Facile and selective electrostatic stabilization of uracil N(1)- anion by a proximate protonated amine: A chemical implication for why uracil N(1) is chosen for glycosylation site

Article abstract of DOI:10.1021/ja972129n

A question of how uracil nitrogen N(1) is selectively activated in enzymes (e.g., for deglycosylation in uracil-DNA glycosylase) has been utterly overlooked, to which we have addressed by a model study with 6-((1,4,7, 10-tetraazacyclododecyl)methyl)uracil

Full text of DOI:10.1021/ja972129n

J. Am. Chem. Soc. 1997, 119, 10909-10919
10909
Facile and Selective Electrostatic Stabilization of Uracil N(1)-
Anion by a Proximate Protonated Amine: A Chemical
Implication for Why Uracil N(1) Is Chosen for Glycosylation
Site
Eiichi Kimura,*,† Hideyuki Kitamura,† Tohru Koike,† and Motoo Shiro§
Contribution from the Department of Medicinal Chemistry, School of Medicine, Hiroshima
UniVersity, Kasumi 1-2-3, Minami-ku, Hiroshima, 734, Japan, and Rigaku Corporation X-ray
Research Laboratory, Matsubaracho 3-9-12, Akishima, Tokyo, 196, Japan
ReceiVed June 26, 1997X
Abstract: A question of how uracil nitrogen N(1) is selectively activated in enzymes (e.g., for deglycosylation in
uracil-DNA glycosylase) has been utterly overlooked, to which we have addressed by a model study with 6-((1,4,7,-
10-tetraazacyclododecyl)methyl)uracil (HL, cyclen-attached uracil). The uracil N(1)H of the diprotonated cyclen-
attached uracil (HL‚2H+) is easily deprotonated to be N(1)- anion form (L-‚2H+) in aqueous solution. The
deprotonation constant (pKa) of 7.14 for HL‚2H+ a L-‚2H+ + H+ was determined by potentiometric pH titration
at 25 °C with I ) 0.10 (NaClO4). The unusually low deprotonation constant (cf. pKa ) 9.9 for 3-methyluracil) is
due to the electrostatic stabilization of the N(1)- anion by a proximate secondary ammonium cation of the diprotonated
cyclen at physiological pH. The X-ray crystal structure of HL‚2H+ as its dipicrate revealed that the uracil N(1)H
is linked by a hydrogen bond network to one of the cyclen secondary ammonium cation through a water. Crystals
of HL‚2H+‚(picrate)2‚H2O (C25H32N12O17) are triclinic, space group P1h (no. 2) with a ) 9.295(4) Å, b ) 19.67(1)
Å, c ) 8.886(6) Å, R ) 94.36(3)°, â ) 102.95(4)°, γ ) 87.04(4)°, V ) 1576(8) Å3, Z ) 2, R ) 0.054, and Rw )
0.081. The electrostatic stabilization of uracil N(1)- anion is reassessed by a comparative study with a zinc(II)
complex with the cyclen-attached uracil, where Zn2+ in the cyclen cavity strongly binds to the uracil N(1)- (localized)
anion. The deprotonation of N(1)H of HL (1 mM) occurred below pH 5 by the effect of equimolar Zn2+, a stronger
acid than two protons. Crystals of the zinc(II) complex (C13H23N6O2Zn‚ClO4‚H2O) are triclinic, space group P1h
(no. 2) with a ) 9.461(3) Å, b ) 13.156(4) Å, c ) 8.687(2) Å, R ) 101.21(2)°, â ) 103.55(2)°, γ ) 73.21(2)°, V
) 997(0) Å3, Z ) 2, R ) 0.063, and Rw ) 0.093. For comparison, we also have investigated the uracil N(1) acidity
with an ethylenediamine-attached uracil and an isomeric cyclen-attached (at C(5)) uracil. The present example of
electrostatic stabilization of N(1)- anion may explain the facile uracil N(1)-alkyl (e.g., glycosyl) bond formation and
cleavage in enzymes.
Introduction
Scheme 1
The enolization of carbonyl compounds is a crucial event in
a wide variety of key reactions in biochemical transformations.
Examples include enzyme-catalyzed C-C bond formation (and
disruption) or isomerization. Of special interest is a question
how weak bases of enzyme (e.g., imidazole or aspartate and
glutamate anions) whose pKa values are less than 7 can
effectively abstract a proton from carbonyl substrates having
much higher pKa values. Cleland et al. proposed that “low-
barrier hydrogen bonds” in the desolvated transition state are
an effective means of increasing the acidity of substrates or
stabilizing the resulting negative charges, which can explain
most of the enzyme catalysis.1,2 On the other hand, Warshel
argued that the stabilization of solvated transition states by
electrostatic interactions is sufficient to account for the major
part of the enzyme catalytic effects.3
proposed reaction mechanism on the basis of X-ray crystal
structure of uracil-bound UDGase, the nucleophilic attack by a
water activated by Asp88 alone seems insufficient to hydrolyze
the uracil N(1)-glycosyl bonding, and some other factor should
be required to help the uracil leave. Namely, the uracil N(1)
needs to be activated, probably by protonation in addition to
electrostatic interaction. The authors speculated that the pro-
tonated His210, which was proven essential by the site-directed
mutagenesis, might protonate directly to N(1) or indirectly to
the carbonyl C(2)dO.
Recently, we were interested in a mechanism of the facile
N(1)-glycosyl bond disruption by the uracil-DNA glycosylase
(UDGase), where a target uracil group may be activated by
electrostatic interaction in the enzyme (Scheme 1).4 In the
† Hiroshima University.
§ Rigaku Corporation.
X Abstract published in AdVance ACS Abstracts, October 15, 1997.
(1) (a) Cleland, W. W.; Kreevoy, M. M. Science 1994, 264, 1887-1890.
(b) Cleland, W. W.; Kreevoy, M. M. Science 1995, 269, 104.
(2) Frey, P. A. Science 1995, 269, 104-106.
(3) Warshel, A.; Papazyan, A.; Kollman, P. A. Science 1995, 269, 102-
104.
This proposed mechanism led us to a similar type of question
that was raised for the carbonyl enolases,5 racemases,6 and
(4) (a) Savva, R.; McAuley-Hecht, K.; Brown, T.; Pearl, L. Nature 1995,
373, 487-493. (b) Slupphang, G.; Mol, C. D.; Kavli, B.; Arvai, A. S.;
Krokan, H. E.; Tainer, J. A. Nature 1996, 384, 87-92.
S0002-7863(97)02129-X CCC: $14.00 © 1997 American Chemical Society

10910 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Kimura et al.
Table 1. Comparison of the Protonation Constantsa of Cyclen Ligands and Zinc(II) Complexation Constants at 25 °C with I ) 0.10 (NaClO4)
6
11
12
cyclen (7a)
HE-cyclen (7b)
log K1
log K2
log K3
log K4
10.83 ( 0.03
9.67 ( 0.02
7.14 ( 0.02c
<2
10.25 ( 0.03
8.81 ( 0.02c
<2
11.62 ( 0.03
9.98 ( 0.03
8.56 ( 0.02c
<2
11.04b
9.86b
<2b
10.72b
9.28b
<2b
<2b
<2b
log K(Zn-L-)
log K(Zn-HL′′)
pKa1
20.2 ( 0.1d
15.3b,e
13.8b,e
12.0 ( 0.1f
6.0 ( 0.1g
10.3 ( 0.1h
pKa2
a Kn ) [HnL-]/[Hn-1L-]aH
.
b From ref 10. c The protonaiton constants correspond to uracil pKa values. d K(Zn-L-) ) [Zn-L-]/[Zn2+][L-],
+
determined by UV spectroscopic pH titration. e The zinc(II) complexation constants are defined as [Zn-7]/[Zn2+][7]. f K(Zn-HL′′) ) [Zn-HL′′]/
[Zn2+][L′′-‚H+], determined by potentiometric pH titration. g pKa1 ) [Zn-L′′-]aH /[Zn-HL′′], determined by potentiometric pH titration. h pKa2
)
+
-
[Zn-L′′2-]aH /[Zn-L′′ ], determined by potentiometric pH titration.
+
Scheme 2
solution) show characteristic UV spectral maximum at 284 and
266 nm, respectively.8 Since deprotonation of the N(3)H almost
overlaps with dissociation of the N(1)H in uracil, there was no
direct way to define the pKa value specific for the N(1)H. It
was puzzling to us why nature specifically selects N(1) for the
alkylation (e.g., glycosylation) site rather than the probably a
little more nucleophilic N(3) site.9 Since the deglycosylation
transiently or intermediately yields N(1)- uracil 3, the stronger
is the acidity of N(1)H (i.e., the easier to generate 3) in UDGase,
the faster would be the catalytic deglycosylation rate. Thus,
we were interested in how a protonated amine (+NH, like the
protonated His210 in UDGase) in the vicinity of uracil would
affect the pKa value of the N(1)H.
Scheme 3
We now have designed 6-((1,4,7,10-tetraazacyclododecyl)-
methyl)uracil (HL, cyclen-attached (at C(6)) uracil, 6). Since
1,4,7,10-tetraazacyclododecane 7a (cyclen) and 1-(2-hydroxy-
ethyl)-1,4,7,10-tetraazacyclododecane 7b (HE-cyclen) are both
in diprotonated species at physiological pH (see Table 1),10 we
might observe the facile and specific deprotonation of the uracil
N(1)H under the possible strong influence of the proximate
protonated amines of the cyclen unit in 8a (HL‚2H+) at
physiological pH. Earlier, we reported that triprotonated
macrocyclic pentaamines and hexaamines facilitated the depro-
tonation of polycarboxylic acids (e.g., citric acid),11 phosphates
(e.g., ATP),12 and catechols13 to form stable 1:1 anion complexes
at neutral pH.14 Recently, Lehn’s group reported an enolase
model with macrocyclic polyamines, where the binding mal-
onate anion to the polyammonium macrocycles accelerates the
H/D exchange at the CH2 group (see 9).15 Anslyn’s group
designed polyazaclefts as a model for enolase and racemase,
which bind to deprotonated 1,3-diketone anions through “tra-
ditional hydrogen bonds” (THBs) in acetonitrile (see 10).16 They
found that THBs between the NH groups of the polyazacleft
and 1,3-cyclohexanedione increase the acidity of the CH2 group
by 1.0 pKa unit.
isomerases.7 How is the developing uracil N(1)- anion (as an
intermediate or transition state at the deglycosylation) stabilized?
This question is translated into how the uracil N(1)H is rendered
more acidic (see Scheme 2)? More specifically, how much the
pKa value for uracil N(1)H can be lowered in the active center
of UDGase? This issue may have far deeper and more
fundamental implications in reactivity of nucleobases in nucleic
acid biochemistry.
Nakanishi et al. showed that uracil 1 deprotonates with a pKa
value of 9.5 (spectrophotometrically determined at 25 °C in
aqueous solution) to form N(3)- species 2, which is in
equilibrium with N(1)- tautomer 3 in ca. 1:1 ratio (see Scheme
3).8 To define the explicit acidities for N(1)H and N(3)H,
3-methyluracil 4 has a pKa value of 10.0 (for N(1)H deproto-
nation) and 1-methyluracil 5 has a pKa value of 9.8 (for N(3)H
deprotonation).8 The conjugate bases of 4 and 5 (in aqueous
(9) In ordinary chemical reactions of uracil, N(3) and N(1) or both are
readily alkylated. For specific N(1) alkylation (e.g., glycosylation), N(3)H
should be protected as a C(4)-O-silylated form, for example. It may be
simply argued that the enzymes stereoselectively hold uracil so that only
the N(1) site is available for a nucleophile.
(5) Anderson, V. E.; Cleland, W. W. Biochemistry 1990, 29, 10498-
10503.
(6) (a) Albery, W. J.; Knowles, J. R. Biochemistry 1986, 25, 2572-
2577. (b) Powers, V. M.; Koo, C. W.; Kenyon, G. L.; Gerlt, J. A.; Kozarich,
J. W. Biochemistry 1991, 30, 9255-9263. (c) Guthrie, J. P.; Kluger, R. J.
Am. Chem. Soc. 1993. 115, 11569-11572. (d) Gerlt, J. A.; Gassman, P. G.
Biochemistry 1993, 32, 11943-11952. (e) Babbitt, P. C.; Mrachko, G. T.;
Hasson, M. S.; Huisman, G. W.; Kolter, R.; Ringe, D.; Petsko, G. A.;
Kenyon, G. L.; Gerlt, J. A. Science 1995, 267, 1159-1161.
(7) (a) Xue, L.; Talalay, P.; Mildvan, A. S. Biochemistry 1990, 29, 7491-
7500. (b) Knowles, J. R. Nature 1991, 350, 121-124. (c) Lodi, P. J. ;
Knowles, J. R. Biochemistry 1993, 32, 4338-4343. (d) Wu, Z. R.;
Ebrahimian, S.; Zawrotny, M. E.; Thornburg, L. D.; Perez-Alvarado, G.
C.; Brothers, P.; Pollack, R. M.; Summers, M. F. Science 1997, 276, 415-
418.
(10) Koike, T.; Kajitani, S.; Nakamura, I.; Kimura, E.; Shiro, M. J. Am.
Chem. Soc. 1995, 117, 1210-1219.
(11) (a) Yatsunami, T.; Sakonaka, A.; Kimura, E. Anal. Chem. 1981,
53, 477-480. (b) Kimura, E.; Sakonaka, A.; Yatsunami, T.; Kodama, M.
J. Am. Chem. Soc. 1981, 103, 3041-3045.
(12) Kimura, E.; Kodama, M.; Yatsunami, T. J. Am. Chem. Soc. 1982,
104, 3182-3187.
(13) Kimura, E.; Watanabe, A.; Kodama, M. J. Am. Chem. Soc. 1983,
105, 2063-2066.
(14) Review: Kimura, E. In Topics in Current Chemistry; Boschke, F.
L., Ed.; Springer-Verlag: New York, 1985; Vol. 128, pp 113-141.
(15) Fenniri, H.; Lehn, J.-M.; Marquis-Rigault. A. Angew. Chem., Int.
Ed. Engl. 1996, 35, 337-338.
(16) (a) Kelly-Rowley, A. M.; Lynch, V. M.; Anslyn, E. V. J. Am. Chem.
Soc. 1995, 117, 3438-3447. (b) Perreault, D. M.; Anslyn, E. V. Angew.
Chem., Int. Ed. Engl. 1997, 36, 432-450.
(8) Nakanishi, K.; Suzuki, N.; Yamazaki, F. Bull. Chem. Soc. Jpn. 1961
34, 53-57. Naturally, there are resonances with O- forms. In the following
discussion, we draw only the representative N- tautomers for brevity.

Electrostatic Stabilization of Uracil N(1)- Anion
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10911
In the present study using our new intramolecular polyam-
monium macrocycle probe 6, we have discovered that the
deprotonation of the uracil group occurs at physiological pH
with a pKa Value of 7.1. Furthermore, the monoanionic charge
localizes predominantly at N(1), unlike the known case of uracil
(2 a 3).8 Herein, we present the synthesis, the pKa value
determination of the N(1)H in the presence of 2H+ (HL‚2H+,
8a) or Zn2+ (Zn-HL, 8b) in cyclen. For comparison, we also
have synthesized 6-(N-(N,N′-dimethylethylenediamino)methyl)-
uracil (11, HL′) and 5-((1,4,7,10-tetraazacyclododecyl)methyl)-
uracil (12, HL′′, an isomer of 6) to investigate the effects of
ammonium and zinc(II) ion on the uracil N(1)H and N(3)H
acidities. The results from the present chemical model would
be of importance in understanding not only the activation
mechanism of the uracil N(1) but also electrostatic effects in
the activation of neutral weak acids in aqueous solution.
Figure 1. Typical titration curves for cyclen-attached (at C(6)) uracil
6 and cyclen-attached (at C(5)) uracil 12 at 25 °C with I ) 0.10
(NaClO4): (a) 1.0 mM 6‚3HBr; (b) 1.0 mM 12‚3HBr; (c) a + 1.0 mM
ZnSO4; (d) b + 1.0 mM ZnSO4. eq(OH-) is the number of equivalents
of base added.
Scheme 4
aqueous HBr in MeOH. The ligand was purified as its 3HBr
salt, as colorless prisms in 75% yield. To compare with 13,
another zinc(II) complex with 12 was synthesized. Treatment
of 12‚3HBr with equimolar Zn(ClO4)2 in aqueous solution at
pH 8 yielded colorless prisms of 1:1 zinc(II) complex 14, as its
monoperchlorate salt. Characterization of 14 is described later.
In order to see the effects of the macrocyclic tetraamine, a
reference uracil derivative 11 (HL′) was synthesized by treating
6-(chloromethyl)uracil with N,N′-dimethylethylenediamine in
EtOH and purified as its dihydrobromic acid salt.
Results
Synthesis of Cyclen-Attached Uracils (6 and 12), Zinc(II)
Complexes of 6 and 12 (13 and 14), and Ethylenediamine-
Attached Uracil 11. The cyclen-attached (at C(6)) uracil 6
(HL) was synthesized by reaction of 3-fold excess amounts of
cyclen with 6-(chloromethyl)uracil in EtOH. The free ligand
6 (HL) was purified by silica gel column chromatography and
isolated as its 3HBr salt (colorless prisms) in 74% yield. To
see a probable stronger electrostatic effect on N(1), zinc(II) was
placed into the cyclen cavity. Treatment of 6‚3HBr with
equimolar Zn(ClO4)2 in aqueous solution at pH 8 yielded
colorless prisms of 1:1 zinc(II) complex 13, as its monoper-
chlorate salt. The structure of 13, where the deprotonated N(1)-
strongly binds to zinc(II), was determined by the UV spectro-
photometric characterization and X-ray crystal analysis, as
described below. The isomeric cyclen-attached (at C(5)) uracil
12 (HL′′) was synthesized by reaction of 2-fold excess amounts
of 1,4,7-tris(tert-butyloxycarbonyl)cyclen (3Boc-cyclen)17 with
5-(bromomethyl)uracil and subsequent deprotection of Boc with
Protonation Constants of Cyclen-Attached (at C(6)) Uracil
6. The protonation constants (Kn) of 6 (HL) were determined
by potentiometric and spectrophotometric (UV and NMR) pH
titrations of 6‚3HBr (HL‚3H+, 1 mM) against 0.10 M NaOH
with I ) 0.10 (NaClO4) at 25 °C (except for NMR study with
20 mM of 6 at 35 °C). A typical potentiometric pH titration
curve is shown in Figure 1a. The titration data were analyzed
+
for the acid-base equilibria 1-4, where aH is the activity of
H+. Table 1 summarizes the protonation constants (K1-4) as
logarithmic values in comparison with those for reference
compounds. The four protonation constants are assigned
according to Scheme 5, where L-‚nH+ species are zwitterionic
forms of 6. This assignment came from the following facts:
(i) Cyclens such as 7a and 7b show two large protonation
constants log K1 of 11.0 and 10.7 and log K2 of 9.9 and 9.3,
respectively, while the remaining two values (log K3 and log
K4) are below 2 (see Table 1).10 (ii) The pH-dependent UV
absorption spectral change for 6 at pH 4.4-9.0 (Figure 2a) is
analogous to that for the monodeprotonation mode of 3-meth-
(17) Kimura, E; Aoki, S.; Koike, T; Shiro, M. J. Am. Chem. Soc. 1997,
119, 3068-3076.

10912 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Kimura et al.
Scheme 5
Figure 2. (a) UV-pH profile for cyclen-attached (at C(6)) uracil 6 (1
mM) at 25 °C with I ) 0.10 (NaClO4): pH 4.4, λmax ) 262 nm (ꢀ
9280); pH 7.1; pH 8.6, λmax ) 285 nm, (ꢀ 10550); pH 12.5, λmax
)
280 nm (ꢀ 4980). (b) The effect of pH upon the 13C chemical shifts of
cyclen-attached (at C(6)) uracil 6 (20 mM) at 35 °C with I ) 0.1
(NaClO4).
yluracil 4 (Figure 3a). Accordingly, log K3 of 7.14 was assigned
to the protonation of uracil N(1)- anion (corresponding to pKa
value of uracil N(1)H).
L- + H+ a L-‚H+
K1 ) [L-‚H+]/[L-]aH
(1)
+
L-‚H+ + H+ a L-‚2H+
K2 ) [L-‚2H+]/[L-‚H+]aH
+
(2)
L-‚2H+ + H+ a HL‚2H+ K3 ) [HL‚2H+]/[L-‚2H+]aH
+
(3)
Figure 3. (a) UV-pH profile for 3-methyluracil (1 mM) at 25 °C with
I ) 0.10 (NaClO4): pH 5.7, λmax ) 258 nm (ꢀ 7150); pH 9.9; pH 11.0,
λmax ) 282 nm, (ꢀ 9890). (b) The effect of pH upon the 13C chemical
shifts of 3-methyluracil (30 mM) at 35 °C with I ) 0.1 (NaClO4).
HL‚2H+ + H+ a HL‚3H+ K4 ) [HL‚3H+]/[HL‚2H+]aH
+
(4)
Further Characterization of the Protonated Forms of 6
in Aqueous Solution. In the UV spectrophotometric titration
in the pH range 4.4-9.0 (Figure 2a), both a decrease in
absorption at 262 nm and an increase at 285 nm with an
isosbestic point at 271 nm allowed the estimation of the same
deprotonation constant (pKa) of 7.1 for HL‚2H+ a L-‚2H+ +
H+, which is almost the same as log K3 value of 7.14 determined
by the potentiometric pH titration. A similar treatment of the
absorbance changes against pH for 3-methyluracil 4 gave a pKa
value of 9.9 for the N(1)H deprotonation (see Figure 3a).
One may extrapolate a UV absorption maximum coefficient
(ꢀ) for the N(1)- form of L-‚2H+ in the same fashion as
Nakanishi’s8 as follows: The neutral form of 3-methyluracil 4
at pH 5.7 (ꢀ 7150 at 258 nm) is converted 100% into the N(1)-
form at pH 11.0 (ꢀ 9890 at 282 nm) (see Figure 3a). Likewise,
if the uracil-undeprotonated species, HL‚2H+ 8a (λmax 262 nm,
ꢀ 9280 at pH 4.4) (see Figure 2a) is completely converted into
the zwitterionic N(1)- forms (i.e., L-‚nH+, where n ) 1 or 2)
at pH 8.6, its maximum ꢀ value should be (9280/7150) × 9890
) 12830. The observed ꢀ value for the uracil anion species,
however, was 10550 at 285 nm (see Figure 2a). The lesser
UV absorption was ascribed to coexistence of the N(3)-
tautomer, which will have an absorption maximum at 262 nm
but a negligible absorption at 285 nm. The composition of the
N(1)- tautomer is thus estimated to be (10550/12830) × 100
) 82%, and the remaining 18% is for N(3)- form (see Scheme
5).
The pH-dependent 13C chemical shifts of the uracil moiety
of 6 (Figure 2b), 3-methyluracil 4 (Figure 3b), 1-methyluracil
5 (Figure 4b), and uracil 1 (Figure 5b) in H2O at 35 °C with I
) 0.1 (NaClO4) are compared. In the case of 4, the pH-
dependent chemical shifts are most vivid at C2 and C6, from
which a protonation constant of 9.9 for 3-methyluracil N(1)-
anion is estimated. In a similar fashion, the pH-dependent
chemical shifts for each uracil carbon of 6 showed a protonation
constant of 7.1 for the twitterionic L-‚2H+ species. Above pH
9 the chemical shifts increase for C4, while it decreases for
C6. These behaviors are somewhat in analogy with those for
C4 and C6 of 5 (Figure 4b). This indicates that, as the two
protons in the cyclen (L+‚2H+) are removed at pH >9, the
heavily localized N(1)- negative charge spreads to N(3), as
depicted in two tautomers for L- in Scheme 5. In consistency,
the UV absorption of 6 at pH 12.5 deviated (see Figure 2a)
from the pattern for 4 to that for anionic uracil tautomers 2 a

Electrostatic Stabilization of Uracil N(1)- Anion
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10913
Figure 6. (a) UV-pH profile for ethylenediamine-attached (at C(6))
uracil 11 (1 mM) at 25 °C with I ) 0.10 (NaClO4): pH 4.2, λmax
)
262 nm (ꢀ 9350); pH 8.8; pH 9.7, λmax ) 283 nm, (ꢀ 6630); pH 12.3,
λmax ) 286 nm (ꢀ 5860). (b) The effect of pH upon the 13C chemical
shifts of ethylenediamine-attached (at C(6)) uracil 11 (20 mM) at 35
°C with I ) 0.1 (NaClO4).
Figure 4. (a) UV-pH profile for 1-methyluracil (1 mM) at 25 °C with
I ) 0.10 (NaClO4): (a) pH 6.1, λmax ) 267 nm (ꢀ 9440); (b) pH 9.7;
(c) pH 11.0, λmax ) 265 nm, (ꢀ 6860). (b) The effect of pH upon the
13C chemical shifts of 1-methyluracil (30 mM) at 35 °C with I ) 0.1
(NaClO4).
Scheme 6
Figure 5. (a) UV-pH profile for uracil (1 mM) at 25 °C with I ) 0.10
(NaClO4): (a) pH 6.8, λmax ) 259 nm (ꢀ 8070); (b) pH 9.3; (c) pH
11.1, λmax ) 283 nm, (ꢀ 5850). (b) The effect of pH upon the 13C
chemical shifts of uracil (30 mM) at 35 °C with I ) 0.1 (NaClO4).
3 (see Figure 5a, at pH 11.1). At pH 12.5 (Figure 2a), the fact
that the major species L- has ꢀ of 4980 (at 280 nm) smaller
than that for the L-‚2H+ species indicates further charge shift
from N(1)- to N(3)-. The ratio of the N(1)- form to N(3)-
form for L- was calculated to be (4980/12830) × 100 ) 39%
to 61% (see Scheme 5).
dependent UV spectra (Figure 6a), as for 6 with reference to
3-methyluracil 4: The hypothetical ꢀ for 100% of the N(1)-
anionic tautomer for L′-‚H+ is assumed to be (9890/7150) ×
9350 ) 12930. Thus, the observed ꢀ ) 6630 at pH 9.718 (at
283 nm) for 11 corresponds to the (6630/12930) × 100 ) 51%
of the complete N(1)- anionic form. Namely, the tautomer ratio
for N(1)- anion to N(3)- anion is ca. 1:1 for L′-‚H+. The
extinction coefficient 6630 at pH 9.7 (at 283 nm) lowered to
5860 at pH 12.3 (at 286 nm), which represents (5860/12930)
× 100 ) 45% of the N(1)- anionic species for L′-. Therefore,
it is concluded that irrespective of the protonated forms L′-‚H+
or L′- the ratio for N(1)- form to N(3)- form remains almost
1:1 with 11.
Protonation Constants of An Isomeric Cyclen-Attached
(at C(5)) Uracil 12. The protonation constants (log K1-4) of
12 were determined by potentiometric pH titration and UV
(Figure 7a) and 13C NMR spectrophotometric pH titrations
(Figure 7b) of 12‚3HBr (HL′′‚3H+) with I ) 0.10 (NaClO4)
(see Table 1). A typical potentiometric pH titration curve is
shown in Figure 1b. The four protonation constants are assigned
according to Scheme 7.
Protonation Constants of Ethylenediamine-Attached (at
C(6)) Uracil 11. The two protonation constants (log K1 ) 10.25
and log K2 ) 8.81) were determined by potentiometric pH
titration at 25 °C with I ) 0.10 (NaClO4) (see Table 1). Taking
together with the UV and 13C NMR results (see Figure 6), the
two protonation equilibria are assigned as shown in Scheme 6.
The pH-dependent UV absorption spectral change for 11 at pH
4.2-12.1 (Figure 6a) is more similar to that for the deproto-
nation mode of uracil 1 than that of 3-methyluracil 5. The pH-
dependent 13C NMR chemical shifts for 11 are sigmoidal in a
similar fashion to those for uracil 1 (Figure 5b) with an inflection
point at pH 8.8 (Figure 6b), which is almost the same as the
log K2 value (corresponding to pKa value for uracil NH).
The ratio for N(1)- anionic to N(3)- anionic tautomer for
L′-‚H+ and L′- forms of 11 were calculated from the pH-

10914 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Kimura et al.
Figure 8. An ORTEP drawing (30% probability ellipsoids) of 8
(picrate)2‚H2O. All hydrogen atoms bound to carbon are omitted for
clarity. The selected intermolecular distances (Å): Ow‚‚‚N1 2.778(3),
N1-H1 0.95, Ow‚‚‚H1 1.85, Ow‚‚‚N13 2.989(3), N13-H13 0.95,
Ow‚‚‚H13 2.18. The selected bond angles (deg): Ow‚‚‚H1-N1 167,
Ow‚‚‚H13-N13 142.
Figure 7. (a) UV-pH profile for cyclen-attached (at C(5)) uracil 12 (1
mM) at 25 °C with I ) 0.10 (NaClO4): pH 4.2, λmax ) 262 nm (ꢀ
8300); pH 8.5; pH 9.3, 288 nm (ꢀ 4430); pH 11.6, λmax ) 288 nm, (ꢀ
7230). (b) The effect of pH upon the 13C chemical shifts of cyclen-
attached (at C(5)) uracil 12 (20 mM) at 35 °C with I ) 0.1 (NaClO4).
X-ray Crystal Structure of Diprotonated Cyclen-Attached
(at C(6)) Uracil 8 as Dipicrate Salt. In an attempt to isolate
and characterize the zwitterionic L-‚2H+ species of 6, the free
form HL in H2O was mixed with an equivalent amount of picric
acid in EtOH and then the solution pH was adjusted to 8.5.
Slow evaporation of the solvents at room temperature precipi-
tated yellow prisms, which turned out to have a formula
HL‚2H+‚(picrate)2 rather than L-‚2H+‚(picrate) in 30% yield.
It has a structure consisting of diprotonated cyclen and neutral
uracil, as finally determined by X-ray crystal analysis. When
2 equiv of picric acid were used without pH adjustment, the
yield rose to 82% (see Experimental Section).
Scheme 7
The crystal structure (Figure 8 with important bond angles
and distances) shows that the cyclen N(13)H and the uracil
N(1)H are linked by hydrogen bonds to the same water mole-
cule, Ow (N(1)‚‚‚Ow, 2.778(3) Å; N(13)‚‚‚Ow, 2.989(3) Å).
Thus, the water oxygen atom looks like taking a tetrahedral
coordination surrounded by four hydrogen atoms. The two pic-
rate O- anions, O7′ and O7′′, lie within hydrogen bond distance
(O7′‚‚‚Ow, 2.868(3) Å; O7′‚‚‚N13, 2.707(4) Å; O7′′‚‚‚N19,
2.568(3) Å).
Zinc(II) Complex of Cyclen-Attached (at C(6)) Uracil 6,
13. The zinc(II) complexation equilibrium with 6 was deter-
mined by potentiometric pH titration at 25 °C with I ) 0.10
(NaClO4). The titration curve with 1 mM 6‚3HBr and equimo-
lar zinc(II) (Figure 1c) revealed formation of a stable 1:1 zinc-
(II) complex 13 (Zn-L-) with a deprotonated uracil N(1)--
coordination at physiological pH, a conclusion being derived
from the observation of a neutralization break at eq(OH-) ) 4.
Since the equilibration for zinc(II) complexation at pH < 6 was
extremely slow, a minimum of 1 h separated each titration point.
Further deprotonation or precipitation of zinc(II) hydroxide was
not observed at eq(OH-) > 4, indicating that the zinc(II)
complex Zn-L- remains stable up to pH 12. The formation
constants of the zinc(II) complex 13 (Zn-L-) could not be
determined, because the buffer pH is too low (i.e., 13 is
extremely stable). Therefore, we resorted to the UV spectral
pH titration under the same conditions (see Figure 9) for
determination of the complexation constant K(Zn-L-) defined
by eq 5. The structure of 13 was confirmed by X-ray crystal
structure analysis, as described below.
In the UV titration in the pH range 4.2-11.6 (Figure 7a),
both a decrease mode at 262 nm and an increase mode at 288
nm gave log K3 of 8.56 for the protonation of uracil N- anion
(corresponding to pKa value of uracil NH). The pH-dependent
ratio of N(3)- form (or more probably C(4)-enolate) to N(1)-
form for 12 was estimated as done for 6 and 11. Using the
maximum ꢀ value of 8300 at 262 nm for 100% HL′′‚2H+ (at
pH 4.2) and ꢀ values for anionic and neutral forms of
3-methyluracil 5 (Figure 5a), one can estimate a hypothetical ꢀ
value of 11 480 () (9890/7150) × 8300) for the 100% N(1)-
species, L′′-‚2H+ of 12. Since the observed ꢀ is 4430 at 288
nm (at pH 9.3), N(1)- form for L′′-‚H+ is present in 39% ()
(4430/11480) × 100). Meanwhile at pH 11.6, ꢀ of 7230
represents (7230/11480) × 100 ) 63% of the total N(1)- form
for L′′- (see Scheme 7). The pH-dependent 13C chemical shifts
are evident at C2 and C4 (Figure 7b) with an inflection point
at pH 8.6, which is almost the same as the pKa value for the
uracil group.
L- + Zn2+ a Zn-L- (13)
K(Zn-L-) ) [Zn-L-]/[L-][Zn2+] (5)
The UV absorption maximum for 13 (Zn-L-) occurs at 278
nm with ꢀ ) 12 330 (pH > 6), which is very close to estimated

Electrostatic Stabilization of Uracil N(1)- Anion
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10915
Figure 10. An ORTEP drawing (30% probability ellipsoids) of 13‚
ClO4‚H2O. Perchlorate anion and water molecules are omitted for
clarity. The selected bond lengths (Å): Zn-N1 1.977(4), Zn-N10
2.207(4), Zn-N13 2.145(4), Zn-N16 2.096(4), Zn-N19 2.160(4). The
selected bond angles (deg): N1-Zn-N10 82.2(1), N1-Zn-N13 115.7-
(2), N1-Zn-N16 138.4(2), N1-Zn-N19 103.8(2), N10-Zn-N13
81.0(1), N10-Zn-N16 139.0(2), N10-Zn-N19 81.5(2), N13-Zn-
N16 83.6(2), N13-Zn-N19 133.7(2), N16-Zn-N19 82.3(2).
Figure 9. UV-pH profile for cyclen-attached (at C(6)) uracil zinc(II)
complex 13 (1 mM) at 25 °C with I ) 0.10 (NaClO4): pH 2.1, λmax
262 nm (ꢀ 9280); pH 3.9; pH 6.6, λmax ) 278 nm, (ꢀ 12 330).
)
Scheme 8
moiety of four nitrogens (N(10), N(13), N(16), N(19)) and a
unidentate uracil N(1)- anion. The Zn-N(1)- bond distance
1.977(4) Å is much shorter than the average Zn-N(cyclen) bond
distance of average 2.15 Å, indicating strong interactions be-
tween the N- anion and zinc(II). Recently, we reported a similar
N5-coordinate (with cyclen N4 and dansylamide N-) zinc(II)
complex 15,20 where the Zn2+-N- bond distance is 1.969 Å.
These facts are in agreement with very strong acidic properties
of zinc(II) in favor of uracil N- anion over neutral nitrogen.
maximum ꢀ values of 12 200 and 12 830 for the N(1)- tautomer
of uracil 38 and the N(1)- form of 6, respectively. As pH is
lowered to pH 2.1, the UV absorption maximum is shifted lower
to a stationary peak with ꢀ ) 9280 at 262 nm, which is identical
to that for the protonated ligand HL‚2H+ 8a (see Figure 9).
Since we observed an isosbestic point (at 268 nm) in this
process, we postulated the equilibrium, as depicted in Scheme
8. Both from the UV absorption decrease at 278 nm and the
increase at 262 nm, we saw occurrence of the deprotonation of
uracil N(1)H of 6 in acidic pH by the effect of equimolar Zn2+
,
which illustrates an extremely strong electrostatic effect by the
proximate Zn2+ in the cyclen cavity. A pH value (pK50) for
50% zinc(II) complexation () 50% deprotonation of uracil
group) is 3.9 with [Zn2+] ) [total ligand] ) 1 mM at 25 °C
with I ) 0.10 (NaClO4). For comparison, we saw the pKa of
7.1 for the same N(1)H deprotonation by the 2H+ cationic effect.
By employing a usual UV spectroscopic pH titration procedure
using the pK50 and ligand protonation constants log Kn,19 we
obtained complexation constant log K(Zn-L-) of 20.2 (see
Table 1).
An X-ray Crystal Structure of Cyclen-Attached (at C(6))
Uracil Zinc(II) Complex (13‚ClO4‚H2O). A colorless prism
of 13‚ClO4‚H2O for X-ray crystallographic study was obtained
by slow recrystalization of 13‚ClO4‚H2O from H2O solution at
room temperature. The elemental analysis (C, H, N), 1H NMR,
and IR data are all compatible with a formula, 13‚ClO4‚H2O.
The X-ray crystal structure provided unequivocal evidence for
the axial coordination of the uracil N- anion, as shown by an
ORTEP drawing with 30% probability thermal ellipsoids in
Figure 10.
Zinc(II) Complexes of an Isomeric Cyclen-Attached (at
C(5)) Uracil 12. The zinc(II) complexation equilibria were
determined by potentiometric pH titration of 12‚3HBr (1 mM)
in the presence of equimolar zinc(II) at 25 °C with I ) 0.10
(NaClO4). Analysis of the pH titration curve for C(5)-isomer
(Figure 1d) revealed the formation of a 1:1 zinc(II) complex
16 (Zn-HL′′) and its monodeprotonated complex 14 (Zn-L′′-)
below pH 7 and a further deprotonated complex 17 (Zn-L′′2-
)
over pH 10 (see Scheme 9). The complexation constant log
K(Zn-HL′′) of 12.0 and two deprotonation constants pKa1 of
6.0 and pKa2 of 10.3 are defined by eqs 6-8 (see Table 1). The
crystalline 1:1 complex 14 was isolated as its monoperchlorate
salt at pH 8. Its UV spectrum (adjusted to pH 8.4 in aqueous
solution) showed λmax 264 nm (ꢀ 4430) (Figure 11), which is
somewhat close to the UV absorptions (λmax 269 nm, ꢀ 5080)
of 4-ethoxyuracil 18.8 We thus assigned the monodeprotonated
complex Zn-L′′- to C(4)-enolate O--bound structure 14.
On acidifying the solution, the absorption at 265 nm increased
with an isosbestic point (at 295 nm), whereby the same pKa1
value of 6.0 (to 14) was obtained spectophotometrically. Further
addition of acid (pH < 4) decomposed Zn-HL′′ (16) into HL′′‚
2H+ and Zn2+, as judged by the stationary peak (λmax 262 nm
and ꢀ 8120 at pH 2.3). On increasing the solution pH, the λmax
shifted to 288 nm with another isosbestic point (at 267 nm),
A somewhat distorted square pyramid (or one may view it
as a trigonal bipyramidal) coordination is evident with the cyclen
(18) The composition for 11 at pH 9.7, at 25 °C with I ) 0.10 is as
follows: HL′‚H+ (9%), L′-‚H+ (71%), L′- (20%).
(19) (a) Kodama, M.; Kimura, E. J. Chem. Soc., Dalton Trans. 1981,
694-700. (b) Conners, K. A. In Binding Constants; John Wiley & Sons:
New York, 1987; Chapter 4, p 141.
(20) Koike, T.; Watanabe, T.; Aoki, S.; Kimura, E.; Shiro, M. J. Am.
Chem. Soc. 1996, 118, 12696-12703.

10916 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Kimura et al.
measure the pKa values of uracil N(1)H group under various
electrostatic environments. It is reasonable that the effects that
lower the pKa value would lower the free energy of the transition
state of the reactions involving the bond formation or cleavage
with uracil N(1).
For biochemical uracil N(1)-glycosylation, nature uses orotic
acid,22 where formation of an activated ribose C(1)-carbonium
ion (in the form of enzyme-ribosylmonophosphate complex) is
involved. The enzyme orotate phosphoribosyl-transferase
(OPRTase) also catalyzes the reverse reaction in the presence
of pyrophosphate. However, how uracil N(1) attacks at the
activated ribose C(1) remains to be totally unelucidated. Since
this phosphoribosylation of orotate requires metal ions (e.g.,
Mg2+), orotate complexes 19 with nickel(II),23 copper(II),24 and
zinc(II)25 were prepared to elucidate the roles played by the
metal ions. How the carboxylate and N(1)--bound form 19 is
linked to the N(1)-phosphoribosylation is yet to be explained.
Major attention was paid to the bidentate chelate effect involving
the facile N(1)H deprotonation, rather than focusing on the
deprotonation propensity of N(1)H by itself.
Figure 11. UV-pH profile for cyclen-attached (at C(5)) uracil zinc(II)
complex (1 mM) at 25 °C with I ) 0.10 (NaClO4): pH 2.3, λmax
)
262 nm (ꢀ 8300); pH 4.6; pH 5.3, λmax ) 265 nm, (ꢀ 7730); pH 6.0;
pH 8.4, λmax ) 264 nm (ꢀ 4430); pH 10.3; pH 11.9, λmax ) 288 nm (ꢀ
9580).
Scheme 9
Although there had been a considerable number of stud-
ies,8,21,26 about dissociation of protons at N(1) and N(3) of
uracils, no study ever tried to correlate the pKa value with the
nucleophilicity. Of these two nucleophilic sites, N(1) is
biochemically more interesting because of glycosylation and
chemically more challenging because of the difficulty in
studying its specific reactivity. By our present new strategy
using 6, we could for the first time achieve the N(1)H abstraction
without prior protection of N(3).27 The pKa of 7.14 for the
N(1)H of HL‚2H+ form 8a illustrates the remarkable lowering
from the pKa value of 9.9 for the N(1)H of 3-methyluracil 4.
Before the deprotonation, the N(1)H is subject to the dipositive
charge effect via a water molecule which, as shown by X-ray
crystal structure (Figure 12a), has the right orientation to form
hydrogen bonds with N(1)H and a cyclen secondary ammonium
group +N(13)H. The H+ removal from N(1)H would release
this electrostatic stress. What is more, the N(1)H (in the less
which permitted estimation of another proton abstraction from
14 (to 17) with a pKa2 value of 10.3, which is identical to the
value determined by potentiometric pH titration. The UV
absorption at pH 11.9 (λmax 288 nm, ꢀ 9580) resembles the
deprotonated form of 18 (λmax 278 nm, ꢀ 6530) (i.e., a red shift
with larger ꢀ value).21
(22) (a) Victor, J.; Greenberg, L. B.; Sloan, D. L. J. Biol. Chem. 1979,
254, 2647-2655. (b) Poulsen, P.; Jensen, K. F.; V-Hansen, P.; Carlsson,
P.; Lundberg, L. G. Eur. J. Biochem. 1983, 135, 223-229. (c) Niedzwicki,
J. G.; Iltzsch, M. H.; Kouni, M. H.; Cha, S. Biochem. Pharmac. 1984, 33,
2383-2395. (d) Bhatia, M. B.; Vinitsky, A.; Grubmeyer, C. Biochemistry
1990, 29, 10480-10487. (e) Grubmeyer, C.; Segura, E.; Dorfman, R. J.
Biol. Chem. 1993, 268, 20299-20304. (f) Bhatia, M. B.; Grubmeyer, C.
Arch. Biochem. Biophys. 1993, 303, 321-325. (g) Aghajari, N.; Jensen, K.
F.; Gajhede, M. J. Mol. Biol. 1994, 241, 292-294. (h) Scapin, G.;
Grubmeyer, C.; Sacchettini, J. C. Biochemistry 1994, 33, 1287-1294.
(23) Sabat, M.; Zglinska, D.; Jezowska-Trzebiatowska. Acta Crystallogr.
1980, B36, 1187.
(24) (a) Arrizabalaga, P.; Castan, P.; Dahan, F. Inorg. Chem. 1983, 22,
2245-2252. (b) Mutikairen, I.; Lumme, P. Acta Crystallogr. 1980, B36,
2233.
(25) (a) Hambley, T. W.; Christopherson, R. I.; Zvargulis, E. S. Inorg.
Chem. 1995, 34, 6550-6552. (b) Karipides, A.; Thoomas, B. Acta
Crystallogr. 1986, C42, 1705. (c) Mutikainen, I. Inorg. Chim. Acta, 1987,
136, 155-158.
L′′-‚H+ + Zn2+ a Zn-HL′′(16)
K(Zn-HL′′) ) [Zn-HL′′]/[L′′-‚H+][Zn2+] (6)
Zn-HL′′ a Zn-L′′- (14) + H+
Ka1 ) [Zn-L′′-]aH+/[Zn-HL′′] (7)
Zn-L′′- a Zn-L′′2- (17) + H+
Ka2 ) [Zn-L′′2-]aH+/[Zn-L′′-] (8)
(26) (a) Wempen, I.; Fox, J. J. J. Am. Chem. Soc. 1964, 86, 2474-
2477. (b) Wierzchowski, K. L.; Litonska, E.; Shugar, D. J. Am. Chem. Soc.
1965, 87, 4621-4629. (c) Psoda, A.; Kazimierczuk, Z.; Shudar, D. J. Am.
Chem. Soc. 1974, 96, 6832-6839. (d) Bensaude, O.; Aubard, J.; Dreyfus,
M.; Dodin, G.; Dubois, J. E. J. Chem. Soc. 1978, 100, 2823-2827.
(27) The pKa value for N(1)H in the N(3)H-protected (as an 4-enolether)
16 was reported to be 10.7 (ref 8).
Discussion
As a means of studying how uracil N(1) is activated in
enzymes such as uracil-DNA glycosylase, we have chosen to
(21) Shugar, D. ; Fox, J. J. Biochim. Biophys. Acta 1952, 9, 199-218.

Electrostatic Stabilization of Uracil N(1)- Anion
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10917
The +2 electrostatic effect on the uracil N(1)H by the two
protons in the cyclen is far surpassed by zinc(II) ion in the
cyclen, as seen with the deprotonation of N(1)H below pH 5
for Zn-HL 8b (to Zn-L- 13, which in the excessive addition
of acid, decomposed into the (protonated) ligand 8a and Zn2+).30
The resulting strong bond formation between Zn2+-uracil N(1)-
is evident in the X-ray crystal structure of Zn-L- 13 (Figure
11) and also in the large stability constant log K(Zn-L-) of
20.2 (Table 1), which is comparable to the stability (log K )
20.8, K ) [15]/[Zn2+][ligand monoanion]) of dansylaminoethyl-
pendant cyclen zinc(II) complex 15.20 It is concluded that
zinc(II) ion is a super acid to replace the weak acid N(1)H of
uracil, as we earlier observed Zn2+-cyclen to deprotonate H2O,31
imides,32 and alcohols10,33 at neutral pH. It may be conceived
that in the orotate phosphoribosyltransferase action metal ions
(e.g., Mg2+), which are acidic and labile, are initially hooked
to the orotate C(6)-carboxylate and then deprotonate N(1)H to
activate it for the subsequent N(1)-glycosylation at physiological
pH.
The cyclen-attached (at C(5)) uracil 12 (HL′′) has offered
another novel reference, which allowed us to compare the
electrostatic effects (by 2H+ and Zn2+) on the enolization at
C(4) accompanied by facile deprotonation either at N(3)H or
at N(1)H. The monodeprotonation from the HL′′‚2H+ form of
uracil (see Scheme 7) occurred with a pKa value of 8.6 (to
L′′-‚2H+), implying that the diprotonated cyclen served to lower
the pKa value of 9.5 (for thymine or 5-methyluracil)8 to facilitate
the C(1)-enolate formation. However, the pKa value is not so
dramatically lowered as in this case of the isomer HL‚2H+ 8a.
It may not clearly be defined about which proton N(1)H or
N(3)H of HL′′‚2H+ (12) dissociates, as was the case for isomeric
HL‚2H+ (8a), in the light of the estimated (from UV absorption
spectra at pH 9.3) close ratio 4:6 for N(1)- to N(3)- species in
the resulting L′′-‚2H+. The zinc(II) ion exerts a far greater
electrostatic effect to yield the enolate in 14 with pKa of 6.0.
The driving force comes from the stable Zn2+-O- (enolate)
bond formation, despite the very tight complex structure.
Figure 12. A proposed mechanism for the facile deprotonation of the
uracil N(1)H by the proximate diprotonated cyclen.
favorable hydrogen bond network (Figure 12a)) is replaced by
the water H by mere rotation of an axis of this water, yielding
now the more stable hydrogen bond network (Figure 12b). This
sort of hydrogen bond shuffling may thermodynamically and
kinetically facilitate the proton transfers specifically at the uracil
N(1). The localization of the negative charge at N(1) (estimated
to be 82% at pH 8.6, see Scheme 5) lends a support to the
conclusion that the N(1)H is more acidic than N(3)H in the
HL‚2H+ form 8a.28 At higher pH where the two protons are
removed from the cyclen, the negative charge at N(1) delocalizes
and the N(3)- anionic tautomer becomes more predominant
(61% at pH 12.5).
It would be of interest to consider another possible tautomer
20 a nonzwitterion HL‚H+ form instead of the zwitterion
L-‚2H+ of Scheme 5, although its participation (if any) was
ruled out from the UV and NMR spectral evidence. From
comparison of the independent pKa values, the acidities for the
uracil N(1)H (e.g., pKa ) 9.9 for 3-methyluracil) and for
cyclen‚2H+ (i.e., log K2 ) 9.7) are nearly the same, and thus
involvement of 20 was theoretically feasible. Then, the reason
why we actually did not see it is ascribed to the much greater
stability of the zwitterionic tautomers L-‚2H+ (in particular,
the uracil N(1)- anionic form L-‚2H+) with the electrostatic
attraction (through space) between the uracil anion and cy-
clen‚2H+.29
Conclusion
We for the first time have demonstrated that uracil N(1)H
may be specifically deprotonated (to yield the stable N(1)-
anionic uracil) in aqueous solution at physiological pH under
certain environments, which may account for why nature
electrochemically chooses the uracil N(1) as a nucleophile for
glycosylation. This fact may also explain how the cleavage of
uracil N(1)-glycosyl bond is aided by proximate protonated
amino acid residues in enzymes such as uracil-DNA glycosylase.
The chemical model that we have designed is a cyclen-attached
(at C(6)) uracil 6. The pKa value was determined to be 7.14
(30) The pKa value for the dansylamide NH of the Zn2+-cyclen-attached
dansylamide 15 was 5.0 (ref 20).
(31) (a) Koike, T.; Kimura, E. J. Am. Chem. Soc. 1991, 113, 8935-
8941. (b) Kimura, E.; Shionoya, M.; Hoshino, A.; Ikeda, T,; Yamada, Y.
J. Am. Chem. Soc. 1992, 114, 10134-10137. (c) Koike, T.; Takamura, M.;
Kimura, E. J. Am. Chem. Soc. 1994, 116, 8443-8449.
A reference compound, an ethylenediamine-attached (at C(6))
uracil 11, gave the pKa value of 8.8 and an almost 1:1 mixture
of N(3)- and N(1)- forms (Scheme 6) and like uracil with pKa
) 9.26 gave a 1:1 mixture of N(3)- and N(1)- forms.8 The
monoprotonated ethylenediamine in HL′‚H+ (11) exerted only
a minor effect on the acidity of uracil moiety, probably because
of the lesser charge of +1 and an unfavorable entropy term.
(32) Thymine derivatives undergo the specific N(3)H deprotonation in
the 1:1 complexes with Zn2+-cyclen, which showed UV absorption
maximum at 265 nm with ꢀ ) 5930. (a) Shionoya, M; Kimura, E.; Shiro,
M. J. Am. Chem. Soc. 1993, 115, 6730-6737. (b) Shionoya, M; Ikeda, T.;
Kimura, E.; Shiro, M. J. Am. Chem. Soc. 1994, 116, 3848-3859. (c)
Shionoya, M.; Sugiyama, M.; Kimura, E. J. Chem. Soc., Chem. Commun.
1994, 1747-1748. (d) Tucker, J. H. R.; Shionoya, M.; Koike, T.; Kimura,
E. Bull. Chem. Soc. Jpn. 1995, 68, 2465-2469. (e) Koike, T.; Takashige,
M.; Kimura, E.; Fujioka, H.; Shiro, M. Chem. Eur. J. 1996, 2, 617-623.
(f) Fujioka, H.; Koike, T.; Yamada, N.; Kimura, E. Heterocycles 1996, 42,
775-787.
(28) This pKa lowering by about 3 may be even greater at an enzyme
active site. In this model, there is an intervening water between the cyclen
secondary ammonium groups and the uracil N(1)- anion in the complexes.
These interactions could be more dramatic in a lower dielectric media at
an enzyme active site.
(29) It is of interest to note that for the diprotonated cyclen-attached
dansylamide (ligand for 15), where the dansylamide also has pKa ca. 10,
the first deprotonation (pKa ) 9.4) occurred from the cyclen‚2H+ but not
from the amide moiety to yield the 20-like species (ref 20).
(33) (a) Kimura, E.; Kodama, Y.; Koike, T.; Shiro, M. J. Am. Chem.
Soc. 1995, 117, 8304-8311. (b) Koike, T.; Inoue, M.; Kimura, E.; Shiro,
M. J. Am. Chem. Soc. 1996, 118, 3091-3099.

10918 J. Am. Chem. Soc., Vol. 119, No. 45, 1997
Kimura et al.
for the uracil N(1)H by potentiometric, UV, and 13C NMR
spectral measurements, which is extremely lowered from the
pKa of 9.87 for 3-methyluracil 4 or 9.26 for uracil 1 (where
N(3)H probably is more readily deprotonated). The great
electrostatic stabilization of the uracil N(1)- anion comes
indirectly through space from the proximate secondary am-
monium group of the diprotonated cyclen. As the solution pH
is raised, the protons in cyclen is removed, whereupon the uracil
anion comes to delocalize to N(3), too. Zinc(II) in the cyclen
(8b) exerts even a stronger effect on the deprotonation of the
uracil N(1)H below pH 5. The resulting zinc(II) complex 13
contains a very stable Zn2+-N(1)- coordination bond. Facile
interaction between the uracil C(4)dO enolate and diprotonated
cyclen was also demonstrated with cyclen-attached (at C(5))
uracil 12. However, its pKa value of 8.6 of the uracil NH group
indicates that it is not so remarkably favorable. Accordingly,
it is concluded that placement of protonated acids near uracil
N(1) would be the most efficient way for activation of uracil at
N(1).
g, 0.69 mmol) was slowly added to an aqueous solution (50 mL) of 6
(0.10 g, 0.32 mmol) in a 100-mL beaker. Yellow prisms of 8‚(picrate)2‚
H2O were obtained by slow evaporation at room temperature followed
by recrystallization from EtOH/H2O (210 mg, yield 82%): mp 210-
211 °C; IR (KBr pellet): 3649, 3348, 3101, 2860, 1714, 1686, 1636,
1615, 1551, 1491, 1456, 1433, 1366, 1333, 1319, 1271, 1165, 1146,
1080, 943, 914, 843, 789, 747, 712, 536 cm-1 1H NMR (0.1 M NaOD
.
in D2O): δ 2.58-2.77 (16H, m, NCH2), 3.38 (2H, s, CH2), 5.78 (1H,
s, uracil CH), 8.95 (4H, s, picrate). 13C NMR (DMSO-d6): δ 41.3,
41.7, 44.4, 47.8, 55.0, 101.3, 124.2, 125.1, 141.8, 151.1, 151.9, 160.8,
163.8. Anal. Calcd for C25H32N12O17: C, 38.87; H, 4.17; N, 21.76.
Found: C, 38.95; H, 4.15; N, 21.62.
Synthesis of Ethylenediamine-Attached (at C(6)) Uracil Dihy-
drobromic Acid Salt, 11‚2HBr. A solution of 6-(chloromethyl)uracil
(0.50 g, 3.1 mmol) and N,N′-dimethylethylenediamine (0.70 mL, 6.5
mmol) in 50 mL of EtOH was refluxed for 12 h. The reaction mixture
was evaporated to dryness. The residue was purified by silica gel
column chromatography (eluent; CH2Cl2/MeOH/28% aqueous NH3 )
5:1:0.1) followed by crystallization from 24% aqueous HBr/MeOH to
obtain colorless prisms of 6-(N-(N,N′-dimethylethylenediamino)methyl)-
uracil dihydrobromic acid salt, 11‚2HBr (0.57 g, 1.5 mmol): dec 233
°C; TLC (eluent; CH2Cl2/MeOH/28% aqueous NH3 ) 5:2:0.5) Rf )
0.22. IR (KBr pellet): 3058, 2849, 2662, 2452, 1705, 1674, 1578,
1474, 1447, 1412, 1362, 1304, 1271, 1234, 1053, 1024, 965, 889, 856,
Experimental Section
General Information. All reagents and solvents used were
purchased at the highest commercial quality and used without further
purification. All aqueous solutions were prepared using deionized and
distilled water. Acetonitrile (CH3CN) was distilled over calcium
hydride. UV spectra were recorded on a Hitachi U-3500 spectropho-
tometer at 25.0 ( 0.1 °C. IR spectra were recorded on a Shimadzu
FTIR-4200 spectrometer at 25 ( 2 °C. 1H (500 MHz) and 13C (125
MHz) NMR spectra at 35.0 ( 0.1 °C were recorded on a JEOL JNM
LA500 spectrometer. Tetramethylsilane in DMSO-d6 and 3-(trimeth-
ylsilyl)propionic-2,2,3,3-d4 acid sodium salt in D2O were used as
internal references for NMR measurements. Elemental analysis was
performed on a Perkin Elmer CHN Analyzer 2400. Thin-layer (TLC)
and silica gel column chromatographies were performed using Merck
Art. 5554 (silica gel) TLC plate and Fuji Silysia Chemical FL-100D
(silica gel), respectively.
797, 775, 628, 588, 534 cm-1 1H NMR (D2O): δ 2.50 (3H, s, CH3),
.
2.77 (3H, s, CH3), 3.05 (2H, t, J ) 6.5 Hz, NCH2), 3.33 (2H, t, J )
6.5 Hz, NCH2), 3.71 (2H, s, CH2), 5.87 (1H, s, uracil CH). 13C NMR
(D2O): δ 36.0, 43.6, 48.8, 55.2, 60.5, 103.6, 155.9, 156.4, 169.8. Anal.
Calcd for C9H18N4O2Br2‚: C, 28.90; H, 4.85; N, 14.98. Found: C,
29.05; H, 4.81; N, 14.93.
Synthesis of Cyclen-Attached (at C(5)) Uracil Trihydrobromic
Acid Salt, 12‚3HBr. A solution of 5-(hydroxymethyl)uracil mono-
hydrate (1.01 g, 3.1 mmol) in 15 mL of 48% aqueous HBr was stirred
at 60 °C for 2 h. After the solution cooled to room temperature,
5-(bromomethyl)uracil was obtained as colorless precipitate, (1.31 g,
2.8 mmol) in 90% yield. TLC (eluent; CH2Cl2/MeOH ) 10:1) Rf )
0.34 (cf. Rf for 5-(hydroxymethyl)uracil ) 0.10). 1H NMR (DMSO-
d6): δ 4.10 (2H, s, CH2), 7.22 (1H, s, uracil CH), 10.64 (1H, s, uracil
NH), 10.98 (1H, s, uracil NH).
Synthesis of Cyclen-Attached (at C(6)) Uracil 6 and Its Trihy-
drobromic Acid Salt. An EtOH solution (100 mL) of cyclen (3.22 g,
18.7 mmol) and 6-(chloromethyl)uracil (1.01 g, 6.3 mmol) was refluxed
for 6 h. The reaction mixture was evaporated to dryness. To the
residue was added 100 mL of dry CH3CN, which was stirred at room
temperature for 24 h. The obtained precipitate was purified by silica
gel column chromatography (eluent; CH2Cl2/MeOH/28% aqueous NH3
) 5:1:0.1) followed by crystallization from MeOH/CH3CN to yield
colorless prisms of 6-((1,4,7,10-tetraazacyclododecyl)methyl)uracil,
6‚0.3CH3CN (1.13 g, 3.7 mmol): dec 220 °C; TLC (eluent; CH2Cl2/
MeOH/28% aqueous NH3 ) 5:2:0.5) Rf ) 0.1. IR (KBr pellet): 3325,
2953, 2841, 1715, 1626, 1566, 1477, 1454, 1410, 1359, 1300, 1138,
A dry CH3CN solution (50 mL) of 5-(bromomethyl)uracil (0.23 g,
1.1 mmol) and 1,4,7-tris(tert-butyloxycarbonyl)-1,4,7,10-tetraazacy-
clododecane (1.09 g, 2.3 mmol) was refluxed for 6 h. After the solution
cooled to room temperature, 5-((4,7,10-tris(tert-butyloxycarbonyl)-
1,4,7,10-tetraazacyclododecyl)methyl)uracil was obtained as colorless
precipitate. To a suspension of the precipitate (0.62 g) in MeOH was
added slowly 48% aqueous HBr (10 mL) at 0 °C. The reaction mixture
was stirred at room temperature for 12 h. After the solvent had been
evaporated, the residue was crystallized from MeOH/H2O to obtain
colorless prisms of 5-((1,4,7,10-tetraazacyclododecyl)methyl)uracil
trihydrobromic acid salt, 12‚3HBr‚H2O (0.43 g, 0.83 mmol): dec 221
°C; TLC (eluent; CH2Cl2/MeOH/28% aqueous NH3 ) 5:2:0.5) Rf )
0.10. IR (KBr pellet): 3408, 2969, 2861, 1713, 1661, 1635, 1578,
1541, 1497, 1441, 1395, 1281, 1219, 1165, 1073, 1096, 978, 947, 775,
1097, 1064, 1016, 966, 891, 819, 777, 724, 642, 553 cm-1 1H NMR
.
(D2O): δ 2.07 (0.9H, s, CH3CN), 2.76-2.93 (16H, m, NCH2), 3.43
(2H, s, CH2), 5.72 (1H, s, uracil CH). 13C NMR (D2O): δ 46.4, 46.7,
48.1, 53.6, 61.0, 100.8, 163.7, 169.6, 173.3, Anal. Calcd for
C13H24N6O2‚0.3CH3CN: C, 52.92; H, 8.13; N, 28.59. Found: C, 52.54;
H, 8.23; N, 28.55.
596, 540 cm-1 1H NMR (D2O): δ 2.89-3.34 (16H, m, NCH2), 3.45
.
(2H, s, CH2), 7.61 (1H, s, uracil CH). 13C NMR (D2O): δ 44.7, 45.2,
47.5, 50.4, 51.7, 110.9, 146.2, 155.9, 169.6. Anal. Calcd for
C13H27N6O3Br3: C, 28.03; H, 5.25; N, 15.18. Found: C, 28.40; H,
5.15; N, 15.13.
Crystallization of 6‚0.3CH3CN (0.84 g, 2.7 mmol) from MeOH/
48% aqueous HBr afforded colorless prisms of 6‚3HBr‚2H2O (1.32 g,
2.3 mmol) in 84% yield: dec 210 °C; IR (KBr pellet): 3428, 3171,
3050, 2793, 1728, 1678, 1496, 1474, 1429, 1389, 1341, 1308, 1267,
Synthesis of Cyclen-Attached (at C(6)) Uracil Zinc(II) Complex,
13. To an aqueous solution (10 mL) of 6‚3HBr‚(H2O)2 (0.58 g, 1.01
mmol) was slowly added an aqueous solution (10 mL) of Zn(ClO4)2‚
6H2O (0.39 g, 1.05 mmol). The solution pH was adjusted to 8 with 1
M NaOH at 50 °C. After the solution cooled to room temperature, the
reaction mixture was concentrated under reduced pressure. The
obtained residue was crystallized from water to afford 13‚ClO4‚H2O
as colorless prisms (0.41 g) in 85% yield: TLC (eluent; MeOH/10%
aqueous NaCl ) 1:1) Rf ) 0.40. IR (KBr pellet): 3445, 3229, 3134,
2964, 1637, 1475, 1404, 1338, 1145, 1116, 1086, 1024, 991, 809, 782,
1234, 1107, 1005, 974, 947, 814, 787, 762, 586, 536 cm-1 1H NMR
.
(D2O): δ 2.93-3.29 (16H, m, NCH2), 3.61 (2H, s, CH2), 5.85 (1H, s,
uracil CH). 13C NMR (D2O): δ 44.4, 44.8, 47.4, 51.1, 57.2, 105.0,
155.6, 156.2, 169.5. Anal. Calcd for C13H31N6O4Br3: C, 27.15; H,
5.43; N, 14.61. Found: C, 27.21; H, 5.63; N, 14.45.
Synthesis of Diprotonated Cyclen-Attached (at C(6)) Uracil
Dipicrate, 8‚(Picrate)2. An EtOH solution (50 mL) of picric acid (0.16
(34) Kodama, M.; Kimura, E. J. Chem. Soc. Dalton Trans. 1978, 1081-
1085.
(35) Martell, A. E.; Motekaitis, R. J. Determination and Use of Stability
Constant, 2nd ed.; VCH: New York, 1992.
626 cm-1
.
1H NMR (D2O): δ 2.65-3.13 (16H, m, NCH2), 3.74 (2H,
s, CH2), 5.71 (1H, s, uracil CH). 13C NMR (D2O): δ 46.1, 47.5, 48.7,

Electrostatic Stabilization of Uracil N(1)- Anion
J. Am. Chem. Soc., Vol. 119, No. 45, 1997 10919
53.8, 58.1, 100.5, 161.6, 167.0, 171.4. Anal. Calcd for C13H25N6O7-
ClZn: C, 32.65; H, 5.27; N, 17.57. Found: C, 32.51; H, 5.53; N,
17.24.
Synthesis of Cyclen-Attached (at C(5)) Uracil Zinc(II) Complex,
14. Colorless prisms of 14‚ClO4‚(H2O)1.5 (143 mg, 82% yield) were
obtained from 12‚3HBr‚H2O (0.20 g, 0.36 mmol) and Zn(ClO4)2‚6H2O
(0.14 g, 0.38 mmol) by the same method for 13: TLC (eluent; MeOH/
10% aqueous NaCl ) 1:1) Rf ) 0.4. IR (KBr pellet): 3298, 3227,
3057, 2928, 1665, 1595, 1483, 1458, 1373, 1287, 1144, 1090, 1024,
Crystal data for 8‚(picrate)2‚H2O: triclinic, space group P1h (no. 2), a
) 9.295(4) Å, b ) 19.67(1) Å, c ) 8.886(6) Å, R ) 94.36(3)°, â )
102.95(4)°, γ ) 87.04(4)°, V ) 1576.84 Å3, Z ) 2, Dcalcd ) 1.627 g
cm-3, 2θmax ) 49.6°, total no. of reflections ) 3995. The non-hydrogen
atoms were refined anisotropically. Hydrogen atoms were included
but not refined. The final cycle of full-matrix least-squares refinement
was based on 3117 observed reflections (I > 3.00σ(I)) and 526 variable
parameters and converged (largest parameter shift was 0.01 times its
esd) with unweighted and weighted agreement factors of R () ∑||Fo|
2
978, 947, 909, 860, 802, 775, 625, 567 cm-1
.
1H NMR (D2O): δ 2.70-
- |Fc||/∑|Fo|) ) 0.054 and Rw () (∑w(|Fo| - |Fc|)2/∑wFo )0.5) ) 0.081.
3.79 (18H, m, NCH2, CH2), 7.48 (1H, s, uracil CH). 13C NMR (D2O):
δ 45.8, 46.8, 47.7, 55.2, 57.1, 112.8, 144.1, 161.1, 175.9. Anal. Calcd
for C13H26N6O7.5ClZn: C, 32.05; H, 5.38; N, 17.25. Found: C, 32.06;
H, 5.43; N, 17.22.
Crystallographic Study of 13‚ClO4‚H2O. An colorless prismatic
crystal of 13‚ClO4‚H2O (C13H25N6O7ClZn, Mr ) 478.21) having
approximate dimensions of 0.40 × 0.40 × 0.25 mm was sealed in a
glass capillary was used for data collection. All measurements were
made on a Rigaku AFC7R diffractometer Ni-filtered Cu-KR radiation.
The data were collected at a temperature of 23 ( 1 °C using the ω-2θ
scan technique to a maximum 2θ value of 120.3°. ω scans of several
intense reflections, made prior to data collection, had an average width
at half-height of 0.28° with a take-off angle of 6.0°. Scans of (1.73 +
0.30 tan θ)° were made at a speed of 16.0°/min (in ω). The weak
reflections (I < 10.0σ(I)) were rescanned (maximum of seven scans),
and the counts were accumulated to ensure good counting statistics.
Stationary background counting time was 2:1. The diameter of the
incident beam collimator was 1.0 mm, and the crystal to detector
distance was 235 mm. The computer-controlled slits were set to 3.0
mm (horizontal) and 4.5 mm (vertical). The structure was solved by
direct methods (SIR 92) and expanded using Fourier techniques
(DIRDIF 94). All calculations were performed using the teXsan
crystallographic software package of Molecular Structure Corporation
(1985, 1992). Crystal data for 13‚ClO4‚H2O: triclinic, space group
P1h (no. 2), a ) 9.461(3) Å, b ) 13.156(4) Å, c ) 8.687(2) Å, R )
101.21(2)°, â ) 103.55(2)°, γ ) 73.21(2)°, V ) 997.0(5) Å3, Z ) 2,
Dcalcd ) 1.593 g cm-3, 2θmax ) 120.3°, total no. of reflections ) 3180.
The non-hydrogen atoms were refined anisotropically. Hydrogen atoms
were included but not refined. The final cycle of full-matrix least-
squares refinement was based on 2436 observed reflections (I > 3.00σ-
(I)) and 271 variable parameters and converged (largest parameter shift
was 0.01 times its esd) with unweighted and weighted agreement factors
of R () ∑||Fo| - |Fc||/∑|Fo|) ) 0.063 and Rw () (∑w(|Fo| - |Fc|)2/
Potentiometric pH Titrations. The electrode system (Orion
Research Expandable Ion Analyzer EA920 and Orion pH Electrode
8102BN) was daily calibrated as follows:34 An aqueous solution (50
mL) containing 4.00 mM of HCl and 96 mM of NaClO4 (I ) 0.10)
was prepared under an argon atmosphere (>99.999% purity) at 25.0
( 0.1 °C, and then the first pH value (pH1) was read. After 4.0 mM
of 0.10 M NaOH (>99% purity) was added to the acidic solution, the
second pH value (pH2) was read. The corresponding theoretical pH
value to pH1 and pH2 are calculated to be pH1′ ) 2.481 and pH2′ )
11.447, respectively, using KW () aH ‚aOH ) ) 10-14.00, K′W ()
+
-
+
[H+][OH-]) ) 10-13.79, and fH (aH /H ) ) 0.825. The correct pH values
+
+
+
(pH ) -log aH ) can be obtained using the following equations: a )
(pH2′ - pH1′)/(pH2 - pH1); b ) pH2′ - a × pH2; pH ) a × (pH-
meter reading) + b.
The potentiometric pH titrations were carried out at 25 °C with I )
0.10 (NaClO4), where at least two independent titration were always
performed. The protonation constants (Kn′ ) [HnL-]/[Hn-1L-][H+])
for ligands, zinc(II) complexation constant K(Zn-HL′′) and deproto-
nation constants (pKa1′) [Zn-L′′-][H+]/[Zn-HL′′], pKa2′) [Zn-L′′2-]-
[H+]/[Zn-L′′-]) were determined by means of the pH-titration program
BEST.35 The σ pH fit values defined in the program are smaller than
0.005 for Kn′ and 0.05 for K(Zn-HL′′), pKa1′, and pKa2′. The mixed
protonation constants Kn, pKa1, and pKa2 are derived from Kn′, pKa1′,
and pKa2′ using [H+] ) aH /fH
.
+
+
Crystallographic Study of 8‚(picrate)2‚H2O. An yellow prismatic
crystal of 8‚(picrate)2‚H2O (C25H32N12O17, Mr ) 772.60) having
approximate dimensions of 0.30 × 0.30 × 0.10 mm was sealed in a
glass capillary and used for data collection. All measurements were
made on a Rigaku RAXIS II imaging plate area detector with graphite
monochromated Mo-KR radiation. Indexing was performed from three
2.0° oscillation images which were exposed for 10.0 min. The data
were collected at a temperature of 25 ( 1 °C to a maximum 2θ value
of 49.6°. A total of 503.5° oscillation images were collected, each
being exposed for 60.0 min. The crystal-to-detector distance was 85.0
mm. The detector swing angle was 0.0°. Readout was performed in
the 105 µm pixel mode. The structure was solved by direct methods
(MULTAN 88) and expanded using Fourier techniques (DIRDIF 94).
All calculations were performed using the teXsan crystallographic
software package of Molecular Structure Corporation (1985, 1992).
2
∑wFo )0.5) ) 0.093.
Acknowledgment. E.K. is grateful to the Grant-in-Aid for
Priority Project “Biometallics” (No. 08249103) from the
Ministry of Education, Science and Culture in Japan.
Supporting Information Available: Tables crystallographic
parameters, atomic coordinates, equivalent isotropic temperature
factors, anisotropic temperature factors, bond distances, and
bond angles in CIF format for 8‚(picrate)2‚H2O and 13‚ClO4‚H2O
(14 pages). See any current masthead page for ordering and
Internet access instructions.
JA972129N