Total synthesis of a mycobactin S, a siderophore and growth promoter of Mycobacterium Smegmatis, and determination of its growth inhibitory activity against Mycobacterium tuberculosis

Article abstract of DOI:10.1021/ja963968x

A general total synthesis of mycobactins, represented by a mycobactin S, was achieved by a convergent approach. Two hydroxamic acid residues, 28 and 40b, were prepared from commercially available N(α)-Cbz-L-lysine via dimethyldioxirane oxidations. Cycliza

Full text of DOI:10.1021/ja963968x

3462
J. Am. Chem. Soc. 1997, 119, 3462-3468
Total Synthesis of a Mycobactin S, a Siderophore and Growth
Promoter of Mycobacterium Smegmatis, and Determination of
its Growth Inhibitory Activity against Mycobacterium
tuberculosis
Jingdan Hu and Marvin J. Miller*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of Notre Dame,
Notre Dame, Indiana 46556
ReceiVed NoVember 15, 1996X
Abstract: A general total synthesis of mycobactins, represented by a mycobactin S, was achieved by a convergent
approach. Two hydroxamic acid residues, 28 and 40b, were prepared from commercially available NR-Cbz-L-lysine
Via dimethyldioxirane oxidations. Cyclization of hydroxylamine 34 to a seven-membered hydroxamic acid, 35, was
mediated by DCC, DMAP, and DMAP‚HCl. The use of a [2-(trimethylsilyl)ethoxy]methyl group as a hydroxyl
protecting group for NR-Cbz-Nꢀ-hydroxy-Nꢀ-palmitoyl-L-lysine methyl ester (28) was critical for this synthesis.
Biological tests indicated that the synthetic mycobactin S was a potent growth inhibitor of Mycobacterium tuberculosis
H37Rv, though it differs in only one stereogenic center from mycobactin T, the siderophore growth promoter of M.
tuberculosis.
Introduction
large amount of mycolic acids with long hydrocarbon chains.
The lengths of the chains produce an exceptionally tightly
packed array with extremely low fluidity, which limits penetra-
tion of antibiotics and chemotherapeutic agents into the cell
walls.7-9 On the other hand, this also causes nutrient acquisition
difficulties for mycobacteria themselves. Studies have shown
that almost all mycobacteria possess a serious problem of iron
acquisition, because the predominant form of iron occurs in its
oxidized ferric [Fe(III)] state, which is highly insoluble at
physiological pH. Also, in animal tissues, most iron is chelated
by host molecules such as lactoferrin and transferrin. In order
to survive, microbes secrete specific low molecular weight iron
chelators termed siderophores (iron carriers) to sequester and
transport iron.10-17 Siderophores usually chelate iron from
extracellular media. In many mammalian infections, sidero-
phores which are produced by microorganisms are even able
to acquire the host’s lactoferrin- or transferrin-bound iron.18
In 1993, the World Health Organization (WHO) declared
tuberculosis (TB) a global emergency with the hope of drawing
the world’s attention to the growing severity of the TB epidemic.
Tuberculosis is a historically chronic, debilitating, and often fatal
disease that normally severely affects respiratory tracts of
humans. After the discovery and utilization of antibiotics such
as streptomycin and isoniazid in the 1940s and 1950s, TB was
considered a controllable disease. However, in the mid-1980s,
a significant resurgence of TB infections occurred, primarily
because of the prevalence of drug-resistant or multiple-drug-
resistant strains, which are potentially incurable.1 The number
of TB cases in the United States in 1994 increased about 10%
relative to 1985, the year with the lowest number of reported
TB cases since national reporting began in 1953.2 The situation
worldwide is even worse. In 1996, the WHO global TB
program estimated that from 1990 to the year 2000, the total
number of TB cases among adults will exceed 40 million.3 The
widespread HIV epidemic is believed to play an important role
in the reemergence of TB. With its contagious nature, TB is
the only major opportunistic infection which can be spread
through the air among people. As expected, HIV-positive
patients with compromised immune systems are especially
susceptible to TB infection. In fact, TB has become the leading
killer of HIV-positive people. One out of three AIDS patients
dies of TB. The medical crisis associated with TB infections,
especially the development of potentially incurable multiple-
drug-resistant (MDR) TB strains, has raised a serious challenge
for current chemotherapeutic treatment.4-6
(7) Connell, N. D.; Nikaido, H. Membrane Permeability and Transport
in Mycobacterium tuberculosis. In Tuberculosis: Pathogenesis, Protection,
and Control; Bloom, B. R., Ed.; ASM Press: Washington, DC, 1994;
p 333.
(8) Liu, J.; Rosenberg, E. Y.; Nikaido, H. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 11254.
(9) Besra, G. S.; Chatterjee, D. Lipid and Carbohydrates of Mycobac-
terium tuberculosis. In Tuberculosis: Pathogenesis, Protection, and Control;
Bloom, B. R., Ed.; ASM Press: Washington, DC, 1994; p 285.
(10) Matzanke, B. F.; Mu¨ller-Matzanke, G.; Raymond, K. N. Sidero-
phore-Mediated Iron Transport. In Iron Carriers and Iron Proteins; Loehr,
T. M., Ed.; VCH: New York, 1989; p 1.
(11) McKee, J. A. Iron Transport Mediated Drug Delivery. I. Synthesis
and Biological Evaluation of Catechol Siderophore-â-Lactam Conjugates.
II. Synthesis and Antifungal Activity of Hydroxamate Siderophore-Based
Antifungal Conjugates. Ph.D. Thesis, University of Notre Dame,
1991, p 4.
(12) Miller, M. J. Chem. ReV. 1989, 89, 1563.
(13) Miller, M. J. Acc. Chem. Res. 1993, 26, 241.
(14) Miller, M. J.; Malouin, F. Siderophore-Mediated Drug Delivery:
The Design, Synthesis, and Study of Siderophore-Antibiotic and Antifungal
Conjugates. In The DeVelopment of Iron Chelators for Clinical Use;
Bergeron, R. J., Brittenham, G. M., Eds.; CRC Press: Boca Raton, FL,
1994; p 275.
One important aspect of drug resistance in strains of TB is
thought to be related to the specific mycobacterial cell wall.
Most cells of mycobacteria, including Mycobacterium tuber-
culosis, are covered by lipid-rich cell walls, which consist of a
X Abstract published in AdVance ACS Abstracts, March 15, 1997.
(1) Lemonick, M. D. The Killers All Around. In Time Magazine;
Lemonick, M. D., Ed.; Time, Inc.: New York, Sept 12, 1994; p 62.
(2) CDC. Tuberculosis Morbidity-United States, 1994; 1995.
(3) WHO. Groups at Risk; 1996.
(15) Yamamoto, S. Farumashia 1995, 31, 588.
(4) Huebner, R. E.; Castro, K. G. Annu. ReV. Med. 1995, 46, 47.
(5) Frieden, T. R.; Sterling, T.; Pablos-Mendez, A.; Kilburn, J. O.;
Cauthen, G. M.; Dooley, S. W. N. Engl. J. Med. 1993, 328, 521.
(6) Jacobs, R. F. Clin. Infect. Dis. 1994, 19, 1.
(16) Winkelmann, G. Biotechnol. AdV. 1990, 8, 207.
(17) Raymond, K. N. Pure Appl. Chem. 1994, 66, 773.
(18) Kretchmar Nguyen, S. A.; Craig, A.; Raymond, K. N. J. Am. Chem.
Soc. 1993, 115, 6758.
S0002-7863(96)03968-6 CCC: $14.00 © 1997 American Chemical Society

Total Synthesis of a Mycobactin S
J. Am. Chem. Soc., Vol. 119, No. 15, 1997 3463
Table 1. General Structures of Mycobactins
Scheme 2
Scheme 1
dihydro-2H-pyran (3). Subsequent synthesis of caprolactam 6
was complicated by N- vs O-alkylation selectivity. Biological
studies of mycobactin S2 revealed that the short alkyl group
(R1 ) CH3) resulted in loss of lipid solubility and siderophore
activity for mycobacteria.23 This indicated that a long lipophilic
side chain is essential for mycobactins to transport iron through
cell membranes and therefore to be potential drug carriers. Thus,
methodology directed toward syntheses of various mycobactins
was explored.
Preparation of an analog of mycobactin T, the siderophore
and growth promoter for M. tuberculosis, with minimal and
defined variations was anticipated to allow us to further test
Snow’s hypothesis that alternate forms of mycobactins might
have antitubercular activity. Thus, mycobactin S (Table 1),
produced by Mycobacterium smegmatis, was chosen to be the
initial target molecule in this study. It is the closest structural
relative of mycobactin T, produced by M. tuberculosis, differing
only in variable substituent R1 and chiral center (d of structure
1). To facilitate the study, we also chose to use a single defined
acyl group [R1 ) CH3(CH2)14] that is similar to the mixture of
acyl groups of both mycobactin S and T.
Retrosynthetically, disconnection of the ester bond of my-
cobactins 1 gives two fragments, mycobactic acids 8, containing
the acyclic hydroxamate residue, and cobactins 9, containing
the cyclic hydroxamate residue (Scheme 2). Further discon-
nections of the amide bonds in fragments 8 and 9 result in four
corresponding components, 10, 11, 12, and 13. Oxazoline
derivative 10 can be prepared from a salicylic acid, 14, and
serine or threonine derivative 15. Hydroxamic acid 11 can be
prepared from 16 by direct amine oxidation using dimethyl-
dioxirane (DMD) oxidation methodology which we recently
reported.24 Some â-hydroxy acids, 12, are commercially
available materials. Nꢀ-Hydroxycyclolysine (13) can also be
Mycobactins 1 are a family of siderophores, which are
isolated from mycobacteria including M. tuberculosis and
Mycobacterium phlei (Table 1).19,20 They promote mycobac-
terial growth Via iron uptake processes. Mycobactins possess
a nearly identical molecular nucleus, which includes two
hydroxamic acids and a 2-(2-hydroxyphenyl)-∆2-1,3-oxazoline
residue serving as iron-chelating components. They vary only
in the stereochemistry of the chiral centers and in the peripheral
groups. After detailed study and structural elucidation of the
mycobactins, Snow20 suggested that alternate or modified forms
of mycobactins might serve as antagonists of mycobacterial
growth and be of therapeutic value.
A mycobactin analog lacking the three hydroxyl groups
necessary for iron chelation was synthesized21 shortly after
structural elucidation of several mycobactins. Biological tests
of these analogs, incapable of chelating iron, revealed no growth
inhibitory or stimulatory activity of mycobacteria, thus confirm-
ing a need for iron complexation. The first synthesis of a
mycobactin analog, mycobactin S2 (2), which contains all iron-
chelating components, but lacks a long lipophilic side chain (R1
) CH3), was accomplished in our group (Scheme 1).22 This
methodology was limited by utilizing an enzymatic resolution
to synthesize intermediate L-ꢀ-hydroxynorleucine (4) from 3,4-
(19) Snow, G. A. Biochem. J. 1965, 97, 166.
(23) Preliminary studies performed by Professor Phillip E. Klebba,
Department of Microbiology, Medical College of Wisconsin, Milwaukee,
WI.
(20) Snow, G. A. Bacteriol. ReV. 1970, 34, 99.
(21) Carpenter, J. G.; Moore, J. W. J. Chem. Soc. C 1969, 12, 1610.
(22) Maurer, P. J.; Miller, M. J. J. Am. Chem. Soc. 1983, 105, 240.
(24) Hu, J.; Miller, M. J. J. Org. Chem. 1994, 59, 4858.

3464 J. Am. Chem. Soc., Vol. 119, No. 15, 1997
Hu and Miller
Scheme 3
Scheme 4
derived from 16 by DMD oxidation and subsequent further
functional group manipulations.25
Scheme 5
Results and Discussion
Synthesis of Mycobactic Acid. Commercially available
methyl salicylate (17) was refluxed in a mixed solvent (methanol
and chloroform) with benzyl bromide in the presence of
potassium carbonate under a stream of argon (Scheme 3).26,27
After filtration and concentration, the residue was saponified
to give 2-(benzyloxy)benzoic acid (18) in 83% yield. Changing
the solvent from chloroform to methylene chloride did not affect
the reaction. However, the transformation was totally sup-
pressed in the absence of methanol. Acid 18 was coupled with
L-serine benzyl ester 19 to afford amide 20 in 90% yield.
Attempted cyclization of amide 20 with thionyl chloride22
resulted in a chloride-substituted derivative 21. Treatment of
20 with Burgess’s reagent28 provided oxazoline 22 in 66% yield.
The corresponding eliminated product 23 also was isolated in
low yield and characterized by 1H NMR and MS spectroscopy.
Hydrogenolytic removal of the benzyl groups from compound
22 gave the corresponding acid 24.
Hydroxylamine 27 was prepared by dimethyldioxirane oxida-
tion of a derivative of amino acid 25 and subsequent hydrolysis
or hydroxylamine exchange.24 Acylation of hydroxylamine 27
with palmitoyl chloride in the presence of NaHCO3 provided
hydroxamic acid 28 (Scheme 4), a key iron-binding component
of the mycobactins. Hydrogenolytic removal of the Cbz group
from hydroxamic acid 28 gave the corresponding amine, which
was then coupled with oxazoline acid 24 using EDC [1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride] to
afford mycobactic acid methyl ester 29 in 59% yield. This
smooth reaction is in contrast to our previous attempts22 related
to coupling reactions of 2-[2-(benzyloxy)phenyl]-∆2-1,3-ox-
azoline-4-carboxylic acid with different amines. Absence of a
large benzyloxy group in acid 24 appeared to be the major factor
in the successful reaction. As a result, this significantly
improved the efficiency of the total synthesis of mycobactin S
(44). Subsequent saponification of 29 with KOH or LiOH in
aqueous THF solution at 0 °C provided mycobactic acid 30
quantitatively.
Synthesis of Cobactin T. NR-Cbz-L-lysine (25) was stirred
in AcOBut in the presence of a catalytic amount of HClO4 in a
sealed flask overnight to give the corresponding tert-butyl ester
31 (Scheme 5). Subsequent oxidation with DMD provided
nitrone 32 in 60% overall yield. After treatment of nitrone 32
with NH2OH‚HCl, followed by basic workup, extraction, and
concentration, the product hydroxylamine 33 was treated with
TFA to yield the corresponding acid 34. Compound 34 was
cyclized using DCC (N,N-dicyclohexylcarbodiimide), DMAP
[4-(dimethylamino)pyridine], and DMAP‚HCl to provide the
desired hydroxamic acid 35, but still in low isolated yield. The
product was perhaps chelated with Fe(III) whose presence could
be traced to silica gel chromatography. The apparent Fe(III)
complex was unable to be chromatographically eluted. The
problem was solved by prior protection of the hydroxamate
hydroxyl group with silyl groups. Treatment of hydroxamic
acid 35 with TBDMSCl (tert-butyldimethylsilyl chloride) or
TBDPSCl (tert-butyldiphenylsilyl chloride) in the presence of
imidazole at 35 °C overnight gave the corresponding azopine
36 or 37 in 50-55% overall yield for the four steps.
(25) Hu, J.; Miller, M. J. Tetrahedron Lett. 1995, 36, 6379.
(26) Farkas, L.; Vermes, B.; Nogradi, M. Tetrahedron 1967, 23, 741.
(27) Schmidhammer, H.; Brossi, A. J. Org. Chem. 1983, 48, 1469.
(28) Wipf, P.; Miller, C. P. J. Org. Chem. 1993, 58, 1575.
With azopines 36 and 37 in hand, synthesis of cobactin T
was subsequently performed. (R)-â-Hydroxybutanoic acid (39)

Total Synthesis of a Mycobactin S
J. Am. Chem. Soc., Vol. 119, No. 15, 1997 3465
Scheme 6
Biological Studies. As indicated earlier, in his excellent
review of the mycobactins in 1970,20 Snow suggested that the
mycobactin structure could be used as a model for the
development of drugs having specific activity against myco-
bacteria, but such a concept “was frustrated by the complexity
of the molecule”. Studies of simplified model compounds
related to mycobactin but incapable of binding iron had no affect
on the growth of M. tuberculosis, prompting the further
statement “Thus, the concept of a mycobactin antagonist seems
to have been sound in principle, though not yet realized in
practice.” We are now pleased to report that, as anticipated by
Snow, biological tests showed that our synthetic mycobactin S
(44) effects greater than 99% inhibition of the growth of M.
tuberculosis H37Rv at the concentration of 12.5 µg/mL. This
strongly indicates that the chiral center (d of structure 1) plays
a critical role in controlling TB growth since mycobactin T
which stereochemically differs only at this one center is a
siderophore and growth promoter of M. tuberculosis. Further
biological studies related to the inibition of the growth of drug-
resistant strains of M. tuberculosis are in progress.
was obtained by treatment of the corresponding sodium salt 38
(Aldrich) with Dowex 50 × 8-200 ion-exchange resin (Scheme
6). After hydrogenolytic removal of the Cbz group from
azopine 36, the resulting amine was coupled with (R)-â-
hydroxybutanoic acid (39) in the presence of DCC, DMAP, and
DMAP‚HCl. TLC analysis indicated that the TBDMS group
of compound 36 did not survive the coupling conditions, perhaps
because of the acidity of butanoic acid 39.
Reactions of TBDPS-protected azopine 37 were attempted
next. The Cbz group of azopine 37 was hydrogenolytically
removed, and the resulting amine was coupled with (R)-â-
hydroxybutanoic acid (39) using DCC, DMAP, and DMAP‚-
HCl to provide O-(tert-butyldiphenylsilyl)cobactin 40a in 63%
yield. The TBDPS group was cleaved using 49% aqueous HF
solution.29 It could also be removed by stirring with Dowex
50 × 8-200 ion-exchange resin in MeOH for a few minutes to
give cobactin T (40b) in 62% yield.
Summary
In conclusion, a general total synthesis of mycobactins was
achieved. This methodology featured applications of DMD
oxidation of primary amines in natural product syntheses, DCC-,
DMAP-, and DMAP‚HCl-mediated cyclization to give azopine
derivatives 36 and 37, and a successful application of SEM
protection for hydroxamic acid 28. The subsequent esterifica-
tion reaction accomplished the total synthesis of a mycobactin
S (44) which was demonstrated to be an effective inhibitor of
clinically important M. tuberculosis.
Synthesis of Mycobactin S. Mycobactic acid 30 and
cobactin 40a, the two major components of mycobactins, were
successfully synthesized by dimethyldioxirane-induced oxidation
of the corresponding primary amines as the key step. Conden-
sation of mycobactic acid 30 with cobactin 40a remained as
the last step in the construction of mycobactin S.
Experimental Section
Instruments and general methods used have been described earlier.35
Optical rotations were measured on a Rudolf Research Autopol III
polarimeter. The ferric chloride test was performed by treating the
TLC plate with 0.3% ferric chloride in aqueous 0.5 N HCl solution.
Anhydrous CH2Cl2 and acetonitrile were freshly distilled from CaH2
before use. Anhydrous tetrahydrofuran was freshly distilled from
sodium and benzophenone. During all hydrogenolysis reactions, the
solvent was purged with nitrogen prior to addition of a catalyst.
Antimycobacterial studies were performed at the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility using their standard
protocol.
Dimethyldioxirane. Dimethyldioxirane was generated by the
method reported by Murray et al.36 The concentration was determined
by iodometric titration.37
2-(Benzoyloxy)benzoic Acid (18). Compound 18 was prepared by
the method of Farkas et al.26 with slight modification. Methylene
chloride was used instead of chloroform. An 83% yield was obtained
with mp 74-76 °C (lit.26 48% yield; mp 76-78 °C)
Our original intention was to determine if it was possible to
effect direct condensation of 30 with 40a without protection of
the hydroxyl groups of 30. Various coupling reagents (DCC,
EDC, and EEDQ) and Mitsunobu conditions were utilized, but
all reactions failed to effect the condensation. Subsequent
protection of the hydroxyl groups of 30 did not provide a useful
synthetic pathway.30 This led to a revised synthetic strategy as
shown in Scheme 7. Readily available hydroxamic acid 28 was
reacted with SEMCl [[2-(trimethylsilyl)ethoxy]methyl chloride]
in the presence of DIEA (N,N-diisopropylethylamine) and
DMAP in toluene to give SEM-protected ester 41 in 91%
yield.31,32 Subsequent hydrogenolytic removal of the Cbz group
from ester 41 gave the corresponding amine, which was then
coupled with unprotected oxazoline 24 using EDC to give
protected mycobactic acid methyl ester 42a in 65% yield.
Treatment of ester 42a with LiOH provided acid 42b quanti-
tatively after acidification. Partially protected mycobactin S 43
was obtained in 49% yield under Mitsunobu conditions.
Treatment of 43 with TFA33,34 in methylene chloride provided
mycobactin S (44) in 56% yield.
N-[2-(Benzyloxy)benzoyl]-L-serine Benzyl Ester (20). To a stirred
solution of 2-(benzyloxy)benzoic acid (18) (3.53 g, 15.5 mmol) and
L-serine benzyl ester hydrochloride (19) (3.27 g, 14 mmol) in CH2Cl2
(70 mL) were added Et3N (2.09 mL, 15 mmol) and EDC [1-ethyl-3-
[3-(dimethylamino)propyl]carbodiimide; 2.96 g, 15.5 mmol]. After
being stirred for 10 h at room temperature under argon, the reaction
mixture was diluted with CH2Cl2 (200 mL), washed with H2O, saturated
NaHCO3 solution, 5% citric acid aqueous solution, H2O, and brine,
dried over Na2SO4, filtered, and concentrated. Recrystallization from
toluene afforded amide 20 (5.12 g, 90%) as white crystals: Rf ) 0.41
(29) Newton, R. F.; Reynolds, D. P. Tetrahedron Lett. 1979, 20, 3981.
(30) Hu, J. Total Syntheses of Mycobactins, Siderophore-Drug Conju-
gates, and Biological Studies. Ph. D. Thesis, University of Notre Dame,
1996, p 71.
(31) Lipshutz, B. H.; Tegram, J. J. Tetrahedron Lett. 1980, 21, 3343.
(32) Pinto, B. M.; Buiting, M. W.; Reimer, K. B. J. Org. Chem. 1990,
55, 2177.
(33) Schlessinger, R. H.; Poss, M. A.; Richardson, S. J. Am. Chem. Soc.
1986, 108, 3112.
(34) Jasson, K.; Frejd, T.; Kihlberg, J.; Magnusson, G. Tetrahedron Lett.
1988, 29, 361.
(EtOAc/CH2Cl2 ) 1/5); mp 116-118 °C; [R]23 ) +24.1° (c ) 1.0,
D
1
CH2Cl2); IR (KBr) 1740, 1620 cm-1; H NMR (300 MHz, CDCl3) δ
(35) Ghosh, A.; Ghosh, M.; Niu, C.; Malouin, F.; Moellmann, U.; Miller,
M. J. Chem. Biol. 1996, 3, 1011.
(36) Murray, R. W.; Jeyaraman, R. J. Org. Chem. 1985, 50, 2847.
(37) Adam, W.; Chan, Y.-Y.; Cremer, D.; Gauss, J.; Scheutzow, D.;
Schindler, M. J. Org. Chem. 1987, 52, 2800.

3466 J. Am. Chem. Soc., Vol. 119, No. 15, 1997
Hu and Miller
Scheme 7
8.81 (d, J ) 6.9 Hz, 1H), 8.19 (dd, J1 ) 7.8 Hz, J2 ) 1.8 Hz, 1H),
7.46-7.25 (m, 11H), 7.10-7.04 (m, 2H), 5.21-5.11 (m, apparently 4
overlapping doublets, 4H), 4.90-4.85 (m, 1H), 3.93-3.89 (m, 2H),
2.32 (br, 1H); 13C NMR (75 MHz, CDCl3) δ 170.13, 165.56, 156.91,
135.50, 135.26, 133.17, 132.31, 128.74, 128.53, 128.33, 128.06, 127.99,
121.48, 121.09, 112.79, 71.23, 67.17, 63.61, 55.38; FABMS m/z 406
(M + 1). Anal. Calcd for C23H21NO5: C, 70.58; H, 5.41; N, 3.58.
Found: C, 70.43; H, 5.60; N, 3.37.
C31H52N2O6: C, 67.85; H, 9.55; N, 5.10. Found: C, 67.83; H, 9.60;
N, 5.16.
Oxazoline-Hydroxamic Acid 29. To a stirred solution of hydrox-
amic acid 28 (44.5 mg, 0.08 mmol) in MeOH (1 mL) at room
temperature was added Pd/C (5 mg, 10%). After being stirred for 1 h
under H2 (1 atm), the mixture was filtered and concentrated to give
the corresponding amine. To another stirred solution of oxazoline 22
(31 mg, 0.08 mmol) in MeOH (1 mL) at room temperature was added
Pd/C (5 mg, 10%). After being stirred for 1 h at room temperature
under H2 (1 atm), the mixture was filtered and concentrated to give
the corresponding acid 24.39 The free amine and acid 24 were then
dissolved in CH2Cl2 (2 mL). To this mixture was added EDC (23 mg,
1.2 mmol, 1.5 equiv). After being stirred for 3 h at room temperature,
the reaction mixture was diluted with EtOAc (5 mL) and then washed
with H2O and brine, dried over Na2SO4, filtered, and concentrated to
give oxazoline-hydroxamic acid 29 (crude product, 43 mg, 92%), as
a light-yellow solid: Rf ) 0.28 (EtOAc/CH2Cl2 ) 1/5); mp 80.0-82.0
°C (recrystallized from CH2Cl2 and hexanes, 59% yield after recrys-
tallization); IR (KBr) 1740, 1650 cm-1; 1H NMR (300 MHz, CD3OD)
δ 7.69 (dd, J1 ) 7.9 Hz, J2 ) 1.5 Hz, 1H), 7.41 (dt, J1 ) 7.8 Hz, J2 )
1.6 Hz, 1H), 6.96 (d, J ) 8.3 Hz, 1H), 6.90 (t, J ) 7.6 Hz, 1H), 5.02
(t, J ) 9.0 Hz, 1H), 4.67-4.60 (m, 2H), 4.44 (dd, J1 ) 8.9 Hz, J2 )
4.9 Hz, 1H), 3.73 (s, 3H), 3.62-2.56 (m, 2H), 2.43 (t, J ) 7.5 Hz,
2H), 1.93-1.27 (m, 32H), 0.90 (t, J ) 6.6 Hz, 3H); 13C NMR (75
MHz, CD3OD) δ 176.18, 173.87, 172.96, 168.55, 161.00, 135.02,
129.51, 119.97, 117.70, 111.45, 70.43, 69.20, 53.85, 52.80, 33.26, 33.06,
31.82, 30.75, 30.62, 30.49, 30.46, 27.10, 25.96, 23.80, 23.72, 14.44;
HRFABMS m/z calcd for C33H53N3O7 604.3961, found 604.3983. Anal.
Calcd for C33H52N3O7: C, 65.64; H, 8.85; N, 6.96. Found: C, 65.58;
H, 8.81; N, 6.75.
(S)-Benzyl 2-[2-(Benzyloxy)phenyl]-∆2-1,3-oxazoline-4-carboxy-
late (22).28 To a stirred solution of N-[2-(benzyloxy)benzoyl]-L-serine
benzyl ester (20) (545 mg, 1.35 mmol) in THF (10 mL) was added
Burgess’s reagent [(methoxycarbonylsulfamoyl)triethylammonium hy-
droxide, inner salt, 360 mg, 1.55 mmol, 1.1 equiv]. After being refluxed
for 30 min at room temperature under argon, the reaction mixture was
diluted with EtOAc (100 mL), washed with H2O and brine, dried over
Na2SO4, filtered, concentrated, and chromatographed on silica gel
eluting with EtOAc/CH2Cl2 (1/15) to yield oxazoline 22 (343 mg, 66%)
as a white, amorphous solid: Rf ) 0.58 (EtOAc/CH2Cl2 ) 1/15); mp
69-71 °C (recrystallized from EtOAc and hexanes); IR (KBr) 1732,
1
1630 cm-1; H NMR (300 MHz, CDCl3) δ 7.81 (dd, J1 ) 7.8 Hz, J2
) 1.8 Hz, 1H), 7.50-7.47 (m, 2H), 7.42-7.26 (m, 9H), 7.00-6.96
(m, 2H), 5.28 (d, J ) 12.3 Hz, 1H), 5.20 (d, J ) 11.7 Hz, 1H), 5.18
(s, 2H), 5.00 (dd, J1 ) 10.5 Hz, J2 ) 8.1 Hz, 1H), 4.68-4.52 (m, 2H);
13C NMR (75 MHz, CDCl3) δ 171.03, 165.65, 157.56, 136.79, 135.40,
132.64, 131.62, 128.50, 128.36, 128.29, 128.25, 127.53, 126.69, 120.67,
117.12, 113.75, 70.55, 69.16, 68.77, 67.12; FABMS m/z 388 (M + 1);
HREIMS m/z calcd for C24H21NO4 387.1471, found 387.1458.
Nr-Cbz-Nδ-hydroxy-Nδ-palmitoyl-L-lysine Methyl Ester (28).38
To a stirred solution of nitrone 2624 (680 mg, 1.94 mmol) in MeOH
(10 mL) at 40 °C was added hydroxylamine hydrochloride salt (203
mg, 2.91 mmol, 1.5 equiv). After the solution was stirred for 10 min
at 40 °C, the solvent was removed. The residue was dissolved in
saturated NaHCO3 solution (15 mL) and then extracted with CH2Cl2.
The combined organic layers were washed with H2O and brine, dried
over Na2SO4, filtered, and concentrated to give the corresponding
hydroxylamine 27. Compound 27 was dissolved in CH2Cl2 (20 mL)
at 0 °C. To this mixture were added NaHCO3 (326 mg, 3.88 mmol, 2
equiv) and palmitoyl chloride (0.706 mL, 2.33 mmol, 1.2 equiv). After
being stirred for 3 h at room temperature, the mixture was filtered,
concentrated, and chromatographed on silica gel eluting with CH2Cl2/
EtOAc (5/2) to give hydroxamic acid 28 (749 mg, 71%) as a white
solid: Rf ) 0.33 (CH2Cl2/EtOAc ) 5/2); mp 66-68 °C (recrystallized
Nitrone 32. To a stirred solution of NR-Cbz-L-lysine (25) (1 g, 3.6
mmol) in AcOBut (50 mL) was added HClO4 (70%, 0.46 mL, 5.3 mmol,
1.5 equiv). The mixture was sealed with a septum and covered with
parafilm. After being stirred overnight, the reaction was extracted with
H2O and 0.5 N HCl solution. The combined aqueous solution was
basified with K2CO3 (10% aqueous solution) to pH 8 and then extracted
with CH2Cl2. The combined organic layers were washed with H2O
and brine, dried over Na2SO4, filtered, and concentrated to give the
corresponding tert-butyl ester, as an oil. The oil was dissolved in
acetone (20 mL) and cooled to -78 °C. To the acetone solution was
added an excess of dimethyldioxirane (50 mL).36 After being stirred
for another 10 min at -78 °C, the mixture was concentrated and
chromatographed on silica gel eluting with EtOAc/MeOH (8/3) to give
nitrone 32 (847 mg, 60%) as a clear oil: Rf ) 0.29 (EtOAc/MeOH )
1
from EtOAc/Skelly B); H NMR (300 MHz, CD3OD) δ 7.35-7.28
(m, 5H), 5.08 (s, 2H), 4.18-4.13 (m, 1H), 3.70 (s, 3H), 3.62-3.56
(m, 2H), 2.44 (t, J ) 7.6 Hz, 2H), 1.83-1.28 (m, 32H), 0.87 (t, J )
6.8 Hz, 3H); 13C NMR (75 MHz, CD3OD) δ 176.09, 174.58, 158.61,
138.13, 129.43, 128.95, 128.72, 67.60, 55.34, 52.63, 33.26, 33.07, 32.08,
30.79, 30.64, 30.50, 27.09, 25.96, 23.81, 23.73, 14.47; HRFABMS m/z
calcd for C31H53N2O6 549.3904, found 549.3897. Anal. Calcd for
1
8/3); IR (neat) 1715 (br) cm-1; H NMR (300 MHz, CDCl3) δ 7.37-
7.31 (m, 5H), 5.41 (d, J ) 7.9 Hz, 1H), 5.10 (s, 2H), 4.28-4.21 (m,
1H), 3.83 (t, J ) 7.2 Hz, 2H), 2.14 (s, 3H), 2.09 (s, 3H), 2.01-1.31
(m, 15H); 13C NMR (75 MHz, CDCl3) δ 171.29, 155.83, 143.50,
(39) Bergeron, R. J.; Liu, C. Z.; McManis, J. S.; Xia, M. X. B.; Algee,
(38) Polonski, T.; Chimiak, A. J. Org. Chem. 1976, 41, 2092.
S. E.; Wiegand, J. J. Med. Chem. 1994, 37, 1411.

Total Synthesis of a Mycobactin S
J. Am. Chem. Soc., Vol. 119, No. 15, 1997 3467
136.28, 128.43, 128.07, 128.02, 82.13, 66.77, 58.51, 54.05, 32.48, 27.92,
26.85, 22.37, 20.36, 19.90; HRFABMS m/z calcd for C21H33N2O5
393.2389, found 393.2400.
(m, 1H), 4.24-4.14 (m, 1H), 3.86-3.69 (m, 2H), 2.45 (dd, J1 ) 15.3
Hz, J2 ) 3.0 Hz, 1H), 2.32 (dd, J1 ) 15.3 Hz, J2 ) 8.6 Hz, 1H), 2.08-
1.42 (m, 6H), 1.23 (d, J ) 6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3)
δ 171.61, 167.06, 64.80, 50.84, 50.31, 43.78, 31.12, 27.72, 25.70, 22.61;
HRFABMS m/z calcd for C10H19N2O4 231.1345, found 231.1329.
TBDPS-Protected Azopine Derivative 37. To a stirred solution
of nitrone 32 (481 mg, 1.23 mmol) in MeOH (5 mL) was added NH2-
OH‚HCl (423 mg, 6.14 mmol, 5 equiv). The solution was stirred at
40 °C for 10 min. After removal of the solvent, the residue was taken
up in saturated NaHCO3 (10 mL). The aqueous solution was extracted
with CH2Cl2. The combined organic layers were dried over Na2SO4,
filtered, and concentrated to afford hydroxylamine 33. Hydroxylamine
33 was then treated with TFA/CH2Cl2 (3 mL/3 mL) for 1.5 h at room
temperature and concentrated to give acid 34. To a refluxing solution
of DCC (1.26 g, 6.14 mmol, 5 equiv), DMAP (749 mg, 6.14 mmol, 5
equiv), and DMAP‚HCl (976 mg, 6.14 mmol, 5 equiv) in CHCl3 (70
mL) was added acid 34 in CHCl3 (70 mL) dropwise over 2 h. The
reaction was refluxed for 1 h after addition. The mixture was
concentrated, taken up in DMF (5 mL), and treated with TBDPSCl
(800 µL, 3.08 mmol, 2.5 equiv) and imidazole (418 mg, 6.14 mmol, 5
equiv). After being stirred at 35 °C under argon overnight, the reaction
was diluted with EtOAc. The EtOAc solution was washed with H2O
and brine, dried over Na2SO4, filtered, concentrated, and chromato-
graphed on silica gel eluting with EtOAc/Skelly B (1/5) to give azopine
37 (339 mg, 54%) as a clear oil: Rf ) 0.19 (EtOAc/Skelly B ) 1/5);
Nr-Cbz-NE-palmitoyl-NE-[[2-(trimethylsilyl)ethoxy]methoxy]-L-
lysine Methyl Ester (41). To a stirred solution of NR-Cbz-Nꢀ-hydroxy-
Nꢀ-palmitoyl-L-lysine methyl ester (28) (3.65 g, 6.67 mmol) in toluene
(100 mL) were added N,N-diisopropylethylamine (23.2 mL, 133 mmol,
20 equiv), DMAP (catalytic amount), and SEMCl (11.8 mL, 66.7 mmol,
10 equiv) under argon. After being stirred for 48 h at 60-70 °C, the
reaction mixture was concentrated and then dissolved in EtOAc (200
mL). The solid in EtOAc was filtered off, and then the organic layer
was washed with H2O and brine, dried over Na2SO4, filtered,
concentrated, and chromatographed on silica gel eluting with EtOAc/
CH2Cl2 (1/15) to give product 41 (4.31 g, 91%) as a clear oil: Rf )
0.19 (EtOAc/CH2Cl2 ) 1/15); IR (neat) 3310 (w), 1720, 1650 cm-1
;
1H NMR (300 MHz, CDCl3) δ 7.36-7.31 (m, 5H), 5.36 (d, J ) 7.8
Hz, 1H), 5.10 (s, 2H), 4.90 (s, 2H), 4.37-4.30 (m, 1H), 3.76-3.62
(m, 7H), 2.38 (t, J ) 7.6 Hz, 2H), 1.86-1.03 (m, 32H), 0.99-0.93
(m, 2H), 0.88 (t, J ) 6.7 Hz, 3H), 0.03 (s, 9H); 13C NMR (75 MHz,
CDCl3) δ 175.36, 172.69, 155.82, 136.18, 128.29, 127.89, 127.85,
99.08, 67.38, 66.68, 53.64, 52.09, 47.16, 32.43, 31.75, 29.52, 29.49,
29.38, 29.30, 29.27, 29.19, 26.10, 24.33, 22.52, 22.14, 18.02, 13.96,
-1.63; HRFABMS m/z calcd for C37H67N2O7Si 679.4718, found
679.4702.
1
IR (neat) 3410, 3330, 1720, 1675 cm-1; H NMR (300 MHz, CDCl3)
δ 7.75-7.71 (m, 4H), 7.45-7.33 (m, 11H), 6.03 (d, J ) 6.3 Hz, 1H),
5.05 (ab q, J1 )17.9 Hz, J2 )12.4 Hz, 2H), 4.05-3.99 (m, 2H), 3.52-
3.45 (m, 2H), 1.89-1.05 (m, 15H); 13C NMR (75 MHz, CDCl3) δ
169.49, 155.26, 136.50, 136.09, 135.99, 132.04, 131.57, 130.19, 130.14,
128.36, 127.90, 127.76, 127.49, 127.44, 66.46, 54.17, 53.06, 31.54,
27.30, 26.85, 25.25, 19.48; HRFABMS m/z calcd for C30H37N2O4Si
517.2523, found 517.2568.
Mycobactic Acid Methyl Ester Analog 42a. Protected hydroxamic
acid 41 (200 mg, 0.295 mmol) was stirred in MeOH (5 mL) in the
presence of Pd/C (10%, 20 mg) under H2 (1 atm) for 1 h. After
filtration, the solvent was removed in Vacuo to provide the correspond-
ing free amine. The free amine was then treated with acid 24 [derived
from ester 22 (137 mg) by hydrogenolysis] and EDC (85 mg, 0.442
mmol, 1.5 equiv) in CH2Cl2 (5 mL). After being stirred for 30 min,
the reaction mixture was concentrated and chromatographed on silica
gel eluting with EtOAc/CH2Cl2 (1/15) to afford compound 42a (140
mg, 65%) as a clear oil: Rf ) 0.19 (EtOAc/CH2Cl2 ) 1/15); IR (neat)
1740, 1650 (br), 1630 cm-1; 1H NMR (300 MHz, CDCl3) δ 7.67 (dd,
J1 ) 7.9 Hz, J2 ) 1.7 Hz, 1H), 7.39 (ddd, J1 ) 8.7 Hz, J2 ) 7.5, J3 )
1.7 Hz, 1H), 7.00 (dd, J1 ) 8.5 Hz, J2 ) 0.8 Hz, 1H), 6.93-6.85 (m,
1H), 4.94 (t, J ) 9.6 Hz, 1H), 4.84 (s, 2H), 4.65-4.61 (m, 2H), 4.57-
4.50 (m, 1H), 3.73 (s, 3H), 3.72-3.66 (m, 2H), 3.60 (t, J ) 7.1 Hz,
2H), 2.33 (d, J ) 7.6 Hz, 2H), 1.91-1.52 (m, 6H), 1.28-1.16 (m,
26H), 0.95-0.89 (m, 2H), 0.83 (t, J ) 6.8 Hz, 3H), -0.003 (s, 9H);
13C NMR (75 MHz, CDCl3) δ 175.45, 172.09, 170.27, 167.68, 159.72,
134.13, 128.47, 118.95, 116.88, 109.94, 99.13, 69.40, 67.97, 67.47,
52.37, 52.04, 47.29, 32.50, 31.82, 31.58, 29.59, 29.56, 29.44, 29.37,
29.26, 26.18, 24.42, 22.59, 22.35, 18.08, 14.02, -1.56; HRFABMS
m/z calcd for C39H68N3O8Si 734.4776, found 734.4764.
TBDMS-Protected Azopine Derivative 36. Compound 36 was
prepared in 54% yield as a clear oil using the procedure described for
the synthesis of 37: Rf ) 0.44 (EtOAc/CH2Cl2 ) 1/15); IR (neat) 1720,
1
1675 cm-1; H NMR (300 MHz, CDCl3) δ 7.44-7.28 (m, 5H), 6.11
(d, J ) 6.5 Hz, 1H), 5.10 (s, 2H), 4.34 (ddd, J1 ) 11.3 Hz, J2 ) 6.6
Hz, J3 ) 1.9 Hz, 1H), 3.83-3.74 (m, 1H), 3.59-3.52 (m, 1H), 2.07-
1.46 (m, 6H), 0.96 (s, 9H), 0.22 (s, 3H), 0.15 (m, 3H); 13C NMR (75
MHz, CDCl3) δ 170.18, 155.43, 136.50, 128.47, 128.01, 127.90, 66.66,
54.36, 53.23, 31.90, 27.58, 25.73, 25.64, 18.04, -4.66, -5.24;
HRFABMS m/z calcd for C20H33N2O4Si 393.2210, found 393.2223.
TBDPS-Protected Cobactin 40a. To a stirred solution of compound
37 (103 mg, 0.199 mmol) in MeOH (4 mL) was added Pd/C (10 mg,
10%). After being stirred for 2 h at room temperature under H2 (1
atm), the reaction mixture was filtered and concentrated to give the
corresponding amine. To a beaker with Dowex 50 × 8-200 resin in
MeOH was added sodium (R)-â-hydroxybutyrate (38; Aldrich, 25 mg).
Acid 39 was obtained after filtration and concentration. To the mixture
of the amine, DCC (103 mg, 0.498 mmol, 2.5 equiv), DMAP (61 mg,
0.498 mmol, 2.5 equiv), and DMAP‚HCl (79 mg, 0.498 mmol, 2.5
equiv) in CHCl3 (10 mL) at 40 °C was added acid 39 in CHCl3 (2 mL)
slowly over 10 min. After being stirred further for 20 min at 40 °C,
the reaction mixture was concentrated and then taken up in EtOAc.
The EtOAc solution was washed with H2O and brine, dried over Na2-
SO4, filtered, concentrated, and chromatographed on silica gel eluting
with EtOAc to give TBDPS-protected cobactin 40a (58 mg 63%) as a
clear oil: Rf ) 0.32 (EtOAc); IR (neat) 3360 (br), 1645 (br) cm-1; 1H
NMR (300 MHz, CDCl3) δ 7.74 (t, J ) 7 Hz, 4H), 7.47-7.35 (m,
6H), 6.78 (d, J ) 6 Hz, 1H), 4.22 (dd, J1 ) 10 Hz, J2 ) 6 Hz, 1H),
4.20-4.10 (m, 1H), 3.90 (br, 1H), 3.56-3.40 (m, 2H), 2.32 (dd, J1 )
15 Hz, J2 ) 3 Hz, 1H), 2.21 (dd, J1 ) 15 Hz, J2 ) 8 Hz, 1H), 1.86-
1.35 (m, 9H), 1.15 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 171.37,
169.29, 136.08, 135.94, 131.94, 131.49, 130.25, 130.21, 127.54, 127.47,
64.69, 54.18, 51.42, 43.48, 30.92, 27.26, 26.84, 25.25, 22.46, 19.50;
HRFABMS m/z calcd for C26H37N2O4Si 469.2523, found 469.2519.
Cobactin T (40b). To a stirred mixture of Dowex resin (50 ×
8-200) in MeOH (1 mL) was added compound 40a (54 mg). After
being stirred for 1 h at room temperature, the reaction mixture was
filtered and concentrated to give a white solid. The solid was
recrystallized from EtOAc/hexanes to give colorless crystals (17 mg,
62%): Rf ) 0.50 (EtOAc/MeOH ) 8/3); mp 137-138 °C; IR (KBr)
1630 cm-1; 1H NMR (300 MHz, CDCl3) δ 6.97 (br, 1H), 4.64-4.58
Protected Mycobactin 43. To a stirred solution of protected
mycobactic acid 42b (obtained from 42a by saponification with LiOH;
72 mg, 0.1 mmol), protected cobactin 40a (48 mg, 0.1 mmol, 1 equiv),
and PPh3 (131 mg, 0.5 mmol, 5 equiv) in freshly distilled THF (4 mL)
was added DEAD (79 µL, 0.5 mmol, 5 equiv)/THF (0.5 mL). After
being stirred for 1 h at room temperature, the reaction was concentrated.
The residue was recrystallized from EtOAc/Skelly B to remove reduced
DEAD and triphenylphosphine oxide. The mother liquor was concen-
trated and chromatographed on silica gel eluting with EtOAc/CH2Cl2
(1/5) to afford protected mycobactin S 43 (57 mg, 49%) as a clear oil:
Rf ) 0.71 (EtOAc/CH2Cl2 ) 1/1); IR (neat) 3320, 1740, 1650 (br),
1
1640 cm-1; H NMR (500 MHz, CDCl3) δ 11.44 (s, 1H), 7.74-7.68
(m, 5H), 7.46-7.35 (m, 7H), 7.02 (dd, J1 ) 8.3 Hz, J2 ) 1.0 Hz, 1H),
6.98 (d, J ) 7.8 Hz, 1H), 6.91-6.87 (m, 2H), 5.26-5.23 (m, 1H),
4.95 (t, J ) 10 Hz, 1H), 4.86 (s, 2H), 4.63 (d, J ) 9.3 Hz, 2H), 4.45-
4.41 (m, 1H), 4.23-4.18 (m, 1H), 3.73-3.69 (m, 2H), 3.64-3.57 (m,
2H), 3.50-3.48 (m, 2H), 2.50 (dd, J1 ) 14.1 Hz, J2 ) 6.8 Hz, 1H),
2.37-2.33 (m, 3H), 1.85-1.51 (m, 12H), 1.28 (d, J ) 6.4 Hz, 3H),
1.26-1.24 (m, 26H), 1.13 (s, 9H), 0.96-0.92 (m, 2H), 0.87 (t, J )
6.8 Hz, 3H), 0.017 (s, 9H); 13C NMR (75 MHz, CDCl3) δ 170.48,
170.24, 169.31, 167.82, 167.48, 159.73, 136.04, 135.91, 134.02, 131.88,
131.49, 130.22, 130.15, 128.43, 127.45, 118.87, 116.88, 109.99, 98.99,
69.36, 69.25, 67.95, 67.50, 54.11, 52.49, 51.58, 42.27, 32.50, 31.82,
31.35, 30.84, 29.58, 29.55, 29.45, 29.39, 29.24, 27.14, 26.80, 26.23,

3468 J. Am. Chem. Soc., Vol. 119, No. 15, 1997
Hu and Miller
25.18, 24.42, 22.59, 22.39, 19.58, 19.46, 18.07, 14.02, -1.54; HR-
FABMS m/z calcd for C64H100N5O11Si2 1170.6958, found 1170.6941.
Mycobactin S (44). Protected mycobactin 43 (180 mg, 0.15 mmol)
was stirred in TFA/CH2Cl2 (2 mL, 1/1) at room temperature for 1 h.
The mixture was concentrated in Vacuo and then taken up in EtOAc.
The solution was washed with H2O and brine, dried over Na2SO4,
filtered, and concentrated to give a colored solid. The solid was
recrystallized from CHCl3/EtOAc to give mycobactin S (44) (83 mg,
68%) as a white, amorphous solid: Rf ) 0.27 (EtOAc/MeOH ) 20/1);
mp 178-180 °C; IR (KBr) 3305 (br), 1735, 1630 (br) cm-1; 1H NMR
(500 MHz, CDCl3 with CD3OD) δ 7.70 (dd, J1 ) 8.0 Hz, J2 ) 1.7 Hz,
1H), 7.44-7.40 (m, 1H), 7.01 (d, J ) 8.3 Hz, 1H), 6.92 (t, J ) 7.5
Hz, 1H), 5.36-5.30 (m, 1H), 5.00 (dd, J1 ) 10.0 Hz, J2 ) 8.7 Hz,
1H), 4.68-4.61 (m, 2H), 4.50 (d, J ) 10.7 Hz, 1H), 4.45 (dd, J1 ) 8.0
Hz, J2 ) 5.0 Hz, 1H), 3.81 (dd, J1 ) 16.0 Hz, J2 ) 11.3 Hz, 1H), 3.70
(dd, J1 ) 16.0 Hz, J2 ) 5.0 Hz, 1H), 3.63-3.50 (m, 2H), 2.59 (dd, J1
) 15.0 Hz, J2 ) 8.7 Hz, 1H), 2.50 (dd, J1 ) 15.0 Hz, J2 ) 4.0 Hz,
1H), 2.43 (dd, J1 ) 8.3 Hz, J2 ) 5.7 Hz, 2H), 2.00-1.48 (m, 12H),
1.34 (d, J ) 6.3 Hz, 3H), 1.30-1.25 (m, 26H), 0.88 (t, J ) 7.0 Hz,
3H); 13C NMR (75 MHz, CDCl3 with CD3OD) δ 174.96, 170.93,
169.05, 168.44, 167.35, 159.11, 133.94, 128.34, 118.96, 116.50, 109.85,
69.33, 69.07, 67.76, 52.33, 52.18, 51.05, 47.15, 42.13, 32.16, 31.67,
30.96, 30.83, 29.44, 29.31, 29.26, 29.19, 29.10, 27.28, 25.65, 25.42,
24.53, 22.41, 22.00, 19.40, 13.75; HRFABMS m/z calcd for C42H68N5O10
802.4966, found 802.4927. Anal. Calcd for C42H67N5O10 C, 62.90;
H, 8.42; N, 8.73. Found: C, 62.70; H, 8.26; N, 8.66.
Acknowledgment. We gratefully acknowledge the NIH and
the Reilly Fellowship to J.H. for support of this research. The
facilities of the Lizzadro Magnetic Resonance Research Center
at the University of Notre Dame are appreciated. Biological
tests of mycobactin S were performed by the Tuberculosis
Antimicrobial Acquisition and Coordinating Facility under
Contract N01-AI-45246 sponsored by the National Institute of
Allergy and Infectious Diseases.
JA963968X