Total synthesis of depsilairdin

Article abstract of DOI:10.1021/jo1009239

(Figure presented) The total synthesis of depsilairdin, a host-selective phytotoxin isolated from Leptosphaeria maculans (the causal agent of blackleg disease of oilseed Brassicas), has been achieved by N-terminal extension of a suitably protected derivative of the hitherto unknown amino acid (2S,3S,4R)-3,4-dihydroxy-3-methyl-proline (Dhmp) followed by esterification with lairdinol A. The latter esterification, complicated by the sterically hindered nature of the carboxyl group, was accomplished by a novel method involving reaction of the 1-hydroxybenzotriazole (HOBt) derived active ester with the bromomagnesium alkoxide of lairdinol A. Three depsilairdin analogues were also prepared by replacing the Dhmp residue with l-proline and cis- and trans-4-hydroxy-l-proline. Phytotoxicity assays showed that the analogues were nontoxic to both blackleg-susceptible (brown mustard) and -resistant (canola) plants, suggesting that the presence of the Dhmp residue in depsilairdin is important for its host-selective toxicity toward brown mustard.

Full text of DOI:10.1021/jo1009239

pubs.acs.org/joc
Total Synthesis of Depsilairdin
Dale E. Ward* and Sandip G. Pardeshi
Department of Chemistry, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada
dale.ward@usask.ca
Received May 11, 2010
The total synthesis of depsilairdin, a host-selective phytotoxin isolated from Leptosphaeria maculans
(the causal agent of blackleg disease of oilseed Brassicas), has been achieved by N-terminal extension
of a suitably protected derivative of the hitherto unknown amino acid (2S,3S,4R)-3,4-dihydroxy-
3-methyl-proline (Dhmp) followed by esterification with lairdinol A. The latter esterification,
complicated by the sterically hindered nature of the carboxyl group, was accomplished by a novel
method involving reaction of the 1-hydroxybenzotriazole (HOBt) derived active ester with the
bromomagnesium alkoxide of lairdinol A. Three depsilairdin analogues were also prepared by
replacing the Dhmp residue with L-proline and cis- and trans-4-hydroxy-L-proline. Phytotoxicity
assays showed that the analogues were nontoxic to both blackleg-susceptible (brown mustard)
and -resistant (canola) plants, suggesting that the presence of the Dhmp residue in depsilairdin is
important for its host-selective toxicity toward brown mustard.
Introduction
Many microbial pathogens produce host-selective toxins
(HSTs) to facilitate infection of plants.1 By definition, these
compounds show selective toxicity to host plants and are
relatively harmless to nonhosts. HSTs are valuable probes to
study the complex metabolic pathways that govern the
interaction of plants with their microbial pathogens.1 Re-
cently, Pedras et al. reported2 that depsilairdin (1) (Figure 1)
is a highly selective toxin produced by the “blackleg” fungus
(Leptosphaeria maculans; asexual stage Phoma Lingam), one
of the most devastating pathogens of the oilseed crops rape-
seed/canola (Brassica napus, B. rapa).3 Recently, strong
evidence has been accumulating that this pathogen is ex-
panding its host range to include mustard (B. juncea), a crop
traditionally resistant to blackleg disease.4 In particular,
FIGURE 1. Structures of depsilairdin (1) and its component residues.
(1) Review: Wolpert, T. J.; Dunkle, L. D.; Ciuffetti, L. M. Annu. Rev.
Phytopathol. 2002, 40, 251–285.
(2) Pedras, M. S. C.; Chumala, P. B.; Quail, J. W. Org. Lett. 2004, 6, 4615–
4617.
(3) (a) Howlett, B. J.; Idnurm, A.; Pedras, M. S. C. Fungal Genet. Biol.
2001, 33, 1–14. (b) Fitt, B. D. L.; Brun, H.; Barbetti, M. J.; Rimmer, S. R. Eur.
J. Plant Pathol. 2006, 114, 3–15.
fungal isolates that produce despilairdin (1) were found to
infect mustard. Plant leaves of mustard treated with depsi-
lairdin (1) showed strong necrotic and chlorotic lesions
similar to those caused by the pathogen, but such symptoms
were not observed on canola or rapeseed leaves.2 Thus, due
to its very selective phytotoxicity, depsilairdin (1) is a most
(4) Pedras, M. S. C.; Yu, Y. Nat. Prod. Commun. 2009, 4, 1291–1304.
5170 J. Org. Chem. 2010, 75, 5170–5177
Published on Web 07/14/2010
DOI: 10.1021/jo1009239
r
2010 American Chemical Society

Ward and Pardeshi
JOCArticle
SCHEME 1. Retrosynthetic Analysis of Depsilairdin (1)
SCHEME 2. Synthesis of the Protected Dhmp Residue 9
during chain extension and chemoselective acylation of the
secondary alcohol in lairdinol A (5) is expected. The tetra-
depsipeptide 6 would be assembled by standard N-terminal
extension of a suitably protected Dhmp residue (e.g., 9).9
Although incorporation of N-methylamino acids is difficult
using standard peptide coupling methods,10a several specia-
lized reagents are available for this purpose.10 Of the re-
quired four residues, 7 and 8 are readily available from D- and
L-valine, respectively,11,12 and we have recently reported13
the synthesis of lairdinol A (5) in 12 steps from (R)-carvone
without the use of protecting groups. As noted above, Dhmp
(4) is a hitherto unknown amino acid. In contemplating possi-
ble strategies to prepare a suitably protected derivative of 4, we
were cognizant of the numerous syntheses of 3,4-dihydroxy-
proline14 and 3-hydroxy-3-methylproline15 derivatives that
have appeared in the literature. Moreover, Blanco and Sardina
disclosed a seemingly straightforward synthesis of the closely
related triol 10 from trans-4-hydroxy-L-proline,16 and easy
access to 9 seemed feasible by adaptation of this route.
desirable compound to probe the susceptibility and resistance
of mustard and canola, respectively, to the blackleg pathogen.
To date these studies have been impeded because insufficient
quantities of 1 are available from fungal cultures. Herein, we
report the total syntheses and relative phytotoxicities of depsi-
lairdin (1) and three analogues where the Dhmp residue is
replaced by proline and (4R)- and (4S)-4-hydroxyproline.
Depsilairdin (1) is a structurally interesting depsipeptide
composed of five residues: (2R)-2-hydroxy-3-methylbuta-
noic acid (Hmb, 2), two N-methyl-L-valines (MeVal, 3),
(2S,3S,4R)-3,4-dihydroxy-3-methyl-proline (Dhmp, 4), and
the sesquiterpene lairdinol A (5) (Figure 1). Dhmp (4) is a
novel amino acid that has not been observed previously.
Similarly, lairdinol A (5) is a novel sesquiterpene that was
isolated5 from the same fungal cultures as 1; ent-5 (cyperusol C)
has been isolated from the plants Cyperous longus6 and Eriger-
on annus.7 Although there are several peptide natural products
that contain a MeVal-MeVal sequence, the MeVal-MeVal-Pro
sequence is rare8 and the Hmb-MeVal-MeVal sequence is
unknown. Finally, (depsi)peptide-sesquiterpene conjugates
are highly unusual among natural products.
Results and Discussion
The synthesis commenced with the preparation of keto-
alcohol 11 that proceeded without incident using the reported
protocols (Scheme 2).17 Surprisingly, attempted protection of
(10) (a) Humphrey, J. M.; Chamberlin, A. R. Chem. Rev. 1997, 97, 2243–
2266. (b) Albericio, F.; Chinchilla, R.; Dodsworth, D. J.; Najera, C. Org.
Prep. Proced. Int. 2001, 33, 203–303. (c) Valeur, E.; Bradley, M. Chem. Soc.
Rev. 2009, 38, 606–631.
Syntheses of linear (depsi)peptides typically proceed by
linear or convergent coupling of suitably protected intact
residues followed by deprotection.9 Our retrosynthetic anal-
ysis of depsilairdin (1) is outlined in Scheme 1. We chose to
disconnect the ester linkage because C-terminal proline
residues in oligopeptide chains are resistant to isomerization
(11) (a) Deechongkit, S.; You, S.-L.; Kelly, J. W. Org. Lett. 2004, 6, 497–
500. (b) Gonzalez, I.; Jou, G.; Caba, J. M.; Albericio, F.; Lloyd-Williams, P.;
Giralt, E. J. Chem. Soc., Perkin Trans. 1 1996, 1427–1433. (c) Wissner, A.;
Grudzinskas, C. V. J. Org. Chem. 1978, 43, 3972–3974. (d) Ward, D. E.;
Vazquez, A.; Pedras, M. S. C. J. Org. Chem. 1999, 64, 1657–1666.
(12) Cheung, S. T.; Benoiton, N. L. Can. J. Chem. 1977, 55, 906–910.
(13) Pardeshi, S. G.; Ward, D. E. J. Org. Chem. 2008, 73, 1071–1076.
(14) Selected recent examples: (a) Taylor, C. M.; Jones, C. E.; Bopp, K.
Tetrahedron 2005, 61, 9611–9617. (b) Soengas, R. G.; Estevez, J. C.; Estevez,
R. J. Tetrahedron: Asymmetry 2003, 14, 3955–3963. (c) Garcia, A. L. L.;
Correia, C. R. D. Tetrahedron Lett. 2003, 44, 1553–1557. (d) Taylor, C. M.;
Barker, W. D.; Weir, C. A.; Park, J. H. J. Org. Chem. 2002, 67, 4466–4474. (e)
Fujii, M.; Miura, T.; Kajimoto, T.; Ida, Y. Synlett 2000, 1046–1048.
(15) Selected recent examples: (a) Yoshitomi, Y.; Makino, K.; Hamada,
Y. Org. Lett. 2007, 9, 2457–2460. (b) Chen, Z.; Ye, T. Synlett 2005, 2781–
2785. (c) Makino, K.; Nagata, E.; Hamada, Y. Tetrahedron Lett. 2005, 46,
8159–8162. (d) Davis, F. A.; Ramachandar, T.; Liu, H. Org. Lett. 2004, 6,
3393–3395. (e) Shen, J.-W.; Qin, D.-G.; Zhang, H.-W.; Yao, Z.-J. J. Org.
Chem. 2003, 68, 7479–7484. (f) Qin, D.-G.; Zha, H.-Y.; Yao, Z.-J. J. Org.
Chem. 2002, 67, 1038–1040.
(5) Pedras, M. S. C.; Chumala, P. B.; Venkatesham, U. Bioorg. Med.
Chem. 2005, 13, 2469–2475.
(6) Xu, F.; Morikawa, T.; Matsuda, H.; Ninomiya, K.; Yoshikawa, M. J.
Nat. Prod. 2004, 67, 569–576.
(7) Li, X.; Yang, M.; Han, Y.-F.; Gao, K. Planta Med. 2005, 71, 268–272.
(8) (a) Pettit, G. R.; Kamano, Y.; Herald, C. L.; Dufresne, C.; Bates,
R. B.; Schmidt, J. M.; Cerny, R. L.; Kizu, H. J. Org. Chem. 1990, 55, 2989–
2990. (b) Horgen, F. D.; Yoshida, W. Y.; Scheuer, P. J. J. Nat. Prod. 2000, 63,
461–467. (c) Luesch, H.; Yoshida, W. Y.; Moore, R. E.; Paul, V. J. J. Nat.
Prod. 2000, 63, 1106–1112. (d) Adams, B.; Poerzgen, P.; Pittman, E.;
Yoshida, W. Y.; Westenburg, H. E.; Horgen, F. D. J. Nat. Prod. 2008, 71,
750–754.
(9) (a) Bodanszky, M. Principles of Peptide Synthesis, 2nd ed.; Springer-
Verlag: Berlin, 1993. (b) Benoiton, N. L. Chemistry of Peptide Synthesis;
CRC Press: Boca Raton, FL, 2005.
(16) Blanco, M.-J.; Sardina, F. J. J. Org. Chem. 1998, 63, 3411–3416.
(17) Blanco, M.-J.; Sardina, F. J. J. Org. Chem. 1996, 61, 4748–4755.
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SCHEME 3. Reaction of MOM-Cl with Imidazole in DMF-d7
(<50% yield after 72 h) and not clean, as previously noted16 by
Blanco and Sardina. Speculating that the apparent higher
reactivity of 11 toward MOM-Cl (5 equiv) under acidic condi-
tions (1 equiv of imidazole) compared to basic conditions
(7 equiv of DIPEA) might be due to protonation of the
tertiary amine in 11, we decided to employ the weaker
tertiary amine base N,N-dimethylaniline (DMA)21 (pKa ≈
5.1) to allow reaction via the conjugate acid of 11. Treatment
of 11 (0.25 M in DMF) with MOM-Cl (5 equiv) and DMA
(7 equiv) gave 12 (65%) along with recovered 11 (30%) after
24 h. Improved conversions were obtained using CH2Cl2 as
solvent, and under optimized conditions, 12 was obtained in
80% isolated yield after 24 h.
the OH group in 11 according to the published procedure (5
equiv each of MOM-Cl and imidazole in DMF)16 failed to give
detectable amounts of the expected MOM ether 12. Varying
the amounts of imidazole (1-7 equiv; MOM-Cl, 5 equiv)
clearly showed that formation of 12 occurred only with excess
MOM-Cl, and apparent high conversions required ratios of
MOM-Cl to imidazole of 3:1 or higher. The reaction of 11
(0.25 M in DMF) with MOM-Cl (5 equiv) and imidazole
Blanco and Sardina reported the methylation of ketone 12
using n-BuLi in presence of HMPA at -78 °C to form the
enolate followed by reaction with CH3I at -78 to 0 °C
(Scheme 2).16 The authors indicated that the resulting 13
was unstable, and consequently they treated the crude with
NaBH4 to obtain 16 (62%), apparently as the sole product.16
This reaction proved very capricious in our hands, and the
reported result could not be reproduced. We verified that
enolate formation was essentially quantitative under the
reported conditions by quenching with D2O (i.e., >95%
deuterium incorporation at C-3). However, similar experi-
ments established that this enolate was unstable above
-50 °C and rapidly decomposed on warming to 0 °C.
Despite extensive experimentation including varying the
reaction time, temperature, solvent, and additives, our best
result was obtained by reaction of the enolate with CH3I at
-50 °C for 4.5 h to give 13 (35%) along with the C-3
diastereomer 14, the O-alkylation product 15, and several
unidentified byproducts. The formation of 14 could result
from methylation of the enolate of 12 from its si face;
alternatively, ent-14 could result from epimerization of 13
at C-2. We did not establish the absolute configuration or
enantiopurity of 14 or 15. Lubell and co-workers have
reported the alkylation of 4-oxoproline derivatives via the
enolate generated by reaction with KHMDS in THF and 1,3-
dimethyltetrahydropyrimidin-2(1H)-one (DMPU).22 We
optimized that procedure for the methylation of 12 and
identified toluene as a superior solvent (cf. THF) and DMPU
as the most effective additive (cf. HMPA and TMEDA).
Thus, reaction of 12 with KHMDS (1.05 equiv) in a 1:1
mixture of toluene and DMPU at -78 °C for 1 h followed by
addition CH3I and reaction for 2 h gave the desired 13
(55-60%) along with 14 (15%) and 15 (8%). In contrast
to the previous report,16 we found that 13 was routinely
isolated and not especially prone to decomposition. Reduc-
tion of 13 with NaBH4 followed by conversion of the
resulting alcohol 16 (dr >10:1) to the corresponding TBS
ether 17a and hydrogenolysis of the 9-phenylfluorenyl (Pf)
group delivered the desired proline fragment 9a.
1
(1 equiv) was complete within 48 h. Although the H NMR
spectrum of the crude product after workup was fairly “clean”
and indicated that full conversion to 12 had been achieved, the
isolated yield after chromatography was only 55%. This
suggested that 12 and/or 11 were converted into unknown
water-soluble compounds under the reaction conditions and
lost upon workup. To investigate this process further, the
reaction of MOM-Cl with imidazole in DMF-d7 was moni-
tored by 1H NMR. Within 10 min, a near equimolar mixture of
MOM-Cl and imidazole (0.25 M) was converted into a ca. 2:1:2
mixture of 18, 19, and 20, respectively (Scheme 3). Using 5
equiv of MOM-Cl compared to imidazole produced a similar
mixture that, over time (72 h), was slowly converted to mainly
18 (>80%). Addition of trans-4-tert-butylcyclohexanol (21b; 1
equiv) to this mixture produced the corresponding MOM ether
21a (ca. 80% after 2.5 h)18 that slowly decomposed over 24 h
(ca. 40% of 21a) by which time MOM-Cl was no longer
present.19 Alternatively, a 3.5:1 of 19 and 18, respectively,
was formed using 2 equiv of imidazole with respect to MOM-
Cl. This mixture was stable over 48 h and, importantly, did not
react with 21b even after 24 h. These experiments explain our
failure to obtain 12 under the reported conditions. Imidazole
rapidly consumes 1 equiv of MOM-Cl and, over time, can
consume 2 equiv of MOM-Cl. The resulting adducts 18 and 19
are not sufficiently reactive to alkylate an alcohol. Thus, with
equimolar amounts imidazole and MOM-Cl (i.e., the reported
protocol)16 or with an excess of imidazole, alkylation of alcohols
does not occur (or is exceedingly slow). Using excess MOM-Cl,
alkylation is possible, but greater than 3 equiv of MOM-Cl
(relative to imidazole) are required for significant conversion.
However, under these conditions the reaction mixture becomes
highly acidic, and this aspect can explain the moderate yields of
12 obtained (e.g., by acid-catalyzed deprotection of the amine,
elimination, etc.). Attempted formation of 12 from 11 using the
standard conditions (MOM-Cl, DIPEA)20 was very sluggish
(21) Scattered reports of the use of DMA/MOM-Cl for preparation of
MOM ethers have appeared: (a) Schmid, G.; Hofheinz, W. J. Am. Chem.
Soc. 1983, 105, 624–625. (b) Cerny, I.; Pouzar, V.; Drasar, P.; Turecek, F.;
Havel, M. Collect. Czech. Chem. Commun. 1986, 51, 128–140. (c) Su, T. L.;
Huang, J. T.; Burchenal, J. H.; Watanabe, K. A.; Fox, J. J. J. Med. Chem.
1986, 29, 709–715. (d) Pouzar, V.; Karaszova, L.; Havel, M. Collect. Czech.
Chem. Commun. 1987, 52, 2735–2743. (e) Su, T. L.; Huang, J. T.; Chou, T. C.;
Otter, G. M.; Sirotnak, F. M.; Watanabe, K. A. J. Med. Chem. 1988, 31,
1209–1215. (f) Iwata, C.; Takemoto, Y.; Kubota, H.; Yamada, M.; Uchida,
S.; Tanaka, T.; Imanishi, T. Chem. Pharm. Bull. 1988, 36, 4581–4584. (g)
Reeder, A. Y.; Joannou, G. E. Steroids 1996, 61, 22–26.
(18) Sawyer, J. S.; Kucerovy, A.; Macdonald, T. L.; McGarvey, G. J. J.
Am. Chem. Soc. 1988, 110, 842–853.
(19) Major byproducts included dimethoxymethane and bis(4-tert-
butylcyclohexyloxy)methane in addition to 18 and 21. For acid-catalyzed
exchange of alcohol groups in methylene acetals, see: Ledneczki, I.; Molnar,
A. Synth. Commun. 2004, 34, 3683–3690.
(20) Wuts, P. G. M.; Greene, T. W. Protective Groups in Organic Synthe-
sis, 4th ed.; Wiley: New York, 2007.
(22) Sharma, R.; Lubell, W. D. J. Org. Chem. 1996, 61, 202–209.
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SCHEME 4. Synthesis of Depsilairdin (1) from Its Component Residues
SCHEME 5. Model Study for Methyl Ester Hydrolysis and
Esterification
Acylation of 9a with 8 to give the dipeptide fragment 22a
proceeded smoothly using bromotri(pyrrolidino)phosphonium
hexafluorophosphate (PyBroP), a reagent known to be effec-
tive for coupling sterically hindered amino acids (Scheme 4).23
Removal of the Cbz protecting group in 22a and iteration of the
above coupling procedure produced the tripeptide fragment
23a. Subjecting 23a to hydrogenolysis followed by reaction
with 7 gave the tetradepsipeptide 24a. Unfortunately, all efforts
to obtain the carboxylic acid 27 by reactions of 24a with LiOH/
25
H2O/MeOH or KOSiMe3/ether24 or Me3SnOH/(CH2Cl)2
were unsuccessful, presumably because of steric hindrance of
the ester carbonyl group. Similar reactions of dipeptide 22a also
failed to hydrolyze the methyl ester (Scheme 5). Hypothesizing
that the ester carbonyl might be activated by intramolecular
hydrogen bonding, we attempted hydrolysis of the ester in 22c.
Gratifyingly, the reaction of 22c with Me3SnOH in (CH2Cl)2 at
80 °C gave the desired acid 22d in good yield.25 To facilitate
application of this hydrolysis strategy to the tetradepsipeptide
24, we prepared the differentially protected derivative 24b by a
route analogous to that used to obtain 24a (Scheme 4).26 The
triethylsilyl group in 24b was selectively removed by reaction
PyBrop/Et3N) resulted in significant isomerization (i.e., 31a:
2-epi-31a, 1-3:1).28 Similarly, esterification of the 2-pyridylthiol
ester 30b by heating with the alcohol in toluene29 or by reaction
with the Li alkoxide (ROH þ BuLi) at ambient temperature also
gave extensive isomerization. Alternatively, reaction of 30b with
the bromomagnesium alkoxide (ROH þ CH3MgBr) in THF
produced 31a (>80%) with minimal isomerization; however,
similar reactions with hindered alcohols (e.g., borneol) were
unproductive at ambient temperature and heating under reflux
resulted in elimination products (31b).30 Reasoning that the use
of activated esters with lower acidity at the R-CH might suppress
isomerization and elimination, we investigated the HOBt ester
30c.31 Gratifyingly, reactions of 30c with the bromomagnesium
with HF pyridine to give 25 that was subjected to ester
3
hydrolysis using Me3SnOH to provide the acid 26 in 85%
yield. Reaction of 26 with TBSOTf followed by hydrolysis of
the intermediate TBS-ester gave acid 27a in high yield.
Given the difficulties experienced in the hydrolysis of the
methyl ester in 24a, it was anticipated that esterification of the
hindered acid in 27a with lairdinol A (5) might prove challenging.
To examine that process, we attempted esterification of the
model dipeptide 30a under a variety of conditions (Scheme 5).
Using simple unhindered alcohols (e.g., (4-methoxyphenyl)-
methanol), attempted esterification of 30a using Mukaiyama’s
reagent (O,O-di(2-pyridyl) thionocarbonate)27 failed, and the
use of more standard reagents (e.g., DCC/DMAP, EDCI, or
(28) The esters 31 were not fully characterized; the relative configurations
at C-2 were established by NOE (e.g., irradiations of HC-2, HC-4, and
H3CC-3).
(23) Coste, J.; Frerot, E.; Jouin, P.; Castro, B. Tetrahedron Lett. 1991, 32,
1967–1970.
(24) (a) Laganis, E. D.; Chenard, B. L. Tetrahedron Lett. 1984, 25, 5831–
5834. (b) Lovric, M.; Cepanec, I.; Litvic, M.; Bartolincic, A.; Vinkovic, V.
Croat. Chem. Acta 2007, 80, 109–115.
(25) Nicolaou, K. C.; Estrada Anthony, A.; Zak, M.; Lee Sang, H.; Safina
Brian, S. Angew. Chem., Int. Ed. 2005, 44, 1378–1382.
(26) Protection of the Hmb hydroxyl group was thought prudent con-
sidering the facile degradation of depsilairdin to form 3,6-diisopropyl-2,5-
morpholindione (ref 2).
(29) Corey, E. J.; Nicolaou, K. C. J. Am. Chem. Soc. 1974, 96, 5614–5616.
(30) For reactions of 2-pyridylthiol esters with Grignard reagents to form
ketones, see: (a) Mukaiyama, T.; Araki, M.; Takei, H. J. Am. Chem. Soc.
1973, 95, 4763–4765. For reactions of 2-pyridylthiol esters with lithium
dialkylcuprates in the presence of oxygen to form esters, see: (b) Kim, S.; Lee,
J. I.; Chung, B. Y. J. Chem. Soc., Chem. Commun. 1981, 1231–1232.
(31) We did not firmly establish whether this product was the O- or N-acyl
1-hydroxybenzotriazole derivative; however, the IR spectrum showed an
absorption at ca. 1825 cm-1, indicative of the O-acyl isomer. For example,
see: (a) Li, P.; Xu, J. C. J. Chem. Soc., Perkin Trans. 2 2001, 113–120. (b)
Katritzky, A. R.; Malhotra, N.; Fan, W.-Q.; Anders, E. J. Chem. Soc., Perkin
Trans. 2 1991, 1545–1547.
(27) This reagent is particularly useful for the synthesis of hindered esters.
Saitoh, K.; Shiina, I.; Mukaiyama, T. Chem. Lett. 1998, 679–680.
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SCHEME 6. Synthesis of Depsilairdin Analogues 36a-c
alkoxides of secondary alcohols (e.g., borneol) in refluxing THF
gave the corresponding esters 31a in good yield.
reported using a natural sample,2 synthetic depsilairdin (1)
caused strong necrotic and chlorotic lesions on leaves of
brown mustard (blackleg-susceptible), whereas canola leaves
(blackleg-resistant) were not affected.33 In contrast, the three
analogues 36a-c did not produce significant lesions on
either brown mustard or canola leaves under the same
conditions. We conclude that the presence of the Dhmp (4)
residue in 1 is important for its selective toxicity.
The HOBt ester 27b was readily prepared from 27a using
the standard method (Scheme 4).31 Reaction of 27b with the
bromomagnesium alkoxide prepared from 5 (2 equiv) and
freshly prepared PhMgBr (1.2 equiv) in refluxing THF gave
the desired ester 28 in 40% yield along with recovered 5
(75%). Deprotection of 28 was achieved by sequential reac-
tion with TBAF in CH2Cl2 (to remove the TBS groups) and
then treatment with Dowex 50 in refluxing aqueous MeOH
(to hydrolyze the MOM ether)32 to give 1 (85%; [R]D -45
(c 0.15, CH2Cl2), lit.2 -65 (c 0.9, CH2Cl2)). 1H and 13C NMR
spectra for synthetic 1 in CDCl3 were essentially super-
imposable with those for natural 1 kindly provided by Prof.
Pedras.33
Using an analogous approach, we prepared some simple
analogues of depsilairdin (1) by replacing the Dhmp (4)
residue with L-proline and cis- and trans-4-hydroxy-L-pro-
line (Scheme 6). Assembly of the tetradepsipeptides 35a-c
proceeded smoothly and with yields comparable to those
obtained in the synthesis of 24. Compared to our difficulties
with 24a, hydrolyses of the methyl esters in 35a-c were
straightforward and could be accomplished with LiOH in
methanolic THF. With 32b and 32c, ester hydrolysis was
accompanied by partial hydrolysis of the proline TBS ether;
the latter was avoided by using Me3SnOH. In sharp contrast
to 27a, esterifications of the resulting acids with lairdinol A
(5) were readily accomplished under standard conditions to
give the corresponding analogues 36a-c in good yields after
removal of the TBS ether(s).34
Summary and Conclusions
In summary, the total synthesis of depsilairdin (1), a host-
selective phytotoxin isolated from L. maculans (the causal
agent of blackleg disease of oilseed Brassicas), has been
achieved by sequential coupling of its five component resi-
dues. The reagents 7 and 8, protected derivatives of the
Hmb (2) and MeVal (3) residues, are readily available from
D- and L-valine, respectively. The (2S,3S,4R)-3,4-dihydroxy-
3-methyl-proline residue (Dhmp; 4) is a previously unknown
amino acid, and the suitably protected Dhmp derivative 9a
was prepared from trans-4-hydroxy-L-proline with signifi-
cant modifications of Sardina’s synthetic route to a related
compound. The synthesis of lairdinol A (5) from (R)-carvone
was described previously. Sequential PyBrop-mediated
N-terminal extension of 9a with 8 followed by reaction with 7
gave the tetradepsipeptide TBS-Hmb-MeVal-MeVal-(4-O-TES)-
(3-O-MOM)Dhmp-OMe (24b) in excellent overall yield.
Hydrolysis of the methyl ester in 24b proved difficult and
could be achieved only by reaction of Me3SnOH with TBS-
Hmb-MeVal-MeVal-(3-O-MOM)Dhmp-OMe (25), a deri-
vative presumably activated by intramolecular hydrogen
bonding. Similarly, esterification of the tetradepsipeptide
acid with lairdinol A (5) was complicated by the sterically
hindered nature of the carboxyl group and required a novel
method involving reaction of the 1-hydroxybenzotriazole
(HOBt) derived active ester with the bromomagnesium
alkoxide of 5. Three depsilairdin analogues 36a-c were
similarly prepared by replacing the Dhmp residue with
L-proline and cis- and trans-4-hydroxy-L-proline; however,
these analogues proved to be biologically inactive. The next
step is to prepare probes (e.g., radiolabeled 1)35 to study the
underlying mechanism(s) responsible for the host-selective
The relative phytotoxicities of depsilairdin (1) and analo-
gues 36a-c (10-4-10-5 M in aqueous methanol) to plants
resistant [canola (B. napus) cv. Westar] and susceptible
[brown mustard (B. juncea) cv. Cutlass] to blackleg isolates
Mayfair 2 and Laird 2 were evaluated by a punctured leaf
assay as previously described.2 Analogous to the results
(32) Seto, H.; Mander, L. N. Synth. Commun. 1992, 22, 2823–2828.
(33) See Supporting Information for additional information.
(34) These conditions (i.e., 1 equiv each of DCC and DMAP in CH2Cl2 at
rt for 15-20 h) produce the HOBt ester in situ. Attempted esterification of
27a with 5 with this protocol (i.e., via 27b), failed; however, heating the
reaction at 50 °C in a sealed tube for 72 h produced an ester (70%). Although
not fully characterized, NOE experiments (ref 28) suggest that this ester is
2-epi-28, and subjecting it to the deprotection sequence used for 28 gave a
product that was clearly different from 1 by 1H NMR.
(35) Pedras, M. S. C.; Zaharia, I. L.; Gai, Y.; Zhou, Y.; Ward, D. E. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 747–752.
5174 J. Org. Chem. Vol. 75, No. 15, 2010

Ward and Pardeshi
JOCArticle
phytotoxiciity of depsilairdin (1) and our results will be
reported in due course.
7.47-7.53 (2H, m), 7.38 (1H, dd, J = 8, 8 Hz), 7.31-7.36 (2H,
m), 7.26-7.31 (4H, m), 7.14 (1H, dd, J = 8, 8 Hz), 4.74 (1H, d,
J = 7.5 Hz), 4.64 (1H, d, J = 12 Hz), 4.60 (1H, d, J = 7.5 Hz),
3.81 (1H, dd, J = 3.5, 12 Hz), 3.53 (1H, d, J = 10.5 Hz), 3.38 (3H,
s), 3.29 (3H, s), 3.21 (1H, dd, J = 3.5, 10.5 Hz), 2.83 (1H, s), 0.89
(3H, s); 13C NMR(125 MHz, CDCl3) δ 176.1, 148.2, 144.7, 142.1,
141.0, 139.4, 129.1, 128.7, 128.6, 127.9, 127.8, 127.7, 127.3, 127.2,
126.5, 120.6, 120.2, 92.8, 83.4, 75.8, 75.5, 69.7, 55.8, 55.0, 51.9,
23.4; HRMSm/z calcd for C28H29NO5 459.2046, found 459.2044.
1H and 13C NMR data (in CD2Cl2) were consistent with those
previously reported.16
Methyl (2S,3S,4R)-3-(Methoxymethoxy)-3-methyl-1-(9-phenyl-
9H-fluoren-9-yl)-4-(triethylsilyloxy)pyrrolidine-2-carboxylate [Pf-
(4-O-TES)(3-O-MOM)Dhmp-OMe] (17b). 2,6-Lutidine (0.110
mL, 0.105 g, 0.980 mmol) and Et3SiOSO2CF3 (0.17 mL, 0.194 g,
0.735 mmol) were sequentially added to a stirred solution of
alcohol 16 (0.225 g, 0.489 mmol) in dry CH2Cl2 (5 mL) at 0 °C.
After 15 min, the mixture was diluted with ethyl acetate and
washed sequentially with satd NaHCO3, 5% aq HCl, satd NaH-
CO3, and brine. The organic layer was dried over Na2SO4,
concentrated, and fractionated by FCC (20% ethyl acetate in
hexane) to afford the titled compound 17b (0.260 g, 93%): [R]D
þ200 (c 1.2, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 7.76 (1H, d,
J = 7.5 Hz), 7.75-7.65 (3H, m), 7.45-7.51 (2H, m), 7.18-7.37
(6H, m), 7.07 (1H, dd, J = 7.5, 7.5 Hz), 5.13 (1H, d, J = 7.5 Hz),
4.56 (1H, d, J = 7.5 Hz), 3.47-3.57 (2H, m), 3.44 (3H, s), 3.28
(1H, dd, J = 6, 10 Hz), 3.21 (3H, s), 2.78 (1H, s), 1.56 (3H, s), 0.90
(9H, t, J = 8 Hz), 0.52 (6H, ap q, J = 8 Hz); 13C NMR (125 MHz,
CDCl3) δ 172.2, 147.6, 146.5, 143.6, 142.6, 139.2, 129.2, 128.58,
128.55, 128.4, 128.1, 127.7, 127.5, 127.1, 125.5, 120.3, 119.8, 92.5,
83.1, 77.9, 77.4, 70.7, 55.0, 54.5, 51.3, 19.9, 6.9, 5.0; HRMS m/z
calcd for C34H43NO5Si 573.2911, found 573.2906 (EI).
Methyl (2S,3S,4R)-3-(Methoxymethoxy)-3-methyl-4-(triethyl-
silyloxy)pyrrolidine-2-carboxylate [(4-O-TES)(3-O-MOM)Dhmp-
OMe] (9b). A stirred suspension of 17b (0.26 g, 0.45 mmol) and
10% Pd/C (0.10 g) in iPrOH (4 mL) was evacuated, and H2 gas was
introduced using a balloon. After 6 h, the reaction mixture was
passed through pad of Celite, and the combined filtrate and
CH2Cl2 washings were concentrated and fractionated by FCC
(5% MeOH in CH2Cl2) to afford the titled compound 9b (0.13 g,
88%) that was somewhat unstable and used immediately in the
next step: [R]D -54 (c 1.1, CH3OH); 1H NMR (500 MHz, CDCl3)
δ: 5.17 (1H, d, J = 7.5 Hz), 4.59 (1H, d, J = 7.5 Hz), 3.99 (1H, dd,
J = 7.5, 9.5 Hz), 3.78 (3H, s), 3.60 (1H, s), 3.26 (3H, s), 3.10-2.99
(2H, m), 2.51 (1H, br s), 1.55 (3H, s), 0.94 (9H, t, J = 8 Hz), 0.58
(6H, ap q, J = 8 Hz); 13C NMR (125 MHz, CDCl3) δ: 171.9, 92.6,
82.3, 79.5, 68.6, 55.2, 52.2, 50.3, 18.8, 6.9, 4.9; HRMS m/z calcd for
C15H31NO5Si 333.1971, found 333.1974 (EI).
Experimental Section33
Methyl (2S,3S)-3-(Methoxymethoxy)-4-oxo-1-(9-phenyl-9H-
fluoren-9-yl)pyrrolidine-2-carboxylate (12). N,N-Dimethylani-
line (1.9 mL, 1.8 g, 15 mmol) and MOM-Cl (0.81 mL, 0.86 g,
11 mmol) were sequentially added to a stirred solution of 11
(0.85 g, 2.1 mmol) in CH2Cl2 (8.5 mL) at room temperature
under argon. After 1 day, the reaction mixture was diluted with
diethyl ether and washed with 10% aq HCl and satd NaHCO3.
The organic layer was dried over Na2SO4, concentrated, and
fractionated by FCC (30% ethyl acetate in hexane) to afford the
title compound as a pale yellow foam (0.75 g, 80%): [R]D -170
(c 1.1, CHCl3) [lit. -158.2 (c 1.1, CHCl3)];16 1H NMR (500
MHz, CDCl3) δ 7.75 (1H, ddd, J = 1, 1, 7.5 Hz), 7.69 (1H, dd,
J = 1, 8 Hz), 7.44 (1H, dd, J = 1, 7.5 Hz), 7.43-7.35 (5H, m),
7.33 (1H, ddd, J = 1, 7.5, 8 Hz), 7.30-7.23 (4H, m), 4.65 (1H, d,
J = 6.5 Hz), 4.57 (1H, d, J = 6.5 Hz), 4.49 (1H, dd, J = 1, 7.5
Hz), 3.98 (1H, d, J = 7.5 Hz), 3.90 (1H, d, J = 17.5 Hz), 3.60
(1H, dd, J = 1, 17.5 Hz), 3.30 (3H, s), 3.11 (3H, s); 13C NMR
(125 MHz, CDCl3) δ 209.3, 170.7, 146.6, 144.9, 141.6, 141.2,
139.9, 129.23, 129.19, 128.9, 128.3, 128.1, 127.7, 126.9, 125.5,
120.4, 120.4, 96.5, 77.7, 75.1, 61.4, 56.2, 52.2, 51.4; HRMS m/z
calcd for C27H25NO5 443.1733, found 443.1717 (EI). 1H and 13
C
NMR data (in CD2Cl2) were consistent with those previously
reported.16
Methyl (2S,3S)-3-(Methoxymethoxy)-3-methyl-4-oxo-1-(9-
phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate (13). DMPU
(5 mL) was added to a stirred solution of KN(SiMe3)2 (0.45 M
in toluene; 3.0 mL, 1.4 mmol) in 5 mL of toluene at 0 °C under
Ar. After 10 min, the mixture was cooled to -78 °C, and
a solution of 12 (0.60 g, 1.4 mmol) in toluene and DMPU (1:1
(v/v); 8 mL) was added. After 1 h, CH3I (0.84 mL, 1.9 g, 1.4 mmol)
was added via syringe. After 2 h, the reaction was quenched by
addition of KH2PO4 (1 M; 20 mL). The mixture was allowed to
warm to ambient temperature and then was extracted with ethyl
acetate. The combined organic layers were washed sequentially
with H2O and brine, dried over Na2SO4, concentrated, and
fractionated by FCC (gradient elution; 10-20% ethyl acetate in
hexane) to afford the diastereomer 14 (0.093 g, 15%) and a 8:1
mixture of the titled compound 13 and 15 (0.42 g, 68%), respec-
tively. The mixture was used in the next step without further
purification. In a smaller scale experiment (50 mg of 12), fractiona-
tion of the crude by PTLC (35% ethyl acetate in hexane; 2 elutions)
gave pure 13 (28 mg, 54%):[R]D -8(c0.3, CH2Cl2);1HNMR(500
MHz, CDCl3) δ 7.75 (1H, ap d, J = 7.5 Hz), 7.70 (1H, ap d, J =
7.5 Hz), 7.21-7.47 (11H, m), 4.91 (1H, d, J = 7.5 Hz), 4.63 (1H, d,
J = 7.5 Hz), 4.08 (1H, d, J = 17.5 Hz), 3.72 (1H, d, J = 17.5 Hz),
3.58 (1H, s), 3.24 (3H, s), 3.00 (3H, s), 1.68 (3H, s); 13C NMR (125
MHz, CDCl3) δ 211.1, 170.8, 146.4, 144.8, 141.5, 141.4, 140.1,
129.2, 129.1, 129.0, 128.3, 128.0, 127.5, 127.4, 126.9, 125.5, 120.50,
120.46, 92.8, 81.7, 74.8, 68.7, 56.0, 52.1, 51.1, 20.7; HRMS m/z
calcd for C28H27NO5 457.1889, found 457.1889.
Methyl (2S,3S,4R)-4-Hydroxy-3-(methoxymethoxy)-3-methyl-
1-(9-phenyl-9H-fluoren-9-yl)pyrrolidine-2-carboxylate [Pf-(3-O-
MOM)Dhmp-OMe] (16). NaBH4 (0.35 g, 0.92 mmol) was added
to a stirred solution of an 8:1 mixture of 13 and 15, respectively,
(0.42 g, 0.092 mmol) in a mixture of CH2Cl2 and MeOH (1:1
(v/v); 9 mL) at -78 °C under argon. After 16 h, the reaction was
quenched by dropwise addition of acetone (5 mL). The mixture
was diluted with CH2Cl2, washed with satd NaHCO3, dried over
Na2SO4, concentrated, and fractionated by FCC (30% ethyl
acetate in hexane) to afford 15 (0.046 g, 11%) and the titled
compound 16 as a foam (0.32 g, 76%): [R]D þ200 (c 0.6, CHCl3)
[lit. 161.7 (c 0.6, CHCl3)];16 1H NMR (500 MHz, CDCl3) δ 7.82
(1H, d, J = 8 Hz), 7.67 (1H, d, J = 8 Hz), 7.60-7.64 (2H, m),
Cbz-MeVal-(4-O-TES)(3-O-MOM)Dhmp-OMe (22b). PyBroP
(0.27 g, 0.58 mmol) and DIPEA (0.14 mL, 0.10 g, 0.78 mmol) were
sequentially added to a stirred solution compound 9b (0.13 g,
0.39 mmol) and Cbz-MeVal-OH (8) (0.13 g, 0.51 mmol) in CH2Cl2
(5.5 mL) at 0 °C. The mixture was stirred for 10 min at 0 °C and
then for 18 h at room temperature. The mixture was diluted with
CH2Cl2, washedsequentiallywithaqcitricacid(0.5M) andsatdaq
NaHCO3, dried over Na2SO4, concentrated, and fractionated by
FCC (30% ethyl acetate in hexane) to give the titled compound 22b
1
(0.020 g, 90%): [R]D -92 (c 1.5, CH2Cl2); H NMR (500 MHz,
CDCl3) (a ca. 5.5:1 mixture of rotamers; signal for the major
rotamer only) δ 7.28-7.39 (5H, m, Ph), 5.14 (d, J = 12.8 Hz), 5.10
(1H, d, J = 12.8 Hz), 5.17 (1H, d, J = 7.5 Hz), 4.60 (1H, d, J =
7.5 Hz), 4.54 (1H, d, J = 11 Hz), 4.38 (1H, dd, J = 7. 9.5 Hz), 4.26
(1H, s), 3.78 (1H, dd, J = 7, 9.5 Hz), 3.75 (3H, s), 3.52 (1H, dd, J =
9.5, 9.5 Hz), 3.28 (3H, s), 2.94 (3H, s), 2.20-2.34 (1H, m), 1.60 (3H,
s), 1.03 (3H, d, J =6.5 Hz), 0.96 (9H, t, J = 8 Hz), 0.88 (3H, d, J =
6.5Hz), 0.62(6H, apq,J=8Hz);13C NMR (125 MHz, CDCl3) (a
ca. 5.5:1 mixture of rotamers; signals for the major rotamer only) δ
170.2, 167.6, 157.2, 136.9, 128.7, 128.2, 128.0, 92.7, 80.7, 77.0, 67.9,
J. Org. Chem. Vol. 75, No. 15, 2010 5175

Ward and Pardeshi
JOCArticle
67.5, 61.5, 55.3, 52.0, 50.1, 29.6, 28.0, 19.6, 19.1, 18.9, 6.9, 4.8;
HRMS m/z calcd for C29H49N2O8Si (M þ H) 581.3253, found
581.3259 (ESI).
TBS-Hmb-MeVal-MeVal-(3-O-MOM)Dhmp-OMe (25). Pyri-
dine (0.12 mL) and HF pyridine (0.12 mL) were added to a stirred
3
solution of 24b (40 mg, 0.052 mmol) in THF (1 mL) at 0 °C. After
1 h, the mixture was quenched by addition of satd NaHCO3 (aq)
(caution: CO2 evolution), and then was diluted with ethyl acetate,
washed sequentially with 2% aq citric acid ( ꢀ 3), satd NaHCO3
(aq) and brine, dried over Na2SO4, concentrated, and fractionated
by FCC (40% ethyl acetate in hexane) to give 25 (32 mg, 95%):
[R]D -90 (c 1.1, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ5.11 (1H,
d, J = 11 Hz), 5.04 (1H, d, J = 11 Hz), 4.82 (1H, d, J = 7.3 Hz),
4.74 (1H, d, J = 7.3 Hz), 4.33 (1H, s), 4.24 (1H, dd, J = 5, 11 Hz),
4.12 (1H, d, J = 11 Hz), 4.10 (1H, d, J = 6.5 Hz), 3.90 (1H, ddd,
J = 5, 5, 11 Hz), 3.81 (1H, dd, J = 5, 11 Hz), 3.79 (3H, s), 3.42 (3H,
s), 3.21 (3H, s), 3.14 (3H, s), 2.22-2.28 (2H, m), 1.90-2.01 (1H,
m), 1.46 (3H, s), 1.01 (3H, d, J = 6.5 Hz), 0.94 (3H, d, J = 6.5 Hz),
0.91 (9H, s), 0.89 (3H, d, J= 6.5 Hz), 0.88 (3H, d, J=6.5Hz), 0.82
(3H, d, J = 6.5 Hz), 0.81 (3H, d, J = 6.5 Hz), 0.04 (3H, s), 0.03
(3H, s); 13C NMR (125 MHz, CDCl3) δ 173.6, 172.5, 170.9, 170.7,
92.7, 82.3, 79.8, 75.7, 67.8, 58.6, 58.5, 56.2, 53.9, 52.7, 32.0, 31.0,
29.9, 27.8, 27.6, 26.0, 22.0, 19.5, 19.3, 19.04, 19.00, 18.8, 18.4, 18.3,
-4.4, -5.0; HRMS m/z calcd for C32H61N3O9Si 659.4177, found
659.4156 (EI).
TBS-Hmb-MeVal-MeVal-(3-O-MOM)Dhmp-OH (26). Me3Sn-
OH (55 mg, 0.30 mmol) was added to a stirred solution of 25 (20mg,
0.030 mmol) in (CH2Cl)2 (0.5 mL), and the reaction mixture was
heated in an oil bath at 80 °C. After 2 days, the mixture was diluted
with ethyl acetate and washed sequentially with 5% aq HCl and
brine. The organic layer was dried over Na2SO4, concentrated, and
fractionated by FCC (10% MeOH in CH2Cl2) to give 26 that was
taken up in CH2Cl2, washed with 5% aq HCl, dried over Na2SO4,
and concentrated to give 26 (17 mg, 87%) (the latter process was
required to obtain material whose NMR spectra showed sharp
signals); [R]D -100 (c 0.4, CH3OH); 1H NMR (500 MHz, CDCl3) δ
5.10 (1H, d, J = 11 Hz), 5.02 (1H, d, J = 11 Hz), 4.87 (1H, d, J =
7.5 Hz), 4.73 (1H, d, J = 7.5 Hz), 4.36 (1H, s, Pro-C-2), 4.22 (1H,
dd, J = 4.5, 11 Hz, Pro-HC-5), 4.10 (1H, d, J = 6.5 Hz), 3.92 (1H,
dd, J =4.5, 4.5 Hz), 3.80 (1H, dd, J = 4.5, 11 Hz), 3.43 (3H, s), 3.20
(3H, s), 3.13 (3H, s), 2.24-2.36 (2H, m), 1.90-1.99 (1H, m), 1.46
(3H, s), 0.98 (3H, d, J = 6.5 Hz), 0.93 (3H, s, J = 6.5 Hz), 0.90 (9H,
s), 0.85-0.89 (6H, m), 0.82 (3H, d, J =6.5 Hz), 0.79 (3H, d, J = 6.5
Hz), 0.03 (3H, s), 0.02 (3H, s); 13C NMR (125 MHz, CDCl3) δ
173.6, 172.5, 171.3, 170.6, 92.5, 82.7, 79.8, 75.7, 67.9, 58.70, 58.67,
58.3, 53.2, 32.0, 31.1, 30.2, 27.7, 27.6, 26.0, 21.5, 19.5, 19.3, 19.1, 19.0,
18.8, 18.4, 18.3, -4.4, -5.0; HRMS m/z calcd for C31H60N3O9Si
(M þ H) 646.4093, found 646.4086 (ESI).
Cbz-MeVal-MeVal-(4-O-TES)(3-O-MOM)Dhmp-OMe (23b).
A stirred suspension of 22b (0.070 g, 0.12 mmol) and 10% Pd/C
(20 mg) in PrOH (1.2 mL) was evacuated, and H2 gas was
i
introduced using a balloon. After 4 h, the reaction mixture was
passed through pad of Celite. The combined filtrate and CH2Cl2
washings were concentrated to give the crude deprotected amine
(0.028 g). PyBroP (0.84 g, 0.18 mmol) and DIPEA 0.042 mL, 0.031
g, 0.24 mmol) were sequentially added to a stirred solution of Cbz-
MeVal-OH (8) (0.041 g, 1.6 mmol) and the above crude amine in
CH2Cl2 (2 mL) at 0 °C under argon. After 10 min, the mixture was
allowed to warm to ambient temperature. After 18 h, the mixture
was diluted with CH2Cl2, washed sequentially with aq citric acid
(0.5 M) and satd aq NaHCO3, dried over Na2SO4, concentrated,
and fractionated by FCC (30% ethyl acetate in hexane) to give 23b
(0.071 g, 85%): [R]D -110 (c 1.4, CH2Cl2); 1H NMR (500 MHz,
CDCl3) (a ca. 2.2:1 mixture of rotamers; signals for the major
rotamer only) δ 7.28-7.38 (5H, m), 5.17-5.20 (3H, m), 5.00 (1H,
d, J = 11 Hz), 4.73 (d, J = 11 Hz), 4.61 (1H, d, J = 7.5 Hz), 4.42
(1H, dd, J = 7, 9.5 Hz), 4.27 (s), 3.76 (3H, s), 3.67-3.74 (2H, m),
3.54 (1H, dd, J = 9.5, 9.5 Hz), 3.28 (3H, s), 3.09 (3H, s), 2.87 (3H,
s), 2.13-2.40 (2H, m), 1.60 (3H, s), 1.03 (d, J = 7 Hz), 0.93-0.99
(9H, m, H3CCSi ꢀ 3), 0.88 (3H, d, J = 7 Hz), 0.86 (3H, d, J =
7 Hz), 0.77 (3H, d, J = 7 Hz), 0.58-0.66 (6H, m); 13C NMR
(125 MHz, CDCl3) (a ca. 2.2:1 mixture of rotamers; signals for the
major rotamer only) δ 171.4, 169.8, 167.6, 157.1, 136.9, 128.7,
128.2, 127.8, 92.7, 80.8, 77.1, 67.9, 67.6, 60.7, 59.2, 55.3, 52.0, 50.3,
30.7, 29.5, 28.1, 27.6, 19.9, 19.7, 19.1, 18.6, 18.3, 6.9, 4.8; HRMS
m/z calcd for C35H59N3O9Si 693.4021, found 693.4010 (EI).
TBS-Hmb-MeVal-MeVal-(4-O-TES)(3-O-MOM)Dhmp-OMe
(24b). Using the modified11d procedure of Wissner,11c oxalyl chlo-
ride (0.013 mL, 0.018 g, 0.15 mmol) was added dropwise to a
solution of tert-butyldimethylsilyl (R)-2-((tert-butyldimethylsilyl)-
oxy)-3-methylbutanoate (0.052 g, 0.15 mmol)11b and DMF (ca.
1 μL, 0.01 mmol) in CH2Cl2 (1 mL) at 0 °C under argon. The
mixture was allowed to warm to ambient temperature and, after
4 h, was diluted with dry hexane (5 mL), and the precipitated solids
were removed by filtration. The combined filtrate and hexane
washings were concentrated to give the crude acid chloride 7.
A stirred suspension of 23b (0.051 g, 0.073 mmol) and 10% Pd/C
i
(9 mg) in PrOH (0.5 mL) was evacuated, and H2 gas was
introduced using a balloon. After 4 h, the reaction mixture was
passed through pad of Celite. The combined filtrate and CH2Cl2
washings were concentrated to give the crude deprotected amine
(0.018 g) that was dissolved in CH2Cl2 (1 mL), and the resulting
solution was added to the above crude 7. DIPEA (0.019 mL, 0.014
g, 0.11 mmol) was added to the stirred mixture at 0 °C. The mixture
was allowed to warm to ambient temperature and after 4 h, diluted
with ethyl acetate, washed sequentially with water, 10% aq citric
acid, satd aq NaHCO3 and brine, dried over Na2SO4, and
concentrated. The resulting yellow oil was fractionated by FCC
(30% ethyl acetate in hexane) to give 24b as a colorless oil (0.048 g,
85%): [R]D -95 (c 2.06, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ
5.13 (1H, d, J = 7.5 Hz), 5.10 (1H, d, J = 11 Hz), 5.02 (1H, d, J =
11 Hz), 4.60 (1H, d, J = 7.5 Hz), 4.43 (1H, dd, J = 6.5, 9.5 Hz),
4.26 (1H, s), 4.10 (1H, d, J = 6.5 Hz), 3.75 (3H, s), 3.71 (1H, dd,
J = 6.5, 9.5 Hz), 3.53 (1H, dd, J = 9.5, 9.5 Hz), 3.28 (3H, s), 3.19
(3H, s), 3.15 (3H, s), 2.22-2.37 (2H, m), 1.91-2.00 (1H, m), 1.60
(3H, s), 1.02 (3H, d, J = 6.5 Hz), 0.97 (9H, dd, J = 7.5, 7.5 Hz),
0.87-0.95 (18H, m), 0.83 (3H, d, J = 6.5 Hz), 0.79 (3H, d, J = 6.5
Hz), 0.63 (6H, ddd, J = 7.5, 7.5, 7.5 Hz), 0.03 (3H, s), 0.02 (3H, s);
13C NMR (125 MHz, CDCl3) δ 173.5, 172.2, 169.7, 167.6, 92.7,
80.7, 79.4, 77.2, 67.8, 58.9, 58.5, 55.3, 52.0, 50.3, 31.9, 30.9, 30.2,
27.9, 27.8, 26.0, 19.8, 19.59, 19.57, 19.0, 18.7, 18.4, 18.2, 6.9, 4.8,
-4.4, -5.0; HRMS m/z calcd for C38H75N3O9Si2 773.5042, found
773.5045 (EI).
TBS-Hmb-MeVal-MeVal-(4-O-TBS)(3-O-MOM)Dhmp-OH
(27a). 2,6-Lutidine (0.021 mL, 0.020 g, 0.18 mmol) and tBuMe2-
SiOSO2CF3 (0.030 mL, 0.035 g, 0.13 mmol) were sequentially
added to a stirred solution of 26 (0.017 g, 0.026 mmol) in dry
CH2Cl2 (0.5 mL) at 0 °C under Ar. After 15 min, the mixture was
diluted with ethyl acetate and washed sequentially with satd
NaHCO3 and brine. The organic layer was dried over Na2SO4
and concentrated to get crude tris-TBS derivative that was taken
up in THF (1 mL). A solution of aq LiOH (0.5 M; 0.70 mL,
0.35 mmol) was added to the above stirred solution at 0 °C. The
reaction mixture was allowed to warm to room temperature and,
after 3 h, was diluted with CH2Cl2 and washed with 10% aq HCl.
The organic layer was dried over Na2SO4, concentrated, and
fractionated by FCC (10% MeOH in CH2Cl2) to give 27a that
was taken up in CH2Cl2, washed with 5% aq HCl, dried over
Na2SO4 and concentrated to give the titled compound (17 mg,
86%) (the latter process was required to obtain material whose
NMR spectra showed sharp signals): [R]D -80 (c 0.5, CH3OH);
1H NMR (500 MHz, CDCl3) δ 5.07-5.13 (2H, m), 5.00 (1H, d,
J = 11 Hz), 4.67 (1H, d, J = 7.5 Hz), 4.40 (1H, dd, J = 6.5, 9.5
Hz), 4.29 (1H, s), 4.09 (1H, d, J = 6.5 Hz), 3.72 (1H, dd, J = 6.5,
9.5 Hz), 3.56 (1H, dd, J = 9.5, 9.5 Hz), 3.32 (3H, s), 3.18 (3H, s),
3.13 (3H, s), 2.21-2.36 (2H, m), 1.90-1.99 (1H, m), 1.58 (3H, s),
5176 J. Org. Chem. Vol. 75, No. 15, 2010

Ward and Pardeshi
JOCArticle
0.97 (3H, d, J= 6.5 Hz), 0.92 (3H, d, J = 6.5 Hz), 0.84-0.91 (24H,
m), 0.82 (3H, d, J = 6.5 Hz), 0.77 (3H, d, J = 6.5 Hz), 0.11 (3H, s),
0.09 (3H, s), 0.03 (3H, s), 0.01 (3H, s); 13C NMR (125 MHz,
CDCl3) δ 173.6, 172.3, 170.5, 170.0, 92.9, 81.1, 79.8, 77.4, 67.6,
58.9, 58.6, 55.6, 50.8, 32.0, 31.0, 30.2, 27.85, 27.75, 26.0, 25.8, 20.1,
19.65, 19.58, 19.1, 19.0, 18.7, 18.4, 18.2, 18.1, -4.4, -4.7, -4.99,
-5.02; HRMS m/z calcd for C37H74N3O9Si2 759.4885 (M þ H)
760.4958, found 760.4967 (ESI).
(1H, s), 4.10 (1H, d, J = 6 Hz), 3.69 (1H, dd, J = 7, 9.5 Hz), 3.57
(1H, dd, J = 9.5, 9.5 Hz), 3.29 (3H, s), 3.20 (3H, s), 3.13 (3H, s),
2.40-2.18 (2H, m), 2.10-1.53 (10H, m), 1.74 (3H, s), 1.57 (3H,
s), 1.40-1.16 (4H, m), 1.13 (3H, s), 1.03 (3H, d, J = 6.5 Hz), 0.99
(3H, s), 0.92 (3H, d, J = 6.5 Hz), 0.90 (9H, s), 0.89 (9H, s), 0.88
(3H, d, J = 6.5 Hz), 0.87 (3H, d, J = 6.5 Hz), 0.82 (3H, d, J =
6.5 Hz), 0.78 (3H, d, J = 6.5 Hz), 0.11 (3H, s), 0.08 (3H, s), 0.03
(3H, s), 0.01 (3H, s); 13C NMR (125 MHz, CDCl3) δ: 173.5,
172.1, 169.8, 166.3, 150.5, 108.6, 92.8, 82.5, 80.7, 79.6, 77.2, 71.6,
68.1, 59.0, 58.6, 56.2, 53.5, 50.5, 45.8, 40.88, 40.83, 38.3, 32.0,
31.1, 30.2, 27.8, 27.8, 26.5, 26.0, 25.93, 25.86, 25.3, 22.9, 21.3,
19.7, 19.6, 19.6, 19.3, 19.0, 18.7, 18.4, 18.1, 18.1, 14.5, -4.4,
-4.6, -5.01, -5.06; HRMS m/z calcd for C52H98N3O10Si2
(M þ H) 980.6785, found 980.6761 (ESI).
TBS-Hmb-MeVal-MeVal-(4-O-TBS)(3-O-MOM)Dhmp-OBt
(27b).31. DCC (16 mg, 0.080 mmol) was added to a stirred
solution of acid 27a (20 mg, 0.026 mmol) and HOBt (5 mg,
0.04 mmol) in CH2Cl2 (0.5 mL). After 6 h the reaction mixture
was diluted with ethyl acetate (0.5 mL). The precipitated DCU
was filtered off, and the combined filtrate and washings were
concentrated and fractionated by PTLC (60% ethyl acetate in
hexane) to give the HOBt ester (21 mg, 91%) as a thick oil: [R]D
-70 (c 0.4, CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.02 (1H, d,
J = 8.5 Hz), 7.72 (1H, d, J = 8.5 Hz), 7.54 (1H, dd, J = 8.5, 8.5
Hz), 7.40 (1H, dd, J = 8.5, 8.5 Hz), 5.30 (1H, d, J = 7.5 Hz), 5.14
(1H, d, J = 11 Hz), 5.10 (1H, d, J = 11 Hz), 4.81 (1H, d, J = 7.5
Hz), 4.63 (1H, s), 4.57 (1H, dd, J = 6.5, 9.5 Hz), 4.12 (1H, d, J =
6.5 Hz), 3.86 (1H, dd, J = 6.5, 9.5 Hz), 3.72 (1H, dd, J = 9.5, 9.5
Hz), 3.45 (3H, s), 3.26 (3H, s), 3.15 (3H, s), 2.41-2.30 (2H, m),
1.96 (1H, dqq, J = 6.5, 6.5, 6.5 Hz), 1.73 (3H, s), 0.94 (3H, d, J =
6.5 Hz), 0.93 (9H, s), 0.92 (9H, s), 0.91 (3H, d, J = 6.5 Hz), 0.90
(3H, d, J = 6.5 Hz), 0.88 (3H, d, J = 6.5 Hz), 0.86 (3H, d, J = 6.5
Hz), 0.79 (3H, d, J = 6.5 Hz), 0.16 (3H, s), 0.14 (3H, s), 0.04 (3H,
s), 0.03 (3H, s); 13C NMR (125 MHz, CDCl3) δ 173.7, 172.4,
170.4, 164.0, 143.6, 128.9 ( ꢀ 2), 124.9, 120.3, 109.6, 93.3, 81.3,
79.7, 77.5, 67.1, 58.9, 58.7, 56.5, 50.4, 32.0, 31.0, 30.3, 27.9, 27.6,
26.0, 25.9, 19.82, 19.76, 19.6, 19.0 ( ꢀ 2), 18.6, 18.4, 18.2 ( ꢀ 2),
-4.4, -4.6, -4.97, -4.99; HRMS m/z calcd for C43H77N6O9Si2
(M þ H) 877.5290, found 877.5259 (ESI).
Depsilairdin (1). TBAF (0.011 g, 0.041 mmol) was added to a
stirred solution of 28 (4 mg, 0.004 mmol) in CH2Cl2 (0.1 mL) at
room temperature. After 2 d, the mixture was diluted with
CH2Cl2 and washed with water. The organic layer was dried
over Na2SO4, concentrated, and fractionated using FCC (50%
ethyl acetate in hexane) to afford corresponding diol (3 mg,
>90%). Dry Dowex 50 (50 mg)32 was added to a solution of the
above diol (3 mg) in a 5:1 (v/v) mixture of MeOH and H2O,
respectively (0.5 mL), and the stirred mixture was heated under
reflux. After 36 h, the mixture was filtered. The combined filtrate
and MeOH washings were concentrated and fractionated by
PTLC (high performance cyano matrix TLC plates, 10 mm ꢀ
10 mm ꢀ 0.2 mm; 40% acetonitrile in H2O) to afford 1 (2.5 mg,
85% from 28) as a colorless oil, [R]D -45 (c 0.15, CH2Cl2) [lit.2
-65 (c 0.9, CH2Cl2)]. 1H and 13C NMR data (in C6D6 solution)
were consistent with those reported previously.2
Acknowledgment. We thank Prof. M. S. C. Pedras and
Dr. P. B. Chumala for conducting phytotoxicity assays and
for providing NMR data for 1 in CDCl3 solution and
Fabiola Becerril-Jimenez (University of Saskatchewan) for
obtaining characterization data for several compounds.
Financial support from the Natural Sciences and Engineering
Research Council (Canada) and the University of
Saskatchewan is gratefully acknowledged.
TBS-Hmb-MeVal-MeVal-(4-O-TBS)(3-O-MOM)Dhmp-Lar
(28). PhMgBr (0.60 M in THF; 0.068 mL, 0.040 mmol) was
added to a stirred solution of lairdinol A (5) (16 mg, 0.068 mmol)
in THF (0.25 mL) at 0 °C. After 30 min, a solution of the 27b
(30 mg, 0.034 mmol) in THF (0.25 mL) was added. The reaction
mixture was allowed to reach ambient temperature and then was
heated under reflux. After 5 h, the mixture was allowed to cool,
quenched by addition of phosphate buffer (pH = 7), and then
extracted with CH2Cl2. The combined organic layers were dried
over Na2SO4, concentrated, and fractionated using PTLC (60%
EtOAc in hexane) to afford lairdinol A (5) (12 mg, 75%) and 28
(13 mg, 40%): [R]D -39 (c 0.20, CH2Cl2); 1H NMR (500 MHz,
CDCl3) δ: 5.10 (1H, d, J = 11 Hz), 5.09 (1H, d, J = 7 Hz), 5.02
(1H, d, J = 11 Hz), 4.72-4.68 (2H, m), 4.65 (1H, d, J = 7 Hz),
4.59 (1H, dd, J = 4, 11.5 Hz), 4.41 (1H, dd, J = 7, 9.5 Hz, 4.19
Supporting Information Available: Experimental proce-
1
dures, characterization data, and copies of H and 13C NMR
spectra for all reported compounds; comparison of NMR data
for natural and synthetic 1; NMR spectra for the reaction of
MOM-Cl with imidazole in DMF-d7; procedures and results for
phytotoxicity assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 75, No. 15, 2010 5177