Synthesis and physical nature of fluorescent photoaffinity probe for the bioorganic studies on tautomycin, a protein phosphatase type 1 selective inhibitor

Article abstract of DOI:10.1246/cl.2004.452

Fluorescent photoaffinity probe, which possesses a dansyl amide functional group on the maleic moiety of tautomycin, was prepared in order to detect the trace amount of labeled peptides. The parent compound dramatically showed fluorescence quenching befor

Full text of DOI:10.1246/cl.2004.452

452
Chemistry Letters Vol.33, No.4 (2004)
Synthesis and Physical Nature of Fluorescent Photoaffinity Probe for the Bioorganic Studies
on Tautomycin, a Protein Phosphatase Type 1 Selective Inhibitor
Masakuni Kurono and Minoru Isobey
yLaboratory of Organic Chemistry, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601
(Received January 23, 2004; CL-040091)
Fluorescent photoaffinity probe, which possesses a dansyl
amide functional group on the maleic moiety of tautomycin,
was prepared in order to detect the trace amount of labeled pep-
tides. The parent compound dramatically showed fluorescence
quenching before the photoreaction due to the exciplex forma-
tion on the basis of the folded conformation. Several benzhydrol
analogs, on the other hand, recovered the strong fluorescence;
thus, this system could be employed for the photoaffinity label-
ing studies.
amine 8, which was successively connected with the activated
ester 6 in the presence of triethylamine (Et3N) to furnish the
compound 9. The protective t-butoxycarbonyl (Boc) group of
9 was removed with TFA in CH2Cl2 to afford the fluorescent
unit 10 as a green oil.8 The coupling reaction was performed
with 11, one of the synthesized photoaffinity probes.7 The mal-
eimidation of 11 with 10 was carried out in 70% N,N0-dimethyl-
acetamide (DMA)/H2O at pH 4, which was successively puri-
fied by HPLC [ODS-UG-5; 250 ꢀ 10:0 mm i.d., 90% CH3CN/
H2O, 4.0 mL/min, Tr ¼ 3:5 (10), 11.5 (11), and 13.5 min (12)]
to furnish the pure fluorescent photoaffinity probe (12) as a light
yellow oil in 83% yield.9 The excess 10 was recovered in 82%
yield. No oxime formation was observed at the 20-ketone posi-
tion under this condition presumably due to the steric conges-
tion.
Tautomycin (TTM, 1) has been known as protein phospha-
tase type 1 (PP1) selective inhibitor,1,2 and the active inhibitor is
not the anhydride (1) but the dicarboxylic acid form (2).3 While
okadaic acid largely inhibits PP2A and weakly does PP1, and ca-
lyculin and microcystin-LR are inhibitors to both phosphatases.2
In 1995 the X-ray crystallographic analysis of PP1–microcystin-
LR complex provided the details of molecular interaction be-
tween the macro molecule and the toxin.4 Successively the X-
ray structures were reported on PP1 complexes with okadaic
acid (2001)5 and with calyculin (2002).6 PP1–TTM complex,
however, has not yet crystallized as a nature of TTM. We plan-
ned, therefore, photoaffinity experiments to study the molecular
interaction.
O
O
O
(a)
O
N
Boc
N
O
Boc
N
O
HO
H
H
O
5
6
N
N
N
N
(b)
(c)
(d)
O
S
S
O
O
O
O
O
O
S
O
S
O O
O
O
O
Boc
HN
HN
Cl
HN
N
N
N
NH2
NH2
H
H
H
9
10
8
7
OH
O
OH
O
OH
H
O
OH
O
OH
O
1'
OH
O
OH
1
O
1'
H
O
O
O
O
10
O
H
N
(e)
> pH 7
< pH 3
14
O
O
4'
5'
4'
5'
OMe
H
6
3
HO
HO
O
O
O
24 22 20 18
OMe
N
O
H
X
O
11
N
10'''
O
O
Anhydride form
Not active
Tautomycin (1)
O
Diacid form
Active
F
3C
N
OH
O
OH
O
OH
H
O
1
O
N
N
5'''
4'
S
O
O
O
O
O
24
20
12
1'
H
N
O
HN
X =
O
O
X =
OMe
X = O
N
N
O
N
H
O
O
OMe
H
1'''
5'
N
H
H
O
1''
9''
N
N
N
H
O
O
O
O
2
syn: 3a
anti: 3b
syn: 4a
anti: 4b
Scheme 2. Synthesis of fluorescent photoaffinity probe: (a) N-
hydroxysuccinimide, EDCꢁHCl, DMF, rt, 36 h, 78%; (b) ethyl-
enediamine, Et3N, CH2Cl2, rt, quant.; (c) 6, Et3N, DMF, rt,
1 h, 68%; (d) TFA/CH2Cl2 (1:1), 0 ꢂC, 1 h, 87%; (e) 10, 70%
DMA/H2O, pH 4, rt, 16 h, then purification by HPLC, 83%.
Scheme 1. Tautomycin and photoaffinity probes.
Recently, we have accomplished the synthesis of two types
of photoaffinity probes (3, 4), which possesses a benzophenone
or a diazirine photophore linking at the 2 position of TTM in or-
der to study the binding site of PP1.7 Further, we designed to in-
troduce a fluorescent unit into tautomycin moiety in order to de-
tect the peptides after the photoreaction. The principles are: (i)
photoaffinity labeling, (ii) tryptic digestion, (iii) acidic transfor-
mation into the maleic anhydride, and (iv) the fluorescent unit
introduction into the anhydride moiety. On the basis of this plan,
we describe the synthesis of a fluorescent photoaffinity probe to
detect the labeled peptides using HPLC–(UV–FL)–ESI–Q–
TOF–MS.
The fluorescent photoaffinity probe (12) surprisingly
showed a dramatic fluorescence quenching (Figure 1a); thus,
the ratio of the fluorescent intensity between 9 and 12 being
9:1. Figure 1b shows the UV–vis absorption of 9, 11, and 12.
The band at around 306 nm of 11 is the absorption of the benzo-
phenone moiety, and the band at 340 nm of 9 is the dansyl amide
moiety. The fluorescent emission wavelength of dansyl amide
moiety does not overlap with the absorption bands of benzophe-
none and dansyl amide moieties. No energy transfer process
would thus be possible. There might be some orbital interaction
at the excited states between the 2 chromophores. The lower in-
tensity of the fluorescence might be due to an exciplex forma-
tion.
Synthesis of the fluorescent photoaffinity probe (12) is sum-
marized in Scheme 2. Dansyl chloride (7) was connected as an
efficient fluorophore with the ethylenediamine to provide the
Copyright Ó 2004 The Chemical Society of Japan

Chemistry Letters Vol.33, No.4 (2004)
453
References and Notes
(a)
(b)
1
For structure of tautomycin, see: a) M. Ubukata, X.-C. Cheng, and
K. Isono, J. Chem. Soc., Chem. Commun., 1990, 244. b) X.-C.
Cheng, M. Ubukata, and K. Isono, J. Antibiot., 43, 809 (1990). c)
M. Ubukata, X.-C. Cheng, M. Isobe, and K. Isono, J. Chem. Soc.,
Perkin Trans. 1, 1993, 617.
A. Takai, K. Sasaki, H. Nagai, G. Mieskes, M. Isobe, K. Isono, and
T. Yasumoto, Biochem. J., 306, 657 (1995).
Y. Sugiyama, I. I. Ohtani, M. Isobe, A. Takai, M. Ubukata, and K.
Isono, Bioorg. Med. Chem. Lett., 6, 3 (1996).
J. Goldberg, H. Huang, Y. Kwon, P. Greengard, A. C. Nairn, and J.
Kuriyan, Nature, 376, 745 (1995).
J. T. Maynes, K. S. Bateman, M. M. Cherney, A. K. Das, H. A. Luu,
C. F. B. Holmes, and M. N. G. James, J. Biol. Chem., 276, 44078
(2001).
1.0
1.0
9
12
2
3
4
5
0.5
0.1
0.5
0.1
9
12
11
400
500
600
700
300
400
500
Wavelength/nm
Wavelength/nm
6
7
8
A. Kita, S. Matsunaga, A. Takai, H. Kataiwa, T. Wakimoto, N.
Fusetani, M. Isobe, and K. Miki, Structure, 10, 715 (2002).
M. Kurono, A. Shimomura, and M. Isobe, Tetrahedron, 60, 1773
(2004).
Figure 1. (a) Fluorescence emission spectra of 9 and 12 in ace-
tonitrile (0:2 ꢀ 10ꢃ4 M) at room temperature. (b) UV–vis ab-
sorption spectra of 9, 11, and 12 in acetonitrile (1:0 ꢀ 10ꢃ4 M)
at room temperature.
Fluorescent unit 10: UV–vis (CH3CN) ꢀmax (") 340 nm (3509). IR
(KBr) ꢀmax 3449, 2926, 1637 cmꢃ1. FL (CH3CN) ꢀex 346 nm, ꢀem
522 nm. 1H NMR (400 MHz, CDCl3) ꢁ 2.89 (6H, s, N(CH3)2), 3.08
(2H, ddd, J ¼ 7:5, 6.0, 4.0, NHCH2CH2NH), 3.39 (2H, ddd, J ¼
7:5, 6.0, 4.0, NHCH2CH2NH), 4.07 (2H, s, COCH2O), 5.53 (1H,
br t, J ¼ 6:0, CH2NH), 5.75 (2H, br s, ONH2), 6.83 (1H, br s,
NHCH2), 7.19 (1H, br d, J ¼ 7:5, ArH), 7.52 (1H, dd, J ¼ 8:5,
7.5, ArH), 7.57 (1H, dd, J ¼ 8:5, 7.5, ArH), 8.23 (1H, dd, J ¼
7:5, 1.5, ArH), 8.26 (1H, br d, J ¼ 8:5, ArH), 8.55 (1H, br d, J ¼
8:5, ArH). ESI–Q–TOF–MS calcd for C16H23N4O4Sþ 367.1440
([M + H]þ); found 367.1499.
20
24
Benzophenone
Maleimide
1'
24
20
1
9
Fluorescent photoaffinity probe 12: UV–vis (CH3CN) ꢀmax (")
333 nm (4809). IR (KBr) ꢀmax 3421, 2927, 1728, 1663, 1540,
1320, 1098, 793 cmꢃ1. FL (CH3CN) ꢀex 347 nm, ꢀem 518 nm. 1H
NMR (600 MHz, CDCl3) ꢁ 0.76 (3H, d, J ¼ 6:8, 7–CH3), 0.85–
1.70 (18H, m), 0.79 (3H, d, J ¼ 7:0 Hz, 13-CH3), 0.95 (3H, d,
J ¼ 7:0 Hz, 25-CH3), 0.95 (3H, d, J ¼ 7:0 Hz, H-26), 0.96 (3H, d,
J ¼ 7:0 Hz, 15-CH3), 1.06 (3H, d, J ¼ 7:0 Hz, 19-CH3), 1.08 (3H,
d, J ¼ 7:0 Hz, 3-CH3), 1.84 (1H, tt, J ¼ 13:0, 5.0 Hz, H-12b),
1.92 (3H, s, H-1), 2.10 (1H, m, H-25), 2.18 (3H, s, 50-CH3), 2.45
(1H, m, H-3), 2.67 (1H, m, H-19), 2.71 (1H, dd, J ¼ 17:5, 4.0 Hz,
H-21a), 2.84 (1H, dd, J ¼ 16:0, 9.0 Hz, H-20a), 2.87 (6H, s,
N(CH3)2), 2.91 (1H, dd, J ¼ 16:0, 4.0 Hz, H-20b), 2.96 (1H, dd,
J ¼ 17:0, 8.0 Hz, H-21b), 3.09 (2H, m, NHCH2CH2NH), 3.14
(1H, t, J ¼ 10:0 Hz, H-6), 3.23 (1H, dd, J ¼ 10:0, 2.0 Hz, H-14),
3.24 (1H, br s, 22-OH), 3.27 (1H, dd, J ¼ 6:0, 2.0 Hz, H-23), 3.33–
3.37 (3H, m, NHCH2CH2NH, 18-OH), 3.40 (3H, s, OCH3), 3.70
(1H, m, H-18), 4.36 (1H, m, H-22), 4.37 (2H, s, H-1000), 4.43 (1H,
br s, 30-OH), 4.56 (1H, d, J ¼ 16:0 Hz, H-100a), 4.60 (1H, d, J ¼
16:0 Hz, H-100b), 5.08 (1H, t, J ¼ 6:0 Hz, H-24), 5.18 (1H, m, H-
30), 5.63 (1H, t, J ¼ 6:0, SO2NHCH2), 7.17 (1H, br d, J ¼ 7:5, H-
9000), 7.44 (1H, t, J ¼ 8:0, H-700), 7.47–7.52 (4H, m, H-600, 1200,
1400, 13000), 7.54 (1H, dd, J ¼ 8:5, 7.5, H-8000), 7.60 (1H, t, J ¼
7:5 Hz, H-1300), 7.80 (3H, m, H-400, 1100, 1500), 7.90 (1H, br t, J ¼
6:0, CH2NHCO), 7.96 (1H, br d, J ¼ 8:0 Hz, H-800), 8.16 (1H, br
s, CONHAr), 8.20 (1H, dd, J ¼ 7:0, 1.0, H-12000), 8.26 (1H, br d,
J ¼ 8:5, H-7000), 8.50 (1H, br d, J ¼ 8:50, H-14000). ESI–Q–TOF–
MS calcd for C72H99N6O18Sþ 1367.6737 ([M + H]þ); found
1367.6808.
Dansyl amide
Spiro ketal
Spiro ketal
Maleic acid
(a)
(b)
Figure 2. Proposed 3-D structures of 2 and 12; (a) One of the
stable conformations of 2; (b) One of the energy-minimized
conformations of 12 on the basis of 2.
Figure 2a shows one of the stable conformations of tautomy-
cin diacid (2), which was reported previously through computer
calculation with Biograf and NMRgraf programs using NOESY
data.3 On the basis of these data, we calculated for 12 to have en-
ergy-minimized conformer with a Macromodel (MMFF force
field).10 One of the resulting six conformers within 12.55 kJ/
mol is shown in Figure 2b. The distance between benzophenone
ꢀ
and dansyl amide moieties is 3.8–5.5 A, which is short enough
for the interaction. These results indicate that the fluorescence
quenching occurred owing to the folded conformation of
TTM. Moreover, reduction of 12 with sodium borohydride in
MeOH led to two benzhydrol derivatives (13, 14), which exhib-
ited the usual fluorescence intensity.11,12 Recovery of the fluores-
cence might be due to the absence of interacting chromophore.
The ratio of fluorescent intensity between of 12 and 13 or 14
was about 1:10 by the fluorophotometer equipped on a HPLC.
This means that the fluorescent photoaffinity labeled peptides
should have the enough intensity, and could be detected by a flu-
orophotometer. Further studies are in progress in order to detect
the labeled peptides through this strategy.
10 Computer calculation was carried out on a Silicon Graphics Octan
computer. Energy-minimization was performed on 10,000 initial
starting conformations with global minimum search program using
Macromodel version 7.1 with MMFF force filed. a) F. Mohamadi,
N. G. J. Richards, W. C. Guida, R. Liskamp, M. Lipton, C. Caufield,
G. Chang, T. Hendrickson, and W. C. Still, J. Comput. Chem., 11,
440 (1990). b) T. A. Halgren, J. Comput. Chem., 20, 730 (1999),
and references cited therein.
11 13: Reduced compound at C900 and C20, ESI–Q–TOF–MS calcd for
C72H104N6O18S2þ 686.359 ([M + 2H]2þ); found 686.332.
12 14: Reduced compound at C900, C20 and C2, ESI–Q–TOF–MS calcd
for C72H106N6O18S2þ 687.367 ([M + 2H]2þ); found 687.341.
This work was financially supported by a Grant-in-Aid from
the Ministry of Education, Culture, Sports, Science and Technol-
ogy, and a grant from Ono Pharm. Co., Ltd. We are grateful to
Prof. K. Isono at ex-Riken Institute and Kaken Pharm. Co.,
Ltd. for supplying a crude tautomycin, Mr. K. Koga for special
NMR spectroscopy, and Prof. N. Harada at Tohoku Univ. for
discussions.
Published on the web (Advance View) March 20, 2004; DOI 10.1246/cl.2004.452