Probing host-selective phytotoxicity: Synthesis and biological activity of phomalide, isophomalide, and dihydrophomalide

Article abstract of DOI:10.1021/jo982376p

The cyclic depsipeptide phomalide [cyclo(Val-(E)-Aba-Hpp-Hmp-(R)-Leu); Aba = 2-amino-2-butenoic acid, Hpp = (2S)-2-hydroxy-3-phenylpropanoic acid, Hmp = (2S)-2-hydroxy-4-methylpentanoic acid] is the host-selective phytotoxin produced by the fungus [Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.] which causes blackleg disease (a devastating disease of several economically important brassica crops). Efficient total syntheses of phomalide, its (Z)-isomer isophomalide, and the two dihydro analogues [(R)-dihydrophomalide and (S)-dihydrophomalide] are described. A [2 + 3] fragment coupling of Cbz-Val-(Z)-Aba with Hpp-Hmp-D-Leu-OBn followed by deprotection and cyclization gave isophomalide which was diastereoselectively isomerized to phomalide by conjugate addition of PhSeH followed by elimination of the corresponding selenoxide. The dihydro analogues were prepared similarly using Cbz-Val-(R)-Abu or Cbz-Val-(S)-Abu (Abu = 2-aminobutanoic acid) in place of Cbz-Val-(Z)-Aba. Biological evaluations of phomalide, isophomalide, and the dihydrophomalides revealed that only phomalide (10^-5 M) caused necrotic, chlorotic, and reddish lesions on canola (Brassica napus and Brassica rapa; susceptible to blackleg) leaves whereas no damage was observed on brown mustard (Brassica juncea; resistant to blackleg) or white mustard (Sinapis alba; resistant to blackleg) leaves, even at significantly higher concentrations (10^-4 M). Thus, both the presence and configuration of the double bond is crucial for selective phytotoxicity. This is the first reported synthesis of an (E)-Aba-containing natural product, and importantly, the (Z) → (E) isomerization approach should be applicable to other (depsi)peptide targets thereby allowing investigation of the effect of the double-bond configuration on various properties.

Full text of DOI:10.1021/jo982376p

J . Org. Chem. 1999, 64, 1657-1666
1657
P r obin g Host-Selective P h ytotoxicity: Syn th esis a n d Biologica l
Activity of P h om a lid e, Isop h om a lid e, a n d Dih yd r op h om a lid e
Dale E. Ward,* Alfredo Va´zquez, and M. Soledade C. Pedras*
Department of Chemistry, University of Saskatchewan, 110 Science Place,
Saskatoon SK S7N 5C9, Canada
Received December 3, 1998
The cyclic depsipeptide phomalide [cyclo(Val-(E)-Aba-Hpp-Hmp-(R)-Leu); Aba ) 2-amino-2-butenoic
acid, Hpp ) (2S)-2-hydroxy-3-phenylpropanoic acid, Hmp ) (2S)-2-hydroxy-4-methylpentanoic acid]
is the host-selective phytotoxin produced by the fungus [Leptosphaeria maculans (Desm.) Ces. et
de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.] which causes blackleg disease (a
devastating disease of several economically important brassica crops). Efficient total syntheses of
phomalide, its (Z)-isomer isophomalide, and the two dihydro analogues [(R)-dihydrophomalide and
(S)-dihydrophomalide] are described. A [2 + 3] fragment coupling of Cbz-Val-(Z)-Aba with Hpp-
Hmp-D-Leu-OBn followed by deprotection and cyclization gave isophomalide which was diastereo-
selectively isomerized to phomalide by conjugate addition of PhSeH followed by elimination of the
corresponding selenoxide. The dihydro analogues were prepared similarly using Cbz-Val-(R)-Abu
or Cbz-Val-(S)-Abu (Abu ) 2-aminobutanoic acid) in place of Cbz-Val-(Z)-Aba. Biological evaluations
of phomalide, isophomalide, and the dihydrophomalides revealed that only phomalide (10-5 M)
caused necrotic, chlorotic, and reddish lesions on canola (Brassica napus and Brassica rapa;
susceptible to blackleg) leaves whereas no damage was observed on brown mustard (Brassica juncea;
resistant to blackleg) or white mustard (Sinapis alba; resistant to blackleg) leaves, even at
significantly higher concentrations (10-4 M). Thus, both the presence and configuration of the double
bond is crucial for selective phytotoxicity. This is the first reported synthesis of an (E)-Aba-containing
natural product, and importantly, the (Z) f (E) isomerization approach should be applicable to
other (depsi)peptide targets thereby allowing investigation of the effect of the double-bond
configuration on various properties.
In tr od u ction
The ability of a fungal pathogen to damage host plant
cells can involve the biosynthesis and release of host-
selective phytotoxins.1 Host-selective phytotoxins are
chemical signals which facilitate pathogen penetration
and colonization of host plant tissues but do not signifi-
cantly affect non host plants. Although many fungal
diseases appear to be mediated by host-selective toxins,
the molecular basis for the selectivity of this process is
not well understood in the majority of the cases.2 One
such disease, blackleg, affects several economically im-
portant brassica crops and can be particularly devastat-
ing for the oilseeds canola (Brassica napus and Brassica
rapa) and rapeseed (B. napus and B. rapa).3 Recently, a
better understanding of the blackleg causing fungus
[Leptosphaeria maculans (Desm.) Ces. et de Not., asexual
stage Phoma lingam (Tode ex Fr.) Desm.] was achieved
with the isolation of phomalide (1), a host-selective toxin
that appears to be involved in disease development.4
Phomalide (1) is a rather unusual cyclic depsipeptide
composed of three R-amino acid and two R-hydroxy acid
residues. The identity of the individual residues and their
sequence were deduced from spectroscopic data.4 The
fragmentation pattern in the mass spectrum of the
derived methyl ester 4 served to corroborate the sequence
suggested by NMR (HMBC). The absolute configurations
of the L-valine (Val), D-leucine (D-Leu), (2S)-2-hydroxy-
4-methylpentanoic acid (Hmp), and (2S)-2-hydroxy-3-
phenylpropanoic acid (Hpp) residues were firmly estab-
lished by acid hydrolysis of 1 and comparison of the
resulting intact residues with authentic samples.4,5 The
(E)-configuration of the 2-amino-2-butenoic (Aba) residue
in 1 was assigned on the basis of the observation of a
positive NOE for the vinylic proton upon saturation of
the enamine N-H. Within this class of natural products,6
phomalide (1) has several noteworthy structural features
including the 15-membered ring, the consecutive ester
(1) For a recent multiauthor review on phytotoxins, see: Graniti,
A. et al. Experientia 1991, 47, 751-826.
(2) Kohomoto, K.; Otani, H. Experientia 1991, 47, 756-764.
(3) For a recent review on the history and control of blackleg disease,
see: Gugel, R. K.; Petrie, G. A. Can. J . Plant Pathol. 1992, 14, 36-45.
(4) Pedras, M. S. C.; Taylor, J . T.; Nakashima, T. T. J . Org. Chem.
1993, 58, 4778-4780.
(5) Pedras, M. S. C. Can. J . Chem. 1997, 75, 314-317.
10.1021/jo982376p CCC: $18.00 © 1999 American Chemical Society
Published on Web 02/13/1999

1658 J . Org. Chem., Vol. 64, No. 5, 1999
Ward et al.
Sch em e 1
linkages, and particularly the unusual (E)-Aba residue.
Numerous peptides and depsipeptides that incorporate
R,â-unsaturated amino acid residues and possess diverse
biological activity have been reported;7,8 where diastereo-
isomerism is possible, those residues are most often
present in the thermodynamically more stable (Z)-con-
figuration.
Because of its host-selective phytotoxicity, phomalide
(1) is an excellent probe for investigating the blackleg
disease resistance of agriculturally important Brassica
species. However, due to significant problems associated
with obtaining sufficient quantities of 1 from fungal
cultures,9,10 its chemical synthesis became an essential
prerequisite for biological studies. The synthetic strategy
typically employed for the preparation of cyclic depsipep-
tides (and peptides) involves linear or convergent cou-
pling of intact hydroxy acid and amino acid fragments
followed by cyclization.11 This approach reduces the key
strategic decisions to the site of cyclization and the order
of the residue coupling. Application of this strategy to
phomalide (1) leaves the introduction of the Aba residue
and control of its stereochemistry as major concerns.
Typically, R,â-unsaturated amino acid residues show
reduced reactivity in both C- and N-terminal acylations
and the free amino forms hydrolyze readily.7,12 As a
consequence, R,â-unsaturated amino acids are usually
not incorporated into peptides as intact residues but
rather are elaborated from suitable precursor residues
after peptide coupling and often late in the synthesis.
Most syntheses of Aba derivatives give the thermo-
dynamically more stable (Z)-isomers selectively7 and
stereoselective syntheses of (Z)-Aba-containing natural
products by incorporating threonine as the surrogate
residue with subsequent â-elimination have been de-
scribed.13 Although the stereoselective preparation of
simple (E)-Aba derivatives has been achieved by syn-
elimination of certain threonine derivatives14 or by anti-
elimination of allo-threonine derivatives,15 these methods
have not been applied to stereocontrolled peptide (or
depsipeptide) synthesis.
It is generally recognized that the presence of R,â-
unsaturated amino acid residues imparts unique reactiv-
ity and conformational properties to peptides. Nonethe-
less, in part due to the lack of access to the (E)-isomers,
the effect of the double-bond configuration on these
properties has not been investigated.7 In fact, to the best
of our knowledge, the synthesis of an (E)-Aba-containing
natural product has not been previously reported. In this
paper we report the stereoselective syntheses of phoma-
lide (1), its (Z)-isomer isophomalide (2), and the two
dihydro analogues (R)-dihydrophomalide [(R)-3] and (S)-
dihydrophomalide [(S)-3].16 In addition, we establish that
the double-bond configuration has a remarkable effect
on the toxicity of these molecules to blackleg resistant
and susceptible plants.
(6) (a) Turner, W. B.; Aldrige, D. C. Fungal Metabolites II; Academic
Press: New York, 1983; pp 436-442. (b) Kleinkauf, H.; von Doheren,
H. Eur. J . Biochem. 1990, 192, 1-15.
(7) For reviews, see: (a) Schmidt, U.; Lieberknecht, A.; Wild, J .
Synthesis 1988, 159-172. (b) Noda, K.; Shimohigashi, Y.; Izumiya, N.
In The Peptides; Gross, E., Meienhofer, J ., Eds.; Academic Press: New
York, 1983; Vol. 5, pp 285-339.
(8) For some recent examples of peptides and depsipeptides contain-
ing the (Z)-Aba residue, see: (a) Bonnard, I.; Rolland, M.; Francisco,
C.; Banaigs, B. Lett. Pept. Sci. 1997, 4, 289-292. (b) Hamann, M. T.;
Otto, C. S.; Scheuer, P. J .; Dunbar, D. C. J . Org. Chem. 1996, 61, 6594-
6600. (c) Ballio, A.; Bossa, F.; Camoni, L.; Di Giorgio, D.; Flamand,
M.-C.; Maraite, H.; Nitti, G.; Pucci, P.; Scaloni, A. FEBS Lett. 1996,
381, 213-216. (d) Namikoshi, M.; Choi, B. W.; Sakai, R.; Sun, F.;
Carmichael, W. W.; Evans, W. R.; Cruz, P. J . Org. Chem. 1994, 59,
2349-2357. (e) Gulavita, N. K.; Pomponi, S. A.; Wright, A. E.; Yarwood,
D.; Sills, M. A. Tetrahedron Lett. 1994, 35, 6815-1688. (f) Ballio, A.;
Bossa, F.; Di Giorgio, D.; Ferranti, P.; Paci, M.; Pucci, P.; Scaloni, A.;
Segre, A.; Strobel, G. A. FEBS Lett. 1994, 355, 96-100. (g) Shigematsu,
N.; Ueda, H.; Takase, S.; Tanaka, H.; Yamamoto, K.; Tada, T. J .
Antibiot. 1994, 47, 311-314. (h) Scaloni, A.; Bachmann, R. C.;
Takemoto, J . Y.; Barra, D.; Simmaco, M.; Ballio, A. Nat. Prod. Lett.
1994, 4, 159-164. For examples containing an (E)-Aba residue, see:
(i) Sano, T.; Kaya, K. Tetrahedron 1998, 54, 463-870. (j) Schummer,
D.; Forche, E.; Wray, V.; Domke, T.; Reichenbach, H.; Hoefle, G. Liebigs
Ann. 1996, 971-978. (k) Bewley, C. A.; He, H.; Williams, D. H.;
Faulkner, D. J . J . Am. Chem. Soc. 1996, 118, 4314-4321. (l) Perga-
ment, I.; Carmeli, S. Tetrahedron Lett. 1994, 35, 8473-8476. (m)
Minami, Y.; Yoshida, K.-i.; Azuma, R.; Urakawa, A.; Kawauchi, T.;
Otani, T. Tetrahedron Lett. 1994, 35, 8001-8004.
(9) For a recent review on phytotoxins of the blackleg fungus, see:
Pedras, M. S. C. Rev. Latinoam. Quim. 1996, 24, 177-182.
(10) Pedras, M. S. C.; Bisenthal, C. J . Can. J . Microbiol. 1998, 44,
547-553
(11) (a) Bodanszky, M. Principles of Peptide Synthesis, 2nd ed.;
Springer-Verlag: Berlin, 1993. (b) Izumiya, N.; Kato, T.; Aoyagi, H.;
Waki, M.; Kondo, M. Synthetic Aspects of Biologically Acitve Cyclic
Peptides; Halsted Press: New York, 1979. (c) Kopple, K. D. J . Pharm.
Sci. 1972, 61, 1345-1356.
(12) Humphrey, J . M.; Chamberlin, A. R. Chem. Rev. 1997, 97,
2243-2266.
Resu lts a n d Discu ssion
Our retrosynthetic analysis of phomalide (1) is pre-
sented in Scheme 1. The choice of the cyclization site is
often crucial to the successful synthesis of a cyclic peptide
or depsipeptide and a number of guidelines for effective
selection have evolved.11c,12,17 Consideration of these ideas
focused our attention on the D-Leu-Val and Hmp-D-Leu
(13) (a) Li, K. W.; Wu, J .; Xing, W.; Simon, J . A. J . Am. Chem. Soc.
1996, 118, 7237-7238. (b) Valentekovich, R. J .; Schreiber, S. L. J . Am.
Chem. Soc. 1995, 117, 9069-9070. (c) Meldal, M.; Bielfeldt, T.; Peters,
S.; J ensen, K. J .; Paulsen, H.; Bock, K. Int. J . Pept. Protein Res. 1994,
43, 529-536. (d) Fukase, K.; Kitazawa, M.; Sano, A.; Shimbo, K.;
Horimoto, S.; Fujita, H.; Kubo, A.; Wakamiya, T.; Shiba, T. Bull. Chem.
Soc. J pn. 1992, 65, 2227-2240. (e) Schmidt, U.; Mundinger, K.;
Mangold, R.; Lieberknecht, A. J . Chem. Soc., Chem. Commun. 1990,
1216-1219. (f) Mihara, H.; Aoyagi, H.; Ueno, T.; Kato, T.; Izumiya, N.
Bull. Chem. Soc. J pn. 1986, 59, 2041-2043.
(14) Rich, D. H.; Tam, J . P. J . Org. Chem. 1977, 42, 3815-3820,
and cited references.
(15) Somekh, L.; Shanzer, A. J . Org. Chem. 1983, 48, 907-908, and
cited references.
(16) For a preliminary communication, see: Ward, D. E.; Vazquez,
A.; Pedras, M. S. C. J . Org. Chem. 1996, 61, 8008-8009.

Probing Host-Selective Phytotoxicity
J . Org. Chem., Vol. 64, No. 5, 1999 1659
Sch em e 2
linkages in 1 as potential sites for cyclization because
lactamization is favored over lactonization and cyclization
between residues of opposite absolute configuration is
particularly favorable.18 The two five-residue linear
precursors associated with these disconnections each
presents the opportunity for convergent synthesis via a
[2 + 3] fragment coupling approach because the Aba-Hpp
linkage can be formed without the usual risk of C-
terminal epimerization. This strategy also allows for
flexibility in the choice of an Aba precursor (e.g., D- or
L-threonine or allo-threonine)19 should it be necessary to
generate this residue later in the synthesis. Despite the
greater steric hindrance, we chose D-Leu-Val site for
cyclization because (i) comparing the fragments required
for preparation of the two possible five-residue linear
precursors, i.e., Val-Aba + Hpp-Hmp-D-Leu versus D-Leu-
Val-Aba + Hpp-Hmp, the former could be prepared by
more direct syntheses with less need for protecting
groups; (ii) if the use of an Aba surrogate was required,
it would be easier to prepare dipeptides with alternate
C-terminal residues (i.e., Val-Xxx) compared to tripep-
tides (i.e., D-Leu-Val-Xxx). Thus our synthetic plan
required the preparation of an N-protected Val-(E)-Aba
dipeptide and a C-protected Hpp-Hmp-D-Leu tridepsipep-
tide followed by fragment coupling, deprotection, and
cyclization; because orthogonal protecting groups could
be unnecessary, our initial synthetic targets were (E)-5
and 6.
The three component residues of the tridepsipeptide 6
are readily available. Silylation20a of Hpp-OH21 gave 11
(Scheme 2). Condensation of Hmp-OH (8)22 with the
p-toluenesulfonic acid salt of D-Leu-OBn (9)23 mediated
by BOP24 gave the didepsipeptide 10 (90%)25 which, in
turn, was acylated with TBSO-Hpp-Cl (12; generated20
in situ from 11) in the presence of DMAP to provide 7 in
95% yield. Removal of the silyl ether protecting group in
7 (HF, CH3CN; 93%) gave the desired the tridepsipeptide
fragment Hpp-Hmp-D-Leu-OBn (6).
Condensation of 2-oxobutanoic acid (13) with Cbz-Val-
NH2 (14)26 gave the dipeptide fragment Cbz-Val-Aba (5)
as a separable 4:1 mixture of (Z)-5 and (E)-5, respectively,
in good yield after optimization of the general procedure
of Rzeszotarska et al.27 (Scheme 2). The Aba configura-
tions were assigned28 on the basis of positive NOE
correlations observed between the vinyl methyl group and
the enamine N-H in (Z)-5 and between the vinyl proton
and the enamine N-H in (E)-5 and are in accord with
the thermodynamic preference for the (Z)-isomer estab-
lished in similar examples.7,27,29 Although this procedure
has been reported to give racemic dipeptides in some
examples,27b 5 was obtained with at least 80% enantio-
meric purity under our conditions.30
(17) (a) Cavelier-Frontin, F.; Achmad, S.; Verducci, J .; J acquier, J .;
Pe`pe, G. J . Mol. Struct. (THEOCHEM) 1993, 286, 125-130. (b)
Cavelier-Frontin, F.; Pe`pe, G.; Verducci, J .; Siri, D.; J acquier, J . J .
Am. Chem. Soc. 1992, 114, 4, 8885-8890. (c) Ueda, K.; Waki, M.;
Izumiya, N. Int. J . Pept. Protein Res. 1987, 30, 33-39, and cited
references.
(18) (a) Brady, S. F.; Varga, S. L.; Friedinger, R. M.; Schwenk, D.
A.; Mendlowski, M.; Holly, F. W.; Veber, D. F. J . Org. Chem. 1979, 44,
3101-3105. (b) Kessler, H.; Kutscher, B. Liebigs Ann. Chem. 1986,
869-892. (c) Kessler, H.; Kutscher, B. Liebigs Ann. Chem. 1986, 914-
931. (d) Ehrlich, A.; Heyne, H.-U.; Winter, R.; Beyermann, M.; Haber,
H.; Carpino, L. A.; Bienert, M. J . Org. Chem. 1996, 61, 8831-8838.
(19) Changing the configuration of one residue in the linear precur-
sor can have a dramatic effect on cyclization.11c,12,17
(20) (a) Wissner, A.; Grudzinskas, C. V. J . Org. Chem. 1978, 43,
3972-3974. (b) Ward, D. E.; Rhee, C. K. Tetrahedron Lett. 1991, 32,
7165-7166.
(21) Urban, F. J .; Moore, B. S. J . Heterocycl. Chem. 1992, 29, 431-
438.
Initial attempts to couple (Z)-5 or (E)-5 with 6 using
DCC/DMAP gave highly variable yields (35-75%) of (Z)-
15 as the only isolated product. To further investigate
(22) Degerbeck, F.; Fransson, B.; Grehn, L.; Ragnarsson, U. J . Chem.
Soc., Perkin Trans. 1 1993, 11-14.
(23) Miller, H. K.; Waelsch, H. J . Am. Chem. Soc. 1952, 74, 1092-
1093.
(24) Abbreviations for reagents: AIBN, 2,2′-azobis(2-methylpro-
panenitrile); (Boc)2O, bis(1,1-dimethylethyl) dicarbonate; BOP, (1H-
benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophos-
phate; BOP-Cl, N,N-bis(2-oxo-3-oxazolidinyl)phosphinic chloride; CSA,
10-camphorsulfonic acid; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;
DCC, dicyclohexylcarbodiimide; DEAD, diethyl azodicarboxylate; DMAP,
4-(dimethylamino)pyridine; DPPA, diphenylphosphoryl azide; EDC,
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; FDPP,
pentafluorophenyl diphenylphosphinate; HOBt, 1-hydroxybenzotria-
zole; TFA, trifluoroacetic acid.
(26) Prepared from Cbz-Val according to Wang’s procedure: Chen,
S.-T.; Wu, S.-H.; Wang, K.-T. Synthesis 1989, 37-38.
(27) (a) Makowski, M.; Rzeszotarska, B.; Kubica, Z.; Wieczorek, P.
Liebigs Ann. Chem. 1984, 920-928. (b) Smelka, L.; Rzeszotarska, B.;
Pietrznski, G.; Kubica, Z. Liebigs Ann. Chem. 1988, 485-486.
(28) Shimohigashi, Y.; Nitz, T. J .; Stammer, C. H. Tetrahedron Lett.
1982, 23, 3235-3236.
(29) (a) Srinivasan, A.; Stephensen, R. W.; Olsen, R. K. J . Org. Chem.
1977, 42, 2256-2260. (b) Nitz, T. J .; Holt, E. M.; Rubin, B.; Stammer,
C. H. J . Org. Chem. 1981, 46, 2667-2673. (c) Goodall, K.; Parsons, A.
F. Tetrahedron Lett. 1995, 36, 3259-3260.
(25) Similar results16 were obtained using DCC/HOBt in place of
BOP and/or with the hydrochloride salt of D-Leu-OBn.23

1660 J . Org. Chem., Vol. 64, No. 5, 1999
Ward et al.
modest to good overall yields from 15: BOP-Cl35 (20%),
DPPA36 (31%), FDDP37 (48%), BOP38 (57%). Improved
yields and better reproducibility were obtained using the
pentafluorophenyl-activated ester method for cycliza-
tion.39 Thus, hydrogenolysis of 15 in the presence40 of di-
tert-butyl dicarbonate gave 17 (90%) which was converted
into the corresponding pentafluorophenyl ester 18 (85%).
The Boc group in 18 was removed by treatment with
TFA, and the resulting trifluoroacetate salt was added
to a two-phase mixture of CHCl3 and aqueous NaHCO3
to give 2 (82%; 62% from 15).39b,c Isophomalide (2) was
clearly differentiated from phomalide (1) by NMR,41 by
HPLC,42 and other properties (vide infra). For example,
conversion of 1 to 4 occurs in aqueous methanol solution
within 24 h at ambient temperature; under the same
conditions, 2 is converted into (Z)-4 with a half-life of >30
days.
Initial attempts to isomerize (Z)-15 were unsuccessful,
and this approach was abandoned after discovering that
cyclization of (E)-1543 via the pentafluorophenyl ester as
described above gave a 1.2:1 mixture of 2 and 1, respec-
tively in 64% yield. Numerous attempts to isomerize 2
into 1 under acidic (TFA, CDCl3, rt; CSA, THF, 66 °C),
basic (Et3N, CDCl3, rt; DBU, benzene, rt; NaH, THF, rt;
BuLi, THF, -78 °C), free radical (HSi(SiMe3)3, AIBN,
PhH, 80 °C),44 or photochemical conditions (PhSSPh, hν)45
failed. Conjugate addition of PhCH2SeH (or PhSeH) to
(Z)-isomers of simple R,â-unsaturated amino acid deriva-
tives followed by oxidation has been reported to give the
corresponding (E)-isomers with fair to good stereoselec-
tivity (2-10:1).46 Reaction of 2 with excess PhSeH (3
equiv) and NaOMe (0.05 equiv) in refluxing THF accord-
ing to the procedure of Mazur and Pilipauskas46 gave a
2.5:1 mixture of 19a and 19c in 38% yield along with
recovered 2 (28%); alternatively, the use of BuLi (0.05
equiv) as base under the same conditions gave the readily
separable selenides 19a (52%), 19b (12%), and 19c (6%).
Oxidation of 19a with H2O2 gave phomalide (1; 94%)
uncontaminated with isophomalide (2).47,48 Similar reac-
Sch em e 3
this process, the condensation of 5 with 21 was examined
in detail (Scheme 3). Reaction of 5 with EDC (or DCC)
at 0 °C rapidly gave the isolable 4-ethylidene-5(4H)-
oxazolone 20 as a 2:1 mixture of (Z)-:(E)-isomers (32%
yield) from (E)-5 or as the (Z)-isomer (90% yield) from
(Z)-5.31 In CDCl3 solution, reaction of (Z)-20 with 21
occurred very slowly if at all (by 1H NMR) in the presence
or absence of DMAP; however, the coupling product 22
could be detected immediately after concentration of the
reaction mixture. The adduct 22 was obtained as a
separable 4:1 mixture of (Z)-:(E)-isomers in 60% yield
from (Z)-20 by allowing the concentrated reaction mix-
ture to stand at room temperature for 30 min prior to
fractionation.32 Similarly, reaction of (Z)-20 or a 2:1
mixture of (Z)-20:(E)-20 with 6 under these conditions
gave, in each case, an approximately 10:1 mixture of (Z)-
15 and (E)-15, respectively (45-60% yield).31 These
results clearly indicated that isomerization of the Aba
residue in 5 occurs both during formation of the oxazolone
and during acylation33 and suggested that incorporation
of the desired (E)-configuration would require a stereo-
selective isomerization of a (Z)-isomer at a later stage in
the synthesis. The yield of the coupling reaction could
be improved by avoiding the isolation of the oxazolone
20 and, under optimized conditions, the DCC/DMAP-
mediated condensation of 6 with the 4:1 mixture of (Z)-
5:(E)-5 gave 15 as a 10-12:1 mixture of (Z)- and (E)-
isomers, respectively, in 91% yield.
Chemoselective removal of the benzyl ester and car-
bamate protecting groups in the presence of the olefin
in 15 required reduction under catalytic transfer hydro-
genation conditions34 to give the putative amino acid 16.
Cyclization of 16 using various coupling reagents under
standard conditions (ca. 1 mM) gave isophomalide (2) in
(35) (a) Diago-Meseguer, J .; Palomo-Coll, A. L. Synthesis 1980, 547-
551. (b) Ishiwata, H.; Sone, H.; Kigoshi, H.; Yamada, K. Tetrahedron
1994, 50, 12853-12882.
(36) (a) Shiori, T.; Ninomuja, K.; Yamada, S. J . Am. Chem. Soc.
1972, 94, 6203-6206 (b) Cavelier, F.; J acquier, R.; Mercadier, J .-L.;
Verducci, J . Tetrahedron 1996, 52, 6173-6186.
(37) (a) Chen, S.; Xu, J . Tetrahedron Lett. 1991, 32, 6711-6714. (b)
Wipf, P.; Fritch, P. C. J . Am. Chem. Soc. 1996, 118, 12358-12367.
(38) Wenger, R. M. Helv. Chim. Acta 1984, 67, 502-525
(39) (a) Schmidt, U.; Lieberknecht, A.; Griesser, H.; Talbiersky, J .
J . Org. Chem. 1982, 47, 3261-3264. (b) Schmidt, U.; Utz, R.; Lie-
berknecht, A.; Griesser, H.; Potzolli, B.; Bahr, J .; Wagner, K.; Fischer,
P. Synthesis 1987, 236-241. (c) Schmidt, U.; Weinbrenner, S. J . Chem.
Soc., Chem. Commun. 1994, 1003-1004.
(40) (a) Sakaitani, M.; Hori, K.; Ohfune, Y. Tetrahedron Lett. 1988,
29, 2983-2984. (b) Bajwa, J . S.; Tetrahedron Lett. 1992, 33, 2955-
2956.
(41) Small differences in the chemical shifts of the signals for the
Aba residues were apparent; otherwise, the 1H and 13C NMR spectra
of 1 were very similar to those of 2 (∆δH < 0.1; ∆δC < 0.5).
(42) Retention times for 1 and 2 were 30.4 and 29.8 min, respec-
tively, with a hypersil ODS column (5-µm particle size; 4.6 × 200 mm)
using MeCN/water gradient elution (1 mL/min; 25-75% MeCN over
35 min).
(43) Obtained in 8% yield by careful fractionation (SiO2; benzene/
ether/acetone, 90:5:5) of a 10:1 mixture of (Z)-15 and (E)-15.
(44) Chatgilialoglu, C.; Ballestri, M.; Ferreri, C.; Vecchi, D. J . Org.
Chem. 1995, 60, 3826-3831.
(45) Thalmann, A.; Oertle, K.; Gerlach, H. Org. Synth. 1984, 63,
192-197.
(46) (a) Mazur, R. H.; Pilipauskas, D. R. Pept.: Synth, Struct.,
Funct., Proc. Am. Pept. Symp., 7th; Rich, D. H.; Gross, E., Eds.; Pierce
Chem. Co.: Rockford, IL, 1981; pp 81-84. (b) Mazur, R. H.; Pilipaus-
kas, D. R. Pept., Proc. Eur. Pept. Symp., 17th; 1982; Blaha, K.; Malon,
p., Eds.; de Gruyter: Berlin, 1983; pp 319-322.
(30) For related examples, the reported27b reaction conditions involve
refuxing in benzene for 4-10 h with p-TsOH (0.2 equiv). In our case,
we examined various conditions and found that refluxing in toluene
for 20-30 min gave improved yields and a much cleaner reaction.
Hydrolysis of 5 (obtained under optimized conditions) by heating
overnight in HCl/HOAc/H2O gave 14 of 80% ee as determined by optical
rotation. That 80% ee is the minimum enantiopurity of 5 is further
supported by obtention of (Z)-15 and (E)-15 in >90% combined yield;
other diastereomers were not detected.
(31) The stereochemistry of 20 was readily assigned by 1H NMR in
analogy with related compounds: (a) Arenal, I.; Bernabe, M.; Fernadez-
Alvarez, E. An. Quim., Ser. C 1981, 77, 56-62. (b) Cativiela, C.; Diaz
De Villegas, M. D.; Mayoral, J . A.; Melendez, E. Synthesis 1983, 899-
902.
(32) The configurations of the Aba residues in (Z)-22 and (E)-22 were
assigned by 1H NMR on the basis of the well-documented trend that
the chemical shifts for the vinyl proton, the vinyl methyl group, and
the enamine N-H are at lower fields in the (E)-isomers than in the
(Z)-isomers. For example, see: (a) Srinivasan, A.; Richards, K. D.;
Olsen, R. K. Tetrahedron Lett. 1976, 891-894. (b) Shin, C.; Hayakawa,
M.; Suzuki, T.; Ohtsuka, A. Bull. Chem. Soc. J pn. 1978, 51, 550-554.
(33) Isomerization during acylation with C-terminal R,â-unsaturated
amino acids is precedented: Makowski, M.; Rzezotarska, B.; Kubica,
Z.; Pietrzynski, G.; Hetper, J . Liebigs Ann. Chem. 1986, 980-991.
(34) Felix, A. M.; Himer, E. P.; Lambros, T. J .; Tzougraki, C.;
Meienhofer, J . J . Org. Chem. 1978, 43, 4194-4196.

Probing Host-Selective Phytotoxicity
J . Org. Chem., Vol. 64, No. 5, 1999 1661
is known to proceed with syn stereoselectivity,53 these
latter results established the absolute configurations for
the selenides 19. Thus, a (2R,3R)-configuration can be
assigned for the 2-amino-3-(phenylselenyl)butanoic acid
residue in 19a because reduction gives (S)-3 and oxida-
tion gives 1; by similar reasoning, the same residues in
19b and 19c are assigned the (2R,3S)- and (2S,3S)-
configurations, respectively. Interestingly, addition of
PhSeH to 1 gave a mixture of the selenides 19 (19a :19b:
19c ) 8:2.5:1 by HPLC) in a ratio similar to that obtained
from 2 (19a :19b:19c ) 7.5:1.5:1 by HPLC). These results
indicate that preferential formation of the like diastere-
omer on addition of PhSeH to the Aba residues in 1 and
2 under these conditions is thermodynamically con-
trolled.54 By contrast, although the like diastereomer
predominated in all cases, addition of selenols to (E)
versus (Z)-isomers of simple R,â-unsaturated amino acid
derivatives reportedly gives very different ratios of adduct
diastereomers suggesting kinetically controlled stereo-
selectivity.46 Nonetheless, the diastereoselective trans-
formation of a (Z)-R,â-unsaturated amino acid residue
into the corresponding (E)-isomer via the like â-selenide
derivative appears to have useful scope. Considering the
mildness of this method and the relatively straight-
forward access to peptides containing (Z)-R,â-unsaturated
amino acid residues,7,13 isomerization of those peptides
is an attractive strategy for the synthesis of the elusive
(E)-isomers.
We have evaluated the phytotoxicity of phomalide (1),
its (Z)-isomer (isophomalide, 2), and the two dihydro
derivatives (R)-3 and (S)-3 to plants susceptible (canola)
and resistant [brown mustard (Brassica juncea) and
white mustard (Sinapis alba)] to blackleg, as previously
reported for other toxins.55 The naturally occurring
phomalide (1) caused necrotic, chlorotic, and reddish
lesions on canola leaves (10-5 M), whereas no damage
was observed on brown or white mustard leaves, even
at significantly higher concentrations (10-4 M). Thus, the
selective phytotoxicity of phomalide (1) appears to mimic
the pathogenicity range of the blackleg fungus; i.e.,
phomalide causes lesions on plants that are susceptible
to blackleg infection (canola), but not on resistant plants
(brown and white mustards). Most importantly, the (Z)-
isomer 2 did not cause lesions on any of the plant leaves
tested, even at 5 × 10-4 M. Interestingly, however, both
dihydrophomalides (R)-3 and (S)-3 (5 × 10-5 M) caused
chlorotic lesions on brown mustard but not on canola or
white mustard. These results indicate that both the
presence and configuration of the double bond in phoma-
lide (1) are important for selective phytotoxicity; however,
it remains to be determined if and how resistant plants
enzymatically convert 1 to less toxic products. Prelimi-
nary results indicate that phomalide (1) is metabolized
by brown mustard leaf tissue to a significantly more polar
compound.
Sch em e 4
tion of 19c also gave 1 (85%) exclusively but only 2 (90%)
was obtained from oxidation of 19b. Synthetic phomalide
was identical in all respects (1H and 13C NMR, [R]D, TLC
and HPLC retention times) with an authentic sample.
The dihydrophomalide analogues (R)-3 and (S)-3 were
prepared from 2-aminobutanoic acid (23) as shown in
Scheme 4. Reactions of 24 with (R)-23 and (S)-23 gave
the dipeptide acids (R)-25 (75%) and (S)-25 (70%) which
were separately esterified (with inversion of configura-
tion) with the tridepsipeptide alcohol 2649 via a Mit-
sunobu reaction50 to give (R)-27 (89%) and (S)-27 (63%),
respectively. The latter fragment coupling procedure
ensured the configurational fidelity of the 2-aminobu-
tanoic acid (Abu) residue. Cyclizations of (R)-27 and (S)-
27 using the pentafluorophenyl-activated ester method
gave (R)-3 (67%) and (S)-3 (44%), respectively. The
considerably higher yields obtained in (R)-series reflect
the greater facility of preparation and cyclization of
(depsi)peptides containing adjacent residues with op-
posite absolute configurations.11c,13,17 The dihydrophoma-
lide diastereomers 3 are readily distinguished by NMR
and have very different chromatographic mobilities.51
Hydrogenation of either 1 or 2 over Pd-C gave (S)-3
diastereoselectively (<5% (R)-3). Alternatively, reduction
(Ph3SnH, AIBN)52 of the selenides 19a or 19b gave (S)-3
(85%); similar reduction of 19c gave (R)-3 (80%). Because
elimination of selenides via the corresponding selenoxides
(47) For other examples of dehydroamino acid syntheses by sele-
noxide fragmentation, see: (a) Walter, R.; Roy, J . J . Org. Chem. 1971,
36, 2561-6563. (b) Reich, H. J .; J asperse, C. P.; Renga, J . M. J . Org.
Chem. 1986, 51, 2981-2988. (c) Hashimoto, K.; Sakai, M.; Okuno, T.;
Shirahama, H. Chem. Commun. 1996, 1139-1140.
(48) Phomalide and isophomalide are separable (PTLC; 30% ethyl
acetate in hexane, multiple development), albeit with considerable
difficulty (phomalide is slightly less polar) and with low efficiency (i.e.,
mainly mixed fractions are obtained).
In summary, we have achieved an efficient synthesis
of the host-selective phytotoxin phomalide (1) and, thereby,
the first synthesis of an (E)-Aba-containing natural
(49) Obtained in 90% yield from 10 and (2R)-2-hydroxy-3-phenyl-
propanoic acid in analogy to the preparation of 6.
(50) (a) Lee, B. H. Tetrahedron Lett. 1997, 38, 757-760. (b) Ohyama,
M.; Iinuma, K.; Suzuki, A. Biosci. Biotech. Biochem. 1994, 58, 1193-
1194. (c) Schmidt, U.; Langner, J . J . Chem. Soc., Chem. Commun. 1994,
2381-2382.
(53) Sharpless, K. B.; Young, M. W.; Lauer, R. F. Tetrahedron Lett.
1973, 1719-1722.
(54) Addition of PhSeH to Aba residues in acyclic peptides also gives
the like diastereomer selectively. For example reactions of Val-(Z)-Aba-
Hpp-Hmp derivatives with PhSeH/BuLi followed by oxidation gave the
corresponding (E)-isomers with ca. 5:1 diastereoselectivity (Ward, D.
E.; Va´zquez, A. Unpublished results).
(51) TLC, SiO2 (50% ethyl acetate in hexane): (R)-3, Rf ) 0.67; (S)-
3, Rf ) 0.26.
(52) Clive, D. L. J .; Chittattu, G. J .; Farina, V.; Kiel, W. A.; Menchen,
S. M.; Russell, C. G.; Singh, A.; Wong, C. K.; Curtis, N. J . J . Am. Chem.
Soc. 1980, 102, 4438-4447.
(55) Pedras, M. S. C.; Se´guin-Swartz, G.; Abrams, S. R. Phytochem-
istry 1990, 29, 777-782.

1662 J . Org. Chem., Vol. 64, No. 5, 1999
Ward et al.
168 °C (MeCN, H2O); [R]D -48 (c 0.19, CHCl3); IR νmax 3282,
3064, 1747, 1693, 1665, 1649 cm-1; 1H NMR (500 MHz) δ 8.00
(1H, br s), 7.30-7.23 (5H, m), 6.80 (1H, q, J ) 7.5 Hz), 6.40
(1H, d, J ) 8 Hz), 6.39 (1H, d, J ) 9 Hz), 5.39 (1H, dd, J ) 5,
8 Hz), 5.33 (1H, dd, J ) 4, 9 Hz), 4.38 (1H, ddd, J ) 7, 8, 8
Hz), 4.24 (1H, dd, J ) 9, 9 Hz), 3.30 (1H, dd, J ) 4, 14.5 Hz),
3.16 (1H, dd, J ) 9, 14.5 Hz), 2.26-2.19 (1H, m), 1.83 (3H, d,
J ) 7.5 Hz), 1.80-1.75 (1H, m), 1.74-1.69 (1H, m), 1.61-1.54
(2H, m), 1.53-1.48 (1H, m), 1.36-1.30 (1H, m), 0.99 (3H, d, J
) 6.5 Hz), 0.94 (3H, d, J ) 7 Hz), 0.92 (3H, d, J ) 6.5 Hz),
0.89 (3H, d, J ) 6.5 Hz), 0.86 (3H, d, J ) 6.5 Hz), 0.83 (3H, d,
J ) 6.5 Hz); 13C NMR (125 MHz) δ 172.3, 170.0, 169.0, 168.2,
166.0, 135.0, 131.8, 129.3 (×2), 128.6 (×2), 127.4, 124.5, 76.1,
72.6, 59.0, 52.1, 39.2, 39.2, 37.0, 27.2, 24.9, 24.1, 22.9, 22.4,
22.3, 21.8, 19.9, 18.0, 14.4; LRMS (EI) m/z (rel intensity) 557
([M]+, 72), 72 (100); HRMS m/z calcd for C30H43N3O7 557.3101,
found 557.3092 (EI). Anal. Calcd for C30H43N3O7‚0.2H2O: C,
64.20; H, 7.79; N, 7.49. Found: C, 64.04; H, 7.61; N, 7.42.
Isop h om a lid e [cyclo(Va l-(Z)-Aba -Hp p -Hm p -(R)-Leu )]
(2). From 19b: Oxidation of 19b (3 mg, 0.004 mmol) with 30%
H2O2 (0.025 mL, 1.16 mmol) in acetone (0.5 mL) at 0 °C as
described above gave 2 (2 mg, 90%). From 18: To a stirred
solution of the pentafluorophenyl ester 18 (308 mg, 0.365
mmol) in CH2Cl2 (6 mL) at 0 °C was added TFA (2 mL). After
1 h, the mixture was concentrated, and the residue was
evacuated at high vacuum (0.5 Torr) for 1 h. The residue was
dissolved in CH2Cl2 (7 mL), and the solution was added over
5 min to a vigorously stirred mixture of CHCl3 (7 mL) and
saturated NaHCO3(aq) (7 mL). Stirring was continued for 2.5
h, and then the aqueous layer was diluted with brine and
extracted with CH2Cl2 (×2). The combined organic layers was
dried over Na2SO4, concentrated, and fractionated by FCC
(30% ethyl acetate in hexane) to give 2 as a white solid (167
mg, 82%; 62% overall yield from 15): mp 168-169 °C (CH2-
Cl2/hexane); [R]D -44 (c 0.14, CHCl3); IR νmax 3334, 3282, 1744,
product. The key step in the synthesis involved a dia-
stereoselective isomerization of the (Z)-isomer isophoma-
lide (2), itself prepared in 45% overall yield in 6 steps
from the readily available 8 and 9 via a [2 + 3] fragment
coupling approach followed by cyclization. The strategy
is suitable for the preparation of analogues as demon-
strated by the synthesis of (R)-3 and (S)-3 and should be
amenable to the preparation of radiolabeled congeners.
Importantly, this (Z) f (E) isomerization approach
should be applicable to other (depsi)peptide targets
thereby allowing an investigation of the effect of the
double-bond configuration on various properties. As
illustrated with 1 and 2, the double-bond configuration
can have a remarkable influence on chemical and biologi-
cal properties, particularly phytotoxicity.
Exp er im en ta l Section
Gen er a l Meth od s. All solvents were distilled prior to use.
Unless otherwise noted, reactions were carried out under an
atmosphere of argon and reaction temperatures refer to the
bath. Concentration refers to removal of volatiles at water
aspirator pressure on a rotary evaporator. Preparative thin-
layer chromatography (PTLC) was carried out on glass plates
(20 × 20 cm) precoated (0.25 mm) with silica gel 60 F254. Flash
column chromatography (FCC), dry flash column chromatog-
raphy (DFC), and medium-pressure chromatography (mpc)
were performed according to the procedures of Still et al.,56
Harwood,57 and Taber,58 respectively. All mixed solvent eluents
are reported as volume-to-volume (v/v) solutions.
Sp ectr a l Da ta a n d An a lysis. High-resolution mass spec-
tra (HRMS) and low-resolution mass spectra (LRMS) were
obtained on a double focusing high-resolution spectrometer;
only partial data are reported. Electron impact (EI) ionization
was accomplished at 70 eV, chemical ionization (CI) at 50 eV
with ammonia as the reagent gas, and fast-atom bombardment
(FAB) in positive ion mode from a glycerol and MeOH matrix.
Infrared spectra were recorded on a Fourier transform inter-
ferometer using a diffuse reflectance cell (DRIFT); only
diagnostic peaks are reported. Unless otherwise noted, NMR
spectra were measured in CDCl3 solution at 300 MHz for 1H
1727, 1683, 1651 cm-1 1H NMR (500 MHz) δ 7.73 (1H, s),
;
7.32-7.24 (5H, m), 6.67 (1H, q, J ) 7 Hz), 6.55 (1H, d, J ) 8.5
Hz), 6.21 (1H, d, J ) 10 Hz), 5.43 (1H, dd, J ) 4, 9.5 Hz), 5.14
(1H, dd, J ) 4, 8.5 Hz), 4.41 (1H, ddd, J ) 7.5, 8, 8.5 Hz), 4.23
(1H, dd, J ) 10, 10 Hz), 3.25 (1H, dd, J ) 14.5, 4 Hz), 3.16
(1H, dd, J ) 14.5, 8.5 Hz), 2.19-2.11 (1H, m), 1.82-1.77 (1H,
m), 1.80 (3H, d, J ) 7 Hz), 1.71-1.65 (2H, m), 1.59-1.45 (3H,
m), 1.15-1.08 (1H, m), 0.98 (3H, d, J ) 6.5 Hz), 0.96 (3H, d,
J ) 7 Hz), 0.91 (3H, d, J ) 6.5 Hz), 0.90 (3H, d, J ) 6.5 Hz),
0.78 (3H, d, J ) 6.5 Hz), 0.77 (3H, d, J ) 6.5 Hz); 13C NMR
(125 MHz) δ 172.0, 169.5, 169.1, 168.1, 166.3, 135.2 (×2), 129.5
(×2), 128.6 (×2), 127.4, 125.5, 75.5, 72.4, 59.3, 51.8, 39.3, 39.3,
37.0, 27.0, 24.9, 23.8, 23.1, 22.5, 22.4, 21.3, 19.7, 18.3, 15.3;
LRMS (FAB) m/z (rel intensity) 558 ([M + 1]+, 34), 119 (100);
HRMS m/z calcd for C30H43N3O7+H 558.3179 (M + H), found
558.3186 (FAB). Anal. Calcd for C30H43N3O7: C, 64.61; H, 7.77;
N, 7.53. Found: C, 64.38; H, 7.86; N, 7.33.
1
and 75 MHz for13C. The H NMR chemical shifts and coupling
constants were determined assuming first-order behavior.
Multiplicity is indicated by one or more of the following: s
(singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br
(broad), ap (apparent). The list of coupling constants (J ;
reported to the nearest 0.5 Hz) corresponds to the order of the
multiplicity assignment. Optical rotations ([R]D) were deter-
mined at ambient temperature using a 1 mL, 10 dm cell; the
units are 10-1 deg cm2 g-1, and the concentrations (c) are
reported in g/100 mL.
(R)-Dih yd r op h om a lid e [cyclo(Va l-(R)-Abu -Hp p -Hm p -
(R)-Leu )] [(R)-3]. From 19c: A stirred solution of Ph3SnH (8
mg, 0.024 mmol), 19c (4 mg, 0.006 mmol), and AIBN (a small
crystal) in toluene (0.50 mL) was heated under reflux under
argon. After 16 h, the cooled (rt) reaction mixture was
concentrated and fractionated by MPC (SiO2, 1.5 g; 50% ethyl
acetate in hexane) to give (R)-3 (2.7 mg, 80%). From (R)-27:
A mixture of (R)-27 (93 mg, 0.12 mmol) and 10% Pd/C (10 mg)
in 95% EtOH (1.5 mL) was stirred under hydrogen (1 atm) at
ambient temperature for 1 h. The mixture was filtered through
Celite, and the combined filtrate and washings was concen-
trated to give a foam (82 mg). DCC (25 mg, 0.12 mmol) was
added to a stirred solution of the above foam and C6F5OH (22
mg, 0.12 mmol) in ethyl acetate (0.6 mL) at 0 °C. After 1 h of
stirring, the mixture was filtered, and the combined filtrate
and washings was concentrated and fractionated by FCC (SiO2;
25% ethyl acetate in hexane) to provide the pentafluorophenyl
ester as a foam (85 mg, 83%). TFA (1.0 mL) was added to a
stirred solution of the active ester (57 mg, 0.066 mmol) in CH2-
Cl2 (1.0 mL) at 0 °C. After 90 min, the mixture was concen-
trated and reconcentrated twice from toluene. The residue was
dissolved in CH2Cl2 (2.0 mL), and the solution was added
dropwise via syringe over 5 min to a vigorously stirred mixture
Ma ter ia ls. (R)- and (S)-Hpp,21 (S)-Hmp,22 D-Leu-OBn‚p-
TsOH,23 Boc-Val, and Cbz-Val were prepared from the corre-
sponding amino acids according to literature procedures. All
other reagents were commercially available and, unless oth-
erwise noted, were used as received.
P h om a lid e [cyclo(Va l-(E)-Aba -Hp p -Hm p -(R)-Leu )] (1).
From 19a : 30% H2O2 (0.100 mL, 1.16 mmol) was added via
syringe to a solution of selenide 19a (98 mg, 0.14 mmol) in
acetone (2 mL) at 0 °C. The reaction mixture was stirred for
30 min at 0 °C and then diluted with water and extracted with
ethyl acetate (×2). The combined organic layers was washed
with brine, dried over Na2SO4, concentrated, and fractionated
by FCC (30% ethyl acetate in hexane) to give phomalide (1)
(72 mg, 94%) as a white solid which was identical in all
respects with an authentic sample. From 19c: Oxidation of
19c (6 mg, 0.008 mmol) as above with 30% H2O2 (0.025 mL)
gave 1 (4 mg, 85%): mp 155-156 °C (CH2Cl2/hexane), 165-
(56) Still, W. C.; Kahn, M.; Mitra, A. J . Org. Chem. 1978, 43, 2923-
2925.
(57) Harwood: L. M. Aldrichimica Acta 1985, 18, 25-25.
(58) Taber, D. F. J . Org. Chem. 1982, 47, 1351-1352.

Probing Host-Selective Phytotoxicity
J . Org. Chem., Vol. 64, No. 5, 1999 1663
of CHCl3 (2.0 mL) and saturated NaHCO3(aq) (2.0 mL). After
12.5 h of stirring at room temperature, the two phases were
separated. The aqueous layer was diluted with brine and
extracted with ethyl acetate (×3). The combined organic layers
was washed with brine, dried over Na2SO4, concentrated, and
fractionated by FCC (SiO2; 30% ethyl acetate in hexane) to
provide (R)-3 as a white solid (31 mg, 84%, 70% overall from
(R)-27): [R]D -19 (c 0.8, CHCl3); IR νmax 3376, 3258, 3068, 1737,
1680, 1642 cm-1; 1H NMR (500 MHz) δ 7.2-7.3 (5H, m), 6.50
(1H, d, J ) 9.5 Hz), 6.27 (1H, d, J ) 9 Hz), 5.99 (1H, d, J ) 10
Hz), 5.42 (1H, dd, J ) 4.5, 9 Hz), 5.15 (1H, dd, J ) 3.5, 9 Hz),
4.60 (1H, ddd, J ) 7, 7.5, 9.5 Hz), 4.49 (1H, ddd, J ) 7.5, 7.5,
9 Hz), 4.04 (1H, ap t, J ) 10, 10 Hz), 3.25 (1H, dd, J ) 3.5,
14.5 Hz), 3.14 (1H, dd, J ) 9, 14.5 Hz), 2.13-2.06 (1H, m),
1.79-1.45 (8H, m), 0.95 (3H, d, J ) 7 Hz), 0.94 (3H, d, J ) 6.5
Hz), 0.91 (3H, d, J ) 6.5 Hz), 0.89 (3H, d, J ) 6.5 Hz), 0.84
(3H, d, J ) 6.5 Hz), 0.82 (3H, d, J ) 6.5 Hz), 0.75 (3H, t, J )
7.5 Hz); 13C NMR (125 MHz) δ 175.2, 172.75, 171.6, 169.5,
168.1, 135.4, 129.7 (×2), 128.8 (×2), 127.6, 75.4, 72.8, 59.6,
53.3, 51.2, 39.5, 39.0, 37.1, 27.4, 26.0, 25.0, 24.3, 23.3, 22.9,
22.5, 21.8, 19.9, 18.7, 10.0; LRMS (EI) m/z (rel intensity) 559
([M]+, 24), 72 (100); HRMS m/z calcd for C30H45N3O7 559.3258,
found 559.3260 (EI). Anal. Calcd for C30H45N3O7: C, 64.38; H,
8.10; N, 7.51. Found: C, 64.48; H, 7.88; N, 7.48.
172.7, 170.2, 169.9, 163.8, 135.5, 131.0, 129.4 (×2), 128.8 (×2),
127.5, 125.2, 74.3, 70.9, 59.2, 52.6, 51.7, 43.7, 40.7, 37.5, 30.6,
25.0, 24.7, 23.6, 23.0, 22.3, 21.5, 19.5, 17.9, 14.5; LRMS (FAB)
m/z (rel intensity) 590 ([M + 1]+, 43), 72 (100); HRMS m/z calcd
for C31H47N3O8+H 590.3441 (M + H), found 590.3413 (FAB).
Hm p -(R)-Leu -Va l-(Z)-Aba -Hp p -OCH3 [(Z)-4]. A solution
of 2 (2.0 mg, 0.004 mmol) in MeOH (2.0 mL) and H2O (1.0
mL) was stirred at room temperature for 56 days. The reaction
mixture was concentrated, extracted with CH2Cl2 (×2), and
the combined organic layers was dried over Na2SO4 and
concentrated. The residue was fractionated by PTLC (50%
ethyl acetate in hexane) to give recovered 2 (1 mg, 50%) and
(Z)-4 (1 mg, 50%): [R]D -15 (c 0.4, CHCl3); IR νmax 3285, 1730,
1656, 1647, 1644 cm-1 1H NMR (500 MHz) δ 7.57 (1H, s),
;
7.33-7.17 (5H, m), 7.00 (1H, d, J ) 7 Hz), 6.89 (1H, d, J ) 9
Hz), 6.85 (1H, q, J ) 7 Hz), 5.25 (1H, dd, J ) 5.5, 7 Hz), 4.44
(1H, ddd, J ) 7, 7.5, 8 Hz), 4.37 (1H, dd, J ) 6, 8.5 Hz), 4.09
(1H, dd, J ) 2.5, 9 Hz), 3.70 (3H, s), 3.25-3.10 (2H, m), 2.90
(1H, bs), 2.34-2.18 (1H, m), 1.85-1.52 (5H, m), 1.73 (3H, d, J
) 7 Hz), 1.49-1.35 (1H, m), 0.99-0.89 (18H, m); 13C NMR
(500 MHz) δ 175.6, 172.5, 170.2, 169.6, 163.8, 136.4, 135.7,
129.5 (×2), 128.8 (×2), 127.4, 125.5, 74.3, 70.9, 58.9, 53.5, 51.9,
43.7, 40.3, 37.6, 30.2, 25.0, 24.7, 23.6, 23.0, 22.4, 21.5, 19.7,
17.8, 15.3; LRMS (EI) m/z (rel intensity) 589 ([M]+, 6), 72 (100);
HRMS m/z calcd for C31H47N3O8: 589.3363, found 598.3364
(EI).
(S)-Dih yd r op h om a lid e [cyclo(Va l-(S)-Abu -Hp p -Hm p -
(R)-Leu )] [(S)-3]. From 1: A mixture of phomalide (15 mg,
0.026 mmol) and 10% Pd/C (5 mg) in ethyl acetate (1.0 mL)
was stirred under H2 (1 atm) at room temperature for 3 h.
The mixture was filtered, and the combined filtrate and
washings was concentrated to give (S)-3 (15 mg, 100%). From
2: Isophomalide (61 mg, 0.11 mmol) was hydrogenated over
10%Pd/C (12 mg) as above to give (S)-3 (51 mg, 84%). From
19a : A stirred solution of PH3SnH (20 mg, 0.056 mmol), 19a
(10 mg, 0.014 mmol), and AIBN (a small crystal) in toluene
(0.50 mL) was heated under reflux under argon. After 23 h,
the cooled (rt) reaction mixture was concentrated and fraction-
ated by MPC (SiO2, 2 g; 50% ethyl acetate in hexane) to give
(S)-3 (7 mg, 88%). From 19b: Selenide 19b (4 mg, 0.006 mmol)
was reduced with Ph3SnH (8 mg, 0.024 mmol) as described
above to yield (S)-3 (2.8 mg, 85%). From (S)-27: As described
above for (R)-27 f (R)-3, (S)-27 (92 mg, 0.12 mmol) was
converted into the corresponding pentafluorophenyl ester (94
mg, 92%). Cyclization of the active ester (57 mg, 0.066 mmol)
gave (S)-3 as a white solid (18 mg, 49%) after fractionation by
FCC (SiO2; 50% ethyl acetate in hexane): [R]D -47 (c 1.0,
Cbz-Va l-(E)-Aba -OH [(E)-5] and Cbz-Va l-(Z)-Aba -OH
[(Z)-5]. A mixture of Cbz-Val-NH2 (14; 375 mg, 1.5 mmol),
2-oxobutanoic acid (230 mg, 2.25 mmol), and p-TsOH‚H2O (57
mg, 0.3 mmol) in dry toluene (2.5 mL) was heated under reflux
with azeotropic water removal for 30 min during which time,
a precipitate formed and the solution became thick. The cooled
(rt) reaction mixture was diluted with toluene, and the
precipitate was filtered off and then suspended in saturated
NaHCO3(aq) (30 mL). The suspension was stirred for 30 min
and then extracted with ethyl acetate (×4). The combined
organic layers was dried over Na2SO4 and concentrated to give
recovered 14 (120 mg, 32%). The aqueous phase was acidified
(pH 3-4) with citric acid and then extracted with ethyl acetate
(×4). The combined organic layers was washed with brine,
dried over Na2SO4, and concentrated to give 5 as a white solid
[a 4:1 mixture of (Z):(E) diastereomers; 232 mg, 46% (68%
based on conversion)] which was used in the next step without
further purification. The 4:1 mixture of isomers (190 mg) was
fractionated by FCC (SiO2; CHCl3:pyridine:HOAc, 84:4:2; dry
loading) to give (E)-5 as a white solid (28 mg; Rf ) 0.4) and
(Z)-5 as a white solid (120 mg; Rf ) 0.26). For (E)-5: [R]D +16
1
CHCl3); IR νmax 3270, 3264, 1747, 1672, 1650, cm-1; H NMR
(500 MHz) δ 7.15-7.35 (5H, m), 6.88 (1H, d, J ) 9 Hz), 6.32
(1H, d, J ) 9.5 Hz), 5.23 (1H, dd, J ) 7, 7 Hz), 5.09 (1H, dd,
J ) 5, 8 Hz), 4.53 (1H, ddd, J ) 6, 8.5, 9 Hz), 4.39 (1H, dd, J
) 4.5, 9.5 Hz), 4.21 (1H, ddd, J ) 7.5, 7.5, 7.5 Hz), 3.18 (1H,
dd, J ) 4.5, 14 Hz), 3.13 (1H, dd, J ) 8.5, 14 Hz), 2.45-2.35
(1H, m), 1.98-1.77 (3H, m), 1.77-1.68 (1H, m), 1.65-1.48 (3H,
m), 1.48-138 (1H, m), 0.97-0.87 (21H, m); 13C NMR (125
MHz) δ 171.6, 171.4, 171.2, 171.1, 168.7, 135.3, 129.6 (×2),
128.8 (×2), 127.5, 75.4, 72.9, 59.6, 53.9, 53.3, 40.5, 38.6, 36.9,
29.3, 25.0, 24.5, 24.2, 22.9, 22.6 (×2), 22.5, 19.8, 17.3, 10.6;
LRMS (EI) m/z (rel intensity) 559 ([M]+, 18), 72 (100); HRMS
m/z calcd for C30H45N3O7 559.3258, found 559.3254 (EI). Anal.
Calcd for C30H45N3O7: C, 64.34; H, 8.10; N, 7.51. Found: C,
64.18; H, 7.85; N, 7.42.
1
(c 0.6, DMF); IR νmax 3617, 3291, 1691, 1659 cm-1; H NMR
(500 MHz) δ 9.25 (1H, s), 7.40-7.29 (5H, m), 6.22 (2H, m),
5.03 (2H, s), 3.87 (1H, m), 1.99 (1H, m), 1.91 (3H, d, J ) 7.5
Hz), 0.88 (3H, d, J ) 7.0 Hz), 0.85 (3H, d, J ) 6.5 Hz); 13C
NMR (125 MHz) δ 169.9, 165.4, 156.2, 137.0, 129.0, 128.4 (×2),
127.7, 127.6 (×2), 123.0, 65.4, 60.5, 30.2, 19.2, 18.1, 13.5; LRMS
(Cl) m/z (rel intensity) 335 ([M + 1]+, 79), 91 (100); HRMS
m/z calcd for C17H22N2O5+H 335.1607 (M + H), found 335.1609
1
(FAB). For (Z)-5: [R]D +24 (c 0.6, DMF); H NMR ((CD3)2SO)
δ 9.09 (1H, s), 7.36-7.21 (6H, m), 6.52 (1H, q, J ) 7 Hz), 5.03
(2H, ap s), 3.98 (1H, dd, J ) 7, 8.5 Hz), 2.03-1.96 (1H, m),
1.63 (3H, d, J ) 7 Hz), 0.93 (3H, d, J ) 6.5 Hz), 0.88 (3H, d,
J ) 6.5 Hz); 13C NMR (500 MHz, (CD3)2SO) δ 170.1, 165.4,
156.1, 137.1, 131.8, 128.3 (×2), 127.7, 127.6 (×2), 124.7, 65.4,
60.2, 30.3, 19.2, 18.1, 13.5; LRMS (CI, NH3) m/z (rel intensity)
Hm p -(R)-Leu -Va l-(E)-Aba -Hp p -OCH3 (4). A solution of
1 (3 mg, 0.005 mmol) in MeOH (2.0 mL) and H2O (1.0 mL)
was stirred at room temperature for 29 h. The reaction mixture
was concentrated and extracted with CH2Cl2 (×2), and the
combined organic layers were dried over Na2SO4 and concen-
trated. The residue was fractionated by PTLC (50% ethyl
acetate in hexane) to provide 4 (3 mg, >90%): [R]D -10 (c 0.4,
335 ([M + 1]+, 25), 227 (100); HRMS m/z calcd for C17H22
-
N2O5+H 335.1607 (M + H), found 335.1590 (FAB). Anal. Calcd
for C17H22N2O5: C, 61.06; H, 6.63; N, 8.37. Found C, 61.26; H,
6.43; N, 8.12.
Hp p -Hm p -(R)-Leu -OBn (6). A solution of 7 (1.04 g, 1.73
mmol) in 5% HF/CH3CN (20 mL) was stirred at room temper-
ature for 2 h. The mixture was concentrated, diluted with CH2-
Cl2, washed with water, saturated NaHCO3(aq), and brine, dried
over Na2SO4, and concentrated. The resulting white solid
residue was fractionated by FCC (30% ethyl acetate in hexane)
to afford 6 as a white solid (783 mg, 93%): mp 83-84 °C (CH2-
Cl2/hexane); [R]D +2.0 (c 1.0, MeOH); IR νmax 3491, 3408, 1734,
1652 cm-1; 1H NMR δ 7.40-7.21 (10H, m), 6.29 (1H, d, J ) 8
CHCl3); IR νmax 3299, 1737, 1656, 1648 cm-1 1H NMR (500
;
MHz) δ 8.0 (1H, s), 7.32-7.14 (6H, m), 7.10 (1H, d, J ) 8.5
Hz), 6.93 (1H, q, J ) 7.5 Hz), 5.37 (1H, dd, J ) 5.5, 7 Hz),
4.49 (1H, ddd, J ) 6, 8, 8 Hz), 4.26 (1H, dd, J ) 6, 8.5 Hz),
4.11 (1H, ddd, J ) 3.5, 6, 10 Hz), 3.71 (3H, s), 3.47 (1H, d, J
) 6 Hz), 3.24 (1H, dd, J ) 5.5, 14.5 Hz), 3.18 (1H, dd, J ) 7,
14.5 Hz), 2.22-2.10 (1H, m), 1.96 (3H, d, J ) 7.5 Hz), 1.71-
1.46 (6H, m), 0.95-0.86 (18H, m); 13C NMR (125 MHz) δ 175.7,

1664 J . Org. Chem., Vol. 64, No. 5, 1999
Ward et al.
Hz), 5.26 (1H, dd, J ) 4.5, 8.5 Hz), 5.21-5.11 (2H, m), 4.68-
4.61 (1H, ddd, J ) 5, 8, 9 Hz), 4.55-4.45 (1H, m), 3.21 (1H,
dd, J ) 14, 4.5 Hz), 2.99 (1H, dd, J ) 14, 7 Hz), 2.63 (1H, d,
J ) 2.5 Hz), 1.79-1.41 (6H, m), 0.96-0.87 (12H, m); 13C NMR
δ 173.0, 172.3, 169.4, 136.0, 135.3, 129.5, 128.6, 128.5, 128.3,
127.1, 73.9, 71.5, 67.2, 50.6, 41.4, 40.6, 40.3, 24.8, 24.4, 23.0,
22.7, 21.9, 21.7; LRMS (FAB) m/z (rel intensity) 484 ([M +
1]+, 100); HRMS m/z calcd for C28H37NO6+H 484.2699 (M +
H), found 484.2714 (FAB). Anal. Calcd for C28H37NO: C, 69.54;
H, 7.71; N, 2.89. Found C, 69.39; H, 7.92; N, 2.88.
(CI, NH3) m/z (rel intensity) 395 ([M + 1]+, 100); HRMS m/z
calcd for C21H38Si2O3+H 395.2438 (M + H), found 395.2421
(CI, NH3). Anal. Calcd for C21H38Si2O3: C, 63.90; H, 9.70.
Found C, 63.90; H, 9.79. The (R)-enantiomer (ent-11) ([R]D +23;
c 1.1, CHCl3) was obtained from (R)-phenyllactic acid using
the same procedure.
Cbz-Va l-NH2 (14). EDC (3.40 g, 17.8 mmol) was added to
a stirred mixture of Cbz-Val (4.26 g, 17.0 mmol) and HOBt
(2.52 g, 18.6 mmol) in CH2Cl2 (80 mL) and DMF (20 mL) at 0
°C. After 30 min, 28% aqueous NH3 (1.4 mL, 22 mmol) was
added dropwise to the reaction mixture (precipitate formation).
The resulting thick mixture was stirred for an additional 1.5
h at 0 °C and 4 h at room temperature and then was filtered.
The combined filtrate and washings was diluted with CH2Cl2,
washed with saturated NaHCO3(aq) and water, and dried over
Na2SO4. The filter cake was triturated with boiling MeCN, and
after removal of the insoluble material, the filtrate was
combined with the previously obtained organic layer. Concen-
tration gave a white solid that was recrystallized from MeCN
to afford Cbz-Val-NH2 as white needles (3.82 g, 90%): mp 200-
202 °C (MeCN); [R]D +23 (c 1.0, DMF); IR νmax 3379, 3317,
TBSO-Hp p -Hm p -(R)-Leu -OBn (7). Oxalyl chloride (0.61
mL, 7.0 mmol) was added dropwise into a solution of 11 (2.37
g, 6.0 mmol) and DMF (0.043 mL, 0.60 mmol) in CH2Cl2 (8
mL) at 0 °C under argon. After 0.5 h of stirring for at 0 °C
and a further 4 h at room temperature, the reaction mixture
was diluted with dry hexane (10 mL), and the resulting solid
was removed by filtration. The combined filtrate and washings
was concentrated, and a solution of 10 (1.68 mg, 5.0 mmol)
and DMAP (0.733 g, 6.0 mmol) in CH2Cl2 (8 mL) was added.
After 2 h of stirring at room temperature, the reaction mixture
was diluted with a 1:1 mixture of hexane and ethyl acetate
and then was washed sequentially with water, 10% citric acid,
saturated NaHCO3(aq), and brine, dried over Na2SO4, and
concentrated. The resulting yellow oil was fractionated by FCC
(10% ethyl acetate in hexane) to give 7 as a colorless oil (2.84
g, 95%): [R]D -26 (c 1.0, CHCl3); IR νmax 3424, 3326, 1753,
1743, 1691, 1667 cm-1; 1H NMR δ 7.35-7.21 (5H, m), 6.31 (1H,
d, J ) 8.5 Hz), 5.28 (1H, dd, J ) 4, 9 Hz), 5.21-5.11 (2H, m),
4.68-4.65 (1H, ddd, J ) 5, 8.5, 8.5 Hz), 4.43 (1H, dd, J ) 4,
8.5 Hz), 3.13 (1H, dd, J ) 4, 13.5 Hz), 2.93 (1H, dd, J ) 8.5,
13.5 Hz), 1.77-1.46 (6H, m), 0.93-0.87 (12H, m), 0.81 (9H,
s), -0.08 (3H, s), -0.22 (3H, s); 13C NMR δ 172.3, 171.9, 169.7,
136.9, 135.3, 129.8, 128.6, 128.4, 128.3, 128.3, 126.8, 73.5, 73.1,
67.1, 50.5, 41.6, 41.4, 40.7, 25.6, 24.8, 24.4, 23.1, 22.7, 21.9,
21.6, 18.1, -5.2, -5.7; LRMS (FAB) m/z (rel intensity) 598 ([M
+ 1]+, 49), 91 (100); HRMS m/z calcd for C34H51NSiO6+H
598.3564 (M + H), found 598.3571 (FAB). Anal. Calcd for
3294, 1657 cm-1 1H NMR ((CD3)2SO) δ 7.34-7.30 (5H, m),
;
7.09 (1H, d, J ) 9 Hz), 6.99 (1H, br s), 5.02 (2H, ap s), 3.80
(1H, dd, J ) 7, 9 Hz), 1.97-1.91 (1H, m), 0.86 (3H, d, J ) 7
Hz), 0.82 (3H, d, J ) 7 Hz); 13C NMR ((CD3)2SO) δ 173.1 (s),
156.1 (s), 137.1 (s), 128.2 (d ×4), 127.6 (d), 65.3 (t), 60.0, 30.1,
19.2, 17.9; LRMS (CI, NH3) m/z (rel intensity) 251 ([M + 1]+,
21), 225 (100); HRMS m/z calcd for C13H18N2O3+H 251.1396
(M + H), found 251.1405 (FAB). Anal. Calcd for C13H18N2O3:
C, 62.38; H, 7.24; N, 11.19. Found C, 62.50; H, 7.28; N, 10.99.
Cbz-Va l-(Z)-Aba -Hp p -Hm p -(R)-Leu -OBn (15). DCC (203
mg, 0.986 mmol) was added to a solution of 6 (330 mg, 0.931
mmol), 5 (a 4:1 mixture of (Z)-:(E)-isomers; 454 mg, 0.939
mmol), and DMAP (120 mg, 0.986 mmol) in CH2Cl2 (3.1 mL).
The reaction mixture was stirred for 1.5 h at 0 °C and then
was filtered (to remove the DCU). The combined filtrate and
washings was concentrated, and the residue allowed to stand
at room temperature overnight. The resulting orange syrup
was taken up in a 1:1 mixture of ethyl acetate and hexane
and filtered to remove the remaining DCU. The combined
filtrate and washings was washed successively with 10% citric
acid, saturated NaHCO3(aq), water, and brine and then was
dried over Na2SO4 and concentrated. The residue was fraction-
ated by FCC (30% ethyl acetate in hexane) to afford 15 as a
foam (a 12:1 mixture of (Z)-:(E)-isomers by HPLC; 687 mg,
91%): [R]D -9.1 (c 1.45, MeOH); IR νmax 3358, 3295, 1731, 1679
cm-1; 1H NMR δ 8.08 (1H, s), 7.35-7.23 (15H, m), 6.97 (1H, q,
J ) 7 Hz), 6.79 (1H, d, J ) 9.5 Hz), 5.55 (1H, d, J ) 9 Hz),
5.28-4.95 (6H, m), 4.77 (1H, m), 4.25 (1H, m), 3.31-3.14 (2H,
m), 2.12-2.04 (1H, m), 1.81 (3H, d, J ) 7 Hz), 1.75-1.45 (5H,
m), 1.22-1.17 (1H, m), 0.95-0.70 (18H, m); 13C NMR δ 174.2,
170.0, 169.3, 168.2, 165.8, 156.4, 137.8, 136.4, 135.2, 135.1,
129.6 (×2), 128.8 (×2), 128.7 (×2), 128.6 (×2), 128.5, 128.2,
128.1 (×2), 128.0 (×2), 127.7, 125.3, 74.8, 73.6, 67.6, 67.1, 60.4,
50.3, 41.6, 40.3, 37.4, 31.5, 24.8, 24.3, 23.2, 23.0, 21.9, 21.6,
19.5, 17.9, 15.4; LRMS (FAB) m/z (rel intensity) 800 ([M +
1]+, 100); HRMS m/z calcd for C45H57N3O10+H 800.4122 (M +
H), found 800.4135 (FAB).
C
34H51NSiO6: C, 68.30; H, 8.59; N, 2.34. Found C, 68.58; H,
8.72; N, 2.32.
Hm p -(R)-Leu -OBn (10). Diisopropylethylamine (1.74 mL,
10.0 mmol) was added dropwise to a stirred mixture of Hmp
(0.661 g, 5.0 mmol), (R)-Leu-OBn‚p-TsOH (9; 1.97 g, 5.0 mmol),
and BOP (2.21 g, 5.0 mmol) in MeCN (33 mL) at room
temperature under argon. After 1.5 h, the reaction mixture
was diluted with brine and extracted with ethyl acetate (×3).
The combined organic layers was washed sequentially with
10% citric acid and brine, dried over Na2SO4, concentrated,
and fractionated by DFC (gradient elution, 5-35% ethyl
acetate in hexane) to afford 10 as a white solid (1.51 g, 90%):
mp 103-104 °C (CHCl3/petroleum ether); [R]D +8.4 (c 1.1,
MeOH); IR νmax 3545, 3292, 1711, 1641 cm-1; 1H NMR δ 7.39-
7.28 (5H, m), 6.84 (1H, d, J ) 8.5 Hz), 5.21-5.11 (2H, m),
4.71-4.64 (1H, m), 4.17 (1H, dd, J ) 3.5, 9.5 Hz), 1.86-1.79
(1H, m), 1.71-1.48 (5H, m), 0.96-0.87 (12H, m); 13C NMR δ
174.5, 172.7, 135.3, 128.6 (×2), 128.4, 128.2 (×2), 70.3, 67.1,
50.5, 43.6, 41.4, 24.9, 24.5, 23.4, 22.8, 21.8, 21.4; LRMS (CI,
NH3) m/z (rel intensity) 336 ([M + 1]+, 21), 228 (100); HRMS
m/z calcd for C19H29NO4 335.2097, found 335.2096 (FAB). Anal.
Calcd for C19H29NO4: C, 68.03; H, 8.71; N, 4.17. Found C,
68.18; H, 8.73; N, 4.01.
TBSO-Hp p -OTBS (11). TBSCl (4.16 g, 27.5 mmol) was
added at once to a solution of (S)-phenyllactic acid (Hpp) (2.22
g, 13.3 mmol) and imidazole (2.72 g, 40.0 mmol) in DMF (8
mL), and the resulting mixture was stirred at room temper-
ature for 21 h. The mixture was partitioned between hexane
and water and the organic layer was washed with saturated
NaHCO3(aq) and brine, dried over Na2SO4, and concentrated.
The resulting residue was distilled under reduced pressure to
give 11 as a colorless oil (4.57 g, 87%): bp 118-120 °C (0.1
mmHg); [R]D -24 (c 1.1, CHCl3): IR νmax 1740, 1617 cm-1; 1H
NMR δ 7.30-7.20 (5H, m), 4.29 (1H, dd, J ) 4, 8.5 Hz), 3.08
(1H, dd, J ) 4, 13.5 Hz), 2.88 (1H, dd, J ) 8.5, 13.5 Hz), 0.93
(9H, s), 0.80 (9H, s), 0.25 (3H, s), 0.19 (3H, s), -0.81 (3H, s),
-0.21 (3H, s); 13C NMR δ 173.2, 137.6, 129.8, 128.1, 126.5,
74.4, 41.7, 25.7, 25.6, 18.2, 17.7, -4.9, -4.9, -5.2, -5.7; LRMS
Cbz-Va l-(E)-Aba -Hp p -Hm p -(R)-Leu -OBn [(E)-15]. Frac-
tionation of 15 (a 10:1 mixture of (Z)-:(E)-isomers; 30 mg) by
PTLC (SiO2; 90:5:5 benzene, ether, acetone; 5 elutions) gave
the more polar (Z)-15 (26 mg, 87%) and (E)-15 (2.5 mg, 8%);
1
IR νmax 3319, 1737, 1676 cm-1; H NMR δ 8.16 (1H, s), 7.33-
7.15 (16H, m), 6.86 (1H, d, J ) 9 Hz), 5.81 (1H, d, J ) 9 Hz),
5.42 (1H, dd, J ) 4, 7.5 Hz), 5.27-5.01 (5H, m), 4.73 (1H, m),
4.28 (1H, dd, J ) 5.5, 8.5 Hz), 3.33 (1H, dd, J ) 4, 14.5 Hz),
3.24 (1H, dd, J ) 8, 14.5 Hz), 2.25-2.10 (1H, m), 1.97 (3H, d,
J ) 7.5 Hz), 1.67-1.50 (5H, m), 1.42-1.30 (1H, m), 0.95 (3H,
d, J ) 6.5 Hz), 0.93-0.87 (9H, m), 0.80 (3H, d, J ) 6.5 Hz),
0.79 (3H, d, J ) 6.5 Hz); 13C NMR δ 173.9, 170.6, 169.5, 168.3,
166.1, 156.6, 136.5, 135.3, 135.3, 132.3, 129.5 (×2), 129.0 (×2),
128.7 (×2), 128.7 (×2), 128.6, 128.4, 128.2 (×4), 127.7, 124.8,
75.5, 74.4, 67.5, 67.2, 60.8, 50.4, 41.7, 40.4, 37.4, 31.5, 24.9,
24.6, 23.3, 23.1, 21.9, 21.8, 19.4, 17.8, 15.0; LRMS (FAB) m/z

Probing Host-Selective Phytotoxicity
J . Org. Chem., Vol. 64, No. 5, 1999 1665
(rel intensity) 800 ([M + 1]+, 12), 91 (100); HRMS m/z calcd
for C45H57N3O10+H 800.4122 (M + H), found 800.4138 (FAB).
Boc-Val-(Z)-Aba-Hpp-Hm p-(R)-Leu (17). 10% Pd/C (0.572
g) was added under an argon atmosphere to a degassed
solution of 15 (0.286 g, 0.36 mmol) and (Boc)2O (0.118 g, 0.54
mmol) in absolute ethanol (2.4 mL), followed by the slow
addition of 1,4-cyclohexadiene (0.34 mL, 3.6 mmol). After 4 h
of stirring at room temperature, the catalyst was filtered off,
and the combined filtrate and washings was concentrated. The
residue was fractionated by MPC (SiO2, 7.5 g; CH2Cl2/MeOH/
AcOH, 95:5:0.2) to provide 17 as a white foam (225 mg, 92%):
1.69 (2H, m), 1.56 (4H, m), 1.31 (3H, d, J ) 7.0 Hz), 0.96-
0.90 (18H, m); 13C NMR δ 171.7, 171.0, 170.8, 168.8, 168.3,
135.2 (×2), 135.0, 129.6 (×2), 129.3 (×2), 128.8 (×2), 128.7,
128.1, 127.5, 77.4, 75.5, 73.5, 59.6, 58.0, 53.2, 40.1, 39.2, 38.9,
36.6, 29.8, 25.0, 24.7, 22.3, 22.6, 22.5 (×2), 19.9, 19.6, 17.6;
LRMS (FAB) m/z (rel intensity) 715 ([M]+, 100); HRMS m/z
calcd for C36H49N3O7Se 715.2736, found 715.2734 (FAB)].
(R)-5-Eth ylid en e-2-(2-m eth yl-1-(ben zyloxyca r bon yla -
m id o)p r op yl)oxa zol-4(5H)-on e (20). EDC (59 mg, 0.31
mmol) was added at once to a suspension of the acid 5 (Z):(E),
4:1; 103 mg, 0.31 mmol) in CH2Cl2 (1.5 mL) at 0 °C. After 30
min of stirring, the reaction mixture was diluted with ethyl
acetate and hexane (1:1) and washed with H2O (×3), dried over
Na2SO4, and concentrated to give 20 as a 5:1 mixture of (Z)-:
[R]D -20 (c 1.4, MeOH); IR νmax 3378, 3291, 1720, 1674 cm-1
;
1H NMR δ {major rotamer} 8.15 (1H, s), 7.31-7.25 (5H, m),
6.83 (1H, q, J ) 7 Hz), 6.77 (1H, d, J ) 9.5 Hz), 5.35-5.21
(2H, m), 4.70 (1H, m), 4.23 (1H, dd, J ) 6.5, 9.5 Hz), 3.30 (1H,
dd, J ) 4, 14.5 Hz), 3.20 (1H, dd, J ) 7.5, 14.5 Hz), 2.04 (1H,
m), 1.81 (3H, d, J ) 7 Hz), 1.72-1.20 (7H, m), 1.42 (9H, s),
0.94 (12H, m), 0.78 (6H, m); 13C NMR δ 175.7, 170.0, 169.2,
168.5, 165.9, 156.9, 136.2, 135.4, 129.6 (×2), 128.8 (×2), 127.5,
125.2, 81.0, 75.2, 73.5, 59.9, 50.2, 42.0, 40.2, 37.5, 31.9, 28.5
(×3), 24.9, 24.3, 23.3, 23.0, 22.0, 21.6, 19.5, 17.8, 15.8; LRMS
(FAB) m/z (rel intensity) 676 ([M + 1]+, 8), 72 (100); HRMS
(E)-isomers (87 mg, 90%): IR νmax 3321, 1805, 1726, 1681 cm-1
;
1H NMR δ {(Z)-isomer} 7.25-7.35 (5H, m), 6.68 (1H, q, J )
7.5 Hz), 5.40 (1H, d, J ) 9 Hz), 5.07-5.16 (2H, m), 4.63 (1H,
dd, J ) 5.5, 9 Hz), 2.20 (1H, m), 2.13 (3H, d, J ) 7.5 Hz), 1.00
(3H, d, J ) 7 Hz), 0.94 (3H, d, J ) 7 Hz); {(E)-isomer} 6.82
(1H, q, J ) 8 Hz), 2.27 (3H, d, J ) 8 Hz); 13C NMR δ {(Z)-
isomer} 166.3, 165.4, 156.2, 136.3, 136.2, 135.6, 128.7 (×2),
128.4, 128.3 (×2), 67.4, 55.4, 31.4, 19.1, 17.6, 14.8; LRMS
(FAB) m/z (rel intensity) 317 ([M + 1]+, 47), 90 (100); HRMS
m/z calcd for C17H20N2O4+H 317.1501 (M + H), found 317.1509
(FAB).
m/z calcd for
C35H53N3O10+H 676.3809 (M + H), found
676.3800 (FAB).
Boc-Va l-(Z)-Aba -Hp p -Hm p -(R)-Leu -OP fp (18). DCC (52
mg, 0.25 mmol) was added to a solution of 17 (171 mg, 0.253
mmol) and C6F5OH (47 mg, 0.25 mmol) in dry ethyl acetate
(2.5 mL) at 0 °C under argon. After the reaction mixture was
stirred for 2.5 h at 0 °C, the DCU was filtered off, the combined
filtrate and washings was concentrated, and the residue was
fractionated by MPC (SiO2, 7.5 g; 25% ethyl acetate in hexane)
to give 18 as a white foam (179 mg, 84%): [R]D -0.10 (c 1.8,
CHCl3); IR νmax 3298, 3287, 1791, 1722, 1671 cm-1; 1H NMR δ
7.54 (1H, s), 7.34-7.22 (5H, m), 6.99 (1H, d, J ) 8 Hz), 6.88
(1H, q, J ) 7 Hz), 5.27 (2H, m), 5.02 (2H, m), 3.76 (1H, dd, J
) 7.5, 8.5 Hz), 3.28 (1H, dd, J ) 4, 14.5 Hz), 3.21 (1H, dd, J )
7, 14.5 Hz), 1.98 (1H, m), 1.80-1.15 (6H, m), 1.78 (3H, d, J )
7 Hz), 1.39 (9H, s), 0.97 (6H, d, J ) 5 Hz), 0.91 (3H, d, J ) 7
Hz), 0.88 (3H, d, J ) 7 Hz), 0.76 (3H, d, J ) 6.5 Hz), 0.74 (3H,
d, J ) 6.5 Hz); 13C NMR δ 170.7, 170.5, 170.0, 168.4, 165.7,
155.8, 137.3, 135.3, 129.7 (×2), 128.9 (×2), 127.6, 125.7, 80.0,
74.8, 73.8, 60.1, 50.1, 41.0, 40.3, 37.4, 31.0, 28.4 (×3), 24.9,
24.3, 23.2, 23.1, 21.7 (×2), 19.3, 18.1, 15.0; LRMS (FAB) m/z
(rel intensity) 842 ([M + 1]+, 9), 72 (100); HRMS m/z calcd for
Cbz-Va l-Aba -Hp p -OMe (22). DCC (62 mg, 0.30 mmol) was
added to a stirred solution of 5 ((Z):(E) 4:1; 100 mg, 0.30 mmol),
21 (54 mg, 0.30 mmol), and DMAP (67 mg, 0.30 mmol) in CH2-
Cl2 (1.0 mL) at 0 °C. After 1 h, the reaction mixture was
concentrated, and after 18 h of standing at room temperature,
the residue was taken up in 1:1 (v/v) mixture of ethyl acetate
and hexane and filtered. The combined filtrate and washings
was concentrated and fractionated by FCC (SiO2; 35% ethyl
acetate in hexane) to provide recovered 21 (8 mg, 15%), (Z)-
22 (88 mg, 59%), and (E)-22 (20 mg, 13%). For (Z)-22: IR νmax
1
3292, 1745, 1691, 1668 cm-1; H NMR δ 7.36-7.18 (11H, m),
6.87 (1H, q, J ) 7 Hz), 5.44 (1H, d, J ) 8.5 Hz), 5.27 (1H, dd,
J ) 6, 6.5 Hz), 5.09 (2H, s), 4.18 (1H, dd, J ) 6, 8.5 Hz), 3.67
(3H, s), 3.23-3.10 (2H, m), 2.21-2.04 (1H, m), 1.74 (3H, d, J
) 7 Hz), 0.99 (3H, d, J ) 7 Hz), 0.93 (3H, d, J ) 7 Hz); 13C
NMR δ 170.0, 169.8, 163.7, 156.7, 136.4 (×2), 135.8, 129.5 (×2),
128.8 (×2), 128.7 (×2), 128.4, 128.3 (×2), 127.4, 125.4, 73.9,
67.4, 60.6, 52.6, 37.6, 31.4, 19.5, 17.9, 15.1; LRMS (EI) m/z
(rel intensity) 496 ([M]+, 3), 91 (100); HRMS m/z calcd for
C
41H52F5N3O10: 841.3573, found 841.3577 (EI).
C
27H32N2O7 496.2210, found 496.2205 (EI). For (E)-22: IR νmax
3294, 1752, 1728, 1687, 1660 cm-1 1H NMR δ 7.56 (1H, s),
;
Cyclo[(2R, 3R)-3-(P h Se)Ab u -H p p -H m p -(R)-Leu -Va l]
7.34-7.18 (11H, m), 5.40 (1H, dd, J ) 5, 7.5 Hz), 5.28 (1H, d,
J ) 8.5 Hz), 5.11 (2H, s), 4.00 (1H, m), 3.74 (3H, s), 3.26 (1H,
dd, J ) 4.5, 14.5 Hz), 3.18 (1H, dd, J ) 8, 14.5 Hz), 2.11 (1H,
m), 2.04 (3H, d, J ) 7.5 Hz), 0.94 (3H, d, J ) 6.5 Hz), 0.88
(3H, d, J ) 7.0 Hz); 13C NMR δ 170.0 (s), 169.7 (s), 163.4 (s),
156.5 (s), 136.4 (s), 135.6 (s), 131.0 (d), 129.4 (d × 2), 128.9 (d
× 2), 128.8 (d × 2), 128.4 (d), 128.3 (d × 2), 127.5 (d), 124.7
(s), 74.1 (d), 67.4 (t), 61.3 (d), 52.8 (q), 37.5 (t), 31.3 (d), 19.4
(q), 17.8 (q), 14.7 (q); LRMS (EI) m/z (rel intensity) 496 ([M]+,
2), 91 (100); HRMS m/z calcd for C27H32N2O7 496.2210, found
496.2211 (EI).
Boc-Va l-OP fp (24). DCC (867 mg, 4.2 mmol) was added
to a stirred solution of Boc-Val (869 mg, 4.0 mmol) and C6F5-
OH (736 mg, 4.0 mmol) in ethyl acetate (4.0 mL) at 0 °C. After
1.5 h, the DCU was removed by filtration, and the combined
filtrate and washings was concentrated and fractionated by
FCC (SiO2; 15% ethyl acetate in hexane) to provide 24 (1.49
g, 97%) as a white solid: [R]D -18.1 (c 1.0, CHCl3); IR νmax
3294, 1786, 1712, 997 cm-1; 1H NMR δ 5.05 (1H, d, J ) 9 Hz),
4.55 (1H, dd, J ) 5, 9 Hz), 2.35-2.29 (1H, m), 1.45 (9H, s),
1.07 (3H, d, J ) 7 Hz), 1.01 (3H, d, J ) 7 Hz); 13C NMR δ
(19a ). BuLi (1 M, 0.011 mL, 0.011 mmol) was added to a
solution of 2 (129 mg, 0.231 mmol) and benzeneselenol (0.073
mL, 0.694 mmol) in THF (0.5 mL) at 0 °C under argon, and
the mixture was heated under reflux for 24 h. The cooled (rt)
reaction mixture was diluted with water and extracted with
ethyl acetate, and the combined organic layers was washed
with water, dried over Na2SO4, concentrated, and fractionated
by FCC (40% ethyl acetate in hexane) to give 19c (oil, 10 mg,
6%) [1H NMR δ 7.62-7.11 (10H, m), 6.83 (1H, d, J ) 9 Hz),
6.29 (1H, d, J ) 9 Hz), 6.11 (1H, d, J ) 10 Hz), 5.36 (1H, dd,
J ) 4.5, 9 Hz), 4.89 (1H, dd, J ) 5, 9 Hz), 4.71 (1H, dd, J ) 4,
7.5 Hz), 4.44 (1H, ddd, J ) 7.5, 7.5, 8 Hz), 4.07 (1H, dd, J )
10, 10 Hz), 3.46 (1H, dq, J ) 5, 7 Hz), 3.01 (1H, dd, J ) 8,
14.5 Hz), 2.92 (1H, dd, J ) 4, 14.5 Hz), 2.13 (1H, m), 1.9-1.4
(6H, m), 1.29 (3H, d, J ) 7 Hz), 0.98-0.89 (12H, m), 0.79 (3H,
d, J ) 7 Hz), 0.76 (3H, d, J ) 6.5 Hz)], 19b (oil, 19 mg, 12%)
[1H NMR (C6D6) δ 7.84 (2H, ap d, J ) 7 Hz), 7.31-7.0 (10H,
m), 6.55 (1H, d, J ) 9 Hz), 5.42-5.28 (2H, m), 5.09 (1H, dd, J
) 6, 6.5 Hz), 4.75 (1H, dd, J ) 5.5, 9.5 Hz), 4.28 (1H, m), 4.02
(1H, m), 3.07 (1H, dd, J ) 7, 14 Hz), 2.97 (1H, dd, J ) 5, 14
Hz), 2.38 (1H, m), 1.78-1.51 (6H, m, J ) 7 Hz), 1.70 (3H, d),
0.97-0.88 (12H, m), 0.84 (6H, d, J ) 6.5 Hz)], and 19a (85
mg, 52%) [[R]D -56 (c 1.3, CHCl3); IR νmax 3269, 1744, 1671,
1655 cm-1; 1H NMR δ 7.63-7.60 (2H, m), 7.38-7.22 (9H, m),
6.97 (1H, d, J ) 9 Hz), 6.40 (1H, d, J ) 9 Hz), 5.32 (1H, dd, J
) 6.5, 7.5 Hz), 5.19 (1H, dd, J ) 7, 7 Hz), 4.83 (1H, dd, J )
7.5, 8.5 Hz), 4.38 (1H, dd, J ) 4.5, 9 Hz), 4.30 (1H, ddd, J )
7.5, 7.5, 7.5 Hz), 3.64 (1H, dq, J ) 7, 7 Hz), 3.21 (1H, dd, J )
6.5, 14.5 Hz), 3.16 (1H, dd, J ) 7.5, 14.5 Hz), 2.40 (1H, m),
168.9, 155.6, 141.4 (dm ×2, J CF ) 252 Hz), 139.9 (dm, J CF
)
254 Hz), 138.1 (dm ×2, J CF ) 249 Hz), 125.0 (br s), 80.6, 58.9,
31.3, 28.4, 19.1, 17.6; LRMS (Cl) m/z (rel intensity) 401 ([M +
18]+, 14), 345 (100); HRMS m/z calcd for C16H18F5NO4+NH4
401.1500 (M + NH4), found 401.1501 (CI, NH3). Anal. Calcd
for C16H18F5NO4: C, 50.14; H, 4.37; N, 3.65. Found C, 50.20;
H, 4.50; N, 3.91.
Boc-Va l-(R)-Abu [(R)-25]. Boc-Val-OPfp (24; 380 mg, 1.0

1666 J . Org. Chem., Vol. 64, No. 5, 1999
Ward et al.
mmol) was added at once to a stirred solution of (2R)-2-
aminobutanoic acid (103 mg, 1.0 mmol) and Na2CO3 (106 mg,
1.0 mmol) in t-BuOH (2.5 mL) and H2O (2.5 mL) at room
temperature. After 13 h, the mixture was acidified (pH ) ca.
3) by addition of citric acid and then was extracted with ethyl
acetate (×3). The combined organic layers was washed with
brine, dried over Na2SO4, concentrated, and fractionated by
FCC (SiO2; CH2Cl2/CH3OH/AcOH, 95:5:0.2) to provide (R)-25
as a white solid (226 mg, 75%): [R]D -10.9 (c 1.0, MeOH); IR
(M + H). Found 484.2701 (FAB). Anal. Calcd for C28H37NO6:
C, 69.54; H, 7.71; N, 2.90. Found C, 60.71; H, 7.62; N, 3.11.
Boc-Va l-(R)-Abu -Hp p -Hm p -(R)-Leu -OBn [(R)-27]. Di-
ethyl azodicarboxylate (0.031 mL, 0.20 mmol) was added
dropwise via syringe over 2 min to a stirred solution of acid
(R)-25 (60 mg, 0.20 mmol), alcohol 26 (91 mg, 0.19 mmol), and
Ph3P (52 mg, 0.20 mmol) in THF (0.9 mL) at 0 °C under argon.
After 19 h of stirring at room temperature, the mixture was
concentrated, and the residue was fractionated by FCC (SiO2;
30% ethyl acetate in hexane) to provide (R)-27 as a white foam
(128 mg, 89%): [R]D -44 (c 1.0, CHCl3); IR νmax 3363, 3317,
ν
max 3310, 1720, 1649 cm-1; 1H NMR ((CD3)2SO) δ 8.02 (1H, d,
J ) 7.5 Hz), 6.59 (1H, d, J ) 9 Hz), 4.12 (1H, ddd, J ) 5, 8, 8
Hz), 3.87 (1H, dd, J ) 7, 8.5 Hz), 1.95-1.87 (1H, m), 1.77-
1.55 (2H, m), 1.38 (9H, s), 0.88-0.80 (9H, m); 13C NMR δ 174.5,
171.9, 156.7, 80.7, 59.6, 53.5, 32.0, 28.4 (×3), 25.5, 19.4, 19.2,
9.4; LRMS (Cl) m/z (rel intensity) 303 ([M + 1]+, 35), 72 (100);
HRMS m/z calcd for C14H26N2O5+H 303.1920 (M + H), found
303.1927 (CI, NH3). Anal. Calcd for C14H26N2O5: C, 55.61; H,
8.67; N, 9.26. Found C, 55.70; H, 8.50; N, 9.31.
Boc-Va l-(S)-Ab u [(S)-25]. Reaction of (2S)-2-amino-
butanoic acid (103 mg, 1.0 mmol) with 24 (383 mg, 1.0 mmol)
as described above gave, after fractionation by FCC (SiO2; 10%
CH3OH in CH2Cl2), (R)-25 as a foam (211 mg, 70%): mp 174-
175 °C (MeOH/ether); [R]D -33 (c 1.0, MeOH); IR νmax 3316,
1720, 1690, 1649 cm-1; 1H NMR ((CD3)2SO) δ 7.88 (1H, d, J )
7.5 Hz), 6.72 (1H, d, J ) 9 Hz), 4.15 (1H, ddd, J ) 5, 7.5, 7.5
Hz), 3.81 (1H, dd, J ) 7, 9 Hz), 2.05-1.85 (1H, m), 1.80-1.50
(2H, m), 1.38 (9H, s), 0.85 (3H, t, J ) 7 Hz), 0.84 (3H, d, J )
7 Hz), 0.81 (3H, d, J ) 7 Hz); 13C NMR δ 175.7 (s), 172.8 (s),
156.6 (s), 80.5 (s), 60.4 (d), 53.6 (d), 30.9 (d), 28.4 (q ×3), 25.2
(t), 19.3 (q), 18.4 (q), 9.7 (q); LRMS (Cl) m/z (rel intensity) 303
([M + 1]+, 84), 203 (100); HRMS m/z calcd for C14H26N2O5+H
303.1920 (M + H), found 303.1920 (CI, NH3).
1
1745, 1718, 1660 cm-1; H NMR δ 7.41-7.22 (10H, m), 6.84
(1H, d, J ) 7 Hz), 6.63 (1H, d, J ) 9 Hz), 5.58 (1H, d, J ) 8.5
Hz), 5.23-5.12 (4H, m), 4.71 (1H, m), 4.63 (1H, m), 4.12 (1H,
m), 3.30 (1H, dd, J ) 3.5, 14.5 Hz), 3.15 (1H, dd, J ) 8.5. 14.5
Hz), 2.30-2.10 (1H, m), 1.90-1.72 (1H, m), 1.72-1.20 (7H,
m), 1.41 (9H, s), 0.98-0.87 (9H, m), 0.86-0.68 (12H, m); 13C
NMR δ 173.9, 173.7, 171.6, 169.7, 168.2, 156.2, 135.4, 135.3,
129.6 (×2), 128.9 (×2), 128.8 (×2), 128.6, 128.3 (×2), 127.6,
79.9, 74.7, 74.3, 67.5, 59.7, 53.5, 50.4, 41.6, 40.3, 37.3, 30.6,
28.5 (×3), 25.6, 24.9, 24.5, 23.2 (×2), 21.7, 21.7, 19.6, 17.6, 9.3;
LRMS (EI) m/z (rel intensity) 767 ([M]+, 2), 72 (100); HRMS
m/z calcd for C42H61N3O10 767.4357, found 767.4359 (EI).
Boc-Va l-(S)-Abu -Hp p -Hm p -(R)-Leu -OBn [(S)-27]. Mit-
sunobu reaction of (S)-25 (100 mg, 0.331 mmol) with 26 (168
mg, 0.348 mmol) as described above gave, after fractionation
by FCC (SiO2; 30% ethyl acetate in hexane), recovered 26 (28
mg, 17%) and (S)-27 as a foam (159 mg, 63%): [R]D -38 (c
1
1.5, CHCl3); IR νmax 3373, 3317, 1745, 1658 cm-1; H NMR δ
7.40-7.20 (10H, m), 7.05 (1H, d, J ) 8 Hz), 6.72 (1H, d, J )
9.5 Hz), 5.30-5.19 (5H, m), 4.82-4.68 (2H, m), 4.06 (1H, dd,
J ) 7.5, 8 Hz), 3.27 (1H, dd, J ) 4, 14.5 Hz), 3.19 (1H, dd, J
) 6.5, 14.5 Hz), 2.01-1.85 (2H, m), 1.85-1.68 (1H, m), 1.68-
1.52 (5H, m), 1.52-1.38 (1H, m), 1.42 (9H, s), 0.96-0.86 (15H,
m), 0.76 (3H, d, J ) 6.5 Hz), 0.74 (3H, d, J ) 6.5 Hz); 13C NMR
δ 174.5, 173.9, 172.0, 169.4, 168.0, 155.9, 135.3, 135.0, 129.9
(×2), 128.8 (×4), 128.6, 128.3 (×2), 127.6, 79.8, 74.9, 73.5, 67.6,
59.8, 53.3, 50.4, 41.6, 40.7, 37.3, 31.6, 28.5 (×3), 25.7, 24.9,
24.0, 23.3, 23.1, 21.9, 21.4, 19.5, 18.0, 10.1; LRMS (EI) m/z
(rel intensity) 767 ([M]+, 1), 121 (100); HRMS m/z calcd for
(R)-Hp p -Hm p -(R)-Leu -OBn (26). Oxalyl chloride (0.244
mL, 2.8 mmol) was added to a stirred solution of ent-11 (947
mg, 2.4 mmol) and DMF (0.018 mL, 0.24 mmol) in CH2Cl2 (4.0
mL) at 0 °C under argon. After 30 min of stirring at 0 °C and
4 h of stirring at room temperature, the mixture was diluted
with hexane (4.0 mL) and filtered, and the combined filtrate
and washings was concentrated. To the resulting residue was
added a solution of 10 (671 mg, 2.0 mmol) and DMAP (293
mg, 2.4 mmol) in CH2Cl2 (4.0 mL) at 0 °C. After 15 min, the
mixture was allowed to warm to room temperature and stirred
at that temperature for 2 h. The reaction mixture was diluted
with ethyl acetate and hexane (1:1) and was washed sequen-
tially with water, 10% citric acid, saturated NaHCO3(aq), and
brine, dried over Na2SO4, and concentrated. The residue was
taken up in 5% HF/CH3CN (20 mL), and after 2.5 h, the
mixture was diluted with ethyl acetate and hexane (1:1) and
was washed sequentially with water, saturated NaHCO3(aq),
and brine, dried over Na2SO4, concentrated, and fractionated
by FCC (SiO2, 30% ethyl acetate in hexane) to afford 26 as a
white solid (873 mg, 90%): mp 100-101 °C (CHCl3/hexane);
C
42H61N3O10+H 768.4435 (M + H), found 768.4436 (FAB).
P h ytotoxicity Bioa ssa ys. Canola, cv. Westar (susceptible
to virulent P. lingam isolates), brown mustard cv. Cutlass
(resistant to virulent P. lingam isolates), and white mustard
cv. Ochre (resistant to virulent P. lingam isolates) plants were
grown under controlled environmental conditions (controlled
environment chamber at 20/18 °C with a 16/8 h day/night
cycle). Leaves of 21-day-old plants were punctured with a
needle, and a 10-µL droplet of the test solution dissolved in
50% aqueous (v/v) methanol was placed on each wound. The
symptoms that developed within 2-5 days varied from little
or no reaction to brownish-yellow lesions. In no case were
lesions observed with 50% aqueous (v/v) methanol.
[R]D -9.4 (c 1.0, CHCl3); IR νmax 3423, 3326, 1750, 1731 cm-1
;
1H NMR δ 7.39-7.22 (10H, m), 6.23 (1H, d, J ) 8 Hz), 5.21
(1H, dd, J ) 4, 8.5 Hz), 5.16 (2H, m), 4.66-4.59 (1H, m), 4.50
(1H, m), 3.16 (1H, dd, J ) 5, 14 Hz), 2.98 (1H, dd, J ) 7.5, 14
Hz), 2.65 (1H, br s), 1.74-1.45 (6H, m), 0.91 (12H, m); 13C NMR
δ 173.5, 172.6, 169.7, 136.4, 135.5, 129.6, 128.8, 128.8, 128.5,
127.2, 73.9, 71.8, 67.4, 50.8, 41.5, 40.8, 40.6, 25.1, 24.6, 23.3,
23.0, 22.1, 21.8; LRMS (FAB) m/z (rel intensity) 484 ([M +
1]+, 38), 91 (100); HRMS m/z calcd for C28H37NO6+H 484.2699
Ackn owledgm en t. Financial support from the Natu-
ral Sciences and Engineering Research Council is grate-
fully acknowledged. A.V. thanks the University of
Saskatchwan and the Universidad Nacional Autonoma
de Me´xico for scholarships.
J O982376P