Bicyclomycin fluorescent probes: Synthesis and biochemical, biophysical, and biological properties

Article abstract of DOI:10.1021/jo030020u

Bicyclomycin (1) is a commercially available antibiotic whose primary site of action in Escherichia coli is the transcription termination factor rho. Key aspects of the 1·rho interaction - K_d, stoichiometry for 1·rho binding, and whether 1 and

Full text of DOI:10.1021/jo030020u

Bicyclom ycin F lu or escen t P r obes: Syn th esis a n d Bioch em ica l,
Biop h ysica l, a n d Biologica l P r op er ties
Andrew P. Brogan,† William R. Widger,*,‡ and Harold Kohn*,†
Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of
North Carolina, Chapel Hill, North Carolina 27599-7360, and Department of Biology and Biochemistry,
University of Houston, Houston, Texas 77204-5934
harold-kohn@unc.edu; widger@uh.edu
Received J anuary 14, 2003
Bicyclomycin (1) is a commercially available antibiotic whose primary site of action in Escherichia
coli is the transcription termination factor rho. Key aspects of the 1‚rho interactionsKd,
stoichiometry for 1‚rho binding, and whether 1 and ATP binding induce conformational changes
in rhosremain unknown. In this study, the design, synthesis, and characterization of a series of
bicyclomycin fluorescent probes (BFP) constructed to sense the 1‚rho interaction are described and
their use documented. We show that dihydrobicyclomycins with medium-to-large C(5a)-substituents
afforded excellent inhibitory activities exceeding those of 1 in the poly(C)-dependent ATPase assay.
The utility of BFP in bicyclomycin-rho binding studies was documented through the use of 5a-
(phenazin-2-ylmethylsulfanyl)dihydrobicyclomycin (15). Excitation (290 nm) of W381 in wild-type
rho in the presence of 15 and ATP led to fluorescence resonance energy transfer (FRET) and gave
a Kd (15) of 9.9 µM. Using ADP in place of ATP or excluding nucleotide did not result in energy
transfer, which suggests that ATP binding induced a conformational change in rho. FRET
measurements provided an approximate weighted average distance (23 Å) between W381 and 15
in the presence of bound ATP. The Kd value for 15‚rho was correlated with ATP binding at the 3
tight ATP binding (Kd(ATP) ) 95 nM) sites in wild-type rho.
The Escherichia coli transcription termination factor
rho is a hexameric protein consisting of identical 47-kDa
protomers.1 Rho plays an integral role in processing gene
products in E. coli and many other Gram-negative
organisms and without rho cell death results.2-5
models are in agreement with experimental results that
have identified the ATP11,12 and the RNA tracking
sites.13,14
The commercially available antibiotic bicyclomycin
(1)15 is the only known natural product inhibitor of the
E. coli rho transcription termination factor.16 We have
provided information concerning the mechanism of the
1‚rho interaction.17-19 Kinetic studies showed that 1 is a
noncompetitive inhibitor with respect to ATP17 and a
mixed inhibitor with respect to short ribonucleotides (i.e.,
ribo(C)10)18 believed to mimic the RNA that rho traverses.
Rho binds to specific rut sites on newly synthesized
RNA and then tracks toward the stalled polymerase
powered by ATP hydrolysis.1 Interaction of rho with the
polymerase leads to mRNA release and transcript ter-
mination. Rho has resisted crystallization, and thus,
detailed structural information is lacking. Rho shows
significant homology with F1-ATP synthase, another
hexameric protein, permitting a structural model of rho
to be generated from sequence comparison with the
â-subunit of F1-ATP synthase and the X-ray crystal-
lographic structures of bovine F1-ATP synthase.6-10 These
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H.; Widger, W. R. Biochemistry 2000, 39, 9077-9083.
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Chem. 1988, 263, 18810-18815.
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30023-30030.
* To whom all correspondence should be addressed.
† University of North Carolina.
(14) Wei, R. R.; Richardson, J . P. J . Biol. Chem. 2001, 276, 28380-
‡ University of Houston.
28387.
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10.1021/jo030020u CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/10/2003
J . Org. Chem. 2003, 68, 5575-5587
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Brogan et al.
Use of bicyclomycin affinity labels10,20,21 and rho muta-
tional studies9,22 permitted us23 and the Richardson
group22 to project the site of 1 binding in rho. We place
bicyclomycin binding between the B and C helices with
the C(1)-triol group in 1 directed toward the outside of
rho and the terminal C(5)-C(5a)-exomethylene unit
pointed toward the ATP terminal γ-phosphate and the
P-loop (residues 175-183) located near the central hole.
ous bicyclomycin structure-activity relationship (SAR)
studies demonstrated that modification of either the 1
C(1)-triol31 or the [4.2.2]piperazinedione ring system32 led
to derivatives with little or no inhibitory activity in rho
functional assays. Correspondingly, placement of small-
and medium-size substituents at the C(5a)-position in 1
provided compounds that inhibited rho-dependent hy-
drolysis of ATP at levels that matched 1.23,33 Accordingly,
we decided to incorporate a fluorophore at the C(5a)-
exomethylene position in 1 and to link the fluorophore
moiety to 1 through a sulfur atom. Our choice of sulfur
was governed by the ease with which thiols add to the
C(5a) site of bicyclomycin derivatives to give the corre-
sponding C(5a)-substituted dihydobicyclomycins and the
excellent inhibitory activities found for select C(5a)-sulfur
substituted dihydrobicyclomycins (see ref 33 for more
details). We began with 5a-phenylsulfanyldihydrobicy-
clomycin (2), a compound we previously reported33 that
functions as an efficient rho inhibitor (I50 ) 75 µM), and
progressively increased the size of the appended aromatic
ring, changed the site of aromatic substitution, and
altered the ring composition. We knew from the outset
that several of the compounds slated for synthesis and
evaluation would not have photophysical properties suit-
able for biochemical measurements, but we were uncer-
tain if C(5a)-substituents larger than a six-membered
ring would efficiently bind to rho and retain sufficient
solubility in aqueous buffer solutions.
Two classes of compounds were prepared. The first
(Table 1, group A, compounds 2-10) contained a thiol
directly attached to the aromatic unit. The second set
(Table 1, group B, compounds 11-16) contained a meth-
ylene spacer placed between the thiol unit and the
aromatic moiety. Both series included monocyclic (2-6,
11), bicyclic (7-9, 12, 13), and tricyclic (10, 14-16) C(5a)-
substituents. We included in these series dihydrobicy-
clomycin C(5a)-moieties expected either to undergo ex-
citation and fluorescence emission or to serve as probes
in FRET experiments.34,35
Syn th esis. The thiol-containing aromatic substituent
was introduced by Michael addition33 to bicyclomycin
2′,3′-acetonide (17)36 (Scheme 1). The Michael acceptor
was the enone formed upon hemiaminal ring opening at
C(6) of 17 under basic conditions. The reactions proceeded
in moderate yields (46-73%) to give diastereomeric
mixtures (∼3:1) of the C(5)-C(5a)-dihydrobicyclomycin
adducts (18-31). The diastereomeric mixtures were not
readily separable and were used directly in the functional
assays, as described in Biochemical and Biological Prop-
erties and BFP-Rho Binding. We observed higher yields
for group B BFP than for group A derivatives, a finding
we expected given the likely increased nucleophilicity for
the thiolate anions used in these syntheses.37 Subsequent
Our findings that bicyclomycin disrupts rho-mediated
ATP hydrolysis and rho-RNA binding17-19 have contrib-
uted to a working model for 1 inhibition of the rho
transcription termination process.10 However, much re-
mains to be learned before this pathway can be verified.
For example, we know the I50 and Ki values for 1 when
rho is stimulated under in vitro conditions17 but do not
know the inherent rho binding constant(s) (Kd) for 1.
Inactivation of one to two subunits in hexameric rho with
bicyclomycin affinity labels is sufficient to halt ATP
hydrolysis,21 but we do not know if 1 binds equally to all
six subunits or selectively to a subset of the six. ATP, by
comparison, binds tightly to only three of the six
protomers,24-27 and a similar situation exists in F1-ATP
synthase.28 Finally, RNA binding to rho induces a con-
formational change in rho seen by altered trypsin diges-
tion, but this technique is too insensitive to monitor
conformational changes upon binding 1 or ATP.18,29,30
In this study, we report the design, synthesis, and
characterization of a series of bicyclomycin fluorescent
probes (BFP) constructed to sense the interaction of 1
with rho. We document that BFP possess useful bio-
chemical and photophysical properties amenable to mecha-
nistic investigations and demonstrate the utility of one
of these compounds.
Resu lts a n d Discu ssion
Ch oice of Com p ou n d s. There are no known BFP, so
we selectively incorporated a fluorescent tag to 1. Previ-
(20) (a) Cho, H.; Park, H.-g.; Zhang, X.; Riba, I.; Gaskell, S. J .;
Widger, W. R.; Kohn, H. J . Org. Chem. 1997, 62, 5432-5440. (b) Riba,
I.; Gaskell, S. J .; Cho, H.; Widger, W. R.; Kohn, H. J . Biol. Chem. 1998,
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(21) Vincent, F.; Widger, W. R.; Openshaw, M.; Gaskell, S. J .; Kohn,
H. Biochemistry 2000, 39, 9067-9076.
(22) Moyse, K. A.; Knight, J . S.; Richardson, J . P. J . Mol. Biol. 2000,
302, 565-579.
(23) Vincent, F.; Srinivasan, J .; Santilla´n, A., J r.; Widger, W. R.;
Kohn, H. J . Org. Chem. 2001, 66, 2251-2264.
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Nature 1994, 370, 621-628. (b) Boyer, P. D. Angew. Chem., Int. Ed.
1998, 38, 8, 2296-2307.
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Chem. 1996, 61, 7750-7755. (b) Zhang, Z.; Kohn, H. J . Am. Chem.
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J r.; Zhang, X.; Hardesty, J .; Widger, W. R.; Kohn, H. J . Med. Chem.
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Org. Chem. 1996, 61, 7764-7776.
(34) Stryer, L. Annu. Rev. Biochem. 1978, 47, 819-846.
(35) Lakowicz, J . R. Principles of Fluorescence Spectroscopy, 2nd ed.;
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5576 J . Org. Chem., Vol. 68, No. 14, 2003

Bicyclomycin Fluorescent Probes
TABLE 1. Sp ectr a l, Bioch em ica l a n d Biologica l P r op er ties for BF P 2-16
a
The number in each entry is the chemical shift value (δ) observed in ppm relative to TMS, followed by the multiplicity of the signal.
All spectra were recorded at 300 MHz, and the solvent used was CD3OD (4:1 CD3OD-DMSO-d6 was used for 14). The entries in italics
correspond to the signals detected for the minor diastereomer observed in the product mixture. The number in each entry is the chemical
b
shift value (δ) observed in ppm relative to TMS. All spectra were recorded at 75 MHz, and the solvent used was CD3OD (DMSO-d6 was
used for 14). The entries in italics correspond to the signals detected for the minor diastereomer observed in the product mixture. c The
number in each entry corresponds to an emission maxima wavelength. The value in parentheses corresponds to the molar extinction
d
coefficient at that maxima. Measurements were performed in water at 25 °C. Fluorescent measurements were performed in water at 25
°C. e Inhibitory properties, poly(C)-dependent ATPase assay42 and filter disk assay.43 f Wavelength of fluorescence excitation (exc) maximum.
g
i
h
Wavelength of fluorescence emission (em) maximum. Quantum yield, determined using tryptophan or quinine sulfate as standards.35
Inhibitory activity measured using the rho poly (C)-dependent ATPase assay.42 The I50 value is the average 50% inhibition concentration
determined from duplicate tests. The corresponding value obtained from bicyclomycin in a concurrently run experiment is provided in
j
parentheses. MIC value is the minimum inhibitory concentration of the compound determined from duplicate tests using W3550 E. coli
in a filter disk assay.43 The corresponding value obtained from bicyclomycin in a concurrently run experiment is provided in parentheses.
k
l
m
n
p
Bcm ) bicyclomycin. Value is from Park et al.33 Value not determined. Compound is not fluorescent. o Not measured. Partially
soluble in water; value not determined.
TFA removal of the 2′,3′-acetonide group in 18-31
provided the desired 3-16, respectively, in 55-99%
yields. In most cases, we isolated and characterized the
intermediate dihydrobicyclomycin acetonide, but for 18,
19, 24, 25, 29, and 31 we found it more expeditious to
directly deprotect the crude acetonide to give 3, 4, 9, 10,
14, and 16, respectively.
Two general procedures were used for the Michael
addition step. We either added thiol38 directly to 17
(Scheme 1, method 1) or mixed the corresponding thio-
acetate derivative with 17 (Scheme 1, method 2) knowing
(37) (a) Edwards, J . O.; Pearson, R. G. J . Am. Chem. Soc. 1962, 84,
16-24. (b) Moffatt, J . G. In Oxidation; Augustine, R. L., Trecker, D.
J ., Eds.; Marcel Dekker: New York, 1971; Vol. 2, pp 1-64.
(38) (a) Shaw, J . E. J . Org. Chem. 1991, 56, 3728-3729. (b) Heffter,
W. Chem. Ber. 1895, 28, 2258-2264.
J . Org. Chem, Vol. 68, No. 14, 2003 5577

Brogan et al.
carbon (δ 28.7-30.8) were observed in the 1H and 13C
NMR spectra, respectively, documenting sulfur substitu-
tion at this site33 (Table 1).
SCHEME 1. P r ep a r a tion of BF P 2-16
Bioch em ica l a n d Biologica l P r op er ties: Str u c-
tu r e-Activity Tr en d s. Dihydrobicyclomycin derivatives
2-16 were evaluated in the rho poly(C)-dependent
ATPase assay (Table 1).42 This test has been shown to
be a reliable assay and has permitted the identification
of bicyclomycin derivatives that inhibit rho.23,31-33 Each
compound was evaluated in duplicate assays using His-
tagged wild-type rho9 and compared with a concurrently
tested 1 sample.
We found that many of the compounds exhibited
inhibitory activity comparable with or better than 1. The
most active compounds were 12, 13, and 15, which were
approximately twice as potent as 1.17 These compounds
are among the most effective bicyclomycin inhibitors in
the rho poly(C)-dependent ATPase assay reported to date.
The number of compounds in this study (15) did not
permit us to deduce definitive SAR patterns. Nonethe-
less, the following may provide useful guidelines for
future studies: (1) The size of the C(5a)-aromatic sub-
stituent did not affect the dihydrobicyclomycin inhibitory
activity in the poly(C)-dependent ATPase assay. In series
A, we observed few differences in activity when proceed-
ing from bicyclomycin derivatives with monocyclic to
bicyclic to tricyclic ring substituents (i.e., 2, 8, and 10).
In series B, excellent activity was also observed for all
compounds except the sparingly soluble 14, irrespective
of the size of the ring system. Interestingly, the data set
did not define the maximum size of the C(5a)-substituent
that still retained potent inhibitory activity. This is an
interesting topological issue since the C(5a)-substituent
is directed toward the approximate 40 Å wide hole within
the rho toroid, the site of RNA translocation. We will
explore this aspect in future studies but indicate here
that larger C(5a)-substituents will require the use of
appended hydrophilic groups to solubilize the bicyclomy-
cin derivative in water. (2) In select cases, inclusion of
heteroatoms (O, N) within the aromatic ring system of
the C(5a)-substituent did not alter the inhibitory proper-
ties of the bicyclomycin derivative. For example, we found
that 2 (I50 ) 75 µM) and 3 (I50 ) 55 µM) displayed
comparable inhibitory activities. Similarly, 12 (I50 ) 25
µM) and 13 (I50 ) 35 µM) were both effective rho
inhibitors. (3) The aromatic ring site of attachment to
the C(5a)-dihydrobicyclomycin may affect rho inhibition.
Diminished activities were found when the point of
attachment was near an aromatic fusion site (i.e., I50
value: 7, 110 µM) compared with a peripheral aromatic
locus (i.e., I50 values (µM): 8, 45; 12, 25; 13, 35; 15, 25;
16, 55). This finding may reflect the structural require-
ments for the C(5a)-substituent for binding to rho.
Together, these findings indicated that medium-to-large
C(5a)-substituted dihydrobicyclomycins serve as excellent
inhibitors of rho poly(C)-dependent ATPase activity and
that the site of aromatic substitution is more important
than the ring size and composition.
that the thioacetyl unit would be removed under the basic
conditions39 employed in the Michael addition reaction.
The arylmethylthioacetates 37-41 were prepared from
the corresponding halides40 by displacement with potas-
sium thioacetate in acetone.41
Ch a r a cter iza tion of BF P . Satisfactory spectral data
1
(IR, H NMR, 13C NMR, and MS) were obtained for all
new compounds. Diagnostic signals for the diastereotopic
5a-methylene protons (δ 1.87-3.12, 3.10-4.14) and 5a-
(39) (a) Patai, S. The Chemistry of the Thiol Group; Wiley: New
York, 1974; p 677. (b) Maskill, H. The Physical Basis of Organic
Chemistry; Oxford University Press: Oxford, 1985; p 159.
(40) (a) Maidwell, N. L.; Rezai, M. R.; Roeschlaub, C. A.; Sammes,
P. G. J . Chem. Soc., Perkin Trans. 2000, 10, 1541-1546. (b) Kaslow,
C. E.; Schlatter, J . M. J . Am. Chem. Soc. 1955, 77, 1054-1055.
(41) For a similar procedure, see: Gryko, D. T.; Clausen, C.; Lindsey,
J . S. J . Org. Chem. 1999, 64, 8635-8647.
The antimicrobial minimal inhibitory concentration
(MIC) values of dihydrobicyclomycins 2-16 were deter-
mined against W3350 E. coli in the filter disk assay43
(42) Sharp, J . A.; Galloway, J . L.; Platt, T. J . Biol. Chem. 1983, 258,
3482-3486.
5578 J . Org. Chem., Vol. 68, No. 14, 2003

Bicyclomycin Fluorescent Probes
(Table 1). None of the compounds exhibited significant
activity (g16 mg/mL). Similar findings have been ob-
served for other C(5a)-substituted dihydrobicyclomy-
cins.33
not shown).45 We observed that in the absence of nu-
cleotide or in the presence of ADP no energy transfer
occurred upon excitation of W381 in the presence of 15.
A modest reduction (e.g., 40 µM 15: ≈15%) in fluorescence
emission was detected with increasing 15 concentration,
P h otop h ysica l P r op er ties. The UV-vis and fluo-
rescent properties of 3-16 were assessed (Table 1). We
did not resynthesize and evaluate 2 since 6 did not
exhibit useful photophysical properties amenable to
fluorescence-based studies. As expected, we observed in
the UV-vis spectra for 3-16 a progressive increase in
the λmax for the highest wavelength absorption as we
proceeded from C(5a)-substituents with an appended
monocyclic ring to those with tricyclic substituents.44 The
longest λmax observed was for 10 (403 nm). The extinction
coefficients for 3-16 were graphically determined from
a set of serially diluted samples and followed Beer’s law.
We found that compounds 7-10 and 12 displayed fluo-
rescent properties. The λmax for fluorescent excitation
ranged from 257 nm (10) to 383 nm (10) while the λmax
for fluorescent emission was from 336 nm (12) to 444 nm
(10). The quantum yield (Φ) for fluorescent emission
ranged from 0.03 to 0.49 with 9 being the most efficient
and 8 the least.
BF P -Rh o Bin d in g. We demonstrated the utility of
BFP in 1‚rho binding studies. Compound 15 was em-
ployed in the initial studies, and we asked if the fluo-
rescence emission generated upon excitation of W381
(λexc ) 290 nm) in rho resulted in fluorescence resonance
energy transfer (FRET) by bound 15. The UV-vis
absorption spectrum (λmax ) 377 nm) for 15 overlaps the
W381 fluorescence emission spectrum (λem,max ) 345
nm).45 W381 is the only tryptophan residue in wild-type
rho and is predicted to be in a region in the C terminus
where the structure is less similar to F1-ATP synthase.
The sequence correspondence of rho and F1-ATP synthase
in this sector is not high, and thus, the projected
coordinates and spatial orientation for W381 are uncer-
tain. Nonetheless, the estimated distance between bicy-
clomycin and W381 on each subunit (21 Å and 35 Å) is
within the range of FRET processes.35 Significantly, our
homology model was built using the F1-ATP synthase
â-subunit with a bound ATP mimic.21b This is important
since in F1-ATP synthase the ATP hydrolysis hinge opens
in the absence of ATP and closes with nucleotide bind-
ing.28 The interaction between the â subunit and the
rotating γ subunit can result in a movement of ap-
proximately 20 Å on the C terminus of the â subunit.28a
The net effect is that the W381-15 distance may be
shorter in the presence of ATP and longer in its absence.
an observation attributed to inner filter effects.35,48
A
similar effect was found using a 1 µM solution of free
tryptophan and 15 (0-40 µM) (data not shown).48 Inclu-
sion of ATP (250 µM) in the reaction solution containing
wild-type rho and 15 led to a pronounced loss of the W381
fluorescence emission when excited at 290 nm that
depended upon 15 concentration (Figure 1A,B). Adding
1 (400 µM) at two different 15 concentrations (20, 40 µM)
led to significant gains in fluorescence emission (20 µM
15: 93%; 40 µM 15: 85%), indicating that 15 and 1 share
a common binding site in rho. After correcting for inner
filter effects,48 we observed a single dissociation constant
for 15 with hexameric rho by Scatchard analysis49 (Kd )
9.9 µM) (Figure 1C).
The FRET studies indicated that the rho W381-15
distance was ATP dependent and demonstrated that ATP
induced a conformational change in rho that permitted
energy transfer provided that 15 binds to rho in the
absence of nucleotide or in the presence of ADP. Previous
attempts to detect an ATP-induced rho conformational
change by limited tryptic digestion were inconclu-
sive.18,29,30 Addition of ATP led to diminished levels of
trypsin cleavage near residue 128.29 Similar findings
were also observed for the adenine nucleotides ADP and
AMPPNP. Our homology model indicated that W381 on
adjacent subunits could potentially contribute to the
observed FRET. Thus, the FRET distance calcula-
tions35,50,51 for the ATP-mediated W381-15 energy trans-
fer revealed that the weighted average W381-15 dis-
tances is approximately 23 Å. Similarly, the lower limit
for W381-15 (if bound) in the presence of ADP or in the
absence of nucleotide is g46 Å.35,50,51 These distance
estimates rest on several assumptions that require
validation in future studies. Among these are that
nucleotide (ATP, ADP) binding to hexameric rho does not
markedly influence 15 complexation to the protein. We
know that 1 lowered the Kd for ATP binding by ap-
proximately 50% at the three tight sites and only
marginally affects ATP binding at the three loose sites,45
but the reverse may not be true. In addition, we have
assumed that nucleotide (ATP, ADP) binding does not
affect the spatial orientation of the dihydrobicyclomycin
C(5a)-phenazin-2-ylmethylsulfanyl moiety. We have done
so because 1 is a reversible, noncompetitive inhibitor with
respect to ATP17 and C(5a)-substituted bicyclomycin
reductive amination probes efficiently modify rho in the
presence of ATP.20,21 Repeated attempts to resolve the
15 diastereomeric mixture by TLC using different solvent
systems (10% MeOH-CHCl3, EtOAc) did not separate
the mixture. HPLC analysis showed two closely spaced
peaks (tR 75, 76 min) indicating that preparative HPLC
may permit separation of the two diastereomers. Future
efforts will be directed to provide purified samples of each
We measured the binding of 15 (0-40 µM) to wild-type
rho46,47 (1 µM based on the monomer) at 25 °C in the
absence of nucleotide and in the presence of either ADP
(400 µM) or ATP (250 µM). Previous studies have shown
that under these conditions rho exists as a hexamer (data
(43) Ericcson, H. M.; Sherris, J . C. Acta Pathol. Microbiol. Scand.
1971, Suppl. 217, 1-90.
(44) Silverstein, R. M.; Bassler, G. C.; Morrill, T. C. Spectrometric
Identification of Organic Compounds, 3rd ed., J ohn Wiley & Sons: New
York, 1974; p 252.
(48) Liu, Y.; Kati, W.; Chen, C.-M.; Tripathi, R.; Molla, A.; Kohl-
brenner, W. Anal. Biochem. 1999, 267, 331-335.
(49) Segal, I. H. Enzyme Kinetics; J ohn Wiley & Sons: New York,
1975; p 218.
(50) Fo¨rster, T. Ann. Physik. 1948, 2, 55-75.
(51) Stryer, L.; Haugland, R. P. Proc. Natl. Acad. Sci. U.S.A. 1967,
58, 719-726.
(45) Xu, Y.; J ohnson, J .; Kohn, H.; Widger, W. R. J . Biol. Chem.
2003, 278, 13719-13727.
(46) Mott, J . E.; Grant, R. A.; Ho, Y.-S.; Platt, T. Proc. Natl. Acad.
Sci. U.S.A. 1985, 82, 88-92.
(47) Nehrke, K. W.; Seifried, S. E.; Platt, T. Nucleic Acids Res. 1992,
20, 6107.
J . Org. Chem, Vol. 68, No. 14, 2003 5579

Brogan et al.
F IGURE 2. Effect of ATP on fluorescence resonance energy
transfer between wild-type rho and 15 (µM). (A) The percent
fluorescence transfer as a function of ATP concentration (0-
120 µM) measured at 25 °C. No significant change in the
plateau was observed for the maximum ATP concentration
(240 µM) used (data not shown). (B) Scatchard analysis of ATP
binding to 15‚rho complex.
are six equal binding sites or a subset of six (e.g., three).
Accordingly, we measured the extent of energy transfer
(345 nm) upon excitation (290 nm) of a solution contain-
ing wild-type rho and 15 (60 µM) as a function of ATP
concentration (0-240 µM) (Figure 2A). We found that low
amounts of ATP (2 µM) led to maximal levels of energy
transfer, and Scatchard analysis indicated a Kd ) 95 nM
for ATP binding (Figure 2B). This value is in agreement
with the estimated (see below) value for the tight ATP
binding site in wild-type rho in the presence of 15. In a
recent study,45 we showed that in the absence of poly-
(C), the rho mutant, F355W, has three tight (Kd1 ) 3 µM)
and three loose (Kd2 ) 58 µM) ATP binding sites and that
adding 1 strengthened the ATP binding at the tight site
(Kd1 ) 1.4 µM), while only modestly affecting the loose
ATP binding site (Kd2 ) 51 µM). Furthermore, we
observed that the Kd1 and Kd2 values for ATP binding to
F355W rho were higher (weaker) than those reported for
wild-type rho where Kd1 ≈ 0.33 µM and Kd2 ≈ 10 µM.25
The I50 value for 15 was approximately one-half that of
1 in the poly(C)-dependent ATPase assay (Table 1),
indicating that 15 binds tighter to rho than 1. Accord-
ingly, we predict that the Kd value for ATP binding in
F IGURE 1. Fluorescence resonance energy transfer between
wild-type rho and 15 in the presence of ATP (250 µM). (A)
Emission spectra at varying 15 concentrations measured at
25 °C. BFP 15 concentrations were as follows: 0, 1.3, 2.5, 5.0,
7.5, 10, 15, 20, 30, 40 µM. The emission spectra were measured
at an exciting wavelength of 290 nm. (B) Saturation concen-
tration curve showing percent fluorescence energy transfer
from wild-type rho (W381) to 15 as a function of 15 concentra-
tion. (C) Scatchard analysis of 15 binding to wild-type rho.
isomer to learn if the stereochemistry at the C(5)-position
affects the I50, Kd, and FRET distances upon 15 binding
to wild-type rho.
Only a single dissociation constant for 15 with hex-
americ rho was observed by Scatchard analysis (Figure
1C). This finding does not permit us to determine if there
5580 J . Org. Chem., Vol. 68, No. 14, 2003

Bicyclomycin Fluorescent Probes
5a -(2-Am in op h en ylsu lfa n yl)d ih yd r obicyclom ycin 2′,3′-
Aceton id e (20). Using general procedure 1, 17 (33 mg, 0.10
mmol) and 2-aminothiophenol (60 mg, 0.48 mmol) gave 20 as
a mixture of diastereomers (∼3:1): yield 48 mg (67%); Rf 0.71
(10% MeOH-CHCl3); FT-IR (KBr) 3312 (br), 2960 (br), 1690,
1392, 1046 cm-1; 1H NMR (CD3OD) for the major diastereomer,
δ 1.35 (s, 3 H, C(2′)CH3), 1.44 (s, 6 H, C(CH3)2), 1.95-2.33 (m,
3 H, C(4)HH′, C(4)HH′, C(5)H), 2.43 (dd, J ) 11.5, 14.3 Hz, 1
H, C(5a)HH′), 3.47 (dd, J ) 2.7, 11.5 Hz, 1 H, C(5a)HH′), 3.70
(d, J ) 8.8 Hz, 1 H, C(3′)HH′), 3.72-4.03 (m, 2 H, C(3)HH′,
C(3)HH′), 4.09 (s, 1 H, C(1′)H), 4.44 (d, J ) 8.8 Hz, 1 H, C(3′)-
HH′), 6.57-6.65 (m, 1 H, C(5′′)H), 6.74 (dd, J ) 1.6, 8.2 Hz, 1
H, C(6′′)H), 7.01-7.09 (m, 1 H, C(4′′)H), 7.29 (dd, J ) 1.6, 8.2
the presence of 15 will be approximately 4-fold lower
(tighter) than 0.33 µM. Significantly, the 15-mediated
FRET measurements at a constant ATP concentration
(250 µM) (Figure 1) correlated with ATP binding to the
three tight sites. Thus, the measured Kd value for 15 (9.9
µM) (Figure 1B) corresponded to only these three ATP
binding sites. Since ATP induced a conformational change
in rho that allowed for the observed energy transfer at
only three of the six sites, we do not know the extent of
15 binding to the remaining three subunits in hexameric
rho, which are governed by loose ATP binding and the
site of 15 occupancy that led to the observed FRET
measurements.
1
Hz, 1 H, C(3′′)H); H NMR (CD3OD) for the minor diastere-
omer, δ 1.36 (s, 3 H, C(2′)CH3), 1.43 (s, 6 H, C(CH3)2), 2.66
(dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′), 4.10 (s, 1 H, C(1′)H),
4.45 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 6.76 (dd, J ) 1.6, 8.2 Hz,
1 H, C(6′′)H), 7.30 (dd, J ) 1.6, 8.2 Hz, 1 H, C(3′′)H), the
remaining peaks overlapped with nearby signals and were not
resolved; the structural assignments were in agreement with
the 1H-1H COSY experiment; 13C NMR (CD3OD) for the major
diastereomer, 24.9, 26.9, 28.3, 30.4, 34.8, 52.4, 63.6, 73.3, 73.4,
83.8, 86.4, 88.7, 111.7, 116.3, 118.4, 119.1, 130.5, 136.2, 150.2,
168.2, 171.7 ppm; 13C NMR (CD3OD) for the minor diastere-
omer, 28.4, 29.8, 33.2, 50.3, 63.6, 89.0, 119.2, 130.7, 136.3,
167.2, 173.6 ppm, the remaining peaks overlapped with nearby
signals and were not resolved; MS (+CI) 468 [M + 1]+; Mr
(+CI) 468.180 70 [M + 1]+ (calcd for C21H30N3O7S 468.180 45).
Con clu sion s
Synthetic procedures were developed to permit incor-
poration of fluorescent labels at the C(5a)-site in dihy-
drobicyclomycins. SAR studies for BFP showed that
incorporation of medium-to-large C(5a)-substituents pro-
vided dihydrobicyclomycin derivatives with excellent
inhibitory activities in the poly(C)-dependent ATPase
assay that exceeded 1. The utility of BFP in bicyclomy-
cin-rho binding studies was documented through the use
of 15. FRET studies with 15 and wild-type rho showed
that Kd ) 9.9 µM in the presence of ATP. There was no
energy transfer when ADP was used in place of ATP or
when nucleotide was excluded, revealing preliminary
information that ATP binding induced a rho conforma-
tional change, provided that 15 binds to rho in the
absence of nucleotide. The FRET measurements for 15
were correlated with ATP binding to the three tight sites
in wild-type rho.
5a -(4-ter t-Bu t ylp h en ylsu lfa n yl)d ih yd r ob icyclom ycin
2′,3′-Aceton id e (21). Using general procedure 1, 17 (45 mg,
0.13 mmol) and 4-tert-butylbenzenethiol (153 mg, 0.92 mmol)
gave 21 as a mixture of diastereomers (∼3:1): yield 48 mg
(72%); Rf 0.71 (10% MeOH-CHCl3); FT-IR (KBr) 3307 (br),
2962 (br), 1691, 1397, 1050 cm-1 1H NMR (CD3OD) for the
;
major diastereomer, δ 1.29 (s, 9 H, C(CH3)3), 1.36 (s, 3 H, C(2′)-
CH3), 1.44 (s, 6 H, C(CH3)2), 1.90-2.33 (m, 3 H, C(4)HH′, C(4)-
HH′, C(5)H), 2.49 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′), 3.61-
4.08 (m, 4 H, C(3′)HH′, C(3)HH′, C(3)HH′, C(5a)HH′), 4.10 (s,
1 H, C(1′)H), 4.44 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 7.27-7.38
(m, 4 H, 2 C(2′′)H, 2 C(3′′)H); 1H NMR (CD3OD) for the minor
diastereomer, δ 2.74 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′),
4.10 (s, 1 H, C(1′)H), 4.45 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), the
remaining peaks overlapped with nearby signals and were not
resolved; the structural assignments were in agreement with
the 1H-1H COSY experiment; 13C NMR (CD3OD) for the major
diastereomer, 24.8, 26.9, 28.3, 30.3, 31.7 (3 C), 34.0, 35.3, 51.9,
63.6, 73.3, 73.4, 83.8, 86.3, 88.7, 111.7, 127.1 (2 C), 130.4 (2
C), 133.6, 150.7, 168.1, 171.6 ppm; 13C NMR (CD3OD) for the
minor diastereomer 28.4, 29.7, 32.2, 49.8, 63.5, 73.5, 83.7, 89.0,
111.7, 127.2 (2 C), 150.6, 167.3, 173.5 ppm, the remaining
peaks overlapped with nearby signals and were not resolved;
MS (+CI) 509 [M + 1]+; Mr (+CI) 509.230 97 [M + 1]+ (calcd
for C25H37N2O7S 509.232 15).
Exp er im en ta l Section
Gen er a l Met h od s. See the Supporting Information and
ref 23.
Gen er a l P r oced u r e 1. P r ep a r a tion of 5a -Su bstitu ted
Dih yd r obicyclom ycin 2′,3′-Aceton id es (Meth od s 1 a n d 2).
To a methanolic solution (3-10 mL) of 17 under Ar was added
the desired nucleophile or substituted thioacetate (2-10 equiv)
and then “pH” was adjusted to 10.5 with aqueous 1.0 M NaOH.
The mixture was stirred at room temperature (2-24 h) until
no starting material remained (TLC analysis). The “pH” of the
mixture was adjusted to 7.0 with aqueous 0.1-1.0 M HCl. The
solvent was removed in vacuo, and the residue was redissolved
in MeOH and filtered when applicable. The solvent was
removed in vacuo, and the residue was purified by preparative
TLC (5-10% MeOH-CHCl3) to afford the desired product.
Gen er a l P r oced u r e 2. P r ep a r a tion of 5a -Su bstitu ted
Dih yd r obicyclom ycin s. To a 50% aqueous methanolic solu-
tion (unless otherwise indicated) (2-10 mL) containing the 5a-
substituted dihydrobicyclomycin 2′,3′-acetonide was added
TFA (2-10 drops), and then the solution was stirred at room
temperature (4-48 h) until no starting material remained
(TLC analysis). The solvent was removed in vacuo and the
residue was purified by preparative TLC (10-20% MeOH-
CHCl3) to afford the desired product.
Gen er a l P r oced u r e 3. P r ep a r a tion of P r ecu r sor Ar yl-
m eth ylth ioa ceta tes 37-41. To an acetone solution (10-20
mL) of the desired aromatic methyl halide (32-36) under Ar
was added potassium thioacetate (1.2 equiv). The mixture was
heated to reflux (1-3 h) until no starting material remained
(TLC analysis) and H2O (15 mL) added. The reaction mixture
was extracted with Et2O (3 × 10 mL), dried (Na2SO4), and
concentrated in vacuo. The product was purified by column
chromatography using 10-50% EtOAc-hexanes.
5a -(Na p h th -1-ylsu lfa n yl)d ih yd r obicyclom ycin 2′,3′-Ac-
eton id e (22). Using general procedure 1, 17 (47 mg, 0.13
mmol) and 1-naphthalenethiol (154 mg, 0.96 mmol) gave 22
as a mixture of diastereomers (∼3:1): yield 46 mg (67%); Rf
0.65 (10% MeOH-CHCl3); FT-IR (KBr) 3296 (br), 2985, 2934,
1689, 1385, 1047 cm-1 1H NMR (CD3OD) for the major
;
diastereomer, δ 1.35 (s, 3 H, C(2′)CH3), 1.43 (s, 6 H, C(CH3)2),
1.96-2.38 (m, 3 H, C(4)HH′, C(4)HH′, C(5)H), 2.66 (dd, J )
11.5, 14.3 Hz, 1 H, C(5a)HH′), 3.69 (d, J ) 8.8 Hz, 1 H, C(3′)-
HH′), 3.72-4.06 (m, 3 H, C(3)HH′, C(3)HH′, C(5a)HH′), 4.09
(s, 1 H, C(1′)H), 4.43 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 7.37-7.57
(m, 3 H, C(3′′)H, C(6′′)H, C(7′′)H), 7.68 (d, J ) 7.1 Hz, 1 H,
C(2′′)H), 7.73 (d, J ) 8.8 Hz, 1 H, C(4′′)H), 7.85 (d, J ) 9.3 Hz,
1 H, C(5′′)H), 8.31 (d, J ) 6.6 Hz, 1 H, C(8′′)H); 1H NMR (CD3-
OD) for the minor diastereomer, δ 1.44 (s, 6 H, C(CH3)2), 2.89
(dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′), 4.10 (s, 1 H, C(1′)H),
4.45 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), the remaining peaks
overlapped with nearby signals and were not resolved; the
structural assignments were in agreement with the 1H-1H
J . Org. Chem, Vol. 68, No. 14, 2003 5581

Brogan et al.
COSY experiment; 13C NMR (CD3OD) for the major diastere-
omer, 24.8, 26.9, 28.2, 30.6, 34.3, 52.0, 63.6, 73.3, 73.5, 83.8,
86.3, 88.7, 111.7, 125.7, 126.8, 127.3, 127.4, 128.2, 128.9, 129.7,
134.1, 134.3, 135.5, 168.1, 171.5 ppm; 13C NMR (CD3OD) for
the minor diastereomer, 28.3, 30.1, 32.7, 63.5, 88.9, 128.4,
129.0, 167.3, 173.4 ppm, the remaining peaks overlapped with
nearby signals and were not resolved; MS (+CI) 503 [M + 1]+;
(m, 3 H, C(3′′)H, C(6′′)H, C(7′′)H), 7.74 (d, J ) 1.1 Hz, 1 H,
C(1′′)H), 7.76-7.83 (m, 3 H, C(4′′)H, C(5′′)H, C(8′′)H); 1H NMR
(CD3OD) for the minor diastereomer, δ 1.44 (s, 3 H, C(CH3)2),
2.30 (dd, J ) 11.5, 15.7 Hz, 1 H, C(5a)HH′), 3.57 (d, J ) 8.8
Hz, 1 H, C(3′)HH′), 4.05 (s, 1 H, C(1′)H), 4.43 (d, J ) 8.8 Hz,
1 H, C(3′)HH′), the remaining peaks overlapped with nearby
signals and were not resolved; the structural assignments were
in agreement with the 1H-1H COSY experiment; 13C NMR
(CD3OD) for the major diastereomer, 24.9, 26.8, 28.2, 30.4,
31.5, 37.6, 52.4, 63.3, 73.2, 73.4, 83.8, 86.4, 88.7, 111.6, 126.8,
127.2, 128.3, 128.6, 128.7, 128.8, 129.3, 134.1, 134.8, 137.3,
168.3, 171.6 ppm; 13C NMR (CD3OD) for the minor diastere-
omer, 26.9, 28.4, 37.5, 50.1, 63.6, 73.5, 83.7, 89.0, 111.7, 128.3,
128.7 ppm, the remaining peaks overlapped with nearby
signals and were not resolved; Mr (+CI) 517.202 04 [M + 1]+
(calcd for C26H33N2O7S 517.200 85).
Mr (+CI) 503.184 37 [M
+
1]+ (calcd for C25H31N2O7S
503.185 20).
5a -(Na p h th -2-ylsu lfa n yl)d ih yd r obicyclom ycin 2′,3′-Ac-
eton id e (23). Using general procedure 1, 17 (52 mg, 0.15
mmol) and 2-naphthalenethiol (122 mg, 0.76 mmol) gave 23
as a mixture of diastereomers (∼3:1): yield 35 mg (46%); Rf
0.56 (10% MeOH-CHCl3); FT-IR (KBr) 3421 (br), 2984 (br),
1690, 1387, 1053 cm-1 1H NMR (CD3OD) for the major
;
diastereomer, δ 1.36 (s, 3 H, C(2′)CH3), 1.45 (s, 6 H, C(CH3)2),
1.90-2.40 (m, 3 H, C(4)HH′, C(4)HH′, C(5)H), 2.63 (dd, J )
11.5, 14.3 Hz, 1 H, C(5a)HH′), 3.70 (d, J ) 8.8 Hz, 1 H, C(3′)-
HH′), 3.72-4.08 (m, 3 H, C(3)HH′, C(3)HH′, C(5a)HH′), 4.10
(s, 1 H, C(1′)H), 4.44 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 7.38-7.49
(m, 3 H, C(3′′)H, C(6′′)H, C(7′′)H), 7.71-7.82 (m, 3 H, C(4′′)H,
5a -(Qu in olin -6-ylm et h ylsu lfa n yl)d ih yd r ob icyclom y-
cin 2′,3′-Aceton id e (28). Using general procedure 1, 17 (51
mg, 0.15 mmol) and 38 (106 mg, 0.50 mmol) gave 28 as a
mixture of diastereomers (∼3:1): yield 43 mg (56%); mp 125-
132 °C; Rf 0.35 (10% MeOH-CHCl3); FT-IR (KBr) 3360 (br),
1
C(5′′)H, C(8′′)H), 7.85 (d, J ) 1.6 Hz, 1 H, C(1′′)H); H NMR
1
2984, 2928, 1689, 1384, 1043 cm-1; H NMR (CD3OD) for the
(CD3OD) for the minor diastereomer, δ 1.33 (s, 3 H, C(2′)CH3),
1.43 (s, 6 H, C(CH3)2), 2.88 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)-
HH′), 3.69 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 4.11 (s, 1 H, C(1′)H),
4.45 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), the remaining peaks
overlapped with nearby signals and were not resolved; the
structural assignments were in agreement with the 1H-1H
COSY experiment; 13C NMR (CD3OD) for the major diastere-
omer, 24.9, 26.9, 28.3, 30.5, 33.3, 51.9, 63.6, 73.3, 73.4, 83.8,
86.4, 88.7, 111.7, 126.7, 127.4, 127.6, 128.0, 128.1, 128.7, 129.6,
133.2, 134.9, 135.4, 168.1, 171.6 ppm; 13C NMR (CD3OD) for
the minor diastereomer, 29.9, 31.8, 50.7, 89.0, 167.1, 173.5
ppm, the remaining peaks overlapped with nearby signals and
were not resolved; MS (+CI) 503 [M +1]+; Mr (+CI) 503.177 14
[M + 1]+ (calcd for C25H31N2O7S 503.177 37).
major diastereomer, δ 1.32 (s, 3 H, C(2′)CH3), 1.34 (s, 3 H,
C(CH3)2), 1.41 (s, 3 H, C(CH3)2), 1.83-2.36 (m, 4 H, C(4)HH′,
C(4)HH′, C(5)H, C(5a)HH′), 3.14 (d, J ) 11.5 Hz, 1 H, C(5a)-
HH′), 3.68 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 3.65-3.97 (m, 2 H,
C(3)HH′, C(3)HH′), 3.89 (s, 2 H, SCH2Ar), 4.07 (s, 1 H, C(1′)H),
4.39 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 7.52 (dd, J ) 4.4, 8.5 Hz,
1 H, C(3′′)H), 7.75-7.92, (m, 2 H, C(5′′)H, C(7′′)H), 7.98 (br d,
J ) 8.5 Hz, 1 H, C(4′′)H), 8.32 (d, J ) 8.2 Hz, 1 H, C(8′′)H),
1
8.80 (dd, J ) 1.6, 4.4 Hz, 1 H, C(2′′)H); H NMR (CD3OD) for
the minor diastereomer, δ 1.45 (s, 3 H, C(CH3)2), 4.07 (s, 1 H,
C(1′)H), 4.44 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), the remaining
peaks overlapped with nearby signals and were not resolved;
the structural assignments were in agreement with the 1H-
1H COSY experiment; 13C NMR (CD3OD) for the major
diastereomer, 24.9, 26.8, 28.2, 30.5, 31.5, 37.0, 52.3, 63.4, 73.2,
73.4, 83.8, 86.3, 88.7, 111.6, 122.8, 128.8, 129.4, 129.6, 132.6,
138.2, 138.9, 148.0, 151.0, 168.3, 171.5 ppm; 13C NMR (CD3-
OD) for the minor diastereomer, 26.9, 28.3, 36.8, 63.6, 73.5,
83.6, 88.9, 111.7, 128.9, 173.6 ppm, the remaining peaks
overlapped with nearby signals and were not resolved; MS
(+CI) 518 [M + 1]+; Mr (+CI) 518.196 04 [M + 1]+ (calcd for
5a -Ben zylsu lfa n yld ih ydr obicyclom ycin 2′,3′-Aceton ide
(26). Using general procedure 1, 17 (40 mg, 0.12 mmol) and
benzyl mercaptan (101 mg, 0.82 mmol) gave 26 as a mixture
of diastereomers (∼3:1): yield 40 mg (73%); Rf 0.55 (10%
MeOH-CHCl3); FT-IR (KBr) 3303 (br), 2958 (br), 1688, 1392,
1
1045 cm-1; H NMR (CD3OD) for the major diastereomer, δ
1.35 (s, 3 H, C(2′)CH3), 1.41 (s, 3 H, C(CH3)2), 1.44 (s, 3 H,
C(CH3)2), 1.82-2.26 (m, 4 H, C(4)HH′, C(4)HH′, C(5)H, C(5a)-
HH′), 3.18 (d, J ) 11.5 Hz, 1 H, C(5a)HH′), 3.63-3.93 (m, 3
H, C(3)HH′, C(3)HH′, C(3′)HH′), 3.68 (s, 2 H, SCH2Ph), 4.08
(s, 1 H, C(1′)H), 4.42 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 7.16-7.38
(m, 5 H, Ph); 1H NMR (CD3OD) for the minor diastereomer, δ
1.45 (s, 3 H, C(CH3)2), 2.28 (dd, J ) 11.5, 15.7 Hz, 1 H, C(5a)-
HH′), 3.16 (d, J ) 11.5 Hz, 1 H, C(5a)HH′), 4.07 (s, 1 H, C(1′)H),
4.44 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), the remaining peaks
overlapped with nearby signals and were not resolved; the
structural assignments were in agreement with the 1H-1H
COSY experiment; 13C NMR (CD3OD) for the major diastere-
omer, 24.9, 26.9, 28.2, 30.4, 31.7, 37.4, 52.4, 63.4, 73.3, 73.4,
83.8, 86.4, 88.7, 111.7, 128.0, 129.5 (2 C), 130.1 (2 C), 140.1
168.3, 171.5 ppm; 13C NMR (CD3OD) for the minor diastere-
omer, 26.9, 28.4, 29.9, 50.1, 63.6, 73.3, 73.6, 83.7, 88.9, 167.2,
173.6 ppm, the remaining peaks overlapped with nearby
signals and were not resolved; MS (+CI) 467 [M + 1]+; Mr
(+CI) 467.185 79 [M + 1]+ (calcd for C22H31N2O7S 467.185 20).
C
25H32N3O7S 518.196 10).
5a -(P h en a zin -2-ylm et h ylsu lfa n yl)d ih yd r ob icyclom y-
cin 2′,3′-Aceton id e (30). Using general procedure 1, 17 (52
mg, 0.15 mmol) and 40 (82 mg, 0.30 mmol) gave 30 as a
mixture of diastereomers (∼3:1): yield 48 mg (56%); mp 134-
140 °C; Rf 0.58 (10% MeOH-CHCl3); FT-IR (KBr) 3283 (br),
1
2982, 2928, 1687, 1379, 1044 cm-1; H NMR (CD3OD) for the
major diastereomer, δ 1.24 (s, 3 H, C(2′)CH3), 1.32 (s, 3 H,
C(CH3)2), 1.36 (s, 3 H, C(CH3)2), 1.83-2.43 (m, 4 H, C(4)HH′,
C(4)HH′, C(5)H, C(5a)HH′), 3.15 (d, J ) 11.5 Hz, 1 H, C(5a)-
HH′), 3.65 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 3.67-4.10 (m, 2 H,
C(3)HH′, C(3)HH′), 3.96 (d, J ) 5.1 Hz, 2 H, SCH2Ar), 4.07 (s,
1 H, C(1′)H), 4.36 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 7.82-7.96
(m, 3 H, C(3′′)H, C(6′′)H, C(7′′)H), 8.02-8.21 (m, 4 H, C(1′′)H,
C(4′′)H, C(5′′)H, C(8′′)H); 1H NMR (CD3OD) for the minor
diastereomer, δ 1.44 (s, 3 H, C(CH3)2), 4.43 (d, J ) 8.8 Hz, 1
H, C(3′)HH′), the remaining peaks overlapped with nearby
signals and were not resolved; the structural assignments were
in agreement with the 1H-1H COSY experiment; 13C NMR
(CD3OD) for the major diastereomer, 24.8, 26.7, 28.2, 30.5,
31.4, 37.2, 52.3, 63.5, 73.2, 73.4, 83.7, 86.3, 88.7, 111.6, 128.9,
130.1, 130.2, 130.4, 131.9, 132.1, 134.0, 143.5, 143.8, 144.2,
144.3, 144.5, 168.1, 171.4 ppm; 13C NMR (CD3OD) for the
minor diastereomer, 26.9, 28.3, 37.3, 50.2, 73.5, 83.6, 88.9,
111.7, 128.8, 143.6, 173.6 ppm, the remaining peaks over-
lapped with nearby signals and were not resolved; MS (+CI)
569 [M + 1]+; Mr (+CI) 569.207 43 [M + 1]+ (calcd for
5a -(Na p h t h -2-ylm e t h ylsu lfa n yl)d ih yd r ob icyclom y-
cin 2′,3′-Aceton id e (27). Using general procedure 1, 17 (24
mg, 0.07 mmol) and 37 (106 mg, 0.49 mmol) gave 27 as a
mixture of diastereomers (∼3:1): yield 26 mg (72%); Rf 0.58
(5% MeOH-CHCl3); FT-IR (KBr) 3283 (br), 2928 (br), 1687,
1383, 1045 cm-1; 1H NMR (CD3OD) for the major diastereomer,
δ 1.30 (s, 3 H, C(2′)CH3), 1.33 (s, 3 H, C(CH3)2), 1.41 (s, 3 H,
C(CH3)2), 1.83-2.25 (m, 4 H, C(4)HH′, C(4)HH′, C(5)H, C(5a)-
HH′), 3.18 (d, J ) 11.5 Hz, 1 H, C(5a)HH′), 3.27-3.82 (m, 3
H, C(3)HH′, C(3)HH′, C(3′)HH′), 3.85 (s, 2 H, SCH2Ar), 4.06
(s, 1 H, C(1′)H), 4.37 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 7.39-7.52
C
28H33N4O7S 569.207 00).
5582 J . Org. Chem., Vol. 68, No. 14, 2003

Bicyclomycin Fluorescent Probes
5a -(2-Am in op h en ylsu lfa n yl)d ih yd r obicyclom ycin (5).
Using general procedure 2, 20 (30 mg, 0.06 mmol) gave 5 as a
mixture of diastereomers (∼3:1): yield 24 mg (89%); mp 140-
145 °C; Rf 0.71 (10% MeOH-CHCl3); FT-IR (KBr) 3296 (br),
2936 (br), 1687, 1403, 1038 cm-1; UV-vis (H2O) 298 nm (ꢀ
793); 1H NMR (CD3OD) for the major diastereomer, δ 1.32 (s,
3 H, C(2′)CH3), 2.08-2.25 (m, 3 H, C(4)HH′, C(4)HH′, C(5)H),
2.46 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′), 3.43 (d, J ) 11.5
Hz, 1 H, C(5a)HH′), 3.52 (d, J ) 8.8 Hz, 1 H, C(3′)HH′), 3.65
(d, J ) 8.8 Hz, 1 H, C(3′)HH′), 3.66-4.06 (m, 2 H, C(3)HH′,
C(3)HH′), 4.01 (s, 1 H, C(1′)H), 6.57-6.65 (m, 1 H, C(5′′)H),
6.75 (dd, J ) 1.6, 8.2 Hz, 1 H, C(6′′)H), 7.01-7.09 (m, 1 H,
C(4′′)H), 7.29 (dd, J ) 1.6, 8.2 Hz, 1 H, C(3′′)H); 1H NMR (CD3-
OD) for the minor diastereomer, δ 2.66 (dd, J ) 11.5, 14.3 Hz,
1 H, C(5a)HH′), 4.03 (s, 1 H, C(1′)H), the remaining peaks
overlapped with nearby signals and were not resolved; the
structural assignments were in agreement with the 1H-1H
COSY experiment; 13C NMR (CD3OD) for the major diastere-
omer, 24.2, 29.9, 34.5, 52.3, 62.1, 68.5, 72.3, 78.1, 83.7, 89.4,
125.7, 126.8, 128.3, 128.9, 134.1, 134.4 ppm, the remaining
peaks overlapped with nearby signals and were not resolved;
MS (+CI) 463 [M + 1]+; Mr (+CI) 463.154 04 [M + 1]+ (calcd
for C22H27N2O7S 463.153 90).
5a -(Na p h th -2-ylsu lfa n yl)d ih yd r obicyclom ycin (8). Us-
ing general procedure 2, 23 (35 mg, 0.07 mmol) gave 8 as a
mixture of diastereomers (∼3:1): yield 19 mg (59%); mp 125-
130 °C; Rf 0.50 (20% MeOH-CHCl3); FT-IR (KBr) 3259 (br),
2935 (br), 1686, 1402, 1038 cm-1; UV-vis (H2O) 283 nm (ꢀ
1
5990); H NMR (CD3OD) for the major diastereomer, δ 1.33
(s, 3 H, C(2′)CH3), 1.99-2.40 (m, 3 H, C(4)HH′, C(4)HH′,
C(5)H), 2.66 (dd, J ) 11.5 Hz, 14.3 Hz, 1 H, C(5a)HH′), 3.53
(d, J ) 11.3 Hz, 1 H, C(3′)HH′), 3.64-4.09 (m, 4 H, C(3′)HH′,
C(3)HH′, C(3)HH′, C(5a)HH′), 4.03 (s, 1 H, C(1′)H), 7.37-7.50
(m, 3 H, C(3′′)H, C(6′′)H, C(7′′)H), 7.72-7.82 (m, 3 H, C(4′′)H,
1
C(5′′)H, C(8′′)H), 7.86 (d, J ) 1.6 Hz, 1 H, C(1′′)H); H NMR
(CD3OD) for the minor diastereomer, δ 2.86 (dd, J ) 11.5, 14.3
Hz, 1 H, C(5a)HH′), 3.69 (d, J ) 11.3 Hz, 1 H, C(3′)HH′), 4.05
(s, 1 H, C(1′)H), 7.84 (d, J ) 1.6 Hz, 1 H, C(1′′)H), the
remaining peaks overlapped with nearby signals and were not
resolved; the structural assignments were in agreement with
the 1H-1H COSY experiment; 13C NMR (CD3OD) for the major
diastereomer, 24.2, 30.0, 32.9, 51.8, 62.1, 68.6, 72.3, 78.2, 83.7,
89.4, 126.7, 127.3, 127.6, 128.0, 128.1, 128.7, 129.5, 133.2,
134.9, 135.4, 168.7, 172.2 ppm; 13C NMR (CD3OD) for the
minor diastereomer, 30.5, 32.1, 72.4, 83.8, 89.7, 127.5, 135.1,
167.7 ppm, the remaining peaks overlapped with nearby
signals and were not resolved; MS (+CI) 463 [M + 1]+; Mr
(+CI) 463.154 91 [M + 1]+ (calcd for C22H27N2O7S 463.153 90).
116.3, 118.4, 119.1, 130.5, 136.1, 150.2, 168.8, 172.1 ppm; 13
C
NMR (CD3OD) for the minor diastereomer, 30.5, 33.6, 50.4,
63.3, 72.4, 83.6, 89.6, 118.7, 119.2, 130.6, 167.6, 174.0 ppm,
the remaining peaks overlapped with nearby signals and were
not resolved; Mr (+CI) 428.149 29 [M + 1]+ (calcd for
C
18H26N3O7S 428.149 15).
5a -(4-t er t -Bu t yl-p h e n ylsu lfa n yl)d ih yd r ob icyclom y-
cin (6). Using general procedure 2, 21 (55 mg, 0.07 mmol) gave
6 as a mixture of diastereomers (∼3:1): yield 41 mg (81%);
mp 128-132 °C; Rf 0.31 (10% MeOH-CHCl3); FT-IR (KBr)
3338 (br), 2958 (br), 1691, 1401, 1045 cm-1; UV-vis (H2O) 255
5a -Ben zylsu lfa n yld ih ydr obicyclom ycin (11). Using gen-
eral procedure 2, 26 (39 mg, 0.08 mmol) gave 11 as a mixture
of diastereomers (∼3:1): yield 29 mg (81%); mp 114-119 °C;
Rf 0.52 (20% MeOH-CHCl3); FT-IR (KBr) 3398 (br), 2928 (br),
1
nm (ꢀ 9760); H NMR (CD3OD) for the major diastereomer, δ
1.29 (s, 9 H, C(CH3)3), 1.33 (s, 3 H, C(2′)CH3), 1.92-2.32 (m, 3
H, C(4)HH′, C(4)HH′, C(5)H), 2.53 (dd, J ) 11.5, 14.3 Hz, 1
H, C(5a)HH′), 3.52 (d, 1 H, J ) 11.6 Hz, 1 H, C(3′)HH′), 3.60-
4.06 (m, 4 H, C(3′)HH′, C(3)HH′, C(3)HH′, C(5a)HH′), 4.02 (s,
1 H, C(1′)H), 7.26-7.36 (m, 4 H, 2 C(2′′)H, 2 C(3′′)H); 1H NMR
(CD3OD) for the minor diastereomer, δ 2.72 (dd, J ) 11.5, 14.3
Hz, 1 H, C(5a)HH′), 3.55 (d, J ) 11.6 Hz, 1 H, C(3′)HH′), 4.04
(s, 1 H, C(1′)H), the remaining peaks overlapped with nearby
signals and were not resolved; the structural assignments were
in agreement with the 1H-1H COSY experiment; 13C NMR
(CD3OD) for the major diastereomer, 24.2, 29.8, 31.7 (3 C),
33.5, 35.3, 51.8, 62.1, 68.5, 72.2, 78.1, 83.7, 89.4, 127.1 (2 C),
130.3 (2 C), 133.6, 150.5, 168.7, 172.2 ppm; 13C NMR (CD3-
OD) for the minor diastereomer, 30.4, 32.6, 63.2, 72.4, 83.6,
89.6, 127.1 (2 C), 130.2 (2 C), 133.8, 167.7, 174.0 ppm, the
remaining peaks overlapped with nearby signals and were not
resolved; Mr (+CI) 469.200 97 [M + 1]+ (calcd for C22H33N2O7S
469.200 85).
1687, 1405, 1038 cm-1 1H NMR (CD3OD) for the major
;
diastereomer, δ 1.32 (s, 3 H, C(2′)CH3), 1.90-2.27 (m, 4 H,
C(4)HH′, C(4)HH′, C(5)H, C(5a)HH′), 3.13 (d, J ) 11.5 Hz, 1
H, C(5a)HH′), 3.50 (d, J ) 12.6 Hz, 1 H, C(3′)HH′), 3.64-3.91
(m, 3 H, C(3′)HH′, C(3)HH′, C(3)HH′), 3.67 (s, 2 H, SCH2Ar),
1
4.01 (s, 1 H, C(1′)H), 7.16-7.39, (m, 5 H, Ar); H NMR (CD3-
OD) for the minor diastereomer, δ 2.28 (dd, J ) 11.5, 15.7 Hz,
1 H, C(5a)HH′), 3.10 (d, J ) 11.5 Hz, 1 H, C(5a)HH′), 3.54 (d,
J
) 12.6 Hz, 1 H, C(3′)HH′), 4.02 (s, 1 H, C(1′)H), the
remaining peaks overlapped with nearby signals and were not
resolved; the structural assignments were in agreement with
the 1H-1H COSY experiment; 13C NMR (CD3OD) for the major
diastereomer, 24.2, 30.1, 31.6, 37.4, 52.3, 62.0, 68.5, 72.4, 78.1,
83.8, 89.4, 128.0, 129.5 (2 C), 130.1 (2 C), 140.1, 168.8, 172.1
ppm; 13C NMR (CD3OD) for the minor diastereomer, 30.4, 30.6,
37.5, 50.2, 63.3, 72.5, 83.6, 89.6, 167.6, 173.7 ppm, the
remaining peaks overlapped with nearby signals and were not
resolved; MS (+CI) 427 [M + 1]+; Mr (+CI) 427.152 66 [M +
1]+ (calcd for C19H27N2O7S 427.153 90).
5a -(Na p h th -1-ylsu lfa n yl)d ih yd r obicyclom ycin (7). Us-
ing general procedure 2, 22 (42 mg, 0.07 mmol) gave 7 as a
mixture of diastereomers (∼3:1): yield 27 mg (59%); mp 130-
135 °C; Rf 0.53 (20% MeOH-CHCl3); FT-IR (KBr) 3249 (br),
2934 (br), 1687, 1394, 1039 cm-1; UV-vis (H2O) 301 nm (ꢀ
5a -(Na p h t h -2-ylm e t h ylsu lfa n yl)d ih yd r ob icyclom y-
cin (12). Using general procedure 2, 27 (35 mg, 0.07 mmol)
gave 12 as a mixture of diastereomers (∼3:1): yield 32 mg
(99%); mp 122-128 °C; Rf 0.52 (20% MeOH-CHCl3); FT-IR
(KBr) 3238 (br), 2926 (br), 1687, 1395, 1032 cm-1; UV-vis
(H2O) 276 nm (ꢀ 4340); 1H NMR (CD3OD) for the major
diastereomer, δ 1.31 (s, 3 H, C(2′)CH3), 1.87-2.35 (m, 4 H,
C(4)HH′, C(4)HH′, C(5)H, C(5a)HH′), 3.15 (d, J ) 11.6 Hz, 1
H, C(5a)HH′), 3.53 (d, J ) 10.5 Hz, 1 H, C(3′)HH′), 3.42-3.88
(m, 3 H, C(3′)HH′, C(3)HH′, C(3)HH′), 3.84 (s, 2 H, SCH2Ar),
4.00 (s, 1 H, C(1′)H), 7.37-7.52 (m, 3 H, C(3′′)H, C(6′′)H,
C(7′′)H), 7.73-7.83 (m, 4 H, C(1′′)H, C(4′′)H, C(5′′)H, C(8′′)H);
1H NMR (CD3OD) for the minor diastereomer, δ 1.28 (s, 3 H,
C(2′)CH3), 4.01 (s, 1 H, C(1′)H), the remaining peaks over-
lapped with nearby signals and were not resolved; the struc-
1
6750); H NMR (CD3OD) for the major diastereomer, δ 1.33
(s, 3 H, C(2′)CH3), 2.00-2.38 (m, 3 H, C(4)HH′, C(4)HH′,
C(5)H), 2.69 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′), 3.53 (d, 1
H, J ) 11.6 Hz, 1 H, C(3′)HH′), 3.67 (d, 1 H, J ) 11.6 Hz, 1 H,
C(3′)HH′), 3.71-4.06 (m, 3 H, C(3)HH′, C(3)HH′, C(5a)HH′),
4.02 (s, 1 H, C(1′)H), 7.38-7.57 (m, 3 H, C(3′′)H, C(6′′)H,
C(7′′)H), 7.68 (d, J ) 7.1 Hz, 1 H, C(2′′)H), 7.73 (d, J ) 8.8 Hz,
1 H, C(4′′)H), 7.85 (d, J ) 9.3 Hz, 1 H, C(5′′)H), 8.31 (d, J )
6.6 Hz, 1 H, C(8′′)H); 1H NMR (CD3OD) for the minor
diastereomer, δ 2.86 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′),
3.54 (d J ) 11.6 Hz, 1 H, C(3′)HH′), 4.04 (s, 1 H, C(1′)H), the
remaining peaks overlapped with nearby signals and were not
resolved; the structural assignments were in agreement with
the 1H-1H COSY experiment; 13C NMR (CD3OD) for the major
diastereomer, 24.2, 30.2, 34.0, 51.9, 62.2, 68.5, 72.4, 78.1, 83.8,
89.4, 125.7, 126.8, 127.3, 127.4, 128.2, 128.8, 129.7, 134.1,
134.3, 135.5, 168.7, 172.1 ppm; 13C NMR (CD3OD) for the
minor diastereomer, 30.7, 33.0, 50.0, 63.2, 72.5, 83.6, 89.6,
1
tural assignments were in agreement with the H-1H COSY
experiment; 13C NMR (CD3OD) for the major diastereomer,
24.3, 30.1, 31.4, 37.6, 52.3, 62.0, 68.5, 72.4, 78.1, 83.8, 89.3,
126.8, 127.2, 128.3, 128.6, 128.7, 128.8, 129.3, 134.1, 134.8,
137.4, 168.8, 172.1 ppm; 13C NMR (CD3OD) for the minor
J . Org. Chem, Vol. 68, No. 14, 2003 5583

Brogan et al.
diastereomer, 30.2, 30.7, 50.2, 63.3, 72.5, 78.1, 83.6, 89.6, 127.2,
128.7 ppm, the remaining peaks overlapped with nearby
signals and were not resolved; Mr (+CI) 477.171 33 [M + 1]+
(calcd for C23H29N2O7S 477.169 55).
8.29 (d, J ) 4.9 Hz, 2 H, 2 C(2′′)H); 1H NMR (CD3OD) for the
minor diastereomer, δ 1.29 (s, 3 H, C(2′)CH3), 2.79 (dd, J )
11.5, 14.3 Hz, 1 H, C(5a)HH′), 3.54 (d, J ) 11.6 Hz, 1 H, C(3′)-
HH′), 4.06 (s, 1 H, C(1′)H), the remaining peaks overlapped
with nearby signals and were not resolved; the structural
5a -(Qu in olin -6-ylm et h ylsu lfa n yl)d ih yd r ob icyclom y-
cin (13). Using general procedure 2, 28 (39 mg, 0.08 mmol)
gave 13 as a mixture of diastereomers (∼3:1) after the “pH”
was adjusted to 8 with aqueous 0.1 M NaOH: yield 30 mg
(83%); mp 133-140 °C; Rf 0.52 (20% MeOH-CHCl3); FT-IR
(KBr) 3337 (br), 2930 (br), 1686, 1400, 1039 cm-1; UV-vis
(H2O) 319 nm (ꢀ 5380); 1H NMR (CD3OD) for the major
diastereomer, δ 1.31 (s, 3 H, C(2′)CH3), 1.90-2.32 (m, 4 H,
C(4)HH′, C(4)HH′, C(5)H, C(5a)HH′), 3.11 (d, J ) 11.6 Hz, 1
H, C(5a)HH′), 3.47 (d, J ) 10.5 Hz, 1 H, C(3′)HH′), 3.56-3.95
(m, 3 H, C(3′)HH′, C(3)HH′, C(3)HH′), 3.88 (s, 2 H, SCH2Ar),
4.01 (s, 1 H, C(1′)H), 7.52 (dd, J ) 4.4, 8.5 Hz, 1 H, C(3′′)H),
7.75-7.92, (m, 2 H, C(5′′)H, C(7′′)H), 7.98 (br d, J ) 8.5 Hz, 1
H, C(4′′)H), 8.32 (d, J ) 8.2 Hz, 1 H, C(8′′)H), 8.80 (dd, J )
1.6, 4.4 Hz, 1 H, C(2′′)H); 1H NMR (CD3OD) for the minor
diastereomer, δ 1.32 (s, 3 H, C(2′)CH3), 4.02 (s, 1 H, C(1′)H),
the remaining peaks overlapped with nearby signals and were
not resolved; the structural assignments were in agreement
1
assignments were in agreement with the H-1H COSY experi-
ment; 13C NMR (CD3OD) for the major diastereomer, 24.2,
30.5, 30.6, 51.5, 62.3, 68.5, 72.2, 78.2, 83.7, 89.5, 122.2 (2 C),
149.5 (2 C), 151.9, 168.5, 171.9 ppm; 13C NMR (CD3OD) for
the minor diastereomer, 29.6, 30.9, 63.2, 72.3, 83.6, 89.7, 122.1,
150.0 ppm, the remaining peaks overlapped with nearby
signals and were not resolved; Mr (+CI) 414.133 62 [M + 1]+
(calcd for C17H24N3O7S 414.133 50).
5a -(P yr id in -2-ylsu lfa n yl)d ih yd r obicyclom ycin (4). Us-
ing general procedure 1, 17 (53 mg, 0.16 mmol) and 2-mer-
captopyridine (120 mg, 1.08 mmol) gave 19 as a crude mixture
(Rf 0.58 (10% MeOH-CHCl3)). The reaction was not purified
but immediately deprotected using general procedure 2 to give
4 as a mixture of diastereomers (∼3:1) after the “pH” was
adjusted to 8 with aqueous 0.1 M NaOH: yield 36 mg (56%);
mp 115-120 °C; Rf 0.32 (20% MeOH-CHCl3); FT-IR (KBr)
3311 (br), 2938 (br), 1688, 1412, 1044 cm-1; UV-vis (H2O) 288
1
with the H-1H COSY experiment; 13C NMR (CD3OD) for the
1
nm (ꢀ 4910); H NMR (CD3OD) for the major diastereomer, δ
major diastereomer, 24.2, 30.1, 31.3, 37.0, 52.2, 62.0, 68.5, 72.3,
78.1, 83.7, 89.3, 122.8, 128.8, 129.4, 129.7, 132.7, 138.2, 139.0,
148.1, 151.0, 168.8, 172.0 ppm; 13C NMR (CD3OD) for the
minor diastereomer, 30.7, 50.1, 63.3, 72.5, 83.6, 89.6, 128.9,
167.6, 174.1 ppm, the remaining peaks overlapped with nearby
signals and were not resolved; MS (+CI) 478 [M + 1]+; Mr
(+CI) 478.164 27 [M + 1]+ (calcd for C22H28N3O7S 478.164 80).
1.34 (s, 3 H, C(2′)CH3), 1.89-2.41 (m, 3 H, C(4)HH′, C(4)HH′,
C(5)H), 2.97 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′), 3.54 (d, 1
H, J ) 11.6 Hz, 1 H, C(3′)HH′), 3.68 (d, 1 H, J ) 11.6 Hz, 1 H,
C(3′)HH′), 3.65-4.14 (m, 3 H, C(3)HH′, C(3)HH′, C(5a)HH′),
4.04 (s, 1 H, C(1′)H), 7.05-7.12 (m, 1 H, C(5′′)H), 7.35 (d, J )
7.7 Hz, 1 H, C(3′′)H), 7.58-7.66 (m, 1 H, C(4′′)H), 8.35 (d, J )
4.9 Hz, 1 H, C(6′′)H); 1H NMR (CD3OD) for the minor
diastereomer, δ 3.12 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′),
3.56 (d, J ) 11.6 Hz, 1 H, C(3′)HH′), 4.06 (s, 1 H, C(1′)H), the
remaining peaks overlapped with nearby signals and were not
resolved; the structural assignments were in agreement with
the 1H-1H COSY experiment; 13C NMR (CD3OD) for the major
diastereomer, 24.3, 30.0, 30.3, 53.3, 62.3, 68.6, 72.4, 78.2, 83.9,
89.4, 121.0, 123.2, 138.1, 150.2, 160.4, 168.9, 171.2 ppm; 13C
NMR (CD3OD) for the minor diastereomer, 28.9, 30.8, 51.0,
63.6, 72.6, 89.7, 121.4, 123.3, 150.3 ppm, the remaining peaks
overlapped with nearby signals and were not resolved; MS
(+CI) 414 [M + 1]+; Mr (+CI) 414.132 71 [M + 1]+ (calcd for
5a -(P h en a zin -2-ylm et h ylsu lfa n yl)d ih yd r ob icyclom y-
cin (15). Using general procedure 2, 30 (42 mg, 0.07 mmol)
gave 15 as a mixture of diastereomers (∼3:1) after the “pH”
was adjusted to 8 with aqueous 0.1 M NaOH: yield 30 mg
(92%); mp 139-145 °C; Rf 0.56 (20% MeOH-CHCl3); HPLC
tR, 75, 76 min; FT-IR (KBr) 3303 (br), 2931 (br), 1686, 1401,
1
1037 cm-1; UV-vis (H2O) 377 nm (ꢀ 17 600); H NMR (CD3-
OD) for the major diastereomer, δ 1.30 (s, 3 H, C(2′)CH3),
1.90-2.41 (m, 4 H, C(4)HH′, C(4)HH′, C(5)H, C(5a)HH′), 3.12
(d, J ) 11.6 Hz, 1 H, C(5a)HH′), 3.46 (d, J ) 10.5 Hz, 1 H,
C(3′)HH′), 3.61 (d, J ) 10.5 Hz, 1 H, C(3′)HH′), 3.64-3.98 (m,
2 H, C(3)HH′, C(3)HH′), 3.96 (d, J ) 5.1 Hz, 2 H, SCH2Ar),
4.01 (s, 1 H, C(1′)H), 7.82-7.96 (m, 3 H, C(3′′)H, C(6′′)H,
C(7′′)H), 8.02-8.21 (m, 4 H, C(1′′)H, C(4′′)H, C(5′′)H, C(8′′)H);
1H NMR (CD3OD) for the minor diastereomer, δ 1.32 (s, 3 H,
C(2′)CH3), 3.54 (d, J ) 10.5 Hz, 1 H, C(3′)HH′), the remaining
peaks overlapped with nearby signals and were not resolved;
the structural assignments were in agreement with the 1H-
1H COSY experiment; 13C NMR (CD3OD) for the major
diastereomer, 24.2, 30.2, 31.4, 37.2, 52.2, 62.2, 68.5, 72.4, 78.1,
83.7, 89.4, 128.9, 130.1, 130.2, 130.4, 131.9, 132.2, 134.1, 143.6,
143.9, 144.2, 144.3, 144.5, 168.7, 171.9 ppm; 13C NMR (CD3-
OD) for the minor diastereomer, 30.4, 30.9, 37.4, 50.4, 63.3,
72.5, 83.5, 89.6 ppm, the remaining peaks overlapped with
nearby signals and were not resolved; MS (+CI) 529
[M + 1]+; Mr (+CI) 529.174 10 [M + 1]+ (calcd for C25H29N4-
O7S 529.175 70).
C
17H24N3O7S 414.133 50).
5a -(4-Met h ylcou m a r in -7-ylsu lfa n yl)d ih yd r ob icyclo-
m ycin (9). Using general procedure 1, 17 (50 mg, 0.15 mmol)
and 7-mercapto-4-methylcoumarin (197 mg, 1.02 mmol) gave
24 as a crude mixture (Rf 0.58 (10% MeOH-CHCl3)). The
reaction was not purified but immediately deprotected using
general procedure 2 to give 9 as a mixture of diastereomers
(∼3:1): yield 9 mg (13%); mp 140-150 °C dec; Rf 0.21 (10%
MeOH-CHCl3); FT-IR (KBr) 3327 (br), 2936 (br), 1692, 1601,
1
1398, 1048 cm-1; UV-vis (H2O) 333 nm (ꢀ 16 900); H NMR
(CD3OD) for the major diastereomer, δ 1.34 (s, 3 H, C(2′)CH3),
1.94-2.38 (m, 3 H, C(4)HH′, C(4)HH′, C(5)H), 2.45 (s, 3 H,
C(4′′)CH3), 2.66 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′), 3.53
(d, 1 H, J ) 11.6 Hz, 1 H, C(3′)HH′), 3.65-4.10 (m, 4 H, C(3′)-
HH′, C(3)HH′, C(3)HH′, C(5a)HH′), 4.04 (s, 1 H, C(1′)H), 6.25
(s, 1 H, C(3′′)H), 7.28-7.35 (m, 1 H, C(6′′)H), 7.38 (d, J ) 1.6
5a -(P yr id in -4-ylsu lfa n yl)d ih yd r obicyclom ycin (3). Us-
ing general procedure 1, 17 (53 mg, 0.155 mmol) and 4-mer-
captopyridine (86 mg, 0.775 mmol) gave 18 as a crude mixture
(Rf 0.33 (10% MeOH-CHCl3)). The reaction was not purified
but immediately deprotected using general procedure 2 to give
3 as a mixture of diastereomers (∼4:1) after the “pH” was
adjusted to 8 with aqueous 0.1 M NaOH: yield 23 mg (37%);
mp 130-140 °C dec; Rf 0.32 (20% MeOH-CHCl3); FT-IR (KBr)
3336 (br), 2935 (br), 1689, 1407, 1038 cm-1; UV-vis (H2O) 263
1
Hz, 1 H, C(8′′)H), 7.65 (d, J ) 7.7 Hz, 1 H, C(5′′)H); H NMR
(CD3OD) for the minor diastereomer, δ 2.84 (dd, J ) 11.5, 14.3
Hz, 1 H, C(5a)HH′), 3.55 (d J ) 11.6 Hz, 1 H, C(3′)HH′), 4.06
(s, 1 H, C(1′)H), the remaining peaks overlapped with nearby
signals and were not resolved; the structural assignments were
in agreement with the 1H-1H COSY experiment; 13C NMR
(CD3OD) for the major diastereomer, 18.6, 24.2, 30.3, 31.8,
51.7, 62.2, 68.5, 72.2, 78.2, 83.7, 89.5, 114.2, 114.9, 118.5, 124.3,
126.4, 144.4, 155.2, 155.3, 162.8, 168.6, 172.0 ppm; 13C NMR
(CD3OD) for the minor diastereomer, 30.7, 31.1, 63.2, 72.4,
83.6, 89.7, 114.3, 115.1, 118.6, 126.5, 173.8 ppm, the remaining
peaks overlapped with nearby signals and were not resolved;
1
nm (ꢀ 13 900); H NMR (CD3OD) for the major diastereomer,
δ 1.33 (s, 3 H, C(2′)CH3), 1.93-2.39 (m, 3 H, C(4)HH′, C(4)-
HH′, C(5)H), 2.65 (dd, J ) 11.5, 14.3 Hz, 1 H, C(5a)HH′), 3.53
(d, 1 H, J ) 11.6 Hz, 1 H, C(3′)HH′), 3.69 (d, 1 H, J ) 11.6 Hz,
1 H, C(3′)HH′), 3.70-4.11 (m, 3 H, C(3)HH′, C(3)HH′, C(5a)-
HH′), 4.04 (s, 1 H, C(1′)H), 7.40 (d, J ) 4.9 Hz, 2 H, 2 C(3′′)H),
Mr (+CI) 495.143 17 [M
495.143 73).
+
1]+ (calcd for C22H27N2O9S
5584 J . Org. Chem., Vol. 68, No. 14, 2003

Bicyclomycin Fluorescent Probes
5a -(An th r a cen -2-ylsu lfa n yl)d ih yd r obicyclom ycin (10).
To a mixture of NaH (60% dispersion on mineral oil, 90 mg,
3.76 mmol) in anhydrous 1-methyl-2-pyrrolidinone (7 mL) was
added 1-butanethiol (338 mg, 3.76 mmol), and then the
mixture was stirred (10 min) under Ar at room temperature.38
To this mixture was added 2-chloroanthracene (200 mg, 0.94
mmol), and the mixture was heated to reflux (18 h). The
mixture was cooled (0 °C), and then H2O (5 mL) was added
and the solution “pH” adjusted to 2 with aqueous 1.0 M HCl.
The mixture was extracted with Et2O (3 × 10 mL), dried (Na2-
SO4), and concentrated in vacuo to obtain 2-mercaptoan-
thracene as a crude yellow solid (Rf 0.51 (10% EtOAc-
hexanes)). The crude product was not purified but used directly
in general procedure 1 with 17 (22 mg, 0.06 mmol) to provide
25 as a crude mixture (Rf 0.47 (10% MeOH-CHCl3)). The
product was then deprotected using general procedure 2
(solvent: 25% methanolic H2O) to give 10 as a mixture of
diastereomers (∼3:1): yield 8 mg (24%); mp 145-150 °C; Rf
0.65 (20% MeOH-CHCl3); FT-IR (KBr) 3259 (br), 2928 (br),
1686, 1403, 1044 cm-1; UV-vis (H2O) 257 (ꢀ 17 600), 362 (ꢀ
1520), 378 (ꢀ 1830), 403 (ꢀ 2230); 1H NMR (CD3OD) for the
major diastereomer, δ 1.34 (s, 3 H, C(2′)CH3), 1.95-2.49 (m, 3
H, C(4)HH′, C(4)HH′, C(5)H), 2.71 (dd, J ) 11.5, 14.3 Hz, 1
H, C(5a)HH′), 3.53 (d, J ) 11.6 Hz, 1 H, C(3′)HH′), 3.63-4.13
(m, 4 H, C(3′)HH′, C(3)HH′, C(3)HH′, C(5a)HH′), 4.04 (s, 1 H,
C(1′)H), 7.36 (dd, J ) 2.2, 9.3 Hz, 1 H, C(3′′)H), 7.42-7.49 (m,
2 H, C(6′′)H, C(7′′)H), 7.89-8.05 (m, 4 H, C(1′′)H, C(4′′)H,
C(5′′)H, C(8′′)H), 8.32 (s, 1 H, C(9′′)H or C(10′′)H), 8.38 (s, 1
H, C(10′′)H or C(9′′)H); 1H NMR (CD3OD) for the minor
diastereomer, δ 1.28 (s, 3 H, C(2′)CH3), 2.90 (dd, J ) 11.5 Hz,
14.3 Hz, 1 H, C(5a)HH′), 3.61 (d, J ) 11.6 Hz, 1 H, C(3′)HH′),
4.06 (s, 1 H, C(1′)H), the remaining peaks overlapped with
nearby signals and were not resolved; the structural assign-
and 41 (125 mg, 0.42 mmol) gave 31 as a crude mixture (Rf
0.60 (10% MeOH-CHCl3)). The reaction was not purified but
immediately deprotected using general procedure 2 to give 16
as a mixture of diastereomers (∼3:1): yield 15 mg (20%); mp
143-155 °C; Rf 0.57 (20% MeOH-CHCl3); FT-IR (KBr) 3320
(br), 2934 (br), 1692, 1398, 1044 cm-1; UV-vis (H2O) 261 (ꢀ
29 900), 377 (ꢀ 4300); 1H NMR (CD3OD) for the major diaster-
eomer, δ 1.31 (s, 3 H, C(2′)CH3), 1.98-2.41 (m, 4 H, C(4)HH′,
C(4)HH′, C(5)H, C(5a)HH′), 3.10 (m, 1 H, C(5a)HH′), 3.48 (d,
J ) 10.5 Hz, 1 H, C(3′)HH′), 3.63 (d, J ) 10.5 Hz, 1 H, C(3′)-
HH′), 3.67-4.03 (m, 4 H, C(3)HH′, C(3)HH′, SCH2Ar), 4.01 (s,
1 H, C(1′)H), 7.79-7.86 (m, 3 H, C(3′′)H, C(6′′)H, C(7′′)H),
8.14-8.26 (m, 4 H, C(1′′)H, C(4′′)H, C(5′′)H, C(8′′)H); 1H NMR
(CD3OD) for the minor diastereomer, δ 1.34 (s, 3 H, C(2′)CH3),
3.54 (d, J ) 10.5 Hz, 1 H, C(3′)HH′), the remaining peaks
overlapped with nearby signals and were not resolved; the
structural assignments were in agreement with the 1H-1H
COSY experiment; 13C NMR (CD3OD) for the major diastere-
omer, 24.2, 30.2, 31.6, 36.8, 52.2, 62.2, 68.5, 72.4, 78.1, 83.7,
89.4, 128.0, 128.1, 128.5, 133.6, 134.8, 134.9, 135.4, 135.5,
136.0, 147.6, 168.7, 172.0, 184.0, 184.1 ppm, the intensity of
the signals at 128.5 and 134.8 ppm were approximately twice
that of nearby peaks; 13C NMR (CD3OD) for the minor
diastereomer, 30.8, 63.3, 72.5, 83.5 ppm, the remaining peaks
overlapped with nearby signals and were not resolved; MS
(-CI) 556 [M]-; Mr (+CI) 557.159 02 [M + 1]+ (calcd for
C
27H29N2O9S 557.159 38).
2-Acetylsu lfa n ylm eth yln a p h th a len e (37). Using general
procedure 3, 32 (500 mg, 2.26 mmol) and potassium thioacetate
(309 mg, 2.71 mmol) gave 37: yield 375 mg (77%); mp 29-30
°C; Rf 0.55 (10% EtOAc-hexanes); FT-IR (KBr) 3374, 2948,
1687, 1129, 1024 cm-1; 1H NMR (CDCl3) δ 2.24 (s, 3 H, C(O)-
CH3), 4.19 (s, 2 H, SCH2Ar), 7.29 (d, J ) 8.2 Hz, 1 H, C(3)H),
7.32-7.40 (m, 2 H, C(6)H, C(7)H), 7.61-7.72 (m, 4 H, C(1)H,
C(4)H, C(5)H, C(8)H); the structural assignments were in
agreement with the 1H-1H COSY experiment; 13C NMR
(CDCl3) 30.0, 33.4, 125.7, 126.0, 126.6, 127.2, 127.4, 127.5,
128.2, 132.3, 133.1, 134.8, 194.5 ppm; MS (+CI) 217 [M + 1]+;
Mr (+CI) 217.068 10 [M + 1]+ (calcd for C13H13OS 217.068 71).
Anal. Calcd for C13H12OS: C, 72.19; H, 5.59; S, 14.82. Found:
C, 71.90; H, 5.64; S, 14.71.
1
ments were in agreement with the H-1H COSY experiment;
13C NMR (CD3OD) for the major diastereomer, 24.3, 30.2, 32.6,
51.8, 62.1, 68.6, 72.3, 78.2, 83.9, 89.4, 126.0, 126.3, 126.4, 126.8,
127.2, 127.6, 129.1, 129.3, 129.8, 131.5, 133.1, 133.4, 133.7,
134.6, 168.8, 172.2 ppm; 13C NMR (CD3OD) for the minor
diastereomer, 30.7, 31.7, 63.3, 72.4, 83.7, 126.1, 126.7, 127.5,
129.9 ppm, the remaining peaks overlapped with nearby
signals and were not resolved; MS (+CI) 512 [M]+; Mr (+CI)
512.162 68 [M]+ (calcd for C26H28N2O7S 512.161 72).
6-Acetylsu lfa n ylm eth ylqu in olin e (38). To a CCl4 solution
of 6-methylquinoline (2.00 g, 14.0 mmol) was added NBS
(freshly recrystallized from H2O, 2.50 g, 14.00 mmol) and a
catalytic amount of benzoyl peroxide (≈50 mg).40a The mixture
was heated to reflux (2 h) until coagulants started to form
(TLC analysis indicated that some starting material re-
mained). The mixture was cooled (rt), filtered, and concen-
trated in vacuo to give 3340b as a crude mixture. The crude
product (Rf 0.43 (50% EtOAc-hexanes)) was not purified but
used immediately in general procedure 3 with potassium
thioacetate (1.92 g, 16.8 mmol) to provide 38: yield 2.20 g
(72%); Rf 0.47 (50% EtOAc-hexanes); FT-IR (neat) 1691, 1588,
1500, 1358, 1126 cm-1; 1H NMR (CDCl3) δ 2.36 (s, 3 H, C(O)-
CH3), 4.28 (s, 2 H, SCH2Ar), 7.36 (dd, J ) 3.8, 8.2 Hz, 1 H,
C(3)H), 7.61 (dd, J ) 2.2, 8.8 Hz, 1 H, C(7)H), 7.72 (s, 1 H,
C(5)H), 8.02-8.10 (m, 2 H, C(4)H, C(8)H), 8.87 (dd, J ) 1.6,
3.8 Hz, 1 H, C(2)H); the structural assignments were in
agreement with the 1H-1H COSY experiment; 13C NMR
(CDCl3) 30.2, 33.2, 121.2, 127.1, 127.9, 129.8, 130.3, 135.6,
135.9, 147.4, 150.2, 194.6 ppm; MS (+CI) 218 [M + 1]+; Mr
(+CI) 218.064 08 [M + 1]+ (calcd for C12H12NOS 218.063 96).
Anal. Calcd for C12H11NOS: C, 66.33; H, 5.10; N, 6.45; S, 14.76.
Found: C, 66.53; H, 5.32; N, 6.21; S, 14.82.
5a -(An th r a cen -9-ylm eth ylsu lfa n yl)d ih yd r obicyclom y-
cin (14). Using general procedure 1, 17 (40 mg, 0.12 mmol)
and 39 (156 mg, 0.59 mmol) gave 29 as a crude mixture (Rf
0.39 (5% MeOH-CHCl3)). The reaction was not purified but
immediately deprotected using general procedure 2 to give 14
as a mixture of diastereomers (∼3:1): yield 17 mg (28%); mp
115-120 °C; Rf 0.51 (20% MeOH-CHCl3); FT-IR (KBr) 3312
(br), 2927 (br), 1686, 1400, 1029 cm-1; 1H NMR (4:1 CD3OD-
DMSO-d6) for the major diastereomer, δ 1.30 (s, 3 H, C(2′)-
CH3), 2.02-2.15 (m, 2 H, C(4)HH′, C(4)HH′), 2.33-2.54 (m, 2
H, C(5)H, C(5a)HH′), 3.48 (d, J ) 11.6 Hz, 1 H, C(3′)HH′),
3.58-3.95 (m, 4 H, C(3)HH′, C(3)HH′, C(3′)HH′, C(5a)HH′),
3.99 (s, 1 H, C(1′)H), 4.75 (d, 1 H, J ) 11.3 Hz, 1 H, CHH′Ar),
4.81 (d, 1 H, J ) 11.3 Hz, 1 H, CHH′Ar), 7.47-7.62 (m, 4 H,
C(2′′)H, C(3′′)H, C(6′′)H, C(7′′)H), 8.06 (d, J ) 8.8 Hz, 2 H,
C(1′′)H, C(8′′)H or C(4′′)H, C(5′′)H), 8.36 (d, J ) 8.8 Hz, 2 H,
C(4′′)H, C(5′′)H or C(1′′)H, C(8′′)H), 8.48 (s, 1 H, C(10′′)H); 1H
NMR (4:1 CD3OD-DMSO-d6) for the minor diastereomer, δ
4.01 (s, 1 H, C(1′)H), the remaining peaks overlapped with
nearby signals and were not resolved; the structural assign-
1
ments were in agreement with the H-1H COSY experiment;
13C NMR (DMSO-d6) for the major diastereomer, 23.7, 28.9,
32.3, 35.6, 51.2, 57.9, 66.6, 70.5, 76.8, 81.9, 87.4, 124.2 (2 C),
125.1 (2 C), 126.0 (2 C), 126.7 (2 C), 128.8 (2 C), 129.1, 129.4,
130.9 (2 C), 168.9, 171.2 ppm; 13C NMR (DMSO-d6) for the
minor diastereomer, 28.7, 81.8, 87.6 ppm, the remaining peaks
overlapped with nearby signals and were not resolved; Mr
(+CI) 527.185 26 [M + 1]+ (calcd for C27H31N2O7S 527.185 20).
9-Acetylsu lfa n ylm eth yla n th r a cen e (39). Using general
procedure 3, 34 (500 mg, 2.20 mmol) and potassium thioacetate
(301 mg, 2.64 mmol) gave 39: yield 525 mg (90%); mp 100-
102 °C; Rf 0.48 (10% EtOAc-hexanes); FT-IR (KBr) 1682,
1433, 1344, 1125 cm-1; 1H NMR (CDCl3) δ 2.31 (s, 3 H, C(O)-
CH3), 5.09 (s, 2 H, SCH2Ar), 7.37-7.42 (m, 2 H, C(2)H, C(7)H
or C(3)H, C(6)H), 7.46-7.51 (m, 2 H, C(3)H, C(6)H or C(2)H,
C(7)H), 7.89 (d, J ) 8.8 Hz, 2 H, C(1)H, C(8)H or C(4)H, C(5)H),
5a -(An th r a qu in on -2-ylm eth ylsu lfa n yl)d ih yd r obicyclo-
m ycin (16). Using general procedure 1, 17 (40 mg, 0.12 mmol)
J . Org. Chem, Vol. 68, No. 14, 2003 5585

Brogan et al.
8.14 (d, J ) 8.8 Hz, 2 H, C(4)H, C(5)H or C(1)H, C(8)H), 8.27
(s, 1 H, C(10)H); the structural assignments were in agreement
with the 1H-1H COSY experiment; 13C NMR (CDCl3) 26.3,
30.1, 123.8 (2 C), 125.0 (2 C), 126.3 (2 C), 127.4, 127.7 (2 C),
129.1 (2 C), 129.9 (2 C), 131.3, 195.5 ppm; MS (+CI) 267 [M +
1]+; Mr (+CI) 266.077 14 [M]+ (calcd for C17H14OS 266.076 54).
Anal. Calcd for C17H14OS ‚0.05 (CH3)2SO: C, 76.01; H, 5.33; S,
12.46. Found: C, 75.76; H, 5.41; S, 12.42.
2-Acet ylsu lfa n ylm et h ylp h en a zin e (40). Using general
procedure 3, 3540a (570 mg, 2.09 mmol) and potassium thio-
acetate (286 mg, 2.51 mmol) gave 40: yield 302 mg (54%); mp
113-115 °C; Rf 0.23 (20% EtOAc-hexanes); FT-IR (KBr) 1680,
1508, 1355, 1116 cm-1; 1H NMR (CDCl3) δ 2.39 (s, 3 H, C(O)-
CH3), 4.33 (s, 2 H, SCH2Ar), 7.66 (d, J ) 9.3 Hz, 1 H, C(3)H),
7.71-7.79 (m, 2 H, C(6)H, C(7)H), 8.01-8.20 (m, 4 H, C(1)H,
C(4)H, C(5)H, C(8)H); the structural assignments were in
agreement with the 1H-1H COSY experiment; 13C NMR
(CDCl3) 30.1, 33.3, 128.2, 129.3, 129.4, 129.7, 130.0, 130.1,
131.4, 140.0, 142.4, 142.9, 143.1, 143.3, 193.9 ppm; MS (+CI)
269 [M + 1]+; Mr (+CI) 269.074 89 [M + 1]+ (calcd for C15H13N2-
OS 269.074 86). Anal. Calcd for C15H12N2OS‚0.2 CH3OH: C,
66.45; H, 4.66; N, 10.20; S, 11.67. Found: C, 66.32; H, 4.65;
N, 9.84; S, 11.72.
2-Acetylsu lfa n ylm eth yla n th r a qu in on e (41). Using gen-
eral procedure 3, 36 (325 mg, 1.08 mmol) and potassium
thioacetate (148 mg, 1.30 mmol) gave 41: yield 302 mg (94%);
mp 139-141 °C; Rf 0.79 (50% EtOAc-hexanes); FT-IR (KBr)
1678, 1588, 1297, 1130 cm-1; 1H NMR (CDCl3) δ 2.39 (s, 3 H,
C(O)CH3), 4.24 (s, 2 H, SCH2Ar), 7.72 (dd, J ) 1.6, 8.2 Hz, 1
H, C(3)H), 7.75-7.82 (m, 2 H, C(6)H, C(7)H), 8.18 (d, J ) 1.6
Hz, 1 H, C(1)H), 8.22 (d, J ) 8.2 Hz, 1 H, C(4)H), 8.24-8.31
(m, 2 H, C(5)H, C(8)H); the structural assignments were in
agreement with the 1H-1H COSY experiment; 13C NMR
(CDCl3) 30.2, 33.1, 127.1, 127.2, 127.3, 127.7, 132.4, 133.4,
134.0, 134.1, 134.4, 144.7, 182.6, 182.8, 194.2 ppm, the
intensity of the signals at 133.4 and 134.0 ppm were ap-
proximately twice that of nearby peaks; MS (+CI) 297 [M +
1]+; Mr (+CI) 297.058 12 [M + 1]+ (calcd for C17H13O3S
297.058 54). Anal. Calcd for C17H12O3S: C, 68.90; H, 4.08; S,
10.82. Found: C, 68.86; H, 4.35; S, 10.52.
(cm3) versus log(1000 × C), where C is the concentration of
the test compound (mg/mL), yielded linear slopes to provide
the minimal inhibitory concentrations (MIC) for bicyclomycin
and bicyclomycin derivatives. Each assay was performed in
duplicate, and the results were averaged.
Gen er a l F lu or escen ce P r oced u r e. Bin d in g Mea su r e-
m en ts Usin g F lu or escen ce Reson a n ce En er gy Tr a n sfer
(F RET). The intrinsic tryptophan fluorescence of wild-type
rho (1 µM based on monomer) was measured in fluorescence
buffer (40 mM Tris-HCl, pH 7.9, 50 mM KCl, 2 mM MgCl2,
0.1 mM EDTA, 0.1 mM DTT) at 25 °C with spectral bandwiths
of 4 nm (excitation) and 16 nm (emission). Rho was equili-
brated in fluorescence buffer (60 min) before fluorescence
measurements. Native rho showed an excitation maximum of
282 nm and an emission maximum of 345 nm. The excitation
wavelength used was 290 nm to minimize inner filter effects
from nucleotides and selectively excite W381. FRET due to
binding of 15 and ATP was measured as a decrease in
fluorescence intensity at 345 nm. To measure percent energy
transfer at 345 nm at each concentration of 15 or ATP the
following equation was used
F345
% transfer345 ) 1 -
100
(1)
(
)
Fmax345
where Fmax345 is the corrected fluorescence emission at 345 nm
in the absence of 15 or ATP, F345 is the corrected fluorescence
emission at each concentration of 15 or ATP. The maximum
% transfer345 was used to indicate complete (observed) ligand
binding binding for Scatchard analysis based on the observa-
tion of only tight ATP binding in our system.
Bin d in g of 5a -(P h en a zin -2-ylm eth ylsu lfa n yl)d ih yd r o-
bicyclom ycin (15) to Wild -Typ e Rh o Usin g F lu or escen ce
Reson a n ce En er gy Tr a n sfer (F RET) a t a Con sta n t ATP
Con cen tr a tion . The general fluorescence procedure was used,
and each sample contained fluorescence buffer, rho (1 µM
based on monomer), ATP (250 µM), and 15 (0-40 µM). Inner
filter effects were corrected for by subtracting the fluorescence
intensity decrease at each concentration of 15 with rho (1 µM
based on monomer) in the absence of ATP.48
In h ibitor y P r op er ties of Bicyclom ycin a n d Bicyclo-
m ycin Der iva t ives in t h e P oly(C)-Dep en d en t ATP a se
Assa y.42 The ability of His-tagged wild-type rho9 to hydrolyze
[γ-32P] ATP was assayed in 100 µL reactions containing
ATPase buffer (40 mM Tris-HCl, pH 7.9, 50 mM KCl, 12 mM
MgCl2), 250 µM ATP, 0.5 µCi of [γ-32P] ATP, 300 nM poly(C),
and 200 nM rho based on monomer. Reactions were preincu-
bated at 32 °C for 90 s prior to the addition of ATP. Aliquots
(1 µL) were removed at various times (15, 30, 45, 60, 75 s)
during the reaction and spotted onto PEI-TLC plates. [γ-32P]
ATP and 32Pi were separated by chromatography on PEI-TLC
plates using 0.75 M KH2PO4, pH 3.5, as the mobile phase. The
developed PEI-TLC plates were used to expose Phosphor-
Imager Plates (Fuji and Molecular Dynamics) (3 h), scanned
on a Storm 860 PC PhosphorImager and analyzed using
Molecular Dynamics’s ImageQuant 5.0. The initial rates of
reactions were determined by plotting the amount of ATP
hydrolyzed versus time. Relative percent activities were
calculated from the initial velocities. Each assay was per-
formed in duplicate, and the results were averaged.
Bin d in g of ATP to Wild -Typ e Rh o Usin g F lu or escen ce
R eson a n ce E n er gy Tr a n sfer (F R E T) a t a Con st a n t 15
Con cen tr a tion . The general fluorescence procedure was used,
and each sample contained fluorescence buffer, rho (1 µM
based on monomer), 15 (60 µM), ATP (0-240 µM). No correc-
tion for inner filter effects was performed since 15 was held
constant throughout the experiment and adenosine nucleotides
had little affect on the inner filter effects at the excitation and
emission wavelengths used.
Dista n ce Mea su r em en t Usin g F lu or escen ce Reso-
n a n ce En er gy Tr a n sfer (F RET). The distance between a
donor/acceptor pair can be measured by Fo¨rster energy
transfer.34,35 The fluorescence intensity of the donor (W381 in
rho) was measured in the presence (FDa) and in the absence
(FD) of acceptor (15), and the resonance energy transfer
efficiency (E) was determined from the equation
FDa
E ) 1 -
(2)
FD
In h ibitor y P r op er ties of Bicyclom ycin a n d Bicyclo-
m ycin Der iva tives in th e An tim icr obia l Assa y.43 Aliquots
(200 µL) from suspensions of overnight LB broth cultures (E.
coli W3350) were diluted into LB broth (2 mL). The suspension
was poured onto 15 mL volume LB agar plates. The solution
was gently rocked to distribute the cells evenly over the plates
surface, and any excess cell solution was removed by pipet.
The plates were incubated at 37 °C (30 min), and an antibiotic-
assay disk (0.25 in. diameter) containing 20 µL to the test
compound (1, 2, 4, 8, 16, 32 mg/mL in 50-100% DMSO) was
placed on the agar surface. The plates were incubated at 37
°C (18 h). Data plots of the zone of inhibited bacterial growth
The distance (R) between the donor (W381 in rho) and the
acceptor (15) was determined by
R ) Ro(E-1 - 1)1/6 in Å
(3)
where Ro is the Fo¨rster distance at which the transfer
efficiency is equal to 50%.50 The distance Ro was calculated
by the equation51
Ro ) 0.211[κ2n-4ΦJ (λ)]1/6 in Å
(4)
5586 J . Org. Chem., Vol. 68, No. 14, 2003

Bicyclomycin Fluorescent Probes
where n is the refractive index of the buffer, assumed to be
1.4 for biomolecules in aqueous solution;35 Φ is the quantum
yield of the donor (W381 in rho); κ2 is the orientation factor, a
value of 0.67 was used assuming that donor and acceptor
groups rotate freely in a short time relative to the excited-
state lifetime of the donor;52 and J (λ) is the spectral overlap
integral in M-1 cm-1 nm4 given by the following equation
to determine Φ of the donor (W381 in rho, Φ ) 0.08) based on
a tryptophan standard35 to correct for inner filter effects.48 The
fluorescence intensity (λexc ) 290 nm) of a solution containing
fluorescence buffer, rho (1 µM based on monomer), 15 (40 µM)
and ATP (50 µM) was used for FDa. ATP was varied in FD and
FDa at a constant 15 (40 µM) concentration to correct for inner
filter effects.48
∞ FD (λ)ꢀA (λ)λ4 dλ
Ack n ow led gm en t. We thank Dr. M. Kawamura
and the Fujisawa Pharmaceutical Co., Ltd., J apan, for
the gift of 1, Dr. T. Platt (University of Rochester) for
the overproducing strain of rho, and Dr. A. Tripathy of
UNC’s Macromolecular Interactions Facility for help in
the FRET experiments. This investigation was sup-
ported by NIH Grant No. GM37934 and the Robert A.
Welch Foundation Grant No. E1381 (W.R.W.).
∫
0
J (λ) )
(5)
∞ FD (λ) dλ
∫
0
where FD(λ) is the corrected fluorescence (FD) of the donor (rho
W381) in the absence of acceptor (15) at λ, ꢀA(λ) is the
extinction coefficient of the acceptor (15) at λ in M-1 cm-1, and
λ is in nanometers. J (λ) was numerically integrated at 1-nm
intervals.
Su p p or tin g In for m a tion Ava ila ble: General methods
along with 1H and 13C NMR spectra for all new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
The fluorescence intensity (λexc ) 290 nm) of a solution
containing fluorescence buffer, rho (1 µM based on monomer)
and 15 (40 µM) was used for FD. This solution was also used
(52) Eftlink, M. E. Methods Biochem. Anal. 1991, 35, 127-205.
J O030020U
J . Org. Chem, Vol. 68, No. 14, 2003 5587