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Fluorescence Reporters for Phosphodiesterase Activity.

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Houte, J. van Thuijl, Org. Muss Spectrom. 1971,5, 1101; d) J. van Thuijl, K. J.
Klebe. J. 3. Houte, ihid. 1972, 6, 1363; e) G. Bouchoux, Y Hoppilliard, ibid.
1977, 12, 196; f) J. Main-Bobo, S. Olesik, W Gase, T. Baer, A. A. Mommers,
J. L. Holmes. J. Am. Chem. Soc. 1986, 108, 106.
[13] K. L. Busch. G. L. Glish, S. A. McLuckey, Muss Spectromerry/Muss Spectronwtrj, Techniques and Applicutions of Tundem Muss Spectrometry, VCH,
New York, 1988.
[14] The VG Analytical ZAB-R mass spectrometer at McMaster University has a
three-sector BEE (B = magnet, E = electric sector) configuration, see: H. F.
van Garderen. P. J. A. Ruttink, P. C. Burgers, G . A. McGibbon, J. K. Terlouw,
Int. J. Muss Spectrom. Ion Processes 1992, 121, 159.
[15] The modified VG ZAB/HF/AMD at the TU Berlin is a four-sector instrument
with a BEBE configuration, see: a) R. Srinivas, D. Siilzle, T. Weiske, H.
Schwdrz. In1 J Muss Spectrom. Ion Proce.wes 1991, 107, 368; b) C. Schalley,
D Schroder. H. Schwarz, ihid. 1996, 153, 173.
[16] B. L. M. van Baar. Ph.D. Thesis. University of Utrecht, 1988, Chapter 1
1171 J. L. Holmes. F. P. Losring, J. K. Terlouw, P. C. Burgers, J. Am. Chem. SOC.
1982. 104, 2931
[18] In NRMS experiments 8 or 10 keV m/z 68 [C,H,NJ+ ions were mass-selected
(by B with the ZAB-R or B,E, with the ZAB/HF/AMD) and then subjected
to neutralizing collisions with N,N-dimethylaniline or xenon vapor present in
a small gas cell located in the field-free region. Afterward, any remaining ions
were removed from the beam by deflection with a positively charged electrode
so that only a beam of fast moving neutral species entered a second gas cell,
therein being (dissociatively) reionized by collisions with oxygen molecules.
For a NR mass spectrum, the survivor and fragment ions were analyzed by
using the next available sector (B2 in Berlin or El at McMaster). Alternatively,
when the recovery ions ( m / z 68) were selectively transmitted by the sector and
then passed through another oxygen-filledcollisioncell(70% transmission) the
resulting CID products could be analyzed by using the last sector. The NR/
CID mass spectrum so obtained is characteristic of only the species surviving
neutralization and reionization. Mass spectra of doubly charged ions (NR/CS)
were also obtained in the same manner. Comparative collision-induced dissociation (CID) and charge-stripping (CS) experiments were performed with the
deflector electrode switched off.
to the extreme importance of this class of enzymes in biochemistry and biotechnology, numerous attempts have been made to
design “artificial phosphodie~terases”.~~~
Independent of the
catalytic mechanism of these systems, highly sensitive assays are
needed for detecting phosphodiesterase activity. So far, UV/Visspectroscopical quantification of nitrophenols liberated from
the corresponding phosphodiesters or 31PNMR spectroscopy
were used in most cases. We found it worthwhile to design a
simple and much more sensitive fluorescence probe for detecting
phosphodiesterase activity. Due to the high sensitivity of fluorescence assays, we reasoned that with such probes phosphodiesterase activity might also be detected in complex mixtures of
biological or synthetic origin.
The mechanism of our reporter molecules is shown in
Scheme 1. In compound A, a fluorophore and a fluorescence
A
4
H~O
B
esterase
C
or
Fluorescence Reporters for
Phosphodiesterase Activity**
Albrecht Berkessel* and Rainer Riedl
Phosphodiesterases catalyze the hydrolysis of phosphodiester
Their most important biological substrates
bonds [Eq. (a)] .I1]
9
R-0-7-OH
R
R-0-7-0-R’
00
f
HO-R’
00
phosphodiesterase
or
b
“20
R-OH
+
(a)
9
HO-7-0-R’
OQ
Scheme 1 Mechanism of the fluorescence reporters
quencher are held in close proximity. The absorption spectrum
of the quencher and the emission spectrum of the fluorophore
should match as well as possible.141Upon irradiation of the
fluorophore, rapid energy transfer to the quencher takes place.
Therefore, this intact phosphodiester does not fluore~ce.‘~’
After hydrolysis of the phosphodiester group, the cleavage
products B and C (or B’ and C‘) can diffuse away from one
another, and the fluorescence is no longer quenched intramolec~ l a r l y . [We
~ ] synthesized two such fluorescence reporters, namely the phosphodiesters 1 and 2. In these compounds a naph-
are the nucleic acids. The manipulation of genetic material (“genetic engineering”) hinges on the sequence-specific cleavage of
DNA by the restriction enzymes,[21which are isolated from
natural sources and, consequently, invariable with respect to
their catalytic parameters and particularly their base-recognition properties (number and type of the bases recognized). Due
[‘I
[‘I
[**I
Prof. Dr A Berkessel,“’ Dipl.-Chem. R. Riedl
Organisch-chemisches Institut der Universitat
Im Neuenheimer Feld 270, D-69120 Heidelberg (Germany)
New addressInstitut fur Organische Chemie der Universitat
Greinstrasse 4. D-50939 Koln (Germany)
Fax: Int code +(221)470-5102
e-mail : berkessel(a uni-koeln.de
This work was supported by the Fonds der Chemischen Industrie
Angew. Chi,m. Int Ed Engl. 1997, 36, No. 13/14
8 VCH VerIagsgeseNschufi mbH. 0-69451
1
2
Wernheim. 1997
0570-0833/97/3613-14~f
8 17.5Nc 5010
1481
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thalene residue acts as the fluorophore, and an azobenzene moiety as the quencher. Since 4-hydroxyazobenzene (pK, =
8.20)['] is a better leaving group than 1-naphthol (pK, = 9.37)[@
or 2-naphthol (pK, = 9.49),16]hydrolysis of 1 and 2 should result in formation of 4-hydroxyazobenzene and the corresponding naphthyl phosphates. We here report the synthesis of 1 and
2 and the activation of their fluorescence by hydrolysis.
As shown in Scheme 2 the preparation of 1 und 2 starts with
the reaction of 1- or 2-naphthol with phosphoryl chloride.[71In
30 7
A,;
71%
I
P0Cl3, pyridine,
benzene, 80"C,
45 rnin
1
77%
I-naphthyl phosphate, I-naphthol is knownL61 to show practically no fluorescence in aqueous solution at 357 nm. When
we employed another commercially available[* phosphodiesterase I (from bovine intestinal mucosa), 1-naphthyl phosphate could not be found at all as the primary hydrolysis
product of 1. HPLC analysis of the (nonfluorescent) reaction mixtures revealed immediate further degradation to 1naphthol.
With this in mind, we subjected the 2-naphthol derivative 2 to
hydrolysis catalyzed by phosphodiesterase I (from bovine intestinal mucosa). In contrast to 1-naphthol, 2-naphthol is
knownE6]to fluoresce strongly in aqueous solution. The results
are summarized in Figure 3. Addition of the enzyme to a solu-
m
0
4
54%
I
I
4-hydroxyazobenzene,
pyridine, toluene,
80"C, 4 h
1
35%
2
Scheme 2. Synthesis of the phosphodiesters 1 and 2.
the second step, the azobenzene moiety is introduced. UV/Vis
spectra of 1 and 2 show absorption bands for the naphthalene
and azobenzene moieties (maxima at shorter and longer wavelength, respectively; Figure 1). Irradiation of the naphthalene
absortion band results in virtually no fluorescence.[8]
0.8
I nrn--+
Figure 2. Fluorescence spectra of a 5.0 x 1 0 - 5 solution
~
of 1 in AMPSO buffer
(0.1 M, pH 8.8), T = 37 "C, V = 1.5mL. A: without enzyme; B: after additlon of
0.05 u phosphodiesterase I (crotalus atrox venom); C: 30 min after addition of the
enzyme; D: 360 rnin after addition of the enzyme; E: 240 rnin after addition of the
enzyme; F: for comparison, 5.0 x ~ O - ' M 1-naphthyl phosphate t5.0x lO-'u
4-hydoxyazobenzene.
500
,
400
0.4
0.3
0.2 .-
I-/
I - 2
- _ _ _1
--- L
0.1 -.
o
o
o
r n ? .- C n
C
~
~
o
o
N CN
9 r n IC
. C n~
Ainrn
o
C
o
~
~
C
g
~
0o~0 C0
C
o
0
N
~
U
Addition of a phosphodiesterase, for example phosphodiesterase I from crotalus atrox
to a solution of 1 in
AMPSO buffer'"] results in a rapid increase in the fluorescence
intensity (Figure 2). The shape of the fluorescence spectrum is
identical to that of an equimolar mixture of 1-naphthyl phosphate and 4-hydroxyazobenzene. However, the intensity of the
control mixture is not reached in the enzyme-catalyzed hydrolysis of 1. Even more remarkably, the fluorescence intensity starts
to decrease after about 4 h.
This unexpected observation results from a minor phosphatase activity in the commercially available enzyme preparat i ~ n . ' HPLC
~]
analysis revealed that 1-naphthyl phosphate initially liberated from 1 slowly degrades to I-naphthol. Unlike
Q VCH Veriagsgeseilschaft mbH, 0.69451 Weinheim,1997
%
S
P
0
~
0
C
9
S
0
r
O
n
0
t
O
Aem;, I nrn
Figure 1. UV/Vis spectra of the fluorescence reporters 1 and 2 (c = 5.0 x 10- M)in
AMPSO buffer (0 1M, pH 8.8), T = 25 "C
1482
~
0
O C 9 C
~
U
~
~
0
b
N
t
0
t
-
t
0
r
~
0
n
W
r
0
n
~
r
0
O
n
Figure 3. Fluorescence spectra of a 5.0 x 10- M solution of 2 in AMPSO buffer
(0.1 M,pH 8.8), T = 37 'C, V = 1.5 mL. A : without enzyme; B: 40 min after addition of 0.05 u phosphodiesterase I (bovine intestinal mucosa); C: 4 h after addition
2-naphthol
of the enzyme (end of reaction); D: for comparison, 5 . 0 ~1 0 - 5 ~
+5.0 x ~ O - ' M 4-hydoxyazobenzene
tion of 2 in AMPSO buffer["] produced a rapid increase in the
fluorescence intensity, which asymptotically approached that of
an equimolar mixture of 2-naphthol and 4-hydroxyazobenzene
(Figure 3). HPLC analysis of the reaction mixtures confirmed
the formation of 4-hydroxyazobenzene and the secondary hydrolysis product 2-naphthol. Hydrolysis of 2 induced an at least
thousandfold increase of the fluorescence intensity!" Furthermore, the progress of the reaction was also indicated by the
yellow color of the 4-hydroxyazobenzene formed (strictly
speaking, of the phenolate anion, I,,, = 421 nm). A plot of the
initial velocities versus the enzyme concentrations (addition of
0S70-0833/97/3613-1482$17.50+ .SO10
Angew. Chern. In[
Ed. Engl. 1997, 36, No. 13/14
N
-
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0.025 -0.1 u) confirmed the expected linear relationship (not
shown, three experiments, p = 0.9984).
The fluorescence reporters 1 and 2 are highly sensitive probes
for detecting phosphodiesterase activity. Their use as test substrates not only allows detection of phosphodiesterase activity,
but also phosphatase activity.
E.xperimental Section
1-Naphthyl phosphoryl chloride (3) and 2-naphthyl phosphoryl chloride (4) were
prepared according to ref [7]
1: Under nitrogen 3 (2.63 g, 10.1 mmol) and 4-hydroxyazobenzene (2.00 g.
10.1 mmol, 1.O equiv) were dissolved in abs toluene (150 mL) with heating, and abs
pyridine (3.19 g. 40.4 mmol, 4.0 equiv) was added dropwise within 30 min. Stirring
was continued for 4 h at 8O'C and overnight at RT. The precipitated pyridinium
hydrochloride was removed by filtration, and the filtrate concentrated on a rotary
evaporator to about 20 mL. The remaining oil was dissolved in pyridine/water (111.
20.0 mL). and the solution stirred at R T for 1 h. Afterwards 10% aqueous K,CO,
(100 mL) and chloroform (100 mL) were added. The biphasic mixture was shaken
vigorously to induce the precipitation of the potassium salt of 1. After 3 h the
precipitate was isolated by filtration and taken up in boiling water (150mL) The
red extract was again filtered. and the filtrate adjusted to pH 1 with concd HCI. The
orange precipitate was isolated by filtration and recrystallized from acetone to
afford 1 as an orange crystalline powder (2 22 g, 54%).
M.p. 167 C (acetone); IR (KBr). i = 1222 [s, v(P=O)], 1204[s, v(C-O)], 1046, 989
[both s, i,(P-O)]. 564cm-' [s, &P-O-Ar)]; ' H N M R (300 MHz, [D,]DMSO):
6 = 7.34 - 7 51 (m. 4H, aryl H ) , 7.51 -7.64 (m. SH, aryl H), 7.70-7.78 (m, 1 H, aryl
H), 7.81 -8.00 (m.5 H, aryl H), 8 03-8 13 (m. 1 H, aryl H), 11.20 (br s, 1 H, OH),
"C NMR (75 MHz, [DJDMSO): b =115.10 (d, 'J(C,P) = 2.82 Hz, aryl CH).
121.00 (d. 'J(C.P) = 5.08 Hz, aryl CH), 121.70 (d, aryl CH), 122.64 (d, aryl CH),
124.32 (d. aryl CH). 124.38 (d, aryl CH), 125.97 (d, 4J(C,P) =1.70 Hz, aryl CH),
126.32 (s. 3J(C.P) = 6.21 Hz, aryl C), 126.44 (d. aryl CH), 126.81 (d, aryl CH),
127 87 (d. aryl CH). 129.60 (d. aryl CH), 131.55 (d, aryl CH), 134.47 (s, aryl C),
147.16 (s. 'J(C.P) =7.35 Hz, aryl C), 148.64 (s, aryl C), 152.06 (s, aryl C), 153.96
(5. 'J(C,P) = 6.78 Hz, aryl C); "P NMR (80 MHz, [D,]DMSO): 6 = -11.68;
LDI-MS n ~ ! :(%) = 403.4 (100) [ M - - HI, 298 (5) [C,,H,,O,P-]. 197.1 (12)
[C,,H,N,O~], 142.9 (SO) [C,,H,O-]; UV/Vis (H,O): i,,, ( E ) = 417 (1322), 330
(14910), 290 (9385). 220 nm (54300); elemental analysis calcd for C,,H,,N,O,P
(404.36):C 65.35. H 4.24, N 6.93. P 7.66; found: C 65.23, H 4 37. N 6.88, P 7.61.
2: Analogous to the preparation of 1, abs pyridine (7.14 g, 90.4 mmol, 4.0 equiv)
was added to a solution o f 4 (5.90 g, 22.6 mmol) and 4-hydroxyazobenzene (4.49 g,
22.6 mmol, 1.O equiv) in abs toluene (300 mL). The mixture was heated to 80 "C for
4 h and stirred overnight at RT. The precipitated pyridinium hydrochloride was
removed by filtration, and the solvent removed on a rotary evaporator. The oily
residue was stirred with pyridine/water (111, 20.0 mL) for 1 h at RT. The solution
was then adjusted to pH 1 by adding concd HCI. A deep-red precipitate formed
overnight. which was isolated by filtration and recrystallized from acetone to afford
2 as orange platelets (3 22 g, 35%).
M.p. 146 C (acetone); FT-IR (KBr): 3 =1228 [s, v(P=O)], 1208 [s, v(C-O)], 976,
946 [both s. v(P-O)j, 551 c m - ' [m. b(P-O-Ar)I; ' H NMR (300 MHz, [D,]DMSO):
6 = 5.05 (br s, 1 H. OH). 7.32-7.51 (m, 5H. aryl H), 7.52-7.63 (rn,3H. aryl H),
7.68-7.73 (br s. 1 H, aryl H), 7.78-7 94 (m, 7H. aryl H): "C N M R (50 MHz,
[DJDMSO): ii =115 79 (d, '4C.P) = 4.58 Hz, aryl CH), 120.77 (d, 'J(C,P) =
4.57 Hz. aryl CH). 121.08 (d, 'J(C,P) = 5.34Hz, aryl CH), 122.56 (d, aryl CH),
124.20 (d. aryl CH). 124.90 (d. aryl CH), 126.59 (d, aryl CH). 127.28 (d, aryl CH),
127.69 (d. aryl CH), 129.34 (d, aryl CH), 129.58 (d, aryl CH). 129 93 (s, aryl C),
131 32(d.aryIC). 133.81 (s,arylC), 147.91 (s,aryl C), I50 21 (s, 'J(C,P) = 6.87 Hz,
aryl C). 152.1 1 (s. aryl C ) , 155.38 (s, 'J(C,P) = 6.87Hz, aryl C); "P NMR
(80 MHz. [DJDMSO). d = - 11 98; LDI-MS: m/r (%) = 403.6 (100) [ M - - HI,
298.4 (7) [C,,H,,O,P-]. 197.4 (7) [C,,H,N,O-]; UViVis (H,O): R,,, ( E ) = 419
(1195). 329 (15280), 286 (7480), 276 (6735). 220nm (63090); elemental analysis
calcd for C,,H,,N,O,P (404.36): C 65.35, H 4.24, N 6.93. P 7.66; found: C 65.24,
H 4.23. N 691. P 7.68. Hydrolyses were carried with a variable-temperature
Shimadzu RF-5000 recording spectrofluorophotometer equipped with a 150 W
xenon lamp
Received: December 11. 1996 [29869IE]
German version: Anfun,. Chem. 1997, 109, 1518-1520
Keywords: analytical methods
troscopy
-
enzymes
- fluorescence spec-
[I] N. Strater. W. N. Lipscomb, T. Klabunde, B. Krebs. A n g e w Chem. 1996, 108,
2158-2190. Angew. Cliem. rnr. Ed. Engl. 1996,35, 2024-2055.
[2] a ) D. M. Skinner, C. A. Holland in Essentials of Molecular Biology, Vol. 2
(Eds . D. Freifelder, G. M Malacinski), Jones and Bartletr, Boston, London,
1993, pp. 262-297: b) D. Kostrewa, F. K. Winkler, Biochemistry 1995, 34,
A n g e w Chem. I n t . Ed. Engl. 1997, 36. No. 13/14
0 VCH
683-696; c) M. Newman, T. Strzelecka, L. E Dorner, I Schildkraut, A. K.
Aggarwal. Science 1995, 269, 656-663; d) I. B. Vipond, S. E Halford, Mol.
Microhiol. 1993, 9,225-231;e) S. E. Halford, J. D. Taylor. C. L. M. Vermote.
I. B. Vipond in Nudere AciaIv and Molecular Biology, Vol 7 (Eds.: F. Eckstein.
D M. J. Lilley), Springer, Berlin, Heidelberg, 1993. pp. 47 -69.
[3] a) D S. Sigman. A. Mazumder, D. M. Perrin, Chem. R r r . 1993, 93. 22952316; b) P. B. Dervan, Science 1986, 232,464-471 ; c) B Linkletter, J. Chin,
Angew. Chem. 1995, 107, 529-531; Angel\,. Chem. I n t . Ed. Enpl. 1995, 34,
472-474; d) W. H. Chapman, Jr., R Breslow, J: Am. Chi,m. SOC.1995, 117$
5462-5469; e) 1. Rammo, R. Hettich, A. Roigk. H.-J. Schneider, Chem. Commun. 1996, 105- 107; f) M.-S. Muche, M. W. Gobel, Angi'ii. Chem. 1996. 108,
2263-2265; Angew. Chem. Int. Ed. Engl. 1996,35, 212667129.
[4] J. R. Lakowicz, Principlm of Fluorescence Spectroscopy. Plenum, New York,
London, 1983.
[5] I. M. Klotz, H. A. Fiess, J. Y. Chen Ho, M. Mellody. J. Am. Chem. Soc. 1954,
76, 513665140,
[6] A. Weller, 2. Elektrochem. 1952,56, 662-668.
[7] 0. M Friedman, A. M. Seligman, 1 Am. Chem. Soc. 1950, 72, 624-625.
[8] When 2 was subjected to hydrolysis, A,,, = 326 nm was used since at this
wavelength there is basically no fluorescence of the enzyme, and the fluorescence intensity of 2-naphthol is not significantly lower than that at
i.,,
=
,290 nm. In the case of 1, j.exc had to be 290 nm since at this excitation
wavelength the fluorescence intensity of 1-naphthyl phosphate is much higher
than that at i,,, = 326 nm.
[9] Phosphodiesterase I (fromcrotalus atrox venom), Sigma Definition ofactivity
by the suppliers: 1 u hydrolyzes 1.0 pmol of bis(p-nitropheny1)phosphate per
rnin at pH 8.8 and 37'C. Concerning the phosphodiesterase activity, the suppliers state 0.16 umg-' protein at pH 8.8 and 37°C. The phosphatase activity
is specified as less than 0.001 umg-' protein at p H 10.4 and 37°C (substrate:
p-nitrophenyl phosphate)
[ 101 AMPS0 : 34 (1,l -dimethyl-2-hydroxyethyl)amino]-2-hydr~~xypropanesulfonic
acid.
[ l l ] a) Phosphodiesterase I (from bovine intestinal mucosa). Sigma. Definition
of activity by the suppliers: 1 u hydrolyzes 1 0 pmol of bis(p-nitrophenyl)
phosphate per minute at pH 8.8 and 37'C The suppliers state "non-specific
phosphatase activity" for enzyme preparation. b) In a control experiment the
rapid dephosphorylation of 1- and 2-naphthyl phosphate could be shown by
HPLC.
[12] Fluorescence intensity at 357 nm at the end of the reaction:fluorescence intensity at 357 nm before addition of enzyme. A higher intensity of excitation
should result in an even higher increase in fluorescence intensity.
N-Bromoacetamide-A New Nitrogen Source
for the Catalytic Asymmetric
Aminohydroxylation of Olefins""
Milan Bruncko, Gunther Schlingloff, and
K. Barry Sharpless*
The a-amino alcohol moiety is one of the most abundant
structural units in biologically active compounds. Recent developments by us[*]and others['] have Ied to viable metal-catalyzed
routes for its asymmetric synthesis. In our recently discovered
asymmetric aminohydroxylation (AA) ,['I olefins are converted
to nonracemic protected amino alcohols in a single step by using
catalytic amounts of osmium(vm) and cinchona alkaloid derivatives (Scheme l), by a process which closely mirrors the wellestablished asymmetric dihydroxylation of olefins (AD) .[31We
have now found conditions that allow amides to join sulfon[*I Prof. K. B. Sharpless, Dr. M. Bruncko, Dr. G. Schlingloff
The Scripps Research Institute
Department of Chemistry BCC 315
10550 North Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax: Int. code +(619) 784-7562
e-maii : sharples@scripps.edu
I**] We thank the National Institutes of Health (GM 28384), the National Science
Foundation (CHE - 9531152). the W M. Keck Foundation, and the Skaggs
Institute for Chemical Biology for financial support. G. S. is grateful for a
postdoctoral fellowshlp grantediby the Swiss National ScienceFoundation and
the Ciba-Geigy Jubilaeums-Stiftung.
Verlagsgese1lschafimbH, 0-69451 Wemheim, 1997
0570-0833197/3613-1483S 17.SOt.50/0
1483
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