High-performance caged nitric oxide: A new molecular design, synthesis, and photochemical reaction

Full text of DOI:10.1021/ja962839d

3840
J. Am. Chem. Soc. 1997, 119, 3840-3841
High-Performance Caged Nitric Oxide: A New
Molecular Design, Synthesis, and Photochemical
Reaction
Shigeyuki Namiki, Tatsuo Arai, and Ken Fujimori*
Department of Chemistry, UniVersity of Tsukuba
Tsukuba, 305 Japan
ReceiVed August 13, 1996
Endogenous nitric oxide is an important messenger molecule
in both the brain1 and blood vessels2 and acts as a cytotoxic
agent in the immune system.3 Since Kaplan reported the
photolytic release of adenosine 5′-triphosphate (ATP) from a
protected derivative that has no bioactivity, i.e., ATP o-
nitrobenzyl ester,4 these photosensitive precursors, called caged
compounds, have become an important tool in the biosciences,
especially in exploring signal transductions in living systems.1b,5,6
The great majority of known caged compounds are o-
nitrobenzyl esters of desired bioactive molecules.5 Soon after
we started this work, Makings and Tsien reported o-nitrobenzyl
ester caged nitric oxides (NOs).7 The quantum yields of NO
formation (ΦNO) for their photochemical uncaging reaction were
very poor (0.02-0.05) and NO chemical yields were only 30-
54%.7 The efficiency of K2Ru(NO)Cl5, used as a caged NO
by Murphy et al., is also very low.6 Here, we report an
extremely high-performance caged NO having a new molecular
design.
Figure 1. Electronic spectra. IA: in 0.1 M sodium phosphate buffer,
pH 7.4. IIA: Transient spectrum obtained by LFP of IA in 0.1 M
sodium phosphate buffer, pH 7.4. IIIA: in 0.1% aqueous phosphoric
acid.
Scheme 1
Scheme 2
The following three factors were considered in our molecular
design of caged NO: (a) Because NO is a free radical, two NO
molecules must be produced from a single caged NO molecule
so that the remaining cage counterpart is a nonradical species
after the photochemical reaction to produce the NO (uncaging).
formation of new C-N π-bondings in the broken-cage residue
(III). Although homolytic cleavage of the N-N bond in mono-
N-nitroso compounds occurs under irradiation, the radical pairs
recombine, providing a starting material or a C-nitroso com-
pound.10,11
(b) Uncaging proceeds in a high ΦNO. To achieve high ΦNO
,
the weaker the bond between the NO and the cage counterpart
in the caged NO, the better. If the bond is very weak, however,
thermal uncaging occurs even at room temperature.8 (c) The
caged NO requires a large molar extinction coefficient.
IA was synthesized by the pathways shown in Scheme 2.12a
IB was prepared by the nitrosation of N,N′-dimethyl-p-phen-
ylenediamine (IVB) with nitrous acid.12b Caged NOs have
strong absorption bands in the UV region (IA λmax ) 300 nm
(ꢀ ) 13.5 mM-1 cm-1) in a phosphate buffer of pH 7.4 (Figure
1); IB λmax ) 299 nm (ꢀ ) 14.05 mM-1 cm-1) in methanol).
Without light, a solution of IA in a buffer of pH 7.4 at 37 °C
initiated no reaction.
Fulfilling the above requirements, we designed N,N′-bis-
(carboxymethyl)-N,N′-dinitroso-p-phenylenediamine (IA) as a
water-soluble caged NO and N,N′-dimethyl-N,N′-dinitroso-p-
phenylenediamine (IB) as a lipid-soluble caged NO.9 The
homolysis of one N-N bond may induce homolysis of the
second N-N bond, producing the second NO either stepwise
or concerted (Scheme 1). The driving force of the expected
reaction is the formation of the two stable free radicals and
HPLC analysis of the reaction mixture after partial photolysis
yielded only one peak besides the peak of the remaining starting
IA under different conditions. The broken cage residue of IA
was identified as IIIA by comparison with the retention time
and electronic spectrum (λmax 290 nm) of the authentic sample
prepared by the oxidation of amino acid IVA with potassium
ferricyanate (Scheme 3).13
Inter- and intracellar NO-mediated signal transductions are
initiated by the coordination of NO with guanylate cyclase, a
heme enzyme.14 To mimic this, IA was photolyzed in a
phosphate buffer solution of pH 7.4 containing mesotetrakis-
(p-sulfonatophenyl)porphinatocobalt (TPPSCo), a heme model,
* Author to whom correspondence should be addressed: kfujimor@staff.
chem.tsukuba.ac.jp (e-mail).
(1) (a) Shibuki, K.; Okada, D. Nature 1991, 349, 326-328. (b) Lev-
Ram, V.; Makings, L. R.; Keitz, P. F.; Kao, J. P. Y.; Tsien, R. Y. Neuron
1995, 15, 407-415. (c) O’Dell, T. J.; Huang, P. L.; Dawson, T. M. Science
1994, 265, 542-546.
(2) (a) Ignarro, L. J.; Byrns, R. E.; Buga, G. M.; Wood, K. S. Circ. Res.
1987, 60, 82-92. (b) Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature
1987, 327, 524-526. (c) Furchgott, R. F. In Vasodilation; Vanhoute, P.
D., Ed.; Paven Press: New York, 1988; pp 401-414. (d) Feelisch, M.;
Poal, M.; Zamora, R.; Deussen, A.; Moncada, S. Nature 1994, 368, 62-
65.
(3) (a) Iyenger, R.; Stuehr, D. J.; Marletta, M. A. Proc. Natl. Acad. Sci.
U.S.A. 1987, 84, 6369-6373. (b) Adams, L. B.; Hibbs, J. B., Jr.; Taintor,
R. R. J. Immunol. 1990, 144, 2725-2729.
(9) In the distribution equilibrium experiments between benzene and 0.1
M sodium phosphate buffer solution at pH 7.4, distribution ratios for IA
(KI ) and IB (KI ) were found to be KI < 10-3 and KI > 5 × 102, where
(4) Kaplan, J. H.; Forbush, B., III; Hoffmann, J. F. Biochemistry 1978,
17, 1929-1935.
A
B
A
B
(5) Adams, S. R.; Tsien, R. Y. Ann. ReV. Physiol. 1993, 55, 755-784.
(6) Murphy, K. P. S. J.; Williams; Bettache, N.; Bliss, T. V. P.
Neuropharmacol. 1994, 33, 1375-1385.
KI ) [I]benzene/[I]water.
(10) Chow, Y. L. In Chemistry of Functional Groups, Supplement F:
The Chemistry of Amino, Nitroso, and Nitro Compounds and their
DeriVatiVes Part 1; Patai, S., Ed.; John Wiley & Sons: Chichester, 1982;
p 181-290.
(7) Makings, L. R.; Tsien, R. Y. J. Biol. Chem. 1994, 269, 6282-6285.
(8) Thionitrite is a good example. Thionitrite photolysis readily produces
NO;8a,b S-N bond homolysis, however, occurs at room temperature.8c,d (a)
Barrett, J.; Debenham, D. F.; Glauser, J. Chem. Commun. 1955, 248-249.
(b) Barrett, J.; Fitzgibbons, L. J.; Glauser, J.; Still, R. H.; Young, P. N. W.
Nature 1966, 211, 848. (c) Arnelle, D. R.; Stamler, J. S. Arch. Biochem.
Biophys. 1995, 318, 279-285. (d) Trace heavy metal ions catalyze
thionitrite decomposition to generate NO: Askew, S. C.; Barnett, D. J.;
McAninly, J.; Williams, D. L. H. J. Chem. Soc., Perkin Trans 2 1995, 742-
745.
(11) The photolysis of N-nitroso-N-methylaniline, for example, initiates
photochromism of the compound through recombination of the intermediary
radical pair: Hoshino, M.; Kokubun, H.; Koizumi, M. Bull. Chem. Soc.
Jpn. 1970, 43, 2796-2800.
(12) (a) For IA: mp 145-6 °C (dec). Anal. Calcd for C10H10N4O6: C,
42.55; H, 3.55; N, 19.86. Found: C, 42.49; H, 3.62; N, 19.55. (b) For IB:
mp 120-1 °C. Anal. Calcd for C8H10N4O2: C, 49.57; H, 5.18; N, 28.83.
Found: C, 49.29; H, 5.20; N, 28.63.
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Communications to the Editor
J. Am. Chem. Soc., Vol. 119, No. 16, 1997 3841
reveal that the photolysis of I occurs stepwise via radical
intermediate II, which disappears via elimination of the second
NO and recombination to regenerate I.
According to this mechanism, II disappears via the kinetic
equation -d[II]/dt ) (kr[NO] + kd)[II]. In the presence of a
large excess NO, the bimolecular reaction predominates over
the unimolecular NO extrusion reaction of II to yield kobsd
)
kr[NO]. From the solubility of NO at 23 °C (1.95 mM)16 and
the kobsd value, the second-order rate constant (kr) for recom-
Figure 2. Difference spectra from photolysis of I in the presence of
porphinatocobalt. (A) Photolysis of IA: [IA]0 ) 1.11 mM, [TPPSCo]0
) 73.8 µM in 0.1 M phosphate buffer of pH 7.4; t ) 0, 2, 4, 6, 8, 10,
and 12 min. (B) Photolysis of IB: [IB]0 ) 0.78 mM, [TPPCo]0 ) 70
µM in THF; t ) 0, 0.5, 1, 1.5, 3, 4, and 5 min.
bination of IIA and NO is calculated to be 1.38 × 108 M-1 s-1
.
Since concentrations of caged NOs are 10-8-10-6 M in
biological applications, the bimolecular recombination of NO
and II should be negligible in practical use.
When the LFP of IB benzene solutions having a constant
initial concentration in the presence of a large excess TPPCo
was performed, time courses of rising absorption at 546 nm
due to the formation of TPPCo(NO) obeyed the first-order
kinetic equation (Figure 3B, trace b). Pseudo-first-order rate
constants correlated linearly with [TPPCo] to obtain the second-
order rate constant for the binding of NO and TPPCo (2.80 ×
109 M-1 s-1), which agrees with previously reported 7 × 108
M-1 s-1 in 2-methyltetrahydrofuran.15 When the benzophenone
T-T band absorbance (ꢀ ) 10 300) at 532.5 nm was used as a
standard,17 the quantum yield for the formation of TPPCo(NO),
Figure 3. (A) Time courses for transient absorption at 405 nm in LFP
of 69.6 µM IA in pH 7.4 sodium phosphate buffer solutions. (a) Under
Ar. (b) Under NO. (B) LFP of IB in benzene under Ar. (a) Time
course for transient absorption at 405 nm, [IB]0 ) 63.6 µM. (b) Time
course for raising absorption of TPPCo(NO) at 546 nm in the presence
of 55 µM TPPCo, [IB]0 ) 14.7 µM. All solid lines are first-order
kinetics simulation curves.
Φ
TPPCo(NO), was determined to be 1.97 ( 0.33 from LFP with
[IB] ) 7.2 µM and [TPPCo] ) 60 µM. In low TPPCo
concentration, not all NOs could be captured by TPPCo. As
was predicted in our molecular design, a single photon generates
almost two NO molecules from these caged NOs.
To further substantiate uncaging efficiency, a solution of IA
(21.3 µM) in a 0.1 M sodium phosphate buffer solution at pH
7.4 was irradiated until all IA was photolyzed under Ar in the
presence of 0.23 mM 2-(4-carboxyphenyl)-3,3,4,4-tetrameth-
ylimidazoline-1-oxyl 3-oxide (carboxy-PTIO), which reacts with
the NO fading blue (λmax 561 nm, ꢀ ) 970 M-1 cm-1).18 Light
having a 300 ( 0.5 nm wavelength was provided from a 500
W xenon lamp through a monochromater. One mole of IA was
found to consume 1.98 ( 0.01 mol of carboxy-PTIO. Similarly,
in irradiation of a tetrahydrofuran solution containing IB and
large excess amounts of 2-phenyl-3,3,4,4-tetramethylimidazole-
1-oxyl 3-oxide (PTIO), 1 mol of IB reacted with 2.05 ( 0.10
mol of PTIO. These observations reveal that 1 mol of caged
NOs quantitatively yields 2 mol of NO upon uncaging.
Scheme 3
in an evacuated, sealed UV cuvette. IB was uncaged in benzene
containing mesotetraphenylporphinatocobalt (TPPCo). The
spectrum change in photolysis (Figure 2) shows difference
spectra obtained by subtracting the initial spectrum from those
recorded at different photolytic uncaging time intervals. Dif-
ference spectra are identical to those obtained using authentic
TPPSCo(NO) and TPPCo(NO) prepared by reacting TPPSCo
and TPPCo with commercially obtained NO gas (λmax 542 nm,
The values of the quantum yield consuming PTIO, ΦPTIO
,
were determined using a potassium ferrioxalate actinometer.19
Values of the experimentally observed ΦPTIO in uncaging were
1.87 ( 0.08 with 300 nm light, 1.6 ( 0.1 with 350 nm light for
IA, and 2.02 ( 0.03 for IB with 300 nm light.20 The quality of
the caged compound is expressed by ꢀΦ. The ꢀΦ values of IA
were 2.7 × 104 M-1 cm-1 with 300 nm light and 6 × 103 M-1
cm-1 with 350 nm light. This is much greater than those for
reported o-nitrobenzyl ester caged NOs (ꢀΦ ) 15-70 M-1
cm-1).7
λ
min 519 nm for TPPSCo(NO) and λmax 545 nm, λmin 520 nm15
for TPPCo(NO)). The photolytic reactions of caged NOs are
clean (Figure 2).
When solutions of IA in pH 7.4 sodium phosphate buffer and
IB in benzene were subjected to laser flash photolysis (LFP)
with 308 nm wavelength and a 20 ns laser pulse at 23 °C,
transient species having visible absorption band (Figure 1)
appeared immediately. The time courses of the decreasing
absorbance at 405 nm consist of fast phases (0, Figure 3, trace
a) and slow phases (O, Figure 3, trace a). Slow phases obeyed
the first-order kinetic equation well (s, Figure 3) to yield kd )
2.96 × 104 s-1 for IA in water and kd ) 2.15 × 105 s-1 for IB
in benzene. Molecular oxygen did not alter the lifetime of the
transient species, but NO accelerated decay. In a NO-saturated
aqueous solution, the slow phase disappeared and the entire
The assay of caged NOs on rat aorta vasorelaxation is
currently underway at this laboratory. Preliminary results reveal
that caged NOs are very high-performance and have no
measurable toxicity.
JA962839D
(16) Lange’s Handbook of Chemistry, 12th ed.; Dean, J. A., Ed.;
McGraw-Hill: New York, 1979; p 10-5.
decay curve fitted a first-order kinetic equation with kobsd
)
2.69 × 105 s-1 (Figure 3A, trace b). These observations and
(17) Land, E. J. Proc. R. Soc. London, Ser. A 1968, 305, 457-471.
(18) Akaike, T.; Yoshida, M.; Miyamoto, Y.; Sato, K.; Kohono, M.;
Sasamoto, K.; Miyazaki, K.; Ueda, S.; Maeda, H. Biochemistry 1993, 32,
827-832.
the NO trapping experiments, below, with TPPCo in benzene
(13) UV spectrum for IIIA (Figure 1) was recorded by a multichannel
detector for HPLC at the height of HPLC peaks. Quinoimines are unstable,
so IIIA was consecutively converted to secondary products. Since we were
not interested in the reactions of IIIA, we did not further examine secondary
products.
(19) Murov, S. L. Handbook of Photochemistry; Marcel Dekker, Inc:
New York, 1973; pp 119-123.
(20) During the 25 ns interval after laser pulse irradiation of an aqueous
A solution, a sharp decay in absorbance accounting for several percent of
I
(14) Traylor, T. G.; Duprat, A. F.; Sharma, V. S. J. Am. Chem. Soc.
1993, 115, 810-811 and references cited therein.
(15) Hoshino, M.; Arai, S.; Yamaji, M.; Hama, Y. J. Phys. Chem. 1986,
90, 2109-2111.
all IIA decay occurred. This may be attributed to the recombination of
radicals pair in the solvent cage to yield IA. No such phenomenon was
observed, however, in LFP of IB in benzene. This may explain the
somewhat lower quantum yield in water than benzene.