A nitroxyl synthase catalytic antibody

Article abstract of DOI:10.1021/ja9538694

An antibody raised against acridinium hapten 1 is shown to catalyze the retro Diels-Alder reaction of the anthracene-HNO cycloadduct 2 to release anthracene 4 and nitroxyl (HNO). Nitroxyl is oxidized to nitric oxide (NO) in the presence of superoxide dism

Full text of DOI:10.1021/ja9538694

3
550
J. Am. Chem. Soc. 1996, 118, 3550-3555
A Nitroxyl Synthase Catalytic Antibody
Nicolaus Bahr, Rolf G u1 ller, Jean-Louis Reymond,* and Richard A. Lerner*
Contribution from the Departments of Molecular Biology and Chemistry,
The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, California 92037
X
ReceiVed NoVember 17, 1995
Abstract: An antibody raised against acridinium hapten 1 is shown to catalyze the retro Diels-Alder reaction of the
anthracene-HNO cycloadduct 2 to release anthracene 4 and nitroxyl (HNO). Nitroxyl is oxidized to nitric oxide
(NO) in the presence of superoxide dismutase. Since the enzyme superoxide dismutase is ubiquitous in vivo, this
catalytic antibody system may be equivalent to NO-synthase. Antibody catalysis is triggered by recognition of the
phenyl rings in hapten 1 at an angle near that of the transition state of the retro Diels-Alder reaction of 2. Acridinium
hapten 1 is in chemical equilibrium with its conjugate Lewis base 9-hydroxyacridane 1-OH (pK ∼ 8.2). Catalytic
-
1
-4
-1
antibody 9D9 (KM(2) ) 100 µM, kcat ) 0.07 min , kuncat ) 3 × 10 min ) is the result of an heterologous
immunization and binds both 1 (Ki ) 16.6 µM at pH 6.1) and 1-OH (Ki ) 0.9 µM at pH 9.0). The more tightly
bound neutral conjugate base 1-OH probably represents a more accurate mimic of the transition state of the retro
Diels-Alder process.
Introduction
Scheme 1. Retro Diels-Alder Reaction and Hapten Used for
Production of Catalytic Antibodies
In the last years a considerable variety of organic transforma-
tions have been catalyzed by monoclonal antibodies, covering
most reaction types from hydrolytic reactions to redox and
pericyclic processes. Catalytic antibodies for Diels-Alder
cycloadditions represent one of the most striking accomplish-
1
2
ments in this field not only because of their ability to control
the enantio- and stereoselectivity of the bond formation but also
because no other protein catalysts were known for this reaction
3
until recently. Antibodies catalyzing such “abiological” pro-
cesses fundamentally broaden the scope of biocatalysis beyond
classical enzymology and offer new opportunities for organic
4
synthesis. They are also very attractive candidates for prodrug
activation in ViVo since interferences by naturally occurring
enzymes are ruled out.5 Herein we report the generation of a
catalytic antibody obtained by immunization against hapten 1
that promotes a retro Diels-Alder reaction releasing nitroxyl
neurons by oxidation of L-arginine to L-citrulline by the enzyme
NO-synthase in response to transient increases in intracellular
(
HNO) as a heterodienophile from the inert prodrug 2 (Scheme
2+
1
). Nitroxyl can in turn be oxidized to nitric oxide (NO) by
Ca and exerts its action by binding to the iron of the prosthetic
heme group in soluble guanylate cyclase, which triggers cGMP
production as a second messenger. The biological effect of
nitric oxide can be mimicked by NO-releasing drugs like
nitroglycerine, which are commonly used to treat angina and
hypertension. However, these drugs decompose rapidly, must
be administered in large doses, and eventually induce tolerance.
The nitroxyl synthase catalytic antibody/SOD couple described
here offers a more controlled and selective route to nitric oxide.
6
the ubiquitous enzyme superoxide dismutase (SOD).
This prodrug release system is of particular biological
significance since nitric oxide acts as a chemical messenger for
a number of fundamental bioregulatory processes, including
blood pressure regulation and memory.7 Originally NO was
identified in biological systems as the active principle of EDRF
(
endothelium-derived relaxing factor).8 NO is produced in
X
Abstract published in AdVance ACS Abstracts, April 1, 1996.
(1) Schultz, P. G.; Lerner, R. A. Science 1995, 269, 1835.
Results and Discussion
(2) (a) Hilvert, D.; Hill, K. W.; Nared, K. D.; Auditor, M.-T. M. J. Am.
Chem. Soc. 1989, 111, 9261. (b) Braisted, A. C.; Schultz, P. G. J. Am.
Chem. Soc. 1990, 112, 7430. (c) Gouverneur, V. E.; Houk, K. N.; de Pacual-
Teresa, B.; Beno, B.; Janda, K. D.; Lerner, R. A. Science 1993, 262, 204.
Substrate Synthesis. Anthracene 4 was prepared starting
from commercially available 10-methylanthracene-9-carboxalde-
hyde (Scheme 2). Wittig reaction with (carbethoxymethylene)-
triphenylphosphorane yielded 93% of trans-ene 5. Hydrogena-
tion of the R,â-unsaturation in ester 5 proved difficult due to
the concurrent reduction of the anthracene moiety. Selective
reduction was achieved employing NaBH4/NiCl2 as described
(
d) Yli-Kauhaluoma, J. T.; Ashley, J. S.; Lo, C.-H.; Tucker, L.; Wolfe, M.
M; Janda, K. D. J. Am. Chem. Soc. 1995, 117, 7041.
3) Oikawa, H.; Katayama, K.; Suzuki, Y.; Ichihara, A. J. Chem. Soc.,
Chem. Commun. 1995, 1321.
4) (a) Reymond, J.-L.; Reber, J.-L.; Lerner, R. A. Angew. Chem., Int.
Ed. Engl. 1994, 33, 475. (b) Sinha, S. C.; Keinan, E. J. Am. Chem. Soc.
995, 117, 3653.
5) (a) Miyashita, H.; Karaki, Y.; Kikuchi, M.; Fujii, I. Proc. Natl. Acad.
(
(
1
(
(7) (a) Culotta, E.; Koshland, D. E., Jr. Science 1992, 258, 1861. (b)
Stamler, J. S.; Singel, D. J.; Loscalzo, J. Science 1992, 258, 1898. (c)
Feldman, P. L.; Griffith, O. W.; Stuehr, D. J. Chem. Eng. News 1993, Dec.
20, 26-38. (d) Snyder, H. Nature 1994, 372, 504.
(8) Feelish, M.; te Poel, M.; Zamora, R.; Deussen, A.; Moncado, S.
Nature 1994, 368, 62.
Sci. U.S.A. 1993, 90, 5337. (b) Campbell, D. A.; Gong, B.; Kochersperger,
L. M.; Yonkovich, S. Y.; Gallop, M. A.; Schultz, P. G. J. Am. Chem. Soc.
1
994, 116, 2165. (c) Smiley, J. A.; Benkovic, S. J. J. Am. Chem. Soc. 1995,
1
17, 3877.
(6) Murphy, M. E.; Sies, H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10860.
0
002-7863/96/1518-3550$12.00/0 © 1996 American Chemical Society

A Nitroxyl Synthase Catalytic Antibody
J. Am. Chem. Soc., Vol. 118, No. 15, 1996 3551
Scheme 3. Synthesis of Hapten 1a
Scheme 2. Synthesis of Substrates 2 and 3a
a
Conditions: (a) SOCl
benzyl ester, 3 equiv of Et
Me SO , toluene, reflux, 2.5 h; (c) 2:1 0.1 N KOH/MeOH, reflux, 20
2
, reflux, 6 h, then 1.3 equiv of â-alanine
a
Conditions: (a) Ph
, 1 equiv of NaBH
ethanolamine, 0.1 equiv of NaCN, EtOH, 20 °C, 36 h; (d) 2 equiv of
3
PdCHCO
2 2 2
Et, CH Cl , reflux, 36 h; (b) 0.3
3
N, CH Cl , 20 °C, 14 h; (b) 1.2 equiv of
2
2
equiv of NiCl
2
4
, EtOH, 20 °C, 6 h; (c) 25 equiv of
2
4
min; (d) 50 equiv of ethanolamine, EtOH, 20 °C, 36 h.
+
-
Me
0%, 8: 30%, recovered 4: 20%; (e) 0.1% TFA in MeOH, 0 °C, 24
3 4
N Bn IO , 2 equiv of HCONHOH, dry DMF, 20 °C, 1.5 h, 7:
3
Scheme 4. Acridinium 1 and Its Pseudo-Base 1-OH as
Transition State Analogs for the Retro Diels-Alder Reaction
of 2
h; (f) 0.1% TFA in MeOH, 20 °C, 12 h.
9
by Suzuki et al. giving an 80% yield of 6 in gram quantities.
Cyanide-catalyzed aminolysis10 of ester 6 gave light yellow,
crystalline 4. Anthracene 4 is stable to air and can be stored at
-
18 °C for months without decomposition.
Diels-Alder reaction of 4 with nitrosoformate, generated in
situ by periodate oxidation of hydroxamic acid, gave a 1:1
mixture of regioisomeric cycloadducts 7 and 8 in moderate
1
1
yield. The isomers were separated by preparative RP-HPLC,
1
and their structure unambiguously resolved by NOE H-NMR
experiments. Thus, a strong NOE was observed between the
CH3 and HCON signals in 8 only. Acid-catalyzed removal of
the formyl group with trifluoroacetic acid in methanol gave 2
or 3 quantitatively. The products were purified by RP-HPLC
and stored at -78 °C as their trifluoroacetate salts as solids or
as 5 mM stock solutions in H2O.
Hapten Design and Synthesis. Catalysis of Diels-Alder
reactions is often effected by Lewis acids in aprotic solvents.
In aqueous environment cycloadditions can be promoted using
2
catalytic antibodies. Catalysis is achieved by binding both
proceeded smoothly using dimethyl sulfate. Saponification of
benzyl ester 10 and acidification furnished hapten 1 as a bright
yellow, crystalline substance in an overall yield of 75%. Hapten
diene and dienophile, leading to a rate enhancement by an
increase in effective molarity of the substrates. Clearly this
effect would not be operative for the targeted unimolecular retro
Diels-Alder reaction. We concentrated instead on the geo-
metrical parameters affected by the reaction. In particular the
dihedral angle (R) between the two phenyl rings changes from
approximately R ) 110° in 2 and 3 to R ) 180° in 4 (Scheme
1
was purified by preparative RP-HPLC and stored as the
trifluoroacetate (TFA) salt. The carboxyl function in 1 was
activated to the sulfo-N-hydroxysuccinimide ester for conjuga-
tion to the carrier proteins BSA (bovine serum albumin) and
1
3
KLH (keyhole limpet hemocyanin). Alternatively, reaction
1
). A compound presenting these two phenyl rings with an
with ethanolamine yielded 11, which was used for competitive
angle between these two values would represent a geometrically
correct transition state mimic. Since immune recognition of
aromatic groups is usually very strong, it was expected that this
design would be suitable for the production of catalytic
antibodies.
Acridinium 1 was used as a transition state analog for
immunization. For its synthesis, acridine-10-carboxylic acid
served as starting material (Scheme 3). Condensation with
â-alanine benzyl ester gave 9.12 Quaternization of 9 to 10
1
4
inhibition studies.
The bright yellow acridinium compounds 1 and 11 are
1
5
classical Lewis acids. In aqueous medium these compounds
are in rapid chemical equilibrium with their conjugate pseudo-
bases 1-OH and 11-OH, which are colorless (Scheme 4). The
apparent pKa values of 1 and 11 were determined by UV
spectroscopy to be approximately 8.2. Consequently, the acid-
(12) (a) Rapaport, E.; Cass, M. W.; White, E. H. J. Am. Chem. Soc.
(
(
9) Satoh, T.; Namba, K.; Suzuki, S. Chem. Pharm. Bull. 1971, 19, 817.
10) H o¨ geberg, T.; Str o¨ m, P.; Ebner, M.; R a¨ msby, S. J. Org. Chem. 1987,
1972, 94, 3153. (b) Rauhut, M. M.; Sheehan, D.; Clarke, R. A.; Roberts,
B. G.; Semsel, A. M. J. Org. Chem. 1965, 30, 3587.
5
2, 2033.
11) (a) Steith, J.; Defoin, A. Synthesis 1994, 1107. (b) Ritter, A. R.;
(13) Li, T.; Hilton, S.; Janda, K. D. J. Am. Chem. Soc. 1995, 117, 2123.
(14) Reymond, J.-L.; Jahangiri, G. K.; Stoudt, C.; Lerner, R. A. J. Am.
Chem. Soc. 1993, 115, 3909.
(15) The apparent pKa was determined by UV spectroscopy to be 8.2.
See: (a) Selby, A. Acridinium Salts and Reduced Acridines, The Chemistry
of Heterocyclic Compounds; Interscience Publishers, J. Wiley & Sons: New
York, 1973; Vol. 9, p 433.
(
Miller, M. J. J. Org. Chem. 1994, 59, 4602. (c) Ensley, H. E.; Mahadevan,
S. Tetrahedron Lett. 1989, 30, 3255. (d) Davies, A. G.; Dang, H.-S. J. Chem.
Soc., Perkin Trans. 2 1991, 721. (e) Corrie, J. E. T.; Kirby, G. W.; Laird,
A. E.; Machinnon, L. W.; Tyler, J. K. J. Chem. Soc., Chem. Commun. 1978,
2
75.

3
552 J. Am. Chem. Soc., Vol. 118, No. 15, 1996
Bahr et al.
base equilibrium at physiological pH ≈ 7.5 allowed a hetero-
logous immunization experiment16 with two compounds (1:
8
4%, 1-OH: 16%) presenting two different orientations of the
phenyl rings either in a completely product-like orientation (1:
R ) 180°) or in bent orientation possibly quite close to the
actual transition state of the reaction (1-OH: R ) 140°, puckered
1
7
boat form). The presence of a positive charge in the more
product-like acidic form 1 was also expected to avoid the
induction of product-inhibited catalysts.
Antibody Catalysis. Twenty-three monoclonal anti-1 anti-
bodies were produced following standard protocols by fusion
and hybridoma selection from spleen cells of Balb/C mice
immunized with the KLH conjugate of 1 (Scheme 1).18 1-BSA
was used in the screening of hybridoma cell supernatants in an
enzyme-linked immunosorbent assay (ELISA). The retro
Diels-Alder reactions of 2 and 3 to give anthracene 4 take place
spontaneously in aqueous buffer above pH ) 4.0 at the rate of
approximately 2% per hour at 20 °C (t1/2(2) ) 36 h). Catalysis
of the reaction was assayed on ELISA plates by following the
appearance of the anthracene fluorescence. Upon excitation
with UV light the anthracene product 4 showed strong fluores-
cence in the visible range (≈460 nm). Individual wells on a
Figure 1. Nitroxyl release revealed by the conversion of Methemo-
globin to nitrosyl-hemoglobin. Measured at 20 °C in 10 mM phosphate,
160 mM NaCl, pH 7.4. (s) Initial conditions: [5] ) 400 µM, [catalytic
antibody 9D9] ) 4 µM, [Met-Hb] ) 6.25 µM. (- -) t ) 55 min.
(- - -) t ) 180 min. (- ‚ -) t ) 270 min.
96-well half-area tissue culture plate were prepared with each
50 µL containing 1.5 mg/mL of purified antibody and 200 µM
2 or 3 in aqueous 10 mM phosphate, 160 mM NaCl, pH 7.4
with 10% v/v DMF to ensure product solubility. Within 2 h at
0 °C, a bright blue fluorescence of characteristic intensity
2
developed that was visually detectable for concentrations of 4
g2 µM by illuminating the ELISA plate in a dark room with a
standard UV lamp (TLC analysis; excitation at 254 nm or 366
nm). Three anti-1 antibodies catalyzed the retro Diels-Alder
reaction of cycloadduct 2. As a control, none of the 120
unrelated monoclonal antibodies catalyzed the cycloreversion.
Catalysis was inhibited by addition of hapten 1 or 11 (Scheme
3
), confirming that the reactions were taking place in the
antibody combining site. One antibody, 9D9, was characterized
in detail.
Antibody 9D9 chemoselectively catalyzed the formation of
anthracene 4 from isomer 2. No activity was detected with 3
(
Scheme 2). The identity of anthracene 4, which was already
Figure 2. pH-rate profile for the retro Diels-Alder reaction. Apparent
rate Vapp with [2 or 3] ) 200 µM substrate at 20 °C, 50 mM NaCl, 50
mM buffer (see Experimental Section): (b) apparent rate under
catalysis by 4 µM Ab 9D9, the horizontal line represents the average
values of all points; (4) background reaction of 2, apparent pK ) 3.0;
(2) background reaction of 3, apparent pK ) 3.60. The lines were
evident from its fluorescence, was further confirmed by RP-
HPLC analysis. The release of nitroxyl (HNO) by antibody
9
D9 was then investigated. The reaction was carried out in
argon-saturated buffer at pH 7.4, under which conditions nitroxyl
-
(
pKa ) 4.7) was released as its conjugate base NO and trapped
III
19
with ferric (Fe ) hemoglobin. The reaction was monitored
by recording UV spectra of the reaction mixture at 30-min
intervals. An increase in absorbance was observed between 530
and 590 nm with two maxima at 545 and 575 nm, a
characteristic signature for nitrosyl-hemoglobin. In the presence
of 400 µM substrate 2, 4 µM catalytic antibody 9D9, and [Met-
Hb] ) 6.25 µM ([heme] ) 25 µM), nitrosyl-hemoglobin was
calculated from the best linear fit of the experimental points as 1/Vapp
+
)
1/Vuncat + (K/Vuncat)[H ].
by nitroxyl generated from Angeli’s salt (Na2N2O3) at 20 °C,
pH 7.0, with [heme] ) 50 µM under rigorously oxygen free
2
0
conditions.
In our experiment there was no decrease of
catalytic activity of the antibody over a period of several hours,
showing that the released nitroxyl did not react with the antibody
itself.
-
1
formed at an apparent rate of 0.06 µM min (Figure 1). Since
the rate of cycloreversion amounts to 0.33 µM min under these
conditions, the efficiency of the NO transfer was approximately
2
oxidation by remaining oxygen. By comparison, a transfer
efficiency of 70% has been reported for the titration of Met-Hb
-1
Catalytic antibody 9D9 was further characterized by kinetic
analysis. Initial rates were determined by following the increase
of anthracene product 4 by HPLC. Catalysis followed Michael-
-
0%. The loss of nitroxyl is probably due to concurrent
-
1
is-Menten kinetics (KM(2) ) 100 µM, kcat ) 0.07 min , kuncat
-
4
-1
)
3 × 10 min ). Antibody 9D9 showed multiple turnover,
(
16) Suga, H.; Ersoy, O.; Williams, S. F.; Tsumuraya, T.; Margolies,
M. N.; Sinskey, A.J.; Masamune, S. J. Am. Chem. Soc. 1994, 116, 6025.
17) Shambu, M. B.; Koganty, R. R.; Digenis, G. A. J. Med. Chem. 1974,
7, 805.
18) (a) K o¨ hler, G.; Milstein, C. Nature 1975, 256, 495. (b) Enguall, E.
Methods Enzymol. 1980, 70, 419.
19) (a) Henry, Y.; Durocq, C.; Drapier, J.-C.; Servent, D.; Pellat, C.;
Guissani, A. Eur. Biophys. J. 1991, 20, 1. (b) Archer, S. FASEB 1993, 7,
49.
and no product inhibition was observed for [4] up to 60 µM.
A pH-rate profile analysis showed that the antibody catalysis
was constant from pH 4.2 to 10.8 (Figure 2). The catalytic
parameters KM, kcat, and kcat/kuncat were essentially identical at
either pH 6.1, 7.4, or 9.0, suggesting that no general acid/base
(
1
(
(
(20) (a) Bazylinski, D. A.; Hollocher, T. C. J. Am. Chem. Soc. 1985,
107, 7982. (b) Di Iorio, E. E. Methods Enzymol. 1981, 76, 57.
3

A Nitroxyl Synthase Catalytic Antibody
J. Am. Chem. Soc., Vol. 118, No. 15, 1996 3553
Figure 3. Dixon plot for competitive inhibition of antibody 9D9
catalyzed retro Diels-Alder reaction of 2 by acridinium 11. Measured
at 20 °C with [2] ) K
M
in 50 mM buffer, 50 mM NaCl, 10% v/v
(2) ) 114 µM, K (1) ) 17 µM;
4) 50 mM bis-Tris propane, pH 9.0, K (2) ) 71 µM, K (1-OH) )
.9 µM. The lines are the best linear fit of the experimental points.
Figure 4. Nitric oxide (NO) released from substrate 2 by Ab9D9/
SOD couple. Amperometric reading from an NO-meter electrode
immersed in 1 mL of 10 mM phosphate, 160 mM NaCl, pH 7.4, stirred
open to air at 20 °C. Upper trace: reaction of 400 µM 2 with 8 µM Ab
9D9 (anti-1) and 32 µM Cu-Zn SOD. A steady NO level is quickly
restored after flushing with either oxygen or nitrogen. Lower trace:
reaction of 125 µM 2 with 8 µM Ab 9D9 (anti-1) and 32 µM Cu-Zn
SOD.
DMF: (2) 50 mM MES pH 6.1, K
M
i
(
0
M
i
The x-coordinate of their intersection point with the horizontal line at
V ) Vmax gives -K
i
.
or ionizable group influenced either substrate binding or
catalysis, in accordance with the hypothesis that the catalyzed
reaction was a concerted cycloreversion triggered by binding
interactions to the two phenyl rings of the substrate.
The rate for the spontaneous, uncatalyzed reaction was equally
constant throughout the pH range and completely insensitive
to catalysis by buffers. A decrease in the rate of spontaneous
cleavage was observed when the pH was lowered below pH
antibody 9D9, which implies that the hapten is bound in its
acidic form 11 and is not hydrated to the conjugate base upon
binding to the antibody. It appears that antibody 9D9 is
genuinely the result of a heterologous immunization with both
immunogens being partially recognized. Although the weaker
binding of acridinium 11 might be related to its positive charge,
the absence of significant product inhibition (Ki(4) > 60 µM)
suggests that its product-like geometry is also not well recog-
nized by antibody 9D9.
4.0. In fact the trifluoroacetate salts of 2 and 3 were both stable,
which greatly facilitated their preparation, handling, and storage.
Thus only the free base forms of either 2 or 3 were reactive for
cycloreversion. The apparent pK values determined from the
pH-rate profile are pK(2) ) 3.0 and pK(3) ) 3.6. The lower
pK for 2 Vs 3 is correlated with the easier deprotection of formyl
derivative 7 vs 8 (Scheme 2), and probably reflects a stronger
inductive effect of the propanamide Vs the methyl substituent.
The inhibition of catalytic antibody 9D9 by 11 (Scheme 4)
was found to be strongly pH dependent. At pH 9.0 where the
neutral base 11-OH (Scheme 4) is predominant (86%), tight
binding was observed with Ki ) 0.9 µM. However, when the
pH was lowered to pH 6.1, where the acidic form 11 is
predominant (99.2%), a much weaker inhibition was observed,
with Ki ) 16.6 µM. Thus catalytic antibody 9D9 binds to the
neutral pseudo-base 11-OH much more tightly than to the acidic
acridinium form 11, which suggests that the pseudo-base is a
much better transition state analog of the reaction. Interestingly,
the dissociation constant of the antibody 9D9-transition state
complex, given by KTS ) KM/(kcat/kuncat), is comparable to the
Ki of pseudo-base 11-OH with KTS ) 0.3 µM at pH 9.0 and
KTS ) 0.5 µM at pH ) 6.1, suggesting that almost all the energy
Nitric Oxide Release. NO- is rapidly converted to nitric
oxide by oxidation with Cu(II)-Zn superoxide dismutase [at
-
pH 7.4: E°(NO/NO ) ) +0.255 V; E°(Cu(II)-Zn SOD/Cu(I)-
Zn SOD) ) +0.42 V]. Nitric oxide release was followed using
an ISO-NO meter. This instrument measures NO concentrations
by selective amperometric oxidation of nitric oxide to nitrate.
The electrode is shielded from solution by a gas-permeable
membrane, and there is no interference from dissolved nitroxyl
-
(
NO ). The experiment was carried out in a stirred solution
open to air. Under these conditions a steady source of nitric
oxide must be present to obtain a durable concentration since
NO is rapidly removed by air oxidation and by diffusion. As
shown in Figure 4, a steady concentration of NO above 100
nM was obtained in the presence of substrate 2, catalytic
antibody 9D9, and Cu(II)-Zn SOD. This concentration is
sufficient to mediate the biological functions of nitric oxide.
While the order of addition did not matter, each of the three
components was necessary to trigger significant NO production.
Steady NO production was also illustrated by the fact that the
NO level was quickly restored after flushing with either nitrogen
or oxygen.
2
1
available in hapten binding has been converted to catalysis.
The observed affinity of catalytic antibody 9D9 for the hapten,
Ki ) 16.6 µM at pH 6.1, is too strong to be attributed to the
fraction of pseudo-base 11-OH at that pH (0.8%), and must
therefore be attributed to binding by the acridinium form itself.
The UV spectrum of acridinium 11 (10 µM in 10 mM
phosphate, 160 mM NaCl, pH 7.4, λmax (ꢀ) ) 366 nm (1700))
is unaffected by addition of up to 100 µM (7.6 mg/mL) of
Conclusion
The first catalytic antibody for a retro Diels-Alder reaction
has been reported. Catalytic antibody 9D9 (anti-1) catalyzes
the release of anthracene derivative 4 and nitroxyl from prodrug
2
with multiple turnovers. The catalytic antibody binds the
(21) Stewart, J. D.; Benkovic, S. J. Nature 1995, 375, 388.
neutral pseudo-base 1-OH, suggesting that this form of the

3
554 J. Am. Chem. Soc., Vol. 118, No. 15, 1996
Bahr et al.
hapten, and not the product-like conjugate acid 1, is a better
transition state mimic for the reaction. As a consequence, there
is no measurable product inhibition of catalysis. Catalysis is
pH-independent and is probably mediated by interactions
between the two phenyl rings of the substrate and the antibody.
Nevertheless, the observed chemoselectivity for substrate 2
suggests that additional factors also influence catalysis, for
example, electrostatic interactions between the antibody and the
transition state if the reaction proceeds via an asynchronous
transition state with partial negative charge on oxygen, or
hydrogen bonding interactions between an antibody residue and
the HNO bridge.
The biological relevance of the reaction has been demon-
strated by the production of physiological concentrations of nitric
oxide in the presence of the enzyme superoxide dismutase.
Releasing nitroxyl by antibody catalysis at a controlled rate
under physiological conditions also offers a unique opportunity
to study its direct biological effect. Most importantly, both the
rate and location of nitroxyl release from prodrug 2 can be
controlled by the localization of the catalytic antibody. Pre-
liminary studies have shown that either 2 or anthracene 4 has
no measurable cytotoxicity at the concentration used for nitric
oxide production, which suggest that the antibody might be a
useful source of nitric oxide in ViVo.
While the rate accelerations we observe are modest, the
importance of the reaction is prompting us to explore the
production of catalytic antibodies with higher efficiencies of
catalysis. Due to the ease of detection of the anthracene product
by fluorescence, large-scale screening is the obvious route
toward catalysis improvement. Since antibody 9D9 appears to
bind tightly to the pseudo-base 1-OH, this form is probably the
most relevant for inducing catalysis. Immunizations with
haptens featuring stabilized forms of pseudo-base 1-OH have
been initiated to generate libraries of catalytic antibodies.
acetate, 30:1) gave 6 (1249 mg, 4.28 mmol, 80%). Colorless needles:
mp 67 °C (methanol). 1H-NMR (CDCl3, 300 MHz): δ 8.36 (m
, 4H),
, 4H), 4.25 (q, J ) 7.2 Hz, 2H), 4.01 (t, J ) 8.5 Hz, 2H), 3.18
s, 3H), 2.82 (t, J ) 8.5 Hz, 2H), 1.34 (t, J ) 7.2 Hz, 3H). FABLRMS
c
7
(
(
.58 (m
c
+
+
3-NBA-NaI): m/e 315 [M + Na] , 292 [M] .
c) 9-[2′-((2′′-Hydroxyethyl)carbamyl)ethyl]-10-methylanthracene
4). The solution of 6 (1000 mg, 3.42 mmol), ethanolamine (5230
(
(
mg, 85.6 mmol), and NaCN (15 mg, 0.30 mmol) in 30 mL of ethanol
was stirred at room temperature for 36 h. CH Cl and aqueous KH PO
4
2
2
2
solution were then added until a clear two-phase system was obtained.
The pH of the aqueous phase was adjusted to ≈7 (2 M HCl). The
2 2
aqueous phase was extracted with CH Cl , the organic phase was
washed (H
Flash chromatography (CH
CHCl ) afforded 4 (893 mg, 2.91 mmol, 85%). Slightly yellow
needles: mp 186 °C (CHCl
2
O) and dried (MgSO
4
), and the solvent was evaporated.
2
Cl /methanol, 20:1) and crystallization
2
(
3
1
3
). H-NMR (CDCl3, 300 MHz): δ 8.32
c c
m , 4H), 7.49 (m , 4H), 5.60 (s br, 1H), 3.95 (t, J ) 7.9 Hz, 2H), 3.48
(
(
t, J ) 4.9 Hz, 2H), 3.23 (q, J ) 4.9 Hz, 2H), 3.04 (s, 3H), 2.63 (t, J
+
)
[
7.9 Hz, 2H). FABLRMS (3-NBA-NaI): m/e 330 [M + Na] , 307
M] . UV (PBS, pH 7.4): λmax (ꢀ) 260 (1.1 × 10 ), 360 (568), 378
+
5
(928), 400 (905).
(d) 9,10-Epoxyimino-12-formyl-9-methyl-10-[2′-((2′′-hydroxyeth-
yl)carbamyl)ethyl]anthracene (7) and 9,10-Epoxyimino-12-formyl-
9-[2′-((2′′-hydroxyethyl)carbamyl)ethyl]-10-methylanthracene (8).
+
-
To the clear solution of 4 (300 mg, 0.98 mmol) and [Me
665 mg, 1.95 mmol) in 2 mL of absolute DMF was added the solution
3 4
N Bn]IO
(
of HCONHOH (119 mg, 1.95 mmol) in 2 mL of absolute DMF over
a period of 30 min at ambient temperature. The mixture turned yellow.
It was stirred for an additional 1 h and then poured on ice. CH
and 0.1 M aqueous Na
phase system was present. The aqueous phase was extracted (CH
and the combined organic phases were washed (NaHCO , H
dried (CaCO ). The solvent was removed in Vacuo at room temper-
2
Cl
2
2 2 5
S O solution were added until a clear two-
2 2
Cl ),
3
2
O) and
3
ature. The oily, slightly yellow residue was taken up in H
2
O/CH
3
CN/
TFA 80/20/0.1 and subjected to preparative RP-HPLC. Besides
recovered 4 (60 mg, 0.20 mmol, 20%), 7 (108 mg, 0.29 mmol, 30%)
and 8 (105 mg, 0.29 mmol, 30%) were isolated as colorless, crystalline
substances. 7: mp 95 °C dec. 1H-NMR (CD
OD, 500 MHz): δ 8.38
, 2H), 7.47 (m , 2H), 7.30 (m , 4H),
.66 (t, J ) 5.7 Hz, 2H), 3.42-3.35 (m, 4H), 2.82 (t, J ) 7.8 Hz, 2H),
3
(
s, 1H), 8.20 (s br, 1H), 7.52 (m
c
c
c
Experimental Section
3
13
General Remarks. Reagents were purchased from Aldrich or Fluka.
Solvents were A.C.S. grade from Fisher. All chromatographies (flash)
were performed with Merck Silicagel 60 (0.040-0.063 mm). Prepara-
tive HPLC was done with Fisher Optima grade acetonitrile and ordinary
deionized water using a Waters prepak cartridge 500 g installed on a
Waters Prep LC 4000 system from Millipore, flow rate 100 mL/min,
3
2.24 (s, 3H). C-NMR (CD OD, 125 MHz): δ 175.0, 152.2, 141.7,
140.3, 129.0, 122.6, 122.5, 121.8, 81.2, 67.1, 61.6, 43.3, 31.7, 23.9,
+
14.9. FABHRMS (3-NBA-NaI): m/e calcd for [C21
367.1658, found 367.1641. 8: mp 97 °C dec. 1H-NMR (CD
MHz): δ 8.27 (s, 1H), 8.15 (s br, 1H), 7.49 (m , 4H), 7.33 (m
3.65 (t, J ) 5.7 Hz, 2H), 3.37 (q, J ) 5.7 Hz, 2H), 3.11 (m , 2H), 2.82
OD, 125 MHz): δ 175.8, 153.7,
141.7, 141.6, 129.0, 122.7, 122.6, 121.8, 83.0, 63.5, 61.5, 43.3, 31.0,
22 2 4
H N O + H]
3
OD, 500
, 4H),
c
c
c
-
1
13
gradient + 0.5% min CH
3
CN, detection by UV at 254 nm. TLC
c 3
(m , 2H), 2.47 (s, 3H). C-NMR (CD
was performed with fluorescent Merck F254 glass plates. NMR spectra
were recorded on a Brucker AM-300 MHz or AM-500 MHz instrument.
25.8, 14.5. FABHRMS (3-NBA-NaI): m/e calcd for [C21
22 2 4
H N O +
3
+
Chemical shift δ are given in ppm and coupling constant J in hertz.
H] 367.1658, found 367.1642.
UV spectra were recorded on a HP-8452A instrument. MS, HRMS
(e) 9,10-Epoxyimino-9-methyl-10-[2′-((2′′-hydroxyethyl)carbam-
yl)ethyl]anthracene (2). The solution of 7 (100 mg, 0.27 mmol) in 5
mL of methanol and 0.5 mL of TFA was stirred at 0 °C for 24 h. The
(high-resolution mass spectra) were provided by the Scripps Research
Institute facility (Gary Siuzdak). NO measurements were performed
with an ISO-NO meter from World Precision Instruments Inc.
2
reaction was stopped by addition of 30 mL of H O, and the crude
(
A) Synthesis. (a) 9-[2′-(Ethoxycarbonyl)vinyl]-10-methylan-
product was purified by RP-HPLC. 2 (117 mg, 0.26 mmol, 95%) was
isolated as its TFA salt and storred at -78 °C. Colorless needles: mp
thracene (5). 10-Methylanthracene-9-carboxaldehyde (2.50 g, 11.4
mmol) and (carbethoxymethylene)triphenylphosphorane (6.38 g, 18.3
64 °C dec. 1H-NMR (CD
3
c c
OD, 300 MHz): δ 7.61 (m , 4H), 7.42 (m ,
mmol) were dissolved in 50 mL of CH
to reflux for 36 h. Removal of the solvent and flash chromatography
hexane/ethyl acetate, 30:1) yielded 5 (3.08 g, 10.6 mmol, 93%).
2
Cl
2
. The mixture was heated
4H), 3.62 (t, J ) 5.6 Hz, 2H), 3.33 (t, J ) 5.6 Hz, 2H), 3.23 (t, J )
6.9 Hz, 2H), 2.91 (t, J ) 6.9 Hz, 2H), 2.28 (s, 3H). FABHRMS (3-
+
+
(
NBA): m/e calcd for [C20
H
22
N
2
O
3
+ H] 339.1709, found 339.1719.
1
Yellow crystals: mp 89 °C (hexane). H-NMR (CDCl3, 300 MHz): δ
(f) 9,10-Epoxyimino-9-[2′-((2′′-hydroxyethyl)carbamyl)ethyl]-10-
methylanthracene (3). The solution of 8 (100 mg, 0.27 mmol) in 5
mL of methanol and 0.5 mL of TFA was stirred at 20 °C for 12 h. The
8
1
.67 (d, J ) 16.5 Hz, 1H), 8.30 (m
c c
, 4H), 7.52 (m , 4H), 6.40 (d, J )
6.5 Hz, 1H), 4.42 (q, J ) 7.3 Hz, 2H), 3.08 (s, 3H), 1.45 (t, J ) 7.3
+
+
Hz, 3H). FABLRMS (3-NBA-NaI): m/e 313 [M + Na] , 290 [M] .
b) 9-[2′-(Ethoxycarbonyl)ethyl]-10-methylanthracene (6).
1550 mg, 5.35 mmol) and NiCl ‚H O (425 mg, 1.79 mmol) were
dissolved in 60 mL of ethanol. NaBH (202 mg, 5.35 mmol) was added
2
reaction was stopped by addition of 30 mL of H O, and the crude
(
5
product was purified by RP-HPLC. 3 (115 mg, 0.25 mmol, 93%) was
isolated as its TFA salt and stored at -78 °C. Colorless needles: mp
(
2
2
63 °C dec. 1H-NMR (CD
OD, 300 MHz): δ 7.61 (m
, 4H), 7.45 (m
3
c
c
,
4
at ambient temperature portionwise over a period of 2 h. The reaction
mixture turned black. It was stirred for an additional 4 h and then
4H), 3.61 (t, J ) 5.7 Hz, 2H), 3.36 (t, J ) 5.7 Hz, 2H), 3.20 (m
c
, 2H),
2.76 (m
c
, 2H), 2.36 (s, 3H). FABHRMS (3-NBA): m/e calcd for
+
acidified with 2 M HCl. The aqueous phase was extracted (CH
and the organic phase was washed (NaHCO , H O) and dried (MgSO
Evaporation of the solvent and flash chromatography (hexane/ethyl
2
Cl
2
),
[C20
H
22
N
2
O
3
+ H] 339.1709, found 339.1715.
3
2
4
).
(g) N-[2′-(Benzyloxycarbonyl)ethyl]-9-acridinecarboxamide (9).
9-Acridinecarboxylic acid hydrate (97%, 1000 mg, 4.34 mmol) was

A Nitroxyl Synthase Catalytic Antibody
J. Am. Chem. Soc., Vol. 118, No. 15, 1996 3555
dissolved in 10 mL of refluxing SOCl
obtained, and all volatiles were removed in Vacuo. The slightly yellow,
crude acid chloride was suspended in 15 mL of CH Cl . A solution of
â-alanine benzyl ester p-toluenesulfonate (1980 mg, 5.64 mmol) and
NEt (1317 mg, 13.02 mmol) in 15 mL of CH Cl was added at room
temperature, and the mixture was stirred for 14 h. The organic phase
was washed (NaHCO , NaCl) and dried (MgSO ). Removal of the
solvent and flash chromatography (hexane/ethyl acetate, 1:1) yielded
(1487 mg, 3.86 mmol, 89%). Light yellow crystals: mp 153 °C
2
. After 6 h a clear solution was
buffer (pH 7.4), and 200 µL of the solution of the activated hapten
was added to 1800 µL of a solution of bovine serum albumin (BSA,
4.4 mg/mL) in 50 mM phosphate buffer (pH 7.4). The mixtures were
kept at 4 °C for 24 h and were then stored at -18 °C.
B. Antibody Assays. (a) Antibodies. Monoclonal antibodies
against the KLH conjugate of 1 were produced using standard
procedures and were obtained from ascites fluid grown from the
individual hybridoma cell lines.17 Each antibody was purified to
homogeneity by ammonium sulfate precipitation followed by anion
exchange and protein G chromatography, dialyzed into 10 mM
phosphate, 160 mM NaCl, pH 7.4, and its concentration estimated by
UV at 280 nm as c (mg/mL) ) Abs/1.4.
2
2
3
2
2
3
4
9
1
(
hexane/CHCl
3
). H-NMR (CDCl
3
, 300 MHz): δ 8.15 (d, J ) 8.4
, 2H), 7.49 (m , 2H), 7.21
Hz, 2H), 7.91 (d, J ) 8.5 Hz, 2H), 7.71 (m
c
c
c
(m , 5H), 6.91 (s br, 1H), 5.11 (s, 2H), 3.98 (q, J ) 6.0, 2H), 2.88 (t,
+
J ) 6.0 Hz, 2H). FABLRMS (3-NBA-NaI): m/e 407 [M + Na] ,
(b) Stock Solutions. Substrate 2 and 3, obtained from HPLC
purification, were stored at -78 °C as 5 mM stock solution in 0.1%
v/v CF COOH in water. 1 and 11 were used as 5 mM stock solutions
3
+
3
84 [M] .
(
h) 9-(N-[2′-(Benzyloxycarbonyl)ethyl]carbamyl)-10-methylacri-
dinium Sulfate (10). 9 (50 mg, 0.13 mmol) and freshly neutralized
CO ) (CH SO (20 mg, 0.16 mmol) were heated in 1 mL of toluene
to reflux for 2.5 h. Removal of all volatiles and flash chromatography
CH Cl /methanol, 5:1) afforded 10 (55 mg, 0.12 mmol, 95%, calcd
as sulfate salt). Bright yellow crystals: mp 158 °C (ether). H-NMR
CD OD, 300 MHz): δ 8.70 (m , 2H), 8.42-8.35 (m, 4H), 7.88 (m
H), 7.42-7.25 (m, 5H), 5.18 (s, 2H), 4.88 (s, 3H), 3.98 (t, J ) 6.3,
of the HPLC purified salt in water.
(K
2
3
)
3 2
4
(c) Kinetic Measurements. Each reaction (200 µL) was initiated
by adding 10 µL of a prediluted substrate solution to 190 µL of the
appropriate buffer (50 or 100 mM buffer, 50 mM NaCl) containing
10% v/v DMF and, when used, the antibody at 0.3-1.5 mg/mL
concentration. The following buffers were used: KCl/HCl for 1.0 e
pH e 2.0; glycine/HCl for 2.2 e pH e 3.6; citrate/phosphate for 2.6
e pH e 4.4; citrate for 3.5 e pH e 6.0; phosphate (PBS: phosphate
buffer saline) for 6.0 e pH e 8.0; bis-Tris propane (1,3-bis[tris-
(hydroxymethyl)methylamino]propane) for 6.5 e pH e 9.5; CAPS (3-
(cyclohexylamino)-1-propanesulfonic acid) for 10.0 e pH e 11.0; MES
(morpholinoethanesulfonic acid) for pH 6.1; HEPES (4-(2-hydroxy-
ethyl)-1-piperazinylethanesulfonic acid) for pH 7.5; Tris (tris(hy-
droxymethyl)aminomethane) for pH 8.0. None of these buffers affected
either the background or the antibody-catalyzed retro Diels-Alder
reaction with 2 or 3.
(
2
2
1
(
3
c
c
,
2
2
+
H), 2.90 (t, J ) 6.3 Hz, 2H). FABLRMS (3-NBA): m/e 399 [M] .
i) 9-(N-[2′-carboxylethyl]carbamyl)-10-methylacridinium Trif-
luoroacetate (1). A solution of 10 (50 mg, 0.11 mmol, sulfate salt) in
(
3
2
mL of 0.1 M aqueous KOH/methanol 2:1 was heated to reflux for
0 min. The colorless mixture was acidified with 2 M HCl (pH 2).
The bright yellow color and fluorescence reappeared. The crude
product was subjected to preparative RP-HPLC. 1 (42 mg, 0.10 mmol,
8
9%) was isolated as its TFA salt. Yellow crystals: mp 203 °C dec. 1:
1
H-NMR (CD
3 c
OD, 300 MHz): δ 8.82 (d, J ) 9.3 Hz, 2H), 8.51 (m ,
4
H), 8.10 (m , 2H), 5.01 (s, 3H), 4.00 (t, J ) 6.3, 2H), 2.88 (t, J ) 6.3
c
For activity screening, the reaction progress was followed visually
by running the reactions in individual wells of a 96 well half-area tissue
culture plate and illuminating the plate in a dark room with a standard
laboratory lamp for TLC at either 254 or 366 nm.
1
Hz, 2H). H-NMR (D
2
O, 300 MHz): δ 8.40 (d, J ) 9.3 Hz, 2H),
8
2
1
.15 (m
c
, 4H), 7.78 (t, J ) 7.6 Hz, 2H), 4.68 (s, 3H), 3.78 (t, J ) 6.4,
13
H), 2.68 (t, J ) 6.4 Hz, 2H). C-NMR (CD
65.9, 155.5, 143.4, 140.5, 140.4, 129.9, 124.0, 120.0, 39.6, 37.4, 34.3.
3
OD, 125 MHz): δ 175.0,
Kinetic measurements were done by following the formation of
anthracene product 4 over a period of 2 to 3 h by RP-HPLC (Microsorb
MV C-18, 300 Å poresize, 0.45 × 22 cm, isocratic elution at 1.5 mL/
min with 35.5% CH CN, 64.4% H O, 0.07% CF COOH, detection by
+
FABLRMS (3-NBA): m/e 309 [M] . FABHRMS (3-NBA): m/e calcd
for [C18
+
1
H
17
N
2
O
3
] 309.1239, found 309.1247. 1-OH: H-NMR (D
2
O/
K
2
CO , 300 MHz): δ 7.10 (m
3
c
, 4H), 6.89 (d, J ) 8.9 Hz, 2H), 6.75 (t,
3
2
3
J ) 7.2 Hz, 2H), 3.28 (t, J ) 6.8, 2H), 3.12 (s, 3H), 2.20 (t, J ) 6.8
Hz, 2H).
UV 254 nm) against propyl-4 hydroxybenzoate as internal standard
(t (4) ) 8.8 min, t (propyl 4-hydroxybenzoate) ) 6.2 min). The net
R
R
(j) 9-(N-[2′-((2′′-Hydroxyethyl)carbamyl)ethyl]carbamoyl)-10-me-
catalytic rate Vcat was obtained by subtracting the apparent rate in a
sample without antibody Vuncat from the apparent rate in an identical
sample with antibody. Lineweaver-Burk analyses were done at [Ab
9D9] ) 0.3 mg/mL, using five different substrate concentrations [2]
) 50, 100, 150, 200, 400 µM. The uncatalyzed rate Vuncat was directly
proportional to the substrate concentration within that range. Plots of
thylacridinium Trifluoroacetate (11). A solution of 10 (45 mg, 0.10
mmol, sulfate salt) and ethanolamine (305 mg, 5.0 mmol) in 3 mL of
ethanol was stirred at room temperature for 36 h. All volatiles were
removed in Vacuo. The oily residue was dissolved in H
2
O/0.1% TFA,
and the crude product was purified by RP-HPLC. 11 (37 mg, 0.08
1
2
mmol, 80%, TFA salt), yellow crystals: mp 184 °C. 11: H-NMR
1/Vcat vs 1/[2] were linear (5 points, r > 0.990). The results were as
-
2
-1
(
8
CD
3
OD, 300 MHz): δ 8.80 (d, J ) 9.2 Hz, 2H), 8.51-8.38 (m, 4H),
follows: at pH 6.1 (MES buffer) k ) 7.2 × 10 min , kuncat ) 3.2
cat
-
4
-1
.02 (t, J ) 7.7 Hz, 2H), 4.92 (s, 3H), 3.97 (t, J ) 6.4, 2H), 3.60 (t,
× 10 min , KM ) 114 µM; at pH 7.4 (PBS buffer) k ) 7.3 ×
cat
10 min-1, kuncat ) 3.3 × 10 min , KM ) 125 µM; at pH 9.0 (bis-
-
2
-4
-1
J ) 5.7, 2H), 3.34 (t, J ) 5.7, 2H), 2.73 (t, J ) 6.4 Hz, 2H).
+
-2
-1
-4
-1
FABLRMS (3-NBA): m/e 352 [M] . FABHRMS (3-NBA-NaI): m/e
Tris propane buffer) k ) 7.0 × 10 min , kuncat ) 2.8 × 10 min
,
cat
+
calcd for [C20
H N
22 3
O
3
]
352.1661, found 352.1668. UV (0.1% TFA/
KM ) 71 µM. Quantitative inhibition studies with hapten 1-OH at pH
9.0 showed that the antibody sample had two catalytic sites per antibody
molecule, with [Ab 9D9] ) 0.3 mg/mL corresponding to 4 µM active
sites. The catalytic activity kcat is reported per active site.
4
H
2
O, pH 2): λmax (ꢀ) 260 (8.4 × 10 ), 366 (1700). 11-OH: UV (CAPS
buffer, pH 10.8): λmax (ꢀ) 285 (1790).
k) Conjugation of Hapten 1 with Carrier Proteins BSA and
KLH. 1-(3-(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride
EDC) (5.4 mg, 0.028 mmol) in 10 µL of H O and N-hydroxysulfos-
uccinimide (Sulfo-NHS) (6.1 mg, 0.028 mmol) in 10 µL of H O were
(
(
2
Acknowledgment. This work was supported in part by the
Humboldt Stiftung, Germany (N.B.). We thank Mary Wolfe
and Diane Schloeder for hybridoma production and Dr. Thomas
Horn for assistance in the NO measurements.
2
added successively to a solution of 1 (8.0 mg, 0.019 mmol) in 400 µL
of DMF. After a 24-h incubation at ambient temperature, 200 µL of
the solution of the activated hapten was added to 1800 µL of a solution
of keyhole limpet hemocyanin (KLH, 4.4 mg/mL) in 50 mM phosphate
JA9538694