Fluorescence emission properties of 8-aza analogues of caffeine, theophylline, and related N-alkyl xanthines

Article abstract of DOI:10.1016/j.bmc.2009.01.007

Fluorescence emission properties of 8-azacaffeine, 8-azatheophylline and other N-alkylated 8-azaxanthines (8-azaXan) have been examined. It is shown that N-methylated 8-azaxanthines, as well as 8-azatheophylline, are highly fluorescent in aqueous medium a

Full text of DOI:10.1016/j.bmc.2009.01.007

Bioorganic & Medicinal Chemistry 17 (2009) 2585–2591
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Fluorescence emission properties of 8-aza analogues of caffeine,
q
theophylline, and related N-alkyl xanthines
a
a,*
b
b,c
Grzegorz M e˛ dza , Jacek Wierzchowski , Borys Kierdaszuk , David Shugar
a
Department of Biophysics, University of Warmia and Mazury, 4 Oczapowskiego Street, PL-10-719 Olsztyn, Poland
Division of Biophysics, Institute of Experimental Physics, University of Warsaw, 93 Zwirki & Wigury St., PL-02-089 Warsaw, Poland
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5a Pawinskiego Street, PL-02-106 Warsaw, Poland
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluorescence emission properties of 8-azacaffeine, 8-azatheophylline and other N-alkylated 8-azaxanth-
ines (8-azaXan) have been examined. It is shown that N-methylated 8-azaxanthines, as well as 8-azathe-
ophylline, are highly fluorescent in aqueous medium as the neutral, and, in some instances, also as the
monoanionic, forms. 8-Azacaffeine exhibits moderate emission, but its isomer, 1,3,8-trimethyl-8-azaXan,
is highly fluorescent. All three 8-azaxanthines monomethylated on the triazole ring, as well as 8-azaxan-
thosine, exhibit increased acidity in the excited state. Some fluorescent pyrazolo[4,3-d]pyrimidine-5,7-
diones, xanthine congeners of pyrazolo[4,3-d]pyrimidines, are also reported. Many of these are good fluo-
rescent probes in enzymatic, receptor binding, and nucleic acid systems, some examples of which are pre-
sented. In particular, 8-azaXan is an excellent fluorescent probe for purine nucleoside phosphorylases, as
a fluorogenic substrate in the reverse, synthetic pathway.
Received 17 September 2008
Revised 31 December 2008
Accepted 7 January 2009
Available online 14 January 2009
Keywords:
8
-Azaxanthine
Caffeine analogues
Fluorescence
Excited-state proton transfer
Fluorescent probes
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
In the course of our studies on the fluorescence of 8-azapu-
1
6,17
rines
we discovered strong room-temperature emission of 8-
Fluorescent nucleic acid base analogues, such as pyrazolopyr-
azaxanthine (8-azaXan) and its 8-methyl derivative in aqueous
1
8
imidines, 8-azapurines (triazolo[4,5-d]pyrimidines), and the corre-
sponding nucleosides and nucleotides are useful as fluorescent
reporter molecules in biological systems, along with a variety of re-
cently introduced dye-tagged nucleosides, nucleotides and oligo-
media. N-methyl derivatives of 8-azaxanthine were long ago
1
9–21
characterized,
but their room-temperature fluorescence has,
to our knowledge, been overlooked in the literature. We here pres-
ent the fluorescence emission properties of other N-methylated 8-
1
–5
22
nucleotides.
In particular, the fluorescent isosteric analogue of
azaxanthines, including 8-azacaffeine, its biologically active iso-
adenosine, formycin A (8-aza-9-deazaadenosine), has been suc-
cessfully applied to study protein–ligand interactions in a number
mer 1,3,8-trimethyl-8-azaXan, and 8-azatheophylline, as well as
some xanthine analogues of pyrazolo[4,3-d]pyrimidines (8-aza-9-
deazapurine, see Scheme 1).
6
–9
of enzyme systems,
and fluorescent purine alkoxyphosphonates,
including those of the 8-azapurine series, have proven useful as
Detailed analysis of spectral features of various substituted
xanthines was found useful in our studies of purine nucleoside phos-
phorylase (PNP)fromvarioussources. Wehavealreadyshown23 that
8-azaxanthine is a moderately good substrate for the Escherichia coli
PNP-II in the reverse, synthetic pathway. Moreover, this reaction
gives at least two products, that is, 8-azaxanthosine, and, addition-
ally, a non-typical, highly fluorescent nucleoside (kmax = 440 nm at
fluorescent probes of purine-nucleoside phosphorylases.1
0,11
Other
examples include studies of the ionic states of catalytic nucleic
acids, such as ribozymes, by monitoring the pH-dependent emis-
sion of 8-azaGuo residues substituted for Guo,12 and many applica-
tions of 2-aminopurine fluorescence to the study of nucleic acid
interactions and dynamics.1
3–15
23
pH 2–11), tentatively identified as the 8-b-
more, the recombinant calf spleen PNP in the same type of reaction
gives another product which is clearly distinct from the 9-b- -ribo-
D-riboside. Further-
Abbreviations: Xan, xanthine; Xao, xanthosine; 8-AzaXan, 8-azaxanthine; 8-
AzaXao, 8-azaxanthosine; /, fluorescence quantum yield; PNP, purine-nucleoside
phosphorylase.
D
side (unpublished). Identification of these products, and factors
affecting the reaction outcome, is important for further applications
of PNPinnucleosidesynthesis, reviewedrecentlybyMikhailopulo.24
Additionally, spectral studies of macromolecular complexes
with fluorescent ligands undergoing ground- and/or excited-state
q
Presented in preliminary form at the XIV Symposium on Chemistry of Nucleic
Acid Components, Cˇ esk y´ Krumlov, June 8–12, 2008 (Collection Symp. Series 10,
4
78–9).
Corresponding author. Tel.: +48 89 3233324; fax: +48 89 3234547.
E-mail address: jacek.wie@uwm.edu.pl (J. Wierzchowski).
*
0
968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.007

2
586
G. M e˛ dza et al. / Bioorg. Med. Chem. 17 (2009) 2585–2591
O
O
O
CH3
O
O
H
N
H
H C
H C
H
3
C
N
N
N
N
N
3
N
N
3
N
HN
1
7
HN
1
7
N
N
8NH
8N
NH
N
O
9
N
9
N
3
N
H
3
N
O
N
CH3
N
CH3
N
O
O
N
O
O
H
CH
3
8
-azaXan (1)
8
-azatheophylline (2)
8-azacaffeine (3)
O
N
O
O
O
CH3
H C
3
N
N
N
N
N
HN
N
N
N
N
N
N
N
HN
HN
HN
N
CH3
N CH3
N
N
O
O
N
H
O
N
H
O
N
H
O
N
H
CH3
bz
ribose
8
-methyl-8-aza-
8-methyl-
7-methyl-
9
-benzyl-
8-azaXao (8)
-
theophylline (4)
8-azaXan (5)
8-azaXan (6)
8
-azaXan (7)
O
O
O
CH CH3
N
N
CH CH3
N
N
2
2
H C
3
N
R
HN
N
HN
N
CH3
O
N
H
O
N
H
O
N
H
R = C H7
R
R
3
7
-ethyl-
1-methyl-7-ethyl-
8-methyl-
8-aza-9-deazaXan (11)
8
-aza-9-deazaXan (9)
8-aza-9-deazaXan (10)
Scheme 1. Fluorescent analogues of xanthine, examined in this paper. Note that purine numbering, instead of UPAC numbering, is maintained throughout. The neutral forms
of 8-azaXan (1) and 8-azatheophylline (2) are shown in their two, experimentally detectable, protomeric forms.
tautomerism has been shown to provide information on hydrogen
with kmax ꢀ 353 nm, and fluorescence quantum yield (/) of 0.18
at pH ꢀ 3 (Table 1, Fig. 1), but non-fluorescent at neutral pH, where
it exists as a monoanion, due to dissociation of the triazole proton
25,26
bonding patterns within the binding site,
and its possible
changes. For example, 6-methylformycin A, complexed with the
E. coli PNP-I, adopts the imino structure in the absence of phos-
phate, but is converted to the amino form in the ternary complex
enzyme–nucleoside-phosphate.7 Tautomerism of the purine sub-
strate in the binding site of the enzyme appears to be an important
factor affecting the site of its enzymatic ribosylation.
(pK
a
ꢀ 4.6). The relatively strong acidity of the triazole proton is
1
9
1
8
typical for 8-azapurines, including 8-azaXan (1, pK
a
ꢀ 4.8 ).
Similar emission (kmax ꢀ 355 nm) is exhibited by 3-methyl-8-aza-
xanthine (Table 1). Both compounds emit only as the neutral spe-
cies, that is, at pH < 5 (Table 1), like the parent 8-azaXan.18
However, the emission spectra of both are blue-shifted by
ꢀ70 nm relative to that of 8-azaXan, as shown in Figure 1 and Ta-
ble 1. This is in accord with the previously postulated phototauto-
meric behavior of 8-azaXan, where the long-wavelength emission
band is due to formation of the rare anionic species, with N(3)H
Additional interest in the foregoing stems from the fact that
many N-alkyl derivatives of 8-azaxanthine are potent antagonists
2
2
of adenosine receptors, and 8-azaXMP is known as a potent
0
28
inhibitor of the yeast orotidine-5 -phosphate decarboxylase.
The parent 8-AzaXan is a good selective inhibitor of urate oxidase
(
2
7
18
uricase). Within a program designed to infer the in vivo efficacy,
dissociated in the excited state (Scheme 2).
specificity and toxicity of chemical leads, NMR metabolic profiling
has recently been employed to demonstrate that 8-azaXan inhibits
hyphal growth of Aspergillus nidulans, and that this occurs via spe-
cific in vivo inactivation of uricase.29 Neutron diffraction is pres-
ently being applied to crystalline complexes of urate oxidase
with 8-azaXan to determine the protonation state of the latter
In highly fluorescent aromatic compounds, the fluorescence
excitation spectrum usually overlaps the UV-absorption spec-
3
3
trum. This does not apply to 8-azaXan, nor to 8-azatheophylline,
where excitation spectra are red-shifted relative to absorption by
ꢀ8 nm in aqueous medium (see Fig. 1). A similar effect was ob-
served in anhydrous alcoholic media (not shown). We interpret
this phenomenon as resulting from ground-state annular N(7)H–
N(8)H tautomerism in 8-azaXan and 8-azatheophylline. The latter,
3
0
within the active site.
The investigated xanthine and xanthosine analogues are conse-
quently promising candidates as fluorescent probes in these, and
other enzymatic systems, including those involved in xanthine
methylation during caffeine biosynthesis, recently reviewed by Ku-
3
4
15
long ago characterized by l’Abbe et al. by means of N NMR
spectroscopy, demonstrated that in DMSO it exists in the neutral
form as a mixture of protomers with 20% as the N(8)H, 80% as
the N(7)H, and no detectable traces of the N(9)H. These findings
are supported by the spectral properties of 8-azatheophylline
derivatives monomethylated on the triazole ring, described below.
3
1
likowska et al.
2
2
. Results and discussion
2
.2. Fluorescence of trimethyl 8-azaxanthines
.1. Fluorescence of 8-azatheophylline and 3-methyl-8-
azaxanthine: comparison with 8-azaxanthine
8
-Azacaffeine (1,3,7-trimethyl-8-azaXan, 3), is only weakly
fluorescent in aqueous medium, with kmax ꢀ 350 nm, and quantum
One of the oldest known purine analogues, 8-azatheophylline
1,3-dimethyl-8-azaXan, 2),32 is a highly fluorescent compound,
yield, / ꢁ 0.015 (Table 1). Its fluorescence excitation spectrum is
(

G. M e˛ dza et al. / Bioorg. Med. Chem. 17 (2009) 2585–2591
2587
Table 1
Spectral parameters and dissociation constants (pK
a
) of 8-azaxanthine, its N-methyl derivatives, and some 8-aza-9-deazaxanthines in aqueous medium
Compound
pK
a
Form
Solvent, pH
Absorbance:
Fluorescence
ꢂ1
ꢂ1
k
max [nm]
e
max [M cm
]
k
max [nm]
/
s
[ns]
8
8
7
-AzaXan (1)
-Methyl- (5)
-Methyl- (6)
4.8; 9.8
Neutrala
Anion
Neutral
5 mM acetic acid, pH 3.5
Phosphate, pH 7
Phosphate, pH 6
263
265
272
299
273
275
303
278
255
268
271
266
280
276
277
255
297
317
296
323
294
316
6500
8500
8700
6450
6030
nd
5600
7600
9100
5400
10,000
13,000
7800
10,000
8350
8750
nd
420
-
420
420
400
350, 430
400
365
365, 430
355
353
-
350
355
363
365, 430
375
367
365
0.22
<0.005
0.5
0.60
0.13
<0.005
0.23
0.11
ꢀ0.1
ꢀ 0.2
0.18
<0.005
0.013
0.44
9.0
-
7.6 (7.3b)
7.3 (7.0b)
a
12
12
4.3
nd
4.9
nd
nd
nd
3.65
-
nd
3.48
nd
nd
nd
nd
nd
nd
nd
nd
Anion
Neutral
10 mM carbonate, pH 10
5 mM acetic acid, pH 3.5
Methanol
10 mM carbonate, pH 10
10 mM phosphate, pH 8
5 mM acetic acid, pH 3.5
5 mM acetic acid, pH 3.5
5 mM acetic acid, pH 3.5
0.001 N KOH
a
Neutral
Anion
Anion
Neutral
Neutral
Neutral
Anion
Neutral
Neutral
Anion
Neutral
Neutral
Anion
Neutral
Anion
9
-Benzyl- (7)
5.4
3
-Methyl-
4.5; 11.4
4.6 (4.5 )
b
1
,3-Dimethyl- (2)
1,3,7-Trimethyl- (3)
1,3,8-Trimethyl- (4)
8-AzaXao (8)
-
-
H
H
2
O
O
2
b
4.65, 4.7
5 mM phosphate, pH 7
1 mM HCl
5 mM phosphate, pH 7
0.001 M KOH
5 mM phosphate, pH 7
0.001 M KOH
5 mM phosphate, pH 7
0.001 M KOH
0.10
ꢀ0.1
0.025
0.15
0.02
0.04
8-Aza-9-deaza-Xan 9-propyl-7-ethyl- (9)
9-Propyl-7-ethyl-1-methyl- (10)
9-Methyl-8-methyl- (11)
9.24
9.5
nd
6950
7850
nd
397
370
375
9.4
Neutral
Anion
0.029
0.19
nd
Fluorescence quantum yields (/) were determined relative to tryptophan (0.15), with excitation at 280 nm. Data for 8-azaXan and 8-methyl-8-azaXan were reported
1
8
earlier.
a
Refers to ground state; on excitation, rapid proton dissociation occurs.
Fluorimetric titration.
b
slightly red-shifted relative to its UV absorption spectrum (Fig. 2),
possibly due to the so-called cage effect.33 By contrast, the isomeric
sion is additionally supported by the near identity of fluorescence
decay times of 8-azatheophylline (2) and 1,3,8-trimethyl-8azaXan
(4, Table 1).
1
,3,8-trimethyl-8-azaXan (4) is highly fluorescent, with a quantum
yield reaching 0.44 (Table 1), and a fluorescence excitation spec-
trum quite similar, but not identical, to the UV absorption spec-
trum (Fig. 2). We were unable to isolate the pure 9-methyl
2.3. Emission properties of 8-azaxanthines methylated on the
triazole ring: increased excited-state acidity of N(3)H
21
derivative of 8-azatheophylline, due to its instability, but preli-
minary inspection of TLC eluates (kmax = 259 nm20) indicate this
compound to be virtually nonfluorescent in aqueous medium.
We therefore conclude that the observed strong emission of 8-
azatheophylline, 8-azaXan and 3-methyl-8-azaXan must be due
to the N(8)H tautomer, since the only strongly fluorescent N-alkyl
In apparent contrast to the foregoing, all three 8-azaXan deriv-
atives, monomethylated on the triazole ring, emit strongly in the
range of pH 2–11, with emission maxima red-shifted relative to
that of 8-azatheophylline (355 nm), but similar to that observed
in 8-azaXan (420 nm). It was earlier demonstrated that the strong
emission of 8-azaXan (1) and 8-methyl-8-azaXan (5) in aqueous
8
-azatheophylline derivative is that alkylated on N(8). This conclu-
wavelength [nm]
1.2
2
50
300
350
400
450
500
A
a
b
1
B
0.8
0.6
0.4
0.2
0
4
4000
39000
34000
wavenumber [cm -1
29000
24000
19000
]
Figure 1. UV absorption (A), fluorescence emission (a), and fluorescence excitation (s s s) spectra of 8-azatheophylline (2); and, for comparison, the previously reported7
UV absorption (B), and fluorescence emission; (b) spectra of 8-azaXan (1), all in 10 mM acetate, pH 3.5. All spectra are normalized to unity.

2
588
G. M e˛ dza et al. / Bioorg. Med. Chem. 17 (2009) 2585–2591
observed emission is strongly solvent-dependent,18 and for 7-
O
O
methyl-8-azaXan virtually disappears in anhydrous alcohols (Table
1), but can be restored by addition of ꢀ0.4 mM triethylamine (not
shown).
N
N
N
N
HN
hv
HN
NH
[O
NH
]
*
O
N
H
N
H
9
-Benzyl-8-azaXan (7) exhibits intense emission at pH 7–9, that
is, as the monoanion (pK
a
= 5.4), centered at ꢀ370 nm (Table 1).
This emission prevails also at pH < 5, due to the postulated N(3)H
photodissociation, but is accompanied by an additional long-wave-
length band, centered at ꢀ430 nm, which exhibits an almost iden-
tical excitation spectrum, virtually in line with UV absorption (data
not shown). The calculated Stokes’ shift for the long-wavelength
-
H+
pK = 4.9
- H+
a
pK* < 0
O
O
N
ꢂ1
N
band is ca. 16,000 cm , pointing to an additional excited-state
HN
N
_
HN
_
N
process, which we tentatively ascribe to keto–enol phototautomer-
ism (under further investigation).
NH ]*
[O
N
O
N
H
N
The proposed N(3)H photodissociation can be verified by calcu-
fluorescent, 420 nm
*
nonfluorescent
lation of the excited-state acidity constant, or pK , of these com-
3
3
pounds, using Foerster’s formula
Scheme 2. Origin of the long-wavelength (kmax = 420 nm) fluorescence of 8-azaXan
in weakly acidic aqueous medium. Note that this emission is not observed in N(3)-
alkylated 8-azaxanthines.
pK ꢂ pK ¼ 2:1 ꢄ 10ꢂ3½
ꢃ
m
00ðA Þ ꢂ
ꢂ
m
00ðAHÞꢅ
ð1Þ
ꢂ
ꢂ1
where
m
00(A ) and
m
00(AH) are the wave numbers (in cm ) of the
0
–0 transitions of the deprotonated and protonated forms, respec-
medium is due to the anions resulting from dissociation of the
tively. The approximate energies of the 0–0 transitions can be esti-
mated using absorption–emission symmetry, as a crossing point
between normalized emission and the lowest-energy absorption
band, assuming that the same species is responsible for absorp-
tion and emission. In the present case, determination of the ‘true’
emission spectrum of the neutral form of 7-methyl-8-azaXan (6)
and 8-azaxanthosine (8), is possible only in non-aqueous medium,
for example, methanol or isopropanol, since in aqueous medium
the photon-induced N(3)H dissociation is nearly complete18 and
emission of the neutral form is not observed (cf. Fig. 3 and Ref. 18).
18
N(3)H which leads to fluorescent anionic species. This anionic
fluorescence is also visible in the weakly acidic pH range, due to
the excited-state dissociation of the N(3)H, observed directly in
3
5
1
8
8
-methyl-8-azaXan, and postulated for 8-azaXan. The same pro-
cess occurs in the 7-methyl and 9-benzyl-derivatives of 8-azaXan,
as shown below.
The emission maxima of both 8-methyl- (5) and 7-methyl-8-
azaXan (6), 420 nm and 400 nm, respectively, are virtually pH-
independent, but the corresponding excitation spectra reveal
marked pH-dependence and roughly coincide with the UV absorp-
tion spectra both at acidic and basic pH (see Fig 3). It follows that
two different ground-state forms, that is, neutral and monoanionic,
lead to a single emitting species, at least in aqueous medium, sup-
ported additionally by identity of the fluorescence decay times at
pH 3 and 9 (Table 1). The emitting species is clearly the monoan-
ion, with N(3)H dissociated, for the following reasons: (a) the
*
The estimated pK values are listed in Table 2 and show very
strong excited-state acidity of all three N-triazole-monomethylat-
ed 8-azaXan derivatives, as well as 8-azaXao (8), which must be as-
cribed to the significantly decreased electron density on the N(3)
1
nitrogen in the S state, relative to the ground state. By contrast,
the excited-state acidity of 8-azatheophylline (2) is somewhat re-
duced relative to that of the ground-state, indicating in this case
some increase of electronegativity of the triazole nitrogens upon
ꢂ1
Stokes shift at pH > 8 is ꢀ8000 cm , while at acidic pH it increases
ꢂ1
to ꢀ15,000 cm , pointing to an excited-state reaction and (b) the
*
electronic excitation. Although pK values calculated according to
wavelength [nm]
300
1.2
250
350
400
a
1
A
0
0
0
0
.8
.6
.4
.2
0
b
B
42000
37000
32000
27000
22000
-
1
wavenumber [cm ]
Figure 2. UV absorption (A), fluorescence emission (a), and fluorescence excitation (s s s) spectra of 8-methyl-8-azatheophylline (4), and UV absorption (B), fluorescence
emission (b) and fluorescence excitation (d d d) spectra of 7-methyl-8-azatheophylline (8-azacaffeine, 3), all in aqueous medium, with kexcc 275 nm. Note that the
fluorescence quantum yield of 8-azacaffeine (3) is about 30-fold lower than that of 8-methyl-8-azatheophylline (4, see Table 1). The UV absorption spectra correspond to
concentrations of 100
lM, and fluorescence to ꢀ10 lM.

G. M e˛ dza et al. / Bioorg. Med. Chem. 17 (2009) 2585–2591
2589
1
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
A a
0
0
0
0
0
0
0
0
0
B b
45000
40000
35000
30000
25000
20000
-1
wavenumber [cm ]
Figure 3. UV absorption (A), fluorescence emission (a) and fluorescence excitation (d d d) spectra of 7-methyl-8-azaXan (6) at pH 3; and B, b and (s s s) at pH 9. The UV
absorption spectra correspond to a concentration of 100 lM.
plete disappearance of the fluorescence of the neutral form in
Table 2
*
Estimated excited-state acidities (pK ) for several 8-azaXan derivatives, based on the
Foerster cycle
aqueous medium. Such low values for the excited state acidity
*
(
pK ) are not uncommon among substituted heterocycles, for
3
6,37
ꢂ
ꢂ1
ꢂ1
*
*
example, hydroxyquinoline derivatives.
Compound, pK
a
m
00(A ) (cm
)
m00(AH) (cm
)
pK –pK
pK
Intermolecular excited-state proton transfer reactions in purine
analogues like formycin A exhibit significant deuterium isotope ef-
fects. And, in fact, for 8-aza-Xan (1) in 0.4 mM HCl/DCl, the inten-
a
8
7
8
1
-Methyl- (5), 7.6
28,800
29,000
32,800
ꢂ8.4
ꢂ7.5
<ꢂ4
+2
ꢂ0.8
ꢀ0
a
-Methyl- (6), 7.3
-AzaXao (8), 4.65
,3-Dimethyl- (2), 4.5
ꢀ32,600
a
6
32,000
>34,000
<0.5
6.5
b
ꢀ33,500
33,000
sity of the long-wavelength emission band in D
2
O is 63% higher
*
35
than in H O. For the neutral form of its 8-methyl congener (5),
2
Note that the errors of the estimated pK values may be as large as ꢀ3 pH units.
a
Estimated on the basis of UV and fluorescence spectra in methanol.
Estimated on the basis of UV and corrected fluorescence spectra in methanol
the isotope effect is smaller and positive (+18%), and close to that
for the anion (+23%), conceivably due to extremely rapid H/D
b
with ꢀ0.4 mM triethylamine.
*
transfer, characteristic for the very low pK (Table 2). By contrast,
with 7-methyl-8-azaXan (6) and 9-benzyl-8-azaXan (7), we ob-
serve negative effects in 0.5 mM HCl/DCl (ꢂ27% and ꢂ21%, respec-
tively), and positive effects in 1 mM NaOH/NaOD (+4% and +23%).
This is in accord with the postulated participation of excited-state
Foerster are only approximate, and errors of up to ꢀ3 pH units may
3
3,35
occur,
the general trend of the acidity changes upon excitation
is quite evident from a comparison of the UV spectra of the neutral
and monoanionic forms of the compounds (see Fig. 4).
proton transfer in the decay of the S state of triazole-alkylated 8-
azaxanthines.
1
The rate constants for the postulated photo-induced N(3)–H
3
1
dissociation can be estimated with the Weller formula, and for
2.4. Emission of 8-azaxanthosine
*
the compounds exhibiting pK < 0, as in 8-methyl-, and possibly
1
0 ꢂ1
7
-methyl-, 8-azaXan, they must be higher than 6 ꢄ 10
s
, leading
The spectral properties, including emission, of 8-azaXao (8) are
very similar, as might be anticipated, to those of 9-benzyl-8-aza-
to picosecond dissociation times and, consequently, virtually com-
2
1
1
1
1
1
00
80
60
40
20
00
8
0
0
0
0
6
4
2
0
0
2
4
6
8
10
12
pH
Figure 4. Fluorimetric titration of 8-azatheophylline (- - - -; 2), monitored at 355 nm (kexc = 275 nm), and of 7-methyl-8-azaXan (––––; 6), monitored at 400 nm, with
excitation at the isosbestic point, 286 nm. The fitted pK values are, approximately, 4.5 for 8-azatheophylline (2) and 7.0 for 7-methyl-8-azaXan (6). Note that at the lower pH
range, <2, dynamic fluorescence quenching occurs, with apparent pK ꢀ 0.6 for 8-azatheophylline and 1.8 for 7-methyl-8-azaXan.
a

2
590
G. M e˛ dza et al. / Bioorg. Med. Chem. 17 (2009) 2585–2591
Xan (7), but with a somewhat lower fluorescence quantum yield
should be useful, amongst others, in studies on the enzymes in-
volved in caffeine biosynthesis.
(Table 1). They differ markedly from those of the parent 8-azaXan
(
1), since the latter exists as a mixture of N(7)H and N(8)H protom-
Some of the investigated compounds undergo excited-state
proton transfer reactions, which makes their spectral properties
very sensitive to micro-environment changes, including isotope,
buffer and solvent effects. They are promising candidates as fluo-
rescent probes.
ers at weakly acidic pH, and at pH 7 as a monoanion with negative
charge on the triazole ring18 (cf. Table 1).
Fluorimetric, as well as spectrophotometric, titrations of the
nucleoside give pK
a
ꢀ 4.65, somewhat lower than that for 9-ben-
a
zyl-8-azaXan (5.4, Table 1), and this pK is most likely due to
N(3)H dissociation, like in Xao.31 Two fluorescence bands, with
maxima at 365 nm and 430 nm, are observed at weakly acidic pH
4
. Experimental
(
pH 3–5, see Fig. 5). At more acidic pH, ꢀ1.5, a dynamic quenching
UV absorption spectra were recorded on a Cary 300 instru-
of the monoanionic fluorescence (365 nm) is observed, accompa-
nied by an increase in intensity of the long-wavelength band
(
neutral, possibly enol, form of the compound, as described above
for 9-benzyl-8-azaXan.
ment, and steady-state fluorescence emission on a Perkin–Elmer
LS-50B, with spectral bandwidth 3–5 nm. Fluorescence quantum
yields were determined relative to tryptophane (0.15). Fluores-
cence yields and fluorescence excitation spectra were measured
in semi-micro cuvettes, with 4 mm pathlength, to avoid inner fil-
ter effect. Sample absorbance never exceeded 0.05 per 4 mm
pathlength. The built-in correction curves were used to correct
the excitation spectra for spectral distribution of the excitation
beam.
430 nm), suggesting that the latter may be due to a non-typical
2
.5. Fluorescence of 8-aza-9-deaza xanthine analogues
We have also observed that the neutral forms of some xanthine
analogues of pyrazolo[4.3-d]pyrimidines (8-aza-9-deazapurines),
compounds 9, 10, 11 (Scheme 1) exhibit moderate, albeit readily
detectable, emission (Table 1). By contrast, the anions of two of
them, 9 and 11, are intense emitters (/ ꢀ 0.15–0.2) and, further-
more, their absorbance extends well to the red of 300 nm (Table
Fluorescence decay times were measured using an Edinburgh
Analytical Instruments (Edinburgh, Scotland) spectrofluorimeter
model FL-900CDT with a discharge nitrogen lamp as light source
(
approximately 2.5 ns impulse width at half-point). Decays were
fitted to single- or double-exponential models, and the proper
1
) permitting of selective excitation in the presence of proteins
2
model was selected depending on the resultant
v value.
and nucleic acids, thus rendering them particularly useful for li-
gand-binding studies. These compounds are easily prepared from
pyrazolo[4,3-d]pyrimidine-7-ones by direct oxidation with bro-
mine water. The highest fluorescence quantum yields are ob-
served for derivatives alkylated on N(8), as found for many 8-
Syntheses of N-methyl 8-azaxanthines were according to
20,21
the literature,
and their identities confirmed by compari-
son of UV spectral and titration data with those published
3
8
19–21
earlier.
8-azaxanthosine was synthesized enzymatically,
on a milligram scale, from 8-azaxanthine, with the aid of pur-
ine-nucleoside phosphorylase II (xanthosine phosphorylase)
from E. coli as catalyst, and 7-methyguanosine as a ribose do-
nor, and purified by HPLC, using Beckman instrument with
1
6,18
azapurine derivatives,
including non-typical nucleosides and
nucleotides, ribosylated at N(8).3
9
3
. Conclusions
23
dC-18 Atlantis (Waters) column, and gradient 0–20% of ace-
tonitrile/water/methanol (1/2/1, v/v/v) in 0.2 M sodium acetate
Various alkylated 8-azaxanthine analogues exhibit strong fluo-
(
pH 6.7).
Alkylated 8-aza-9-deazaxanthines (pyrazolo[4,3-d]pyrimidine-
,7-diones) were synthesized from the corresponding 7-ones by
rescence in aqueous medium, which has been overlooked in the
chemical literature. Their fluorescence has been shown to be help-
ful, for example, for product identification in enzymatic ribosyla-
tion by various forms of purine-nucleoside phosphorylase, and
5
38
treatment with bromine water, and addition of 3–5% DMF to in-
crease solubility of starting compounds, at room temperature.
wavelength [nm]
0
0
0
0
0
0
0
0
0
.9
.8
.7
.6
.5
.4
.3
.2
.1
0
3
00
400
500
B b
A a
c
43000
39000
35000
31000
wavenumber [cm ]
27000
23000
19000
-1
Figure 5. UV absorption (A), fluorescence emission (a), and fluorescence excitation (d d d) spectra of 8-azaxanthosine (8) in aqueous medium pH 3, and (B, b, s s s) at pH
8
8
. Emission was recorded with an excitation wavelength 290 nm for pH 8 and 260 nm for pH 3, at concentration <10
lM. The UV spectra are normalized to concentration
5
lM. Note that, although the UV absorption is independent of pH in the range 1–3, there is a marked change in emission from pH 3–1.5 (curve c), and lower pH, hence due
to excited-state processes.

G. M e˛ dza et al. / Bioorg. Med. Chem. 17 (2009) 2585–2591
2591
Their spectral parameters were virtually identical to those pub-
lished elsewhere.
17. Wierzchowski, J.; Bzowska, A.; Stepniak, K.; Shugar, D. Z. Naturforsch. Sect., C
2004, 59c, 713.
4
0
18. Wierzchowski, J.; Sepiol, J.; Sulikowski, D.; Kierdaszuk, B.; Shugar, D. J.
Photochem. Photobiol. A 2006, 179, 276.
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