motuporin is not essential for the inhibition
of PP1. Compound 48 is a marginally weaker inhibitor
of PP1 than 5-[L-Ala]-motuporin (41), with IC50 ) 37.8
nM. Because compounds 41 and 48 differ in potency by
only a factor of 2, these data do not provide any further
insight into the structures of these molecules. However,
the observation that both 41 and 48 are potent inhibitors
of PP1 bolsters our conclusion regarding the contribution
of the N-methyldehydrobutyrine residue to PP1 inhibi-
tion and indicates that diastereomers of motuporin (and
motuporin analogues) also may be effective PP1 inhibi-
tors.
Con clu sion s
In conclusion, none of the above approaches to motu-
porin are entirely satisfactory. In each case, a major
hurdle is presented by the purification of the final product
following saponification of the methyl esters, which
appears to occur with multiple side reactions. Small
samples of motuporin have been obtained, although none
with greater than approximately 95% purity. Notably,
saponification of macrocycle 39 containing an L-threonine
residue appears to be significantly less satisfactory than
saponification of the equivalent macrocycle containing a
D-threonine residue as prepared by Valentkovich and
Schreiber. 5-[L-Ala]-Motuporin has been prepared from
its dimethyl ester, and the yields for all except the final
two steps are generally better than for motuporin.
However, the cyclization gives a mixture of two products,
and the final saponification is not a clean reaction,
resulting in a difficult purification. Synthetic 5-[L-Ala]-
motuporin is a potent inhibitor of PP1, indicating that
the replacement of N-methyldehydrobutyrine by N-
methylalanine is not severely detrimental to the activity
of nodularin and motuporin cyclic peptides. We suggest
that future approaches to motuporin and nodularin cyclic
peptides should avoid synthesis of the methyl esters and
instead rely on acid-labile carboxyl protecting groups
such as tert-butyl esters. Moreover, our results indicate
that motuporin analogues lacking the dehydroamino acid
should retain inhibitory activity against protein phos-
phatases.
Sch em e 17
Exp er im en ta l Section
LR inhibits PP1 activity with IC50 ) 0.25 nM. Synthetic
motuporin displays an IC50 ) 4.0 nM, compared to
literature values of IC50 < 1 nM vs PP1 for the natural
product1 and IC50 ) 1.1 nM vs PP2A for synthetic
motuporin prepared by Valentkovich and Schreiber.31
The small discrepancy in these values probably reflects
differences in enzyme source and the assay conditions
employed. Whereas rat adipocyte lysates were used in
our experiments, previous workers have employed the
purified PP1 catalytic subunit isolated from rabbit skel-
etal muscle.79
Microcystin-LR was obtained from Calbiochem. All other
reagents used were of the highest purity available from
Aldrich. Solvents were HPLC grade. Anhydrous THF was
distilled from sodium/benzophenone. Dichloromethane and
acetonitrile were distilled from calcium hydride. Triethylamine
and diisopropylethylamine were distilled and stored over 4 Å
molecular sieves. NMR spectra are reported for solutions in
CDCl3 unless otherwise indicated. Residual chloroform was
used as a reference and set at 7.26 ppm. Optical rotations were
recorded at ambient temperature (23 °C).
(2R,3S)-3-Hyd r oxy-2-m et h yl-4-p h en ylbu t a n -(N-m et h -
yl-O-m eth yl)-h yd r oxa m id e. To the hydrochloride salt of
N,O-dimethyl hydroxylamine (1.3 g, 13.34 mmol, 2.2 equiv)
in CH2Cl2 (10 mL) under N2 at -20 °C was added trimethyl-
aluminum (2 M in CH2Cl2, 6.67 mL, 13.34 mmol, 2.2 equiv).
After 5 min, a solution of 15 (2.14 g, 6.06 mmol) in CH2Cl2 (20
mL + 2 × 4 mL rinses) precooled to -60 °C was added via a
cannula. The mixture was allowed to warm to 0-4 °C and
5-[L-Ala]-Motuporin is a slightly weaker inhibitor of
(78) Brady, M. J .; Nairn, A. C.; Saltiel, A. R. J . Biol. Chem. 1997,
47, 29698-29703.
(79) Craig, M.; Mcready, T. L.; Luu, H. A.; Smillie, M. A.; Dubord,
P.; Holmes, C. F. B. Toxicon 1993, 31, 1541-1549.

Total Synthesis of Motuporin and 5-[L-Ala]-Motuporin
J . Org. Chem., Vol. 64, No. 8, 1999 2721
stirred for 3 h. Upon completion of the reaction, the CH2Cl2
solution was poured into a mixture of ice and saturated sodium
bicarbonate solution, and the aqueous layer was extracted with
copious CH2Cl2. The combined organic extracts were washed
with H2O and then with brine and dried over anhydrous
MgSO4. Chromatography on SiO2 (30-50% ethyl acetate in
hexanes) provided the product as a yellow oil (1.12 g, 4.72
mmol, 78%): Rf ) 0.25 (1:1, ethyl acetate/hexanes); [R]D -14.1°
was quenched at 0 °C by the addition of 10% EtOH in THF
(10 mL). The solution was concentrated to half volume, then
supplemented with hexanes, and filtered to remove the tri-
phenyl phosphine oxide. The filtrate was washed with water
and then brine and dried over anhydrous magnesium sulfate.
Column chromatography on SiO2 (2-10% ethyl acetate in
hexanes) provided the product as a yellow oil (690 mg, 3.12
mmol, 80%): Rf ) 0.39 (1:19, ethyl acetate/hexanes); [R]D -2.6°
(c 2.0, CHCl3); IR (film) 3028, 2968, 2928, 2871, 2825, 1496,
1453, cm-1; 1H NMR (360 MHz) δ 7.18-7.32 (5H, m, aromatic),
5.07 (1H, d, J ) 8 Hz, H-3), 3.25 (3H, s, OCH3), 3.14-3.19
(1H, m, H-5), 2.86 (1H, dd, J ) 4, 14 Hz, H-6), 2.68 (1H, dd, J
) 11, 14 Hz, H-6), 2.48-2.54 (1H, m, H-4), 1.73 (3H, s, vinylic
CH3), 1.56 (3H, s, vinylic CH3), 1.02 (3H, d, J ) 7 Hz, CH3);
13C NMR (90 MHz) δ 139.8, 131.3, 129.4, 128.0, 127.7, 125.8,
87.4, 58.5, 38.2, 36.6, 25.9, 17.9, 16.7; MS m/z (rel int) 218
(0.6, M+), 135 (100); HRMS (EI) calcd for C15H22O 218.1671,
found 218.1668.
(2E,4S,5S)-5-Meth oxy-2,4-d im eth yl-6-p h en ylh ex-2-en -
1-ol (18). A solution of (2E,2S,3S)-5-methoxy-2,3-dimethyl-6-
phenylhex-2-ene (690 mg, 3.17 mmol) and SeO2 (702 mg, 6.33
mmol, 2 equiv) in EtOH (12 mL) was heated under reflux for
2 h. Additional SeO2 (350 mg, 3.17 mmol, 1 equiv) was added,
and heating was continued for an additional 5 h. The mixture
was allowed to cool and then filtered through a glass wool plug
to remove the black precipitate that had formed. The filtrate
was cooled to 0 °C and CeCl3‚7H2O (1.18 g, 3.17 mmol, 1 equiv)
was added, creating a red solution. NaBH4 (360 mg, 9.51 mmol,
3 equiv) was added portionwise, causing vigorous efferves-
cence. This mixture was stirred for 30 min at 0 °C, diluted
with EtOAc, and washed with saturated aqueous NaHCO3
solution and then brine. The organic layer was dried over
anhydrous MgSO4, filtered, and evaporated to give a yellow
oil (520 mg, 2.22 mmol, 70%): Rf ) 0.21 (1:1, ethyl acetate:
hexanes); [R]D -3.8° (c 1.0, EtOH); IR (film) 3425, 2827, 1496
cm-1; 1H NMR (360 MHz) δ 7.14-7.30 (5H, m, aromatic), 5.32
(1H, d, J ) 11 Hz, H-3), 3.99 (2H, s, H-1), 3.25 (3H, s, OCH3),
3.17-3.29 (1H, m, H-5), 2.80 (1H, dd, J ) 4, 14 Hz, H-6), 2.70
(1H, dd, J ) 7, 14 Hz, H-6), 2.50-2.61 (1H, m, H-4), 1.57 (3H,
s, vinylic CH3), 1.02 (3H, d, J ) 7 Hz, CH3); 13C NMR (90 MHz)
δ 139.6, 134.7, 129.4, 128.6, 128.1, 125.9, 87.0, 68.8, 58.5, 38.1,
36.0, 16.4, 13.8; MS m/z (rel int) 252 (17, M + NH4+), 185 (100),
135 (92), 120 (20), 91 (23); HRMS (CI) calcd for C15H26NO2
252.1963, found 252.1968.
(c 1, EtOH); IR (film) 3419, 2938, 1653, 1636, 1495, 1456, cm-1
;
1H NMR (360 MHz) δ 7.21-7.39 (5H, m, aromatic), 4.13 (1H,
ddd, J ) 4, 7, 10 Hz, H-3), 3.7 (1H, br s, OH), 3.49 (3H, s,
OCH3), 3.17 (3H, s, NCH3), 2.91 (1H, dd, J ) 8, 14 Hz, H-4),
2.90 (1H, br s, H-2), 2.74 (1H, dd, J ) 7, 14 Hz, H-4), 1.27
(3H, d, J ) 7 Hz, CH3); 13C NMR (90 MHz) δ 178, 138.3, 129.1,
128.3, 126.2, 72.82, 61.1, 40.0, 37.3, 31.7, 10.0; MS m/z (rel
int) 238 (100, M + 1); HRMS (CI) calcd for C13H20NO3 (M +
H) 238.1443, found 238.1426. Anal. Calcd for C13H19NO3: C,
65.80; H, 8.07; N, 5.89. Found: C, 64.79; H, 8.06; N, 5.75. The
chiral auxiliary (4R,5S)-(-)-4-methyl-5-phenyl-2-oxazolidinone
also was recovered from this reaction; it elutes from the silica
gel column after the hydroxamide product.
(2R,3S)-3-Met h oxy-2-m et h yl-4-p h en ylbu t a n -(N-m et h -
yl-O-m eth yl)-h yd r oxa m id e (16). To a solution of 3-hydroxy-
2-methyl-4-phenylbutan-(N-methyl-O-methyl)-hydroxamide (1.1
g, 4.64 mmol) in dry THF (20 mL) at 0 °C was added sodium
hydride (60% in oil, 186 mg, 4.64 mmol, 1 equiv) in one portion.
Methyl iodide (2.9 mL, 46.4 mmol, 10 equiv) was filtered into
the reaction mixture through a pad of sodium carbonate. After
48 h of stirring at room temperature, the reaction mixture was
poured into ice-cold saturated NH4Cl solution, and the organic
components were extracted into ethyl acetate. The combined
organic extracts were washed with brine and dried over
anhydrous MgSO4. Column chromatography on SiO2 (40%
ethyl acetate in hexanes) provided the title compound as a
yellow oil (890 mg, 3.55 mmol, 76%): Rf ) 0.38 (1:1, ethyl
acetate/hexanes); [R]D -5.5° (c 1.0, CHCl3); IR (film) 2974,
1
2937, 1660, 1455 cm-1; H NMR (300 MHz) δ 7.17-7.30 (5H,
m, aromatic), 3.61, (1H, ddd, J ) 3, 9, 9 Hz, H-3), 3.46 (3H, s,
OCH3), 3.25 (3H, s, OCH3), 3.15, (3H, s, NCH3), 2.85-2.91 (2H,
m, H-4, H-2), 2.74 (1H, dd, J ) 8, 15 Hz, H-4), 1.22 (3H, d, J
) 9 Hz, CH3); 13C NMR (75 MHz) δ 139.1, 129.6, 128.1, 126.0,
84.2, 61.1, 58.9, 40.2, 39.0, 32.5, 13.6; MS m/z (rel int) 252
(26, M + H), 223 (24), 222 (100), 190 (20). Anal. Calcd for
C
14H21NO3: C, 66.91; H, 8.42; N, 5.57. Found: C, 66.75; H,
8.46; N, 5.56.
(2R)-3-P en tyn -2-yl N-Boc-Glycin a te. To a solution of
(2R)-3-pentyn-2-ol (1.5 g, 17.9 mmol) in CH2Cl2 (80 mL) was
added N-Boc glycine (2.84 g, 16.2 mmol), DCC (3.69 g, 17.9
mmol), and DMAP (199 mg, 1.62 mmol), and the reaction
mixture was stirred at room temperature for 3 h. The solid
dicyclohexylurea was removed by filtration and washed with
CH2Cl2 (2 × 50 mL). The filtrate was washed with 1 M HCl,
saturated aqueous sodium bicarbonate, and brine. The organic
layer was dried (MgSO4), concentrated, and purified by chro-
matography on SiO2 (8:1, hexane/ethyl acetate) to provide the
title compound as a colorless oil (3.57 g, 91%): Rf ) 0.26 (4:1,
hexane/ethyl acetate); [R]D +69.1° (c 3.49, CHCl3); IR (film)
3348, 2980, 2938, 2255, 1757, 1515 cm-1; 1H NMR (300 MHz)
δ 1.40 (9H, s), 1.49 (3H, s), 1.84 (3H, d, J ) 2.0 Hz), 3.93 (2H,
d, J ) 4.8 Hz), 5.00 (1H, br s), 5.46-5.50 (1H, m); 13C NMR
(75 MHz) δ 21.5, 28.2 (3C), 42.6, 61.7, 76.6, 77.2, 79.7, 81.3,
155.3, 169.0; HRMS calcd for C12H19NO4 [M + H]+ 242.1392,
found 242.1394. Anal. Calcd for C12H19O4N: C, 59.74; H, 7.94;
N, 5.81. Found: C, 59.45; H, 7.87; N, 5.87.
Z-(2R)-3-P en ten -2-yl N-Boc-Glycin a te (12). To a solution
of (2R)-3-pentyn-2-yl N-Boc-glycinate (3.48 g, 14.4 mmol) in
ethyl acetate (100 mL) was added Pd/BaSO4 (348 mg) and
quinoline (348 mg), and the reaction mixture was hydroge-
nated at atmospheric pressure for 1.5 h. The catalyst was
removed by filtration through a pad of Celite and rinsed with
ethyl acetate (2 × 50 mL). The filtrates were concentrated and
purified by chromatography on SiO2 (8:1, hexane/ethyl acetate)
to give 12 as a colorless oil (3.42 g, 97%): Rf ) 0.33 (4:1,
hexane/ethyl acetate); [R]D -13.6° (c 1.94, CHCl3); IR (film)
3388, 2981, 2931, 1754, 1715, 1511 cm-1; 1H NMR (300 MHz)
δ 1.24 (3H, d, J ) 5.9 Hz), 1.40 (9H, s), 1.73 (3H, d, J ) 1.3
(2E ,2S ,3S )-5-Me t h oxy-2,3-d im e t h yl-6-p h e n ylh e x-2-
en e (17). To a solution of (2R,3S)-3-methoxy-2-methyl-4-
phenylbutan-(N-methyl-O-methyl)-hydroxamide (1 g, 3.98 mmol)
in dry THF (40 mL) at 0 °C under nitrogen was added lithium
aluminun hydride (151 mg, 3.98 mmol, 1 equiv) in portions.
This mixture was stirred at room temperature for 1 h and then
recooled to 0 °C. Acetone (5 mL) was added and the stirring
continued for 10 min. The whole mixture was then poured into
an ice-cold mixture of 1:1 0.1 M citric acid (aqueous) and
hexanes and stirred vigorously at 0 °C for 30 min. The aqueous
and organic layers were separated, and the organic layer was
washed with brine and then dried over anhydrous sodium
sulfate. Filtration and evaporation gave (2R,3S)-3-m eth oxy-
2-m eth yl-4-p h en ylbu ta n a l as a colorless oil: Rf ) 0.67 (2:3,
ethyl acetate/hexanes); 1H NMR (300 MHz) δ 9.65 (1H, d, J )
1 Hz, CHO), 7.17-7.29 (5H, m, aromatic), 3.86 (1H, ddd, J )
3, 6, 6 Hz, H-3), 3.27 (3H, s, OCH3), 2.96 (1H, dd, J ) 6, 14
Hz, H-4), 2.69 (1H, dd, J ) 8, 14 Hz, H-4), 2.13-2.39 (1H, m,
H-2), 1.18 (3H, d, J ) 9 Hz, CH3). This aldehyde was used
without further purification. In a separate flask, isopropyl-
triphenylphosphonium iodide (1.72 g, 1 equiv) was suspended
in dry THF (15 mL) under nitrogen and treated with nBuLi
(2.49 mL, 1.6 M, 1 equiv) at -10 °C. After 30 min, a red-brown
solution was obtained. This solution was cooled to -78 °C and
added dropwise via a cannula to a solution of the aldehyde in
dry THF (15 mL) under nitrogen at -78 °C. The color of the
ylid initially dissipated upon addition to the aldehyde but
eventually was retained. The resulting mixture was stirred
at -78 °C for 1 h, removed to a -10 °C cold bath, and allowed
to warm slowly to room temperature overnight. The reaction

2722 J . Org. Chem., Vol. 64, No. 8, 1999
Samy et al.
Hz), 3.88 (2H, t, J ) 5.2 Hz), 5.00 (1H, br s), 5.34-5.41 (1H,
m), 5.57-5.63 (1H, m), 5.71-5.76 (1H, m); 13C NMR (75 MHz)
δ 13.0, 20.3, 28.1 (3C), 42.5, 67.9, 79.4, 127.5, 129.6, 155.4,
169.2; HRMS calcd for C12H21O4N [M + H]+ 244.1549, found
244.1541. Anal. Calcd for C12H21O4N: C, 59.24; H, 8.70.
Found: C, 59.20; H, 8.52; N, 5.80.
(4:1, hexane/ethyl acetate); [R]D -19.5° (c 0.74, CHCl3); IR
1
(film) 3416 (br), 2095, 1778, 1645, 1518 cm-1; H NMR (360
MHz) δ 1.37 (3H, d, J ) 7.1 Hz), 1.46 (9H, s), 3.56-3.60 (1H,
m), 3.76 (3H, s), 4.79 (1H, dd, J ) 3.4, 8.3 Hz), 5.40 (1H, d, J
) 8.3 Hz); 13C NMR (90 MHz) δ 12.7, 28.2 (3C), 41.5, 52.6,
55.0, 80.4, 136.4, 138.1, 139.2, 139.5, 140.9, 142.3, 155.6, 169.8,
170.5; HRMS calcd for C17H18O6NF5 [M + H]+ 428.1135, found
428.1141.
E-(2S,3R)-2-(N-Boc-a m in o)-3-m et h yl-4-h exen oic Acid
(13). nBuLi (19.20 mL, 30.7 mmol, 1.6 M in hexane) was added
to a solution of diisopropylamine (5.10 mL, 36.3 mmol) in THF
(30 mL) at -78 °C under N2 and stirred at this temperature
for 20 min. Compound 12 (3.40 g, 13.9 mmol) in THF (20 mL)
was added via a cannula. After 5 min, a solution of ZnCl2 (2.28
g, 16.7 mmol) in THF (20 mL) was added, and the reaction
mixture was allowed to warm slowly to room temperature
overnight. The yellow reaction mixture was treated with 1 M
HCl (5 mL) and concentrated under vacuum to give an oily
residue, which was dissolved in ether and washed with 1 M
HCl. The ether layer was extracted with 1 M NaOH. The
combined aqueous extracts were acidified with 1 M HCl and
then back-extracted with ether. The ether extracts were dried
(MgSO4) and concentrated to provide 13 as a colorless oil (3.04
g, 89%): [R]D +5.8° (c 0.6, CHCl3); IR (film) 3423, 2981, 2931,
Dip ep tid e (8). To a solution of the activated ester 26b (427
mg, 1 mmol) in CH2Cl2 (20 mL) containing DMAP (140 mg,
1.14 mmol) was added a solution containing the TFA salt of
L-valine allyl ester (310 mg, 1.14 mmol) and diisopropylethyl-
amine (0.55 mL, 3.12 mmol) in CH2Cl2 (5 mL). After 48 h of
stirring at room temperature, the reaction mixture was diluted
with CH2Cl2 and washed with 10% aqueous citric acid solution,
saturated aqueous sodium bicarbonate, and brine. The organic
extracts were dried (MgSO4), concentrated, and purified by
chromatography on SiO2 (2:1, hexane/ethyl acetate) to give 8
as a white solid (320 mg, 80%): Rf ) 0.40 (1:1, hexane/ethyl
acetate); [R]D -5.5° (c 0.64, CHCl3); IR (film) 3533, 2971, 2933,
1748, 1716, 1658, 1499 cm-1; 1H NMR (300 MHz) δ 0.83 (3H,
d, J ) 6.9 Hz), 0.86 (3H, d, J ) 6.9 Hz), 1.22 (3H, d, J ) 7.2
Hz), 1.36 (9H, s), 2.07-2.14 (1H, m), 3.08-3.12 (1H, m), 3.64
(3H, s), 4.33 (1H, dd, J ) 3.7, 9.6 Hz), 4.43 (1H, dd, J ) 4.9,
8.5 Hz), 4.54 (2H, t, J ) 7.4 Hz), 5.18 (1H, d, J ) 10.4 Hz),
5.25 (1H, d, J ) 13.4 Hz), 5.76-5.89 (2H, m), 6.45 (1H, d, J )
8.6 Hz); 13C NMR (75 MHz) δ 15.0, 17.6, 18.8, 28.2 (3C), 31.0,
41.6, 52.2, 56.1, 57.0, 65.6, 79.5, 118.6, 131.4, 155.9, 171.1,
171.6, 173.4; HRMS calcd for C19H32O7N2 [M + H]+ 401.2288,
found 401.2284. Anal. Calcd for C19H32O7N2; C, 56.99; H, 8.05;
N, 6.99. Found: C, 57.24; H, 7.98; N, 6.84.
1
1715, 1504, cm-1; H NMR (300 MHz) δ 1.11 (3H, d, J ) 6.9
Hz), 1.46 (9H, s), 1.69 (3H, d, J ) 6.3 Hz), 2.68-2.78 (1H, m),
4.23-4.28 (1H, m), 4.91 (1H, d, J ) 8.3 Hz), 5.28-5.35 (1H,
m), 5.54-5.61 (1H, m); 13C NMR (75 MHz) δ 16.9, 17.9, 28.3
(3C), 39.0, 58.1, 80.2, 127.6, 130.2, 155.8, 176.8; HRMS calcd
for C12H21O4N [M + H]+ 244.1549, found 244.1542.
Meth yl
E-(2S,3R)-2-(N-Boc-Am in o)-3-m eth yl-4-h ex-
en oa te (19). To a solution of acid 13 (1.80 g, 7.4 mmol) in
DMF (40 mL) was added solid NaHCO3 (1.25 g, 14.8 mmol)
and methyl iodide (2.30 mL, 37.0 mmol), and the mixture was
stirred at room temperature for 24 h. Following dilution with
water, the crude product was extracted with ethyl acetate, and
the organic extracts were dried (MgSO4), concentrated, and
purified by chromatography on SiO2 (8:1, hexane/ethyl acetate)
to give the title compound as a colorless oil (1.82 g, 95%): Rf
) 0.36 (4:1, hexane/ethyl acetate); [R]D -6.0° (c 1.74, CHCl3);
(2R,3R)-2-(N-Boc-Am in o)-3-m eth ylh ex-4-en ol. Lithium
aluminum hydride (412 mg, 10.88 mmol) was added portion-
wise to a solution of ester 19 (1.40 g, 5.44 mmol) in ether (75
mL) at 0 °C, and this reaction mixture was allowed to stir at
room temperature for 1.5 h. After recooling to 0 °C, the reaction
was quenched by addition of water (0.5 mL), 1 M NaOH (0.5
mL), and water (0.5 mL). Excess MgSO4 was added to the
reaction mixture, and stirring was continued for 1 h. The solids
were removed by filtration and washed several times with
ether. The combined filtrates were concentrated to obtain the
alcohol as a colorless oil (1.12 g, 90%): Rf ) 0.34 (1:1, hexane/
ethyl acetate); [R]D -0.8° (c 1.3, CHCl3); IR (film) 3416 (br),
IR (film) 3381, 2963, 2925, 1750, 1500 cm-1 1H NMR (300
;
MHz) δ 1.07 (3H, d, J ) 6.6 Hz), 1.45 (9H, s), 1.67 (3H, d, J )
5.9 Hz), 2.67-2.69 (1H, m), 3.74 (3H, s), 4.21-4.23 (1H, m),
4.91 (1H, d, J ) 6.7 Hz), 5.22-5.30 (1H, m), 5.48-5.52 (1H,
m); 13C NMR (75 MHz) δ 16.8, 17.8, 28.2 (3C), 39.3, 51.7, 58.1,
79.5, 127.1, 130.4, 155.4, 172.2; HRMS calcd for C13H23O4N
1
2130, 1634 cm-1; H NMR (300 MHz) δ 0.98 (3H, d, J ) 6.8
[M + H]+ 258.1705, found 258.1698. Anal. Calcd for C13H23
-
Hz), 1.39 (9H, s), 1.62 (3H, d, J ) 6.2 Hz), 2.34-2.38 (1H, m),
3.45-3.60 (3H, m), 4.74 (1H, d, J ) 8.3 Hz), 5.25-5.33 (1H,
m), 5.41-5.49 (1H, m); 13C NMR (75 MHz) δ 17.4, 17.8, 28.3
(3C), 37.9, 57.0, 63.9, 79.3, 126.2, 132.2, 156.5; HRMS calcd
for C12H23O4N [M + H]+ 230.1756, found 230.1748.
O4N: C, 60.68; H, 9.01; N, 5.44. Found: C, 60.63; H, 8.88; N,
5.29.
r-Meth yl (2R,3S)-N-Boc-3-Meth yla sp a r ta te (26a ). To a
solution of alkene 19 (3.0 g, 11.66 mmol) in CCl4 (25 mL), CH3-
CN (25 mL), and H2O (35 mL) was added sodium periodate
(10.0 g, 46.6 mmol) and ruthenium(III) chloride (54 mg, 2.2
mol %). This mixture was stirred at room temperature
overnight. Upon completion of the reaction, the mixture was
diluted with CH2Cl2 (100 mL), and the phases were separated.
The aqueous phase was extracted with CH2Cl2 (2 × 50 mL),
dried (MgSO4), concentrated, and purified by chromatography
on SiO2 (10% methanol in dichloromethane) to yield 26a as a
colorless oil (2.61 g, 86%): [R]D -8.9° (c 1.82, CHCl3); IR (film)
3438, 2095, 1652, 1504 cm-1; 1H NMR (300 MHz) δ 1.29 (3H,
d, J ) 7.2 Hz), 1.46 (9H, s), 3.32 (1H, m), 3.75 (3H, s), 4.54
(1H, dd, J ) 3.2, 9.2 Hz), 5.45 (1H, d, J ) 9.5 Hz); 13C NMR
(75 MHz) δ 13.5, 28.3 (3C), 41.4, 52.4, 55.2, 80.2, 155.9, 171.2,
178.1; HRMS calcd. for C11H19O6N [M + H]+ 262.1290, found
262.1280.
r-Meth yl â-P en taflu or oph en yl (2R,3S)-N-Boc-3-Meth yl-
a sp a r ta te (26b). To a solution of acid 26a (840 mg, 3.21 mmol)
in CH2Cl2 (75 mL) was added DCC (662 mg, 3.21 mmol),
DMAP (79 mg, 0.642 mmol), and pentafluorophenol (1.18 g,
6.42 mmol), and the mixture was stirred at room temperature
overnight. The solid dicyclohexylurea was removed by filtration
and washed with CH2Cl2 (2 × 50 mL). The filtrate was washed
with 1 M HCl, saturated aqueous NaHCO3, 1 M NaOH, and
brine. The organic extracts were dried (MgSO4), concentrated,
and purified by chromatography on SiO2 (8:1, hexane/ethyl
acetate) to give 26b as a white solid (1.16 g, 85%): Rf ) 0.35
(2R,3R)-2-(N-Boc-Am in o)-3-m et h ylh ex-4-en ol
ter t-
Bu tyld im eth ylsilyl eth er (20). To a solution of (2R,3R)-2-
(N-Boc-amino)-3-methylhex-4-enol (950 mg, 4.36 mmol) in
DMF (15 mL) was added imidazole (445 mg, 6.54 mmol) and
TBDMSCl (723 mg, 4.80 mmol). The mixture was allowed to
stir at room temperature for 15 h, diluted with (1:1) benzene/
ethyl acetate (75 mL), and washed with 10% aqueous citric
acid solution and brine. The organic extracts were dried
(MgSO4), concentrated, and purified by chromatography on
SiO2 (3:1, ether/hexane) to obtain the product as a colorless
oil (1.42 g, 96%): Rf ) 0.58 (4:1, hexane/ethyl acetate); [R]D
+13.3° (c 1.28, CHCl3); IR (film) 3416 (br), 2951, 2836, 1708,
1497 cm-1 1H NMR (300 MHz) δ 0.42 (6H, s), 0.91 (9H, s),
;
1.00 (3H, d, J ) 6.9 Hz), 1.43 (9H, s), 1.66 (3H, d, J ) 5.6 Hz),
2.41-2.48 (1H, m), 3.46-3.64 (3H, m), 4.53 (1H, d, J ) 8.1
Hz), 5.31-5.49 (2H, m); 13C NMR (75 MHz) δ -5.4 (2C), 17.4,
17.9, 18.3, 25.9 (3C), 28.3 (3C), 37.4, 55.9, 63.2, 78.9, 125.7,
132.6, 155.7; HRMS calcd for C18H37O3NSi [M + H]+ 344.2621,
found 344.2608.
Meth yl (2S,3R)-3-(N-Boc-Am in o)-4-(ter t-bu tyld im eth -
ylsilyloxy)-2-m eth yl-bu ta n oa te (21). Ozone was bubbled
through a solution of alkene 20 (1.52 g, 4.42 mmol) in CH2Cl2
(80 mL) and 2.5 M methanolic NaOH (8.8 mL) at -78 °C. After
135 min, the yellow reaction mixture acquired the character-
istic blue color of ozone. The reaction mixture was diluted with
ether and water and then extracted with CH2Cl2 (Note:

Total Synthesis of Motuporin and 5-[L-Ala]-Motuporin
J . Org. Chem., Vol. 64, No. 8, 1999 2723
allowing the reaction mixture to warm to room temperature
results in epimerization at C-2). The organic extracts were
washed with brine, dried (MgSO4), and concentrated to obtain
the product ester as a colorless oil (1.44 g, 90%): Rf ) 0.52
(4:1, hexane/ethyl acetate); [R]D +13.6° (c 1.16, CHCl3); IR
(film) 3416.4 (br), 2924, 2854, 1715, 1645, 1490 cm-1; 1H NMR
(300 Hz) δ 0.04 (6H, s), 0.88 (9H, s), 1.23 (3H, d, J ) 6.6 Hz),
1.45 (9H, s), 2.85-2.88 (1H, m), 3.52-3.58 (2H, m), 3.68 (3H,
s), 3.77-3.79 (1H, m), 5.34 (1H, d, J ) 9.5 Hz); 13C NMR (75
MHz) δ -5.6 (2C), 14.9, 18.2, 25.8 (3C), 28.4 (3C), 39.5, 51.5,
54.1, 63.5, 79.1, 155.6, 175.6; HRMS calcd for C17H35O5NSi [M
+ H]+ 362.2363, found 362.2359.
gel chromatography (8:1, hexane/ethyl acetate) gave E,E-9
(43.5 mg, 51%).
N-Boc-Ad d a (6). To N-Boc-Adda methyl ester (102 mg, 0.23
mmol) in THF (6 mL) was added 1 M LiOH (1.15 mL), and
the reaction mixture was stirred at room temperature for 68
h, acidified with 1 M HCl, and extracted with ether. The
organic extracts were dried (MgSO4) and concentrated to
obtain the product acid (97 mg, 98%), which was used without
further purification: [R]D -14.2° (c 1.84, CHCl3); IR (film) 3416
1
(br), 2129, 1645 cm-1; H NMR (300 MHz) δ 1.02 (3H, d, J )
6.5 Hz), 1.25 (3H, d, J ) 5.8 Hz), 1.44 (9H, s), 1.60 (3H, s),
2.55-2.83 (4H, m), 3.12-3.20 (1H, m), 3.23 (3H, s), 4.37 (1H,
br s), 5.27-5.50 (3H, m), 6.20 (1H, d, J ) 15.6 Hz), 7.12-7.26
(5H, m); 13C NMR (75 MHz) δ 12.7, 14.5, 16.2, 28.4 (3C), 36.7,
38.3, 44.1, 54.4, 58.6, 79.5, 87.0, 124.9, 125.9, 128.1, 129.3,
132.4, 135.9, 136.4, 139.3, 155.6, 179.1; HRMS calcd for
Met h yl (2S,3R)-3-(N-Boc-Am in o)-4-oxo-2-m et h ylb u -
ta n oa te (11). NBS (730 mg, 4.10 mmol) was added to
compound 20 (1.35 g, 3.73 mmol) in DMSO (10 mL) and water
(0.4 mL) at 0 °C. The mixture was allowed to stir at 10 °C for
2 h, then diluted with ether, and washed with brine. After
drying (MgSO4), the solution was concentrated and purified
by chromatography on SiO2 (4:1, ether/hexane) to provide a
white solid (770 mg, 84%): Rf ) 0.22 (1:1, hexane/ethyl
acetate); [R]D +2.8° (c 1.62, CHCl3); IR (film) 3416.4 (br), 2369,
1771.1, 1680 cm-1; 1H NMR (360 MHz) δ 1.26 (3H, d, J ) 7.2
Hz), 1.45 (9H, s), 2.86-2.89 (1H, m), 3.67 (2H, d, J ) 5.1 Hz),
3.70 (3H, s), 5.39 (1H, br s); 13C NMR (75 MHz) δ 14.7, 28.3
(3C), 39.8, 51.7, 54.8, 63.9, 79.6, 156.2, 175.6; HRMS calcd for
C
25H37O5N [M + H]+ 432.2750, found 432.2728.
N-Boc-Ad d a P en ta flu or op h en yl Ester (35). To acid 6 (33
mg, 0.0765 mmol) in CH2Cl2 (3 mL) was added pentafluo-
rophenol (43 mg, 0.230 mmol), DCC (24 mg, 0.115 mmol), and
DMAP (2 mg), and the mixture was stirred at room temper-
ature for 2 h. After dilution with CH2Cl2, the solution was
washed with 1 M HCl, saturated aqueous sodium bicarbonate,
and brine, dried (MgSO4), and concentrated. Chromatography
on SiO2 (8:1, hexane/ethyl acetate) gave the pentafluorophenyl
ester as a colorless oil (40 mg, 88%): Rf ) 0.55 (4:1, hexane/
ethyl acetate); [R]D -11.0° (c 1.16, CHCl3); IR (film) 3416 (br),
C
11H21O5N [M + H]+ 248.1498, found 248.1508.
DMSO (107 µL, 1.5 mmol) was added to a solution of oxalyl
1
chloride (375 µL, 0.75 mmol) in CH2Cl2 (2 mL) at -78 °C. After
15 min, the alcohol prepared above (124 mg, 0.5 mmol) in CH2-
Cl2 (5 mL) was added. The resultant cloudy mixture was
allowed to stir at -78 °C for 1 h, triethylamine (279 µL, 2
mmol) was added, and the reaction mixture was allowed to
stir for 45 min at -78 °C. Following dilution with CH2Cl2, the
solution was washed with saturated aqueous NH4Cl, saturated
aqueous NaHCO3, and brine and dried (MgSO4). Evaporation
of the solvent provided aldehyde 11 (123 mg), which was used
without further purification: Rf ) 0.48 (1:1, hexane/ethyl
2095, 1645 cm-1; H NMR (360 MHz) δ 1.03 (3H, d, J ) 6.7
Hz), 1.38 (3H, d, J ) 7.1 Hz), 1.46 (9H, s), 1.62 (3H, s), 2.56-
2.83 (4H, m), 3.16-3.20 (1H, m), 3.24 (3H, s), 4.56 (1H, br s),
5.05 (1H, br s), 5.42-5.52 (2H, m), 6.27 (1H, d, J ) 15.6 Hz),
7.18-7.28 (5H, m); 13C NMR (90 MHz) δ 12.6, 13.8, 13.9, 16.1,
28.3 (3C), 36.7, 38.2, 43.8, 43.9, 54.3, 58.6, 79.8, 86.8, 123.4,
129.5, 132.2, 136.3, 136.4, 147.5, 139.3, 139.6, 139.8, 142.3,
155.2, 170.8; HRMS calcd for C31H36O5NF5 [M + H]+ 598.2592,
found 598.2604.
(2S,3R)-N-Boc-N-m et h yl-O-b en zyl-L-Th r eon in e Tr i-
m eth ylsilyleth yl Ester (27). N-Boc-N-methyl-O-benzyl-L-
threonine (1.18 g, 3.65 mmol) and 4-(dimethylamino)pyridine
(75 mg, 0.37 mmol, 0.15 equiv) were dissolved in dry THF and
cooled to 0 °C under nitrogen. Trimethylsilylethanol (1.05 mL,
7.31 mmol, 2 equiv) was added dropwise, followed by 1,1′-
dicyclohexylcarbodiimide (580 mg, 4.75 mmol, 1.3 equiv) in one
portion. The solution was allowed to warm to room tempera-
ture and stirred for 36 h, during which time a white precipitate
formed. This mixture was filtered; the filtrate was concen-
trated and redissolved in ethyl acetate then washed with
saturated aqueous NaHCO3, saturated aqueous NH4Cl, and
brine; and dried over anhydrous MgSO4. Column chromatog-
raphy on SiO2 (10% ethyl acetate in hexanes) provided the
product as a colorless oil (0.86 g, 2.03 mmol, 56%): Rf ) 0.6
(1:2, ethyl acetate/hexanes); [R]D +10.5° (c 1.0, CHCl3); IR (film)
2957, 2933, 2902, 2874, 2864, 1733, 1693 cm-1; 1H NMR (300
MHz, 3:1 mixture of rotamers) δ 7.20-7.40 (5H, m, arom), 4.9
(0.6H, d, J ) 5 Hz, H-2), 4.58-4.63 (1.3H, m, CH2, H-2 min.),
4.38-4.44 (1H, m, CH2), 4.09-4.31 (3H, m, CH2O, H-3), 2.99
(0.6H, s, NCH3), 3.00 (0.3H, s, NCH3), 1.49 (6H, s, Boc), 1.45
(3H, s, Boc), 1.21-1.28 (3H, m, H-4), 0.90-1.01 (2H, m, CH2-
Si), 0.40 (3H, s, (CH3)3Si), 0.30 (6H, s, (CH3)3Si); 13C NMR (75
MHz) δ 128.1, 127.2, 79.7, 75.3, 74.8, 71.5, 71.0, 63.5, 63.1,
33.1, 28.4, 17.6, 16.4, -1.5; MS m/z (rel int) 424 (19, M+H),
368 (35), 324 (50), 296 (100), 282 (31), 160 (35), 91 (38). Anal.
Calcd for C22H37NO5Si: C, 62.38; H, 8.81; N, 3.31. Found: C,
62.42; H, 8.64; N, 3.24.
1
acetate); H NMR (300 MHz) δ 1.26 (3H, d, J ) 7.2 Hz), 1.44
(9H, s), 3.26-3.30 (1H, m), 3.66 (3H, s), 4.32 (1H, dd, J ) 3.4
and 9.4 Hz), 5.59 (1H, d, J ) 9.1 Hz), 9.63 (1H, s).
N-Boc-Ad d a Meth yl Ester (E,E-9). To phosphonium salt
25 (420 mg, 0.75 mmol) in THF (5 mL) at -78 °C was added
n-butyllithium (470 µL, 0.75 mmol, 1.6 M in hexane), and this
mixture was stirred at -78 °C for 15 min and then at 0 °C for
30 min. Aldehyde 11 (184 mg, 0.75 mmol) in THF (5 mL) was
added, and the reaction mixture was allowed to warm to room
temperature over 20 h. The reaction was quenched by the
addition of saturated aqueous ammonium chloride and then
extracted with ether. The combined organic extracts were
washed with brine, dried (MgSO4), concentrated, and purified
by chromatography on SiO2 (8:1, hexane/ethyl acetate) to give
the product as a colorless oil (138 mg, 41%): Rf ) 0.38 (4:1,
hexane/ethyl acetate); [R]D -13.3° (c 0.73, CHCl3); IR (film)
3416 (br), 2973, 1708, 1650, 1497 cm-1; 1H NMR (300 MHz) δ
1.03 (3H, d, J ) 6.7 Hz), 1.22 (3H, d, J ) 7.2 Hz), 1.45 (9H, s),
1.60 (3H, s), 2.57-2.79 (4H, m), 3.17-3.21 (1H, m), 3.23 (3H,
s), 3.66 (3H, s), 4.37 (1H, br s), 5.29 (1H, br s), 5.37-5.47 (2H,
m), 6.19 (1H, d, J ) 15.6 Hz), 7.12-7.26 (5H, m); 13C NMR
(75 MHz) δ 12.7, 14.3, 16.2, 28.4 (3C), 36.8, 38.4, 44.1, 51.5,
54.7, 58.5, 79.3, 86.9, 125.1, 125.8, 128.1, 129.3, 132.4, 135.9,
136.0, 136.2, 139.4, 155.4, 175.1; HRMS calcd for C26H39O5N
[M + H]+ 446.2906, found 446.2887. The isomeric product
Z,E-9 was isolated in 20% yield: Rf ) 0.40 (4:1, hexane/ethyl
1
acetate); H NMR (360 MHz) δ 1.03 (3H, d, J ) 6.7 Hz), 1.20
(3H, d, J ) 7.2 Hz), 1.44 (9H, s), 1.73 (3H, s), 2.53-2.74 (3H,
m), 2.86-2.90 (1H, dd, J ) 3.5, 14.1 Hz), 3.23 (3H, s), 3.23-
3.25 (1H, m), 3.67 (3H, s), 4.83 (1H, br s), 5.19-5.25 (2H, m),
5.35 (1H, d, J ) 9.6 Hz), 5.93 (1H, d, J ) 1.8 Hz), 7.15-7.28
(5H, m).
N-Boc-D-γ-Glu t a m yl-(r-m et h yl
est er )-N-m et h yl-O-
ben zyl-L-th r eon in e Tr im eth ylsilyleth yl Ester (28). To
N-Boc-N-Me-O-Bn-L-threonine trimethylsilyl ethyl ester (119
mg, 0.28 mmol) in CH2Cl (5 mL) was added HCl-saturated
2
ether (5 mL), and the solution was stirred at room temperature
Ad d a Isom er iza tion . To Z,E-9 (85 mg, 0.19 mmol) in CH2-
Cl2 (38 mL) at -78 °C was added I2 (10 mg, 0.038 mmol), and
the mixture was photolyzed using a tungsten lamp for 110 min.
The reaction mixture was diluted with CH2Cl2 (30 mL), washed
with 10% aqueous Na2S2O3 solution (2.25 mL) followed by
brine (15 mL), and then dried (MgSO4). Purification by silica
for 90 min and then concentrated 3 times from CH2Cl to
2
provide the hydrochloride salt. This salt and NMM (154 µL,
1.4 mmol) were dissolved in CH2Cl2 (5 mL) and added to a
solution of N-Boc-D-glutamate R-methyl ester (74 mg, 0.28
mmol) in CH2Cl (3 mL) at 0 °C. BOP (137 mg, 0.308 mmol)
2
was added, and the reaction mixture was allowed to warm to

2724 J . Org. Chem., Vol. 64, No. 8, 1999
Samy et al.
room temperature overnight. Following dilution with CH2Cl 2
(30 mL), the mixture was washed with 1 M HCl (5 mL),
saturated aqueous sodium bicarbonate, and brine and then
dried (MgSO4). Removal of the solvents in vacuo and chroma-
tography on SiO2 (1:1, hexane/ethyl acetate) gave the title
compound as a colorless oil (136 mg, 86%): Rf ) 0.46 (1:1,
hexane/ethyl acetate); [R]D +8.1° (c 0.96, CHCl3); IR (film) 3424
(33 mg, 125 µmol) was added, and the solution was stirred for
2 h at 0 °C. Additional TBAF‚H2O (5 mg) was added, and the
mixture was stirred for 5.5 h at room temperature. After
dilution with ethyl acetate, the solution was washed with 0.1
M HCl and brine and dried (Na2SO4). The drying agent was
removed, and the solvents were evaporated to give the crude
acid, which was used without further purification. Dipeptide
8 (46 mg, 115 µmol) was dissolved in 1:2 TFA/CH2Cl2 (3 mL)
and stirred at room temperature for 30 min. The solvents were
evaporated to provide amine salt 33. This salt was redissolved
in CH2Cl2 (1 mL) and NMM (42 µL, 384 µmol) was added. The
resulting solution was added to the crude acid prepared above
dissolved in CH2Cl2 (2 mL), and the mixture was cooled to 0
°C under nitrogen. BOP reagent (47 mg, 106 µmol) was added
in one portion, and the reaction mixture was allowed to warm
to room temperature and stirred overnight. Following the
addition of more CH2Cl2, the solution was washed with 10%
citric acid (aqueous), saturated aqueous NaHCO3, and brine
and dried (Mg2SO4). Following removal of the drying agent
and evaporation of the solvents, chromatography on SiO2
provided 5a (43 mg, 46%) as a colorless solid: Rf ) 0.38 (ethyl
1
(br), 2102, 1650, 1455 cm-1; H NMR (300 MHz) δ 0.03 (9H,
s), 0.94 (2H, t, J ) 8.3 Hz), 1.18 (3H, d, J ) 6.2 Hz), 1.26 (2H,
t, J ) 7.1 Hz), 1.43 (9H, s), 2.15-2.19 (2H, m), 2.44-2.55 (2H,
m), 3.13 (3H, s), 3.72 (3H, s), 4.40 (1H, d, J ) 11.6 Hz), 4.61
(1H, d, J ) 11.6 Hz), 5.31 (1H, br s), 5.45 (1H, d, J ) 3.9 Hz),
7.27-7.33 (5H, m); 13C NMR (75 MHz, mixture of rotamers) δ
-1.6 (3C), 16.2, 17.5, 27.9, 28.3 (3C), 29.4, 34.1, 52.0, 53.6,
60.5, 63.2, 71.5, 75.3, 79.6, 127.1, 127.3, 127.4, 127.5, 128.1,
128.2, 138.3, 155.2, 169.7, 172.6, 172.6, 173.3; HRMS calcd
for C28H46O8N2Si [M + H]+ 567.3102, found 567.3082.
N-Boc-D-γ-Glu ta m yl-(r-m eth yl ester )-N-m eth yl-L-th r e-
on in e Tr im eth ylsilyleth yl Ester (7). To dipeptide 28 (480
mg, 0.85 mmol) was added 10% Pd/C (100 mg) and ethanol
(25 mL). The suspension was placed under hydrogen at
atmospheric pressure for 5 h and then filtered through a pad
of Celite, rinsing several times with ethyl acetate. The
combined filtrates were concentrated, and the product was
purified by chromatography on SiO2 (10% MeOH in CH2Cl2)
to give 7 as a colorless oil (350 mg, 86%): Rf ) 0.49 (10% MeOH
in CH2Cl2); [R]D -26.3° (c 1.08, CHCl3); IR (film) 3416 (br),
2095, 1645 cm-1; 1H NMR (360 MHz) δ 0.04 (9H, s), 1.02 (2H,
t, J ) 8.9 Hz), 1.25 (3H, d, J ) 6.1 Hz), 1.44 (9H, s), 1.88-
1.93 (1H, m), 2.27-2.57 (3H, m), 3.11 (3H, s), 3.75 (3H, s),
4.21-4.28 (4H, m), 4.31-4.36 (1H, m), 4.52 (1H, t, J ) 6.3
Hz), 4.60 (1H, br s); 13C NMR (90 MHz, mixture of rotamers)
δ -1.7 (3C), 17.3, 19.9, 27.6, 28.1 (3C), 29.2, 35.6, 52.2, 52.8,
63.5, 64.1, 65.5, 79.8, 155.5, 169.9, 172.9, 173.1; HRMS calcd
for C21H40O8N2Si [M + H]+ 477.2632, found 477.2623.
1
acetate); H NMR (360 MHz) δ 0.83 (3H, d, J ) 7 Hz), 0.88
(3H, d, J ) 7 Hz), 1.13 (3H, d, J ) 7 Hz), 1.23 (3H, d, J ) 7
Hz), 1.38 (9H, s), 1.62 (3H, s), 1.74-1.88 (1H, m), 1.92-2.03
(1H, m), 2.10-2.21 (1H, m), 2.31-2.44 (2H, m), 2.50-2.72 (4H,
m), 2.80 (1H, dd, J ) 4, 1 Hz), 2.88 (0.5H, s), 2.97 (0.5H, s),
3.05 (1H, s), 3.10-3.28 (1H, m), 3.22 (2H, s), 3.46-3.53 (0.5H,
m), 3.89-4.00 (1H, m), 3.73 and 3.74 (5H, two singlets), 4.36-
4.50 (2H, m), 4.52-4.70 3H, m), 4.70-4.85 (1H, m), 5.23 (1H,
dd, J ) 1, 11 Hz), 5.29 (3H, s), 5.33-5.36 (1H, m), 5.60-5.70
(1H, m), 5.84-5.96 (1H, m), 6.04-6.21 (1H, m), 6.59-6.70 (1H,
m), 6.71-6.81 (1H, m), 7.16-7.26 (5H, m), 7.38 (1H, d, J ) 9
Hz), 7.53 (0.5H, d, J ) 5 Hz); 13C NMR (90 MHz) δ 12.75, 13.49,
14.98, 15.71, 16.15, 17.63, 18.73, 18.91, 19.56, 23.75, 27.47,
28.39, 30.71, 33.57, 36.58, 38.18, 41.12, 51.28, 52.40, 52.55,
53.38, 54.81, 56.70, 57.06, 57.35, 58.53, 65.81, 66.00, 86.83,
118.77, 124.91, 125.94, 128.14, 129.34, 131.68, 132.54, 135.95,
137.04, 139.32, 155.67, 171.10, 172.61, 173.29.
Dip ep tid e 30. Dipeptide 7 (40 mg, 100 µmol) was dissolved
in 1:1 TFA/CH2Cl2 (0.5 mL) and stirred at room temperature
for 15 min. Following evaporation of the solvents, the resulting
salt was dissolved in saturated aqueous sodium bicarbonate
solution (10 mL) and extracted twice with ethyl acetate. The
combined extracts were washed with brine and then concen-
trated to provide the crude product (26 mg, 87%), which was
used without further purification.
Tetr a p ep tid e (34). To dipeptide 8 (80 mg, 0.20 mmol) in
CH2Cl2 (3 mL) was added HCl-saturated ether (3 mL), and
the solution was stirred at room temperature for 1 h. The
solvents were removed under reduced pressure and reconcen-
trated repeatedly from ether to remove excess HCl and provide
the amine salt 33b. Dipeptide 7 (95.3 mg, 0.20 mmol) was
dissolved in THF (3 mL) and cooled to 0 °C. TBAF‚H2O (78.4
mg, 0.30 mmol) was added, and the mixture was allowed to
stir at room temperature overnight. Following acidification
with 1 M HCl, the mixture was extracted with ethyl acetate
and the combined organic extracts were washed with brine
and dried (MgSO4). Removal of the drying agent and evapora-
tion of the solvent gave the free carboxylic acid, which was
used without further purification. To this acid was added 33a
in DMF (2.5 mL), and the mixture was cooled to 0 °C. Collidine
(80 µL, 0.60 mmol) was added, followed by HATU (91 mg, 0.24
mmol). The reaction mixture was stirred at 0 °C for 1 h and
then at room temperature for 20 h. Following dilution with
1:1 benzene/ethyl acetate (20 mL), the solution was washed
with 1 M HCl, saturated aqueous NaHCO3, and brine and
dried (MgSO4). Concentration followed by chromatography on
SiO2 (5% hexane in ethyl acetate) gave tetrapeptide 34 (77 mg,
59%) as a colorless oil: Rf ) 0.29 (5% hexane in ethyl acetate);
[R]D -13.8° (c 0.73, CHCl3); IR (film) 3416, 2088, 1736, 1645
Tr ip ep tid e 31. To dipeptide 30 (102 mg, 0.27 mmol) was
added N-Boc-Adda (6) (97 mg, 0.225 mmol) in CH2Cl2 (2 mL).
The solvent was evaporated and replaced with dry DMF (2
mL). Collidine (89 µL, 0.765 mmol) was added dropwise, and
the solution was cooled to 0 °C. HATU (103 mg, 0.27 mmol)
was added, and the reaction mixture was allowed to stir for
14.5 h at room temperature. Following dilution of the reaction
mixture with 1:1 benzene/ethyl acetate, it was washed with
10% citric acid (aqueous), saturated aqueous sodium bicarbon-
ate, and brine and dried over anhydrous magnesium sulfate.
Removal of the drying agent and evaporation of the solvent
followed by chromatography on SiO2 (95:5, CHCl3/CH3OH)
provided the tripeptide (140 mg, 79%) as a colorless oil: Rf )
0.32 (1:1, ethyl acetate/hexanes); [R]D -26.0° (c 0.5, CHCl3);
1H NMR (300 MHz) δ(mixture of conformers) 0.01-0.10 (10H,
m), 1.00 (6H, d, J ) 6 Hz), 1.12-1.33 (10H, m) 1.40-1.50 (11H,
m), 1.60 (3.5H, s), 1.78-2.09 (1H, m), 21.-2.27 (1H, m), 2.30-
2.45 (1H, m), 2.39 (2H, s), 2.49-2.74 (4H, m), 2.80 (1H, dd, J
) 5, 14 Hz), 3.08 (1.5H, d, J ) 7 Hz), 3.11-3.25 (1H, m), 3.23
and 3.24 (4.5H two singlets), 3.7-3.75 (3H, m), 4.13-4.30 (3H,
m), 4.4-4.78 (2H, m), 5.19 (1H, dd, J ) 5, 7 Hz), 5.39 (1H, d,
J ) 11 Hz), 5.42-5.43 (1H, m), 6.19 (1H, dd, J ) 7, 16 Hz),
7.15-7.30 (8H, m); 13C NMR (90 MHz, mixture of rotamers) δ
12.73, 14,13, 15.31, 16.96, 17.10, 19.56, 26.65 (br), 28.86, 30.29
(br), 35.68 (br), 36.53, 38.18, 44.53 (br), 51.15, 52.37, 52.47,
55.27 (br), 58.55, 58.55, 60.32, 63.39, 63.68, 63.90, 64.39, 65.89,
79.07, 86.95, 125.76, 125.9, 128.11, 129.35, 132.43, 132.62,
135.75, 139.36, 155.87, 169.79, 172.41, 173.47, 174.94; MS m/z
(rel int) 790 (14), 135 (100); HRMS calcd for C41H67N3O10Si
[M]+ 790.4674, found 790.4626.
cm-1 1H NMR (360 MHz, mixture of rotamers) δ 0.88 (6H,
;
dd, J ) 6.6 and 12.6 Hz), 1.15 (3H, d, J ) 6.2 Hz), 1.22 (3H,
d, J ) 7.1 Hz), 1.42 (9H, s), 1.55-1.59 (1H, m), 2.14-2.19 (2H,
m), 2.48-2.57 (2H, m), 3.12 (3H, s), 3.69 (3H, s), 3.72 (3H, s),
4.29-4.47 (3H, m), 4.56-4.72 (3H, m), 5.07 (1H, d), 5.24 (1H,
d, J ) 10.4 Hz), 5.32 (1H, d, J ) 15.3 Hz), 5.48 (1H, d, J ) 7.9
Hz), 5.64 (1H, d, J ) 7.5 Hz), 5.82-5.94 (1H, m), 6.44 (1H, d,
J ) 7.8 Hz), 7.40 (1H, d, J ) 8.7 Hz); 13C NMR (90 MHz,
mixture of rotamers), δ 13.4, 15.4, 17.5, 18.9, 19.6, 23.6, 27.3,
28.2 (3C), 29.2, 30.9, 34.1, 40.1, 52.6, 54.5, 57.0, 61.4, 63.1,
66.0, 66.5, 80.0, 119.0, 131.5, 155.6, 155.8, 171.1, 171.5, 172.9,
173.3, 173.7, 173.9, 174.0; HRMS calcd for C30H50O12N4 [M +
H]+ 659.3503, found 659.3406.
P en ta p ep tid e 5a fr om 31. Tripeptide 31 (76 mg, 96 µmol)
was dissolved in THF (5 mL) and cooled to 0 °C. TBAF‚H2O

Total Synthesis of Motuporin and 5-[L-Ala]-Motuporin
J . Org. Chem., Vol. 64, No. 8, 1999 2725
P en ta p ep tid e 5a . Tetrapeptide 34 (51 mg, 0.0744 mmol)
was dissolved in 1:1 TFA/CH2Cl2 (4 mL) and stirred at room
temperature for 1 h. The solvents were removed under reduced
pressure, and the residue was redissolved in CH2Cl2 and
reconcentrated repeatedly to remove excess TFA. The remain-
ing salt was combined with DIPEA (37 µL, 0.21 mmol) and
DMAP (1 mg, 0.00744 mmol) in CH3CN (3 mL) and added to
a solution of N-Boc-Adda pentafluorophenyl ester (42 mg, 0.070
mmol) in CH3CN (3 mL). The reaction mixture was stirred at
room temperature for 94 h, diluted with CH2Cl2 (30 mL), and
washed with 1 M HCl, then brine, and dried (MgSO4). Removal
of the solvent in vacuo followed by chromatography on SiO2
(5% MeOH in ethyl acetate) gave pentapeptide 5a (47 mg, 69%)
as a colorless oil: Rf ) 0.54 (10% MeOH in CH2Cl2); [R]D -19.6°
(c 0.5, CHCl3); IR (film) 3416, 2073, 1645, 1436 cm-1; 1H NMR
(360 MHz, CD3OD, mixture of rotamers) δ 0.95 (6H, d, J )
6.9 Hz), 1.03 (3H, d, J ) 6.8 Hz), 1.14 (3H, d, J ) 7.0 Hz),
1.18 (3H, d, J ) 7.1 Hz), 1.22 (3H, d, J ) 7.2 Hz), 1.46 (9H, s),
1.64 (3H, d, J ) 6.8 Hz), 1.98-2.01 (2H, m), 2.13-2.17 (2H,
m), 2.48-2.69 (6H, m), 2.94 (2H, d, J ) 10.5 Hz), 3.10 (3H, s),
3.23 (3H, s), 3.69 (3H, s), 3.72 (3H, s), 4.21-2.33 (2H, m), 4.62
(2H, d, J ) 4.6 Hz), 5.23 (1H, d, J ) 10.4 Hz), 5.35 (1H, d, J
) 17.1 Hz), 5.40 (1H, d, J ) 9.5 Hz), 5.41-5.49 (1H, m), 5.92-
5.99 (1H, m), 6.21 (1H, d, J ) 15.1 Hz), 7.15-7.26 (5H, m);
13C NMR (90 MHz, CD3OD, mixture of rotamers) δ 13.1, 13.2,
15.6, 16.3, 16.7, 18.6, 19.7, 20.9, 21.1, 21.3, 27.8, 28.9, 30.5,
30.7, 31.9, 33.5, 34.1, 37.8, 39.2, 41.8, 41.9, 45.5, 45.6, 52.9,
53.2, 55.7, 57.1, 58.9, 59.1, 63.2, 64.1, 64.3, 66.1, 66.9, 67.2,
80.5, 88.6, 119.1, 127.2, 129.4, 130.7, 133,6, 134.1, 134.2, 136.8,
137.1, 137.6, 140.8, 157.8, 171.9, 172.5, 172.6, 173.7, 173.8,
175.4, 175.8, 176.4, 177.3, 177.5; HRMS calcd for C50H77N5O14
[M]+ 972.5545, found 972.5535.
N-Boc-D-γ-Glu ta m yl-(r-m eth yl ester )-Z-N-m eth yld eh y-
d r obu t-2-en oic Acid Tr im eth ylsilyleth yl Ester (29). To
the dipeptide alcohol 28 (130 mg, 0.273 mmol) in DMF (10
mL) was added a solution of Martin sulfurane (367 mg, 0.546
mmol) in DMF (5 mL). This mixture was stirred at 120 °C
overnight, cooled to room temperature, diluted with 1:1
benzene/ethyl acetate (30 mL), and washed with water fol-
lowed by 10% aqueous NaOH solution. The organic layer was
dried (MgSO4), concentrated, and purified by chromatography
on SiO2 (2:1, hexane/ethyl acetate) to give 29 (88 mg, 70%) as
a colorless oil: Rf ) 0.36 (1:1, hexane/ethyl acetate); [R]D -7.7°
(c 0.75, CHCl3); IR (film) 3431, 2362, 2341, 1645 cm-1; 1H NMR
(360 MHz, 50 °C) δ 0.07 (9H, s), 1.05 (2H, t, J ) 8.3 Hz), 1.81
(3H, d, J ) 7.0 Hz), 1.90-2.18 (4H, m), 2.98 (3H, s), 3.72 (3H,
s), 4.22-4.31 (3H, m), 5.18 (1H, br s), 7.00 (1H, q, J ) 7.0 Hz);
13C NMR (90 MHz, mixture of rotamers), δ -1.5 (3C), 13.5,
17.4, 27.5, 27.8, 28.3 (3C), 29.1, 29.4, 34.7, 52.2, 53.0, 63.4,
63.9, 79.7, 134.6, 136.7, 139.0, 139.3, 155.4, 163.9, 172.1, 172.8;
HRMS calcd for C21H38N2O7Si [M + H]+ 459.2527, found
459.2531.
N-Boc-D-γ-glu ta m yl-(r-m eth yl ester )-Z-N-m eth yld eh y-
d r obu t-2-en oic a cid (36). To dipeptide 29 (26 mg, 0.057
mmol) in THF (3 mL) at 0 °C was added TBAF‚H2O (22 mg,
0.086 mmol). The mixture was allowed to warm to room
temperature overnight. Acidification with 1 M HCl was
followed by extraction with ethyl acetate, then the organic
extracts were washed with brine, dried (MgSO4) and concen-
trated to give acid 36 (20 mg, 100%) as a colorless oil, which
was used without further purification. [R]D -4.1° (c 1.1,
CHCl3); IR (film) 3445, 2081, 1694, 1638, 1518 cm-1; 1H NMR
(360 MHz, 50 °C) δ 1.43 (9H, s), 1.83-1.86 (3H, m), 1.90-
2.03 (2H, m), 2.15-2.21 (2H, m), 3.02 (3H, s), 3,72 (3H, s),
4.16-4.22 (1H, m), 5.32-5.34 (1H, m), 7.13 (1H, q, J ) 7.0
Hz); HRMS calcd for C16H26N2O7 [M + H]+ 359.1818, found
359.1821.
Tetr a p ep tid e 37. Dipeptide 8 (52 mg, 0.13 mmol) was
dissolved in 1:3 TFA/CH2Cl2 (4 mL) and stirred at room
temperature for 20 min. The solvents were removed under
reduced pressure, and the residue was redissolved in CH2Cl2
and reconcentrated repeatedly to remove excess TFA. The dry
sample was stored under high vacuum overnight to give the
TFA salt (54 mg), which was used without further purification.
This salt was combined with dipeptide acid 36 (46.5 mg, 0.13
mmol) in DMF (2 mL) at 0 °C, and collidine (52 µL, 0.42 mmol)
was added, followed by HATU (54 mg, 0.154 mmol). The
reaction mixture was stirred at 0 °C for 1 h and then at room
temperature for 22 h. Following dilution with 1:1 benzene/
ethyl acetate (20 mL), the solution was washed with 1 M HCl,
saturated aqueous NaHCO3, and brine and dried (MgSO4).
Concentration followed by chromatography on SiO2 (5% hexane
in ethyl acetate) gave tetrapeptide 37 (39 mg, 47%) as a
colorless oil: Rf ) 0.31 (5% MeOH in CH2Cl2); [R]D -15.3° (c
1
1.1, CHCl3); IR (film) 3457, 2101, 1734, 1642, 1555 cm-1; H
NMR (360 MHz, 50 °C) δ 0.88 (3H, d, J ) 7.1 Hz), 0.91-0.97
(3H, m), 1.25 (3H, d, J ) 7.2 Hz), 1.41 (9H, s), 1.75 (3H, d, J
) 7.0 Hz), 2.01-2.21 (3H, m), 2.37-2.42 (1H, m), 3.03 (3H, s),
3.26-3.28 (1H, m), 3.70 (3H, s), 3.73 (3H, s), 4.41-4.46 (1H,
m), 4.67 (2H, d, J ) 6.9 Hz), 5.25 (1H, d, J ) 10.4 Hz), 5.34
(1H, d, J ) 17.2 Hz), 5.86-5.96 (1H, m), 6.16 (1H, d, J ) 7.3
Hz), 6.31 (1H, br s), 6.99 (1H, q, J ) 6.7 Hz), 7.19 (1H, d, J )
9.1 Hz), 7.35 (1H, d, J ) 8.1 Hz); 13C NMR (90 MHz, mixture
of rotamers), δ 13.1, 13.2, 14.5, 15.3, 17.0, 17.4, 18.9, 19.3, 26.8,
26.9, 28.3 (3C), 29.2, 29.5, 30.5, 30.6, 34.1, 34.6, 40.6, 52.2,
52.3, 52.9, 53.0, 53.4, 53.6, 54.7, 54.9, 57.0, 57.5, 66.0, 66.4,
19.4, 118.7, 118.8, 131.5, 131.7, 135.6, 135.9, 136.2, 136.6,
156.2, 163.4, 171.3, 172.2, 172.3, 173.4, 173.5; HRMS calcd
for C30H48N4O11 [M + H]+ 641.3398, found 641.3387.
P en ta p ep tid e 38. Tetrapeptide 37 (38 mg, 0.0593 mmol)
was dissolved in 1:3 TFA/CH2Cl2 (4 mL) and stirred at room
temperature for 35 min. The solvents were removed under
reduced pressure, and the residue was redissolved in CH2Cl2
and reconcentrated repeatedly to remove excess TFA. The dry
sample was stored under high vacuum overnight to provide
the TFA salt (39 mg, 100%), which was used without further
purification. This salt was combined with DIPEA (31 µL, 0.178
mmol) and DMAP (1.5 mg, 0.0119 mmol) in CH3CN (2 mL)
and added to a solution of N -Boc-Adda pentafluorophenyl ester
(35) (35 mg, 0.0593 mmol) in CH3CN (2 mL). The reaction
mixture was stirred at room temperature for 96 h, diluted with
CH2Cl2 (25 mL), washed with 1 M HCl and then brine, and
dried (MgSO4). Removal of the solvent in vacuo followed by
chromatography on SiO2 (5% MeOH in ethyl acetate) gave
pentapeptide 38 (30 mg, 53%) as a colorless oil: Rf ) 0.32 (5%
MeOH in CH2Cl2); [R]D -18.4° (c 0.56, CHCl3); IR (film) 3339,
1
2966, 2938, 1743, 1666, 1497 cm-1; H NMR (500 MHz, CD3-
OD, 50 °C) δ 0.94 (3H, d, J ) 7.0 Hz), 0.96 (3H, d, J ) 7.0 Hz),
1.03 (3H, d, J ) 7.0 Hz), 1.13 (3H, d, J ) 7.5 Hz), 1.24 (3H, d,
J ) 6.0 Hz), 1.44 (9H, s), 1.62 (3H, s), 1.78 (3H, d, J ) 7.5
Hz), 1.99-2.02 (2H, m), 2.10-2.17 (2H, m), 2.58-2.70 (6H,
m), 2.81 (2H, dd, J ) 5.0 and 13.5 Hz), 3.06 (3H, s), 3.21 (3H,
s), 3.68 (3H, s), 3.72 (3H, s), 4.16-4.19 (1H, m), 4.32 (1H, d, J
) 6.0 Hz), 4.62 (2H, d, J ) 6.0 Hz), 5.22 (1H, d, J ) 10.4 Hz),
5.32 (1H, d, J ) 16.5 Hz), 5.39 (1H, t, J ) 9.0 Hz), 5.48-5.54
(1H, m), 5.90-5.97 (1H, m), 6.18 (1H, d, J ) 15.5 Hz), 6.90
(1H, q, J ) 6.5 Hz), 7.14-7.26 (5H, m); 13C NMR (90 MHz,
CD3OD) δ 13.0, 13.6, 15.4, 16.6, 16.5, 18.4, 19.5, 27.6, 27.8,
28.0, 28.8 (3C), 30.6, 30.7, 31.8, 35.3, 37.7, 39.1, 41.3, 45.2,
52.8, 53.1, 53.3, 56.4, 56.5, 58.8, 58.9, 66.7, 80.4, 88.5, 118.9,
127.1, 129.2, 130.6, 133.4, 133.9, 134.1, 136.7, 136.9, 137.1,
137.6, 140.6, 157.7, 172.5, 173.5, 174.6, 176.5, 177.1; HRMS
calcd for C50H75N5O13 [M]+ 954.5439, found 954.5346.
Com p ou n d 39 via Ma cr ocyliza tion Usin g F DP P . To the
pentapeptide 5a (47 mg, 0.0484 mmol) in THF (4.7 mL) was
added dimedone (20.3 mg, 0.1452 mmol) and Pd(PPh3)4 (5.6
mg, 4.8 µmol), and the mixture was stirred at room temper-
ature for 45 h. Following removal of the solvent in vacuo, the
residue was purified by chromatography on SiO2 (ethyl acetate
and then methanol) to obtain the pentapeptide acid 5b (32 mg,
71%). This acid was dissolved in 1:1 TFA/CH2Cl2 (3 mL) and
stirred at room temperature for 1 h. Evaporation of the
solvents was followed by repeated reconcentration from CH2-
Cl2 to remove the excess TFA. The oily residue was stored
under high vacuum overnight to obtain the pentapeptide
amine as its TFA salt (5c). To this amine salt (32.5 mg, 0.0343
mmol) in DMF (34.3 mL) was added NMM (38 µL, 0.343 mmol)
and FDPP (26.4 mg, 0.0686 mmol), and the solution was
stirred at room temperature for 24 h. Concentration and

2726 J . Org. Chem., Vol. 64, No. 8, 1999
Samy et al.
purification by chromatography on SiO2 (5% MeOH in ethyl
acetate) provided macrocycle 39 as a colorless oil (22 mg, 79%).
Com p ou n d 40. To the pentapeptide 38 (50 mg, 0.0524
mmol) in THF (6 mL) was added dimedone (22 mg, 0.157
mmol) and Pd(PPh3)4 (6 mg, 5.24 × 10-3 mmol), and the
mixture was stirred at room temperature for 19 h. Following
removal of the solvent under reduced pressure, chromatogra-
phy on SiO2 (ethyl acetate/methanol) gave the pentapeptide
acid (48 mg, 100%). To this acid (48 mg, 0.0524 mmol) in ethyl
acetate (3 mL) was added pentafluorophenol (29 mg, 0.157
mmol), DCC (16.2 mg, 0.0786 mmol), and DMAP (1.3 mg,
0.0105 mmol), and the mixture was stirred at room temper-
ature overnight. Removal of the solvent under reduced pres-
sure followed by chromatography on SiO2 (5% MeOH in ethyl
acetate) gave the activated ester (44 mg, 78%). This material
was dissolved in CH2Cl2 (5 mL), and TFA (2 mL) was added.
After stirring at room temperature for 1 h, the reaction
mixture was concentrated, redissolved in CH2Cl2, and recon-
centrated repeatedly to remove the excess TFA. The residue
was kept under high vacuum overnight. To the dry sample
(45 mg, 0.041 mmol) was added CH3CN (41 mL) followed by
DIPEA (143 µL, 0.82 mmol) and DMAP (1.0 mg, 0.008 mmol).
The dilute solution was stirred at room temperature for 5 d
and then concentrated under reduced pressure. Chromatog-
raphy on SiO2 (5% MeOH in ethyl acetate) provided the
macrocycle 40 (4.1 mg, 13%): Rf ) 0.34 (5% MeOH in CH2-
1, hexane/ethyl acetate) gave 42 (750 mg, 89%) as a colorless
oil: Rf ) 0.43 (4:1, hexane/ethyl acetate); [R]D -26.2° (c 1.1,
CHCl3); IR (film) 2952, 2362, 1743, 1701, 1462 cm-1; 1H NMR
(500 MHz) (mixture of rotamers) δ 0.05 (9H, s), 1.00 (2H, t, J
) 8.0 Hz) 1.39 (3H, d, J ) 5.5 Hz), 1.45 (9H, s), 1.47 (9H, s),
2.80 (3H, s), 2.86 (3H, s), 4.22 (2H, t, J ) 8.5 Hz), 4.42 (1H, q,
J ) 6.5 Hz), 4.82 (1H, J ) 7.0 Hz); 13C NMR (90 MHz, 50 °C)
δ -1.8 (3C), 14.6, 17.2, 28.1 (3C), 30.4, 53.5, 62.7, 79.5, 171.2;
HRMS calcd for C14H29NO4Si [M + H]+ 304.1944, found
1
304.1959. Note: the H NMR spectrum at room temperature
exhibits two sets of signals due to rotamers. These signals
coalesce into one set of signals at 323 K, as is clearly evident
from signals due to the N-Boc and N-Me groups appearing as
singlets at 1.47 and 2.83 ppm, respectively. The R-proton is
not clearly visible at 323 K, although on expanding the
spectrum one observes two broad singlets at 4.45 and 4.81
ppm, respectively.
N-Boc-D-Glu t a m yl-(r-m et h yl est er )-N-m et h yl-L-a la -
n in e Tr im eth ylsilyleth yl Ester (43). N-Boc-N-methyl-L-
alanine trimethylsilylethyl ester (167 mg, 0.55 mmol) was
dissolved in 1:2 TFA/CH2Cl2 (3 mL) and stirred at room
temperature for 30 min. The reaction mixture was concen-
trated, and the residue was dissolved in CH2Cl2 and recon-
centrated repeatedly to remove the excess TFA. The dry
sample was stored under high vacuum overnight to give the
product TFA salt (175 mg, 100%), which was used without
purification. N-Boc-D-Glutamic acid-R-methyl ester (131 mg,
0.50 mmol) and the TFA salt were dissolved in DMF (7 mL)
at 0 °C and collidine (198 µL, 1.50 mmol) was added, followed
by HATU (209 mg, 0.55 mmol). The reaction mixture was
stirred at 0 °C for 1 h and then at room temperature for 19 h.
After dilution with 1:1 benzene/ethyl acetate (25 mL), the
mixture was washed with 1 M HCl, saturated aqueous
NaHCO3, and then brine and dried (MgSO4). Chromatography
on SiO2 (1:1, hexane/ethyl acetate) gave the title compound
as a colorless oil (160 mg, 72%): Rf ) 0.42 (1:1, hexane/ethyl
acetate); [R]D -34.7° (c 0.53, CHCl3); IR (film) 3423, 2362, 2334,
Cl2); [R]D -16.2° (c 1.2, CHCl3); IR (film) 3423, 2095, 1638 cm-1
;
1H NMR (500 MHz, CD3OD), δ(50 °C) 0.83 (3H, d, J ) 6.5
Hz), 0.87 (3H, d, J ) 7.0 Hz), 1.00 (3H, d, J ) 6.5 Hz), 1.10
(3H, d, J ) 7.0 Hz), 1.30 (3H, d, J ) 7.0 Hz), 1.60 (3H, s), 1.76
(3H, d, J ) 7.5 Hz), 2.01-2.14 (2H, m), 2.36-2.39 (2H, m),
2.56-2.62 (2H, m), 2.68 (2H, dd, J ) 7.5, 14.0 Hz), 2.75-2.82
(1H, m), 3.01-3.07 (2H, m), 3.09 (3H, s), 3.23 (3H, s), 3.24-
3.27 (2H, m), 3.70 (3H, s), 3.70-3.76 (1H, m), 3.87 (3H, s), 4.32
(1H, br s), 4.38 (2H, t, J ) 3.0 Hz), 4.48-4.50 (1H, m), 4.69
(1H, dd, J ) 4.0, 6.5 Hz), 5.43 (1H, d, J ) 9.5 Hz), 5.57 (1H,
dd, J ) 8.5, 16.0 Hz), 6.25 (1H, d, J ) 16.0 Hz), 6.98 (1H, q, J
) 7.0 Hz), 7.15-7.21 (5H, m); 13C NMR (90 MHz, CD3OD), δ
12.8, 13.3, 16.4, 16.5, 16.7, 17.0, 19.6, 19.8, 26.0, 27.6, 29.5,
29.9, 34.7, 35.1, 37.7, 38.9, 39.8, 45.3, 52.4, 52.7, 53.1, 53.8,
55.8, 57.5, 58.1, 58.7, 88.3, 126.1, 127.1, 129.2, 130.5, 133.7,
137.0, 137.4, 138.2, 139.1, 140.5, 165.6, 171.2, 173.6, 174.3,
175.2, 176.3, 176.6; HRMS calcd for C42H61N5O10 [M + H]+
796.4497, found 796.4491.
Motu p or in (1). To macrocycle dimethyl ester 40 (4.1 mg,
5.15 µmol) in THF (0.5 mL) at 0 °C was added 1 M LiOH (26
µL, 0.026 mmol), and the solution was stirred for 1 h. The
reaction mixture was acidified with Amberlyte IR-120 ion-
exchange resin, diluted with ethyl acetate, filtered through a
plug of glass wool, and then concentrated. The crude product
(4 mg) was purified by reverse-phase HPLC (C-18, 70:30
MeOH/H2O, 0.1% TFA) to obtain motuporin 1 as a white
solid: [R]D (disodium salt) ) -12.3° (c 0.4, CH3OH), lit. [R]D
-73° (c 0.00718, CH3OH); IR (film) 3431, 2917, 2854, 2369,
2334, 1645 cm-1; 1H NMR (500 MHz, CD3OD) δ 0.77 (3H, d, J
) 7.0 Hz), 0.85 (3H, d, J ) 7.0 Hz), 0.92 (3H, d, J ) 6.5 Hz),
1.00 (3H, d, J ) 6.5 Hz), 1.22 (3H, d, J ) 7.0 Hz), 1.63 (3H, s),
1.74 (3H, d, J ) 7.0 Hz), 2.18-2.30 (2H, m), 2.46-2.55 (2H,
m), 2.57-2.61 (2H, m), 2.67 (1H, dd, J ) 7.5 and 14.0 Hz),
2.82 (1H, dd, J ) 4.5 and 14.0 Hz), 3.02-3.07 (2H, m), 3.07
(3H, s), 3.23 (3H, s), 3.23-3.27 (2H, m), 4.24-4.28 (2H, m)
4.42 (2H, J ) 3.5 Hz), 5.39 (1H, d, J ) 10.0 Hz), 5.62 (1H, dd,
J ) 9.0 and 16.0 Hz), 6.22 (1H, d, J ) 16.0 Hz), 6.94 (1H, q, J
) 7.0 Hz), 7.14-7.25 (5H, m); HRMS calcd for C40H57N5O10
[M + H]+ 768.4184, found 768.4220.
1722, 1645 cm-1 1H NMR (360 MHz, 50 °C) δ 0.01 (9H, s),
;
0.96 (2H, t, J ) 8.5 Hz), 1.34 (3H, d, J ) 7.3 Hz), 1.41 (9H, s),
1.94-2.04 (1H, m), 2.12-2.21 (1H, m), 2.89 (3H, s), 3.70 (3H,
s), 4.17 (2H, t, J ) 8.0 Hz), 4.21-4.25 (1H, m), 5.17 (1H, q, J
) 7.3 Hz); 13C NMR (90 MHz 50 °C) (major rotamer) δ -1.7
(3C), 14.3, 17.4, 27.6, 28.2 (3C), 29.6, 31.3, 52.0, 52.3, 55.1,
63.3, 79.7, 155.4, 171.8, 172.0, 172.7; HRMS calcd for
C
20H38N2O7Si [M + H]+ 447.2527, found 447.2544.
Tetr a p ep tid e 44. Dipeptide 43 (155 mg, 0.35 mmol) was
dissolved in THF (5 mL) at 0 °C, and TBAF‚H2O (139 mg,
0.525 mmol) was added. The reaction mixture was allowed to
warm to room temperature overnight, acidified with 1 M HCl,
and extracted with ethyl acetate. The organic extracts were
washed with brine, dried (MgSO4), and concentrated to give
the acid (121 mg, 100%) as a colorless oil, which was used in
the next step without further purification: [R]D -33.7° (c 1.3,
1
CHCl3); IR (film) 3452, 2095, 1645 cm-1; H NMR (360 MHz,
50 °C) δ 1.37-1.40 (3H, d, hidden by Boc group), 1.40 (9H, s),
1.93-1.97 (1H, m), 2.17-2.22 (1H, m), 2.36-2.52 (2H, m), 2.92
(3H, s), 3.71 (3H, s), 4.25-4.29 (1H, m), 5.18-5.22 (1H, m),
5.41 (1H, d, J ) 7.9 Hz); HRMS calcd for C15H26N2O7 [M +
H]+ 347.1818, found 347.1819. A sample of the acid (49 mg,
0.14 mmol) was combined with 33b (0.14 mmol) in DMF (2
mL). The solution was cooled to 0 °C, and collidine (56 µL,
0.42 mmol) was added, followed by HATU (59 mg, 0.154 mmol).
The reaction mixture was stirred at 0 °C for 1 h and then at
room temperature for 22 h. After dilution with 1:1 benzene/
ethyl acetate (20 mL), the solution was washed with 1 M HCl,
saturated aqueous sodium bicarbonate, and brine and then
dried (MgSO4). Chromatography on SiO2 (5% hexane in ethyl
acetate) gave the tetrapeptide (76 mg, 86%) as a colorless oil:
Rf ) 0.32 (5% MeOH in CH2Cl2); [R]D -31.0° (c 0.9, CHCl3);
N-Boc-N-Met h yl-L-a la n in e Tr im et h ylsilylet h yl E st er
(42). To a solution of N-Boc-N-Me-L-alanine (560 mg, 2.76
mmol) in CH2Cl2 (10 mL) at 0 °C was added 2-trimethylsilyl
ethanol (0.436 mL, 3.04 mmol), DCC (627 mg, 3.04 mmol), and
DMAP (68 mg, 0.552 mmol). The reaction mixture was allowed
to warm to room temperature overnight. The solid dicylohexyl-
urea was removed by filtration and washed with CH2Cl2 (2 ×
25 mL). The combined filtrates were washed with 10% aqueous
citric acid, saturated aqueous sodium bicarbonate, and brine,
dried (MgSO4), and concentrated. Chromatography on SiO2 (8:
IR (film) 3445, 2095, 1736, 1645, 1511 cm-1 1H NMR (360
;
MHz, 50 °C) δ 0.89 (3H, d, J ) 6.9 Hz), 0.93 (3H, d, J ) 6.9
Hz), 1.24 (3H, d, J ) 7.2 Hz), 1.35 (3H, d, J ) 7.1 Hz), 1.45
(9H, s), 2.05-2.07 (1H, m), 2.19-2.27 (2H, m), 2.38-2.44 (1H,
m), 2.55-2.61 (1H, m), 2.94 (3H, s), 3.11-3.13 (1H, m), 3.71
(3H, s), 3.75 (3H, s), 4.29-4.31 (1H, m), 4.50-4.52 (1H, m),

Total Synthesis of Motuporin and 5-[L-Ala]-Motuporin
J . Org. Chem., Vol. 64, No. 8, 1999 2727
4.65 (2H, d, J ) 6.9 Hz), 5.26-5.43 (4H, m), 5.86-5.96 (1H,
m), 6.19 (1H, d, J ) 6.7 Hz); 13C NMR (90 MHz) δ 13.2, 15.4,
17.5, 18.3, 27.1, 28.2 (3C), 29.5, 30.5, 31.1, 38.6, 41.0, 51.8,
52.2, 52.5, 53.1, 54.3, 56.7, 65.9, 79.7, 118.4, 131.4, 155.6, 171.2,
171.3, 171.8, 172.7, 172.8, 173.7; HRMS calcd for C29H48N4O11
[M + H]+ 629.3397, found 629.3427.
7.14-7.26 (5H, m); HRMS calcd for C41H61N5O10 [M + H]+
784.4497, found 784.4490 and 47 (5.9 mg, 27%): Rf ) 0.32 (5%
MeOH in CH2Cl2); [R]D -56.0° (c 0.6, CHCl3); IR (film): 3438,
1
2102, 1638 cm-1; H NMR (500 MHz, CD3OD, 50 °C) δ 0.93
(3H, d, J ) 6.5 Hz), 0.95 (3H, d, J ) 7.0 Hz), 1.02 (3H, d, J )
7.0 Hz), 1.09 (3H, d, J ) 7.0 Hz), 1.24 (3H, d, J ) 7.0 Hz),
1.31 (3H, d, J ) 7.0 Hz), 1.66 (3H, s), 1.90-1.93 (1H, m), 2.27-
2.43 (2H, m), 2.59-2.70 (2H, m), 2.74-2.77 (2H, m), 2.78-
2.83 (2H, dd, J ) 5.0 and 14.0 Hz), 2.92 (3H, s), 3.17-3.22
(2H, m), 3.23 (3H, s), 3.23-3.27 (2H, m), 3.68-3.73 (1H, m),
3.73 (3H, s), 3.75 (3H, s), 3.82 (1H, d, J ) 7.0 Hz), 4.17 (2H, t,
J ) 7.0 Hz), 4.56 (1H, d, J ) 3.0 Hz), 5.33-5.38 (1H, m), 5.43
(1H, d, J ) 10 Hz), 5.78 (1H, dd, J ) 1.0 and 15.5 Hz), 6.25
(1H, d, J ) 15.5 Hz), 7.15-7.26 (5H, m); 13C NMR (90 MHz,
CD3OD) δ 13.0, 13.3, 15.3, 16.5, 17.5, 18.6, 19.8, 27.7, 29.6,
30.0, 30.7, 31.4, 37.6, 39.0, 40.7, 45.2, 52.8, 53.1, 53.2, 56.1,
57.4, 58.8, 62.1, 88.4, 126.5, 127.1, 129.2, 130.5, 133.0, 133.1,
133.7, 134.0, 137.1, 138.2, 140.6, 172.6, 173.1, 173.9, 174.9,
176.7, 177.3; HRMS calcd for C41H61N5O10 [M + H]+ 784.4497,
found 784.4490.
P en ta p ep tid e 45. Tetrapeptide 44 (138 mg, 0.22 mmol)
was dissolved in 1:2.5 TFA/CH2Cl2 (7 mL) and stirred at room
temperature for 45 min. After removal of the solvents under
reduced pressure, the residue was redissolved in CH2Cl2 and
reconcentrated repeatedly to remove the excess TFA. The dry
sample was stored under high vacuum overnght to give the
TFA salt (141.3 mg, 100%), which was used without further
purification. This salt was combined with DMAP (5.4 mg, 0.044
mmol) and DIPEA (115 µL, 0.66 mmol) in CH3CN (5 mL) and
added to a solution of N-Boc-Adda pentafluorophenyl ester (35)
(131.5 mg, 0.22 mmol) in CH3CN (5 mL). This mixture was
stirred at room temperature for 90 h, diluted with CH2Cl2 (25
mL), washed with 1 M HCl and then brine, and dried (MgSO4).
Concentration followed by purification by chromatography on
SiO2 (5% MeOH in ethyl acetate) gave unreacted N-Boc-Adda
pentafluorophenyl ester (14.2 mg) and pentapeptide 45 (150
mg, 81% based on recovered starting material) as a colorless
oil: Rf ) 0.33 (5% MeOH in CH2Cl2); [R]D -31.6° (c 0.9, CHCl3);
5-[L-Ala ]-Motu p or in (41). To macrocycle dimethyl ester 46
(27 mg, 0.0344 mmol) in THF (2 mL) at 0 °C was added 1 M
LiOH (172 µL, 0.172 mmol). The mixture was stirred for 2 h
and then acidified with Amberlyte IR-120 ion-exchange resin.
The suspension was filtered through a plug of glass wool and
then concentrated and purified by reverse-phase HPLC (C-
18, 70:30 MeOH/H2O, 0.1% TFA) to obtain 5-[L-Ala]-motuporin
41 as a white solid: [R]D -29° (c 0.1, CH3OH); IR (film) 3438,
IR (film) 3445, 2060, 1736, 1645, 1504 cm-1 1H NMR (500
;
MHz, CD3OD, 50 °C) δ 0.93-0.96 (6H, m), 1.02 (3H, d, J )
6.7 Hz), 1.16 (3H, d, J ) 6.9 Hz), 1.21 (3H, d, J ) 6.9 Hz),
1.31 (3H, d, J ) 7.1 Hz), 1.44 (9H, s), 1.62 (3H, s), 1.96-1.99
(2H, m), 2.12-2.24 (2H, m), 2.46-2.49 (3H, m), 2.57-2.69 (3H,
m), 2.80 (2H, dd, J ) 6.5, 14.0 Hz), 2.94 (3H, s), 3.21 (3H, s),
3.68 (3H, s), 3.72 (3H, s), 4.19 (1H, d, J ) 5.0 Hz), 4.31 (1H, d,
J ) 6.5 Hz), 4.47 (1H, dd, J ) 5.0. 9.0 Hz), 4.53 (1H, d, J )
5.0 Hz), 4.62 (2H, d, J ) 5.7 Hz), 5.22 (1H, dd, J ) 1.2, 10.4
Hz), 5.33 (1H, d, J ) 1.5, 17.2 Hz), 5.39 (1H, d, J ) 9.4 Hz),
5.53 (1H, dd, J ) 6.5, 15.5 Hz), 5.89-5.97 (1H, m), 6.19 (1H,
d, J ) 15.6 Hz), 7.13-7.25 (5H, m); 13C NMR (90 MHz, CD3-
OD) δ 13.1, 13.6, 15.4, 16.2, 16.5, 18.5, 19.5, 27.8, 28.8 (3C),
30.8, 31.6, 31.9, 37.6, 38.9, 41.4, 45.4, 52.8, 53.0, 53.6, 55.9,
58.7, 58.9, 66.6, 80.2, 88.3, 118.9, 126.8, 127.0, 129.2, 130.5,
133.4, 133.7, 136.9, 137.4, 140.5, 157.6, 172.4, 173.5, 173.6,
174.8, 176.4, 177.2; HRMS calcd for C49H75N5O13 [M]+ 942.5439,
found 942.5413.
1
2847, 2362, 2341, 1645 cm-1; H NMR (500 MHz, CD3OD) δ
0.95 (3H, d, J ) 6.0 Hz), 1.01 (6H, d, J ) 7.0 Hz), 1.14 (3H, d,
J ) 7.0 Hz), 1.20 (3H, d, J ) 7.5 Hz), 1.37-1.40 (3H, m), 1.62
(3H, s), 1.91-1.94 (1H, m), 2.25-2.40 (2H, m), 2.57-2.69 (2H,
m), 2.78-2.83 (2H, m), 2.89-2.94 (2H, m), 3.03 (3H, s), 3.18-
3.23 (2H, m), 3.23 (3H, s), 3.23-3.26 (2H, m), 3.23-3.26 (2H,
m), 3.82 (1H, t, J ) 7.5 Hz), 4.38-4.53 (3H, m), 5.21-5.24 (1H,
m), 5.39 (1H, d, J ) 9.5 Hz), 5.52 (1H, dd, J ) 7.0 and 16.0
Hz), 6.24 (1H, d, J ) 15.5 Hz), 7.14-7.26 (5H, m); 13C NMR
(90 MHz, CD3OD), δ 12.9, 13.9, 16.1, 16.5, 19.5, 19.7, 27.5,
30.4, 30.6, 31.2, 37.7, 38.9, 40.6, 46.2, 54.3, 55.4, 56.2, 58.8,
61.4, 88.4, 126.3, 127.1, 129.2, 130.5, 133.8, 136.9, 138.1, 140.5,
172.9, 173.5, 174.2, 175.5, 175.6, 176.8; HRMS calcd for
C39H57N5O10 [M + H]+ 756.4184, found 756.4187.
Com p ou n d 46. Ma cr ocyliza tion via th e P en ta flu or o-
p h en yl Ester . Pentapeptide 45 (148 mg, 0.157 mmol) was
dissolved in THF (16 mL), and dimedone (66 mg, 0.471 mmol)
and Pd(PPh3)4 (18 mg, 0.0157 mmol) were added. The solution
was stirred at room temperature for 21 h. After removal of
the solvents under reduced pressure, chromatography on SiO2
(ethyl acetate/methanol) provided the pentapeptide acid (117
mg, 82%). To a portion of this acid (31.5 mg, 0.035 mmol) in
ethyl acetate (3 mL) was added pentafluorophenol (19.3 mg,
0.105 mmol), DCC (11.0 mg, 0.053 mmol), and DMAP (0.7 mg,
0.007 mmol), and the solution was stirred at room temperature
overnight. Concentration and chromatography on SiO2 (5%
MeOH in ethyl acetate) gave the activated ester (30 mg, 80%).
This ester was dissolved in CH2Cl2 (5 mL), and TFA (2 mL)
was added. After stirring at room temperature for 1 h, the
reaction mixture was concentrated, redissolved in CH2Cl2, and
re-evaporated repeatedly to remove all of the excess TFA. This
sample was kept under high vacuum overnight. To the dry
cyclization precursor was added CH3CN (28 mL) followed by
DIPEA (98 µL, 0.56 mmol) and DMAP (0.7 mg, 5.6 × 10-3
mmol). This dilute solution was stirred at room temperature
for 7 d. Evaporation of the solvent followed by chromatography
on SiO2 (5% MeOH in ethyl acetate) gave 46 (9.3 mg, 42%):
P r otein P h osp h a ta se Assa y. Protein phosphatase assays
were performed in reaction mixtures containing 0.5 µL 3T3-
L1 cell lysate,78 4 µL PP1 buffer (50 mM Hepes, pH 7.4, 2 mM
EDTA, 2 mg mL-1 glycogen, 0.2% 2-mercaptoethanol, 0.1 mM
PMSF, 1 mM benzamidine, and 10 µg mL-1 aprotinen), 15 µL
PP1 buffer containing 6 nM okadaic acid, and 0.5 µL of
inhibitor solution or an appropriate control. These mixtures
were incubated at 37 °C for 1 min before the addition of 10 µL
32P-labeled phosphorylase (15 µM in 50 mM Hepes, pH 7.4,
15 mM caffeine, 0.1 mM EDTA, 0.2% 2-mercaptoethanol;
specific activity 2000-3000 cpm pmol-1) to initiate the reac-
tion. After 6 min, the reactions were terminated by the
addition of 90 µL ice-cold 20% trichloroacetic acid, and 5 µL
2% BSA in H2O was added to aid protein precipitation. The
samples were allowed to stand on ice for 10 min, and then the
proteins were pelleted by centrifugation at 15 000g for 2 min.
The amount of radioactive phosphate released into the super-
natant was measured by scintillation counting. All measure-
ments were performed in duplicate in at least two separate
assays.
1
Rf ) 0.3 (5% MeOH in CH2Cl2); H NMR (500 MHz, CD3OD,
Ack n ow led gm en t. This work was supported by the
donors to the Petroleum Research Fund administered
by the American Chemical Society and by NSF grant
CHE-932133. Technical assistance was provided by
Karin Stein, Erika Taylor, and Robert Shurmer. We
thank Professor Raymond Anderson for supplying a
sample of authentic motuporin and relevant NMR
spectra and Dana Dejohn and J im Windak for mass
spectral analyses.
50 °C) δ 0.95 (3H, d, J ) 7.5 Hz), 1.01 (3H, d, J ) 6.5 Hz),
1.12 (3H, d, J ) 6.5 Hz), 1.15 (3H, d, J ) 7.0 Hz), 1.23 (3H, d,
J ) 7.0 Hz), 1.40 (3H, d, J ) 6.5 Hz), 1.62 (3H, s), 1.90-1.92
(1H, m), 2.25-2.40 (2H, m), 2.59-2.69 (4H, m), 2.78-2.82 (2H,
dd, J ) 4.5 and 14.0 Hz), 3.13-3.19 (2H, m), 3.03 (3H, s), 3.21-
3.24 (2H, m), 3.23 (3H, s), 3.66-3.72 (1H, m), 3.72 (3H, s), 3.74
(3H, s), 3.77 (1H, d, J ) 6.5 Hz), 4.47-4.50 (2H, m), 4.55 (1H,
d, J ) 3.0 Hz), 5.17-5.19 (1H, m), 5.40 (1H, d, J ) 9.5 Hz),
5.50 (1H, dd, J ) 7.5 and 15.0 Hz), 6.25 (1H, d, J ) 16.0 Hz),

2728 J . Org. Chem., Vol. 64, No. 8, 1999
Samy et al.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
for compound 48. This material is available free of charge via
the Internet at http://pubs.acs.org.
1
spectra for compounds 7-9, 29, 39, 40, 43, and 47; H NMR
1
spectrum and HPLC trace for compound 1; H NMR spectra
for compounds 41 and 46; and 1H NMR and mass spectrum
J O982145I