Synthesis and characterization of photolabile o-nitrobenzyl derivatives of urea

Article abstract of DOI:10.1021/jo0201373

We present here the synthesis and characterization of four photolabile derivatives of urea in which α-substituted 2-nitrobenzyl groups are covalently attached to the urea nitrogen. These derivatives photolyze readily in aqueous solution to release free ur

Full text of DOI:10.1021/jo0201373

Syn th esis a n d Ch a r a cter iza tion of P h otola bile o-Nitr oben zyl
Der iva tives of Ur ea
Raymond Wieboldt,† Doraiswamy Ramesh,‡ Evelyn J abri,§ P. Andrew Karplus,|
Barry K. Carpenter, and George P. Hess*
Molecular Biology and Genetics, 217 Biotechnology Building, Cornell University,
Ithaca, New York 14853-2703
gph2@cornell.edu
Received February 27, 2002
We present here the synthesis and characterization of four photolabile derivatives of urea in which
R-substituted 2-nitrobenzyl groups are covalently attached to the urea nitrogen. These derivatives
photolyze readily in aqueous solution to release free urea. The R-substituents of the 2-nitrobenzyl
group strongly influence the rate of the photolysis reaction measured with transient absorption
spectroscopy. Rates of photolysis at pH 7.5 and room temperature (approximately 22 °C) for N-(2-
nitrobenzyl)urea, N-(R-methyl-2-nitrobenzyl)urea, N-(R-carboxymethyl-2-nitrobenzyl)urea, and N-(R-
carboxy-2-nitrobenzyl)urea are, respectively, 1.7 × 104, 8.5 × 104 , 4.0 × 104, and 1.1 × 105 s-1. The
quantum yields determined by measurement of free urea following irradiation by a single laser
pulse at 308 nm were 0.81 for N-(2-nitrobenzyl)urea, 0.64 for N-(R-methyl-2-nitrobenzyl)urea, and
0.56 for N-(R-carboxy-2-nitrobenzyl)urea. The caged N-(R-carboxy-2-nitrobenzyl)urea is not a
substrate of the enzyme urease, while the photolytically released urea is. Also, neither this
caged urea nor its photolytic side products inhibit hydrolysis of free urea by urease. Thus, the
R-carboxy-2-nitrobenzyl derivative of urea is suitable for mechanistic investigations of the enzyme
urease.
In tr od u ction
glycine,10,11 NMDA,12 kainate,13 γ-aminobutyric acid,14,15
and glutamate16 initiates fast transmembrane ion cur-
rents mediated by neurotransmitter receptors on cell
surfaces. The ability to spatially direct release of free
products is useful where diffusional barriers exist that
prevent transport of substrates to their targets. For
example, caged calcium is widely employed to activate
calcium-mediated processes occurring inside cells.2 Caged
carbamoylcholine photolysis has been used to determine
the location of receptors on isolated BC3H1 cells which
express the muscle-type acetylcholine receptor.17 Caged
glutamate was employed to map glutamate-sensitive
synaptic connections between neurons within a layer of
mammalian neuronal tissue18 and to probe functions of
the neural network in the pharynx of a living organism,
the nematode Caenorhabditis elegans.19
Photolabile derivatives (“caged compounds”) of many
biochemically important molecules are now available and
are exploited in applications for the temporal and spatial
resolution that is achieved by their photolysis.1-6 Many
caged compounds release free products on a millisecond
or even microsecond time scale when photolyzed by a
pulse of UV light. This is important for activation of pro-
cesses which are inherently rapid. For example, photoly-
sis of caged compounds such as carbamoylcholine,7-9
* To whom correspondence should be addressed. Phone: (607) 255-
4809. Fax: (607) 255-6249.
† Present address: 100 Abbott Park Rd., Abbott Park, IL 60064-
3500.
‡ Present address: Calbiochem-Novabiochem Corp., La J olla, CA
92121.
§ Present address: Department of Chemistry, Indiana University,
Bloomington, IN 47405-7102.
(10) Wilcox, M.; Viola, R. W.; J ohnson, K. W.; Billington, A. P.;
Carpenter, B. K.; McCray, J . A.; Guzikowski, A. P.; Hess, G. P. J . Org.
Chem. 1990, 55, 1585.
| Department of Biochemistry and Biophysics, Oregon State Uni-
versity, Corvallis, OR 97331-7303.
Department of Chemistry and Chemical Biology, Cornell Univer-
sity, Ithaca, NY 14853.
(11) Ramesh, D.; Wieboldt, R.; Niu, L.; Carpenter, B. K.; Hess, G.
P. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11074.
(12) Gee, K. R.; Niu, L.; Schaper, K.; Hess, G. P. J . Org. Chem. 1995,
60, 4260.
(13) Niu, L.; Gee, K. R.; Schaper, K.; Hess, G. P. Biochemistry 1995,
35, 2030.
(14) Gee, K. R.; Wieboldt, R.; Hess, G. P. J . Am. Chem. Soc. 1994,
116, 8366.
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Biochemistry 1994, 33, 1526.
(1) Kaplan, J . H. Annu. Rev. Physiol. 1990, 52, 897.
(2) Adams, S. R.; Tsien, R. Y. Annu. Rev. Physiol. 1993, 55, 755.
(3) Corrie, J . E. T.; Trentham, D. R. In Bioorganic Photochemistry;
Corrie, J . E. T., Trentham, D. R., Eds.; Wiley: New York, 1993; p 243.
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291, 30.
(7) Milburn, T.; Matsubara, N.; Billington, A. P.; Udgaonkar, J . B.;
Walker, J . W.; Carpenter, B. K.; Webb, W. W.; Marque, J .; Denk, W.;
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1995, 92, 12008.
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10.1021/jo0201373 CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/20/2002
J . Org. Chem. 2002, 67, 8827-8831
8827

Wieboldt et al.
SCHEME 1
Photochemical protection used with many caged mol-
ecules involves coupling with a 2-nitrobenzyl group, and
this strategy has now been used for a variety of functional
groups, including carbamates,7 amines,15,20 carboxy-
lates,14,16 phosphates,21 phenols,22 and amides.23 Photo-
protection of amide precursors has also been used in
general synthetic procedures24 and has been applied in
solid-phase peptide synthesis.25,26 The temporal and
spatial resolution achievable with photolysis of caged
compounds is potentially useful in time-resolved X-ray
crystallography,25-29 and caged compounds have been
used to release substrates by photolysis of precursors
inside a protein crystal.30 For example, synchrotron
radiation and Laue diffraction were used to monitor
structural changes that occur when free GTP is released
in a crystal of a GTP-binding protein by photolysis of a
caged phosphate ester precursor of GTP.31,32
A photolabile precursor of urea may be applicable in
crystallographic and kinetic investigations of urease, an
enzyme found in plants, fungi, and bacteria.33 This nickel
metalloenzyme catalyzes the hydrolysis of urea to am-
monia and bicarbonate.34,35 Urease from Klebsiella aero-
genes has been crystallized and the structure determined
at 2.0 Å resolution.36 Models for the binding and hydroly-
sis of urea have been proposed and recently reviewed.37
The goal of the work described here was to develop
photolabile urea derivatives that rapidly release free urea
upon photolysis. We report here the synthesis and
properties of four urea analogues that release urea when
F IGURE 1. UV-vis spectra of the photolytic conversion of
compound 3 to products. A 60 µL aliquot of a 1.0 mM solution
of compound 3 in 100 mM phosphate buffer adjusted to pH
7.5 was photolyzed with 337 nm light produced by a pulsed
nitrogen laser at room temperature. The laser was focused into
a 2 × 2 mm cuvette to provide a beam with dimensions of
approximately 2 × 6 mm. The energy of each shot was 4.5 mJ ,
and the individual spectra correspond to 0, 5, 20, 40, 60, 90,
and 120 pulses. The optical path lengths for the laser and
monitoring beams in the cuvette were 0.2 cm. The dark line
(lower trace at 350 nm) corresponds to the spectrum of the
starting material, and the isosbestic points are located at 258
and 295 nm. The photolysis was continued until no further
spectral changes occurred; the dashed line is the final spectrum
and corresponds to essentially complete conversion to products.
exposed to a pulse of UV light. N-(R-Carboxy-2-nitroben-
zyl)urea (compound 3), which was soluble in aqueous
solution, photolyzed rapidly, exhibited a high quantum
yield, and was tested for compatibility with urease.
Neither the caged compound itself nor its photolysis side
products inhibit urease in solution. The caged compound
is, therefore, expected to be useful in further studies of
this enzyme.
Resu lts a n d Discu ssion
(20) Muralidharan, S.; Maher, G. M.; Boyle, W. A.; Nerbonne, J . M.
Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 5199.
The structures of four photolabile urea derivatives and
their synthesis are presented in Scheme 1. The deriva-
tization is performed in a single step with the addition
of an R-substituted 2-nitrobenzylamine to isocyanate. The
reaction occurs under acidic conditions, and the deriva-
tized urea product precipitates readily from the reaction
mixture. The derivatives photolyze in aqueous solutions
buffered at pH 7.4 and release free urea and a presumed
nitroso side product38 when exposed to a pulse of UV light
in the range of 300-340 nm. The general mechanism for
photohydrolysis of 2-nitrophenyl compounds in aqueous
media has been discussed.38,39 Figure 1 shows the changes
which occur in the UV-vis spectrum of compound 3, in
which the urea nitrogen is protected by the R-carboxyni-
trobenzyl group,7 as the photolysis reaction proceeds from
starting material to products at pH 7.4. The spectral
changes reflect the transformation of the 2-nitrobenzyl
derivative (absorbance maximum at 268 nm) to the
products, which for compound 3 are a nitrosophenyl
(21) Kaplan, J . H.; Forbush, B.; Hoffman, J . F. Biochemistry 1978,
32, 1929.
(22) Walker, J . W.; Martin, H.; Schmitt, F. R.; Barsotti, R. J .
Biochemistry 1993, 32, 1338.
(23) Ramesh, D.; Wieboldt, R.; Billington, A. P.; Carpenter, B. K.;
Hess, G. P. J . Org. Chem. 1993, 58, 4599.
(24) Henriksen, D. B.; Breddam, K.; Møller, J .; Buchardt, O. J . J .
Am. Chem. Soc. 1992, 114, 1876.
(25) Ajayaghosh, A.; Pillai, V. N. R. J . Org. Chem. 1990, 55, 2826.
(26) Pillai, V. N. R. Synthesis 1980, 1.
(27) J ohnson, L. N. Protein Sci. 1992, 1, 1237.
(28) Corrie, J . E. T.; Katayama, Y.; Reid, G. P.; Anson, M.;
Trentham, D. R. Philos. Trans. R. Soc. London, A 1992, 340, 233.
(29) Moffat, K. Annu. Rev. Biophys. Biosphys. Chem. 1989, 18, 309.
(30) Hadju, J .; Andersson, I. Annu. Rev. Biophys. Biomol. Struct.
1993, 22, 467.
(31) Schlichting, I.; Almo, S. C.; Rapp, G.; Wilson, K.; Petratos, K.;
Lentfer, A.; Wittinghofer, A.; Kabsch, W.; Pai, E. F.; Petsko, G. A.;
Goody, R. S. Nature 1990, 345, 309.
(32) Howell, P. L.; Almo, S. C.; Parsons, M. R.; Hajdu, J .; Petsko,
G. A. Acta Crystallogr., Sect. B: Struct. Sci. 1992, 48, 200.
(33) Sumner, J . B. J . Biol. Chem. 1926, 69, 435.
(34) Zerner, B. Biorg. Chem. 1991, 19, 116.
(35) Hausinger, R. P. In Biochemistry of Nickel; Hausinger, R. P.,
Ed.; Plenum Press: New York, 1993; p 23.
(36) J abri, E.; Carr, M. B.; Hausinger, R. P.; Karplus, P. A. Science
1995, 268, 998.
(37) Hausinger, R. P.; Karlus, P. A.; In Handbook of Metalloproteins;
Messerschmidt, A. et al., Eds.; J ohn Wiley & Sons: Chichester, 2001;
pp. 867-879,
(38) McCray, J . A.; He´rbette, L.; Kihara, T.; Trentham, D. R. Proc.
Natl. Acad. Sci. U.S.A. 1980, 77, 7237.
(39) Schupp, H.; Wong, W. K.; Schnabel, W. J . J . Photochem. 1987,
36, 85.
8828 J . Org. Chem., Vol. 67, No. 25, 2002

Urea Photolabile Precursors (Caged Urea)
TABLE 1. Ur ea Der iva tives: P h otolysis Ra te a n d
P r od u ct Yield a t p H 7.4 a n d Room Tem p er a tu r e
product
product
photolysis quantum
photolysis quantum
compd ratea (s-1
)
yieldb
compd ratea (s-1
)
yieldb
1
2
1.7 × 104
0.81
0.64
3
4
1.1 × 105
0.56
N.D.
8.5 × 104
4.0 × 104
a
Measured with excitation by a pulsed excimer laser at 308
b
nm, 30 mJ per pulse. Measured with excitation by a pulsed
excimer laser at 337 nm, 5 mJ per pulse. Duplicate determinations
were performed for each reported value; the relative error is
estimated at 20%. N.D. not determined.
F IGURE 2. Absorbance transient produced by photolysis of
compound 3. The trace was produced by photolysis of a 3 mM
solution of the caged urea in pH 7.5 phosphate buffer by a 10
ns pulse of 308 nm UV light (30 mJ per pulse) from an excimer
laser at room temperature. The path length of the monitoring
beam in the cuvette was 0.2 cm. The effective bandwidth of
the detection system was 500 kHz. The absorbance decay of
the photolysis intermediate was measured at 430 nm, near
the absorbance maximum for the intermediate.
SCHEME 2
F IGURE 3. Spectral dispersion of the transient absorbance
signal of compound 3. Absorbance was measured at discrete
wavelengths between 380 and 500 nm immediately after
excitation by the laser pulse. The concentration, pH of the
solution, and other conditions are those given in the caption
for Figure 2.
absorbance signals at wavelengths between 350 and 500
nm measured 2 µs after the laser was triggered. The
intermediates for compounds 2 and 4 have absorbance
maxima at 420 nm, whereas 1 and 3 have maxima close
to 430 nm. The spectra are consistent with the appear-
ance of aci-nitro intermediates observed with similar
2-nitrobenzyl caged compounds39 and are distinct from
the spectra of the caged compounds themselves and the
products of the photolysis reaction. aci-Nitro intermediate
decay rates have been shown to be directly related to the
rate of product release,41,42 and free urea is, therefore,
produced on a time scale of 10-100 µs for compounds
1-4.
The photolysis rate is also dependent on the pH. A plot
of the aci-nitro intermediate decay rate for compound 3
versus pH is shown in Figure 4. The accelerated rates at
lower pH are qualitatively similar to rates observed for
a series of (2-nitrobenzyl)glutamine compounds which
employed an analogous amide linkage.23 However, com-
pound 3 photoyzed 10-100 times faster than the corre-
sponding (R-carboxy-2-nitrobenzyl)glutamine derivative
at each pH where measurements were made. Quantum
yield determinations for compounds 1-3 were performed
in aqueous pH 7.4 buffer, and the results are presented
in Table 1. Both the photolysis rates and quantum yields
R-keto acid (absorbance maximum at 338 nm) and free
urea (Scheme 2). The spectra share clear isosbestic
points, so the spectral changes reflect the formation of
the nitroso side product and no other absorbing species.
The rate and quantum yield of the photolysis reaction
are important criteria in the selection of the most
appropriate derivative for use in time-resolved crystal-
lography. Photolysis of 2-nitrophenyl derivatives proceeds
through an aci-nitro intermediate which has a charac-
teristic near-UV absorbance.38 This intermediate forms
on a submicrosecond time scale immediately following
excitation and then decays to products in 10-100 µs. The
intermediate has a characteristic absorbance profile with
a maximum between 420 and 440 nm,38,40 and the
intermediate disappearance can be followed spectropho-
tometrically to determine the reaction rate. Figure 2
shows the decay transient observed when compound 3
is photolyzed in buffer at pH 7.4; the half-life of the decay
is 6.3 µs. Transient decay rates of the series of urea
derivatives were determined by fitting a single-exponen-
tial function to the absorbance data and are shown in
Table 1. Figure 3 shows the spectrum of this transient
for compound 3 determined from the maximum of the
(41) Walker, J . W.; Reid, G. P.; McCray, J . A.; Trentham, D. R. J .
Am. Chem. Soc. 1988, 110, 7170.
(40) Walker, J . W.; McCray, J . A.; Hess, G. P. Biochemistry 1986,
25, 1799.
(42) Wootton, J . F.; Trentham, D. R. NATO ASI Ser. C 1989, 272,
277.
J . Org. Chem, Vol. 67, No. 25, 2002 8829

Wieboldt et al.
or degradation of the caged compound itself. Thus,
compound 3 is neither a substrate nor an inhibitor of K.
aerogenes urease.
It was important to determine that urease is able to
hydrolyze the free urea released by photolysis of com-
pound 3. To test this, a large-volume aliquot of compound
3 (0.5 mL, 10 mM solution in urease assay buffer) was
photolyzed with the nitrogen laser. In contrast to the
experiments shown above for determination of the quan-
tum yield, many laser shots during an extended 4 min
irradiation time were required for photolysis of the larger
sample and complete conversion was not possible. The
photolysis products absorb at the 337 nm nitrogen laser
wavelength and may also undergo a photoreaction them-
selves. Nevertheless, a product of photolysis of compound
3 was turned over by urease to produce the color reaction
of the assay (Table 2), which is consistent with release
of free urea by the photolysis.
The rate of hydrolysis is only 5% of that expected if
each photolyzed molecule produced one molecule of urea.
The low activity is not due to inhibition of urease by other
photolysis products since the presence of photolyzed
products of compound 3 does not slow urease hydrolysis
of added free urea (line 3 vs line 6 of Table 2). These
results indicate that urease hydrolyzes urea released by
photolysis of compound 3 and that byproducts of the
photolysis reaction do not inhibit hydrolysis of urea by
the enzyme.
F IGURE 4. Variation of photolysis rate with pH at room
temperature (22 °C). Solutions of 3 mM compound 3 in 100
mM buffers were photolyzed by 308 nm, 30 mJ pulsed light
from the excimer laser, and the rates of the transient inter-
mediate absorbance decay were determined from exponential
approximations to the decay data. The solid line represents a
spline fit to the data points. The buffers used were phosphate
at pH 6.5 and 7.5, borate at pH 8.5 and 9.8, and bicarbonate
at pH 11.0.
TABLE 2. Activity of Ur ea sea in th e P r esen ce of
Com p ou n d 3b
specific
activity
(U/mg)
normalized
activity
(%)
Hadfield and Hadju43 have proposed criteria which are
desirable for a caged compound used in crystallographic
experiments, and compound 3 conforms to several of
these requirements: (1) it is water soluble at concentra-
tions up to at least 50 mM, (2) photolysis occurs between
300 and 340 nm, which is above the typical optical
absorbance of proteins, (3) it has a quantum yield of 0.6,
and (4) the photolysis products do not inhibit urease.
These characteristics of compound 3 are sufficient to
justify its application in crystallographic studies of ure-
ase. However, because the absorbance of the photolysis
products is greater than for the caged compound over the
range of 300-400 nm (Figure 1), the extent to which the
compound may be photolyzed in a crystal under condi-
tions of continuous irradiation may be limited.
The caged urea derivatives shown here also serve as
models for similar compounds which could be useful in
situations where rapid, high-yield photolytic release of
a free amide is required. For instance, it may be possible
to adapt the procedures used here to produce light-
activatable derivatives of hydroxyurea, an inhibitor of
ribonuclease reductase used in clinical treatments of
malignancies and potentially as an HIV virus inhibitor.44
It may also be useful in studies of hydroxyurea in the
treatment of sickle cell anemia.45
substrate
free urea
compound 3
118 ( 2
0
100 ( 2
0
compound 3 + free urea
113 ( 8
96 ( 7
free urea
photolyzed compound 3
photolyzed compound 3 + free urea
94 ( 3
5 ( 3
106 ( 5
100 ( 3
5 ( 3
110 ( 5
a
Urease used in these assays was stored for 2 months at 4 °C
and had a specific activity of 110 ( 10 U/mg at the start of the
caged urea assays and 95 ( 10 U/mg at the beginning of the
b
photolyzed caged urea assays. When present, urea and/or caged
urea were each at 10 mM. Given the efficiency of photolysis, the
photolyzed samples would be expected to contain about 5 mM free
urea and side products and 5 mM unreacted caged compound.
Freshly prepared K. aerogenes urease has a specific activity of
∼1800 U/mg (1 unit of urease is equivalent to the amount of
enzyme required to hydrolyze 1 µmol of urea/min under the assay
conditions). Upon storage at 4 °C for extended lengths of time,
the enzyme activity decreases with no effect on Km.
of the caged urea derivatives were greater than values
found previously for an analogous series of amide deriva-
tives of glutamine, asparagine, and glycine.23
Caged urea derivatives may be useful in investigations
of the interaction of urea with the active sites in crystals
of the enzyme urease. Compound 3 was chosen for
evaluation with the enzyme because of its high photolysis
rate and good quantum yield. To assess the compatibility
of caged urea with urease, the specific activity of the
enzyme in the presence of compound 3 and its photolysis
products was measured. Table 2 shows that the activity
of the enzyme is unchanged in the presence of 10 mM
(R-carboxy-2-nitrobenzyl)urea, indicating that compound
3 does not itself inhibit urease. Furthermore, when
compound 3 was incubated with the enzyme in the
absence of urea, there was no indication that either urea
or free ammonia was released by enzymatic hydrolysis
Exp er im en ta l Section
For NMR, chemical shifts are referenced to the residual
proton peak in D2O at 4.64 ppm, and coupling constants (J )
are measured in hertz. Elemental analysis was performed by
(43) Hadfield, A.; Hadju, J . J . Mol. Biol. 1994, 236, 995.
(44) Lori, F.; Malykh, A.; Cara, A.; Sun, D.; Weinstein, J . N.;
Lisziewicz, J .; Gallo, R. C. Science 1994, 266, 801.
(45) Orringer, E. P.; Abraham, D. J .; Parker, J . C. In Sickle Cell
Disease: Basic Principles and Clinical Practice; Embury, S. H., et al.,
Eds.; Raven Press: New York, 1994; pp 861-871.
8830 J . Org. Chem., Vol. 67, No. 25, 2002

Urea Photolabile Precursors (Caged Urea)
a microanalytical laboratory, and high-resolution mass spectra
were provided by the Mass Spectrometry Laboratory at the
University of Illinois, Champaign. The amine hydrochlorides
used as starting materials for the synthesis either were
obtained from commercial sources or were synthesized accord-
ing to reported methods for 1-amino-1-(2-nitrophenyl)ethane40
and 3-amino-3-(2-nitrophenyl)propanoic acid.46
nitrogen laser at 337 nm (5 mJ per pulse) at 5 Hz for
approximately 4 min at room temperature. An 80 µL sample
of this solution was then added to the reaction mixture. The
change in the absorbance spectrum (Figure 1) was used to
estimate the extent of photolysis and indicated that 53% of
the starting caged compound was photolyzed under these
conditions.
The synthetic procedure is depicted in Scheme 1. Portions
of sodium cyanate (3.0 mmol) were added over a period of 6 h
to a stirred mixture of the appropriate benzylamine hydro-
chloride complex (1.0 mmol), glacial acetic acid (3.0 mmol), and
water (20 mL). A precipitate formed after 3 h, and stirring
was continued overnight. For isolation of compound 1, the
reaction mixture was cooled on ice; the solid was filtered,
washed with a water and acetic acid (1:1) mixture, and dried.
For compounds 2-4, the solutions were lyophilized and passed
through a Sephadex LH-20 column twice using water as
eluent.
Com p ou n d 1, N-(2-Nitr oben zyl)u r ea . Yield: 60%. Mp:
145 °C. 1H NMR (D2O, DMSO-d6): 7.97 (d, 1H, J ) 8.4, C3-H),
7.60 (m, 1H), 7.42 (m, 2H) and 4.47 (s, 2H, CH2). Anal. Calcd
for C8H9N3O3: C, 49.23; H, 4.62; N, 21.59. Found: C, 49.46;
H, 4.61; N, 21.25.
La ser -F la sh P h otolysis. Instrumentation used for tran-
sient absorption spectroscopy has been described.23 A single
30 mJ pulse of 308 nm light from a XeCl excimer laser was
used to initiate photolysis, and the decay of the transient
intermediate absorbance was recorded by digitizing the pho-
tomultiplier output at rates up to 2 MHz. All measurements
of photolysis rates and quantum yields were performed at room
temperature. Nonlinear least-squares fitting of single-expo-
nential decays were fit to the data to determine the photolysis
rate constants. The remainder of the experiments used 5 mJ ,
337 nm pulsed light produced by an excimer laser charged with
N2 and He gas instead of the Xe/HCl/He excimer mixture.
Qu a n tu m Yield Deter m in a tion . Free urea produced by
the photolysis reaction was determined by reversed-phase
HPLC.49 Urea was eluted under isocratic conditions from a
4.5 × 300 mm C-18 column50 using buffer consisting of 50 mM
phosphate at pH 6.4 and 2 mM tetraethylammonium chloride;
free urea was detected by absorbance at 205 nm. Peak areas
were compared to concentration standards in the range from
0 to 20 mM urea. Solutions (20 mM) of the compounds in 50
mM phosphate buffer at pH 7.4 were photolyzed by single 30
mJ pulses of 308 nm light from the XeCl excimer laser at room
temperature. The energy of the laser light absorbed in the
solution was measured with a pyroelectric thermopile energy
meter.23 Aliquots of the photolyzed solution were combined
with HPLC running buffer and used for the chromatographic
analysis. The quantum yield was calculated as the number of
free urea molecules liberated by photolysis divided by the
number of photons absorbed.
Com p ou n d 2, N-(r-Meth yl-2-n itr oben zyl)u r ea .47 Yield:
48%. Mp: 125-126 °C. 1H NMR (D2O, DMSO-d6): 7.79 (d, 1H,
J ) 8.4 Hz), 7.53 (m, 1H), 7.33 (m, 2H), 5.05 (q, 1H, J ) 6.8),
1.36 (d, 2H, J ) 6.9). High-resolution mass spectrum (m/z):
(M + H)+ calcd for C9H12N3O3 210.0879, found 210.0888.
Com p ou n d 3, N-(r-Ca r boxy-2-n itr oben zyl)u r ea . Syn-
thesized according to Davis et al.51 Yield: 55%. Mp: 138-145
1
°C dec. H NMR (D2O): 7.90 (d, 1H, J ) 7.72, C3-H), 7.47 (m,
1H), 7.42 (m, 2H) 5.46 and 5.45 (1H, CH). High-resolution
mass spectrum (m/z): (M + H)+ calcd for C9H9N3O5 240.0620,
found 240.0607.
Com p ou n d 4, N-(r-Ca r boxym eth yl-2-n itr oben zyl)u r ea.
1
Yield: 60%. Mp: 162-4 °C. H NMR (D2O): 7.85 (d, 1H, J )
7.9, C3-H), 7.52 (m, 1H), 7.35 (m, 2H), 5.33 (m, 1H, CHCH2),
2.56 (m, 2H, CHCH2). High-resolution mass spectrum (m/z):
(M + H)+ calcd for C11H13N3O6 254.0777, found 254.0763.
Ur ea se Assa y. The Bertholet ammonia assay36,48 was used
as a measure of enzyme activity achieved when urea is
released from a caged precursor by photolysis and also to test
whether urease is inhibited by compound 3. The urease
concentration used in each assay was 2 × 10-4 mg/mL as
determined by the bicinchoninic acid protein staining method.
All activity assays were carried out in triplicate, and steady-
state initial rates were based on measurements made at 0.5,
10, 15, and 20 min. To provide aliquots of photolyzed caged
compound, 500 µL of a 50 mM solution of compound 3 (in 10
mM phosphate buffer at pH 7.4) was irradiated with a pulsed
Ack n ow led gm en t. This work was supported by
National Institutes of Health Grant GM04842 awarded
to G.P.H. E.J . and P.A.K. were supported under USDA
Grant 9303870 awarded to P.A.K. and R. P. Hausinger.
We also thank Dr. Robert Hausinger for providing K.
aerogenes urease, and Molecular Probes, Inc. for a gift
of some of the N-(R-carboxy-2-nitrobenzyl)urea (com-
pound 3) used.
J O0201373
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