Study of pH-dependent zinc(II)-carboxamide interactions by zinc(II)-carboxamide-appended cyclen complexes (cyclen = 1,4,7,10-tetraazacyclododecane)

Article abstract of DOI:10.1021/ic020087k

To elucidate intrinsic recognition of carboxamides by zinc(II) in carbonic anhydrase (CA) (as inhibitors) and carboxypeptidase A (CPA) (as substrates), a new series of Zn²⁺-carboxamide-appended cyclen complexes have been synthesized and characterized (cyclen = 1,4,7,10-tetraazacyclododecane). Two types of Zn²⁺-carboxamide interactions have been found. In the first case represented by a zinc(II) complex of carbamoylmethyl-l,4,7,10-tetraazacyclododecane (L¹), the amide oxygen binds to zinc(II) at slightly acidic pH (to form ZnL¹), and the deprotonated amide N^- binds to zinc(II) at alkaline pH (to form ZnH_-1,L¹) with pK_a = 8.59 at 25°C and l = 0.1 (NaNO₃), as determined by potentiometric pH titrations, infrared spectral changes, and ¹³C and ¹H NMR titrations. The X-ray crystal structure of ZnH_-1L³ (where L³ = N-(4-nitrophenyl)carbamoylmethyl cyclen, pK_a = 7.01 for ZnL³ ? ZnH_-1L³) proved that the zinc(II) binds to the amidate N^-(Zn-N^- distance of 1.974(3) A) along with the four nitrogen atoms of cyclen (average Zn-N distance 2.136 A). Crystal data: monoclinic, space group P2₁/n (No. 14) with a = 10.838(1) A, b = 17.210(2) A, c = 12.113(2) A, b = 107.38(1)°, V = 2156.2(5) A³, Z = 4, R = 0.042, and R_w = 0.038. These model studies provide the first chemical support that carboxamides are CA^- inhibitors by occupying the active Zn²⁺ site both in acidic and alkaline pH to prevent the occurrence of the catalytically active Zn²⁺-OH^- species. In the second case represented by a zinc(II) complex of 1-(N-acetyl)aminoethylcyclen, ZnL⁶, the pendant amide oxygen had little interaction with zinc(II) at acidic pH. At alkaline pH, the monodeprotonation yielded a zinc(II)-bound hydroxide species ZnL⁶(OH^-) (pK_a = 7.64) with the amide pendant remaining intact. The ZnL⁶(OH^-) species showed the same nucleophilic activity as Zn²⁺-cyclen-OH^-. The second case may mimic the Zn²⁺-OH^- mechanism of CPA, where the nucleophilic Zn²⁺-OH^- species does not act as a base to deprotonate a proximate amide.

Full text of DOI:10.1021/ic020087k

Inorg. Chem. 2002, 41, 3239−3248
Study of pH-Dependent Zinc(II)−Carboxamide Interactions by
Zinc(II)−Carboxamide-Appended Cyclen Complexes (Cyclen )
1,4,7,10-Tetraazacyclododecane)
,†
Eiichi Kimura,* Teruhiro Gotoh,† Shin Aoki,† and Motoo Shiro‡
Department of Medicinal Chemistry, Faculty of Medicine, Hiroshima UniVersity, Kasumi 1-2-3,
Minami-ku, Hiroshima, 734-8551 Japan, and Rigaku Corporation X-ray Research Laboratory,
Matsubaracho 3-9-12, Akishima, Tokyo, 196-8666 Japan
Received January 29, 2002
To elucidate intrinsic recognition of carboxamides by zinc(II) in carbonic anhydrase (CA) (as inhibitors) and
carboxypeptidase A (CPA) (as substrates), a new series of Zn2+−carboxamide-appended cyclen complexes have
been synthesized and characterized (cyclen ) 1,4,7,10-tetraazacyclododecane). Two types of Zn2+−carboxamide
interactions have been found. In the first case represented by a zinc(II) complex of carbamoylmethyl-1,4,7,10-
tetraazacyclododecane (L1), the amide oxygen binds to zinc(II) at slightly acidic pH (to form ZnL1), and the
deprotonated amide N- binds to zinc(II) at alkaline pH (to form ZnH-1L1) with pK ) 8.59 at 25 °C and I ) 0.1
a
(NaNO ), as determined by potentiometric pH titrations, infrared spectral changes, and 13C and 1H NMR titrations.
3
The X-ray crystal structure of ZnH-1L3 (where L3 ) N-(4-nitrophenyl)carbamoylmethyl cyclen, pK ) 7.01 for ZnL3
a
a ZnH-1L3) proved that the zinc(II) binds to the amidate N- (Zn−N- distance of 1.974(3) Å) along with the four
nitrogen atoms of cyclen (average Zn−N distance 2.136 Å). Crystal data: monoclinic, space group P21/n (No. 14)
3
with a ) 10.838(1) Å, b ) 17.210(2) Å, c ) 12.113(2) Å, b ) 107.38(1)°, V ) 2156.2(5) Å , Z ) 4, R ) 0.042,
and Rw ) 0.038. These model studies provide the first chemical support that carboxamides are CA- inhibitors by
occupying the active Zn2+ site both in acidic and alkaline pH to prevent the occurrence of the catalytically active
Zn2+−OH- species. In the second case represented by a zinc(II) complex of 1-(N-acetyl)aminoethylcyclen, ZnL6,
the pendant amide oxygen had little interaction with zinc(II) at acidic pH. At alkaline pH, the monodeprotonation
yielded a zinc(II)-bound hydroxide species ZnL6(OH-) (pK ) 7.64) with the amide pendant remaining intact. The
a
ZnL6(OH-) species showed the same nucleophilic activity as Zn2+−cyclen−OH-. The second case may mimic the
Zn2+−OH- mechanism of CPA, where the nucleophilic Zn2+−OH- species does not act as a base to deprotonate
a proximate amide.
Introduction
CPA catalyzes the hydrolysis of hydrophobic C-terminal
amino acids from polypeptide substrates,3 and its active-site
zinc(II) is bound to His69, Glu72, His196, and a water
molecule hydrogen bonding to Glu270 (Scheme 1). In a
widely accepted Zn2+-hydroxide mechanism for CA and
CPA, the zinc(II)-bound waters that are generated at neutral
pH are activated to attack at the electrophilic sites of
Carbonic anhydrase (CA, EC 4.2.1.1) and carboxypepti-
dase A (CPA, EC 3.4.17.1) are mechanistically the two most
typical zinc(II) enzymes.1 CA catalyzes the reversible
hydration of CO2 to bicarbonate ion,2 and its active-site zinc-
(II) is bound to His94, His96, His119, and a water molecule.
* To whom correspondence should be addressed. E-mail: ekimura@
hiroshima-u.ac.jp.
(2) (a) Botre`, F.; Gros, G.; Storey, B. T. Carbonic Anhydrase; VCH:
Weinheim, 1991. (b) Bertini, I.; Luchinat, C. Acc. Chem. Res. 1983,
16, 272-279. (c) Silverman, D. N.; Lindskog, S. Acc. Chem. Res.
1988, 21, 30-36. (d) Liljas, A.; Håkansson, K.; Jonsson, B. H.; Xue,
Y. Eur. J. Biochem. 1994, 219, 1-10. (e) Christianson, D. W.; Flerke,
C. A. Acc. Chem. Res. 1996, 29, 331-339.
(3) (a) Quiocho, F. A.; Lipscomb, W. N. AdV. Protein Chem. 1971, 25,
1-78. (b) Christianson, D. W.; Lipscomb, W. N. Acc. Chem. Res.
1989, 22, 62-69.
† Hiroshima University.
‡ Rigaku Corporation.
(1) (a) Walsh, C. Enzymatic Reaction Mechanisms; Freeman and Co.:
New York, 1979. (b) Voet, D.; Voet, J. G. Biochemistry; Wiley &
Sons: New York, 1990. (c) Lipscomb, W. N.; Stra¨ter, N. Chem. ReV.
1996, 96, 2375-2433. (d) Coleman, J. E. Curr. Opin. Chem. Biol.
1998, 2, 222-234.
10.1021/ic020087k CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/17/2002
Inorganic Chemistry, Vol. 41, No. 12, 2002 3239

Kimura et al.
nitrogen of acetazolamide is deprotonated.10 The zinc(II) ion
in the [12]aneN3 complex lowered the pKa value of an
intramolecular sulfonamide from 11.2 to <7 to yield a very
stable sulfonamidate complex 3 at neutral pH,11 which lost
the catalytic activity. Later, a zinc(II) complex of 1,4,7,10-
tetraazacyclododecane (cyclen), 4a, also produced a nucleo-
philic and simultaneously basic zinc(II)-OH- species (4b)
with pKa of 7.9 at 25 °C10 and acted as another CA model.12
A greater advantage of the macrocyclic ligand cyclen over
[12]aneN3 is that the zinc(II) is more firmly held with higher
thermodynamic and kinetic stability. A dansylamide-pendant
cyclen was designed as a model for fluorescent dansylamide
binding to CA, which made an efficient and selective Zn2+
fluorosensor in the form of 5 (cyclen ) 1,4,7,10-tetraaza-
cyclododecane).13 The Zn2+-cyclen complex 4 catalytically
hydrolyzed an activated carboxamide in â-lactam ring of
penicillin as a model for zinc(II)-containing â-lactamase II.14
Scheme 1
polarized carbonyl substrates. It is interesting that carboxa-
mides are substrates for CPA and inhibitors for CA.4 The
visible spectral study of binding of iodoacetamide (Ki ) 40
mM, at pH 8.1) to cobalt(II)-substituted CA I suggested N-
coordination of the amidate anion to cobalt(II).4b Until now,
however, there have been few chemical models for this type
of amide or amidate association with Zn2+. More funda-
mentally, the pKa values of the amide a amidate in the
vicinity of Zn2+ were not reported.5 On the other hand,
aromatic sulfonamides, which are stronger CA inhibitors than
carboxamides, are now well established to bind to zinc(II)
as amidate anions at physiological conditions.6
How do Zn2+ ions work differently toward the carboxa-
mides in CA and CPA? It may be helpful to consider that
the acidity of zinc(II) is probably higher in CA with the
ligation of three neutral donors (His)3 than in CPA with
(His)2(Glu-). This argument is compatible with a fact that
the pKa of 6.8 for the zinc-bound water in the wild-type CA
II is increased to g9.6 in a mutant CA II with His94 f
Asp-, as kinetically determined by hydrolysis of 4-nitro-
phenyl acetate.7 The more acidic Zn2+ in CA may be more
favorable in attracting a carboxamide at the fourth coordina-
tion site than the less acidic Zn2+ in CPA. Then, intrinsic
properties of Zn2+ to be questioned are (1) how do the
carboxamide inhibitors and the nucleophile hydroxide com-
pete for Zn2+ in CA? (2) what are the pKa values for
deprotonating the Zn2+-bound carboxamides? and (3) why
does the catalytically active Zn2+-OH- species in CPA (see
Scheme 1) not work as a base toward substrate carboxamides
to turn it from a substrate to an inhibitor?
Accordingly, different modes of recognition of carboxa-
mides by CA and CPA might be mimicked by using
carboxamide-appended cyclens, such as carbamoylmethyl-
cyclen 6 (L1), N-benzylcarbamoylmethylcyclen 7 (L2), N-(4-
nitrophenyl)carbamoylmethylcyclen 8 (L3), N,N′-diethylcar-
In our earlier zinc enzyme model studies, zinc(II)-1,5,9-
triazacyclododecane (Zn2+-[12]aneN3) complex 1 has been
shown to be one of the most suitable mechanistic models
for CA in aqueous solution.8 The water at the fourth co-
ordination site of 1a can be deprotonated to 1b with a very
low pKa value of 7.3 at 25 °C.9a Compound 1 has revealed
the intrinsic role of zinc(II) of CA in catalyzing the reversible
CO2 hydration and the carboxyester hydrolysis.9b Moreover,
a typical CA inhibitor acetazolamide reacted with 1 to yield
1:1 complex 2 at physiological pH, wherein the sulfonamide
(8) For review: (a) Kimura, E. In Progress in Inorganic Chemistry; Karlin,
K. D., Ed.; John Wiley & Sons: New York, 1994; Vol. 41, pp 443-
491. (b) Kimura, E.; Koike, T. In AdVances in Inorganic Chemistry;
Sykes, A. G., Ed.; Academic Press: New York, 1997; Vol. 44, pp
229-261. (c) Kimura, E.; Koike, T. In ComprehensiVe Supramolecular
Chemistry; Reinhoudt, D. N., Ed.; Pregamon: Tokyo, 1996; Vol. 10,
pp 429-444. (d) Kimura, E.; Koike, T. J. Chem. Soc., Chem. Commun.
1998, 1495-1500. (e) Kimura, E.; Koike, T. In Bioinorganic Catalysis;
Reedijik, J., Bouwman, E., Ed.; Marcel Dekker, Inc.: New York, 1999;
pp 33-54. (f) Kimura, E.; Kikuta, E. J. Biol. Inorg. Chem. 2000, 5,
139-155. (g) Kimura, E. Acc. Chem. Res. 2001, 34, 171-179.
(9) (a) Kimura, E.; Shiota, T.; Koike, T.; Shiro, M.; Kodama, M. J. Am.
Chem. Soc. 1990, 112, 5805-5811. (b) Zhang, X.; van Edlik, R.;
Koike, T.; Kimura, E. Inorg. Chem. 1993, 32, 5749-5755.
(10) Koike, T.; Kimura, E. J. Am. Chem. Soc. 1991, 113, 8935-8941.
(11) Koike, T.; Kimura, E.; Nakamura, I.; Hashimoto, Y.; Shiro, M. J.
Am. Chem. Soc. 1992, 114, 7338-7345.
(4) (a) Whitney, P. L. Eur. J. Biochem. 1970, 16, 126-135. (b) Rogers,
J. I.; Mukherjee, J.; Khalifah, R. G. Biochemistry 1987, 26, 5672-
5679. (c) Liang, J. Y.; Lipscomb, W. N. Biochemistry 1989, 28, 9724-
9733.
(5) Sigel, H.; Martin, R. B. Chem. ReV. 1982, 82, 385-426.
(6) (a) Eriksson, A. E.; Kylsten, P. M.; Jones, T. A.; Liljas, A. Proteins
1988, 4, 283-293. (b) Boriack, P. A.; Christianson, D. W.; Kingery-
Wood, J.; Whitesides, G. M. J. Med. Chem. 1995, 38, 2286-2291.
(7) Kiefer, L. L.; Ippolito, C. A.; Christianson, D. W. J. Am. Chem. Soc.
1993, 115, 12581-12582.
(12) Zhang, X.; van Edlik, R. Inorg. Chem. 1995, 34, 5606-5614.
(13) (a) Koike, T.; Watanabe, T.; Aoki, S.; Kimura, E.; Shiro, M. J. Am.
Chem. Soc. 1996, 118, 12696-12703. (b) For review: Kimura, E.;
Koike, T. Chem. Soc. ReV. 1998, 27, 179-184.
(14) Koike, T.; Takamura, M.; Kimura, E. J. Am. Chem. Soc. 1994, 116,
8443-8449.
3240 Inorganic Chemistry, Vol. 41, No. 12, 2002

Zinc(II)-Carboxamide Interactions
bamoylmethylcyclen 9 (L4), 2-(carbamoyl)ethylcyclen 10
(L5), 2-(N-acetylamino)ethylcyclen 11 (L6), and 2-((4-ni-
trobenzoyl)amino)ethylcyclen 12 (L7).
6‚3HCl‚3H2O (58% yield): Mp 250-251 °C dec. IR (KBr):
3322, 2739, 1688, 1605, 1478, 1441, 1370, 1316, 1267, 1020 cm-1
.
1H NMR (D2O): δ 3.01-3.19 (16H, m), 3.48 (2H, s). 13C NMR
(D2O): δ 45.26, 45.70, 47.33, 52.82, 57.77, 179.00. Anal. Calcd
for C10H32N5O4Cl3: C, 30.58; H, 8.21; N, 17.83. Found: C, 30.57;
H, 8.13; N, 17.81. 7‚3HCl‚3H2O (75% yield): Mp 217-219 °C.
IR (KBr): 3441, 2965, 2934, 1657, 1545, 1453, 1370, 702 cm-1
.
1H NMR (D2O): δ 3.00-3.19 (16H, m), 3.51 (2H, s), 4.42 (2H,
s), 7.34-7.43 (5H, m). 13C NMR (D2O): δ 43.27, 43.70, 45.39,
50.81, 56.46, 128.27, 128.58, 129.85, 138.65, 174.07. Anal. Calcd
for C17H38N5O4Cl3: C, 42.28; H, 7.93; N, 14.50. Found: C, 42.31;
H, 8.11; N, 14.51. 8‚3HCl‚0.5H2O (54% yield): Mp 242-244 °C
dec. IR (KBr): 3001, 2643, 1701, 1614, 1597, 1555, 1505, 1410,
1343, 1300, 1258 cm-1. 1H NMR (D2O): δ 3.07-3.27 (16H, br),
3.70 (2H, s), 7.62 (2H, d, J ) 9.2 Hz), 8.17 (2H, d, J ) 9.2 Hz).
13C NMR (D2O): δ 45.13, 45.69, 47.44, 52.85, 59.15, 123.28,
128.02, 146.20, 146.47, 175.21. Anal. Calcd for C16H30N6O3.5Cl3:
C, 40.99; H, 6.40; N, 17.93. Found: C, 41.17; H, 6.37; N, 17.94.
Zinc(II) Complex of 1-Carbamoylmethyl-1,4,7,10-tetraaza-
cyclo-dodecane, 17(ClO4)2. A MeOH solution (2 mL) of 6‚3HCl‚
3H2O (392 mg, 1.0 mmol) was mixed with 1 M NaOMe in MeOH
(3 mL). After insoluble inorganic salts were filtered off, the filtrate
was concentrated under reduced pressure to obtain the free ligand
6. To an EtOH solution (10 mL) of 6 was added a EtOH (10 mL)
solution of Zn(ClO4)2‚6H2O (372 mg, 1.0 mmol) at room temper-
ature, and the whole was stirred for 1 h. After the solvent was
evaporated, the remaining solids were recrystallized from CH3CN/
EtOH to obtain 17(ClO4)2 (356 mg, 72% yield) as colorless needles
(although we have not experienced the explosion of ClO4 salts of
zinc complexes, the standard warning of their hazards should be
noted): Mp 227-228 °C dec. IR (KBr): 3237, 3127, 2924, 1686,
Experimental Section
General Information. All reagents and solvents were purchased
at the highest commercial quality and used without further
purification. Anhydrous acetonitrile (CH3CN) was obtained by
distillation from calcium hydride. All aqueous solutions were
prepared using deionized and distilled water. The Good’s buffers
(Dojindo) were commercially available: MOPS (3-(N-morpholino)-
propanesulfonic acid, pKa ) 7.2), HEPES (N-(2-hydroxyethyl)-
piperazine-N′′-2-ethanesulfonic acid, pKa ) 7.5), EPPS (3-(4-(2-
hydroxyethyl)-1-piperazinyl)propanesulfonic acid, pKa ) 8.0),
TAPS (N-(tris(hydroxymethyl)methylamino)-3-propanesulfonic acid,
pKa ) 8.4), and CHES (2-(cyclohexylamino)ethanesulfonic acid,
pKa ) 9.5). N-(4-Nitrophenyl)chloroacetamide and N-benzylchlo-
roacetamide were prepared by standard procedure from the corre-
sponding acid chloride and amine. UV spectra were recorded on a
Hitachi U-3500 spectrophotometer equipped with a temperature
controller unit at 25 ( 0.1 °C. IR spectra were recorded on a Horiba
1
1422, 1327, 1144, 1115, 1090, 1013, 993, 627 cm-1. H NMR
1
FTIR-710 spectrophotometer at room temperature. H (500 MHz)
(D2O): δ 2.65-3.10 (16H, m), 3.31 (2H, s). 13C NMR (D2O): δ
45.77, 46.27, 47.22, 55.86, 56.67, 177.69. Anal. Calcd for
C10H23N5O9Cl2Zn: C, 24.33; H, 4.70; N, 14.19. Found: C, 24.19;
H, 4.57; N, 14.23.
and 13C (125 MHz) NMR spectra at 35 ( 0.1 °C were recorded on
a JEOL Delta 500 spectrometer. 3-(Trimethylsilyl)propionic-2,2,3,3-
d4 acid sodium salt in D2O and tetramethylsilane in CD3CN were
used as internal references for H and 13C NMR measurements.
1
Zinc(II) Complex of Carbamoylmethylamidate, 17a(ClO4)‚
H2O. To a solution of 17(ClO4)2 (247 mg, 0.5 mmol) in CH3CN
(10 mL) was added 0.5 mL of 1 M NaOMe in MeOH (0.5 mmol).
After the reaction mixture was concentrated under reduced pressure,
the remaining solids were recrystallized from CH3CN to afford
17a(ClO4)‚H2O (102 mg, 50% yield) as colorless needles: Mp >
270 °C. IR (KBr): 3185, 2921, 1584, 1447, 1406, 1248, 1144, 1117,
1090, 999, 627 cm-1. 1H NMR (D2O): δ 2.73-2.84 (8H, m), 2.96-
3.07 (8H, m), 3.29 (2H, s). 13C NMR (D2O): δ 46.31, 47.39, 48.36,
55.27, 60.26, 179 (carbonyl carbon, as determined by HMQC and
HSQC). Anal. Calcd for C10H24N5O6ClZn: C, 29.20; H, 5.88; N,
17.00. Found: C, 29.13; H, 5.67; N, 17.04.
The pD values in D2O were corrected for a deuterium isotope effect
using pD ) (pH-meter reading) + 0.40. Elemental analyses were
performed on a Perkin-Elmer CHN 2400 analyzer. Thin-layer (TLC)
and silica gel column chromatographies were performed using a
Merck 5554 (silica gel) TLC plate and Fuji Silysia Chemical FL-
100D, respectively.
1-Carbamoylmethyl-1,4,7,10-tetraazacyclododecane Trihy-
drochloric Acid Salt (6‚3HCl‚3H2O), 1-(N-Benzylcarbamoyl-
methyl)-1,4,7,10-tetraazacyclododecane Trihydrochloric Acid
Salt (7‚3HCl‚3H2O), and 1-(N-(4-Nitrophenyl)carbamoyl-
methyl)-1,4,7,10-tetraazacyclododecane Trihydrochloric Acid
Salt (8‚3HCl‚0.5H2O). A solution of 3Boc-cyclen (3.8 g, 8.0
mmol)15 and the corresponding 2-bromo- (or 2-chloro-) acetamide
derivatives (9.6 mmol) (2-bromoacetamide for 6, N-benzyl-2-
chloroacetamide for 7, or 2-chloro-N-(4-nitrophenyl)acetamide for
8, respectively) in CH3CN (50 mL) was stirred in the presence of
Na2CO3 (9.6 mmol) and NaI (9.6 mmol) at 80 °C under an argon
atmosphere for 1 day. After insoluble inorganic salts were filtered
off, the filtrate was concentrated under reduced pressure. The
remaining residue was purified by silica gel column chromatography
(hexane/AcOEt) to obtain 14, 15, or 16. These compounds were
deprotected with 4 M HCl in 1,4-dioxane for 0.5-2 h at 0 °C to
room temperature, and precipitates were recrystallized from H2O
to afford 6‚3HCl‚3H2O, 7‚3HCl‚3H2O, or 8‚3HCl‚0.5H2O.
Zinc(II) Complex of 1-(N-(4-Nitrophenyl)carbamoylmethyl)-
1,4,7,10-tetraazacyclododecane, 19(ClO4)2‚H2O. An aqueous
solution (10 mL) of 8‚3HCl‚0.5H2O (230 mg, 0.49 mmol) was
added to a 3 N NaOH aqueous solution, and the solution was
extracted with CHCl3 (50 mL × 5). After the combined organic
layers were dried over anhydrous Na2SO4, the solvent was
concentrated under reduced pressure to obtain the free ligand 8 as
a yellow oil. To a EtOH (10 mL) solution of the free 8 was added
Zn(ClO4)2‚6H2O (186 mg, 0.50 mmol) at room temperature, and
the whole was stirred for 1 h. After the solvent was evaporated,
the remaining solids were crystallized from CH3CN/EtOH to obtain
19(ClO4)2‚H2O (179 mg, 58% yield) as pale yellow needles: Mp
249-250 °C. IR (KBr): 1663, 1626, 1597, 1579, 1563, 1512, 1345,
1
1121, 1109, 1092, 888, 627 cm-1. H NMR (CD3CN): δ 2.78-
(15) Kimura, E.; Aoki, S.; Koike, T.; Shiro, M. J. Am. Chem. Soc. 1997,
119, 3068-3076.
3.09 (16H, m), 3.69 (2H, s), 7.84 (2H, d, J ) 9.3 Hz), 8.29 (2H,
Inorganic Chemistry, Vol. 41, No. 12, 2002 3241

Kimura et al.
d, J ) 9.3 Hz), 9.48 (1H, br). 13C NMR (CD3CN): δ 45.64, 45.99,
47.43, 55.81, 57.97, 121.87, 126.00, 143.40, 145.87, 174.20. Anal.
Calcd for C16H30N6O13Cl2Zn: C, 30.36; H, 4.46; N, 13.29. Found:
C, 30.38; H, 4.61; N, 13.23.
slurry in water), and 1N NaOH (8.0 mL, 8.0 mmol) in EtOH (80
mL) was stirred under H2 (20 atm) at room temperature for 2 days.
After Raney nickel was filtered off with Celite (No. 545), the filtrate
was evaporated. The remaining residue was dissolved in CH3CN
(20 mL), to which was added Na2CO3 (0.79 g, 7.45 mmol) and
acetyl chloride (0.78 g, 9.9 mmol). The reaction mixture was stirred
at room temperature under an argon atmosphere for 4 h. After
insoluble inorganic salts were filtered off, the filtrate was concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography (CH2Cl2/MeOH). After evaporation of
the solvent, the remaining colorless amorphous solid was dissolved
in MeOH (20 mL), to which 37% aqueous HCl (10 mL) was slowly
added at 0 °C. After the reaction mixture was stirred for 4 h at
room temperature, the solvent were evaporated, and the remaining
solids were recrystallized from H2O/EtOH to obtain colorless prisms
of 11‚4HCl‚H2O (467 mg, 14% yield): Mp 177-178 °C. IR
Zinc(II) Complex of N-(4-Nitrophenyl)carbamoylmethyla-
midate, 19a(ClO4)‚H2O. To a solution of 19(ClO4)2‚H2O (189 mg,
0.30 mmol) in CH3CN (3 mL) was added 3 mL of 0.1 M NaOH.
After the reaction mixture was concentrated under reduced pressure,
the remaining solids were recrystallized from CH3CN/H2O to obtain
yellow prisms of 19a(ClO4)‚H2O (122 mg, 76% yield): Mp > 270
°C. IR (KBr): 3295, 1615, 1572, 1491, 1319, 1181, 1119, 1107,
1
955, 855, 625 cm-1. H NMR (CD3CN): δ 2.67-2.81 (8H, m),
2.97-3.05 (8H, m), 3.35 (2H, s), 7.55 (2H, d, J ) 9.3 Hz), 8.11
(2H, d, J ) 9.3 Hz). 13C NMR (CD3CN): δ 44.46, 45.52, 47.01,
53.36, 60.49, 124.76, 125.38, 142.79, 155.71, 173.98. Anal. Calcd
for C16H27N6O8ClZn: C, 36.08; H, 5.11; N, 15.84. Found: C, 36.00;
H, 4.93; N, 15.70.
1
(KBr): 1668, 1577, 1498, 1442, 1375, 1286, 1099, 950 cm-1. H
NMR (D2O): δ 2.00 (3H, s), 2.73 (2H, t, J ) 6.0 Hz), 2.94-2.99
(8H, m), 3.18-3.20 (8H, m), 3.34 (2H, t, J ) 6.0 Hz). 13C NMR
(D2O): δ 25.18, 39.40, 44.82, 45.15, 47.36, 51.48, 55.80, 177.80.
Anal. Calcd for C12H33N5O2Cl4: C, 33.89; H, 7.82; N, 16.47.
Found: C, 34.19; H, 8.20; N, 16.71.
1-(N,N-Diethylcarbamoylmethyl)-1,4,7,10-tetraazacyclodode-
cane Tetrahydrochloric Acid Salt, 9‚4HCl‚H2O. To a solution
of 1,4,7,10-tetraazacyclododecane (1.3 g, 7.3 mmol) in EtOH (15
mL) was slowly added a solution of N,N-diethylchloroacetamide
(570 mg, 3.8 mmol) in EtOH (15 mL). The reaction mixture was
stirred at 80 °C for 8 h and then reacted with (Boc)2O (6.6 g, 30
mmol) and triethylamine (3.1 g, 30 mmol) at room temperature
for 8 h. After the reaction mixture was concentrated under reduced
pressure, the residue was purified by silica gel column chroma-
tography (AcOEt). After evaporation of the solvent, the residue
was dissolved in MeOH (10 mL), to which was slowly added 37%
aqueous HCl (10 mL) and stirred for 2 h at room temperature. After
the solvents were evaporated, the residue was crystallized from
MeOH to obtain 9‚4HCl‚H2O (922 mg, 54% yield) as colorless
prisms: Mp 188 °C dec. IR (KBr): 3002, 2820, 2762, 2672, 1642,
1576, 1478, 1458, 1431 cm-1. 1H NMR (D2O): δ 1.11 (3H, t, J )
7.2 Hz), 1.18 (3H, t, J ) 7.2 Hz), 3.02-3.15 (16H, m), 3.32-3.38
(4H, m), 3.66 (2H, s). 13C NMR (D2O): δ 14.96, 15.85, 44.53,
44.71, 45.61, 45.99, 47.48, 53.33, 57.36, 174.58. Anal. Calcd for
C14H37N5O2Cl4: C, 37.42; H, 8.30; N, 15.59. Found: C, 37.19; H,
8.61; N, 15.39.
1-(2-(Carbamoyl)ethyl)-1,4,7,10-tetraazacyclododecane Ditri-
fluoroacetic Acid Salt (10‚2CF3COOH). Compound 10‚2CF3-
COOH was synthesized from 1,4,7,10-tetraazacyclododecane and
2-bromopropionamide using the same method as 9‚4HCl‚H2O and
isolated as ditrifluoroacetic acid salt. IR (KBr): 1667, 1200, 1176,
1129, 798, 720 cm-1. 1H NMR (D2O): δ 2.50 (2H, t), 2.89-3.20
(18H, m). 13C NMR (D2O): δ 31.73, 42.21, 42.56, 44.72, 48.42,
48.67, 178.25. Anal. Calcd for C15H27N5O5F6: C, 34.90; H, 6.08;
N, 15.66. Found: C, 34.75; H, 6.21; N, 15.39.
Zinc(II) Complex of N-1-(2-Acetylaminoethyl)-1,4,7,10-tet-
raazacyclododecane, 27(ClO4)2. Compound 11‚4HCl‚H2O (100
mg, 0.24 mmol) was passed through an anion exchange column
(Amberlite IRA-400, OH- form) with water to obtain the free ligand
11. To an EtOH solution (10 mL) of the free 11 was added Zn-
(ClO4)2‚6H2O (102 mg, 0.27 mmol), and the whole was stirred at
room temperature for 1 h. After the solvents were evaporated, the
remaining solids were crystallized from CH3CN/EtOH to obtain
17(ClO4)2 (68 mg, 55% yield) as colorless needles: Mp > 270 °C.
IR (KBr): 2933, 1643, 1579, 1377, 1143, 1115, 1090, 627 cm-1
.
1H NMR (D2O): δ 2.02 (3H, s), 2.81-2.99 (16H, m), 3.09-3.11
(2H, m), 3.46 (2H, t, J ) 6.7 Hz). 13C NMR (D2O): δ 22.09, 34.77,
42.66, 43.98, 44.77, 50.49, 51.58, 175.19. Anal. Calcd for
C12H27N5O9Cl2Zn: C, 27.63; H, 5.22; N, 13.43. Found: C, 27.70;
H, 5.22; N, 13.37.
1-(2-(4-Nitrobenzoyl)amino)ethyl-1,4,7,10-tetraazacyclodode-
cane Trihydrochloric Acid Salt, 12‚3HCl‚2H2O. Compound 12
was synthesized from 3Boc-cyclen (4.3 g, 9.1 mmol) and 4-ni-
trobenzoyl chloride (1.86 g, 10 mmol) using the same method used
for the synthesis of 11‚4HCl‚H2O. Recrystallization from H2O gave
pale yellow needles of 12‚3HCl‚2H2O (2.2 g, 48% yield): Mp 237-
238 °C. IR (KBr): 1651, 1601, 1523, 1446, 1344, 1302, 877, 843,
1
714 cm-1. H NMR (D2O): δ 2.86-2.91 (6H, m), 2.98 (4H, br),
3.16-3.21 (8H, m), 3.60 (2H, t, J ) 6.1 Hz), 8.00 (2H, d, J ) 8.9
Hz), 8.36 (2H, d, J ) 8.9 Hz). 13C NMR (D2O): δ 39.77, 44.60,
45.07, 47.24, 51.19, 55.46, 127.02, 127.22, 131.36, 131.49, 141.76,
152.75, 171.74. Anal. Calcd for C17H35N6O5Cl3: C, 40.04; H, 6.93;
N, 16.49. Found: C, 40.22; H, 7.01; N, 16.36.
1-Cyanomethyl-4,7,10-tris(tert-butylcarbonyl)-1,4,7,10-tet-
raazacyclododecane, 24. Bromoacetonitrile (2.0 g, 16.9 mmol) was
reacted with 3Boc-cyclen (4.2 g, 8.9 mmol) in CH3CN (30 mL) in
the presence of Na2CO3 (1.2 g, 11.3 mmol) at 80 °C under an argon
atmosphere for 2 days. After insoluble inorganic salts were removed,
the filtrate was concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (hexane/AcOEt)
to afford 24 as a colorless amorphous solid (3.6 g, 87%). IR
Potentiometric pH Titration. The preparation of the test
solutions and the calibration method of the electrode system
(Potentiometric Automatic Titrator AT-400 and Auto Piston Buret
APB-410 (Kyoto Electronics Manufacturing, Co. Ltd.) with Orion
Research Ross Combination pH Electrode 8102BN) were described
earlier.9-11,13-17 The theoretical pH values to pH1 and pH2 are
calculated to be pH1′ ) 2.481 and pH2′ ) 11.447, using Kw-
(KBr): 2231, 1678, 1458, 1363, 1252, 1171 cm-1 1H NMR
.
(CDCl3): δ 1.44 (18H, s, C(CH3)3), 1.47 (9H, s, C(CH3)3), 2.78-
2.87 (4H, br), 3.28-3.53 (12H, m), 3.29-3.33 (2H, br). 13C NMR
(CDCl3): δ 28.52, 28.77, 46.50, 47.40, 49.93, 50.17, 53.98, 54.49,
79.62, 79.94, 80.25, 114.55, 155.15, 155.96, 156.12.
1-(2-Acetylamino)ethyl-1,4,7,10-tetraazacyclododecane Tet-
rahydrochloric Acid Salt, 11‚4HCl‚H2O. A mixture of 24 (3.6
g, 7.8 mmol), Raney nickel (Aldrich (Raney 2800 Nickel), 50%
(aH+aOH ) ) 10-14.00, Kw′([H+][OH-]) ) 10-13.79, and fH ) 0.825.
-
+
+
The correct pH values (pH ) -log aH ) can be obtained using the
(16) Shionoya, M.; Ikeda, T.; Kimura, E.; Shiro, M. J. Am. Chem. Soc.
1994, 116, 3848-3859
(17) Aoki, S.; Kawatani, H.; Goto, T.; Kimura, E.; Shiro, M. J. Am. Chem.
Soc. 2001, 123, 1123-1132.
3242 Inorganic Chemistry, Vol. 41, No. 12, 2002

Zinc(II)-Carboxamide Interactions
Scheme 2
following equations: a ) (pH2′ - pH1′)/(pH2 - pH1); b ) pH2′ -
a × pH2; pH ) a × (pH-meter reading) + b. The calibration method
provides an experimental confidence limit of (0.01 pH unit. For
the potentiometric pH titration, the experiment was carried out (0.1
N aq NaOH was used as a base) with I ) 0.10 (NaNO3 or NaClO4),
and at least two independent titrations were performed. Protonation
constants (Kn′ ) [HnL]/[Hn-1L][H+]) of 6-11, the deprotonation
constants (pKa′ ) log([ZnL]/ [ZnL-OH- (ZnH-1L)][H+])) of zinc-
(II) complexes (17-19, 21, 23, 27, and 28), the metal complexation
constants (K(ZnL) ) [ZnL]/[Zn2+][L]), and the succinimide anion
(A-) complexation constants (K(ZnL-A-) ) [ZnL-A-]/[ZnL][A-])
were determined by means of the pH-titration program BEST.18
+
The mixed constants (Kn ) [HnL]/[Hn-1L]aH and pKa) were
calculated from Kn′ and pKa′ with [H+] ) aH /fH . The speciation
distribution values (%) against pH ()-log[H+]; 0.084) were
obtained using the program SPE.18
+
+
Crystallographic Study of 19a(ClO4)‚H2O. A yellow prism of
19a(ClO4)‚H2O (C16H27N6O8ClZn, Mr ) 532.26), having ap-
proximate dimensions 0.30 × 0.10 × 0.08 mm3, was mounted in
a glass capillary. All measurements were made on a Rigaku RAXIS
IV imaging plate area detector with graphite monochromated Mo
KR radiation (µ ) 13.20 cm-1) at -70 ( 1 °C. Indexing was
performed from two oscillations which were exposed for 1 min. A
total of 55 images, corresponding to 220° oscillation angles, were
collected with two different goniometer settings. Exposure time was
0.20 min/°. The camera radius was 127.40 nm. Readout was
performed in the 100 µm pixel mode. The structure was solved by
direct methods (SIR 97) and expanded by means of Fourier
techniques (DIRDIF 94). All calculations were performed with the
teXsan crystallographic software package developed by the Mo-
lecular Structure Corp. (1985, 1992).
Scheme 3
Scheme 4
Kinetics for 4-Nitrophenyl Acetate Hydrolysis by 16 in
Aqueous Solution. The 4-nitrophenyl acetate (NA) hydrolysis was
measured by an initial slope method (following the increase in 400
nm absorption of released 4-nitrophenolate) in 10% (v/v) CH3CN
aqueous solution at 25.0 ( 0.5 °C. Buffer solutions containing 20
mM Good’s buffers (MOPS, pH 7.1; HEPES, pH 7.5; EPPS, pH
7.9; TAPS, pH 8.5) were used, and the ionic strength was adjusted
to 0.10 with NaClO4. For the initial rate determination, the following
typical procedure was employed: After NA (0.5 mM) and a zinc-
(II) complex (0.5 mM) were mixed in the buffer solution, the UV
absorption increase was immediately recorded until ∼1% formation
of 4-nitrophenolate, where log ꢀ values for 4-nitrophenolate were
3.97 (pH 7.1), 4.12 (pH 7.5), 4.22 (pH 7.9), and 4.25 (pH 8.5) at
400 nm. The first-order rate constant kobsd (s-1) was calculated from
the decay slope ([4-nitrophenolate]/[NA]). The value of kobsd/[total
form a six-membered chelate with coordination of the
carbonyl oxygen or amidate N-. The second type of ligands,
11 and 12, may yield five-membered chelates by coordination
of the amidate N-, or seven-membered chelates with the
carbonyl oxygen.
Ligands 6-8 and their zinc(II) complexes 17-19 were
prepared from 1,4,7-tris(tert-butyloxycarbonyl)cyclen 13
(3Boc-cyclen),15 as shown in Scheme 2. N,N-Diethylcar-
bamoylmethylcyclen, 9, was synthesized via 20 by treating
cyclen with N,N-diethylchloroacetamide in EtOH (Scheme
3), and its Zn2+ complex 21 was isolated as a perchlorate
salt. A similar method was used to synthesize 2-(carbamoyl)-
ethylcyclen, 10, and the corresponding Zn2+ complex 23
(Scheme 4). Acylaminoethylcyclens 11 and 12 were syn-
thesized as shown in Scheme 5. The zinc(II) complex 27
with 11 was isolated as a perchlorate salt, while 28 with 12
was prepared in situ. For details, see the Experimental
Section.
Zn2+ complex] gave the second-order rate constant k′NA (M-1 s-1
)
for NA hydrolysis. The second-order rate constant kNA was
determined from the maximum k′NA values.
Results and Discussion
Design and Synthesis of New Ligands 6-12 and Their
Zinc(II) Complexes. Basically, two kinds of amide pendants
were designed; one was a carboxyamide, type 6-10, and
the other an acylaminoethyl type 11 and 12. The ligands 6
and 7 could produce five-membered chelates by coordination
of the carbonyl oxygen or amidate N-. The ligand 9 has no
dissociable amide hydrogen and could form a five-membered
chelate only with the carbonyl oxygen. The ligand 10 could
Protonation and Zinc(II) Complexation Constants for
6-12. The protonation constants (Kn) of new ligands 6-12
(all represented by L) were determined by potentiometric
pH titrations (1.0 mM) with 0.10 M NaOH solution contain-
ing I ) 0.10 (NaClO4 or NaNO3) at 25.0 °C (for a typical
pH titration curve for 1 mM 8, see the Supporting Informa-
tion). The titration data were analyzed for equilibria 1, where
(18) Martell, A. E.; Motekaitis, R. J. Determination and Use of Stability
Constants, 2nd ed; VCH: New York, 1992.
aH is the activity of H+. The log K1 and K2 values are
+
Inorganic Chemistry, Vol. 41, No. 12, 2002 3243

Kimura et al.
Table 1. Comparison of Ligand Protonation Constants (Kn),a,b Zinc(II)
Complexation Constants (K(ZnL)),c and Deprotonation Constants (pKa),d
and Apparent Complexation Constants Kapp (M-1) at pH 7.0e at 25 °C
with I ) 0.10 (NaNO3 or NaClO4)
Scheme 5
N-methyl-
cyclenb,f
6b,g
7b,h
8b,h
log K1
10.7
9.7
15.1
7.68
8.6
10.57
9.31
14.4
8.59
9.8
10.42
9.22
14.2
7.92
10.0
10.62
9.22
14.0
7.01
9.6
log K2
log K (ZnL)c
d
pKa
e
log Kapp
9b,g
10b,h
11b,h
12b,g
log K1
11.10
9.63
15.5
9.92
8.9
11.00
9.09
14.5
8.19
8.4
10.67
8.82
13.4
7.64
8.2
10.88
8.67
13.3
7.48
8.0
log K2
log K (ZnL)c
d
pKa
e
separated from log K3 and K4 values, the trend being the
same as those of N-methylcyclen (log K1 ) 10.7, log K2 )
9.7, log K3 < 2, log K4 < 2).16 Deprotonation of the amide
hydrogens was not recognized for the free ligands 6-12.
log Kapp
a Kn ) [HnL]/[Hn-1L]aH (M-1). Experimental errors are (0.03. b For
+
all ligands shown, log K3 and log K4 are <2. c K(ZnL) ) [ZnL]/[Zn][L]
(M-1). Experimental errors are (0.1. d Ka ) [ZnL(OH-) (or ZnH-1L)]aH
+
/
e
[ZnL] (M). Kapp ) [ZnL]/[Zn]free[L]free (M-1) at pH 7.0, where [ZnL] )
[ZnL(OH2)] + [ZnL(OH-)] and [L]free)[L] + [HL] + [H2L] + [H3L] +
[H4L]. Experimental errors are ( 0.1. f From ref 16. g I ) 0.10 (NaClO4).
h I ) 0.10 (NaNO3).
Hn-1
L
(n-1)+ + H+ a HnLn+
Kn ) [HnL]/[Hn-1L]aH
+
(1)
nitrogens of cyclen and two carbonyl oxygens of amides.19
The 1:1 zinc(II) complexation equilibria were determined
by the potentiometric pH titration of protonated ligands 6-12
(1.0 mM) in the presence of an equimolar amount of zinc-
(II) ion with I ) 0.10 (NaClO4 or NaNO3) at 25.0 °C (for a
typical pH titration curve for 1 mM 8 + 1 mM Zn2+, see
the Supporting Information). The titration curves revealed
two distinct equilibria: the first is for the ZnL complex
formation (see eq 2), and the second is for monodeprotona-
tion from ZnL (see eq 3). The first equilibration was slow
and took more than 2 h for each titration point, while the
following deprotonation process was fast within 5 min at
each titration point. For the monodeprotonated species, either
a hydroxide-bound structure ZnL-OH- or an amidate-N--
bound structure ZnH-1L may be considered.
Kapp ) [ZnL]/[Zn]free[L]free (M-1)
(4)
(5)
[ZnL]free ) [ZnL(OH2)] + [ZnL(OH-)]
[L]free ) [L] + [HL] + [H2L] + [H3L] + [H4L] (6)
Zn2+ + L a ZnL
K(ZnL) ) [ZnL]/[L][Zn2+] (2)
ZnL(H2O) a ZnL(OH-) (or ZnH-1L) + H+
Amide Oxygen-Coordinating ZnL (17-19) and Amidate
N--Coordinating ZnH-1L (17a-19a) with Carbamoyl-
Pendant Cyclens (L ) 6, 7, and 8). The monodeprotonation
occurred in 17, 18, and 19 with pKa of 8.59, 7.92, and 7.01,
respectively. The monodeprotonated complexes 17a and 19a
were isolated as perchlorate salts. The deprotonated species
were all assigned to the amidate N--bound zinc(II) com-
plexes (ZnH-1L), 17a, 18a, and 19a, rather than the Zn2+-
OH- complexes, 17b, 18b, and 19b (Scheme 6). The first
evidence for the amidate N--coordinating structures is the
remarkable lowering of the amide stretching frequency νCd
O: from 1686 cm-1 (KBr pellet) or 1645 cm-1 (in D2O) for
17 to 1576 cm-1 (in KBr pellet) or 1576 cm-1 (in D2O) for
17a, from 1640 cm-1 (in D2O) for 18 to 1570 cm-1 (in D2O
solution) for 18a, and from 1663 cm-1 (KBr pellet) for 19
to 1572 cm-1 (in KBr pellet) for 19a. An amidate N--bound
zinc(II) in 31 showed a similar νCdO at 1570 cm-1 (in KBr
Ka ) [Zn(OH-) (or ZnH-1L)]aH+/[ZnL(H O)] (3)
2
All of the obtained values for ligands 6-12 are sum-
marized in Table 1, along with a reference N-methylcyclen
ligand. For practical comparison, apparent stability constants
Kapp (defined by eqs 4-6) at pH 7.0 are also included. The
log Kapp values for the carbamoyl-pendant cyclens 6-9 are
9.8, 10.0, 9.6, and 8.9, respectively, which are larger than
the values of 8.2 and 8.0 for the acetylamino-pendant cyclens
11 and 12, or 8.6 for N-methylcyclen. The comparison
implies that the higher stability of the former type of ZnL
complexes 6-9 is due to the coordination of the pendant
amide oxygen to form the stable five-membered chelates. A
six-membered chelation by an analogous carbamoyl pendant
of 10 (to 23) is less likely, and the log Kapp remains to be
8.4. Earlier, the five-membered chelations by pendant
carbamoyls were reported in 29 and 30. In 29, zinc(II) is
7-coordinate with four nitrogens of cyclen and three carbonyl
oxygens of amides.17 In 30, zinc(II) is 6-coordinate with four
(19) Maumela, H.; Hancock, R. D.; Carlton, L.; Reibenspies, J. H.;
Wainwright. J. Am. Chem. Soc. 1995, 117, 6698-6707.
3244 Inorganic Chemistry, Vol. 41, No. 12, 2002

Zinc(II)-Carboxamide Interactions
Scheme 6
Figure 1. ORTEP drawing (50% probability ellipsoids) of 19a(ClO4)‚
H2O. Bond distances (Å): Zn(1)-N(15) 1.974(3), Zn(1)-N(1) 2.178(3),
Zn(1)-N(4) 2.127(2), Zn(1)-N(7) 2.141(3), Zn(1)-N(10) 2.097(3). Bond
angles (deg): N(1)-Zn(1)-N(4) 81.9(1), N(1)-Zn(1)-N(10) 82.7(1),
N(4)-Zn(1)-N(7) 82.3(1), N(7)-Zn(1)-N(10) 83.2(1), N(1)-Zn(1)-
N(15) 85.0(1).
17a or 19a in solution, we have studied hydrolysis of
4-nitrophenyl acetate (NA) by 17 or 19 at pH 7.0-8.5 and
35 °C with I ) 0.1 (NaNO3 or NaClO4) and found no
hydrolysis over 24 h. The lack of the nucleophilic activity
indicates a negligible proportion of the nucleophilic zinc-
(II)-OH- species. Our conclusion is that the reactiVe sites
on the Zn2+-cyclen 17-19 are occupied by the carboxamide
oxygens in acidic pH and carboxamidato nitrogens in basic
pH. Therefore, the nucleophilic zinc(II)-OH- species could
not be generated in acidic to alkaline pH in 17-19 (for the
distribution diagram for 19, see the Supporting Information).
This study provided with the first chemical model of the
amide inhibition of CA, just as we earlier designed 5 as a
model for the sulfonamide inhibition of CA.13
Amide-Coordinating ZnL, 19, and a Hydroxide-Bound
ZnL(OH-), 21b, with L ) N,N-Diethylcarbamoylmeth-
ylcyclen 9. The zinc(II) complex 21 of N,N-diethylcarbam-
oylmethylcyclen 9 has the highest stability constant logK(ZnL)
of 15.5 among the carbamoyl-cyclen complexes, implying
the strongest amide coordination. Despite its absence of
dissociable amide hydrogen, the potentiometric pH titration
of 21 showed monodeprotonation with a high pKa value of
9.92. The IR spectra of 21 and its deprotonated species (pD
11.2) showed the same νCdO (in D2O) at 1617 cm-1. The
13C NMR spectra of 26 (pD 5.9) and its monodeprotonated
species showed the carbonyl carbon signals at δ ) 174 and
172, respectively. In the NA hydrolysis reaction with 21,
the catalysis was found at high pH (CHES buffer, pH 9.3).
Thus, the deprotonated species was assigned to a zinc(II)-
OH- complex, 21a (Scheme 7). The pKa value of 9.92 for
21 a 21a is much higher than that of 7.68 for the
N-methylcyclen complex, 32a a 32b,22 because of the
competitive amide coordination. Moreover, we saw a H-D
exchange for the methylene adjacent to the carbonyl in
pellet) or 1577 cm-1 (in D2O).20 Other evidence came from
the UV absorption change for 19 a 19a. The absorption
maximum λmax ) 302 nm (ꢀ ) 14500) for 19 red-shifted to
354 nm (ꢀ ) 14800) for 19a in acetonitrile. The UV spectral
change with an isosbestic point at 325 nm was seen in the
titration of 19 (0.5 mM) with 1,8-diazabicyclo[5.4.0]-7-
undecene (DBU) (0-0.5 mM) to 19a in acetonitrile. The
13C NMR spectra of 19 and 19a in CD3CN showed the
carbonyl carbon signals at almost the same δ ) 174 ppm,
1
while the H NMR spectra (in CD3CN) showed the amide
hydrogen at δ ) 9.5 for 19 disappeared for 19a.
The final conclusive evidence for the amidate N--bound
structure was obtained by X-ray crystal analysis of 19a.
Figure 1 shows ORTEP drawing of 19a with 50% probability
thermal ellipsoids.21 The zinc(II) ion is coordinated by four
nitrogen atoms (N(1), N(4), N(7), and N(10)) of cyclen, and
amidate anion nitrogen N(15). The Zn-N bond distances
are 2.178(3) Å for Zn-N(1), 2.127(2) Å for Zn-N(4), 2.141-
(3) Å for Zn-N(7), and 2.097(3) Å for Zn-N(10), while
the Zn-N-(15) bond distance is 1.974(3) Å. For comparison,
the reported amidate anion-bound zinc(II) complex 31
showed the Zn-N- bond distance being 2.035(4) Å, which
is shorter than the Zn-N bonds (2.09-2.12 Å, where N are
three secondary amines of macrocyclic ring).20
To examine if the electrostatically equivalent zinc(II)-
OH- species (17b or 19b) is present in equilibration with
(20) Kimura, E.; Koike, T.; Shiota, T.; Iitaka, Y. Inorg. Chem. 1990, 29,
4621-4629.
(21) Crystallographic data of 19a are listed in the Supporting Information.
(22) Koike, T.; Kajitani, S.; Nakamura, I.; Kimura, E.; Shiro, M. J. Am.
Chem. Soc. 1995, 117, 1210-1219.
Inorganic Chemistry, Vol. 41, No. 12, 2002 3245

Kimura et al.
Scheme 7
Scheme 9
Scheme 8
tested to see the hydrolysis of external NA by 27a at pH
7.1-8.5. The second-order rate constant kNA was determined
to be (4.6 ( 0.2) × 10-2 M-1 s-1, a value almost the same
as 4.7 × 10-2 M-1 s-1 reported for 32b.22 We consider that
these Zn2+-cyclen-OH- species are nucleophilic enough
for carboxyesters, but not for carboxamides.
alkaline D2O solution (pD 11.2), implying equilibration with
an enolate Zn2+ complex 21b. In a relevant carbonyl-bound
Zn2+ complex, 33, reported as a model for zinc-containing
class II aldolases (Scheme 8),23 deprotonation occurred with
lower pKa of 8.4 to yield a mixture of an enolate-bound Zn2+
complex 33a and a hydroxide-bound complex 33b (in a ratio
of 23%:77% at 25 °C). In analogy to 21, the H-D exchange
occurred at the methylene adjacent to the carbonyl.
Amide-Noncoordinating ZnL (27 and 28) and Hydrox-
ide-Bound ZnL(OH-) (27a and 28a) with L ) N-
Acetylaminoethylcyclen, 11, and N-Nitrobenzoylamino-
ethylcyclen, 12. The lower K(ZnL) values with the
acetylamino-pendant 11 and 12 than those with the carbam-
oyl-pendant cyclens 6-9 should reflect unfavorable seven-
membered chelations with N-acetylamino pendants (Table
1). The deprotonation constants pKa of their zinc(II) com-
plexes 27 and 28 were 7.64 and 7.48, values similar to 7.68
for the zinc(II)-bound water in 32a.23 The IR spectra showed
constant νCdO (in D2O) at 1624 cm-1 (for 22) and 1636 cm-1
(for 23). These IR changes differed from those found for
the earlier amidate-bound zinc(II) complexes 17a, 18a, and
19a. Little UV spectral changes at varing pH 7-9 disproved
formation of 4-nitrobenzoylamino anion 28b. Accordingly,
the present deprotonated structures were assigned to the
Zn2+-OH- species 27a and 28a (Scheme 9).
If acting as a nucleophile, these Zn2+-OH- species might
attack at the intramolecular amides for hydrolysis. However,
we saw no amide hydrolysis at pD 7.5-8.5 and 60 °C in 72
h, that is, no occurrence of 4-nitrocarboxylate and zinc(II)
complex of aminoethylcyclen, 34.13a Earlier, we found
extremely fast ester hydrolysis of an acetate-pendant by the
intramolecular attack of Zn2+-OH- in 35.22 We then have
Different Succinimide Affinity to Carbamoyl-Pendant
Complex 17 and Acetylamino-Pendant Complex 25. It is
now understood that the higher pKa value (8.59) for 17 a
17a with respect to those for 32 ()7.68) (Scheme 7) or 27
()7.64) (Scheme 9) is due to competitive occupation of a
vacant Zn2+ site by the carbamoyl amide pendant. We then
have studied whether the carbamoyl pendants may be
displaced by an external succinimidate. In Zn2+-cyclen
complex 4a, a succinimide is a strong ligand to yield 1:1
Zn2+-cyclen-succinimidate complex ZnL-A-, 36 (Scheme
10).14 The succinimide binding constants K(A-) ()[ZnL-
A-]/[ZnL][A-] (M-1), where A- is deprotonated imide N-
anion species of succinimide for 17 and 27) were determined
by potentiometric pH titrations of 17 and 27 with the aid of
the pH-titration program BEST.18 The results indeed fit to
the formation of ZnL-A- species 37 and 38. The log K(A-)
value for 17 a 37 was 4.0, which is smaller than the values
of 5.8 for 27 a 38 and 5.6 for 4a a 36.14 It is concluded
that, although carbamoyl groups bind to Zn2+ either with
oxygen or nitrogen, they can be replaced by other stronger
ligands. The similar log K(A-) values for 4a and 27 support
the proposed little coordination of the acetylamino group in
27.
Comparison with Other Metal Ion-Amide Complexes.
(23) Kimura, E.; Gotoh, T.; Koike, T.; Shiro, M. J. Am. Chem. Soc. 1999,
121, 1267-1274.
It is instructive to compare the present pH-dependent Zn2+-
3246 Inorganic Chemistry, Vol. 41, No. 12, 2002

Zinc(II)-Carboxamide Interactions
Scheme 10
Scheme 11
amide binding manners with previous metal models including
well studied Co3+-tetraamine N4 systems.24-27 With respect
to the Zn2+-amide hydrogens, higher acidities (i.e., lower
pKa values) for the Co3+-bound amide hydrogens are
anticipated. Surprisingly, however, the Co3+-coordinated
amide protons are much less acidic with pKa of 11.4 for
[Co3+(en)2(glyNH2)]3+ (39a)25b and 10.1 (by kinetic measure-
ment)25b or 9.4 (by potentiometric titration)24 for [Co3+(trien)-
(glyglyOCH3)]3+ (39b), with respect to the pKa value of 8.6
for the comparable Zn2+-amide complex (17) (Scheme 11).
The solvent hydroxide at pH below the pKa values (namely
∼8 < pH < ∼10) acted as an external nucleophile to attack
at the Co3+-chelated amide carbons in 39 at 25 °C to yield
the hydrolysis products 40. However, a following study25c
indicated that hydroxide seemed to simultaneously attack at
Co3+ in Co3+(en)2 complexes, as monodeprotonated Co3+-
hydroxide complex cis-[Co3+(en)2(glyNR1R2)(OH-)] (43)
was identified from pH 9∼14 solution of cis-[Co3+(en)2-
(glyNR1R2)Br] (where R1 ) R2 ) H, R1 ) H and R2 ) CH3,
R1 ) R2 ) CH3). The glycine amide hydrolysis by the Co3+-
OH- in 43 was 10 times more efficient than by the external
OH- in 39. Thus, the Zn2+-OH species in our models did
not seem to be as nucleophilic as Co3+-OH- for the
intramolecular carboxamide hydrolysis. It is interesting to
note that the same Co3+-bound glycine amide hydrogen in
[Co3+(NH3)4(glyNH2)] (41) was extremely acidic with a pKa
value of ∼0.4 to yield [Co3+(NH3)4(glyNH-)] (42), which
was inert to the amide hydrolysis.25a
very electrophilic Pd2+-bound carboxamides or the internal
attack of Pd2+-OH2 at the proximate amides was proposed
for the peptide hydrolysis mechanism in very acidic pH <
2. At pH > 2, the amide hydrogens were deprotonated to
bind to Pd2+. As a consequence, peptides did not undergo
amide hydrolysis at neutral pH.
In a Gd3+ complex of tetraamide derivatives of cyclen (44),
the deprotonation first yielded a hydroxide complex, 45, with
pKa of 7.90 at 25 °C and then the amide NH deprotonated
complexes with pKa of 11.02 and 11.89.29 Some of these
lanthanide complexes promote RNA cleavage, apparently by
the catalytically active lanthanide-bound hydroxide species
such as 45.30
Summary and Conclusions
Two different pH-dependent Zn2+-amide interactions in
the newly designed zinc(II) complexes of carboxamide-
pendant cyclens were revealed. The zinc(II)-cyclen com-
plexes with carbamoyl-pendants 17, 18, and 19 possessed
amide-coordinating structures at acidic to neutral pH. Mon-
odeprotonation occurred with pKa values of 8.59 for 17, 7.92
for 18, and 7.01 for 19 at 25 °C, and the resulting products
were all the amidate N--bound zinc(II) complexes 17a, 18a,
and 19a, respectively. By the preferred amidate N- formation
from 17, 18, and 19, nucleophilic Zn2+-OH- species were
In a recent work of peptide hydrolysis at very acidic pH
< 2 by Pd2+ complexes,28 the external attack of H2O at the
(24) Collman, J. P.; Kimura, E. J. Am. Chem. Soc. 1967, 89, 6096-
6103.
(25) (a) Buckingham, D. A.; Foster, D. M.; Sargeson, A. M. J. Am. Chem.
Soc. 1969, 91, 3451-3456. (b) Buckingham, D. A.; Davis C. E.;
Foster, D. M.; Sargeson, A. M. J. Am. Chem. Soc. 1970, 92, 5571-
5579. (c) Buckingham, D. A.; Foster, D. M.; Sargeson, A. M. J. Am.
Chem. Soc. 1970, 92, 6151-6158.
(26) For review: Sutton, P. A.; Buckingham, D. A. Acc. Chem. Res. 1987,
20, 357-364.
(27) Chin, J. Acc. Chem. Res. 1991, 24, 145-152.
(28) (a) Chen, X.; Zhu, L.; Yan, M.; You, X.; Kostic, N. M. J. Chem.
Soc., Dalton Trans. 1996, 2653-2658. (b) Parac, T. N.; Kostic, N.
M. J. Am. Chem. Soc. 1996, 118, 51-58. (c) Zhu, L.; Bakhiar, R.;
Kostic, N. M. J. Biol. Inorg. Chem. 1998, 3, 381-391. (d) Parac, T.
N.; Ullmann, G. M.; Kostic, N. M. J. Am. Chem. Soc. 1999, 121,
3127-3135. (e) Kaminskaia, N. V.; Johnson, T. W.; Kostic, N. M. J.
Am. Chem. Soc. 1999, 121, 8663-8664.
(29) Aime, S.; Barge, A.; Bruce, I. I.; Botta, M.; Boward, J. A. K.; Moloney,
J. M.; Parker, D.; de Sausa, A. S.; Woods, M. J. Am. Chem. Soc.
1999, 121, 5762-2771.
(30) (a) Chin, K. O. A.; Morrow, J. R. Inorg. Chem. 1994, 33, 5036-
5041. (b) Amin, S.; Voss, D. A., Jr.; Harrocks, W. DeW.; Lake, C.
H.; Churchill, M. R.; Morrow, J. R. Inorg. Chem. 1995, 34, 3294-
3300.
Inorganic Chemistry, Vol. 41, No. 12, 2002 3247

Kimura et al.
not generated. It is thus chemically feasible that carboxamides
strongly bind to zinc(II) both in acidic pH and basic pH as
inhibitors in some Zn2+ enzymes such as CA. In another
type of Zn2+-cyclen complexes with acetylamino pendants
11 and 12, the carboxamides did not interact with Zn2+,
because of the unfavorable seven-membered chelation. In
alkaline pH, hydroxide ion preferentially bound to Zn2+ to
yield nucleophilic zinc(II)-OH- complexes 27a and 28a
with pKa values of 7.64 and 7.48, respectively. Hence, the
Zn2+-amidate N- species, 27b and 28b, were not produced.
This situation may somewhat mimic the carboxamide
substrate interaction to the active center of CPA. The Zn2+-
OH- species in 27a was demonstrated to hydrolyze 4-ni-
trophenyl acetate, although it failed to hydrolyze the in-
tramolecular carboxamides, as known to CPA. In our model,
Zn2+-OH- species alone was not sufficient to hydrolyze a
proximate carboxamide.
Acknowledgment. We are grateful to the Ministry of
Education, Science and Culture in Japan for financial support
through a Grant-in-Aid (12470479 for E.K and 12771355
and 13557195 for S.A.). S.A. is also thankful to Asahi Glass
Foundation and the Research Foundation for Pharmaceutical
Sciences. T.G. thanks the Japan Society for the Promotion
of Science for Graduate Scholarship.
Supporting Information Available: Titration curves for the
1.0 mM 8 and the 1.0 mM 8/1.0 mM Zn2+ mixture and specia-
tion diagram for the 1.0 mM 8/1.0 mM Zn2+ mixture as a func-
tion of pH, Tables of crystallographic parameters, atomic coordi-
nates, equivalent isotropic temperature factors, anisotropic temp-
erature factors, bond distances, bond angles, and torsion angles
in CIF format of the X-ray structure report for 19a(ClO4)2‚H2O.
This material is available free of charge via the Internet at
http://pubs.acs.org.
IC020087K
3248 Inorganic Chemistry, Vol. 41, No. 12, 2002