Synthesis of the second generation photoaffinity probes of tautomycin

Article abstract of DOI:10.1016/j.tet.2007.01.047

Five photoaffinity probes of tautomycin, which possess an aromatic azide with linker attached to the 2-position of tautomycin, were prepared in order to study the binding site of tautomycin with protein phosphatase 1γ. The photoaffinity probes were synthe

Full text of DOI:10.1016/j.tet.2007.01.047

Tetrahedron 63 (2007) 2593–2603
Synthesis of the second generation photoaffinity
probes of tautomycin
*
Magne O. Sydnes and Minoru Isobe
Laboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University,
Chikusa, Nagoya 464-8601, Japan
Received 20 January 2007; accepted 23 January 2007
Available online 25 January 2007
Abstract—Five photoaffinity probes of tautomycin, which possess an aromatic azide with linker attached to the 2-position of tautomycin,
were prepared in order to study the binding site of tautomycin with protein phosphatase 1g. The photoaffinity probes were synthesized by
selectively introducing the photolabeling units onto the 2-position of tautomycin by using oxime chemistry.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
The X-ray crystallographic structure of the complex of PP1
and the three later natural products, namely microcystin-
LR,5 okadaic acid,6 and calyculin A,7 have provided detailed
information of the interaction between the protein and the
toxins. However, no such X-ray crystallography data has
yet been reported for the PP1–TTM complex. An artifact
probably caused by the non-crystalline nature of TTM.
Tautomycin (TTM, 1, Scheme 1) was first isolated in 19871
and structurally elucidated in 1990 by Isono et al.2 TTM
was found to be a specific inhibitor of protein phosphatase
(PP) 1 and 2A, which are two of in total four major enzymes
that dephosphorylate serine/threonine residues of proteins in
eukaryotic cells.3 Three other natural products, namely
microcystin-LR (4), okadaic acid (5), and calyculin A (6)
(see Fig. 1), have also been found to inhibit PP1 and
PP2A.4 These three compounds are stronger inhibitors of
PP2A than PP1, while TTM was found to inhibit PP1 more
selectively than PP2A.4
Our group has for some time been involved in work toward
the elucidation of the binding site of PP1–TTM. We recently
reported the synthesis of five photoaffinity probes of tauto-
mycin, which possessed a benzophenone or a diazirine pho-
tophore attached to the 2-position of the natural product.8
OH
O
O
O
OH
O
OH
O
OH
H
1
O
O
14
3
10
6
> pH 7
< pH 3
3'
1'
3'
1'
24
22
20
18
O
HO
HO
O
O
O
H
O
OMe
5'
5'
O
Tautomycin (TTM, 1a)
Tautomycin diacid (TTMDA, 1b)
Not active
Inhibitor
> pH 9
OH
O
O
O
OH
H
1
O
3'
1'
14
OH
3
+
10
6
O
24
22
20
18
HO
O
H
O
5'
OMe
O
2
3
Scheme 1. Tautomycin (TTM) and tautomycin diacid (TTMDA).
Keywords: Tautomycin; Photoaffinity probe; Photolabeling; Protein phosphatase.
* Corresponding author. Tel.: +81 52 789 4109; e-mail: isobem@agr.nagoya-u.ac.jp
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.01.047

2594
M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
O
HO
COOH
O
O
O
O
O
N
O
HN
HO
O
O
OH
O
O
O
OH
OH
OMe
NH
Okadaic acid (5)
H
N
H
N
O
OH
O
PO(OH)2
N
N
OMe
OH N(Me)2
O
O
COOH
O
H
O
CN
O
HN
HN
Microcystin-LR (4)
O
OH OH OMe
NH2
Calyculin A (6)
Figure 1. The structure of microcystin-LR (4), okadaic acid (5), and calyculin A (6).
A synthesis of 13C-labeled tautomycin has also been
achieved and the interaction between PP1 and the natural
product has been studied by 13C NMR.9 However, these
studies have not, as of yet, enabled us to elucidate the bind-
ing site for TTM with PP1, mainly due to low chemical yield
for the photochemical reaction.
pH 8).15a However, under alkaline conditions (MeOH,
20% Cs2CO3, pH 9) TTM (1) is unstable and undergoes
trans-esterification followed by elimination resulting in the
formation of acid 2 and alcohol 3 (Scheme 1).2,15a Basic con-
ditions should therefore be avoided when introducing the
photolabeling unit into tautomycin. From our previous
work8a we knew that an oxime linker would work well since
the coupling with TTM (1a) can be achieved at pH 6.17
Herein, we report the synthesis of a series of new photoaffin-
ity probes of tautomycin (compounds 7–9), which possess an
aryl fluoro azide photophore10,11 attached to the 2-position
of TTM through linkers with various length and functional-
ity (Scheme 2). These compounds will be used in order to
study the binding interaction of TTM–PP1 using different
MS techniques. Photoaffinity probe 9 also incorporates a di-
sulfide bond, that can be cleaved by a number of reducing
agents including dithiothreitol (DTT),12 thus reducing the
molecular weight of the bound photoaffinity probes with
PP1 making it more suitable for MS analysis.
2. Synthesis of the photolabeling units containing the
azide photophore
Synthesis of the photolabeling units 10–12 started with ethyl
4-fluorobenzoate (13), which was converted to acid 14 over
four steps using chemistry well established within the
group.18 The specifics of this chemistry is outlined in Scheme
3 and started with nitration of ethyl 4-fluorobenzoate giving
compound 15 in good yield (90%). The nitro group within the
latter compound was then reduced to the amine upon treat-
ment with hydrogen over Pd/C (10%), thus affording the cor-
responding amine 16 in 93% yield. Diazotization of substrate
16 with sodium nitrite, followed by Sandmeyer reaction with
sodium azide utilizing a procedure reported by Pinney and
Katzenellenbogen19 gave the desired compound 17 in excel-
lent yield (94%). Hydrolysis of the ester functionality within
the former compound gave the desired acid 14 in 97% yield.
The first generation photoaffinity probes8 and the photo-
affinity probes described herein were designed taking into ac-
count the structure–activity relationship reported for natural
and synthetic derivatives of TTM.13–16 Briefly, the active in-
hibitor is the dicarboxylic acid form of TTM (1b, TTMDA)
and not the anhydride form (1a) (see Scheme 1).15a,16a The
hydroxyl groups at C22 and C30 are also necessary for the
bioactivity15b,16a and the hydrophobic spiroketal moiety con-
tributes significantly to the selective inhibition of PP.15b Thus,
we have introduced the photolabeling units into the 2-position
of TTM in order to retain the activity of the natural product.
Acid 14 was then converted to the corresponding activated
ester 18 by using a method analogous to the one reported pre-
viously by us.8a By such means, and after a simple purifica-
tion by flash chromatography, compound 18 could be
isolated in 84% yield. Substrate 18 was then converted to
TTM (1) exists predominantly as the diacid (TTMDA, 1b)
under mild alkaline conditions (CH3CN, 3% NaHCO3,
F
OH
3'
O
1'
O
OH
3'
O
1'
OH
22
O
OH
18
H
1
O
O
N3
14
3
10
6
> pH 7
< pH 3
24
20
O
HO
HO
O
O
O
O
H
N
OMe
O
H
N
5'
5'
O
O
O
O
N
H
O
n
7a, n = 1
8a, n = 2
7b, n = 1
8b, n = 2
O
O
F
OH
O
1'
OH
3'
O
1'
OH
22
O
OH
18
H
1
O
N3
14
3
10
6
> pH 7
< pH 3
3'
24
20
O
HO
HO
O
O
H
N
OMe
O
H
N
5'
5'
S
O
O
S
N
H
O
9a
9b
Scheme 2. Photoaffinity probes.

M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
2595
F
F
F
F
F
NO2
NH2
N3
N3
a
b
c
d
CO2Et
CO2Et
CO2Et
CO2Et
CO2H
13
15 90%
16 93%
17 94%
14 97%
e
F
F
F
N3
N3
N3
g
f
O
BocHN
O
H
N
H2N
N
O
O
N
O
N
H
O
n
n
H
O
O
18 84%
10, n = 1, 69%
11, n = 2, 68%
19, n = 1, 94%
20, n = 2, 98%
h
F
F
N3
N3
g
BocHN
O
H
N
S
H2N
S
S
N
H
O
S
N
H
O
O
12 54%
21 97%
Scheme 3. Synthesis of the photolabeling units (compounds 10–12). Reagents and conditions: (a) HNO3, H2SO4, 0 ꢀC, 45 min; (b) H2, Pd/C (10%), EtOH, rt,
23 h; (c) NaNO2, NaN3, TFA, 0 ꢀC, 10 min; (d) 2 N KOH/EtOH solution, Et2O, 0 ꢀC, 15 min; (e) N-hydroxysuccinimide, EDC$HCl, DMF, rt, 4.5 h; (f) 1,2-
diaminoethane for n¼1 and 1,3-diaminopropane for n¼2, MeOH, 0 ꢀC, 7 min; (g) N-tert-butoxycarbonyl-aminooxy acetic acid, EDC$HCl, CH2Cl2, THF, rt,
overnight; (h) 2,20-diaminoethyl disulfide dihydrochloride, Et3N, MeOH, 0 ꢀC, 40 min.
amines 19 and 20 upon treatment with 1,2-diaminoethane
and 1,3-diaminopropane, respectively (Scheme 3).8a This
gave, after purification, the two compounds 19 (94%) and
20 (98%) in excellent yields. The two amines were then cou-
F
H
pled with N-tert-butoxycarbonyl-aminooxy acetic acid using
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-
chloride (EDC$HCl) as the coupling reagent giving protected
amines 10 and 11 in 69% and 68% yield, respectively. Finally
the third photolabeling unit, namely compound 12, was pre-
pared in two steps from activated ester 18. The free amine 21
(97% yield) was first formed using a slightly modified proce-
dure compared to the one used previously for the synthesis of
compounds 19 and 20 (see Section 5 for details) followed by
coupling with N-tert-butoxycarbonyl-aminooxy acetic acid
using the same method as previously described. This gave
substrate 12 in 54% yield after a simple purification on silica.
Despite the low yield for the final coupling reaction, namely
the formation of compound 12, the synthetic strategy out-
lined here still provides the desired photolabeling unit in
a serviceable yield.
N
OH
CF3
BocHN
O
H
N
N
H
O
F
O
O
22
H
N
OH
CF3
O
BocHN
N
H
N
H
O
23
F
H
N
OH
BocHN
BocHN
CF3
O
H
N
S
S
N
O
The photo-reactivity of compounds 10–12 was tested at this
stage in order to verify their activity before attaching them to
tautomycin. All three substrates worked satisfactory and re-
sulted in C–H insertion (compounds 22–24 in Fig. 2) when
irradiated in 2,2,2-trifluoroethanol as evident from extensive
ESI-Q-TOF-MS and -MS/MS analysis.20
H
O
24
F
H
N
OH
CF3
O
H
N
SH HS
N
H
O
O
25
Next the product mixture from the photolysis experiment of
disulfide 12 was concentrated and ca. half of the product
mixture (ca. 0.3 mg) was treated with excess DTT in aceto-
nitrile at room temperature for 16 h. This experiment was
executed in order to verify if the disulfide bond within this
26
m/z = 273 (M + Na)+
m/z = 335 (M + Na)+
Figure 2. Photolysis products (22–24) and structure of the two compounds
(25 and 26) formed upon treating disulfide 24 with DTT in acetonitrile.

2596
M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
compound could easily be cleaved under such conditions.
The resulting product mixture was analyzed by ESI-Q-
TOF-MS, which revealed that no starting material remained.
Furthermore, a strong signal at m/z 273 [(M+Na)+] and
a weak signal at m/z 335 [(M+Na)+] corresponding to the
masses of the two expected products, namely compounds
25 and 26 (Fig. 2), further proved the positive outcome of
this reaction. Moreover, strong signals at m/z 217
[(MꢁC4H8+Na)+] and m/z 173 [(MꢁC5H8O2+Na)+] result-
ing from fragmentation of amine 25, which corresponds to
fragmentation patterns associated with Boc-protected
amines, serve as further proof of successful formation of
the two expected reaction products.
corresponding aminooxy compounds 27–29 formed were
used in the next step without further purification.
Tautomycin diacid (1b) for the synthesis of the photoaffinity
probes was obtained by converting TTM (1a) to TTMDA
(1b) by treating acetonitrile/water (4:1) solution of the anhy-
dride 1a with aqueous NaHCO3 according to our previously
reported method.8a A solution of aminooxy 27 in N,N-dimeth-
ylacetoamide (DMA)/H2O (1:1), which prior to addition had
been adjusted to pH 6 by adding base (0.1 N NaOH solu-
tion), was then added to a solution of TTMDA (1b) in
DMA/H2O (1:1) at room temperature. The resulting reaction
mixture was stirred in the dark for 49 h before being poured
into a ice-cooled solution of 1.0 N hydrochloric acid/aceto-
nitrile (1:1) (the solution obtained after addition to acid had
pH 3). The ice bath was then removed and the reaction mix-
ture was stirred at room temperature for 13.5 h. After a sim-
ple work-up, the crude product was purified by HPLC giving
syn-7a and anti-7b in 2% (4% yield at 58% conversion) and
26% (45% yield at 58% conversion) yield, respectively,
together with some recovered starting material (42%).
3. Synthesis of the photoaffinity probes bearing
the azide photophore
After proving that our photolabeling units (compounds 10–
12) would undergo the desired photochemical reaction focus
could now shift toward the formation of the desired photo-
affinity probes. This was implemented using the same
strategy as in our previous work8a and the details are outlined
in Scheme 4. The protection group within substrates 10, 11,
and 12 was removed upon treatment with trifluoroacetic
acid (TFA) in dichloromethane (Scheme 4). Thus, the
The syn and anti-oxime configurations were determined
from H NMR spectroscopy on the basis of Karabatsos
1
report, thus the deshielding effect (N–O group) or shielding
effect (N lone pair) was exerted by the oxime moiety.21 In the
F
N3
a
10, n = 1
11, n = 2
H2N
O
H
N
N
H
O
H
n
O
27, n = 1
28, n = 2
b
F
OH
3'
O
OH
22
O
20
OH
1
O
O
N3
14
3
10
6
1'
24
18
O
O
Ar-2H
O
H
N
OMe
O
1''
H
N
5'
O
N
H
O
syn-7a, n = 1, 2% (4% at 58% conversion)
n
O
anti-7a, n = 1, 26% (45% at 58% conversion)
anti-8a, n = 2, 27% (45% at 61% conversion)
F
N3
a
H2N
O
12
H
N
S
S
N
H
O
O
29
b
F
OH
O
1'
OH
O
20
OH
18
H
1
O
O
N3
14
3
10
6
3'
24
22
O
O
O
H
N
Ar-6H
OMe
O
1''
H
5'
N
S
O
S
N
O
3''
5''
syn-9a, 4% (7% at 63% conversion)
anti-9a, 33% (49% at 63% conversion)
H
O
Scheme 4. Synthesis of the photoaffinity probes. Reagents and conditions: (a) TFA, CH2Cl2, 0 ꢀC, 2–4.5 h; (b) DMA/H2O (1:1), rt, 48–49 h then 1 N HCl/
CH3CN (1:1), 0 ꢀC–rt, 13.5–17 h (see Section 5 for specifics).

M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
2597
syn isomer (syn-7a) the H-3 chemical shift (d 3.36 ppm) was
down-field by 0.98 ppm in comparison with the correspond-
ing H-3 chemical shift (d 2.38 ppm) for the anti isomer (anti-
7a). Furthermore, the H-1 proton is more shielded by
0.11 ppm in the syn isomer (d 1.76 ppm) than the corre-
sponding signal in the anti isomer (d 1.87 ppm).
recorded on either a Bruker ARX-400 operating at
400 MHz for proton and 101 MHz for carbon, a JEOL
JNM-A600 operating at 600 MHz for proton or a Bruker
AMX2600 operating at 151 MHz for carbon. Chemical
shifts were recorded as d values in parts per million (ppm)
using tetramethylsilane (d¼0.00 ppm) and residual chloro-
form (d¼77.0 ppm) as internal standard for proton and car-
bon NMR, respectively. The assignment of signals in various
1H NMR spectra was assisted by conducting a range of 2D
NMR experiments (COSY, NOESY, ROESY, and HO-
HAHA). Fluorine (19F) NMR spectra were recorded on
a JEOL JNM-A400 operating at 376 MHz for fluorine and
spectra were referenced externally to 1,1,1-trifluorotoluene
at 0.00 ppm. Low- and high-resolution mass spectra, EI
and FAB, were recorded on a JEOL JMN-700. ESI mass
spectra were recorded on a Q-TOF mass spectrometer (Mi-
cromass, Manchester, UK) equipped with a Z-spray type
ESI source. Optical rotations were measured with a JASCO
P-1010-GT polarimeter at the sodium D-line (589 nm) and
the concentrations (c) (g/100 mL) indicated using spectro-
scopic grade CHCl3. Elemental analyses were performed
by Mr. S. Kitamura in the Analytical Laboratory at Bioagri-
cultural Sciences, Nagoya University. Unless otherwise
noted, the reaction flask was wrapped with aluminum foil
in order to protect the reaction from light, and non-aqueous
reactions were carried out under an argon atmosphere. Reac-
tions were monitored by thin-layer chromatography (TLC)
carried out on 0.25 mm silica gel coated glass plates
60F254 using UV light as visualizing agent and 12-molybdo-
(VI)phosphoric acid n-hydrate, p-anisaldehyde or basic
KMnO4 solution followed by heating as developing agents.
Silica gel 60 (particle size 0.063–0.2 mm ASTM) was used
for flash chromatography. Tautomycin (1) was purified ac-
cording to our previously published procedure.22 The prog-
ress of the coupling reaction between tautomycin and the
photolabeling units was monitored by HPLC using a Devel-
osil C30-UG-5 column (4.6ꢂ250 mm i.d.), CH3CN/H2O 4:1
with a flow rate of 1.0 mL/min, detected at 254 nm.
The acid treatment used at the final stage of the reaction
serves two purposes: It converts the product(s) from the di-
acid form to the anhydride form, thus simplifying purifica-
tion by HPLC (the diacid form has almost the same
retention time as the solvent shock on a reversed phase col-
umn), and it also protonates the aminooxy compound, which
is present in excess in the reaction mixture. The latter is nec-
essary in order to avoid the aminooxy reacting with the an-
hydride functionality, thus forming compound(s) with two
photoprobes attached.8a The acid treatment also causes the
syn-oxime to isomerize to the corresponding anti-oxime
isomer, which is the thermodynamic most stable form of
the two.
The remaining two aminooxy compounds (28 and 29) were
converted to the corresponding photoaffinity probes by sim-
ilar means. Thus forming the desired compounds anti-8a,
syn-9a, and anti-9a in 27% (45% at 61% conversion), 4%
(7% at 63% conversion), and 33% (49% at 63% conversion)
yields, respectively. In the second experiment we could see
that also the syn product (syn-8a) was formed prior to acid
treatment, as evident from HPLC analysis. However, after
acid treatment only the anti product could be detected and
isolated. Furthermore, we did not isolate any products result-
ing from reaction with the carbonyl functionality at C20 in
any of these experiments, a result that is most likely due to
the steric congestion around this ketone.
4. Conclusion
The synthesis of five photoaffinity probes (syn-7a, anti-7a,
anti-8a, syn-9a, and anti-9a), which possess an aromatic
azide photophore attached to the 2-position of tautomycin,
has been accomplished through the selective reaction of pho-
tolabeling units (27–29) with tautomycin diacid (1b). Efforts
directed toward quantifying the PP1g inhibitory activity of
these photoaffinity probes as well as studies directed toward
elucidating the binding site with PP1 are now in progress.
Analysis of modified protein and peptides derived from the
later study will be executed by using matrix assisted laser
desorption ionization (MALDI)-TOF-MS as well as nano-
HPLC-ESI-Q-TOF-MS and -MS/MS. The outcome of these
studies will be reported in due course.
5.1.1. Ethyl 4-fluoro-3-nitrobenzoate (15). HNO3 (1.5 mL
of a 60% solution) was added dropwise over a 5 min period
to a stirred solution of ethyl 4-fluorobenzoate (13) (3.03 g,
18.02 mmol) in H2SO4 (13 mL) at 0 ꢀC. The resulting reac-
tion mixture was then stirred at 0 ꢀC for 45 min before being
diluted with EtOAc (15 mL) and poured into ice water
(30 mL). The water phase was extracted with EtOAc
(3ꢂ30 mL) and the combined organic fractions were washed
with water (2ꢂ30 mL) and brine (1ꢂ30 mL) before being
dried (Na2SO4). Filtration and concentration in vacuo gave
a yellow oil, which was subjected to flash chromatography
(silica, hexane/Et2O 3:2 elution). Concentration of the rele-
vant fractions (Rf 0.36) gave 3.45 g (90%) of the title com-
pound 1523 as a light-yellow solid, mp 43.5–44 ꢀC. IR
1
5. Experimental
nmax 1726, 1618, 1543, 1353, 1285, 1112 cmꢁ1; H NMR
(CDCl3, 400 MHz) d 8.73 (dd, J¼2.2 and 7.0 Hz, 1H, Ar-
2H), 8.32 (ddd, J¼2.2, 4.2, and 8.8 Hz, 1H, Ar-5H), 7.38
(dd, J¼8.8 and 10.0 Hz, 1H, Ar-6H), 4.44 (q, J¼7.2 Hz,
2H, CH2), 1.43 (t, J¼7.2 Hz, 3H, CH3); 13C NMR (CDCl3,
5.1. General experimental
Melting points were measured on a Yanaco MP-S3 melting
point apparatus and are uncorrected. Infrared spectra (IR)
were recorded on a JASCO FT/IR-6100 spectrophotometer
and are reported in wave number (cmꢁ1). UV spectra were
recorded on a JASCO V-570 UV/VIS/NIR spectrophoto-
meter. Proton (1H) and carbon (13C) NMR spectra were
101 MHz) d 163.6, 158.0 (d, JC–F¼272 Hz), 136.5 (d, JC–F
¼
9 Hz), 127.8, 127.6 (3), 127.6 (0), 118.7 (d, JC–F¼22 Hz),
62.1, 14.2; 19F NMR (CDCl3, 376 MHz) d ꢁ48.1; MS
ꢃ
(EI+) m/z 213 (M+ , 16%), 185 (60), 168 (100), 122 (67),
94 (50), 83 (14).

2598
M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
5.1.2. Ethyl 3-amino-4-fluorobenzoate (16). 10% Pd/C
(183 mg) was added to a solution of ethyl 4-fluoro-3-nitro-
benzoate (15) (3.42 g, 16.04 mmol) in EtOH (100 mL) at
room temperature. The reaction mixture was then stirred
vigorously under an atmosphere of H2 for 23 h before being
filtered through a plug of Celite and washed after with EtOH
(2ꢂ10 mL). Concentration of the filtrate under reduced pres-
sure gave a dark orange oil, which was subjected to flash
chromatography (silica, hexane/Et2O 2:1 elution). Concen-
tration of the relevant fractions (Rf 0.24) gave 2.73 g
(93%) of ethyl 3-amino-4-fluorobenzoate (16)24 as a yellow
oil. IR nmax 3470, 3373, 2983, 1711, 1631, 1596, 1514, 1442,
organic phases were washed with water (1ꢂ30 mL) before
being dried (Na2SO4). Filtration and concentration under
reduced pressure gave 2.37 g (97%) of the title acid 1425
as a yellow solid, mp 156–157 ꢀC (hexane/Et2O). IR nmax
2922 (br), 2132, 1704, 1437, 1290, 1084, 962, 763 cmꢁ1
;
1H NMR (CDCl3+two drops of DMSO-d6, 400 MHz)
d 8.23–7.86 (br s, 1H, COOH), 7.85–7.81 (m, 2H, Ar-2H
and Ar-5H), 7.15 (d, 1H, J¼9.2 and 10.4 Hz, Ar-6H); 13C
NMR (CDCl3+two drops of DMSO-d6, 101 MHz) d 167.1,
157.3 (d, JC–F¼257 Hz), 128.2 (d, JC–F¼11 Hz), 127.9 (d,
JC–F¼4 Hz), 127.7 (d, JC–F¼8 Hz), 122.8 (d, JC–F¼2 Hz),
116.6 (d, JC–F¼20 Hz); 19F NMR (CDCl3+two drops of
ꢃ
1310, 1254, 1203, 1102, 765 cmꢁ1
;
1H NMR (CDCl3,
DMSO-d6, 376 MHz) d ꢁ54.7; MS (EI+) m/z 181 (M+ ,
400 MHz) d 7.50 (dd, J¼2.2 and 8.8 Hz, 1H, Ar-2H), 7.42
(ddd, J¼2.2, 4.8, and 8.4 Hz, 1H, Ar-5H), 7.02 (dd, J¼8.4
and 10.8 Hz, 1H, Ar-6H), 4.35 (q, J¼7.2 Hz, 2H, CH2),
3.78 (br s, 2H, NH2), 1.38 (t, J¼7.2 Hz, 3H, CH3); 13C
25%), 153 (72), 138 (18), 124 (26), 108 (30), 97 (100), 82
ꢃ
(35), 70 (26), 57 (17); HRMS (EI+) found: M+ , 181.0317.
ꢃ
C7H4FN3O2 requires M+ , 181.0288.
NMR (CDCl3, 101 MHz) d 166.1, 154.3 (d, JC–F
¼
5.1.5. 3-Azido-4-fluorobenzoic acid 2,5-dioxopyrrolidin-
1-yl ester (18). A solution of EDC$HCl (2.74 g,
247 Hz), 134.6 (d, JC–F¼14 Hz), 127.0 (d, JC–F¼3 Hz),
120.5 (d, JC–F¼8 Hz), 118.1 (d, JC–F¼5 Hz), 115.1 (d,
JC–F¼19 Hz), 61.0, 14.3; 19F NMR (CDCl3, 376 MHz)
14.29 mmol) in DMF (200 mL) was added to a stirred solu-
tion of acid 14 (2.34 g, 12.92 mmol) and N-hydroxysuccin-
imide (1.57 g, 13.64 mmol) in DMF (50 mL) at room
temperature. The reaction mixture was then stirred at room
temperature for 4.5 h before being concentrated under re-
duced pressure. The residue thus obtained was dissolved in
EtOAc (60 mL) and washed with water (2ꢂ20 mL) and
brine (1ꢂ20 mL) before being dried (Na2SO4). Filtration
and concentration under reduced pressure gave a yellow
oil, which was subjected to flash chromatography (silica,
hexane/EtOAc 4:1/3:1/2:1/1:1 gradient elution).
Concentration of the relevant fractions (Rf 0.5 in hexane/
EtOAc 1:1) gave 3.01 g (84%) of the title compound 18 as
an off-white solid, mp 135–136 ꢀC. IR nmax 2119, 1768,
1735, 1596, 1506, 1415, 1368, 1315, 1203, 1071, 1017,
ꢃ
d ꢁ65.9; MS (EI+) m/z 183 (M+ , 90%), 155 (38), 138
(100), 110 (41), 83 (17).
5.1.3. Ethyl 3-azido-4-fluorobenzoate (17). NaNO2
(2.03 g, 29.42 mmol) followed by NaN3 (2.87 g,
44.14 mmol) was added to a stirred solution of amine 16
(2.68 g, 14.63 mmol) in trifluoroacetic acid (30 mL) main-
tained at 0 ꢀC. The reaction mixture was stirred at 0 ꢀC for
10 min before being diluted with Et2O (20 mL) and the
resulting solution was poured into ice water (30 mL).
The water phase was extracted with Et2O (2ꢂ30 mL) and
the combined organic fractions were washed with water
(2ꢂ20 mL) and brine (1ꢂ20 mL) before being dried
(Na2SO4). Concentration under reduced pressure gave
a dark orange oil, which was subjected to flash chromato-
graphy (silica, hexane/hexane/Et2O 9:1/8:2 gradient
elution). Concentration of the relevant fractions (Rf 0.5 in
hexane/Et2O 8:2) gave 2.89 g (94%) of azide 17 as a orange
oil. IR nmax 2123, 1724, 1603, 1506, 1418, 1306, 1418, 1306,
1253, 1109, 763 cmꢁ1; 1H NMR (CDCl3, 400 MHz) d 7.80
(ddd, J¼2.0, 4.8, and 8.4 Hz, 1H, Ar-5H), 7.78 (dd, J¼2.0
and 8.4 Hz, 1H, Ar-2H), 7.14 (dd, J¼8.4 and 10.4 Hz, 1H,
Ar-6H), 4.38 (q, J¼7.2 Hz, 2H, CH2), 1.40 (t, J¼7.2 Hz,
3H, CH3); 13C NMR (CDCl3, 101 MHz) d 164.8, 157.4 (d,
645 cmꢁ1 1H NMR (CDCl3, 400 MHz) d 7.90 (ddd,
;
J¼2.4, 4.4, and 8.4 Hz, 1H, Ar-5H), 7.85 (dd, J¼2.4 and
8.4 Hz, 1H, Ar-2H), 7.24 (dd, J¼8.4 and 10.4 Hz, 1H, Ar-
6H), 2.90 (s, 4H, 2ꢂCH2); 13C NMR (CDCl3, 101 MHz)
d 169.0, 160.3, 158.6 (d, JC–F¼260 Hz), 129.3 (d, JC–F
¼
11 Hz), 128.5 (d, JC–F¼9 Hz), 123.5 (d, JC–F¼3 Hz), 122.2
(d, JC–F¼4 Hz), 117.4 (d, JC–F¼20 Hz), 25.6; 19F NMR
ꢃ
(CDCl3, 376 MHz) d ꢁ52.2; MS (EI+) m/z 278 (M+ ,
22%), 250 (47), 164 (98), 136 (27), 108 (100), 81 (22), 57
ꢃ
(19); HRMS (EI+) found: M+ , 278.0443. C11H7FN4O4 re-
ꢃ
quires M+ , 278.0451. Anal. Found: C 47.59, H 2.31, N
19.85%. C11H7FN4O4 requires: C 47.49, H 2.54, N 20.14%.
JC–F¼257 Hz), 128.4 (d, JC–F¼11 Hz), 127.7 (d, JC–F
¼
4 Hz), 127.4 (d, JC–F¼8 Hz), 122.5 (d, JC–F¼2 Hz), 116.6
(d, JC–F¼20 Hz), 61.4, 14.2; 19F NMR (CDCl3, 376 MHz)
5.1.6. N-(2-Amino-ethyl)-3-azido-4-fluorobenzamide
(19). 1,2-Diaminoethane (2.30 mL, 34.37 mmol) was added
to a stirred solution of activated ester 18 (193.1 mg,
0.694 mmol) in MeOH (200 mL) maintained at 0 ꢀC. The re-
action mixture was stirred for 7 min at 0 ꢀC before being
concentrated under reduced pressure and the residue thus ob-
tained was purified by flash chromatography [silica, CH2Cl2/
MeOH/ammonia solution (28% aq solution) 95:4.25:0.75
elution]. Evaporation of the relevant fractions (Rf 0.07)
gave 146.2 mg (94%) of the title compound 19 as a yellow
oil, which slowly solidified upon standing, mp 117–
118 ꢀC. IR nmax 3294, 2124, 1643, 1597, 1548, 1502,
ꢃ
d ꢁ56.6; MS (EI+) m/z 209 (M+ , 33%), 181 (90), 164
(39), 152 (71), 124 (90), 108 (100), 97 (78), 81 (28), 57
ꢃ
(24); HRMS (EI+) found: M+ , 209.0625. C9H8FN3O2 re-
ꢃ
quires M+ , 209.0601. Anal. Found: C 51.68, H 3.75, N
19.92%. C9H8FN3O2 requires: C 51.68, H 3.85, N 20.09%.
5.1.4. 3-Azido-4-fluorobenzoic acid (14). A solution of
KOH/EtOH (10 mL of a 2 N solution) was added dropwise
to a stirred solution of ester 17 (2.83 g, 13.53 mmol) in
Et2O (20 mL) maintained at 0 ꢀC. The reaction mixture
was then stirred at 0 ꢀC for 15 min before being allowed to
heat to room temperature. After stirring at room temperature
for 5 h the reaction mixture was cooled to 0 ꢀC and acidified
to pH 1 with HCl (1 N aq solution). The resulting solution
was extracted with EtOAc (3ꢂ30 mL) and the combined
1
1323, 1232, 1092, 964 cmꢁ1; H NMR (CDCl3, 400 MHz)
d 7.58 (dd, J¼2.4 and 8.8 Hz, 1H, Ar-2H), 7.53 (ddd,
J¼2.4, 4.4, and 8.8 Hz, 1H, Ar-5H), 7.26 (br s, 1H, NH),
7.10 (dd, J¼8.8 and 10.4 Hz, 1H, Ar-6H), 3.47 (q,

M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
2599
J¼5.6 Hz, 2H, CH2), 2.94 (t, J¼5.6 Hz, 2H, CH2), 1.83 (br s,
2H, NH2); 13C NMR (CDCl3, 101 MHz) d 165.8, 156.4 (d,
131.4 (d, JC–F¼4 Hz), 128.5 (d, JC–F¼11 Hz), 124.5 (d,
JC–F¼8 Hz), 120.7 (d, JC–F¼2 Hz), 116.7 (d, JC–F¼20 Hz),
83.4, 76.4, 41.8, 38.9, 28.1; 19F NMR (CDCl3, 376 MHz)
d ꢁ59.1; MS (FAB+) m/z 397 (M++H, 5%), 297 (14);
HRMS (FAB+) found: M++H, 397.1663. C16H22FN6O5 re-
quires M++H, 397.1636. Anal. Found: C 48.48, H 5.01, N
20.90%. C16H21FN6O5 requires: C 48.48, H 5.34, N 21.20%.
JC–F¼255 Hz), 131.7 (d, JC–F¼4 Hz), 128.3 (d, JC–F
¼
11 Hz), 124.3 (d, JC–F¼7 Hz), 120.4 (d, JC–F¼2 Hz), 116.5
(d, JC–F¼20 Hz), 42.3, 41.0; 19F NMR (CDCl3, 376 MHz)
ꢃ
d ꢁ59.0; MS (EI+) m/z 223 (M+ , 3%), 194 (97), 181 (45),
166 (83), 137 (98), 109 (99), 108 (100), 82 (61), 81 (57),
ꢃ
57 (47); HRMS (EI+) found: M+ , 223.0828. C9H10FN5O
ꢃ
requires M+ , 223.0869. Anal. Found: C 48.53, H 4.42, N
31.34%. C9H10FN5O requires: C 48.43, H 4.52, N 31.38%.
5.1.9. N-[3-(2-Aminooxy-acetylamino)-propyl]-N-tert-
butoxycarbonyl-3-azido-4-fluorobenzamide (11). EDC$
HCl (39.2 mg, 0.204 mmol) was added to a stirred solution
of amine 20 (31.7 mg, 0.134 mmol) and N-tert-butoxy-
carbonyl-aminooxy acetic acid (30.1 mg, 0.157 mmol) in
CH2Cl2 (2.7 mL) and THF (0.3 mL) at room temperature.
The resulting reaction mixture was stirred at room tempera-
ture overnight before being diluted with CH2Cl2 (20 mL),
washed with water (1ꢂ10 mL) and brine (1ꢂ10 mL), and
dried over Na2SO4. Filtration and concentration gave a
yellow oil, which was subjected to flash chromatography
(silica, EtOAc/hexane/Et3N 69.8:30:0.2/79.8:20:0.2
gradient elution). Concentration of the relevant fractions (Rf
0.4 in EtOAc/hexane 8:2) gave 37.4 mg (68%) of the title
compound 11 as a colorless oil. UV (MeOH): lmax (log 3)
284 (3.29); IR nmax 3334 (br), 2962, 2926, 2855, 2123,
1691, 1652, 1600, 1542, 1504, 1322, 1260, 1168,
5.1.7. N-(3-Amino-propyl)-3-azido-4-fluorobenzamide
(20). 1,3-Diaminopropane (2.40 mL, 28.83 mmol) was
added to a stirred solution of activated ester 18 (165.0 mg,
0.593 mmol) in MeOH (165 mL) maintained at 0 ꢀC. The
reaction mixture was stirred for 7 min at 0 ꢀC before being
concentrated in vacuo. The residue thus obtained was sub-
jected to flash chromatography [silica, CH2Cl2/MeOH/
ammonia solution (28% aq solution) 95:4.25:0.75 elution].
Concentration of the relevant fractions (Rf 0.08) gave
137.8 mg (98%) of the desired amine 20 as a yellow oil,
which solidified upon standing to a waxy solid, mp 37–
40 ꢀC. IR nmax 3280, 2125, 1642, 1597, 1549, 1503, 1323,
1091, 963 cmꢁ1; 1H NMR (CDCl3, 400 MHz) d 8.26 (br s,
1H, NH), 7.60 (dd, J¼2.4 and 8.4 Hz, 1H, Ar-2H), 7.53
(ddd, J¼2.4, 4.4, and 8.4 Hz, 1H, Ar-5H), 7.11 (dd, J¼8.4
and 10.4 Hz, 1H, Ar-6H), 3.57 (q, J¼6.0 Hz, 2H, CH2),
2.95 (br s, 2H, NH2), 1.74 (quintet, J¼6.0 Hz, 4H, CH2);
1
1092 cmꢁ1; H NMR (CDCl3, 400 MHz) d 8.47 (br s, 1H,
NH), 7.96 (s, 1H, NH), 7.75 (br t, J¼5.6 Hz, 1H, NH),
7.72 (dd, J¼2.2 and 8.0 Hz, 1H, Ar-2H), 7.62 (ddd, J¼2.2,
4.4, and 8.4 Hz, 1H, Ar-5H), 7.14 (dd, J¼8.4 and 10.6 Hz,
1H, Ar-6H), 4.36 (s, 2H, CH2), 3.45 (quintet, J¼6.0 Hz,
2ꢂCH2), 1.78 (quintet, J¼6.0 Hz, 2H, CH2), 1.48 [s, 9H,
C(CH3)3]; 13C NMR (CDCl3, 101 MHz) d 170.3, 165.4,
158.0, 156.4 (d, JC–F¼255 Hz), 131.7 (d, JC–F¼3 Hz), 128.4
13C NMR (CDCl3, 101 MHz) d 165.3, 156.3 (d, JC–F
¼
255 Hz), 131.9 (d, JC–F¼3 Hz), 128.3 (d, JC–F¼12 Hz),
124.3 (d, JC–F¼9 Hz), 120.4 (d, JC–F¼2 Hz), 116.6 (d,
JC–F¼20 Hz), 41.0, 40.0, 30.6; 19F NMR (CDCl3,
ꢃ
376 MHz) d ꢁ59.5; MS (EI+) m/z 237 (M+ , 38%), 220
(12), 209 (22), 194 (35), 192 (35), 167 (28), 151 (27), 138
(70), 137 (50), 108 (100), 97 (15), 82 (18), 81 (17), 57
(d, JC–F¼12 Hz), 124.3 (d, JC–F¼8 Hz), 120.7 (d, JC–F¼
2 Hz), 116.6 (d, JC–F¼19 Hz), 83.3, 76.1, 35.7, 35.5, 29.0,
28.0; 19F NMR (CDCl3, 376 MHz) d ꢁ59.4; MS (FAB+)
m/z 411 (M++H, 13%), 311 (29); HRMS (FAB+) found:
M++H, 411.1785. C17H24FN6O5 requires M++H, 411.1792.
ꢃ
(32), 56 (34); HRMS (EI+) found: M+ , 237.1038.
ꢃ
C10H12FN5O requires M+ , 237.1026. Anal. Found: C
50.73, H 5.37, N 29.59%. C10H12FN5O requires: C 50.63,
H 5.10, N 29.52%.
5.1.10. N-(2-Aminoethyl disulfide 20-ethyl)-3-azido-4-flu-
orobenzamide (21). A solution of 2,20-diaminoethyl di-
sulfide dihydrochloride (1.07 g, 4.75 mmol) in MeOH
(20 mL), which prior to addition had been treated with tri-
ethylamine (1.32 mL, 9.47 mmol), was added to a stirred so-
lution of activated ester 18 (53.1 mg, 0.191 mmol) in MeOH
(80 mL) maintained at 0 ꢀC. The reaction mixture was then
stirred at 0 ꢀC for 40 min before being concentrated under
reduced pressure. The resulting residue was subjected to
flash chromatography [silica, CH2Cl2/MeOH/ammonia
solution (28% aq solution) 95:4.25:0.75 elution]. Concentra-
tion of the appropriate fractions (Rf 0.1) gave 58.6 mg (97%)
of the title compound 21 as a viscous yellow oil. IR nmax
3292 (br), 2925, 2123, 1644, 1599, 1549, 1502, 1323,
5.1.8. N-[2-(2-Aminooxy-acetylamino)-ethyl]-N-tert-bu-
toxycarbonyl-3-azido-4-fluorobenzamide (10). EDC$HCl
(27.0 mg, 0.141 mmol) was added to a stirred solution of
amine 19 (21.3 mg, 0.0954 mmol) and N-tert-butoxycar-
bonyl-aminooxy acetic acid (20.9 mg, 0.1093 mmol) in
CH2Cl2 (2.0 mL) and THF (0.2 mL) at room temperature.
The resulting reaction mixture was stirred at room tempera-
ture overnight before being diluted with CH2Cl2 (20 mL),
washed with water (1ꢂ10 mL) and brine (1ꢂ10 mL), and
dried over Na2SO4. Filtration and concentration gave a
yellow oil, which was subjected to flash chromatography
(silica, EtOAc/Et3N 99.8:0.2 elution). Concentration of the
relevant fractions (Rf 0.4 in EtOAc) gave 25.9 mg (69%)
of the title compound 10 as a colorless oil. UV (MeOH):
lmax (log 3) 286 (3.12). IR nmax 3302 (br), 2979, 2934,
2125, 1725, 1655, 1601, 1550, 1503, 1325, 1288, 1258,
1230 cmꢁ1 1H NMR (CDCl3, 400 MHz) d 7.58 (dd,
;
J¼2.0 and 8.0 Hz, 1H, Ar-2H), 7.51 (ddd, J¼2.0, 4.4, and
8.8 Hz, 1H, Ar-5H), 7.14 (dd, J¼8.8 and 10.4 Hz, 1H,
Ar-6H), 6.91 (br s, 1H), 3.79 (apparent q, J¼6.0 Hz, 2H,
CH2), 3.04 (apparent br s, 2H, CH2), 2.92 (t, J¼6.2 Hz,
CH2), 2.80 (t, J¼6.2 Hz, CH2), 1.73 (br s, 2H, NH2); 13C
1237, 1166, 1115 cmꢁ1 1H NMR (CDCl3, 400 MHz)
;
d 8.58 (br s, 1H, NH), 7.93 (br s, 1H, NH), 7.65 (dd,
J¼2.0 and 8.0 Hz, 1H, Ar-2H), 7.64 (br s, 1H, NH), 7.57
(ddd, J¼2.0, 4.4, and 8.8 Hz, 1H, Ar-5H), 7.12 (dd, J¼8.8
and 10.4 Hz, 1H, Ar-6H), 4.35 (s, 2H, CH2), 3.59 (m, 4H,
CH2), 1.46 [s, 9H, C(CH3)3]; 13C NMR (CDCl3,
101 MHz) d 170.7, 165.9, 158.0, 156.5 (d, JC–F¼255 Hz),
NMR (CDCl3, 101 MHz) d 165.8, 156.6 (d, JC–F
¼
255 Hz), 131.5 (d, JC–F¼4 Hz), 128.6 (d, JC–F¼11 Hz),
124.3 (d, JC–F¼8 Hz), 120.5 (d, JC–F¼2 Hz), 116.8 (d,
JC–F¼19 Hz), 42.2, 40.6, 39.0, 37.6; 19F NMR (CDCl3,

2600
M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
376 MHz) d ꢁ58.6; MS (FAB+) m/z 316 (M++H, 100%);
HRMS (FAB+) found: M++H, 316.0723. C11H15FN5OS2
requires M++H, 316.0702. Anal. Found: C 42.71, H 4.34,
N 20.79%. C11H14FN5OS2$1/4C4H8O2 requires: C 42.72,
H 4.78, N 20.76%.
fractions were concentrated under reduced pressure. The
crude yellow oil was subjected to HPLC purification [Devel-
osil C30-UG-5 (4.6ꢂ250 mm i.d.), CH3CN/H2O 4:1, 1 mL/
min, 254 nm] thus affording three fractions, A–C.
Concentration of fraction A (tR 12.55 min) gave 7.1 mg
(42% recovery) of TTM (1a), which was identical, in all
respects, with an authentic sample.
5.1.11. N-[2-(Aminooxy-acetylamino)-2-aminoethyl di-
sulfide 20-ethyl]-N-tert-butoxycarbonyl-3-azido-4-fluoro-
benzamide (12). EDC$HCl (27.1 mg, 0.141 mmol) was
added to a stirred solution of amine 21 (29.3 mg,
0.093 mmol) and N-tert-butoxycarbonyl-aminooxy acetic
acid (30.5 mg, 0.160 mmol) in CH2Cl2 (2.0 mL) and THF
(0.4 mL) at room temperature. The resulting reaction
mixture was stirred at room temperature overnight before
being diluted with CH2Cl2 (20 mL), washed with water
(1ꢂ10 mL) and brine (1ꢂ10 mL), and dried (Na2SO4).
Filtration and concentration gave a yellow oil, which was
subjected to flash chromatography (silica, hexane/EtOAc/
Et3N 70:29.8:0.2/50:49.8:0.2 gradient elution). Concen-
tration of the relevant fractions (Rf 0.45 in hexane/EtOAc
1:1) gave 24.5 mg (54%) of the title compound 12 as a color-
less oil. UV (MeOH): lmax (log 3) 284 (3.30); IR nmax 3293
(br), 2978, 2927, 2122, 1724, 1652, 1600, 1548, 1502, 1322,
Concentration of fraction B (tR 14.79 min) gave 0.5 mg [2%
(4% at 58% conversion)] of compound syn-7a as a white
solid, mp 63–65 ꢀC. [a]D22 ꢁ33.9 (c 0.04, CHCl3); IR nmax
3340 (br), 2960, 2929, 2871, 2126, 1766, 1654, 1547,
1261, 1229, 1096, 1076, 757 cmꢁ1 1H NMR (CDCl3,
;
600 MHz) d 7.64 (dd, J¼2.2 and 7.9 Hz, 1H, Ar-2H), 7.59
(br s, 1H, NH), 7.52 (ddd, J¼2.2, 4.4, and 8.6 Hz, 1H, Ar-
5H), 7.14 (dd, J¼8.6 and 10.4 Hz, 1H, Ar-6H), 6.78 (appar-
ent t, J¼4.4 Hz, 1H, NH), 5.20 (apparent br d, J¼9.2 Hz, 1H,
H-30), 5.09 (t, J¼6.2 Hz, 1H, H-24), 4.63 (br s, 1H, 30-OH),
4.48 (s, 2H, H-100), 4.36 (m, 1H, H-22), 3.75–3.45 (m, 7H,
H-18, H-300, H-400, 18-OH, 22-OH), 3.44 (s, 3H, 23-OCH3),
3.36 (apparent q, J¼7.3 Hz, 1H, H-3), 3.27 (dd, J¼2.1 and
5.7 Hz, 1H, H-23), 3.23 (dd, J¼2.0 and 10.3 Hz, 1H,
H-14), 3.19 (dt, J¼2.4 and 8.3 Hz, 1H, H-6), 2.99 (dd,
J¼8.5 and 17.5 Hz, 1H, H-21b), 2.92 (dd, J¼3.4 and
16.2 Hz, 1H, H-20b), 2.78 (dd, J¼9.8 and 16.2 Hz, 1H,
H-20a), 2.69–2.65 (m, 2H, H-19, H-21a), 2.26 (d, J¼1.3 Hz,
3H, 50-CH3), 2.10 (m, 1H, H-25), 2.02–1.95 (m, 1H, H-12b),
1.87–1.23 (m, 19H), 1.76 (s, 3H, H-1), 1.10 (d, J¼7.0 Hz,
3H, 19-CH3), 1.06 (d, J¼7.0 Hz, 3H, 3-CH3), 0.99 (d,
J¼6.0 Hz, 3H, 15-CH3), 0.98 (d, J¼6.6 Hz, 3H, H-26),
0.96 (d, J¼7.0 Hz, 3H, 25-CH3), 0.88 (d, J¼7.0 Hz, 3H,
13-CH3), 0.82 (d, J¼6.3 Hz, 3H, 7-CH3); 19F NMR
(CDCl3, 376 MHz) d ꢁ58.9; HRMS (ESI+) found:
[(M+H)+], 1045.5518. C52H78FN6O15 requires [(M+H)+],
1045.5509.
1288, 1257, 1233, 1164, 1114 cmꢁ1 1H NMR (CDCl3,
;
400 MHz) d 8.55 (br s, 1H, NH), 7.92 (s, 1H, NH), 7.70
(dd, J¼2.0 and 8.0 Hz, 1H, Ar-2H), 7.63 (ddd, J¼2.0, 4.6,
and 8.4 Hz, 1H, Ar-5H), 7.56 (br t, J¼5.4 Hz, 1H, NH),
7.13 (dd, J¼8.4 and 10.4 Hz, 1H, Ar-6H), 4.33 (s, 2H,
CH2), 3.74 (q, J¼6.0 Hz, CH2), 3.67 (q, J¼6.8 Hz, CH2),
3.01 (t, J¼6.0 Hz, CH2), 2.87 (t, J¼6.8 Hz, CH2), 1.48 [s,
9H, C(CH3)3]; 13C NMR (CDCl3, 101 MHz) d 169.6,
165.9, 157.9, 156.4 (d, JC–F¼255 Hz), 131.4 (d, JC–F
¼
4 Hz), 128.3 (d, JC–F¼12 Hz), 124.9 (d, JC–F¼8 Hz), 120.6
(d, JC–F¼2 Hz), 116.6 (d, JC–F¼20 Hz), 83.3, 76.2, 39.4,
38.8, 38.3, 37.2, 28.1; 19F NMR (CDCl3, 376 MHz)
d ꢁ59.0; MS (FAB+) m/z 489 (M++H, 12%), 389 (18);
HRMS (FAB+) found: M++H, 489.1382. C18H26FN6O5S2
requires M++H, 489.1390.
Concentration of fraction C (tR 16.58 min) gave 5.9 mg
[26% (45% at 58% conversion)] of compound anti-7a as
white solid, mp 77–79 ꢀC. [a]D23 ꢁ7.7 (c 0.12, CHCl3); IR
nmax 3335 (br), 2960, 2929, 2876, 2126, 1766, 1645, 1542,
5.1.12. Synthesis of photoaffinity probes syn-7a and anti-
7a. Trifluoroacetic acid (1.2 mL) was added dropwise over
a 10 min period to a stirred solution of amine 10 (19.0 mg,
0.0479 mmol) in CH2Cl2 (1.2 mL) maintained at 0 ꢀC. The
resulting reaction mixture was stirred at 0 ꢀC for 2 h before
being concentrated under reduced pressure. In order to
remove trace amounts of TFA the residue was dissolved in
water (5.0 mL) and concentrated in vacuo thus providing
aminooxy 27 (Rf 0.34 in EtOAc). The crude product was
used directly in the coupling reaction with tautomycin 1b
without further purification. MS (ESI+) m/z 297 [(M+H)+].
1497, 1261, 1229, 1096, 757 cmꢁ1 1H NMR (CDCl3,
;
600 MHz) d 7.63 (dd, J¼2.2 and 7.9 Hz, 1H, Ar-2H),
7.54–7.51 (m, 2H, Ar-5H, NH), 7.15 (dd, J¼8.6 and
10.4 Hz, 1H, Ar-6H), 6.86 (apparent t, J¼5.5 Hz, 1H,
NH), 5.20 (br d, J¼10.1 Hz, 1H, H-30), 5.09 (t, J¼6.2 Hz,
1H, H-24), 4.73 (br s, 1H, 30-OH), 4.47 (s, 2H, H-100), 4.38
(m, 1H, H-22), 3.71 (dt, J¼2.2 and 8.6 Hz, 1H, H-18),
3.59–3.49 (m, 6H, H-300, H-400, 18-OH, 22-OH), 3.44 (s,
3H, 23-OCH3), 3.28 (d, J¼2.4 and 11.9 Hz, 1H, H-23),
3.27 (d, J¼2.0 and 6.0 Hz, 1H, H-14), 3.16 (apparent t,
J¼9.5 Hz, 1H, H-6), 2.99 (dd, J¼8.6 and 17.0 Hz, 1H, H-
21b), 2.91 (dd, J¼3.5 and 16.1 Hz, 1H, H-20b), 2.77 (dd,
J¼9.7 and 16.1 Hz, 1H, H-20a), 2.70–2.66 (m, 2H, H-21a,
H-19), 2.38 (m, 1H, H-3), 2.25 (d, J¼1.3 Hz, 3H, 50-CH3),
2.10 (m, 1H, H-25), 2.02–1.95 (m, 1H, H-12b), 1.87 (s, 3H,
H-1), 1.81 (m, 1H, H-13), 1.72–1.22 (m, 17H), 1.07 (d,
J¼7.0 Hz, 3H, 19-CH3), 1.05 (d, J¼7.0 Hz, 3H, 3-CH3),
0.99 (d, J¼6.4 Hz, 3H, 15-CH3), 0.97 (d, J¼6.8 Hz, 3H,
H-26), 0.96 (d, J¼6.8 Hz, 3H, 25-CH3), 0.86 (d, J¼7.0 Hz,
3H, 13-CH3), 0.81 (d, J¼6.4 Hz, 3H, 7-CH3); 13C NMR
(CDCl3, 101 MHz) d 215.1, 172.8, 169.4, 165.9, 164.7,
164.5, 157.4, 142.8, 142.1, 131.2, 124.3 (d, JC–F¼8 Hz),
A solution of aminooxy 27 in DMA/H2O (0.8 mL of a 1:1
solution), which carefully had been adjusted to pH 6 with
NaOH solution (0.1 N aq solution), was added to a stirred so-
lution of tautomycin 1b26 (17.1 mg, 0.0218 mmol) in DMA/
H2O (2.0 mL of a 1:1 solution) at room temperature. The re-
sulting reaction mixture was stirred in the dark at room tem-
perature for 49 h before being poured into an ice-cooled
solution of HCl/CH3CN (3.0 mL of a 1 N HCl aq/CH3CN
1:1 solution) and stirred vigorously for 10 min. The ice
bath was then removed and stirring was continued at room
temperature for 13.5 h. The resulting reaction mixture was
extracted with CHCl3 (3ꢂ15 mL) and the combined organic

M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
2601
120.6, 116.7 (d, JC–F¼20 Hz), 95.5, 80.8, 74.8, 74.1, 73.5,
72.3, 66.4, 63.8, 59.0, 52.5, 45.8, 41.4, 40.8, 39.2, 38.8,
36.0, 34.9 (4), 34.9 (0), 31.6, 30.3, 30.1, 29.4, 28.6, 28.0,
27.6, 27.2, 26.7, 19.3, 17.8, 17.6, 17.1, 16.7, 13.4, 11.3,
10.9, 10.0 (three signals obscured or overlapping); 19F
NMR (CDCl3, 376 MHz) d ꢁ58.7; HRMS (ESI+) found:
[(M+H)+], 1045.5505. C52H78FN6O15 requires [(M+H)+],
1045.5509.
50-CH3), 2.10 (sextet, J¼6.6 Hz, 1H, H-25), 2.02–1.95 (m,
1H, H-12b),1.89 (s, 3H, H-1), 1.82 (m, 1H, H-13), 1.74
(quintet, J¼6.0 Hz, 2H, H-400), 1.69–1.25 (m, 17H), 1.07
(0) (d, J¼7.0 Hz, 3H, 19-CH3), 1.06 (7) (d, J¼7.1 Hz,
3H, 3-CH3), 0.99 (d, J¼6.4 Hz, 3H, 15-CH3), 0.97 (d,
J¼6.8 Hz, 3H, H-26), 0.95 (d, J¼7.0 Hz, 3H, 25-CH3),
0.86 (d, J¼7.0 Hz, 3H, 13-CH3), 0.81 (d, J¼6.4 Hz, 3H, 7-
CH3); 13C NMR (CDCl3, 101 MHz) d 215.2, 169.4, 165.5,
164.4, 142.9, 142.1, 124.2, 120.7, 116.7 (d, JC–F¼20 Hz),
95.5, 80.6, 74.8, 74.0, 73.5, 72.2, 66.3, 63.8, 59.0, 52.3,
45.9, 40.9, 38.9, 36.0, 35.8, 35.4, 35.0, 34.8, 31.7, 30.3,
30.1, 29.5, 29.3, 28.6, 28.0, 27.5, 27.0, 26.7, 19.3, 17.9,
17.8, 17.3, 16.8, 13.5, 11.5 (nine signals obscured or over-
lapping); 19F NMR (CDCl3, 376 MHz) d ꢁ59.1; HRMS
(ESI+) found: [(M+H)+], 1059.5601. C53H80FN6O15 re-
quires [(M+H)+], 1059.5666.
5.1.13. Synthesis of photoaffinity probe anti-8a. Trifluoro-
acetic acid (1.2 mL) was added dropwise over a 10 min pe-
riod to a stirred solution of amine 11 (19.7 mg, 0.048 mmol)
in CH2Cl2 (1.2 mL) maintained at 0 ꢀC. The resulting reac-
tion mixture was stirred at 0 ꢀC for 4.5 h before being con-
centrated under reduced pressure. In order to remove trace
amounts of TFA the residue was dissolved in water
(5.0 mL) and concentrated in vacuo thus providing amino-
oxy 28 (Rf 0.29 in EtOAc/hexane 4:1). The crude product
was used directly in the coupling reaction with tautomycin
1b without further purification. MS (ESI+) m/z 311
[(M+H)+].
5.1.14. Synthesis of photoaffinity probes syn-9a and anti-
9a. Trifluoroacetic acid (1.2 mL) was added dropwise over
a 10 min period to a stirred solution of amine 12 (22.6 mg,
0.046 mmol) in CH2Cl2 (1.2 mL) maintained at 0 ꢀC. The
resulting reaction mixture was stirred at 0 ꢀC for 2 h before
being concentrated under reduced pressure. In order to
remove trace amounts of TFA the residue was dissolved in
water (5.0 mL) and concentrated in vacuo thus providing
aminooxy 29 (Rf 0.6 in EtOAc). The crude product was
used directly in the coupling reaction with tautomycin 1b
without further purification. MS (ESI+) m/z 389 [(M+H)+].
A solution of aminooxy 28 in DMA/H2O (0.8 mL of a 1:1
solution), which carefully had been adjusted to pH 6 with
NaOH solution (0.1 N aq solution), was added to a stirred so-
lution of tautomycin 1b26 (17.1 mg, 0.0218 mmol) in DMA/
H2O (2.0 mL of a 1:1 solution) at room temperature. The re-
sulting reaction mixture was stirred in the dark at room tem-
perature for 48 h before being poured into an ice-cooled
solution of HCl/CH3CN (3.0 mL of a 1 N HCl aq/CH3CN
1:1 solution). The resulting reaction mixture was stirred vig-
orously for 10 min at 0 ꢀC before the stirring was continued
at room temperature in the dark for 13.5 h. The reaction
mixture was then extracted with CHCl3 (3ꢂ15 mL) and
the combined organic phases were concentrated under re-
duced pressure. The resulting yellow oil was purified by
HPLC [Develosil C30-UG-5 (4.6ꢂ250 mm i.d.), CH3CN/
H2O 4:1, 1 mL/min, 254 nm] thus affording two fractions,
A and B.
A solution of aminooxy 29 in DMA/H2O (0.8 mL of a 1:1
solution), which carefully had been adjusted to pH 6 with
NaOH solution (0.1 N aq solution), was added to a stirred so-
lution of tautomycin 1b26 (17.1 mg, 0.0218 mmol) in DMA/
H2O (2.0 mL of a 1:1 solution) at room temperature. The re-
sulting reaction mixture was stirred in the dark at room tem-
perature for 48 h before being poured into an ice-cooled
solution of HCl/CH3CN (3.0 mL of a 1 N HCl aq/CH3CN
1:1 solution). The resulting reaction mixture was stirred vig-
orously for 10 min at 0 ꢀC before the stirring was continued
at room temperature in the dark for 17 h. The reaction mix-
ture was then extracted with CHCl3 (3ꢂ15 mL) and the com-
bined organic phases were concentrated under reduced
pressure. The resulting yellow oil was purified by HPLC
[Develosil C30-UG-5 (4.6ꢂ250 mm i.d.), CH3CN/H2O
4:1, 1 mL/min, 254 nm] thus affording three fractions, A–C.
Concentration of fraction A (tR 12.55 min) gave 6.6 mg
(39% recovery) of TTM (1a), which was identical, in all
respects, with an authentic sample.
Concentration of fraction B (tR 18.95 min) gave 6.3 mg
[27% (45% at 61% conversion)] of compound anti-8a as
a white solid, mp 94–96 ꢀC. [a]D23 ꢁ4.1 (c 0.07, CHCl3);
IR nmax 3407 (br), 2960, 2929, 2858, 2122, 1725, 1658,
Concentration of fraction A (tR 12.55 min) gave 5.5 mg
(33% recovery) of TTM (1a), which was identical, in all
respects, with an authentic sample.
1640, 1560, 1461, 1261, 1073, 801 cmꢁ1
;
1H NMR
(CDCl3, 600 MHz) d 7.68 (dd, J¼2.2 and 7.8 Hz, 1H, Ar-
2H), 7.59 (ddd, J¼2.2, 4.4, and 8.7 Hz, 1H, Ar-5H), 7.51
(apparent t, J¼5.9 Hz, 1H, NH), 7.16 (dd, J¼8.7 and
10.3 Hz, 1H, Ar-6H), 6.70 (apparent t, J¼6.2 Hz, 1H,
NH), 5.20 (br d, J¼10.3 Hz, 1H, H-30), 5.09 (t, J¼6.2 Hz,
1H, H-24), 4.52 (s, 1H, 30-OH), 4.50 (s, 2H, H-100), 4.35
(m, 1H, H-22), 3.72 (dt, J¼2.2 and 8.6 Hz, 1H, H-18),
3.60–3.40 (m, 6H, H-300, H-500, 18-OH, 22-OH), 3.44 (s,
3H, 23-OCH3), 3.27 (dd, J¼1.8 and 11.0 Hz, 1H, H-23),
3.26 (dd, J¼2.0 and 6.3 Hz, 1H, H-14), 3.17 (apparent t,
J¼10.0 Hz, 1H, H-6), 2.98 (dd, J¼8.5 and 17.4 Hz, 1H, H-
21b), 2.92 (dd, J¼3.3 and 16.2 Hz, 1H, H-20b), 2.77 (dd,
J¼9.7 and 16.2 Hz, 1H, H-20a), 2.70–2.66 (m, 2H, H-21a,
H-19), 2.41 (apparent q, J¼6.7 Hz, 1H, H-3), 2.26 (s, 3H,
Concentration of fraction B (tR 21.98 min) gave 1.1 mg [4%
(7% at 63% conversion)] of compound syn-9a as a white
solid, mp 132–134 ꢀC. [a]D24 ꢁ44.4 (c 0.01, CHCl3); IR
nmax 3349 (br), 2960, 2929, 2876, 2126, 1766, 1658, 1542,
1502, 1261, 1229, 1096, 1073, 757 cmꢁ1
;
1H NMR
(CDCl3, 600 MHz) d 7.69 (dd, J¼2.2 and 7.8 Hz, 1H, Ar-
2H), 7.61 (ddd, J¼2.2, 4.4, and 8.6 Hz, 1H, Ar-5H), 7.33
(br s, 1H, NH), 7.14 (dd, J¼8.6 and 10.4 Hz, 1H, Ar-6H),
6.68 (t, J¼4.4 Hz, 1H, NH), 5.20 (apparent br d,
J¼9.2 Hz, 1H, H-30), 5.09 (t, J¼6.1 Hz, 1H, H-24), 4.56
(br s, 1H, 30-OH), 4.47 (s, 2H, H-100), 4.35 (m, 1H, H-22),
3.77–3.44 (m, 7H, H-300, H-600, H-18, 18-OH, 22-OH),
3.44 (s, 3H, 23-OCH3), 3.37 (apparent q, J¼7.3 Hz, 1H,

2602
M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
H-3), 3.27 (dd, J¼2.1 and 5.7 Hz, 1H, H-23), 3.23 (dd,
J¼2.0 and 9.9 Hz, 1H, H-14), 3.19 (m, 1H, H-6), 3.01–
2.97 (m, 3H, H-500, H-21b), 2.92 (dd, J¼3.5 and 16.3 Hz,
1H, H-20b), 2.85 (t, J¼7.0 Hz, 2H, H-400), 2.78 (dd, J¼9.8
and 16.3 Hz, 1H, H-20a), 2.67–2.63 (m, 2H, H-19, H-21a),
2.27 (d, J¼1.3 Hz, 3H, 50-CH3), 2.10 (m, 1H, H-25), 2.02–
1.95 (m, 1H, H-12b), 1.85 (m, 1H, H-13), 1.80 (s, 3H, H-1),
1.65–1.25 (m, 17H), 1.10 (d, J¼7.1 Hz, 3H, 19-CH3), 1.07
(d, J¼7.0 Hz, 3H, 3-CH3), 0.99 (d, J¼6.6 Hz, 3H,
15-CH3), 0.98 (d, J¼6.8 Hz, 3H, H-26), 0.97 (d, J¼6.8 Hz,
3H, 25-CH3), 0.89 (d, J¼7.0 Hz, 3H, 13-CH3), 0.82 (d,
J¼6.6 Hz, 3H, 7-CH3); 19F NMR (CDCl3, 376 MHz)
d ꢁ58.8; HRMS (ESI+) found: [(M+H)+], 1137.5349.
C54H82FN6O15S2 requires [(M+H)+], 1137.5264.
and Kaken Phermaceutical Co. Ltd. for kindly providing
a sample of crude tautomycin.
References and notes
1. Cheng, X.-C.; Kihara, T.; Kusakabe, H.; Magae, J.; Kobayashi,
Y.; Fang, R.-P.; Ni, Z.-F.; Shen, Y.-C.; Ko, K.; Yamaguchi, I.;
Isono, K. J. Antibiot. 1987, 40, 907–909.
2. (a) Ubukata, M.; Cheng, X.-C.; Isono, K. J. Chem. Soc., Chem.
Commun. 1990, 244–246; (b) Cheng, X.-C.; Ubukata, M.;
Isono, K. J. Antibiot. 1990, 43, 809–819; (c) Ubukata, M.;
Cheng, X.-C.; Isobe, M.; Isono, K. J. Chem. Soc., Perkin
Trans. 1 1993, 617–624.
3. (a) Cohen, P. Annu. Rev. Biochem. 1989, 58, 453–508; (b)
Shenolikar, S.; Nairn, A. C. Adv. Second Messenger
Phosphoprotein Res. 1991, 23, 1–121.
4. Takai, A.; Sasaki, K.; Nagai, H.; Mieskes, G.; Isobe, M.; Isono,
K.; Yasumoto, T. Biochem. J. 1995, 306, 657–665.
5. Goldberg, J.; Huang, H.; Kwon, Y.; Greengard, P.; Nairn, A. C.;
Kuriyan, J. Nature 1995, 376, 745–753.
Concentration of fraction C (tR 23.48 min) gave 8.2 mg
[33% (49% at 63% conversion)] of compound anti-9a as
a white solid, mp 50–51 ꢀC. [a]D24 ꢁ0.6 (c 0.27, CHCl3);
IR nmax 3362 (br), 2965, 2929, 2876, 2126, 1766, 1654,
1542, 1502, 1261, 1229, 1096, 757 cmꢁ1
;
1H NMR
(CDCl3, 600 MHz) d 7.66 (dd, J¼2.2 and 7.9 Hz, 1H, Ar-
2H), 7.58 (ddd, J¼2.2, 4.4, and 8.6 Hz, 1H, Ar-5H), 7.15
(1) (t, J¼5.5 Hz, 1H, NH), 7.15 (0) (dd, J¼8.6 and
10.3 Hz, 1H, Ar-6H), 6.69 (t, J¼6.0 Hz, 1H, NH), 5.21 (br
d, J¼8.2 Hz, 1H, H-30), 5.09 (t, J¼6.2 Hz, 1H, H-24), 4.66
(br s, 1H, 30-OH), 4.47 (d, J¼2.6 Hz, 2H, H-100), 4.35 (m,
1H, H-22), 3.75 (q, J¼6.0 Hz, 2H, H-300), 3.72–3.60 (m,
4H, H-600, H-18, 18-OH), 3.44 (s, 3H, 23-OCH3), 3.38–
3.20 (m, 3H, H-23, H-14, 22-OH), 3.17 (apparent t,
J¼9.8 Hz, 1H, H-6), 2.99–2.95 (m, 3H, H-500, H-21b), 2.91
(dd, J¼3.3 and 16.1 Hz, 1H, H-20b), 2.84 (t, J¼6.7 Hz,
2H, H-400), 2.77 (dd, J¼9.8 and 16.1 Hz, 1H, H-20a), 2.71–
2.65 (m, 2H, H-21a, H-19), 2.41 (apparent q, J¼6.8 Hz,
1H, H-3), 2.27 (d, J¼1.3 Hz, 3H, 50-CH3), 2.10 (sextet,
J¼6.7 Hz, 1H, H-25), 2.02–1.95 (m, 1H, H-12b), 1.85 (s,
3H, H-1), 1.85–1.23 (m, 18H), 1.08 (d, J¼7.1 Hz, 3H, 19-
CH3), 1.07 (d, J¼7.0 Hz, 3H, 3-CH3), 1.00 (d, J¼6.4 Hz,
3H, 15-CH3), 0.98 (d, J¼6.8 Hz, 3H, H-26), 0.96 (d,
J¼6.8 Hz, 3H, 25-CH3), 0.88 (d, J¼7.0 Hz, 3H, 13-CH3),
0.81 (d, J¼6.6 Hz, 3H, 7-CH3); 13C NMR (CDCl3,
151 MHz) d 215.2, 171.2, 169.4, 165.8, 164.8, 164.4,
156.5 (d, JC–F¼255 Hz), 142.9, 142.1, 131.3, 128.4, 124.6
(d, JC–F¼8 Hz), 120.6, 116.7 (d, JC–F¼20 Hz), 95.5, 80.6,
74.8, 74.0, 73.6, 72.3, 66.3, 63.8, 59.0, 52.4, 45.9, 40.9,
39.2, 38.8, 38.3, 37.8, 37.0, 36.0, 35.0, 34.9, 31.7, 30.3,
30.1, 29.4, 28.6, 28.0, 27.5, 27.2, 26.7, 19.3, 18.2, 17.9,
17.8, 17.3, 16.8, 13.5, 11.3, 10.9, 10.1 (one signal was
obscured or overlapping); 19F NMR (CDCl3, 376 MHz)
d ꢁ58.7; HRMS (ESI+) found: [(M+H)+], 1137.5283.
C54H82FN6O15S2 requires [(M+H)+], 1137.5264.
6. Maynes, J. T.; Bateman, K. S.; Cherney, M. M.; Das, A. K.;
Luu, H. A.; Holmes, C. F. B.; James, M. N. G. J. Biol. Chem.
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7. Kita, A.; Matsunaga, S.; Takai, A.; Kataiwa, H.; Wakimoto,
T.; Fusetani, N.; Isobe, M.; Miki, K. Structure 2002, 10,
715–724.
8. (a) Kurono, M.; Shimomura, A.; Isobe, M. Tetrahedron 2004,
60, 1773–1780; (b) Kurono, M.; Isobe, M. Chem. Lett. 2004,
33, 452–453.
9. Isobe, M.; Kurono, M.; Tsuboi, K.; Takai, A. Chem. Asian J.,
in press.
10. Poe, R.; Schnapp, K.; Young, M. J. T.; Grayzar, J.; Platz, M. S.
J. Am. Chem. Soc. 1992, 114, 5054–5067.
11. Karney, W. L.; Borden, T. J. Am. Chem. Soc. 1997, 119, 3347–
3350.
12. Li, H.; Liu, Y.; Fang, K.; Nakanishi, K. Chem. Commun. 1999,
365–366.
13. For Isono’s structure–activity investigations, see: Nishiyama,
U.; Ubukata, M.; Magae, J.; Kataoka, T.; Erdodi, F.;
Hartshorne, D. J.; Isono, K.; Nagai, K.; Osada, H. Biosci.
Biotechnol. Biochem. 1996, 60, 103–107.
14. For Oikawa and Ichihara’s structure–activity investigations,
see: (a) Kawamura, T.; Matsuzawa, S.; Mizuno, Y.; Kikuchi,
K.; Oikawa, H.; Oikawa, M.; Ubukata, M.; Ichihara, A.
Biochem. Pharmacol. 1998, 55, 995–1003; (b) Oikawa, H.
Curr. Med. Chem. 2002, 9, 2033–2054.
15. For Isobe’s structure–activity investigations, see: (a)
Sugiyama, Y.; Ohtani, I. I.; Isobe, M.; Takai, A.; Ubukata,
M.; Isono, K. Bioorg. Med. Chem. Lett. 1996, 6, 3–8; (b)
Takai, A.; Tsuboi, K.; Koyasu, M.; Isobe, M. Biochem. J.
2000, 350, 81–88.
Acknowledgements
16. For Chamberlin’s structure–activity investigations, see: (a) Liu,
W.; Sheppeck, J. E., II; Colby, D. A.; Huang, H.-B.; Nairn,
A. C.; Chamberlin, A. R. Bioorg. Med. Chem. Lett. 2003, 13,
1597–1600; (b) Colby, D. A.; Liu, W.; Sheppeck, J. E., II;
Huang, H.-B.; Nairn, A. C.; Chamberlin, A. R. Bioorg. Med.
Chem. Lett. 2003, 13, 1601–1605.
17. (a) Jencks, W. P. Prog. Phys. Org. Chem. 1964, 2, 63–128;
(b) Shao, J.; Tam, J. P. J. Am. Chem. Soc. 1995, 117,
3893–3899.
Financial support from Grant-in-aid for Specially Promoted
Research (16002007) from the Ministry of Education,
Sports, Science and Technology (MEXT), Japan is grate-
fully acknowledged. M.O.S. is grateful for the provision of
an Inoue Fellowship from the Inoue Foundation for Science.
We thank Dr. M. Kuse and Mr. K. Koga for acquisition of
certain MS and NMR data, respectively, and Mr. I. Doi for
expert assistance with the operation of ESI-Q-TOF-MS.
We would also like to thank Dr. K. Isono (ex-Riken Institute)
18. Kuse, M.; Doi, I.; Kondo, N.; Kageyama, Y.; Isobe, M.
Tetrahedron 2005, 61, 5754–5762.

M. O. Sydnes, M. Isobe / Tetrahedron 63 (2007) 2593–2603
2603
19. Pinney, K. G.; Katzenellenbogen, J. A. J. Org. Chem. 1991, 56,
3125–3133.
23. Ethyl 4-fluoro-3-nitrobenzoate is also available from several
chemical suppliers.
20. The details of these studies are the subject of a separate publi-
cation, which is in preparation.
24. Hopff, H.; Valkanas, G. J. Org. Chem. 1962, 27, 2923–
2924.
21. (a) Karabatsos, G. J.; Taller, R. A.; Vane, F. M. J. Am. Chem.
Soc. 1963, 85, 2327–2328; (b) Karabatsos, G. J.; His, N.
Tetrahedron 1967, 23, 1079–1095; (c) Karabatsos, G. J.;
Taller, R. A. Tetrahedron 1968, 24, 3347–3360.
25. Cogan, D. A.; Hao, M.-H.; Qian, K. C.; Swinamer, A. D. U.S.
Patent 2004102492, 2004; Chem. Abstr. 2004, 141, 7121.
26. TTMDA (1b) was obtained by treating TTM (1a) with base
according to our previously reported method. For details see
Ref. 8a.
22. Kurono, M.; Isobe, M. Tetrahedron 2003, 59, 9609–9617.