Highly selective recognition of thymidine mono- and diphosphate nucleotides in aqueous solution by ditopic receptors zinc(II)-bis(cyclen) complexes (cyclen = 1,4,7,10-tetraazacyclododecane)

Article abstract of DOI:10.1021/ja994537s

Highly selective and efficient recognition of thymidine and uridine nucleotides such as 3'-dTMP (thymidine 3'-monophosphate), 5'-dTMP (thymidine 5'-monophosphate), 2'-UMP (uridine 2'-monophosphate), 3'-UMP (uridine 3'- monophosphate), 5'-UMP (uridine 5'-monophosphate), 5'-dTDP (thymidine 5'- diphosphate), 5'-dTTP (thymidine 5'-triphosphate), AZTMP (3'-azido-3'- deoxythymidine 5'-monophosphate), and AZTDP (3'-azido-3'-deoxythymidine 5'- diphosphate) with ditopic dimeric zinc(II) complexes of macrocyclic 12- membered tetramines, meta- and para-xylyl-bis(Zn²⁺-cyclen)s (Zn₂L⁴ and Zn₂L⁵) (cyclen = 1,4,7,10-tetraazacyclododecane) has been studied by potentiometric pH titration, isothermal titration calorimetry, UV spectrophotometric titration, and NMR titration. The apparent 1:1 complexation constants for 5'-dTMP, K(app) (= [(Zn₂L)-(S^--OPO₃^2- )]/[Zn₂L](free)[S-OPO₃^2-](free) (M^-1)), where S^- denotes the imide- deprotonated thymine part), with Zn₂L⁴ or Zn₂L⁵ determined by potentiometric pH titration showed a more stable complex with Zn₂L⁵ (log K(app) = 6.4) than with Zn₂L⁴ (log K(app) = 5.5) at pH 7.6 with I = 0.1 (NaNO₃) and 25 °C. These values are much greater than log K(app) (K(app) = [ZnL³-dT^-]/[ZnL³](free)[dT](free) (M^-1)) of 3.2 for a nucleoside thymidine (dT) complex with a monomeric Zn²⁺-benzylcyclen (ZnL³). The 1:1 complexation was confirmed by the FAB mass spectroscopic data for Zn₂L with 3'- and 5'-dTMP. The combined data from the spectrophotometric UV titration and ¹H NMR measurements of 5'-dTMP and 5'-dTDP with Zn₂L⁵ in D₂O at pD 7.8 and 35 °C indicated that the terminal phosphate dianion interacted with one of the (Zn²⁺-cyclen)s and the imide anion of dT bound to the other Zn²⁺-cyclen.

Full text of DOI:10.1021/ja994537s

4542
J. Am. Chem. Soc. 2000, 122, 4542-4548
Highly Selective Recognition of Thymidine Mono- and Diphosphate
Nucleotides in Aqueous Solution by Ditopic Receptors
Zinc(II)-Bis(cyclen) Complexes (Cyclen )
1,4,7,10-Tetraazacyclododecane)
Shin Aoki and Eiichi Kimura*
Contribution from the Department of Medicinal Chemistry, Faculty of Medicine, Hiroshima UniVersity,
Kasumi 1-2-3, Minami-ku, Hiroshima, 734-8551, Japan
ReceiVed December 30, 1999
Abstract: Highly selective and efficient recognition of thymidine and uridine nucleotides such as 3′-dTMP
(thymidine 3′-monophosphate), 5′-dTMP (thymidine 5′-monophosphate), 2′-UMP (uridine 2′-monophosphate),
3′-UMP (uridine 3′-monophosphate), 5′-UMP (uridine 5′-monophosphate), 5′-dTDP (thymidine 5′-diphosphate),
5′-dTTP (thymidine 5′-triphosphate), AZTMP (3′-azido-3′-deoxythymidine 5′-monophosphate), and AZTDP
(3′-azido-3′-deoxythymidine 5′-diphosphate) with ditopic dimeric zinc(II) complexes of macrocyclic 12-
membered tetramines, meta- and para-xylyl-bis(Zn2+-cyclen)s (Zn2L4 and Zn2L5) (cyclen ) 1,4,7,10-
tetraazacyclododecane) has been studied by potentiometric pH titration, isothermal titration calorimetry, UV
spectrophotometric titration, and NMR titration. The apparent 1:1 complexation constants for 5′-dTMP, Kapp
2-
() [(Zn2L)-(S--OPO32-)]/[Zn2L]free[S-OPO3
]
free (M-1)), where S- denotes the imide-deprotonated thymine
part), with Zn2L4 or Zn2L5 determined by potentiometric pH titration showed a more stable complex with
Zn2L5 (log Kapp ) 6.4) than with Zn2L4 (log Kapp ) 5.5) at pH 7.6 with I ) 0.1 (NaNO3) and 25 °C. These
values are much greater than log Kapp (Kapp ) [ZnL3-dT-]/[ZnL3]free[dT]free (M-1)) of 3.2 for a nucleoside
thymidine (dT) complex with a monomeric Zn2+-benzylcyclen (ZnL3). The 1:1 complexation was confirmed
by the FAB mass spectroscopic data for Zn2L with 3′- and 5′-dTMP. The combined data from the
spectrophotometric UV titration and H NMR measurements of 5′-dTMP and 5′-dTDP with Zn2L5 in D2O at
1
pD 7.8 and 35 °C indicated that the terminal phosphate dianion interacted with one of the (Zn2+-cyclen)s and
the imide anion of dT bound to the other Zn2+-cyclen.
Introduction
nucleotide phosphates would be of great help to increase the
efficacy of the drugs.
Among various nucleoside anti-HIV drugs, typically pre-
scribed now are 3′-azido-3′-deoxythymidine (AZT), 2′,3′-
dideoxycytidine (ddC), and 2′,3′-dideoxyadenosine (ddA).1-3
These drugs go into cells mainly by simple passive diffusion4
and then undergo phosphorylation to each ultimate nucleotide
triphosphate derivative to be active against HIV reverse tran-
scriptase.5 The step from their monophosphate to diphosphate
esters is rate-determining in the phosphorylation,5a and some
kinase-deficient cells such as macrophages become reservoirs
for HIV.6 Therefore, development of lipophilic carriers for these
Until now, a number of artificial carriers for nucleotides have
been reported.7-13 Examples include polyammonium cations7-9
(e.g., 17), a guanidino cation receptor,10 cationic cyclophane
(7) For review, see: (a) Kimura, E.; Koike, T. J. Chem. Soc., Chem.
Commun. 1998, 1495-1500. (b) Kimura, E.; Kodama, M.; Yatsunami, T.
J. Am. Chem. Soc. 1982, 104, 3182-3187. (c) Kimura, E.; Watanabe, A.;
Nihira, H. Chem. Pharm. Bull. 1983, 31, 3264-3268. (d) Kimura, E.;
Fujioka, H.; Yatsunami, A.; Nihira, H.; Kodama, M. Chem. Pharm. Bull.
1985, 33, 655-661. (e) Kimura, E.; Kuramoto, Y.; Koike, T.; Fujioka, H.;
Kodama, M. J. Org. Chem. 1990, 55, 42-46. (f) Hosseini, M. W.; Blacker,
A. J.; Lehn, J.-M. J. Am. Chem. Soc. 1990, 112, 3896-3904. (g) Aguilar,
J. A.; Garc´ıa-Espana, E.; Guerrero, J. A.; Luis, S. V.; Llinares, J. M.;
Miravet, J. F.; Ram´ırez, J. A.; Soriano, C. J. Chem. Soc., Chem. Commun.
1995, 2237-2239. (h) Bencini, A.; Bianchi, A.; Bianchi, A.; Fusi, V.;
Giorgi, C.; Granchi, A.; Paoletti, P.; Valtancoli, B. J. Chem. Soc., Perkin
Trans. 2 1997, 775-781.
* To whom correspondence should be addressed. E-mail: ekimura@
ipc.hiroshima-u.ac.jp.
(1) Martin, J. C., Ed. Nucleotide Analogues as AntiViral Agents; American
Chemical Society: Washington, DC, 1989.
(2) Merigan, T. C., Jr.; Bartlett, J. G.; Bolognesi, D. Textbook of AIDS
Medicine, 2nd ed.; Williams & Williams: Baltimore, 1999.
(3) For review, see: (a) Huryn, D. M.; Okabe, M. Chem. ReV. 1992, 92,
1745-1768. (b) De Clercq, E. J. Med. Chem. 1995, 38, 2491-2517.
(4) Zimmerman, T. P.; Mahony, W. B.; Prus, K. L. J. Biol. Chem. 1987,
262, 5748-5754.
(5) (a) Furman, P. A.; Fyfe, J. A.; St. Clair, M. H.; Weinhold, K.; Rideout,
J. L.; Freeman, G. A.; Lehrman, S. N.; Bolognesi, D. P.; Broder, S.; Mitsuya,
H.; Barry, D. W. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8333-8337. (b)
Bourdais, J.; Biondi, R.; Sarfati, S.; Guerreiro, C.; Lascu, I.; Janin, J.; Ve´ron,
M. J. Biol. Chem. 1996, 271, 7887-7890.
(8) (a) Tabushi, I.; Kobuke, Y.; Imuta, J. J. Am. Chem. Soc. 1980, 102,
1744-1745. (b) Tabushi, I.; Kobuke, Y.; Imuta, J. J. Am. Chem. Soc. 1981,
103, 6152-6157.
(9) (a) Li, T.; Diederich, F. J. Org. Chem. 1992, 57, 3449-3455. (b) Li,
T.; Krasne, S. J.; Persson, B.; Kaback H. R.; Diederich, F. J. Org. Chem.
1993, 58, 380-384.
(10) (a) Deslongchamps, G.; Gala´n, A.; de Mendoza, J.; Rebek, J., Jr.
Angew. Chem., Int. Ed. Engl. 1992, 31, 61-63. (b) Kato, Y.; Conn, M.;
Rebek, J., Jr. J. Am. Chem. Soc. 1994, 116, 3279-3284.
(11) (a) Odashima, K.; Koga, K. In ComprehensiVe Supramolecular
Chemistry; Vo¨gtle, F., Ed.; Elsevier Science: Oxford, U.K., 1996; Vol. 2,
Chapter 5, pp 143-194. (b) Odashima, K.; Bu¨hlmann, P.; Sugawara, M.;
Tohda, K.; Koga, K. Umezawa, Y. In AdVances in Supramolecular
Chemistry; Gokel, G. W. Ed.; JAI Press Inc.: Greenwich, 1997; pp 211-
285.
(6) (a) Richman, D. D.; Kornbluth, R. S.; Carson, D. A. J. Exp. Med.
1987, 166, 1144-1149. (b) Magnani, M.; Casabianca, A.; Fraternale, A.;
Brandi, G.; Gessani, S. Williams, R.; Giovine, M.; Damonte, G.; De Flora,
A. Benatti, U. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 4403-4408.
10.1021/ja994537s CCC: $19.00 © 2000 American Chemical Society
Published on Web 05/02/2000

Recognition of Thymidine Nucleotides
J. Am. Chem. Soc., Vol. 122, No. 19, 2000 4543
(QCP66),11 bis(intercaland) receptors,12 and mono- and dica-
tionic sapphyrins (e.g., monocationic 2).13 However, most of
these were monotopic receptors for phosphate anions and lacked
base selectivities. Exceptionally, the ditopic receptor 2 succeeded
in extracting 5′-AMP and 5′-GMP from an aqueous phase of
pH 5.0-7.0 to CHCl3 phase due to recognition of A and G by
the cytosine moiety and of the phosphate dianion part by the
protonated sapphyrin.13a On the other hand, selective carriers
for thymidine (dT) or uridine (U) nucleotides are unknown,
which, if available, would be extremely useful, for instance, in
effective administration of AZT.
(phenyl) phosphate were 3.0 and 3.6, respectively, at pH 7.6
and 25 °C with I ) 0.1 (NaNO3).18,19
It is now of interest to investigate whether bis(Zn2+-cyclen)
complexes, m-dimer Zn2L4 6 and p-dimer Zn2L5 7, that we
synthesized earlier as ditopic receptors for dianionic barbital20
could be good ditopic receptors15j,21 for dT nucleotides to
produce 1:1 complexes such as 8-11. Moreover, 6 and 7 and
their free ligands were discovered to possess an extremely potent
anti-HIV activity.22 Herein we present the selective recognition
of thymidine 3′-monophosphate (3′-dTMP), thymidine 5′-
monophosphate (5′-dTMP), uridine 2′-monophosphate (2′-
UMP), uridine 3′-monophosphate (3′-UMP), uridine 5′-mono-
phosphate (5′-UMP), thymidine 5′-diphosphate (5′-dTDP),
thymidine 5′-triphosphate (5′-dTTP), AZT 5′-monophosphate
(AZTMP), and AZT 5′-diphosphate (AZTDP) by 6 and 7
(Scheme 1).
We earlier discovered that Zn2+-cyclen complex 3a (ZnL1)
acted as a monotopic receptor for dT and U among all of the
nucleosides at physiological pH in aqueous solution, yielding
stable 1:1 complexes 4 by a Zn2+-imide N- anion bonding and
two complementary hydrogen bonds (cyclen ) 1,4,7,10-
tetraazacyclododecane).14,15 Recently, a lipophilic hexadecyl-
cyclen 3b (ZnL2)16 was developed as a new type of dT-selective
transporter of dT, U, AZT, and the relevant nucleoside
compounds.17 We also found that 3a acted as a good monotopic
receptor for dianionic phosphate monoesters to produce 5 in
aqueous solution.18 For example, the 1:1 complexation constants,
log Kapp (Kapp ) [5a (or 5b)]/[3c]free[phosphate]free (M-1)), of
3c with dianions of mono(4-nitrophenyl) phosphate and mono-
Results and Discussion
FAB (Fast Atom Bombardment) Mass Study of 1:1 dTMP
Complexes with Zn2L4 6 and Zn2L5 7. To confirm the 1:1
complexation of dTMPs with bis(Zn2+-cyclen)s, we ran FAB
mass (positive) experiment for a mixture of 3′- or 5′-dTMP (5
mM) and 6 or 7 (5 mM) in H2O (pH 7.5 ( 0.1). The
experimental mass spectra for 1:1 6/(3′-dTMP)3- and 1:1 7/(5′-
dTMP)3- (both at m/z 895 with Zn isotopic peaks at m/z 893,
897, and 899 etc.) fit to the theoretical mass distribution spectra
(C34H58N10O8PZn2) for 8 and 11 (see Supporting Information).
The Potentiometric pH Titration of 3′-dTMP, 5′-dTMP,
3′-UMP, and 5′-UMP with Zn2L4 6 and Zn2L5 7. The
potentiometric pH titration of thymidine mononucleotide (3′-
and 5′-dTMP) and uridine mononucleotide (3′- and 5′-UMP)
with 6 and 7 at 25 °C with I ) 0.10 (NaNO3) was studied to
(12) (a) Lehn, J.-M. Supramolecular Chemistry: Concepts and Perspec-
tiVes VCH: Weinheim, 1995. (b) Cudic, P.; Zinic, M.; Tomisci, V.; Simeon,
V.; Vigneron, J.-P.; Lehn, J.-M. J. Chem. Soc., Chem. Commun. 1995,
1073-1075. (c) Teulade-Fichou, M.-P.; Vigneron, J.-P.; Lehn, J.-M. J.
Chem. Soc., Perkin Trans. 2 1996, 2169-2175.
(13) (a) Kra´l, V.; Sessler, J. L.; Furuta, H. J. Am. Chem. Soc. 1992, 114,
8704-8705. (b) Sessler, J. L.; Furuta, H.; Kra´l, V. Supramol. Chem. 1993,
1, 209-220. (c) Kra´l, V.; Andrievskyu, A.; Sessler, J. L. J. Chem. Soc.,
Chem. Commun. 1995, 2349-2351. (d) Kra´l, V.; Furuta, H.; Shreder, K.;
Lynch, V.; Sessler, J. L. J. Am. Chem. Soc. 1996, 118, 1595-1607. (e)
Iverson, B. L.; Shreder, K.; Kra´l, V.; Sansom, P.; Lynch, V.; Sessler, J. L.
J. Am. Chem. Soc. 1996, 118, 1608-1616.
(14) For review, see: (a) Kimura, E.; Shionoya, M. In Transition Metals
in Supramolecular Chemistry; Fabbrizzi, L., Poggi, A., Eds.; Kluwer
Academic Publishers: Dordrecht, 1994, 245-259. (b) Kimura, E.; Shionoya,
M. In Metal Ions in Biological Systems; Sigel, A., Sigel, H., Eds.; Marcel
Dekker: New York, 1996; Vol. 33, pp 29-52.
(15) (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.; Kimura, E.;
Hayashida, H.; Petho, G.; Marzilli, L. G. Supramol. Chem. 1993, 2, 173-
176. (d) Shionoya, M.; Sugiyama, M.; Kimura, E. J. Chem. Soc., Chem.
Commun. 1994, 1747-1748. (e) Kimura, E.; Ikeda, T.; Shionoya, M. Pure
Appl. Chem. 1997, 69, 2187-2195. (f) Koike, T.; Goto, T.; Aoki, S.;
Kimura, E.; Shiro, M. Inorg. Chim. Acta 1998, 270, 424-432. (g) Kimura,
E.; Ikeda, T.; Aoki, S.; Shionoya, M. J. Biol. Inorg. Chem. 1998, 3, 259-
267. (h) Kikuta, E.; Murata, M.; Katsube, N.; Koike, T.; Kimura, E. J. Am.
Chem. Soc. 1999, 120, 5126-5436. (i) Kikuta, E.; Katsube, N.; Kimura,
E. J. Biol. Inorg. Chem. 1999, 4, 431-440. (j) Kimura, E.; Kikuchi, M.;
Kitamura, H.; Koike, T. Chem. Eur. J. 1999, 5, 3113-3123.
(16) (a) Kimura, E.; Hashimoto, H.; Koike, T. J. Am. Chem. Soc. 1996,
118, 10963-10970. (b) Kimura, E.; Koike, T. In Bioinorganic Catalysis;
Reedijk, J., Bouwman, E., Eds.; Marcel Dekker: New York, 1999; pp 33-
54.
(17) Aoki, S.; Honda, Y.; Kimura, E. J. Am. Chem. Soc. 1998, 120,
10018-10026.
(19) For other examples of artificial phosphate receptors, (a) Kneeland,
D. M.; Ariga, K.; Lynch, V. M.; Huang, C.-Y.; Anslyn, E. V. J. Am. Chem.
Soc. 1993, 115, 10042-10055. (b) Niikura, K.; Metzger, A.; Anslyn, E. V.
J. Am. Chem. Soc. 1998, 120, 8533-8534. (c) Schmidtchen, F. P.; Berger,
M. Chem. ReV. 1997, 97, 1609-1646. (d) Berger, M.; Schmidtchen, F. P.
J. Am. Chem. Soc. 1999, 121, 9986-9993. (e) Antonisse, M. M. G.;
Reinhoudt, D. N. J. Chem. Soc., Chem. Commun. 1998, 443-448. (f)
Chrisstoffels, L. A. J.; de Jong, F.; Reinhoudt, D. N.; Sivelli, S.; Gazzola,
L.; Casnati, A.; Ungaro, R. J. Am. Chem. Soc. 1999, 121, 10142-10151.
(g) Bazzicalupi, C.; Bencini, A.; Bianchi, A.; Cecchi, M.; Escuder, B.; Fusi,
V.; Garc´ıa-Espana, E.; Giorgi, C.; Luis, S. V.; Maccagni, G.; Marcelino,
V.; Paoletti, P.; Valtancoli, B. J. Am. Chem. Soc. 1999, 121, 6807-6815.
(h) Wong, C.-H.; Hendrix, M.; Manning, D. D.; Rosenbohm, C.; Greenberg,
W. A. J. Am. Chem. Soc. 1998, 120, 8319-8327.
(20) Koike, T.; Takashige, M.; Kimura, E.; Fujioka, H.; Shiro, M. Chem.
Eur. J. 1996, 2, 617-623.
(21) Aoki, S.; Sugimura, C.; Kimura, E. J. Am. Chem. Soc. 1998, 120,
10094-10102.
(22) For review, see: (a) Kimura, E.; Koike, T.; Inouye, Y. Macrocyclic
Polyamines and Their Metal Complexes: A Novel Type of Anti-HIV Agent.
In PerspectiVes in Bioinorganic Chemistry; Hay, R. W., Dilworth, J. R.,
Nolan, K. B., Eds.; JAI Press Inc.: Stamford, 1999; Vol. 4, pp 145-164.
(b) Inouye, Y.; Kanamori, T.; Yoshida, T.; Bu, X.; Shionoya, M.; Koike,
T.; Kimura, E. Biol. Pharm. Bull. 1994, 17, 243-250. (c) Inouye, Y.;
Kanamori, T.; Sugiyama, M.; Yoshida, T.; Koike, T.; Shionoya, M.;
Enomoto, K.; Suehiro, K.; Kimura, E. AntiViral Chem. Chemother. 1995,
6, 337-344. (d) Inouye, Y.; Kanamori, T.; Yoshida, T.; Koike, T.; Shionoya,
M.; Fujioka, H.; Kimura, E. Biol. Pharm. Bull. 1996, 19, 456-458.
(18) (a) Koike, T.; Kimura, E. J. Am. Chem. Soc. 1991, 113, 8935-
8941. (b) Koike, T.; Kajitani, S.; Nakamura, I.; Kimura, E.; Shiro, M. J.
Am. Chem. Soc. 1995, 117, 1210-1219. (c) Fujioka, H.; Koike, T.; Yamada,
N.; Kimura, E. Heterocycles 1996, 42, 775-787. (d) Kimura, E.; Aoki, S.;
Koike, T.; Shiro, M. J. Am. Chem. Soc. 1997, 119, 3068-3076.

4544 J. Am. Chem. Soc., Vol. 122, No. 19, 2000
Aoki and Kimura
Figure 2. Speciation diagram for the 5′-dTMP (1 mM) and Zn2L5 (7)
(1 mM) as a function of pH at 25 °C with I ) 0.1 (NaNO3). Other
species which exist in less than 5% are omitted for clarity.
Figure 1. Typical pH titration curves of (a) 1 mM 5′-dTMP, (b) 1
mM Zn2L5 (7), and (c) 1 mM 5′-dTMP + 1 mM Zn2L5 (7) at 25 °C
with I ) 0.1 (NaNO3), where equiv(OH-) is the number of equivalents
of base added.
6.5 ( 0.1, and 10.2 ( 0.1, respectively. The intrinsic complex-
ation constant, log Ks defined by eq 6, at 25 °C with I ) 0.10
(NaNO3) was determined to be 9.6 ( 0.1 by the program
Scheme 1
“BEST”.23 An apparent complex formation constant, log Kapp
,
defined by eqs 7-9, at pH 7.6 was then calculated to be 6.4.
Zn2L(OH2)2 a Zn2L(OH2)(OH-) + H+:
K1 ) [Zn2L(OH2)(OH-)]aH+/[Zn L(OH ) ] (1)
2
2 2
Zn2L(OH2)(OH-) a Zn2L(OH-)2 + H+:
K2 ) [Zn2L(OH-)2]aH+/[Zn L(OH )(OH-)] (2)
2
2
S-OPO3H2 a S-OPO3H- + H+:
K3 ) [S-OPO3H-]aH+/[S-OPO H ] (3)
3
2
S-OPO3H- a S-OPO32- + H+:
K4 ) [S-OPO32-]aH+/[S-OPO H-] (4)
3
S-OPO32- a S--OPO32- + H+:
K5 ) [S--OPO32-]aH+/[S-OPO 2-] (5)
3
Zn2L + S--OPO32- a Zn2L-(S--OPO32-):
Ks ) [(Zn2L)-(S--OPO32-)]/[Zn2L(OH2)2][S--
OPO32-](M-1) (6)
Kapp ) [(Zn2L)-(S--OPO32-)]/[Zn2L]free[S-OPO3
]
free
2-
(at designated pH)(M-1) (7)
[Zn2L]free ) [Zn2L(OH2)2] + [Zn2L(OH2)(OH-)] + [Zn2L
(OH-)2] (8)
free ) [S-OPO3H2] + [S-OPO3H-] + [S-
OPO32-] + [S--OPO32-] (9)
see details in the 1:1 complexation. Figure 1 shows the typical
titration curves for 1 mM 5′-dTMP (curve a), 1 mM 7 (curve
b), and 1 mM 5′-dTMP + 1 mM 7 (curve c).
2-
[S-OPO3
]
The deprotonation constants of Zn(II)-bound waters in 7, pK1
and pK2 (defined by eqs 1 and 2), were determined from Figure
1b to be 7.2 and 7.9 at 25 °C with I ) 0.10 (NaNO3), as earlier
reported.20 From Figure 1a, the deprotonation constants of 5′-
dTMP, pK3, pK4, and pK5 (defined by eqs 3-5, where S is
thymine part and S-OPO3H2, S-OPO3H-, S-OPO32-, and
S--OPO32- denote free, mono-deprotonated, di-deprotonated,
and tri-deprotonated species of 5′-dTMP, respectively) were <2,
Figure 2 depicts a speciation diagram for 5′-dTMP (1 mM)
and 7 (1 mM) as a function of pH at 25 °C with I ) 0.1
(NaNO3), which shows that the population of the 1:1 complex
(11) is over 95% at 6.6 < pH < 8.8.
The log Ks and log Kapp values for the 1:1 complexes, 8 (6-
(3′-dTMP) or 6-(3′-UMP)), 9 (7-(3′-dTMP) or 7-(3′-UMP)),
10 (6-(5′-dTMP) or 6-(5′-UMP)), and 11 (7-(5′-dTMP) or

Recognition of Thymidine Nucleotides
J. Am. Chem. Soc., Vol. 122, No. 19, 2000 4545
Table 1. Apparent Complexation Constants (log Kapp) for
Imide-Containing Nucleotides with Zinc(II)-cyclen Complexes at pH
7.6 and 25 °C Determined by Potentiometric pH Titration,a
Isothermal Titration Calorimetry (in 50 mM HEPES Buffer),b and
UV Titration (in 50 mM HEPES Buffer)c with I = 0.1 (NaNO3)
3c
6
7
dT
3.2a (5.7)d
3.2, 3.2b
3.4c
3.3c
(6:dT ) 1:2)
c-dTMP
5′-CMP
3′-dTMP
3.5, 3.5b
(6:c-dTMP ) 1:2)
3.2a (4.3)d
3.4b
3.3a (3.7)d
3.3b
5.2a (8.6)d
5.3b
5.9a (8.9)d
5.8b,e
5.4c,f
5.8c,f
Figure 3. Isothermal titration calorimetry curves for (a) 6 (0.2 mM)
+ 3′-dTMP, (b) 7 (0.2 mM) + 3′-dTMP, (c) 6 (0.2 mM) + 5′-dTMP,
and (d) 7 (0.2 mM) + 5′-dTMP at pH 7.6 (50 mM HEPES with I )
0.1 (NaNO3)) and 25 °C. Equiv(dTMP) is the number of equivalents
of [dTMP] added against [Zn2L].
5′-dTMP
3.4, 3.4b
5.5a (9.3)d
6.4a (9.6)d
(3c:5′-dTMP ) 2:1) 5.5b
> 6b,e
5.7c,f
5.7b
> 6c,f
2′-UMP
3′-UMP
4.8a (7.8)d
5.5a (8.5)d
5.2c,f
5.7c,f
the potentiometric pH titrations, the ITC gave only the Kapp
values at the given pH in the buffer, but it was simpler to
measure. Typical titration curves for 3′-dTMP and 5′-dTMP
(both 0.2 mM) with 6 and 7 (both 10.0 mM) are shown in Figure
3, in which cumulative heats (mJ) of the complexation reaction
are plotted (the reaction was exothermic). The obtained values
(5.3 ( 0.1, 5.8 ( 0.1, 5.5 ( 0.1, and >6 for the 1:1 complexes,
8, 9, 10, and 11, respectively)28 for log Kapp (defined by eq 7)
at pH 7.6 in 50 mM HEPES buffer matched well with the
corresponding log Kapp values (5.2, 5.9, 5.5, and 6.4) obtained
by the potentiometric pH titration data (see Table 1).
The present log Kapp value of 3.4 for the 1:1 complexation
of a nucleoside dT with ZnL3 3c was identical to the one
determined earlier by the potentiometric pH titration.15,21 The
1:1 complexation of thymidine 3′,5′-cyclic-monophosphate (c-
dTMP) with 3c occurred only through a coordination bond
between the imide-N- and zinc(II) cation.15 As a consequence,
it gave log Kapp of 3.3, almost the same value for the 1:1 3c-
dT- complex. Thus, the monoanionic cyclic phosphate part
would have little interaction with Zn2+-cyclen.29 The 1:2
complexation of dT with 6 occurred independently and the log
Kapp for the first and second binding were both 3.2.
The ITC for 3′-dTMP, 5′-dTMP, 5′-dTDP, 5′-dTTP, AZTMP,
and AZTDP with the ditopic receptors showed all the 1:1
complexations with log Kapp values in the range of 5.0-5.6 with
6 and more than 5.6 with 7. It is most significant that the
complexation of those nucleotides with the ditopic receptors is
∼40-1000 times more favorable than that (log Kapp ) 3.4) of
dT with the monotopic 3c, due to the additive binding effect of
the dibasic phosphate2- to the second Zn2+-cyclen moieties in
6 and 7. Furthermore, the p-isomer 7 was generally a better
5′-UMP
5.4a (8.3)d
5.5c,f
6.2a (8.8)d
> 6b
> 6c,f
> 6b
5′-dTDP
5.6b
5.5c,f
5.0b
5.5b
5.7c,f
5.3b
5.5c
> 6c,f
5′-dTTP
5′-AZTMP
5.6b
> 6b,e
> 6c,f
5.9b
5′-AZTDP
> 6c,f
a,b,c For the definition of Kapp and experiment conditions, see the text.
The same titration was carried out at least twice, and the experimental
errors were (3%. d The intrinsic complexation constants Ks defined
by eq 6 in the text. e Titrations were carried out at [5′-dTMP] ) 0.2
mM and 0.1 mM, and the average values are listed. Titrations were
carried out at [nucleotide] ) 0.1 mM and 50 µM, and the average values
f
are listed.
7-(5′-UMP)) determined by the similar potentiometric pH
titrations are also included in Table 1.24,25
The potentiometric pH titration of 5′-CMP (non-dT mono-
nucleotide)26 with 6 gave the log Kapp value of 3.2 ( 0.1 at pH
7.6 (log Ks ) 4.3 ( 0.1), almost the same as log Kapp of 3.5
(log Ks ) 4.6) for the 1:1 complex 12 from 6 and mono(phenyl)
phosphate at pH 7.6.18c
Isothermal Titration Calorimetry of dT (U) Nucleotides
with Zn2L4 6 and Zn2L5 7. We measured the complexation
constants of all the dT nucleosides and nucleotides with a
monotopic ZnL3 (3c) and the ditopic receptors 6 and 7 by
isothermal titration calorimetry (ITC)27 at pH 7.6 (50 mM
HEPES with I ) 0.1 (NaNO3)) and 25 °C. In comparison to
(27) (a) Freire, E.; Mayorga, O. L.; Straume, M. Anal. Chem. 1990, 62,
950a-959a, 1254a. (b) Wadso¨, I. Chem. Soc. ReV. 1997, 79-86.
(28) From our standpoint it appears that the apparent complexation
constants (Kapp) for zinc(II) complexes, 3c, 6, and 7, with dT, 3′-dTMP,
5′-dTMP, 3′-UMP, and 5′-UMP were most accurately determined by
potentiometric pH titration, which were then checked by the ITC and UV
titrations. The ITC experiments with lower concentrations of [Zn2L] < 0.1
mM) gave too many experimental errors in the measured heat. We carried
out the UV titrations of 3′- and 5′-dTMP, 3′- and 5′-UMP, 5′-dTDP,
AZTMP, and AZTDP with 6 and 7 at two different concentrations of
nucleotide ([nucleotide] ) 0.1 mM and 50 µM) and obtained almost the
same Kapp values at these two concentrations (the average values are listed
in Table 1). As for the extremely strong complexation of 7-(5′-dTMP) (see
Figure 3d for ITC and Figure 4d for UV titration) and 7-(5′-dTDP) (not
shown), the log Kapp, values were barely calculable at 6.3 ( 0.1 and 6.7 (
0.1, respectively, from both ITC and UV titration. However, it would be a
safer estimate to put them at >6.
(23) (a) Martell, A. E.; Motekaitis, R. J. Determination and Use of
Stability Constants, 2nd ed.; VCH: New York, 1992. (b) Martell, A. E.;
Hancock, R. D. Metal Complexes in Aqueous Solutions.; Plenum Press:
New York, 1996.
(24) The pK3, pK4, and pK5 values for 3′-dTMP (<2, 6.1 ( 0.1, and 9.9
( 0.1, respectively), 3′-UMP (<2, 5.8 ( 0.1, and 9.5 ( 0.1, respectively),
and 5′-UMP (<2, 6.2 ( 0.1, and 9.5 ( 0.1, respectively) were determined
in this study.
(25) By HPLC using ODS columns, it was confirmed that any chemical
conversion of nucleotides did not occur during the potentiometric pH
titration, ITC, and UV experiments.
(29) It is of interest to compare with Sessler’s protonated sapphyrin which
interacted with c-dTMP through the monobasic phosphate- chelation with
Kapp of ∼102 (M-1) at pH 6.1 (10 mM bis-Tris buffer) (ref 13d, e).
(26) The pK3 and pK4 values for 5′-CMP were 4.5 ( 0.1 and 6.6 ( 0.1,
respectively.

4546 J. Am. Chem. Soc., Vol. 122, No. 19, 2000
Aoki and Kimura
Figure 4. The change of ꢀ values of 3′-dTMP and 5′-dTMP at 267
nm on increasing concentration of Zn2L at pH 7.6 (50 mM HEPES
with I ) 0.1 (NaNO3)) and 25 °C: (a) 3′-dTMP (50 µM) + 6; (b)
3′-dTMP (50 µM) + 7; (c) 5′-dTMP (50 µM) + 6; (d) 5′-dTMP (50
µM) + 7. Equiv(Zn2L) is the number of equivalents of [Zn2L] added
against [dTMP].
receptor than the m-isomer 6, probably due to the appropriate
distance for the better interaction. Such a strong ditopic
complexation was also seen between 2′-UMP and the m-dimer
6, but not with the p-isomer 7, possibly because the distance
between the two zinc(II) cations in 7 is too long for the
simultaneous binding to 2′-phosphate and the uracil moiety.
Among 5′-dTMP, 5′-dTDP, and 5′-dTTP, the diphosphate 5′-
dTDP seemed to form the most stable complex with 7.28
The phosphorylation of AZTMP to AZTDP catalyzed by
thymidylate kinase (ATP:dTMP phosphotransferase) is rate-
determining in the metabolic pathway of AZT.5 Therefore, the
most effective administration form may be AZTDP or AZTTP
rather than AZT, the former nucleotide, however, being difficult
in cell permeation due to the highly ionic characters. Because
6 and 7 are good hosts for AZTMP and AZTDP, their
derivatization into lipophilic forms may make a new effective
AZT administration form.
The UV Spectrophotometric Titration of dT (or U)
nucleotides with 6 and 7. The UV spectrophotometric titration
of 3′-dTMP and 5′-dTMP served to confirm the deprotonated
imide functionality in the 1:1 complexes as depicted by 8-11.
Earlier, we saw that the imide deprotonation caused a decrease
in the ꢀ values of dT in the UV absorption at 267 nm.15a Figure
4 shows the decreases in the ꢀ267 values of 3′- and 5′-dTMP
(initial concentration ) 50 µM) at pH 7.6 (50 mM HEPES with
I ) 0.10 (NaNO3)) and 25 °C.
Figure 5. 1H NMR (500 MHz) spectral change of 5′-dTMP (1 mM)
in D2O (pD 7.8 ( 0.1) with increasing concentration of 7 at 35 °C.
The ratio of 5′-dTMP:7 is (a) 0:1, (b) 1:0, (c) 1:0.2, (d) 1:0.6, (e) 1:1,
and (f) 1:2, respectively. The dashed arrows indicate peaks of the
uncomplexed species, and the plain arrows are peaks of complexed
species. Peak 1 is aromatic protons (ArH) of uncomplexed 7. Peaks 2,
3, 4, 5, and 6 are H(6), H(1′), H(2′â), H(2′R), and Me(5) of
uncomplexed 5′-dTMP, respectively (for numbering, see Scheme 2).
Peaks 7 and 8 are ArH of 7 complexed with 5′-dTMP. Peaks 9, 10, 11,
12 and 13 correspond to H(6), H(1′), H(2′R), H(2′â), and Me(5) of
complexed 5′-dTMP, respectively.
us more about structure of the complex 11. Figure 5a and b
show the aromatic and anomeric region, and methyl region for
7 (1 mM) and 5′-dTMP (1 mM), respectively. With addition of
7 (0.2 and 0.6 mM) to 1 mM 5′-dTMP (Figure 5c and d), two
independent sets of peaks appeared, indicating that (1) the
quantitative 1:1 complexation of 5′-dTMP with 7 occurred at
the millimolar order of concentration and that (2) the 1:1
From curves as shown in Figure 4, the log Kapp values for
6-(3′-dTMP), 7-(3′-dTMP), 6-(5′-dTMP), and 7-(5′-dTMP),
(these titrations were carried out at two different concentrations
with [nucleotide] ) 0.1 mM and 50 µM and [6 or 7] ) 5 mM)
were calculated to be 5.4 ( 0.1, 5.8 ( 0.1, 5.7 ( 0.1, and >6,
respectively, which agreed with the values obtained by the
potentiometric pH titration and ITC measurements. It should
be remarked that the ꢀ267 values of 5′-dTMP decreased by 31
and 36% upon 1:1 complexation with 6 and 7, respectively,
which were significantly greater than the ꢀ267 decrease of dT
upon 1:1 complexation with 3c (16% decrease).15a The differ-
ence might come from the tighter binding of the dT moiety
(i.e., greater deprotonation) with Zn2+-cyclen in 11 than in 4.
Similarly, the log Kapp values for 6-(3′-UMP), 7-(3′-UMP),
6-(5′-UMP), and 7-(5′-UMP), 6-(5′-dTDP), 7-(5′-dTDP),
6-AZTMP, 7-AZTMP, 6-AZTDP, and 7-AZTDP were
determined, which showed the identical behaviors as 3′- and
5′-dTMP (see Table 1).
complex was kinetically inert on the NMR time scale (10-2
10-3 sec).30,31
-
Furthermore, the initial singlet peak of the aromatic protons
of the uncomplexed 7 (peak 1 in Figure 5a) changed to two
doublet of doublets-like (peaks 7 and 8 in Figure 5c-f
presumably corresponding to HA and HB, respectively, in
Scheme 2), implying that the two Zn2+-cyclen moieties of 7
became non-equivalent in the complex 11.
The NOE cross-peaks were observed between CH3(5) of 5′-
dTMP (peak 13 in Figure 5) and 5′-dTDP and aromatic protons
(peak 8 () HB in Scheme 2)) of 7 (1 and 2%, respectively) in
11, suggesting that the thymine ring and the aromatic ring of 7
(30) Lian, L.-Y.; Roberts, G. C. K. In NMR of Macromolecules; Roberts,
G. C. K., Ed.; IRL Press: New York, 1993; pp 153-182.
(31) A singlet peak of phosphorus of 5′-dTMP (3.0 mM) appeared at δ
6.3 and 6.9 (ppm) in the absence and presence of 1 equiv of 7, respectively,
on the 31P NMR in D2O at pD 7.8 ( 0.1 and 5 °C. In the presence of 0.6
equiv (1.8 mM) of 7, these two peaks were observed separately, supporting
that phosphorus oxygen is bound to Zn2+-cyclen in the kinetically inert
1:1 complex 11 (see Supporting Information).
1H NMR Titration of 5′-dTMP with 7 in Aqueous
1
Solution. The H NMR (500 MHz) spectra of the 5′-dTMP
complex with 7 in D2O at pD 7.8 ( 0.1 and 35 °C might inform

Recognition of Thymidine Nucleotides
J. Am. Chem. Soc., Vol. 122, No. 19, 2000 4547
Scheme 2
mechanistically different kinds of anti-HIV active agents, AZT
nucleotides and the bis(Zn2+-cyclen) (and their derivatives), may
produce a new cocktail for AIDS treatment.
Experimental Section
General Information. All reagents and solvents used were of the
highest commercial quality and used without further purification. The
zinc(II) complexes 3c,18c 6,18c,d and 720,21 were synthesized as we
previously described. 3′-dTMP, 5′-dTMP, 5′-dTDP, 5′-dTTP, c-dTMP,
2′-UMP, 3′-UMP, 5′-UMP, AZTMP, and 5′-CMP were purchased from
1
Sigma, and their purity was checked by HPLC (ODS columns), H
and/or 31P NMR, or potentiometric pH titration. All aqueous solutions
were prepared using deionized and redistilled water. The buffer solution
for ITC and UV titration was HEPES (2-[4-(2-hydroxyethyl)-1-
piperazinyl]ethanesulfonic acid, (pKa ) 7.6 at 20 °C). The ionic strength
of the buffer was adjusted to 0.10 with NaNO3. IR spectra for AZTDP
were recorded on a Shimadzu FTIR-4200 spectrometer by applying
the sample on IR cards (type 61, 3M Co. LTD). 1H NMR spectra were
recorded on a JEOL Lambda (500 MHz) or Alpha (400 MHz)
spectrometer. 3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt
(tsp) were used as an internal reference for 1H and 13C NMR
measurements in D2O. A D2O solution of 85% phosphoric acid was
used as an external reference for 31P HMR. The external The pD values
in D2O were corrected for a deuterium isotope effect using pD ) [pH-
meter reading] + 0.40. FAB mass (positive) spectra were recorded on
JEOL JMS-SX-101 using glycerin as a matrix.
are closely fixed as depicted in Scheme 2. The π-π stacking
interactions between the benzene ring and thymine ring in 11
may also contribute to the observed hypochromic effect in ꢀ267
values in UV titration.
The 1H NMR spectrum of 1:1 complex 11 of 5′-dTMP with
7 (0.5 mM) did not collapse by addition of 100 equiv of Na2-
HPO4 in D2O at pD 7.8 ( 0.1 and 35 °C, implying that 5′-
2-
dTMP was far better guests than HPO4 for the host 7 and
thus large excess of phosphate anions did not hinder the efficient
recognition of 5′-dTMP. This fact may be important in case
the bis (Zn2+-cyclen)s were used for AZTMP (or AZTDP)
transporter in biological conditions.
3′-Azido-3′-deoxythymidine-5′-diphosphate (AZTDP).5b Phos-
phorus oxychloride (55 µL, 0.59 mmol) was added to a mixture of
AZT34 (124 mg, 0.47 mmol) and 1,8-bis(dimethylamino)naphthalene
(Proton Sponge) (154 mg, 0.72 mmol) in anhydrous trimethyl phosphate
(0.5 mL) at 0 °C, and the whole was stirred at 0-5 °C.35 After 3 h, a
suspension of tetra-n-butylammonium phosphate (809 mg, 2.4 mmol)
and tri-n-butylamine (0.40 mL, 1.7 mmol) in DMF (6 mL) was added
quickly at 0 °C and the reaction mixture was turned into a colorless
clear solution. After 2 min, 10 mL of 0.2 M triethylammonium
carbonate buffer (pH 7.5) was added to the reaction mixture and stirred
for 10 min. The whole was evaporated in vacuo, and then 5 mL of
liquid NH3 was added at 0 °C. After stirring overnight at room
temperature, the whole was evaporated. The residue was purified by
column chromatography on QAE-Sephadex A-25 with triethylammo-
nium bicarbonate buffer (0.2-0.5 M, pH 7.5). The final purification
was carried out by reversed-phase HPLC (PREP-ODS, GL Science,
Inc.) with the gradient 0-10% MeOH in 10 mM triethylammonium
bicarbonate buffer (pH 7.5). Concentration of AZTDP in a stock
aqueous solution was determined by 31P NMR in the presence of Na2-
HPO4 (22% yield). IR (IR card) 2988, 2951, 2106 (N3), 1702, 1692,
Conclusions
The m- and p-bis(Zn2+-cyclen) complexes Zn2L4 6 and Zn2L5
7 have been shown to be the first highly selective ditopic
receptors for various derivatives of dT (and U) nucleotides
including AZTMP and AZTDP at physiological pH in aqueous
solution. The resulting complexes 8-11 in general are kineti-
cally inert (on the NMR time scale) and thermodynamically
much more stable than each labile component of 3c-dT- (4)
and 3c-phosphate2- (5) at pH 7.6. For instance, the log Kapp
values were 5.5-5.7 and ∼6.4 for 5′-dTMP with 6 and 7,
respectively, at pH 7.6. It is due to the simultaneous bindings
of the thymine moieties and dianionic phosphate moieties to
the two Zn2+-cyclen units in Zn2L. Although the log Kapp values
for 1:1 complexation of dT (U) nucleotide and ditopic receptors
at pH 7.6, which are nearly the sum of log Kapp values for 3c-
dT- and 3c-phosphate2-, may not be so surprising, the selective
recognition of dT (U) nucleotides by the gain in complex
stability by a factor of 102-103 is important, especially in
aqueous solution. The fact that the speciation of 11 is over 95%
at wide pH range (6.6-8.8) in Figure 2 is noteworthy. The
p-isomer Zn2L5 7 in general formed more stable complexes than
the m-dimer Zn2L5 6. Appropriate attachment of lipophilic
functions to 6 or 7 would make novel transport agents for dT-
nucleotide drugs. These derivatives also would be useful in
1
1667, 1454, 1400, 1248, 1110, 1060, 959, 837, 515 cm-1. H NMR
(400 MHz, D2O/tsp) δ 1.25 (9H, t, J ) 7.2 Hz, HN(CH2CH3)3), 1.91
(3H, s, CH3(5)), 2.42-2.48 (2H, m, H(2′)), 3.18 (6H, q, J ) 7.2 Hz,
HN(CH2CH3)3), 4.14-4.24 (3H, m, H(3′) and H(5′)), 4.53-4.56 (1H,
m, H(4′)), 6.25 (1H, dd, H(1′), J ) 6.6, 6.8 Hz), 7.73 (1H, s, H(6)).
31C NMR (100 MHz, D2O) δ 9.14, 12.49, 37.17, 47.65, 61.81, 66.30,
83.94 (d C(5′), JC-P ) 9.1 Hz), 85.83, 112.73, 138.19, 152.61, 167.43.
31P NMR (162 MHz, D2O/85% H3PO4) δ -8.3 (d, R-P, JP-P ) 22.2
Hz), -7.6 (d, â-P, JP-P ) 22.2 Hz).
separation and detection of various nucleotides.32 The bis(Zn2+
-
Potentiometric pH Titrations. The preparation of the test solutions
and the calibration method of the electrode system (Orion Research
Expandable Ion Analyzer EA920 and Orion Research Ross Combination
pH Electrode 8102BN) were described earlier.15,16,18 All of the test
solutions (50 mL) were kept under an argon (>99.999% purity)
atmosphere. The potentiometric pH titrations were carried out with I
) 0.10 (NaNO3) at 25.0 ( 0.1 °C, and at least two independent titrations
were performed. Deprotonation constants and intrinsic complexation
cyclen) 6 and 7 and their free ligands possess potent anti-HIV
activities.22 It was proposed that bis(macrocyclic tetraamine)s
work as specific inhibitors of the interaction between HIV gp120
and a coreceptor of T cell (chemokine receptors such as
CXCR4), thereby blocking the invasion of HIV into T cells.33
It will be interesting to see if the combination of the two
(32) Gosselin, G.; Imbach, J.-L.; Sommadossi, J.-P. Bull. Inst. Pasteur
1994, 92, 181-196.
(33) (a) De Clercq, E.; Yamamoto, N.; Pauwels, R.; Baba, M.; Schols,
D.; Nakashima, H.; Balzarini, J.; Debyser, Z.; Murrer, B. A.; Schwartz,
D.; Thornton, D.; Gridger, G.; Fricker, S.; Henson, G.; Abrams, M.; Picker,
D. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 5286-5290. (b) Bridger, G. J.;
Skerlj, R. T.; Padmanabhan, S.; Martellucci, S. A.; Henson, G. W.; Struyf,
S.; Witvrouw, M.; Schols, D.; De Clercq, E. J. Med. Chem. 1999, 42, 3971-
3981 and references therein.
(34) (a) Horwitz, J. P.; Urbanski, J. A.; Chua, J. J. Org. Chem. 1962,
27, 3300-3302. (b) Fox, J. J.; Miller, N. C. J. Org. Chem. 1963, 28, 936-
941. (c) Horwitz, J. P.; Chua, J.; Noel, M. J. Org. Chem. 1964, 29, 2076-
2078. (d) Lin, T.-S.; Prusoff, W. H. J. Med. Chem. 1978, 21, 109-112.
(35) (a) Kova´cs, T.; O¨ tvo¨s, L. Tetrahedron Lett. 1988, 29, 4525-4528.
(b) Sakthivel, K.; Barbas, C. F., III. Angew. Chem., Int. Ed. 1998, 37, 2872-
2875.

4548 J. Am. Chem. Soc., Vol. 122, No. 19, 2000
Aoki and Kimura
constants defined in the text were determined by means of the program
BEST.23 All of the sigma fit values defined in the program are smaller
analysis of UV titration to determine apparent complexation constants,
Kapp, for which the decreases in ꢀ267 values (for dT and AZT derivatives)
or those in ꢀ262 values (for U derivatives) were used instead of the heat
of reaction. The molar absorption coefficients (ꢀ) (M-1‚cm-1) of the
nucleotides at pH 7.6 and 25 °C used for determination of their
concentrations in aqueous buffer solutions were as follows: 3′-dTMP,
λmax 267 nm (ꢀ 9.5 × 103); 5′-dTMP, λmax 267 nm (ꢀ 9.7 × 103), 3′-
UMP, λmax 262 nm (ꢀ 1.0 × 104); 5′-UMP, λmax 262 nm (ꢀ 1.0 × 104);
5′-dTDP, λmax 267 nm (ꢀ 9.7 × 103); AZTMP, λmax 267 nm (ꢀ 9.4 ×
103), and AZTDP, λmax 267 nm (ꢀ 9.4 × 103), respectively.
than 0.05. The KW () aH ‚aOH ), K′W () [H+][OH-]) and fH values
+
-
+
used at 25 °C are 10-14.00, 10-13.79, and 0.825. The corresponding mixed
constants, K2 () [HO--bound species]aH /[H2O-bound species]), are
+
derived using [H+] ) aH /fH . The species distribution values (%)
against pH () -log[H+] + 0.084) were obtained using the program
SPE.23
+
+
Isothermal Calorimetric Titrations.27 The heats of 1:1 complex-
ation of nucleosides and nucleotides with zinc(II) complexes were
recorded on a Calorimetry Science Corporation Isothermal Titration
Calorimeter 4200 at 25.0 ( 0.1 °C and pH 7.6 (50 mM HEPES buffer
with I ) 0.10 (NaNO3)). The calorimeter was calibrated by heat (474.7
mJ) of protonation of tris(hydroxymethyl)aminomethane (250 mM, 1.0
mL) by 10 µL injection of 1.00 mM aqueous HCl at 25.0 °C. The
solution (1.0 mL) of 3c (2.0 mM), 6, (0.2 or 0.5 mM) or 7 (0.1, 0.2, or
0.5 mM, depending on the affinity) in 50 mM HEPES was put into a
calorimeter cell. After the cell temperature had become constant at 25.0
°C, the solution of a nucleoside (50 mM), a phosphate (50 mM), or a
nucleotide (10 or 50 mM) in 50 mM HEPES was portionwise loaded.
The titrations were run at least twice. The obtained calorimetric data
Acknowledgment. E.K. is thankful for the grant from the
Grant-in-Aid for Priority Project “Biometallics” (No. 08249103)
from the Ministry of Education, Science and Culture in Japan.
S.A. is thankful for the grant from the Grant-in-Aid for Young
Scientists (No. 10771249) and the grant from Nissan Science
Foundation and Uehara Memorial Foundation. We thank the
Research Center for Molceular Medicine (RCMM), Faculty of
Medicine, Hiroshima University, for the use of NMR instru-
ments (a JEOL Alpha (400 MHz)).
was analyzed for ∆H values and apparent complexation constants, Kapp
,
using the program Data Works and Bind Works (Calorimetry Sciences
Corp).
Supporting Information Available: 1H, 13C, and 31P NMR
spectra of AZTDP, FAB mass spectra of 6-(3′-dTMP) and
7-(5′-dTMP) complexes with the theoretical mass distribution
spectra, and 31P NMR spectra of 5′-dTMP in the absence and
the presence of 7 in D2O at pD 7.8 ( 0.1 and 5 °C (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
UV Titrations. UV spectra were recorded on a Hitachi U-3500
spectrophotometer at 25.0 ( 0.1 °C. The solution (2.3-2.5 mL) of a
nucleotide ([nucleotide] ) 0.1 mM or 50 µM) in 50 mM HEPES (pH
7.6 with I ) 0.1 (NaNO3)) was put into a quartz cell. After the cell
temperature had become constant at 25.0 °C, the solution of a zinc(II)
complex (concentration was 5.0 mM) in 50 mM HEPES was portion-
wise added. The titrations were run at least twice. The program Bind
Works (Calorimetry Sciences Corp) mentioned above was used for
JA994537S