Preparation and photophysical properties of a caged kynurenine

Article abstract of DOI:10.1016/j.bmcl.2012.02.097

We have prepared l-kyurenine 4-hydroxyphenacyl ester, a caged derivative of l-kynurenine. N^α-tBOC-l-tryptophan was reacted with 4-hydroxyphenacyl bromide in DMF with K₂CO₃ as the base to give the N^α-tBOC 4-hydroxyphenacyl ester. The ester was then treated with O₃ in MeOH at -20 °C, followed by trifluoroacetic acid in CH₂Cl₂, then aqueous HCl to obtain the caged kynurenine as the dihydrochloride salt. The caged kynurenine is stable as a dry solid in the dark at -78 °C, but in aqueous solutions in phosphate buffer at pH 7-8 hydrolyzes rapidly (t_1/2 ～5 min). Solutions in Tris at pH 7 are more stable (t_1/2 >30 min), and solutions in 1 mM HCl are stable for several hours. As expected, the ester is cleaved in microseconds with laser pulses at 355 nm. The caged kynurenine may be useful for preparation of substrate complexes for crystallography or in biological studies on kynurenine.

Full text of DOI:10.1016/j.bmcl.2012.02.097

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2734–2737
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preparation and photophysical properties of a caged kynurenine
Chandan Maitrani a, Derren J. Heyes b, Sam Hay b, Selvanathan Arumugam a, Vladimir V. Popik a,
Robert S. Phillips a,c,
⇑
a Department of Chemistry, University of Georgia, Athens, GA 30602, USA
b Manchester Interdisciplinary Biocentre and Faculty of Life Science, University of Manchester, Manchester, UK
c Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA
a r t i c l e i n f o
a b s t r a c t
a
Article history:
We have prepared L-kyurenine 4-hydroxyphenacyl ester, a caged derivative of L-kynurenine. N -tBOC-L-
Received 11 January 2012
Revised 24 February 2012
Accepted 28 February 2012
Available online 6 March 2012
tryptophan was reacted with 4-hydroxyphenacyl bromide in DMF with K2CO3 as the base to give the
a
N -tBOC 4-hydroxyphenacyl ester. The ester was then treated with O3 in MeOH at ꢀ20 °C, followed by
trifluoroacetic acid in CH2Cl2, then aqueous HCl to obtain the caged kynurenine as the dihydrochloride
salt. The caged kynurenine is stable as a dry solid in the dark at ꢀ78 °C, but in aqueous solutions in
phosphate buffer at pH 7–8 hydrolyzes rapidly (t1/2 ꢁ5 min). Solutions in Tris at pH 7 are more stable
(t1/2 >30 min), and solutions in 1 mM HCl are stable for several hours. As expected, the ester is cleaved
in microseconds with laser pulses at 355 nm. The caged kynurenine may be useful for preparation of
substrate complexes for crystallography or in biological studies on kynurenine.
Keywords:
Tryptophan metabolism
Photochemistry
Caged compound
Ó 2012 Elsevier Ltd. All rights reserved.
Kynurenine is an intermediate in the eponymous metabolic
pathway of tryptophan in mammals and some fungi and bacteria.
The kynurenine pathway has been found to be involved in
regulation of the immune response1 and in neurodegenerative
diseases.2–4 Furthermore, kynurenine has been shown recently to
have endothelial relaxing properties due to activation of guanylate
cyclase, the target of NO.5 Recently, kynurenine has been shown to
be an endogenous ligand for the Aryl Hydrocarbon (AH) receptor.6
Thus, enzymes in the kynurenine pathway are of interest as possi-
ble drug targets. Kynureninase, one of the enzymes in the pathway,
catalyzes the hydrolytic cleavage of kynurenine in bacteria, or
ð1Þ
Caged compounds, which contain a protecting group that can be
rapidly removed by irradiation with light, have been prepared for
over three decades. The first generation caged compounds used
o-nitrobenzyl groups as the cage,11–16 but they suffer from rela-
tively slow uncaging (milliseconds) and potentially deleterious
nitrosobenzaldehyde products. 7-Nitroindoline derivatives17 over-
come some of these problems, as do 4-methylcoumarins.18–25 More
recently, Givens popularized the 4-hydroxyphenacyl group as a
cage,26,27 which has fast photochemistry and releases relatively
innocuous 4-hydroxyphenylacetate by a photochemical Favorskii
rearrangement. The 4-hydroxyphenacyl group has been used previ-
ously to cage carboxylic acids, thiols and phosphates.28–30 Hence,
we decided to use the 4-hydroxyphenacyl group to prepare a caged
3-hydroxykynurenine in mammals, to give L-alanine and anthrani-
late or 3-hydroxyanthranilate, respectively (Eq. 1). Previously, we
have determined the structures of Pseudomonas fluorescens7 and
human kynureninase8, and a complex of human kynureninase with
3-hydroxyhippurate,9 by X-ray crystallography. We attempted to
soak crystals of human kynureninase with substrate to obtain a
structure of the complex, but the crystals dissolved upon substrate
addition. In addition, in stopped-flow experiments, we found that
the quinonoid intermediate, absorbing at 494 nm, formed within
the dead time of the instrument,10 precluding measurement of
the rate constant for its formation. Thus for determination of a
substrate structure by kinetic crystallography, as well as for mech-
anistic experiments, it would be useful to have a caged derivative of
L
-kynurenine 4-hydrophenacyl ester.
The synthesis of the caged kynurenine was performed as shown
a
in Scheme 1. First, commercially available N -tBOC-
L
-tryptophan
was reacted with 4-hydroxyphenacyl bromide in DMF with
a
L-kynurenine.
K2CO3 as the base to give the N -tBOC 4-hydroxyphenacyl ester
in 98% isolated yield.31 Treatment of 4-hydroxyacetophenone
under the standard conditions of refluxing CHCl3 and EtOAc32 in
our hands gave a product contaminated with the
However, we found that 4-hydroxyphenacyl bromide was cleanly
a,a-dibromide.
⇑
Corresponding author. Tel.: +1 706 542 1996; fax: +1 706 542 9454.
E-mail addresses: rsphillips@chem.uga.edu, plp@uga.edu (R.S. Phillips).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.097

C. Maitrani et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2734–2737
2735
Scheme 1. Synthesis of L-kynurenine 4-hydroxyphenacyl ester dihydrochloride.
prepared in high yield by the reaction of 4-hydroxyacetophenone
with CuBr2 in MeOH for 4 h at room temperature. The product,
N-tBOC-L-tryptophan 4-hydroxyphenacyl ester, was then subjected
to ozonolysis at ꢀ20 °C in MeOH to provide the tBOC-protected N-
formyl kynurenine. The tBOC and formyl groups were removed
with TFA in CH2Cl2, and treatment of the residue after evaporation
with aqueous HCl, followed by lyophilization, gave the caged kyn-
urenine as the hygroscopic dihydrochloride salt. High resolution
MS of the product gave a mass of 343.1294 (M+1, C18H19N2O5).33
The caged kynurenine is stable indefinitely as the dry solid at
ꢀ78 °C in the dark, and at least for 15 days as a solution in dry
DMSO in the dark. Aqueous solutions are much less stable, releas-
ing L-kynurenine by hydrolysis with a t1/2 of less than 5 min in
phosphate buffer, pH 7 or 8, at room temperature. Solutions in
1 mM HCl at room temperature or below are much more stable,
showing no decomposition for up to 3 h. We also checked the sta-
bility of the caged compound in pH 7 Tris buffer containing 55 mM
MgCl2 and 25% w/v polyethylene glycol (PEG), the buffer used in
crystallization of human kynureninase.8,9 We have found that the
caged compound shows no sign of decomposition for about
30 min in this buffer, which should allow sufficient time to soak
and freeze enzyme crystals.
Figure 1. Caged kynurenine photophysics. A. The absorbance spectrum of 50
kynurenine 4-hydroxyphenacyl ester in 0.05 M potassium phosphate, pH 8.0 (black
line). The red line is the absorbance spectrum of 50 -kynurenine in 0.05 M
potassium phosphate, pH 8.0. B. The black trace shows photolysis of a 100
solution of caged kynurenine at 320 nm. The red trace was measured at 494 nm in
the presence of kynureninase. In both cases, single traces are shown.
lM L-
lM L
l
M
The photophysics of the caged kynurenine were examined in a
combined laser flash photolysis and stopped-flow system (Applied
Photophyhsics LKS.60 with SX.1 accessory and Quantel Brilliant B
Nd:YAG laser). Solutions of caged kynurenine were prepared in
1 mM HCl and kept on ice until use. The compound was then
mixed with pH 8.0 phosphate buffer in the stopped-flow instru-
ment, and immediately flashed (within 1 s of mixing) with a laser
pulse at 355 nm (6–8 ns pulse; ꢁ200 mJ). Uncaging was monitored
by the decrease in absorbance at 320 nm (Fig. 1A), the absorption
amounts. However, ꢁ40% of the transient absorbance decrease at
320 nm is recovered with a lifetime of 14 ls (Fig. 1B), suggesting
that a reversible process is competitive with the uncaging reaction.
When P. fluorescens kynureninase was added to the buffer, no evi-
dence for reaction on this time-scale was observed at 494 nm
(where reaction of the enzyme with substrate forms a quinonoid
maximum (
e
ꢁ10,000 Mꢀ1 cmꢀ1 at pH 8.0) of the phenolate ion
intermediate9) upon laser flashing, suggesting that the ꢁ1–2
lM
of the 4-hydroxyphenacyl cage. Absorbance decreases occurred
concentration of kynurenine released after a single flash was not
sufficient to trigger an observable amount of enzymatic reaction.
The enzyme absorption at 355 nm in this experiment was less than
0.1, so the lack of reaction could not be due to an inner filter effect.
A possible mechanism for the uncaging of kynurenine 4-hydrox-
yphenacyl ester is shown in Scheme 2. Absorption of a photon
within 1
is rapid and that about 1.7
l
s of the laser pulse, showing that the uncaging kinetics
M of un-caged kynurenine is liberated
l
in each pulse (Fig. 1B). Consistent with this, HPLC measurements
after 100 pulses of laser irradiation showed the quantitative forma-
tion of kynurenine and 4-hydroxyphenylacetate in equimolar

2736
C. Maitrani et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2734–2737
Scheme 2. Mechanism for uncaging of
L
-kynurenine 4-hydroxyphenacyl ester.
2. Heyes, M. P.; Brew, B. J.; Saito, K.; Quearry, B. J.; Price, R. W.; Lee, K.; Bhalla, R.
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results in excitation of the phenolate to a singlet state, which
rapidly crosses over to a triplet state. It is possible that the kynuren-
ine chromophore, with kmax at 360 nm (Fig. 1A), can act as an anten-
na to assist in the excitation, since kynurenine is only weakly
fluorescent.34 The triplet state of the cage has been shown to under-
go elimination of the leaving group,35–37 resulting in the Favorskii
intermediate, which undergoes rapid hydrolysis to give the prod-
uct, 4-hydroxyphenylacetate. Tautomerization of the triplet state
may also occur, giving the quinone methide, which converts back
to the ester by intersystem crossing and tautomerization rather
than release of the amino acid (Scheme 2). This latter process
may account for the apparent reversibility of uncaging seen in Fig-
ure 1B. The quantum yield for photolysis of 4-hydroxyphenacyl es-
ters decreases dramatically at pH values above the pKa of the
phenol, ca. 7.5, so we expect the efficiency of uncaging to be 10%
or less under our reaction conditions at pH 8.
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We have successfully synthesized a caged form of L-kynurenine,
the 4-hydroxyphenacyl ester, and demonstrated that it can be un-
caged in microseconds with 355 nm laser pulses. Although the
stopped-flow-flash experiments did not appear to liberate suffi-
cient quantities of uncaged kynurenine to observe formation of
reaction intermediates of kynureninase, the compound still may
be useful where continuous illumination can be used, or to prepare
substrate complexes of kynureninase for crystallography. In addi-
tion, it may find application in studies of the biological effects of
kynurenine.
Acknowledgment
S.H. is a Biotechnology and Biological Sciences Research Council
(BBSRC) David Phillips Fellow.
References and notes
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C. Maitrani et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2734–2737
2737
31. Mp 195–198 °C; 1H NMR: CDCl3, 7.85 (d, 2H), 7.75 (d, 1H), 7.54 (d, 1H), 7.32 (t,
1H), 7.25 (t, 1H), 7.1 (d, 2H), 7.05 (s, 1H), 5.62 (s, 2H), 4.57 (m, 1H), 3.57 (dd,
2H), 2.95 (s, 9H); 13C NMR: CDCl3, 27.6, 28.9, 55.3, 67.1, 78.9, 110.8, 112.2,
116.2, 118.7, 119.2, 121.7, 124.6,126.2, 127.8, 131.1, 136.9, 156.2, 163.4, 172.9,
191.3.
two 15 ml portions of EtOAc. The combined organic layer was washed with
15 ml of 2N HCl. The acid wash was combined with the aqueous layer, treated
with activated charcoal, filtered through celite, and lyophilized. Yield: 0.20 g
(21%). HPLC analysis showed the compound to be 99% pure. 1H NMR: MeOH-d4,
7.82 (d, 2H), 7.75 (d, 1H), 7.25 (t, 1H), 6.92 (d, 2H), 6.89 (d, 2H), 6.58 (t, 1H),
5.52 (d, 2H), 4.51 (t, 1H), 3.73 (dd, 2H); 13C NMR: MeOH-d4, 38.3, 48.2, 68.1,
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L
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115.7, 119.7, 120.6, 121.2, 124.9, 131.1, 131.6, 136, 142.8, 162.1, 169.8, 194.2,
D
methanol by warming on a water bath, then cooled in a dry ice- acetone bath
to about ꢀ15 to ꢀ20 °C. Ozone gas was bubbled through the cold reaction
mixture, and progress was monitored by TLC. The reaction was quenched with
a solution of 4 g sodium bisulfite in 20 ml water. The mixture was then
concentrated under vacuum below 30 °C to remove methanol. Water (20 ml)
was then added, and the mixture was extracted with two 30 ml portions of
CH2Cl2 and the combined organic extract was dried over anhydrous sodium
sulfate. Trifluoroacetic acid (3 ml) was added, giving a clear yellow solution,
and it was stirred overnight. The reaction mixture was concentrated under
vacuum below 25 °C, then water (15 ml) was added, and it was extracted with
198.5; [
a
]
ꢀ14.2° (c = 1.3 in water).
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