Incremental conversion of outer-membrane permeabilizers into potent antibiotics for gram-negative bacteria

Article abstract of DOI:10.1021/ja982938m

Cholic acid derivatives appended with amine groups have been prepared for use as permeabilizers of the outer membranes of Gram-negative bacteria. These compounds interact synergistically with antibiotics such as erythromycin and novobiocin to inhibit grow

Full text of DOI:10.1021/ja982938m

J. Am. Chem. Soc. 1999, 121, 931-940
931
Incremental Conversion of Outer-Membrane Permeabilizers into
Potent Antibiotics for Gram-Negative Bacteria
Chunhong Li,† Loren P. Budge,† Collin D. Driscoll,† Barry M. Willardson,†
Glenn W. Allman,‡ and Paul B. Savage*,†
Contribution from the Departments of Chemistry and Biochemistry and Microbiology,
Brigham Young UniVersity, ProVo, Utah 84602
ReceiVed August 17, 1998
Abstract: Cholic acid derivatives appended with amine groups have been prepared for use as permeabilizers
of the outer membranes of Gram-negative bacteria. These compounds interact synergistically with antibiotics
such as erythromycin and novobiocin to inhibit growth of Gram-negative bacteria. When a hydrophobic chain
is included on the permeabilizers, they can be converted into potent antibiotics. The role of the hydrophobic
chain is to facilitate self-promoted transport of the cholic acid derivatives across the outer membrane of Gram-
negative bacteria. These compounds share activities found with polymyxin B and derivatives.
Introduction
The outer membrane common to Gram-negative bacteria is
unique among lipid bilayers in its structure and function. The
outer leaflet of the outer membrane of most species of Gram-
negative bacteria is comprised primarily of lipid A (1;1 Figure
1). Lipopolysaccharide (LPS) or Gram-negative endotoxin
consists of lipid A linked to a core oligosaccharide, which is
typically bonded to a polysaccharide.2 Lipid A molecules are
believed to form intermolecular noncovalent bridging inter-
actions via divalent cations (Mg2+ and Ca2+), providing a
substantial permeability barrier to hydrophobic molecules,
lysozymes, and proteases.3 Disruption of the lipid A cross
bridging significantly increases the permeability of the outer
membrane. Cross bridging can be disrupted by metal ion
chelators such as EDTA,4 by amines including tris(hydroxy-
methyl)aminomethane (TRIS) at high concentrations (>30
mM),5 or at much lower concentrations by compounds that
selectively bind LPS.6-8
Figure 1. (A) Structure of lipid A from E. coli. (B) Schematic
representation of the permeability barrier provided by lipid A-divalent
cation interactions.
Compounds known to bind LPS and increase the permeability
of the outer membrane of Gram-negative bacteria include
bactericidal/permeability increasing protein,6 a number of small
cationic peptides,7 the polymyxin family of antibiotics, and
polymyxin derivatives.8 Many of these compounds have been
shown to bind the lipid A portion of LPS.9 Polymyxin B1 and
B2 (used as a mixture and collectively termed PMB) (2; Figure
2) are the most commonly used members of the polymyxin
† Department of Chemistry and Biochemistry.
‡ Department of Microbiology.
(1) Galanos, C.; Luderitz, O.; Rietshel, E. T.; Westphal, O.; Brade, H.;
Brade, L.; Freudenberg, M.; Schade, U.; Imoto, M.; Yoshimura, H.;
Kusumoto, S.; Shiba, T. Eur. J. Biochem. 1985, 148, 1.
(2) Galanos, C.; Lu¨deritz, O.; Rietschel, E. T.; Westphal, O. Int. ReV.
Biochem. 1977, 14, 239.
(3) (a) Vaara, M. Microbiol. ReV. 1992, 56, 395. (b) Nikaido, H.; Vaara,
M. Microbiol. ReV. 1985, 49, 1. (c) Hancock, R. E. W. Annu. ReV. Microbiol.
1984, 38, 237.
(4) For examples see: (a) Beacham, I. R.; Kahana, R.; Levy, L.; Yagil,
E. J. Bacteriol. 1973, 116, 957. (b) Brown, M. R. W.; Richards, R. M. E.
Nature 1965, 207, 1391.
(5) For examples see: (a) Hancock, R. E. W.; Raffle, V. J.; Nicas, T. I.
Antimocrob. Agents Chemother. 1981, 19, 777. (b) Reynolds, B. L.; Pruul,
H. Infect. Immun. 1971, 3, 365.
(8) (a) Morris, C. M.; George, A.; Wilson, W. W.; Champlin, F. R. J.
Antibiot. 1995, 48, 67. (b) Ofek, I.; Cohen, S.; Rahmani, R.; Kabha, K.;
Tamarkin, D.; Herzig, Y.; Rubenstein, E. Antimicrob. Agents Chemother.
1994, 38, 374. (c) Vaara, M. Antimicrob. Agents Chemother. 1993, 37, 354.
(d) Kimura, Y.; Matsunaga, H.; Vaara, M. J. Antibiot. 1992, 45, 742. (e)
Viljanen, P.; Matsunaga, H.; Kimura, Y.; Vaara, M. J. Antibiot. 1991, 44,
517. (f) Vaara, M. Drugs Exp. Clin. Res. 1991, 17, 437. (g) Peterson, A.
A.; Hancock, R. E. W.; McGroarty, E. J. J. Bacteriol. 1985, 164, 1256. (h)
Vaara, M.; Vaara, T. Antimicrob. Agents Chemother. 1983, 24, 107. (i)
Vaara, M.; Vaara, T. Antimicrob. Agents Chemother. 1983, 24, 114. (j)
Vaara, M.; Vaara, T. Nature 1983, 303, 526.
(9) (a) Taylor, A. H.; Heavner, G.; Nedelman, M.; Sherris, D.; Brunt,
E.; Knight, D.; Ghrayeb, J. J. Biol. Chem. 1995, 270, 17934. (b) Guzzano-
Santoro, H.; Parent, J. B.; Grinna, L.; Horwitz, A.; Parsons, T.; Theofan,
G.; Elsbach, P.; Weiss, J.; Conlon, P. J. Infect. Immun. 1992, 60, 4754. (c)
David, S. A.; Balasubramanian, K. A.; Mathan, V. I.; Balaram, P. Biochim.
Biophys. Acta 1992, 1165, 147. (d) Moore, R. A.; Bates, N. C.; Hancock,
R. E. W. Antimicrob. Agents Chemother. 1986, 29, 496.
(6) Weiss, J.; Elsbach, P.; Olson, K.; Odeberg, H. J. Biol. Chem. 1978,
253, 2664.
(7) (a) Vaara, M.; Porro, M. Antimicrob. Agents Chemother. 1996, 40,
1801. (b) Maloy, W. L.; Kari, U. P. Biopolymers 1995, 37, 105. (c) Hancock,
R. E. W.; Falla, T.; Brown, M. AdV. Microb. Physiol. 1995, 37, 135.
10.1021/ja982938m CCC: $18.00 © 1999 American Chemical Society
Published on Web 01/26/1999

932 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Li et al.
Figure 3. Compounds 6-14 with varied side chain lengths.
Figure 2. Polymyxin B2 (2), deacyl polymyxin B (3), polymyxin B
nonapeptide (4), and polymyxin B heptapeptide (5).
Scheme 1
family of antibiotics which includes polymyxins A, B1, B2, D1,
E1, and E2, circulin A, and octapeptins A1, A2, A3, B1, B2, B3,
and C1. PMB is bactericidal to Gram-negative strains of bacteria,
and its proposed mechanism of action involves association with
lipid A9c,d on the surface of bacteria, self-promoted transport
through the outer membrane, and disruption of the cytoplasmic
membrane, resulting in cell death.3a,10 In contrast, deacylpoly-
myxin B (DAPB) (3; Figure 2),8e polymyxin B nonapeptide
(PMBN) (4; Figure 2),8j and polymyxin B heptapeptide (PMBH)
(5; Figure 2)8d are much less bactericidal than PMB or not
bactericidal at all. Nevertheless, DAPB, PMBN, and PMBH
retain the ability to bind LPS and increase the permeability of
the outer membranes of Gram-negative bacteria to hydrophobic
antibiotics.8 On the basis of the behaviors of PMB and its
derivatives, two distinct activities of these compounds have been
categorized as (1) sublethal permeabilization of the outer
membrane (a result of LPS binding) and (2) lethal disruption
of the cytoplasmic membrane (requiring self-promoted transport3c
through the outer membrane).3a PMB is capable of both types
of activity, while DAPB, PMBN, and PMBH are primarily
limited to permeabilization of the outer membrane.
We recently reported the development of cholic acid-derived
compounds that effectively increase the permeability of the outer
membrane of Gram-negative bacteria.11 These compounds were
designed to provide nonpeptide mimics of the structure of
PMBH, the LPS binding domain of PMB. Importance was
placed on reproducing in our cholic acid derivatives the relative
spatial arrangement of the primary amines of the diaminobutyric
acid residues in PMBH which are conserved among the
polymyxin family of antibiotics. Our continued efforts have
yielded compounds that permeabilize not only the outer
membrane of Gram-negative bacteria, but also specific deriva-
tives that rival PMB in bactericidal activity. These compounds
exhibit the two activities demonstrated by PMB and derivatives,
i.e., sublethal permeabilization of the outer membrane and/or a
second lethal activity which appears to be disruption of the
cytoplasmic membrane. Through our design process we have
incrementally converted cholic acid-derived permeabilizers into
potent antibiotics by varying the hydrophobic nature of the side
chain extending from C-17 of the steroid nucleus as in series
6-14 (Figure 3). The length of the side chain appears to
influence the ability of the compounds to pass through the outer
membrane, and variations in the chain yield compounds with a
range of combinations of sublethal and lethal activity. These
variations contrast the abrupt structural changes and activities
observed upon conversion of PMB to DAPB, PMBN, or PMBH.
Results and Discussion
Our initial design included primary amine groups attached
to a steroid nucleus derived from cholic acid as a mimic of the
structure of PMBH. However, no function was anticipated for
the carbon chain extending from C-17. Nevertheless, we
observed dramatic effects in the antibacterial activity of the
cholic acid derivatives upon altering the nature of the steroid
side chain. Consequently, to fully explore the role of the steroid
side chain in sublethal and lethal activities, we prepared a series
of compounds in which the side chain was incrementally varied
in length.
Preparation of compounds in which the steroid side chain
was lengthened from C-24 (11-14) was straightforward (Scheme
1). Reaction of alcohol 1511 with methyl iodide, propyl bromide,
pentyl bromide, or octyl bromide followed by reduction gave
11-14. Synthesis of compounds with truncated side chains (6-
10) required oxidative cleavage of carbons from the steroid side
chain, and published methods were used to prepare starting
materials 1712 and 2513 (Schemes 2 and 3). For the synthesis of
7 (Scheme 2), the ketone of 17 was protected followed by
(10) Cerny, G.; Teuber, M. Arch. Mikrobiol. 1971, 78, 166.
(11) Li, C.; Peters, A. S.; Meredith, E. L.; Allman, G. W.; Savage, P. B.
J. Am. Chem. Soc. 1998, 120, 2961.

ConVersion of Permeabilizers into Antibiotics
J. Am. Chem. Soc., Vol. 121, No. 5, 1999 933
Scheme 2
Scheme 3
installation of the propylene-linked azides, giving 23. Reduction
of 23 provided 7 as a 9:1 mixture of epimers at C-20. Compound
7 was tested as the mixture. Truncation to C-17 yielding 6 was
achieved using a Baeyer-Villiger oxidation of ketone 23,
followed by deprotection and reduction. Preparation of 9
(Scheme 3) employed ozonolysis of 25 and reduction to give
alcohol 26. After hydrolysis of the esters in 26, the pathway
used to prepare 1011 was followed to give 9. The side chain of
29 was also shortened by a carbon, via ozonolysis of alkene
30, to give 8 after reduction.
The compounds in the series 6-14 were tested for the ability
both to act as antibiotics alone and to permeabilize the outer
membrane of Gram-negative bacteria, causing sensitization to
hydrophobic antibiotics that ineffectively cross the outer
membrane. We anticipated that variations in the length of the
steroid side chain would have a direct effect on antibiotic
behavior and that this effect would differ from that observed
on the permeabilization behavior.
derivatives was not species dependent, we also measured the
activity of selected compounds with Pseudomonas aeruginosa
(ATCC 27853). To characterize the activity of the cholic acid
derivatives, we measured minimum inhibition concentration
(MIC) and minimum bactericidal concentration (MBC) values.
To measure permeabilization of the outer membrane of the
bacteria, we used erythromycin and novobiocin. These antibiot-
ics are very active against Gram-positive bacteria,14 but, due to
the permeability barrier of the outer membrane, are much less
active against Gram-negative organisms. We measured the MICs
of erythromycin and novobiocin against E. coli (ATCC 10798)
as 70 and >500 µg/mL, respectively. Our threshold measure
of permeabilization was the concentration of the cholic acid
derivatives required to lower the MIC of either erythromycin
or novobiocin to 1 µg/mL.15
Results of the MIC, MBC, and permeabilization (with
erythromycin) measurements are plotted in Figure 4. Note that
the MIC and MBC values of the compounds dropped dramati-
cally as the steroid side chain length increased. In contrast, the
We focused our studies on Escherichia coli K-12 strain ATCC
10798; however, to demonstrate that activity of the cholic acid
(14) Conte, J. E., Jr. Manual of Antibiotics and Infectious Diseases;
Williams & Wilkins: Philadelphia, 1995.
(15) Many clinically useful antibiotics against Gram-negative bacteria
have MIC values of ca.1 µg/mL (see ref 14).
(12) Barton, D. H. R.; Wozniak, J.; Zard, S. Z. Tetrahedron 1989, 45,
3741.
(13) Meystre, C.; Wettstein, A. HelV. Chim. Acta 1947, 30, 1256.

934 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Li et al.
Table 1. MIC, MBC, Permeabilization, and FIC Data for 6-14
with E. Coli (ATCC 10798)
MIC MBC
a
b
d
compd (µg/mL) (µg/mL) (µg/mL) (µg/mL) FICc (µg/mL) FICe
6
7
8
140
140
70
70
36
28
11
3.0
3.0
>200
>160
130
130
60
30
20
5.0
4.0
2.0
1.0
0.6
1.5
3.0
160
180
140
80
50
25
4.0 0.069
2.5 0.51
3.0 1.0
0.23
0.16
0.086
0.071
0.070
0.050
60
40
14
12
0.43
0.29
0.20
0.17
0.11
0.11
0.093
0.27
9
10
11
12
13
14
4.0
33
14
5.0
10
3.0
1.0
0.8
ndf
ndf
Figure 4. Compound number in order of increasing steroid side chain
length vs (2) concentration required to lower the MIC of erythromycin
from 70 to 1 µg/mL (note that PMBN (4) is included), ([) MIC, (9)
MBC with E. coli (10798).
a Concentration required to lower the MIC of erythromycin from
b
c
70 to 1 µg/mL. MBC with 1 µg/mL erythromycin. FIC values with
d
erythromycin. Concentration required to lower the MIC of novobiocin
e
f
from >500 to 1 µg/mL. FIC values with novobiocin. nd ) not
determined.
concentrations required to permeabilize the outer membrane
reached a near minimum with shorter side chain lengths than
are required for minimum MIC or MBC values. The data
describing permeability include a measurement using PMBN.16
Table 2. MIC, Permeabilization, and FIC Data for 6, 8, 11, and 13
with P. Aeruginosa (ATCC 27853)
compd
MIC (µg/mL)
a
FICb
Permeabilization of the outer membrane of Gram-negative
bacteria alone does not cause cell death,17 suggesting that the
cholic acid derivatives must pass through the outer membrane
to kill the bacteria. Because longer side chains result in lower
MIC and MBC values, the apparent role of the hydrophobic
steroid side chain is to facilitate membrane insertion and self-
promoted transport after initial association with the outer
membrane (Figure 5). This mechanism of action is similar to a
model proposed for the activity of PMB and derivatives:3a,18
binding to the outer membrane increases permeability while self-
promoted transport, requiring a hydrophobic side chain, is
required for antibacterial action.
In our results, we found a limited correlation between side
chain length and permeabilization; i.e., some side chain is
required for maximal permeabilization as seen in the difference
in activity of compounds 6-8. We propose that the side chains
in compounds such as 8-11 initiate limited membrane insertion,
and that membrane insertion yields greater membrane perme-
ability than association with LPS on the cell surface alone (see
Figure 5). A similar effect is observed with PMB: in studies
with reconstituted outer membranes of Gram-negative bacteria,
Seydel and co-workers19 found that PMB, which is capable of
self-promoted transport, transiently increased the permeability
of the membrane to ions, whereas PMBN caused no increase
in ion permeability.
6
8
11
13
150
70
10
125
13
2.5
2.0
0.83
0.19
0.25
1.0
2.0
a Concentration required to lower the MIC of novobiocin from 70
to 1 µg/mL. FIC values with novobiocin.
b
display FIC values of less than 0.5 with erythromycin, with some
values near 0.05 (Table 1).
Details from studies with novobiocin are also shown in Table
1. The fact that results with erythromycin and novobiocin were
comparable demonstrates that the activity of the cholic acid
derivatives is not antibiotic dependent. Suggesting that the
activity is not species dependent, similar trends were observed
with E. coli (ATCC 10798) and P. aeruginosa (ATCC 27853),
although, as expected,21 P. aeruginosa proved to be more
resistant than E. coli (Table 2).
Though the cholic acid derivatives were designed to mimic
the behavior of PMB and derivatives, they share some structural
features with squalamine (31; Figure 6), an antibiotic isolated
from sharks.22 However, the ionic topology of squalamine differs
markedly from the cholic acid derivatives we prepared; under
physiological conditions squalamine offers termini of opposite
charges with a hydrophobic core, while the cholic acid deriva-
tives are facially amphiphilic. Nevertheless, a proposed mech-
anism of action for squalamine involves membrane disruption,
and a proposed active conformation of squalamine and mimics
has an intramolecular salt bridge between the terminal amine
of the spermidine group and the sulfate at C-24.23 This salt
bridge results in positioning of amines above a face of the
steroid. This conformation minimally resembles the arrangement
of amines found in the cholic acid derivatives; however, the
number and positioning of the amines are different in the two
types of steroid derivatives. To observe whether including a
sulfate at C-24 in our cholic acid derivatives would increase
the activity of the compounds, we prepared 32. Treatment of
15 with SO3-pyridine complex and reduction of the azides with
triphenylphosphine gave 32 in 66% yield. The MIC of 32 with
E. coli (ATCC 10798) was measured as 60 µg/mL. The
To quantify the synergistic behavior of our compounds with
erythromycin and novobiocin, fractional inhibition concentration
(FIC) values were calculated.20 An FIC value is a standard
measure of the ability of two antibiotics to inhibit bacterial
growth synergistically and is defined as FIC ) [A]/MICA
+
[B]/MICB where [A] and [B] are the concentrations of com-
pounds A and B that in combination inhibit bacterial growth
and MICA and MICB are the MICs of compounds A and B,
respectively. Synergism between antibiotics is indicated by FIC
values of less than 0.5. With the exception of 14, the compounds
(16) Obtained from Boehringer Mannheim as 95% pure PMBN and used
as received.
(17) Permeabilization is classified as a “sublethal action”; see ref 3a.
DAPB (3), PMBN (4), PMBH (5), and 6-11 can cause increases in
permeability at concentrations much lower than their corresponding MBC
values.
(18) Wiese, A.; Mu¨nsterman, M.; Gutsmann, T.; Linder, B.; Kawahara,
K.; Za¨hringer, U.; Seydel, U. J. Membr. Biol. 1998, 162, 127.
(19) Scho¨der, G.; Brandenburg, K.; Seydel, U. Biochemistry 1992, 31,
631.
(20) Elion, G. B.; Singer, S.; Hitchings, G. H. J. Biol. Chem. 1954, 208,
477.
(21) P. aeruginosa strains are typically more antibiotic resistant than
strains of E. coli; see ref 14.
(22) Moore, K. S.; Wehrli, S.; Roder, H.; Rogers, M.; Forrest, J. N., Jr.;
McCrimmon, D.; Zasloff, M. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1354.
(23) (a) Kikuchi, K.; Bernard, E. M.; Sadownik, A.; Regen, S. L.;
Armstrong, D. Antimicrob. Agents Chemother. 1997, 41, 1433. (b) Deng,
G.; Dewa, T.; Regen, S. L. J. Am. Chem. Soc. 1996, 118, 8975.

ConVersion of Permeabilizers into Antibiotics
J. Am. Chem. Soc., Vol. 121, No. 5, 1999 935
Figure 5. Proposed mechanism for action of cholic acid derivatives and PMB (see refs 2a and 16). (A) Association of cholic acid derivatives or
PMB with LPS disrupts the lipid A cross bridging and increases the permeability of the membrane. (B) A hydrophobic chain (if present) inserts into
the membrane, facilitating incorporation of the remainder of the molecule into the membrane. (C) Insertion of the molecule into the membrane
further increases the permeability of the membrane and allows self-promoted transport. (D) As the compound passes through the outer membrane,
it gains access to the cytoplasmic membrane.
Figure 6. Squalamine (31) and 32.
concentration required to lower the MIC of erythromycin to 1
µg/mL was 4.0 µg/mL with the same strain. As compared to
Figure 7. Compounds 33-35.
the parent alcohol (10), the antibiotic and permeabilization
activities of 32 are decreased by addition of a sulfate at C-24.
To develop more potent antibiotics, we noted that PMB
contains amines in the exocyclic chain, and therefore, we
included an amine in the side chain of two cholic acid
derivatives: one with primary amines (33) and the other with
guanidine groups (34) (Figure 7). As observed earlier,11
incorporation of guanidine groups increases the activity of the
cholic acid derivatives as compared to compounds containing
primary amines. As a control, we prepared 35, lacking a
hydrophobic side chain. The three compounds were prepared
from 15, via mesylation of the alcohol followed by reaction
with octylamine and reduction to give 33 in 65% yield from
the mesylate. Compound 33 was reacted with aminoimino-
methanesulfonic acid to give 34 in 91% yield. Reaction of the
mesylate of 15 with sodium azide and reduction gave 35 in
61% yield.
The MIC of the control (35) was relatively high, as expected,
as was the MBC (Figure 8). In contrast, the MICs of 33 and 34
were very low; in fact they rival PMB in activity. Notably, the
MBCs of 33, 34, and PMB were very similar to the MICs; that
is, at a threshold concentration these compounds kill all of the
bacteria in solution. Through a range of concentrations (1-
100 µg/mL), we found no evidence of aggregation with 33 or
34. These results are consistent with the model proposed for
activity in Figure 5: a hydrophobic chain is necessary for self-
promoted transport, and the compounds must traverse the outer
membrane to effect cell death.
Figure 8. MIC (hatched bars) and MBC (solid bars) values for 33-
35 and PMB (2) measured with E. coli (ATCC 10798).
As an additional means of demonstrating the membrane-
disrupting capabilities of the cholic acid derivatives, we used a
luciferin/luciferase-based cell lysis assay.24 In this assay E. coli,
containing an inducible luciferase coding plasmid, was incubated
with the inducing agent (toluene) and then treated with a lysis
buffer containing either PMB or one of the cholic acid
derivatives and Triton X-100, followed by addition of luciferin
and ATP. Cell lysis resulted in luminescence. The concentrations
of the membrane-disrupting agents (PMB and the cholic acid
derivatives) were varied, and the resulting luminescence was
measured. In the absence of the membrane-disrupting agents
(24) (a) Willardson, B. M.; Wilkins, J. F.; Rand, T. A.; Schupp, J. M.;
Hill, K. K.; Keim, P.; Jackson, P. J. Appl. EnViron. Microbiol. 1998, 64,
1006. (b) Schupp, J. M.; Travis, S. E.; Price, L. B.; Shand, R. F.; Keim, P.
Biotechniques 1995, 19, 18.

936 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Li et al.
chromatography (10% EtOAc in hexanes) afforded the desired product
(92 mg, 90% yield) as a pale yellow oil: 1H NMR (CDCl3, 500 MHz)
δ 3.68-3.64 (m, 1 H), 3.61-3.57 (m, 1 H), 3.52 (t, J ) 6.1 Hz, 2 H),
3.49 (br s, 1 H), 3.46-3.35 (m, 10 H), 3.25 (d, J ) 2.4 Hz, 1 H),
3.23-3.19 (m, 1 H), 3.16-3.11 (m, 1 H), 3.09-3.03 (m, 1 H), 2.17-
2.03 (m, 3 H), 1.95-1.55 (m, 17 H), 1.51-1.40 (m, 4 H), 1.38-1.17
(m, 5 H), 1.11-0.96 (m, 3 H), 0.93-0.89 (m, 9 H), 0.65 (s, 3 H); 13
C
NMR (CDCl3, 75 MHz) δ 80.64, 79.79, 76.08, 72.67, 71.59, 65.01,
64.44, 64.33, 49.04, 48.94, 48.75, 46.61, 46.40, 42.68, 42.00, 39.83,
35.72, 35.45, 35.30, 35.10, 32.38, 29.81, 29.77, 29.72, 29.09, 27.88,
27.76, 27.65, 26.52, 23.55, 23.12, 23.04, 22.87, 18.06, 12.60, 10.79;
HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M + Na]+) 708.4910
(23.5%), calcd 708.4920.
Figure 9. MIC values (solid bars) with E. coli (ATCC 10798) and
concentrations required for half-maximal luminescence (hatched bars)
(see the text) for 33, 34, and PMB (2).
11-13. Representative Procedure. Preparation of 12. (Compound
14 was reported in ref 11.) Compound 16b (0.092 g, 0.134 mmol) was
dissolved in THF (10 mL) followed by the addition of LiAlH4 (0.031
g, 0.81 mmol). The suspension was stirred under N2 for 12 h. Na2-
SO4‚10H2O (∼1 g) was then carefully added. After the gray color in
the suspension dissipated, anhydrous Na2SO4 was added, and the
precipitate was removed by filtration. Concentration and silica gel
chromatography (CH2Cl2/MeOH/28% NH3‚H2O, 12:6:1 and then 10:
5:1) yielded a glass which was dissolved in 1 M HCl (2 mL). The
resulting clear solution was washed with Et2O (2 × 10 mL). A 20%
NaOH solution was added to the aqueous phase until the solution
became strongly basic. CH2Cl2 (3 × 10 mL) was used to extract the
basic solution. The combined extracts were dried over anhydrous Na2-
SO4 and concentrated in vacuo to give the desired product (0.045 g,
no luminescence was observed. In Figure 9, comparison is made
of the MICs of 33, 34, and PMB as compared to the
concentrations required for half-maximal luminescence (lumi-
nescence maxima were similar for all compounds tested). The
luminescence data correlate better with MIC values than with
permeabilization of the outer membrane as measured with
erythromycin and novobiocin. For example, the concentration
required for half-maximal luminescence of 7 (115 µg/mL) is
comparable to its MIC with E. coli (10798) (140 µg/mL), while
the concentration at which 7 lowered the MIC of erythromycin
to 1 µg/mL (20 µg/mL) is much lower. Notably, the concentra-
tion at which PMBN caused half-maximal luminescence was
>100 µg/mL. In the luminescence assay as in MIC values,
compounds 33 and 34 rival PMB in activity.
1
55% yield) as a white glass. Data for 12: H NMR (∼20% CDCl3 in
CD3OD, 500 MHz) δ 4.73 (br s, 6 H), 3.74-3.70 (m, 1 H), 3.65-3.61
(m, 1 H), 3.55 (t, J ) 6.3 Hz, 2 H), 3.42-3.38 (m, 4 H), 3.33-3.30
(m, 2 H), 3.16-3.10 (m, 2 H), 2.83-2.73 (m, 6 H), 2.18-2.06 (m, 3
H), 1.96-1.20 (series of multiplets, 26 H), 1.12-0.98 (m, 3 H), 0.95-
0.92 (m, 9 H), 0.70 (s, 3 H); 13C NMR (∼20% CDCl3 in CD3OD, 75
MHz) δ 81.67, 80.49, 77.04, 73.44, 72.28, 67.77, 67.71, 67.06, 47.74,
47.08, 43.75, 42.82, 41.21, 40.60, 40.56, 40.12, 36.47, 36.19, 36.04,
35.74, 34.09, 33.82, 33.78, 33.16, 29.49, 28.87, 28.43, 27.18, 24.22,
23.66, 23.49, 23.40, 18.64, 13.04, 11.03; HRFAB-MS (thioglycerol +
Na+ matrix) m/e ([M + H]+) 608.5348 (100%), calcd 608.5330. Data
Conclusions
We have demonstrated that by appropriately arranging amine
or guanidine groups on a steroid nucleus, it is possible to prepare
compounds that demonstrate activities similar to those of PMB
derivatives. Furthermore, by including elements found in the
exocyclic chain of PMB in the cholic acid derivatives, we have
incrementally converted membrane permeabilizers into potent
antibiotics. This process has demonstrated the role of a
hydrophobic chain in initiating self-promoted transport through
the outer membrane of Gram-negative bacteria. Compounds
lacking the chain permeabilize the outer membrane, but are
ineffective in killing the cell. A sufficient hydrophobic chain
facilitates traversal of the outer membrane, leading to effective
bactericidal activity.
1
for 11: H NMR (∼20% CDCl3 in CD3OD, 500 MHz) δ 4.79 (br s,
6H), 3.74-3.71 (m, 1 H), 3.66-3.62 (m, 1 H), 3.55 (t, J ) 6.1 Hz, 2
H), 3.52 (br s, 1 H), 3.38-3.28 (series of multiplets, 4 H), 3.33 (s, 3
H), 3.16-3.10 (m, 2H), 2.83-2.72 (m, 6 H), 2.19-2.07 (m, 3 H),
1.97-1.62 (series of multiplets, 15 H), 1.58-1.20 (series of multiplets,
9 H), 1.13-0.98 (m, 3 H), 0.95 (d, J ) 6.3 Hz, 3 H), 0.93 (s, 3 H),
0.70 (s, 3 H); 13C NMR (∼20% CDCl3 in CD3OD, 75 MHz) δ 81.82,
80.65, 77.20, 74.43, 67.85, 67.18, 58.90, 47.80, 47.22, 43.91, 43.01,
41.31, 40.78, 40.69, 40.22, 36.63, 36.35, 36.18, 35.86, 34.27, 33.97,
33.26, 29.60, 29.03, 28.58, 28.53, 27.14, 24.33, 23.61, 23.45, 18.68,
13.06; HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M + Na]+)
602.4855 (100%), calcd 602.4873. Data for 13: 1H NMR (∼50% CDCl3
in CD3OD, 500 MHz) δ 4.08 (br s, 6 H), 3.71-3.67 (m, 1 H), 3.62-
3.58 (m, 1 H), 3.53 (t, J ) 6.3 Hz, 2 H), 3.49 (br s, 1 H), 3.43-3.38
(m, 4 H), 3.31-3.27 (m, 2 H), 3.14-3.07 (m, 2 H), 2.83-2.73 (m, 6
H), 2.16-2.03 (m, 3 H), 1.93-1.17 (series of multiplets, 30 H), 1.10-
0.96 (m, 3 H), 0.93-0.89 (m, 9 H), 0.67 (s, 3 H); 13C NMR (∼50%
CDCl3 in CD3OD, 75 MHz) δ 80.51, 79.35, 75.85, 71.29, 70.83, 66.73,
66.62, 65.96, 46.68, 45.98, 42.59, 41.63, 40.20, 39.53, 39.43, 39.21,
35.34, 35.04, 35.00, 34.71, 33.11, 32.90, 32.82, 32.00, 29.15, 28.49,
28.15, 27.75, 27.35, 26.22, 23.18, 22.60, 22.45, 22.34, 17.77, 13.75,
12.22; HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M + H]+)
636.5679 (100%), calcd 636.5669.
Effects of the cholic acid derivatives on Gram-positive
bacteria and on mammalian cells are being determined and will
be communicated in due course. Additionally, the ability of the
cholic acid derivatives to inhibit the effects of LPS on human
monocytes is being actively investigated.
Experimental Section
General Procedures. 1H and 13C NMR spectra were recorded on a
Varian VXR 500 (500 MHz) or Varian Unity 300 (300 MHz)
spectrometer and are referenced to TMS, residual CHCl3 (1H), residual
CHD2OD (1H), CDCl3 (13C), or CD3OD (13C). IR spectra were recorded
on a Perkin-Elmer 1600 FTIR instrument. Mass spectrometric data were
obtained on a JEOL SX 102A spectrometer. THF was dried over Na°/
benzophenone, and CH2Cl2 was dried over CaH2 prior to use. Other
reagents and solvents were obtained commercially and used as received
unless otherwise noted.
16a-d. Representative Procedure. Preparation of 16b. (Com-
pound 16d was reported in ref 9.) NaH (0.06 g, 60% in mineral oil,
1.49 mmol) and propyl bromide (0.136 mL, 1.49 mmol) were added
to a DMF solution of compound 1511 (0.096 g, 0.149 mmol). The
suspension was stirred under N2 for 24 h. H2O (20 mL) was added,
and the mixture was extracted with hexanes (3 × 10 mL). The combined
extracts were dried over Na2SO4 and concentrated in vacuo. Silica gel
18. A mixture of 1712 (1.00 g, 2.10 mmol), ethylene glycol (3.52
mL, 63 mmol), and p-TsOH (20 mg, 0.105 mmol) was refluxed in
benzene under N2 for 16 h. Water formed during the reaction was
removed by a Dean-Stark moisture trap. The cooled mixture was
washed with saturated aqueous NaHCO3 (50 mL), and the aqueous
phase was extracted with Et2O (50 mL, 2 × 30 mL). The combined
extracts were washed with brine (75 mL) and dried over anhydrous
Na2SO4. Removal of the solvent in vacuo gave the desired product
(1.09 g, 100%) as a glass: 1H NMR (CDCl3, 300 MHz) δ 5.10 (t, J )
2.7 Hz, 1 H), 4.92 (d, J ) 2.7 Hz, 1 H), 4.63-4.52 (m, 1 H), 3.98-

ConVersion of Permeabilizers into Antibiotics
J. Am. Chem. Soc., Vol. 121, No. 5, 1999 937
3.80 (m, 4 H), 2.32 (t, J ) 9.5 Hz, 1 H), 2.13 (s, 3 H), 2.08 (s, 3 H),
2.05 (s, 3 H), 2.00-1.40 (series of multiplets, 15 H), 1.34-0.98 (m, 3
H), 1.20 (s, 3 H), 0.92 (s, 3 H), 0.82 (s, 3 H); 13C NMR (CDCl3, 75
MHz) δ 170.69, 170.63, 170.47, 111.38, 75.07, 74.23, 70.85, 64.95,
63.43, 49.85, 44.73, 43.39, 41.11, 37.37, 34.84, 34.80, 34.52, 31.42,
29.18, 27.02, 25.41, 24.16, 22.72, 22.57, 22.44, 21.73, 21.63, 13.40;
HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M + H]+) 521.3106
(38.6%), calcd 521.3114.
desired product (0.509 g, 98% yield) as a clear oil: 1H NMR (CDCl3,
300 MHz) δ 3.83-3.72 (m, 8 H), 3.66 (t, J ) 5.6 Hz, 2 H), 3.54 (br
s, 2 H), 3.43-3.28 (m, 4 H), 3.24-3.12 (m, 2 H), 2.26-2.00 (m, 4
H), 2.08 (s, 3 H), 1.98-1.50 (series of multiplets, 15 H), 1.42-0.96
(series of multiplets, 6 H), 0.90 (s, 3 H), 0.62 (s, 3 H); 13C NMR (CDCl3,
75 MHz) δ 210.49, 78.87, 76.30, 66.86, 66.18, 65.69, 61.74, 61.43,
60.71, 55.31, 48.05, 43.02, 41.58, 39.53, 35.28, 35.09, 34.96, 32.77,
32.70, 32.31, 31.12, 28.72, 27.88, 27.14, 23.47, 22.75, 22.47, 22.34,
13.86; HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M + Na]+)
547.3624 (100%), calcd 547.3611.
19. Compound 18 (1.09 g, 2.10 mmol) was dissolved in MeOH (50
mL). NaOH (0.84 g, 21 mmol) was added to the solution. The
suspension was refluxed under N2 for 24 h. MeOH was removed in
vacuo, and the residue was dissolved in Et2O (100 mL), washed with
H2O (100 mL) and brine (100 mL), and dried over anhydrous Na2SO4.
The desired product (0.80 g, 96% yield) was obtained as white solid
23. Et3N (0.168 mL, 1.20 mmol) was added to the solution of 22
(0.18 g, 0.344 mmol) in dry CH2Cl2 (10 mL) at 0 °C followed by the
addition of mesyl chloride (0.088 mL, 1.13 mmol). After 10 min, H2O
(3 mL) and brine (30 mL) were added. The mixture was extracted with
EtOAc (30 mL, 2 × 10 mL), and the extracts were washed with brine
(50 mL) and dried over anhydrous Na2SO4. After removal of solvent,
the residue was dissolved in DMSO (5 mL), and NaN3 (0.233 g, 3.44
mmol) was added. The suspension was heated to 50 °C under N2 for
12 h. H2O (50 mL) was added to the cooled suspension. The mixture
was extracted with EtOAc (30 mL, 2 × 10 mL), and the extracts were
washed with brine (50 mL) and dried over anhydrous Na2SO4. SiO2
column chromatography (EtOAc/hexanes, 1:5) afforded the product
(0.191 g, 88% yield) as a pale yellow oil: 1H NMR (CDCl3, 300 MHz)
δ 3.72-3.64 (m, 2 H), 3.55-3.24 (series of multiplets, 11 H), 3.18-
3.02 (m, 2 H), 2.22-2.02 (m, 4 H), 2.08 (s, 3 H), 1.95-1.46 (series of
multiplets, 15 H), 1.38-0.96 (series of multiplets, 6 H), 0.89 (s, 3 H),
0.62 (s, 3 H); 13C NMR (CDCl3, 75 MHz) δ 210.36, 79.69, 79.22,
75.98, 65.08, 64.80, 64.53, 55.31, 48.93, 48.86, 48.76, 48.06, 43.03,
41.91, 39.66, 35.44, 35.31, 35.12, 31.04, 29.77, 29.69, 29.67, 28.99,
28.10, 27.65, 23.60, 22.99, 22.95, 22.50, 14.00; HRFAB-MS (thioglyc-
erol + Na+ matrix) m/e ([M + Na]+) 622.3820 (100%), calcd 622.3805.
7. Compound 23 (0.191 g, 0.319 mmol) was dissolved in THF (20
mL) followed by the addition of LiAlH4 (60.4 mg, 1.59 mmol). The
gray suspension was stirred under N2 for 12 h. Na2SO4‚10 H2O (∼1 g)
powder was carefully added. After the gray color in the suspension
disappeared, anhydrous Na2SO4 was added, and the precipitate was
removed by filtration. After the removal of solvent in vacuo, the residue
was purified by column chromatography (SiO2, MeOH/CH2Cl2/28%
NH3‚H2O, 3:3:1). The relevant fractions were collected, and 1 M HCl
(2 mL) was added to dissolve the residue. The resulting clear solution
was washed with Et2O (2 × 10 mL). To the aqueous phase was added
20% NaOH solution until the solution became strongly basic. The
solution was extracted with CH2Cl2 (20 mL, 2 × 10 mL), and the
combined extracts were dried over anhydrous Na2SO4. Removal of
solvent in vacuo gave the desired product (0.115 g, 69% yield) as a
colorless oil. From 1H NMR, it is apparent that the product is a mixture
of two stereoisomers at C20 in a ratio of 9:1. No attempt was made to
separate the stereoisomers; they were tested as a mixture: 1H NMR
(20% CDCl3 in CD3OD, 300 MHz) δ 4.69 (br s, 7 H), 3.76-3.69 (m,
1 H), 3.63-3.53 (m, 5 H), 3.50-3.40 (m, 1 H), 3.29 (br s, 1 H), 3.18-
3.07 (m, 2 H), 2.94-2.83 (m, 1 H), 2.81-2.66 (m, 5 H), 2.23-2.06
(m, 4 H), 1.87-1.50 (series of multiplets, 15 H), 1.39-0.96 (series of
multiplets, 6 H), 1.11 (d, J ) 6.1 Hz, 3 H), 0.93 (s, 3 H), 0.75 (s, 3 H);
13C NMR (20% CDCl3 in CD3OD, 75 MHz) δ 81.46, 80.67, 77.32,
70.68, 67.90, 67.66, 67.18, 50.32, 47.17, 43.30, 43.06, 40.74, 40.64,
40.38, 40.26, 36.31, 36.28, 35.93, 34.30, 34.02, 33.29, 29.63, 29.31,
28.43, 26.10, 24.67, 24.09, 23.96, 23.50, 13.30 for the major isomer;
HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M + H]+) 524.4431
(64.2%), calcd 524.4427.
1
after removal of solvent in vacuo: mp 199-200 °C; H NMR (10%
CD3OD in CDCl3, 300 MHz) δ 4.08-3.83 (series of multiplets, 9 H),
3.44-3.34 (m, 1 H), 2.41 (t, J ) 9.3 Hz, 1 H), 2.22-2.10 (m, 2 H),
1.96-1.50 (series of multiplets, 12 H), 1.45-0.96 (series of multiplets,
4 H), 1.32 (s, 3 H), 0.89 (s, 3 H), 0.78 (s, 3 H); 13C NMR (10% CD3-
OD in CDCl3, 75 MHz) δ 112.11, 72.35, 71.57, 68.09, 64.54, 63.24,
49.36, 45.90, 41.48, 41.45, 39.18, 38.79, 35.29, 34.71, 34.45, 29.90,
27.26, 26.60, 23.65, 22.54, 22.44, 22.35, 13.46; HRFAB-MS (thioglyc-
erol + Na+ matrix) m/e ([M + Na]+) 417.2622 (87.3%), calcd 417.2617.
20. To a round-bottom flask were added 19 (0.80 g, 2.03 mmol)
and dry THF (100 mL) followed by NaH (60% in mineral oil, 0.81 g,
20.3 mmol). The suspension was refluxed under N2 for 30 min, and
allyl bromide (1.75 mL, 20.3 mmol) was added. After 48 h at reflux,
another 10 equiv of NaH and allyl bromide were added. After an
additional 48 h, cold water (50 mL) was added to the cooled suspension.
The resulting mixture was extracted with Et2O (60 mL, 2 × 30 mL).
The combined extracts were washed with brine (100 mL) and dried
over anhydrous Na2SO4. Silica gel chromatography (6% EtOAc in
hexanes) gave the desired product (0.94 g, 90% yield) as a pale yellow
oil: 1H NMR (CDCl3, 300 MHz) δ 6.02-5.84 (m, 3 H), 5.31-5.04
(m, 6 H), 4.12-4.05 (m, 2 H), 4.01-3.81 (m, 7 H), 3.70 (dd, J )
12.9, 5.6 Hz, 1 H), 3.55 (t, J ) 2.6 Hz, 1 H), 3.33 (d, J ) 2.9 Hz, 1
H), 3.18-3.08 (m, 1 H), 2.65 (t, J ) 10.0 Hz, 1 H), 2.32-2.14 (m, 3
H), 1.84-1.45 (series of multiplets, 10 H), 1.41-1.22 (m, 3 H), 1.27
(s, 3 H), 1.14-0.92 (m, 2 H), 0.89 (s, 3 H), 0.75 (s, 3 H); 13C NMR
(CDCl3, 75 MHz) δ 136.38, 136.07, 136.00, 116.31, 115.54, 115.38,
112.34, 80.07, 79.22, 75.05, 69.83, 69.34, 68.82, 65.14, 63.24, 48.80,
45.96, 42.47, 42.15, 39.40, 35.55, 35.16, 35.15, 29.04, 28.22, 27.52,
24.21, 23.38, 23.11, 22.95, 22.58, 13.79; HRFAB-MS (thioglycerol +
Na+ matrix) m/e ([M + Na]+) 537.3549 (100%), calcd 537.3556.
21. To a solution of 20 (0.94 g, 1.83 mmol) in THF (50 mL) was
added 9-BBN (0.5 M solution in THF, 14.7 mL, 7.34 mmol). The
mixture was stirred under N2 for 12 h followed by the addition of 20%
NaOH solution (4 mL) and 30% H2O2 solution (4 mL). The resulting
mixture was refluxed for 1 h followed by the addition of brine (100
mL). The mixture was extracted with EtOAc (4 × 30 mL), and the
combined extracts were dried over anhydrous Na2SO4. After the removal
of solvent in vacuo, the residue was purified by SiO2 column
chromatography (EtOAc followed by 10% MeOH in CH2Cl2) to give
the product (0.559 g, 54% yield) as a colorless oil: 1H NMR (CDCl3,
300 MHz) δ 4.02-3.52 (series of multiplets, 17 H), 3.41-3.35 (m, 1
H), 3.29 (d, J ) 2.4 Hz, 1 H), 3.22-3.15 (m, 3 H), 2.58 (t, J ) 10.0
Hz, 1 H), 2.27-1.95 (m, 3 H), 1.83-1.48 (series of multiplets, 16 H),
1.40-0.93 (series of multiplets, 5 H), 1.27 (s, 3 H), 0.90 (s, 3 H), 0.75
(s, 3 H); 13C NMR (CDCl3, 75 MHz) δ 112.41, 80.09, 79.09, 76.31,
66.70, 66.02, 65.93, 64.80, 63.26, 61.53, 61.25, 60.86, 48.59, 45.80,
42.51, 41.72, 39.10, 35.36, 35.02, 34.98, 32.87, 32.52, 32.40, 28.88,
27.94, 27.21, 24.33, 23.02, 22.84 (2 C’s), 22.44, 13.69; HRFAB-MS
(thioglycerol + Na+ matrix) m/e ([M + Na]+) 591.3881 (100%), calcd
591.3873.
24. Compound 23 (0.256 g, 0.489 mmol) was dissolved in CH2Cl2
(10 mL), and cooled to 0 °C followed by the addition of Na2HPO4
(0.69 g, 4.89 mmol) and urea-hydrogen peroxide complex (UHP)
(0.069 g, 0.733 mmol). Trifluoroacetic anhydride (TFAA) (0.138 mL,
0.977 mmol) was then added dropwise. The suspension was stirred for
12 h, and additional UHP (23 mg, 0.25 mmol) and TFAA (0.069 mL,
0.49 mmol) were added. After another 12 h, H2O (30 mL) was added,
and the resulting mixture was extracted with EtOAc (3 × 20 mL). The
combined extracts were washed with brine (50 mL), dried over
anhydrous Na2SO4, and concentrated in vacuo. SiO2 chromatography
(EtOAc/hexanes, 1:5) afforded the desired product (0.145 g, 55% yield)
as a colorless oil: 1H NMR (CDCl3, 300 MHz) δ 5.21 (dd, J ) 9.3
22. To a solution of 21 (0.559 g, 0.98 mmol) in acetone (40 mL)
and water (4 mL) was added PPTS (0.124 g, 0.49 mmol), and the
solution was refluxed under N2 for 16 h. The solvent was removed in
vacuo. Water (40 mL) was added to the residue, and the mixture was
extracted with EtOAc (40 mL, 2 × 20 mL). The combined extracts
were washed with brine, dried over MgSO4, and concentrated in vacuo.
SiO2 column chromatography (8% MeOH in CH2Cl2) afforded the

938 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Li et al.
and 7.3 Hz, 1 H), 3.70-3.57 (m, 2 H), 3.55 (t, J ) 6.0 Hz, 2 H),
3.43-3.37 (m, 6 H), 3.32-3.25 (m, 3 H), 3.17-3.02 (m, 2 H), 2.28-
2.05 (m, 4 H), 2.03 (s, 3 H), 1.86-1.19 (series of multiplets, 19 H),
0.97 (dd, J ) 14.5 and 3.3 Hz, 1 H), 0.90 (s, 3 H), 0.78 (s, 3 H); 13C
NMR (CDCl3, 75 MHz) δ 171.08, 79.71, 78.03, 75.72, 75.53, 65.41,
65.04, 64.53, 48.79, 48.70, 46.49, 41.92, 39.44, 37.81, 35.45, 35.22,
35.10, 29.73, 29.63, 28.89, 28.33, 27.50, 27.34, 23.39, 22.97, 22.92,
21.28, 12.72; HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M - H]+)
614.3798 (24.5%), calcd 614.3778.
solid was dissolved in DMF (40 mL) followed by the sequential addition
of NEt3 (1.12 mL, 8.02 mmol), DMAP (16.3 mg, 0.13 mmol), and
trityl chloride (1.49 g, 5.34 mmol). The suspension was stirred under
N2 for 12 h and then heated to 50 °C for 24 h. H2O (100 mL) was
added to the cooled suspension, and the mixture was extracted with
EtOAc (3 × 50 mL). The combined extracts were washed with brine
(100 mL), dried over anhydrous Na2SO4, and concentrated in vacuo.
Silica gel chromatography (EtOAc) afforded the product (1.20 g, 72%
yield) as a pale yellow glass. To this glass were added dry THF (80
mL) and NaH (60% in mineral oil, 0.77 g, 19.3 mmol). The suspension
was refluxed under N2 for 1/2 h before the introduction of allyl bromide
(1.67 mL, 19.3 mmol). After 48 h at reflux, another 10 equiv of NaH
and allyl bromide were introduced. After another 48 h, the reaction
mixture was cooled, and H2O (100 mL) was slowly added. The resulting
mixture was extracted with hexanes (3 × 50 mL), and the combined
extracts were washed with brine (100 mL) and dried over anhydrous
Na2SO4. Silica gel chromatography (5% EtOAc in hexanes) afforded
the product (1.27 g, 64% yield for all three steps) as a clear glass: 1H
NMR (CDCl3 , 300 MHz) δ 7.46-7.43 (m, 6 H), 7.29-7.16 (m, 9 H),
5.98-5.81 (m, 3 H), 5.29-5.18 (m, 3 H), 5.14-5.03 (m, 3 H), 4.11-
3.97 (m, 4 H), 3.75-3.67 (m, 2 H), 3.49 (br s, 1 H), 3.32-3.13 (d, J
) 2.4 Hz, 1 H), 3.20-3.13 (m, 2 H), 3.00 (m, 1 H), 2.33-2.12 (m, 3
H), 2.03-0.92 (series of multiplets, 19 H), 0.88 (s, 3 H), 0.78 (d, J )
6.6 Hz, 3 H), 0.65 (s, 3 H); 13C NMR (CDCl3, 75 MHz) δ 144.71,
136.08, 136.04, 135.94, 128.80, 127.76, 126.86, 116.30, 115.57, 86.53,
80.77, 79.20, 74.96, 69.42, 69.34, 68.81, 62.00, 46.87, 46.48, 42.67,
42.11, 39.90, 36.15, 35.50, 35.14, 35.10, 33.23, 28.99, 28.09, 27.75,
27.56, 23.36, 23.32, 23.12, 18.24, 12.66; HRFAB-MS (thioglycerol +
Na+ matrix) m/e ([M + Na]+) 765.4875 (100%), calcd 765.4859.
28. To a THF (40 mL) solution of 27 (1.27 g, 1.71 mmol) was added
9-BBN (0.5 M solution in THF, 17.1 mL). The mixture was stirred for
12 h before the addition of NaOH (20% solution, 10 mL) and H2O2
(30% solution, 10 mL). The resulting mixture was refluxed for 1 h
followed by the addition of brine (100 mL) and extraction with EtOAc
(4 × 30 mL). The combined extracts were dried over anhydrous Na2-
SO4 and concentrated in vacuo. Silica gel chromatography (5% MeOH
in CH2Cl2) afforded the product (1.26 g, 93% yield) as a clear glass:
1H NMR (5% CD3OD in CDCl3 , 300 MHz) δ 7.46-7.43 (m, 6 H),
7.32-7.20 (m, 9 H), 3.94 (s, 3 H), 3.78-3.56 (m, 10 H), 3.48 (br s, 1
H), 3.32-3.26 (m, 2 H), 3.24-3.12 (m, 3 H), 3.00 (dd, J ) 8.2 and
6.1 Hz, 1 H), 2.23-1.96 (m, 3 H), 1.90-0.95 (series of multiplets, 25
H), 0.90 (s, 3 H), 0.77 (d, J ) 6.6 Hz, 3 H), 0.66 (s, 3 H); 13C NMR
(5% CD3OD in CDCl3, 75 MHz) δ 144.52, 128.64, 127.64, 126.76,
86.43, 80.55, 79.31, 77.65, 77.23, 76.80, 76.06, 66.17, 66.01, 65.41,
61.93, 61.20, 60.73, 60.39, 47.29, 46.08, 42.65, 41.62, 39.49, 36.02,
35.10, 34.89, 34.77, 32.89, 32.71, 32.41, 32.26, 28.68, 27.70, 27.51,
27.19, 23.26, 22.66, 22.50, 18.23, 12.34; HRFAB-MS (thioglycerol +
Na+ matrix) m/e ([M + Na]+) 819.5169 (100%), calcd 819.5099.
29. To a CH2Cl2 (50 mL) solution of compound 28 (1.26 g, 1.58
mmol) at 0 °C was added Et3N (0.92 mL, 6.60 mmol) followed by
mesyl chloride (0.47 mL, 6.05 mmol). After 15 min, H2O (10 mL)
was added followed by brine (80 mL). The mixture was extracted with
EtOAc (60 mL, 2 × 30 mL), and the combined extracts were dried
over anhydrous Na2SO4. After removal of solvent in vacuo, the residue
was dissolved in DMSO (10 mL), and NaN3 (1.192 g, 18.3 mmol)
was added. The suspension was heated to 60 °C under N2 overnight.
H2O (100 mL) was added, and the mixture was extracted with EtOAc
(3 × 40 mL). The combined extracts were washed with brine and dried
over anhydrous Na2SO4. Removal of the solvent in vacuo afforded a
pale yellow oil. The oil was dissolved in MeOH (10 mL) and CH2Cl2
(20 mL), and TsOH (17.4 mg, 0.092 mmol) was added. After 12 h,
saturated aqueous NaHCO3 (20 mL) and brine (50 mL) were added,
and the mixture was extracted with EtOAc (3 × 40 mL).The combined
extracts were washed with brine (50 mL) and dried over anhydrous
Na2SO4. Silica gel chromatography (EtOAc/hexanes, 1:3) afforded the
desired product (0.934, 94%) as a pale yellow oil: 1H NMR (CDCl3,
500 MHz) δ 3.75-3.70 (m, 1 H), 3.68-3.63 (m, 2 H), 3.62-3.57 (m,
1 H), 3.53 (t, J ) 6.1 Hz, 2 H), 3.50 (br s, 1 H), 3.46-3.38 (m, 6 H),
3.26 (d, J ) 2.4 Hz, 1 H), 3.24-3.20 (m, 1 H), 3.16-3.12 (m, 1 H),
3.10-3.04 (m, 1 H), 2.17-2.04 (m, 3 H), 1.96-1.63 (m, 14 H), 1.53-
1.45 (m, 3 H), 1.35-1.20 (m, 7 H), 1.08-1.00 (m, 1 H), 0.97-0.88
6. Compound 24 (0.145 g, 0.236 mmol) was dissolved in CH2Cl2 (2
mL) and MeOH (1 mL). A 20% NaOH solution (0.2 mL) was added.
The mixture was stirred for 12 h, and anhydrous Na2SO4 was used to
remove water. After concentration in vacuo, the residue was purified
by silica gel chromatography (EtOAc/hexanes, 1:3) to afford the desired
product (0.124 g, 92% yield) as a colorless oil. 1H NMR (CDCl3 , 300
MHz) δ 4.29 (br s, 1 H), 3.69-3.60 (m, 2 H), 3.52 (t, J ) 6.0 Hz, 2
H), 3.45-3.32 (m, 8 H), 3.26 (d, J ) 2.7 Hz, 1 H), 3.17-3.02 (m, 2
H), 2.19-1.94 (m, 4 H), 1.90-1.62 (series of multiplets, 13 H), 1.57-
1.20 (series of multiplets, 7 H), 0.97 (dd, J ) 14.3 and 3.1 Hz, 1 H),
0.90 (s, 3 H), 0.73 (s, 3 H); 13C NMR (CDCl3, 75 MHz) δ 79.69, 78.03,
75.47, 73.38, 65.46, 65.00, 64.47, 48.87, 48.68, 46.83, 41.93, 39.71,
37.87, 35.43, 35.20, 35.09, 29.96, 29.69, 29.59, 29.53, 28.89, 28.44,
27.48, 23.72, 22.91, 22.71, 11.77. The alcohol (0.124 g, 0.216 mmol)
was dissolved in dry THF (20 mL) followed by the addition of LiAlH4
(33 mg, 0.866 mmol). The gray suspension was stirred under N2 for
12 h. Na2SO4‚10H2O (∼2 g) was carefully added. After the gray color
in the suspension dissipated, anhydrous Na2SO4 was added, and the
precipitate was removed by filtration. After the removal of solvent,
the residue was purified by column chromatography (SiO2, MeOH/
CH2Cl2/28% NH3‚H2O, 2.5:2.5:1). After concentration of the relevant
fractions, 1 M HCl (2 mL) was added to dissolve the milky residue.
The resulting clear solution was washed with Et2O (2 × 10 mL). To
the aqueous phase was added 20% NaOH solution until the solution
became strongly basic. CH2Cl2 (20 mL, 2 × 10 mL) was used to extract
the basic solution. The combined extracts were dried over anhydrous
Na2SO4, and removal of solvent gave the desired product (0.050 g,
47% yield) as a colorless oil: 1H NMR (20% CDCl3 in CD3OD, 300
MHz) δ 4.77 (s, 7 H), 4.25 (t, J ) 8.5 Hz, 1 H), 3.75-3.68 (m, 1 H),
3.66-3.58 (m, 1 H), 3.55 (t, J ) 6.1 Hz, 2 H), 3.48-3.41 (m, 1 H),
3.34 (br s, 1 H), 3.30 (d, J ) 3.6 Hz, 1 H), 3.17-3.08 (m, 2 H), 2.86-
2.70 (m, 6 H), 2.20-1.91 (m, 4 H), 1.88-1.16 (series of multiplets,
19 H), 1.00 (dd, J ) 14.2 and 3.0 Hz, 1 H), 0.93 (s, 3 H), 0.73 (s, 3
H); 13C NMR (20% CDCl3 in CD3OD, 75 MHz) δ 80.62, 79.12, 76.74,
73.77, 68.50, 67.79, 67.17, 47.69, 43.04, 40.76, 40.64, 40.62, 40.22,
39.01, 36.32, 36.25, 35.94, 34.27, 33.97, 33.72, 30.13, 29.53, 28.43,
24.48, 23.58, 23.40, 12.38; HRFAB-MS (thioglycerol + Na+ matrix)
m/e ([M + H]+) 496.4108 (100%), calcd 496.4114.
26. Compound 2513 (2.30 g, 3.52 mmol) was dissolved in MeOH
(50 mL) and CH2Cl2 (100 mL). A small amount of Et3N was added,
and the solution was cooled to -78 °C. Ozone was bubbled through
the solution until a blue color persisted. Me2S (4 mL) was introduced
followed by the addition of NaBH4 (0.266 g, 0.703 mmol) in MeOH
(10 mL). The resulting solution was allowed to warm and stir overnight.
The solution was concentrated in vacuo, and brine (60 mL) was added.
The mixture was extracted with EtOAc (40 mL, 2 × 30 mL), and the
combined extracts were washed with brine and dried over anhydrous
Na2SO4. Silica gel chromatography (EtOAc) afforded the product (1.24
1
g, 76% yield) as a white solid: mp 219-220 ° C; H NMR (CDCl3 ,
300 MHz) δ 5.10 (t, J ) 2.8 Hz, 1 H), 4.90 (d, J ) 2.7 Hz, 1 H),
3.73-3.59 (m, 2 H), 3.56-3.44 (m, 1 H), 2.13 (s, 3 H), 2.09 (s, 3 H),
2.07-0.95 (series of multiplets, 23 H), 0.91 (s, 3 H), 0.83 (d, J ) 6.3
Hz, 3 H), 0.74 (s, 3 H); 13C NMR (CDCl3, 75 MHz) δ 170.84, 170.82,
75.63, 71.77, 71.03, 60.73, 48.10, 45.26, 43.54, 41.16, 38.78, 37.89,
35.00, 34.43, 32.26, 31.50, 30.60, 29.07, 27.50, 25.70, 22.96, 22.71,
21.81, 21.63, 18.18, 12.35; HRFAB-MS (thioglycerol + Na+ matrix)
m/e ([M + H]+) 465.3197 (20%), calcd 465.3216.
27. Compound 26 (1.24 g, 2.67 mmol) was dissolved in MeOH (30
mL), and NaOH (0.54 g, 13.4 mmol) was added. The suspension was
refluxed under N2 for 24 h. The MeOH was removed in vacuo followed
by the addition of H2O (50 mL). The precipitate was filtered, washed
with H2O, and then dried in vacuo to give a white solid (1.02 g). This

ConVersion of Permeabilizers into Antibiotics
J. Am. Chem. Soc., Vol. 121, No. 5, 1999 939
(m, 1 H), 0.94 (d, J ) 6.8 Hz, 3 H), 0.89 (s, 3 H), 0.67 (s, 3 H); 13C
NMR (CDCl3, 75 MHz) δ 80.64, 79.81, 76.06, 65.05, 64.49, 64.34,
61.03, 49.02, 48.98, 48.78, 46.93, 46.53, 42.76, 42.01, 39.83, 39.14,
35.46, 35.33, 35.12, 32.97, 29.79, 29.73, 29.10, 27.90, 27.68, 23.56,
23.06, 22.88, 18.24, 12.60; HRFAB-MS (thioglycerol + Na+ matrix)
m/e ([M + Na]+) 652.4285 (100%), calcd 652.4295.
1.45 (m, 3 H), 1.40-1.08 (m, 5 H), 1.03 (d, J ) 6.8 Hz, 3 H), 1.02-
0.96 (m, 1 H), 0.93 (s, 3 H), 0.72 (s, 3 H); 13C NMR (∼10% CD3OD
in CDCl3, 75 MHz) δ 81.74, 80.64, 77.23, 67.95, 67.87, 67.18, 47.32,
44.59, 43.72, 43.01, 41.26, 40.80, 40.71, 40.23, 40.02, 36.36, 36.20,
35.87, 34.27, 33.99, 33.90, 29.60, 29.05, 28.58, 28.08, 24.49, 23.62,
23.46, 16.84, 13.12; HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M
+ H]+) 538.4578 (4.7%), calcd 538.4584.
9. Compound 29 (0.245 g, 0.391 mmol) was dissolved in THF (30
mL) followed by the addition of LiAlH4 (59 mg, 1.56 mmol). The gray
suspension was stirred under N2 for 12 h. Na2SO4‚10H2O powder (∼1
g) was carefully added. After the gray color in the suspension dissipated,
anhydrous Na2SO4 was added, and the precipitate was removed by
filtration. After the removal of solvent, the residue was purified by
silica gel chromatography (CH2Cl2/MeOH/28% NH3‚H2O, 10:5:1 and
then 10:5:1.5). The solvent was removed from relevant fractions, and
1 M HCl (4 mL) was added to dissolve the residue. The resulting clear
solution was extracted with Et2O (3 × 10 mL). A 20% NaOH solution
was added until the solution became strongly basic. CH2Cl2 (4 × 10
mL) was used to extract the basic solution. The combined extracts were
dried over anhydrous Na2SO4, and removal of solvent in vacuo gave
the desired product (0.15 g, 71% yield) as a colorless oil: 1H NMR
(∼20% CD3OD in CDCl3, 500 MHz) δ 4.73 (br s, 7 H), 3.74-3.70
(m, 1 H), 3.65-3.60 (m, 2 H), 3.56-3.52 (m, 4 H), 3.31-3.28 (m, 2
H), 3.16-3.09 (m, 2 H), 2.82-2.71 (m, 6 H), 2.19-2.06 (m, 3 H),
1.97-1.66 (series of multiplets, 15 H), 1.58-1.48 (m, 3 H), 1.38-
0.98 (m, 7 H), 0.96 (d, J ) 6.8 Hz, 3 H), 0.93 (s, 3 H), 0.71 (s, 3 H);
13C NMR (∼20% CD3OD in CDCl3, 75 MHz) δ 81.80, 80.60, 77.17,
67.88, 67.86, 67.18, 60.73, 48.11, 47.28, 43.93, 42.99, 41.34, 40.76,
40.72, 40.24, 39.70, 36.33, 36.18, 35.86, 34.29, 33.99, 33.96, 33.83,
29.60, 29.00, 28.57, 28.54, 24.33, 23.59, 23.48, 18.86, 13.04; HRFAB-
MS (thioglycerol + Na+ matrix) m/e ([M + H]+) 552.4756 (100%),
calcd 552.4772.
30. o-NO2C6H4SeCN (0.094 g, 0.21 mmol) and Bu3P (0.095 mL,
0.38 mmol) were stirred in dry THF (5 mL) at 0 °C for 1/2 h followed
by the addition of compound 29 (0.10 g, 0.159 mmol) in THF (2 mL).
The suspension was stirred for 1 h followed by the addition of H2O2
(30% aqueous solution, 2 mL). The mixture was stirred for 12 h
followed by extraction with hexanes (4 × 10 mL). The combined
extracts were dried over anhydrous Na2SO4. The desired product (0.035
g, 36% yield) was obtained as a pale yellow oil after silical gel
chromatography (10% EtOAc/hexanes): 1H NMR (CDCl3, 500 MHz)
δ 5.73-5.66 (ddd, J ) 17.1, 10.2, 8.3 Hz, 1 H), 4.90 (dd, J ) 17.1,
2.0 Hz, 1 H), 4.82 (dd, J ) 10.2 Hz, 1.96 Hz, 1 H), 3.68-3.64 (m, 1
H), 3.62-3.58 (m, 1 H), 3.54-3.26 (m, 9 H), 3.25-3.22 (m, 2 H),
3.15-3.11 (m, 1 H), 3.10-3.04 (m, 1 H), 2.17-1.62 (series of
multiplets, 18 H), 1.51-1.43 (m, 2 H), 1.35-1.18 (m, 4 H), 1.06-
0.91 (m, 2 H), 1.02 (d, J ) 6.3 Hz, 3 H), 0.90 (s, 3 H), 0.68 (s, 3 H);
13C NMR (CDCl3, 75 MHz) δ 145.50, 111.72, 80.60, 79.82, 76.09,
65.06, 64.50, 64.45, 49.05, 48.97, 48.79, 46.43, 46.13, 42.76, 42.03,
41.30, 39.84, 35.49, 35.34, 35.15, 29.82, 29.80, 29.75, 29.11, 28.00,
27.84, 27.68, 23.56, 23.08, 22.95, 19.79, 12.87; HRFAB-MS (thioglyc-
erol + Na+ matrix) m/e ([M + Na]+) 634.4167 (90.6%), calcd 634.4169.
8. Compound 30 (0.105 g, 0.172 mmol) was dissolved in CH2Cl2 (5
mL) and MeOH (5 mL) at -78 °C. O3 was bubbled into the solution
for ca. 20 min. Me2S (1 mL) was added , and the solvent was removed
in vacuo. The residue was dissolved in THF (15 mL), and LiAlH4 (0.033
g, 0.86 mmol) was added. The suspension was stirred for 12 h. Na2-
SO4‚10H2O (∼2 g) was carefully added. After the gray color of the
suspension dissipated, anhydrous Na2SO4 was added, and the precipitate
was removed by filtration. Concentration and silica gel chromatography
(CH2Cl2/MeOH/28% NH3‚H2O, 10:5:1.5 and then 9:6:1.8) yielded a
white glass. To this material was added 1 M HCl (4 mL). The resulting
clear solution was washed with Et2O (3 × 10 mL). A 20% NaOH
solution was added to the aqueous phase until the solution became
strongly basic. CH2Cl2 (4 × 10 mL) was used to extract the basic
solution. The combined extracts were dried over anhydrous Na2SO4,
and removal of solvent gave the desired product (0.063 g, 68% yield)
as a colorless oil: 1H NMR (∼10% CD3OD in CDCl3 , 500 MHz) δ
4.76 (br s, 7 H), 3.75-3.71 (m, 1 H), 3.66-3.62 (m, 1 H), 3.58-3.52
(m, 4 H), 3.33-3.29 (m, 2 H), 3.22 (dd, J ) 10.5 and 7.6 Hz, 1 H),
3.15-3.09 (m, 2 H), 2.81 (t, J ) 6.8 Hz, 2 H), 2.76-2.71 (m, 4 H),
2.19-2.08 (m, 3 H), 2.00-1.66 (series of multiplets, 14 H), 1.58-
32. Compound 15 (0.118 g, 0.183 mmol) was dissolved in dry CH2-
Cl2 (10 mL), and SO3‚pyridine complex (0.035 g, 0.22 mmol) was
added. The suspension was stirred for 12 h. The solvent was removed
in vacuo to give a white powder. To the white powder was added 1 M
HCl (10 mL), and the resulting mixture was extracted with CH2Cl2 (4
× 10 mL). The combined extracts were dried over anhydrous Na2SO4.
The desired product (0.11 g, 84%) was obtained as a pale yellow oil
after silica gel chromatography (10% MeOH in CH2Cl2): 1H NMR
(∼10% CD3OD in CDCl3, 500 MHz) δ 4.03 (t, J ) 6.8 Hz, 2 H),
3.69-3.65 (m, 1 H), 3.62-3.58 (m, 1 H), 3.55 (t, J ) 6.1 Hz, 2 H),
3.51 (br s, 1 H), 3.46-3.38 (m, 6 H), 3.27 (d, J ) 2.4 Hz, 1 H), 3.26-
3.21 (m, 1 H), 3.18-3.07 (m, 2 H), 2.18-2.03 (m, 3 H), 1.95-1.47
(series of multiplets, 19 H), 1.40-0.96 (series of multiplets, 9 H), 0.92
(d, J ) 6.8 Hz, 3 H), 0.91 (s, 3 H), 0.66 (s, 3 H); 13C NMR (∼10%
CD3OD in CDCl3, 75 MHz) δ 80.43, 79.68, 75.87, 69.30, 64.82, 64.32,
64.14, 48.78, 48.73, 48.50, 46.44, 46.21, 42.49, 41.76, 39.61, 35.36,
35.17, 35.06, 34.85, 31.73, 29.53, 29.46, 29.44, 28.84, 27.68, 27.48,
27.38, 25.91, 23.30, 22.75, 22.66, 17.70, 12.32; HRFAB-MS (thioglyc-
erol + Na+ matrix) m/e ([M - H + 2Na]+) 768.3831 (100%), calcd
768.3843. The azides were reduced by treating the triazide (0.11 g,
0.15 mmol) with Ph3P (0.20 g, 0.77 mmol) in THF (10 mL) and H2O
(1 mL). The mixture was stirred for 3 days. The solvent was removed
in vacuo, and the residue was purified by silica gel chromatography
(CH2Cl2/MeOH/28% NH3‚H2O, 12:6:1 and then 10:5:1.5) to afford the
desired product (0.077 g, 78% yield) as a glass. HCl in Et2O (1 M, 0.5
mL) was added to the glass to give the corresponding HCl salt: 1H
NMR (∼10% CDCl3 in CD3OD, 500 MHz) δ 4.81 (s, 10 H), 4.07-
3.97 (m, 2 H), 3.82 (br s, 1 H), 3.71 (br s, 1 H), 3.65 (t, J ) 5.2 Hz,
2 H), 3.57 (br s, 1 H), 3.37-3.30 (m, 2 H), 3.22-3.02 (m, 8 H), 2.12-
1.71 (series of multiplets, 17 H), 1.65-1.01 (series of multiplets, 13
H), 0.97 (d, J ) 6.8 Hz, 3 H), 0.94 (s, 3 H), 0.73 (s, 3 H); 13C NMR
(∼10% CDCl3 in CD3OD, 75 MHz) δ 81.89, 80.58, 77.50, 70.04, 66.71,
66.56, 66.02, 47.11, 46.76, 44.20, 42.66, 40.50, 39.60, 39.40, 36.24,
36.11, 35.89, 35.67, 32.28, 29.38, 29.23, 29.10, 28.94, 28.49, 26.06,
24.21, 23.46, 23.30, 18.50, 12.86; HRFAB-MS (thioglycerol + Na+
matrix) m/e ([M + Na]+) 668.4271 (100%), calcd 668.4258.
33. The mesylate derived from 1511 (0.19 g, 0.264 mmol) was stirred
with excess octylamine (2 mL) at 80 °C for 12 h. After removal of
octylamine in vacuo, the residue was chromatographed (silica gel,
EtOAc/hexanes, 1:4, with 2% Et3N) to afford the desired product (0.19
g, 95% yield) as a pale yellow oil: 1H NMR (CDCl3, 300 MHz) δ
3.69-3.37 (series of multiplets, 11 H), 3.26-3.00 (m, 4 H), 2.61-
2.53 (m, 4 H), 2.20-2.02 (m, 3 H), 1.98-0.99 (series of multiplets,
40 H), 0.92-0.85 (m, 9 H), 0.65 (s, 3H); 13C NMR (CDCl3, 75 MHz)
δ 80.60, 79.74, 76.05, 64.97, 64.40, 64.28, 50.79, 50.25, 49.00, 48.90,
48.71, 46.47, 46.34, 42.65, 41.96, 39.80, 35.77, 35.41, 35.27, 35.05,
33.73, 31.96, 30.25, 29.76, 29.74, 29.67, 29.39, 29.05, 27.84, 27.61,
27.55, 26.70, 23.50, 23.00, 22.82, 22.79, 18.06, 14.23, 12.54; HRFAB-
MS (thioglycerol + Na+ matrix) m/e ([M + H]+ ) 755.6012 (100%),
calcd 755.6024. Τhe triazide (0.18 g, 0.239 mmol) was dissolved in
THF (10 mL) and EtOH (10 mL). Lindlar catalyst (44 mg) was added,
and the suspension was shaken under H2 (50 psi) for 12 h. After removal
of the solvent in vacuo, the residue was purified by silica gel
chromatography (CH2Cl2/MeOH/28% NH3‚H2O, 10:5:1 and then 10:
5:1.5). To the product was added 1 M HCl (2 mL), and the resulting
clear solution was extracted with Et2O (2 × 10 mL). A 20% NaOH
solution was added until the solution became strongly basic. CH2Cl2
(20 mL, 2 × 10 mL) was used to extract the basic solution. The
combined extracts were dried over anhydrous Na2SO4, and removal of
solvent in vacuo gave the desired product (0.114 g, 68% yield) as a
clear oil: 1H NMR (∼20% CDCl3 in CD3OD, 500 MHz) δ 4.79 (br s,
7 H), 3.74-3.70 (m, 1 H), 3.66-3.61 (m, 1 H), 3.56-3.51 (m, 3 H),
3.31-3.29 (m, 2 H), 3.16-3.09 (m, 2 H), 2.88-2.72 (m, 6 H), 2.59-
2.51 (m, 4 H), 2.18-2.07 (m, 3 H), 1.97-1.66 (series of multiplets,

940 J. Am. Chem. Soc., Vol. 121, No. 5, 1999
Li et al.
14 H), 1.62-0.97 (series of multiplets, 25 H), 0.95 (d, J ) 6.3 Hz, 3
H), 0.93 (s, 3 H), 0.89 (t, J ) 6.8 Hz, 3 H), 0.70 (s, 3 H); 13C NMR
(∼20% CDCl3 in CD3OD, 75 MHz) δ 81.82, 80.63, 77.23, 67.85, 67.19,
51.20, 50.69, 47.82, 47.24, 43.92, 43.01, 41.30, 40.80, 40.68, 40.22,
36.74, 36.38, 36.20, 35.87, 34.66, 34.15, 33.87, 32.90, 30.54, 30.39,
30.30, 29.64, 29.03, 28.59, 28.41, 26.96, 24.37, 23.65, 23.48, 18.75,
14.63, 13.09; HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M + H]+
) 677.6309 (46.6%), calcd 677.6309.
of the solvent in vacuo, the residue was purified by silica gel
chromatography (CH2Cl2/MeOH/28% NH3‚H2O, 5:3:1 and then 2:2:
1). To the product was added 1 M HCl (2 mL), and the resulting solution
was washed with Et2O (2 × 10 mL). A 20% NaOH solution was added
to the aqueous phase until the solution became strongly basic. CH2Cl2
(10 mL, 2 × 5 mL) was used to extract the basic solution. The combined
extracts were dried over anhydrous Na2SO4, and concentration in vacuo
gave the desired product (0.044 g, 64% yield) as a colorless oil: 1H
NMR (∼20% CDCl3 in CD3OD, 500 MHz) δ 4.79 (br s, 8 H), 3.74-
3.70 (m, 1 H), 3.66-3.62 (m, 1 H), 3.56-3.52 (m, 3 H), 3.31-3.27
(m, 2 H), 3.16-3.10 (m, 2 H), 2.82-2.70 (m, 6 H), 2.64-2.54 (m, 2
H), 2.19-2.07 (m, 3 H), 1.99-1.66 (series of multiplets, 14 H), 1.58-
0.96 (series of multiplets, 13 H), 0.96 (d, J ) 6.6 Hz, 3 H), 0.93 (s, 3
H), 0.70 (s, 3 H); 13C NMR (∼20% CDCl3 in CD3OD, 75 MHz) δ
81.96, 90.76, 77.33, 67.92, 67.26, 47.84, 47.33, 44.04, 43.24, 43.15,
41.40, 40.91, 40.78, 40.29, 36.82, 36.48, 36.28, 35.96, 34.39, 34.11,
30.59, 29.69, 29.13, 28.68, 28.64, 24.43, 23.69, 23.48, 18.77, 13.06;
HRFAB-MS (thioglycerol + Na+ matrix) m/e ([M + H]+ ) 565.5041
(100%), calcd 565.5057.
MIC and MBC Measurements. A known concentration of bacteria
(∼106 cells/mL) was incubated with varied concentrations of a
compound of interest for 24 h in a nutrient broth (tryptic soy, Difco).
Bacterial growth was monitored via cell counting and turbidity
measurements. The MIC value was the concentration of the studied
compound at which the number of bacteria remained constant or
decreased during incubation. The MBC value was determined as the
concentration at which fewer than 0.1% of the bacteria survived
incubation with the compound of interest for 24 h. For an example see
ref 25.
34. Compound 33 (0.08 g, 0.12 mmol) was dissolved in CHCl3 (5
mL) and MeOH (5 mL), aminoiminosulfonic acid (0.045 g, 0.36 mmol)
was added, and the suspension was stirred for 12 h. The solvent was
removed in vacuo, and the residue was dissolved in 1 M HCl (6 mL)
and H2O (10 mL). The solution was washed with Et2O (3 × 5 mL),
and a 20% NaOH solution was then added dropwise until the solution
became strongly basic. The basic mixture was extracted with CH2Cl2
(4 × 5 mL). The combined extracts were dried over anhydrous Na2-
SO4 and concentrated in vacuo to give the desired product (0.087 g,
91% yield) as a white glass: 1H NMR (∼20% CDCl3 in CD3OD, 500
MHz) δ 4.96 (br s, 13 H), 3.74-3.68 (m, 1 H), 3.65-3.50 (m, 4 H),
3.38-3.18 (series of multiplets, 10 H), 2.60-2.50 (m, 4 H), 2.15-
1.99 (m, 3 H), 1.88-1.72 (m, 14 H), 1.60-0.99 (series of multiplets,
25 H), 0.94 (br s, 6 H), 0.89 (t, J ) 6.6 Hz, 3 H), 0.71 (s, 3 H); 13C
NMR (∼20% CDCl3 in CD3OD, 75 MHz) δ 159.00, 158.87, 158.72,
81.68, 79.93, 76.95, 66.59, 65.93, 65.45, 50.82, 50.40, 47.64, 46.94,
43.67, 42.27, 40.18, 39.25, 36.19, 35.66, 35.40, 34.21, 32.45, 30.51,
30.26, 30.18, 30.10, 29.86, 29.35, 28.71, 28.15, 28.00, 26.87, 23.94,
23.44, 23.23, 23.12, 18.61, 14.42, 12.98; HRFAB-MS (thioglycerol +
Na+ matrix) m/e ([M + H]+ ) 803.6958 (18.4%), calcd 803.6953.
35. The mesylate derived from 1511 (0.092 g, 0.128 mmol) was
dissolved in DMSO (2 mL) followed by the addition of NaN3 (0.0167
g, 0.256 mmol). The suspension was heated to 70 °C for 12 h. H2O
(20 mL) was added to the cooled suspension, and the mixture was
extracted with EtOAc/hexanes (1:1) (20 mL, 3 × 10 mL). The combined
extracts were washed with brine (30 mL), dried over anhydrous Na2-
SO4, and concentrated in vacuo to give the product (0.081 g, 95% yield)
as a pale yellow oil: 1H NMR (CDCl3, 300 MHz) δ 3.69-3.36 (m, 11
H), 3.25-3.02 (m, 6 H), 2.20-2.02 (m, 3 H), 1.97-1.60 (m, 15 H),
1.55-0.98 (m, 13 H), 0.92 (d, J ) 6.3 Hz, 3 H), 0.89 (s, 3 H), 0.66 (s,
3 H); 13C NMR (CDCl3, 75 MHz) δ 80.59, 79.77, 76.03, 65.01, 64.46,
64.30, 52.12, 48.99, 48.95, 48.76, 46.44, 46.42, 42.70, 41.99, 39.82,
35.56, 35.44, 35.31, 35.09, 33.09, 29.79, 29.77, 29.71, 29.08, 27.88,
27.78, 27.66, 25.65, 23.53, 23.03, 22.85, 18.00, 12.58; HRFAB-MS
(thioglycerol + Na+ matrix) m/e ([M + Na]+ ) 691.4512 (100%), calcd
691.4496. The tetraazide (0.081 g, 0.12 mmol) was dissolved in THF
(5 mL) and EtOH (10 mL). Lindlar catalyst (30 mg) was added, and
the suspension was shaken under H2 (50 psi) for 12 h. After removal
Luciferin/Luciferase-Based Cell Lysis Assay. The assay was
performed as published,24 with the following exceptions: trans-1,2-
diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA) was not used,
0.1% Triton X 100 was used instead of 4%, and cells were incubated
in the lysis buffer for 5 min before being exposed to luciferin and ATP.
Acknowledgment. Financial support from the National
Institutes of Health (Grant GM 54619) is gratefully acknowl-
edged.
Supporting Information Available: IR spectral data and1H
and 13C spectra for compounds 6-14 and 32-35. This material
is available free of charge via the Internet at http://pubs.acs.org.
JA982938M
(25) Pitt, W. G.; McBride, M. O.; Lunceford, J. K.; Roper, R. J.; Sagers,
R. D. Antimicrob. Agent Chemother. 1994, 38, 2577.