Synthesis of caged NAD(P)+ coenzymes: Photorelease of NADP+

Full text of DOI:10.1021/ja962904j

J. Am. Chem. Soc. 1997, 119, 3403-3404
3403
Synthesis of Caged NAD(P)+ Coenzymes:
Scheme 1
Photorelease of NADP+
Charles P. Salerno,* Marianne Resat, Douglas Magde, and
Joseph Kraut
Department of Chemistry and Biochemistry
UniVersity of California, San Diego
La Jolla, California 92093-0506
Scheme 2a
ReceiVed August 19, 1996
The initiation of enzyme activity can be accomplished through
the photolytic release of essential substrates or cofactors from
biologically inactive “caged” precursors. There is a growing
list of cofactors and substrates which have been caged.1 These
include ATP,2 GTP,3 cAMP,4 Ca 2+,5 and several neurotrans-
mitters.6
The emerging field of time-resolved crystallography has
utilized caged molecules to rapidly initiate catalysis within
crystals.7 Complexation of caged GTP with H-Ras P21 was
one of the first examples of such an approach.3 Exposure of a
crystallized caged GTP H-Ras P21 complex to ultraviolet light
followed by rapid X-ray crystallographic data collection using
synchrotron radiation provided a real-time picture of the catalytic
cycle. More recently, crystalline isocitrate dehydrogenase was
activated via the photorelease of caged isocitrate.8
Our current investigations have involved the development of
caged NAD(P)+ analogs for use in time-resolved crystal-
lographic studies. The nicotinamide dependent oxido-reductases
comprise some one-sixth of all known enzymes, but general
methods for their photoactivation are lacking. To date there
have been no examples of caged NAD+ or NADP+ analogs.
We now report the synthesis of both NAD+ and NADP+
modified at the carboxamide nitrogen with a photolabile
o-nitrobenzyl group. The use of o-nitrobenzyl groups for the
photorelease of the carboxamide functionality has been previ-
ously demonstrated under aqueous conditions for several
biologically active amides, including glutamine and asparagine.6a
The rate of release of o-nitrobenzyl groups under aqueous
conditions is typically pH dependent.1,6a The quantum efficency
of product release is influenced by substitents at either the
nitrobenzyl benzylic or aromatic groups.1
1
a Reaction conditions: (a) MeONH3Cl, pyridine/ethanol, reflux; (b)
borane (1 M), THF, reflux; (c) nicotinoyl chloride hydrochloride, DMF,
triethylamine.
with similiary placed bulky additions.9 Furthermore, the
carboxamide has generally been shown to be a marginal
contibutor to cofactor binding when compared with the high
binding contributions of the pyrophposphate and adenosine
moieties.10
Synthesis of caged NAD(P)+ was carried out by a combina-
tion of enzymatic and synthetic organic methods. Incorporation
of the nitrobenzyl group into the NAD(P)+ cofactors was
achieved by the use of NADase glycohydrolase from porcine
brain, for the stereoselective exchange of the nicotinamide goup
of NAD+ or NADP+ with N-(2-nitrobenzyl)nicotinamide (1)
(Scheme 1).11 Pig brain NADase has shown a tolerance for
the exchange of a wide spectrum of substituted nicotinamides
and circumvents the use of involved synthetic routes.12
Synthesis of N-(2-nitrobenzyl)nicotinamide was carried out
through the coupling of 2-nitrobenzylamine with nicotinoyl
chloride (Scheme 2). 2-Nitrobenzylamine was synthesized by
the conversion of o-nitrobenzaldehyde to the oxime ether
followed by borane reduction to the amine in 60% yield.13,14
Incoproration of 1 into NAD+ and NADP+ proceeded by the
reaction of an excess of 1 and NADase for reaction times of 4
and 7.5 h, respectively. Yields of 10% for caged NAD+ and
20% for caged NADP+ (2) were obtained following purifica-
tion.15 Neither yield was optimized although the modest water
solubility of 1 limits the use of an excess of 1 to drive the
exchange further to completion. Characterization of caged
NAD(P)+ was accomplished by electrospray mass spectrometry,
1H NMR, UV spectroscopy, and HPLC.16 The purity of 2 was
determined to be 95% by HPLC analysis and the purity of caged
NAD+ to be 97%.
The incorporation of caged NAD(P)+ into crystalline enzyme
complexes demands placement of the nitrobenzyl group in a
region of the cofactor molecule which eliminates biological
activity without seriously compromising binding. The place-
ment of the nitrobenzyl group at the carboxamide was encour-
aged by the negligible biological activities of NAD(P)+ analogs
The photorelease of NADP+ was verified under aqueous
conditions by 1H NMR, HPLC, electrospray mass spectrometry,
* Author to whom correspondance should be addressed.
(1) (a) Adams, S. R.; Tsien, R. Y. Annu. ReV. Physiol. 1993, 55, 755-
84. (b) Givens, R. S.; Kueper, L. W. Chem. ReV. 1993, 93, 55-66.
(2) (a) Kaplan, J. N.; Forbush, B.; Hoffman, J. F. Biochemistry 1978,
17, 1929-1935. (b) Walker, J. W.; Reid, G. P.; McCray, J. A.; Trentham,
D. R. J. Am. Chem. Soc. 1988, 110, 7170-7177.
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R. S. Nature 1990, 345, 309-315.
(4) Givens, R. S.; Athey, P. S.; Kueper, L. W.; Matuszewski, B.; Xue,
J. Y. J. Am. Chem. Soc. 1992, 114, 8708-8710.
(9) Anderson, B. M. The Pyridine Nucleotide Coenzymes; Everse, J.,
Anderson, B., You, K., Eds.; Academic Press: New York, 1982; pp 91-
126.
(10) Anderson, B. M.; Kaplan, N. O. In Pyridine Nucleotide Coenzymes,
Chemical, Biochemical, and Medical Aspects, Part A; Dolphin, D., Poulson,
R., Avramovic, O., Eds.; John Wiley & Sons: New York, 1987; pp 569-
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(11) Anderson, B. M.; Ciotti, C. J.; Kaplan, N. O. J. Biol. Chem. 1959,
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(5) (a) Adams, S. R.; Kao, J. P.; Tsien, R. Y. J. Am. Chem. Soc. 1989,
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A.; Tsien, R. Y. J. Am. Chem. Soc. 1988, 110, 3212-3220.
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Ramesh, D.; Carpenter, B. K.; Hess, G. P. Biochemistry 1994, 33, 1526-
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(13) Feuer, H.; Braunstein, D. M. J. Org. Chem. 1969, 34, 1817-1820.
(14) This route to o-nitrobenzylamines has also proved an efficent means
of converting o-nitroacetophenone and 2,6-dinitrobenzaldehyde to the
corresponding amines in 50 and 60% yields, respectively.
(15) Exchange of the nicotinamide group of NAD(P)+ with water by
NADase to form the corresponding ADPR or ATPR products is the
dominant pathway under conditions of low N-(2-nitrobenzyl)nicotinamide
concentration.
(7) Hadju, J. Annu. ReV. Biophys. Biomol. Struct. 1993, 22, 467-498.
(8) Brubaker, M. J.; Dyer, D. H.; Stoddard, B.; Koshland, D. E.
Biochemistry 1996, 35, 2854-2864.
(16) See Supporting Information for detailed analysis.
S0002-7863(96)02904-6 CCC: $14.00 © 1997 American Chemical Society

3404 J. Am. Chem. Soc., Vol. 119, No. 14, 1997
Communications to the Editor
Scheme 3
stacked form places the o-nitrobenzyl group in close proximity
to both the adenine N-1 and N-6, which have been shown to be
reactive with a variety of alkylating agents.21 The 1-(2-
nitrophenyl)ethyl group has been used as a caging group to
enhance yields of release by virture of its less reactive
o-nitrosoacetophenone byproduct.2a Incorporation of similar
groups into NAD(P)+ is currently being pursued.
ultraviolet spectroscopy, and biological assay.16 Compound 2
was photolyzed by exposure to an excimer laser at 308 nm
(Scheme 3). The yield of NADP+ measured after complete
disappearance of 2 was 40 ( 2.5% over the pH range of 6.2-
8.0.17 This relatively low final yield of conversion to NADP+
was considered as being related to the nitroso aldehyde
byproduct. The o-nitroso aldehyde has been demonstrated to
be biologically reactive.18 The use of thiols to form less reactive
adducts with nitroso aldehydes has been applied to increase
yields and minimize adverse biological effects.2a Additon of
dithioerythritol (DTE) to photolysis experiments, however,
results in only a modest increase of 50% NADP+ released.19
Several minor byproducts could be observed using HPLC
analysis. The limited response to DTE addition may have a
conformational origin. The solution conformation of NADP+
places the nicotinamide and adenine groups in an equilibrium
between open and stacked forms.20 Both forms are known to
exist in appreciable amounts at 25 °C and possess distinctive
chemistries with regard to addition by nucleophiles.20 The
The quantum efficency of NADP+ photorelease was measured
at 308 nmn using excimer laser photolysis. Potassium ferri-
oxolate actinometry was used to measure laser intensity and
HPLC to assess the amount of NADP+ released.22 A value of
Φapp ) 0.19 ( 0.02 for the appearance of NAD was obtained
and Φdis ) 0.30 ( 0.02 for the disappearence of 2.16
The biological inactivity of caged NADP+ was established
for several NADP+ dependent dehydrogenases.16 Inactivity
relative to controls under conditions of high enzyme activity
was demonstrated for glucose 6-phosphate dehydrogenase,
aldehyde dehydrogenase, ethanol dehydrogenase, and aromatic
alcohol dehydrogenase. The absence of enzymatic reduction
of caged NADP+ was established by monitoring for absorption
increases in the 300-400 nm range corresponding to the 1,4-
dihydropyridine chromophore (λmax ) 335 nm, ꢀ ) 6220) of
NADPH. The biological activity of photoreleased NADP+ was
ascertained by the addition of aldehyde dehydrogenase to
completely photolyzed samples of 2. The new absorption
maximum at 335 nm produced upon addition of aldehyde
dehydrogenase corresponds to the enzymatic reduction of
photoreleased NADP+ to NADPH.16 Values of 55-60%
NADP+ released are measured by this method. The photolysis
of 2 was also carried out in the presence of ethanol dehydro-
genase, demonstrating reduction of released NADP+ com-
mensurate with exposure to ultraviolet irradiation.16
(17) Conditions: 0.5 mL of 0.1 mM 2 in aqueous solution with
appropriate buffer (pH ) 6.0-7.1, 150 mM ammonium formate; pH )
7.1-8.0, 75 mM Tris) was placed in a 1 cm quartz cuvette with magnetic
stirrer at 22 °C. Photolysis carried out under standard atmospheric conditions
as net yields of conversion demonstrated no oxygen dependence. The
samples were uniformly irradiated with 120 pulses from an excimer laser
(308 nm, 8 mJ/pulse). NADP+ present in photolyzed samples was measured
by HPLC using a YMC C18 analytical column. Amounts of NADP+ released
were quantified by using an HPLC standard of NADP+ (99%, Sigma, ꢀ )
18 000, 260 nm) to correlate HPLC peak integrations to moles of NADP+
released. See Section 2b of the Supporting Information for details of HPLC
conditions.
In summary, the incorporation of the o-nitrobenzyl group into
the NAD(P)+ cofactors has been demonstrated. The biological
inactivtiy of caged NADP+ has been verified as well the release
of NADP+ upon excimer laser irradiation and quantum effi-
cencies of release. The incorporation of other photolabile groups
into NAD(P)+ is currently being investigated.
(18) McCray, J. R.; Trentham, D. R. Ann. ReV. Biophys. Biophys. Chem.
1989, 18, 239-270.
(19) Conditions: 0.5 mL of 0.1 mM 2 in aqueous solution with 150
mM ammonium formate (pH ) 7.1, 22 °C) was placed in a 1 cm quartz
cuvette with magnetic stirrer. DTE concentrations ranged from 0.2-25 mM.
Solutions were degassed with argon. The samples were uniformly irradiated
with 120 pulses from an excimer laser (308 nm, 8 mJ/pulse). NADP+ present
in photolyzed samples was measured by the methods described in ref 17.
Maximum yield of 50% obtained for concentrations between 0.5 and 10
mM DTE.
Acknowledgment. This work was supported by Grant CA17374
from the National Institutes of Health. We thank Prof. Stanley L. Miller
for use of the HPLC apparatus and Jason Dworkin and Matthew Levy
of the Department of Chemistry and Biochemistry, University of
California, San Diego, for assistance with HPLC analysis.
(20) Populations of the folded form range from 25 to 50% under normal
physiological conditions. (a) Oppenheimer, N. J. In Pyridine Nucleotide
Coenzymes, Chemical, Biochemical, and Medical Aspects, Part A; Dolphin,
D., Poulson, R., Avramovic, O., Eds.; John Wiley & Sons: New York,
1987; pp 185-230. (b) Oppenheimer, N. J.; Arnold, L. J.; Kaplan, N. O.
Proc. Natl. Acad. Sci. 1971, 68, 3200-3205.
(21) (a) Windmuller, H. G.; Kaplan, N. O. J. Biol. Chem. 1961, 236,
2716-2726. (b) Burton, R. M.; Kaplan, N. O. Arch. Biochem. Biophys.
1963, 101, 139-149.
Supporting Information Available: Experimental procedures and
spectra (34 pages). See any current masthead page for ordering and
Internet instructions.
JA962904J
(22) Hatchard, C. G.; Parker, C. A. Proc. R. Soc. London, A 1956, 235,
518-536.