Unsymmetrical dizinc complexes as models for the active sites of phosphohydrolases

Article abstract of DOI:10.1039/b925563j

The unsymmetrical dinucleating ligand 2-(N-isopropyl-N-((2-pyridyl)methyl) aminomethyl)-6-(N-(carboxylmethyl)-N-((2-pyridyl)methyl)aminomethyl) -4-methylphenol (IPCPMP or L) has been synthesized to model the active site environment of dinuclear metallohydrolases. It has been isolated as the hexafluorophosphate salt H₄IPCPMP(PF₆)₂· 2H₂O (H₄L), which has been structurally characterized, and has been used to form two different Zn(ii) complexes, [{Zn₂(IPCPMP) (OAc)}₂][PF₆]₂ (2) and [{Zn₂(IPCPMP) (Piv)}₂][PF₆]₂ (3) (OAc = acetate; Piv = pivalate). The crystal structures of 2 and 3 show that they consist of tetranuclear complexes with very similar structures. Infrared spectroscopy and mass spectrometry indicate that the tetranuclear complexes dissociate into dinuclear complexes in solution. Potentiometric studies of the Zn(ii):IPCPMP system in aqueous solution reveal that a mononuclear complex is surprisingly stable at low pH, even at a 2:1 Zn(ii):L ratio, but a dinuclear complex dominates at high pH and transforms into a dihydroxido complex by a cooperative deprotonation of two, probably terminally coordinated, water molecules. A kinetic investigation indicates that one of these hydroxides is the active nucleophile in the hydrolysis of bis(2,4-dinitrophenyl)phosphate (BDNPP) enhanced by complex 2, and mechanistic proposals are presented for this reaction as well as the previously reported transesterification of 2-hydroxypropyl p-nitrophenyl phosphate (HPNP) promoted by Zn(ii) complexes of IPCPMP.

Full text of DOI:10.1039/b925563j

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www.rsc.org/dalton | Dalton Transactions
Unsymmetrical dizinc complexes as models for the active sites of
phosphohydrolases†
Martin Jarenmark,a Edit Csapo´,b Jyoti Singh,a Simone Wo¨ckel,c Etelka Farkas,b Franc Meyer,c
Matti Haukkad and Ebbe Nordlander*a
Received 4th December 2009, Accepted 11th May 2010
First published as an Advance Article on the web 4th August 2010
DOI: 10.1039/b925563j
The unsymmetrical dinucleating ligand 2-(N-isopropyl-N-((2-pyridyl)methyl)aminomethyl)-6-(N-
(carboxylmethyl)-N-((2-pyridyl)methyl)aminomethyl)-4-methylphenol (IPCPMP or L) has been
synthesized to model the active site environment of dinuclear metallohydrolases. It has been isolated as
the hexafluorophosphate salt H4IPCPMP(PF6)2·2H2O (H4L), which has been structurally
characterized, and has been used to form two different Zn(II) complexes, [{Zn2(IPCPMP)(OAc)}2]-
[PF6]2 (2) and [{Zn2(IPCPMP)(Piv)}2][PF6]2 (3) (OAc = acetate; Piv = pivalate). The crystal structures
of 2 and 3 show that they consist of tetranuclear complexes with very similar structures. Infrared
spectroscopy and mass spectrometry indicate that the tetranuclear complexes dissociate into dinuclear
complexes in solution. Potentiometric studies of the Zn(II) : IPCPMP system in aqueous solution reveal
that a mononuclear complex is surprisingly stable at low pH, even at a 2 : 1 Zn(II) : L ratio, but a
dinuclear complex dominates at high pH and transforms into a dihydroxido complex by a cooperative
deprotonation of two, probably terminally coordinated, water molecules. A kinetic investigation
indicates that one of these hydroxides is the active nucleophile in the hydrolysis of
bis(2,4-dinitrophenyl)phosphate (BDNPP) enhanced by complex 2, and mechanistic proposals are
presented for this reaction as well as the previously reported transesterification of 2-hydroxypropyl
p-nitrophenyl phosphate (HPNP) promoted by Zn(II) complexes of IPCPMP.
one of the most commonly used to catalyse hydrolysis reactions
Introduction
in nature due to its high Lewis acidity, flexible coordination
sphere and lack of associated redox chemistry.6,8 Examples of
multinuclear zinc enzymes that catalyse phosphoester cleavage
are phosphotriesterase,9 alkaline phosphatase,10 nuclease P111 and
phospholipase C.12 A schematic drawing of the structures of the
active sites of the two latter enzymes is shown in Fig. 1. To help
in the elucidation of the mechanisms involved in phosphoester
cleavage, small synthetic coordination complexes have been used
as structural and functional models for the active sites of zinc-
dependent enzymes.1,13–18 In addition to revealing details of the
function of dinuclear sites, these model complexes may also be
The storage and processing of genetic information is of utmost
importance for all living organisms. Nature has chosen phospho-
diesters for this task due to their resistance to hydrolytic cleavage,
with estimated half lives of tens to hundred of thousand to hundred
of billions of years for DNA and about 110 years for RNA.1
To regulate and express this genetic information, organisms need
efficient enzymes for cleavage and formation of phosphodiester
bonds. In such enzymes, metals are frequently necessary as
cofactors.2–4 Several enzymes have evolved to make use of two
and even three metal ions in a cooperative manner to maximize
efficiency.4–6 Among the “biological” metals,7 zinc stands out as
aInorganic Chemistry Research Group, Chemical Physics, Center for Chem-
istry and Chemical Engineering, Lund University, Box 124, SE-221 00, Lund,
Sweden. E-mail: Ebbe.Nordlander@chemphys.lu.se
bDepartment of Inorganic and Analytical Chemistry, University of Debrecen,
P.O.B. 21, H-4010, Debrecen, Hungary
cInstitute for Inorganic Chemistry, Georg-August-Universita¨t Go¨ttingen,
Tammannstrasse 4, D-37077, Go¨ttingen, Germany
dDepartment of Chemistry, University of Joensuu, Box 111, FI-80101,
Joensuu, Finland
† Electronic supplementary information (ESI) available: An overlay plot of
the molecular structures of complexes 2 and 3 (Fig. S1). Titration curves
including fitted curves for the titration of H4IPCPMP(PF6)2 (H4L) and
mixture of ZnCl2 and L to ratios of 1.5 : 1 and 2 : 1 (Fig. S2). Spectra
from ESI-MS+ of mixtures of ZnCl2 and L (2 : 1) with calculated isotope
patterns for the species ZnL, Zn2L, and Zn2L(OH), and an ESI-MS
spectrum of complex 2 (Fig. S3 and S4). Kinetic traces for the HPNP
transesterification and BDNPP hydrolysis at various pH. Table of hydrogen
bonds in the crystal structure of H4IPCPMP (Table S1). CCDC reference
numbers 659751, 667079 and 755949. For ESI and crystallographic data
in CIF or other electronic format see DOI: 10.1039/b925563j
Fig. 1 Schematic drawing of the active site structures of phospholipase
C and nuclease P1. The only difference in donors between the two active
sites is the substitution of the glutamate for an aspartate in nuclease P1.
The metal–metal distances are given for the phospholipase C active site.
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designed to act as artificial restriction enzymes capable of selective
cleavage of phosphodiester bonds in DNA or RNA for use in
molecular biology.14,15 Further potential uses include their use
as pharmaceuticals directed towards genetic and viral diseases14
and for detoxification of phosphotriesters and derivatives of these
found in some pesticides and chemical warfare reagents.15
Despite great efforts, consensus on the exact mechanisms for
the various polynuclear metallohydrolases and their models has
not yet been reached. The exact roles of the metal ions regarding
binding of the substrate, stabilisation of the transition state, and
identity of the nucleophile are not perfectly understood although
much progress has been made.6,19,20 The three main proposals for
the nucleophilic attack on the phosphorus centre by dinuclear
Zn(II) enzymes and biomimetic complexes are shown in Fig. 2,
along with the two most commonly discussed suggestions for the
binding of the substrate.
Fig. 3 The four related dinucleating ligands of concern in our studies.
R = 2-pyridyl for IPCPMP (a) and BCPMP (b). R = 1-methylimidazolyl
for ICIMP (a) and BCIMP (b).
Scheme 1 Transesterification of HPNP (top) and hydrolysis of BDNPP
(bottom)
Fig. 2 Mechanistic proposals for the nucleophilic attack on the phos-
phorus centre in phosphodiester cleavage by dinuclear metalloenzymes
and model compounds (active nucleophiles identified by numerals 1–3).19
All interactions with protein residues have been omitted.
speciation of complex 2, and a detailed kinetic investigation
of the transesterification of HPNP and the hydrolysis of the
organophosphoester 2,4-bis-dinitrophenol-phosphate (BDNPP,
Scheme 1) by complex 2. Parts of these results have been published
in an earlier communication.22
We have developed a number of symmetric and unsym-
metric dinucleating ligands (Fig. 3) that include carboxylate
groups, a type of donor moiety that is frequently found in
the active sites of metallohydrolases. The unsymmetric lig-
ands incorporate a non-coordinating group on one side that
(potentially) leaves a vacant coordination site on one of the
metals. In enzyme active sites, vacant or solvent-coordinated
sites are necessary for facile coordination of the substrate and
for activation of solvent-derived nucleophiles.21 Reaction of
Zn(X)2·2H2O (X = acetate or pivalate) with the dinucleating lig-
ands BCPMP and IPCPMP (cf . Fig. 3 for structures of the ligands)
leads to the formation of Na3[Zn2(BCPMP)(OAc)2]2PF6 ·6H2O
(1), [{Zn2(IPCPMP)(OAc)}2][PF6]2 (2) and [{Zn2(IPCPMP)-
(O2CCMe3)}2][PF6]2 (3).22 The solid state structures of 2 and 3
contain tetranuclear “dimer of dimer” complexes, as has been
observed for analogous complexes of the ICIMP‡ ligand.23–25
In situ synthesized zinc complexes of IPCPMP also showed
higher activity towards transesterification of 2-hydroxypropyl p-
nitrophenyl phosphate (HPNP, Scheme 1) than 1 did.22 Here we
present the complete procedures for the syntheses of the ligand
IPCPMP and complexes 1–3, an investigation of the solution
Results and discussion
Synthesis and structure
The ligand IPCPMP (L) was assembled via modifications of a
convergent synthetic protocol (Scheme 2).22,25,26 The asymmetry
of L is introduced by the selective oxidation of one of the benzyl
‡ Ligand abbreviations used in this paper: BCPMP
{N-(carboxymethyl)-N-(2-pyridylmethyl)amine}methyl]-4-methylphenol,
IPCPMP
2-[N-isopropyl-N-{(2-pyridyl)methyl}aminomethyl]-6-[N-
(carboxylmethyl)-N -{(2-pyridyl)methyl}aminomethyl]-4-methylphenol
(also denoted L), BCIMP
2,6-bis[N-{N-(carboxymethyl)-N-(1-
2-[N-
=
2,6-bis[N-
=
=
methylimidazolyl)amine}methyl]-4-methylphenol, ICIMP
=
isopropyl-N-{(1-methylimidazolyl-pyridyl)methyl}aminomethyl]-6 - [N-
(carboxylmethyl) - N - {(1 - methylimidazolyl)methyl}aminomethyl] - 4-
methylphenol
Scheme 2 Synthetic pathway for the ligand IPCPMP
8184 | Dalton Trans., 2010, 39, 8183–8194
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Table 1 Crystallographic data for H4IPCPMP(PF6)2. 1.25H2O (H4L) and
[{Zn2IPCPMP(OAc)}2][PF6]2 (2)
˚
Table 2 Bond distances (A) of the ligand framework in
H4IPCPMP(PF6)2, [{Zn2(IPCPMP)(OAc)}2](PF6)2 (2) and [{Zn2-
(IPCPMP)(Piv)}2](PF6)2 (3)
H4L
2
H4L
2
3
Empirical formula
Fw
C104H146F48N16O17P8
3052.13
120(2)
0.71073
Triclinic
¯
P1
14.2670(9)
14.3112(10)
18.2423(12)
67.849(4)
87.856(3)
69.826(3)
3219.7(4)
1
1.574
0.246
48041
11283
C56H66F12N8O10P2Zn4
1562.59
120(2)
0.71073
Monoclinic
P21/c
12.5983(2)
19.4790(3)
12.5532(4)
90
97.187(2)
90
3056.38(12)
2
1.698
1.704
53314
6951
1.056
0.0375
0.0349
O1–C8
O2–C8
O3–C16
O4–C27
O5–C27
N1–C1
N1–C5
N2–C6
N2–C7
N2–C9
N3–C17
N3–C18
N3–C21
N4–C22
N4–C26
C1–C2
C2–C3
C3–C4
C4–C5
C5–C6
1.232(7)
1.310(7)
1.373(7)
—
1.262(3)
1.250(3)
1.359(3)
1.259(3)
1.259(3)
1.350(3)
1.343(3)
1.471(3)
1.476(3)
1.486(3)
1.503(3)
1.518(3)
1.484(3)
1.336(3)
1.350(3)
1.383(4)
1.382(4)
1.382(4)
1.392(3)
1.510(3)
1.520(3)
1.502(3)
1.398(3)
1.410(3)
1.392(3)
1.509(3)
1.388(4)
1.400(3)
1.403(3)
1.509(3)
1.520(4)
1.515(4)
1.504(4)
1.395(4)
1.376(5)
1.389(5)
1.378(4)
1.503(4)
1.265(5)
1.247(5)
1.355(5)
1.250(5)
1.265(5)
1.336(6)
1.349(5)
1.471(5)
1.468(5)
1.489(5)
1.504(5)
1.508(5)
1.486(5)
1.342(5)
1.346(5)
1.383(6)
1.387(7)
1.378(8)
1.386(6)
1.499(7)
1.521(5)
1.503(5)
1.390(6)
1.398(5)
1.397(6)
1.521(6)
1.386(6)
1.391(6)
1.402(5)
1.503(5)
1.525(5)
1.529(5)
1.508(5)
1.391(6)
1.377(6)
1.381(6)
1.382(6)
1.524(6)
1.469(10)
1.479(12)
1.482(11)
1.501(12)
1.565(12)
1.626(12)
1.654(11)
T/K
˚
l/A
Crystal system
Space group
—
˚
1.335(8)
1.359(8)
1.474(8)
1.451(7)
1.468(8)
1.518(7)
1.553(7)
1.478(8)
1.327(7)
1.334(8)
1.352(10)
1.382(10)
1.368(9)
1.362(8)
1.479(8)
1.477(8)
1.489(8)
1.385(8)
1.405(8)
1.381(8)
1.502(8)
1.394(8)
1.380(8)
1.386(8)
1.509(8)
1.512(9)
1.497(9)
1.510(8)
1.381(9)
1.367(9)
1.375(9)
1.378(9)
a/A
˚
b/A
˚
c/A
a/◦
b/◦
g /◦
V/A
Z
3
˚
rc/Mg m-3
m(Mo-Ka)/mm-1
No. reflns.
Unique reflns.
GOOF (F2)
Rint
1.028
0.1336
0.0863
0.1691
a
R1 (I ≥ 2s)
C7–C8
b
wR2 (I ≥ 2s)
0.0897
C9–C10
C10–C11
C10–C16
C11–C12
C12–C13
C12–C14
C14–C15
C15–C16
C15–C17
C18–C19
C18–C20
C21–C22
C22–C23
C23–C24
C24–C25
C25–C26
C27–C28
C28–C31A
C28–C30B
C28–C29A
C28–C29B
C28–C31B
C28–C30A
C33–C34
ꢀ
ꢀ
ꢀ
ꢀ
a R1 = ꢀFo| - |Fcꢀ/ |Fo|. b wR2 = [ [w(Fo - Fc2)2]/ [w(Fo2)2]]1/2
.
2
alcohol moieties using MnO2. In previous syntheses,25,26 the side
arms were attached sequentially by chlorination of the alcohol
group and nucleophilic substitution, followed by reduction of the
aldehyde and a second chlorination and substitution to afford
the unsymmetric ligand. We have modified this procedure by
introducing the first side arm by reductive amination of the alde-
hyde group using sodium tris(acetoxy)borohydride.27 This saves
two steps although the overall yield is not improved significantly.
The yields for the attachment of the ester-containing sidearm
are reduced due to the difficulty of separating the product from
starting materials. In the final step, the ester group is hydrolysed in
aqueous alkali solution. The unsymmetric ligand was isolated by
acidification and precipitation as H4IPCPMP(PF6)2·H2O (H4L).
X-Ray quality crystals were grown from a mixture of methanol
and water. Crystallographic data are shown in Table 1 and selected
distances for the ligand framework are shown in Table 2 (cf. Table
S1 in the ESI for hydrogen bonding distances†). The structure
of H4L is displayed in Fig. 4, showing the hydrogen-bonding
network where two water molecules form bridges between two
ligands related by inversion symmetry.
As already mentioned, dinuclear zinc complexes can be formed
with IPCPMP in methanol solution if the ligand is depro-
tonated first. Two complexes were prepared, one containing
acetate ([{Zn2(IPCPMP)(OAc)}2][PF6]2, 2) and the other pivalate
(trimethylacetate) bridges ([{Zn2(IPCPMP)(O2CCMe3)}2][PF6]2,
3). Crystals of X-ray quality could be grown for both complexes.
The structure of 3 was briefly presented in a previous report22 while
crystallographic data for 2 can be found in Table 1 and relevant
bond distances for both 2 and 3 are shown in Tables 2 and 3. As
seen in Fig. 5(a), complex 2 consists of two phenolate-bridged
dinuclear entities related by inversion symmetry and bound
together by the endogenous (IPCPMP-derived) carboxylates of
the ligand. The Zn ions are coordinated in a distorted trigonal
bipyramidal geometry (For 2 t: Zn1 0.77, Zn2 0.52 and for
a To allow comparison with H4L, the atom labels of 2 and 3 have been
renumbered compared to the CIF files already published.22
3 t: Zn1 0.78, Zn2 0.63, cf. ref. 26 for a definition of the
t index).22,28,29 The carboxylates that bridge the two dinuclear
entities bind in a syn,anti-m-1,3 manner with the more basic syn
position30 coordinated to the other dinuclear part. The structure
of 2 is closely related to that of 3 (a structure overlay is shown
in Fig. S1 of the ESI†), and similar complexes have previously
been structurally characterized for the ligand ICIMP (Fig. 3).23–25
Overall, the two complexes have quite similar bond lengths, but
there is an interesting difference in the bond length for Zn(2)–
O(5), which is the bond holding the two dinuclear units together.
This bond is much longer in 2 (2.0545(17) A) than in 3 (1.990 A),
contrary to what may be expected on steric grounds since the
less bulky acetates should permit closer approach of the two
dinuclear entities. As the difference in bond distances is related
to the strength of interaction, this suggests that 2 might be more
˚
˚
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◦
˚
Table 3 Bond distances (A) and angles ( ) around the Zn ions in
[{Zn2(IPCPMP)(OAc)}2](PF6)2 (2) and [{Zn2(IPCPMP)(Piv)}2](PF6)2 (3)
2a
3a
Zn1–N1
Zn1–N2
Zn1–O1
Zn1–O3
Zn1–O5
Zn2–N3
Zn2–N4
2.112(2)
2.087(3)
2.221(3)
2.022(3)
1.956(3)
2.006(3)
2.102(3)
2.147(3)
2.1736(19)
2.0394(17)
1.9680(16)
1.9860(17)
2.1132(19)
2.111(2)
Zn2–O2
Zn2–O3
2.0543(17)
2.0246(16)
2.0741(17)
3.4019(4)
4.9326(4)
4.7630(4)
116.86(8)
77.78(8)
115.31(7)
80.15(7)
119.90(7)
92.28(7)
121.01(7)
100.63(7)
94.92(8)
167.07(8)
93.67(7)
81.24(8)
115.42(7)
93.27(7)
1.990(3)
2.004(2)
2.069(3)
Zn2–O4
Zn1–Zn2b
3.3949(6)
5.1398(7)
4.9327(6)
118.02(13)
77.81(13)
114.95(12)
79.55(11)
122.24(12)
89.96(11)
117.80(11)
100.99(11)
94.94(13)
168.96(11)
96.32(11)
81.30(12)
122.30(11)
97.04(12)
138.36(11)
86.17(12)
99.32(11)
90.08(11)
89.11(11)
94.95(12)
175.98(12)
Zn1–Zn1b
Zn1–Zn2ib
Zn1–O3–Zn2
N1–Zn1–N2
O1–Zn1–N1
O1–Zn1–N2
O3–Zn1–N1
O3–Zn1–N2
O3–Zn1–O1
O3–Zn1–O5
O5–Zn1–N1
O5–Zn1–N2
O5–Zn1–O1
N3–Zn2–N4
O2–Zn2–N3
O2–Zn2–N4
O2–Zn2–O3
O2–Zn2–O4
O3–Zn2–N3
O3–Zn2–N4
O3–Zn2–O4
O4–Zn2–N3
O4–Zn2–N4
146.06(7)
84.47(7)
98.50(7)
90.88(7)
89.96(7)
101.44(7)
177.04(7)
a To allow comparison with H4L, the atom labels of 2 and 3 have been
renumbered compared to the CIF files already published.22 b Determined
using Diamond v3.1 (www.crystalimpact.com/diamond/).
Fig.
5
MERCURY72 plots of the dimeric structure of
[{Zn2IPCPMP(OAc)}2]2+
2 (a) and one of the dinuclear entities
(2*) of the dimer (b). Solvent and counterions have been omitted
for clarity and the ellipsoids are drawn at 50% probability. To allow
comparison with H4L, the atom labels of 2 and 3 have been renumbered
compared to the CIF files already published.22
˚
O3 1.96880(16), Zn2–O3 2.0246(16) A and 3: Zn1–O3 1.956(3),
˚
Zn2–O3 2.004 A). The only bonds that are longer to Zn1 than
Zn2 are those from the tertiary amines (2: Zn1–N2 2.1736(19),
˚
Zn2–N3 2.1132(19) A and for 3: Zn1–N2 2.221(3), Zn2–N4
2.147(3) A). Que and coworkers31 have argued that such elongation
˚
of metal–tertiary amine bonds may be due to strain imposed by
coordination of the chelating arms of the ligand. In the case
of the unsymmetric ligand IPCPMP, Zn1 is involved in three
coordination rings (compared to two for Zn2). In agreement
with the above-mentioned argument regarding strain, the tertiary
amine (N2) bound to Zn1 is further away from the metal than
that bound to Zn2 (N3), where only two coordination rings exist.
The resulting weaker interaction between N2 and Zn1 might also
explain the shorter bonds to the other groups from Zn1 relative to
Zn2 and hence the unsymmetrical bridging.
Fig. 4 Mercury72 plot of H4IPCPMP(PF6)2·H2O (H4L). Counter-ions
have been omitted for clarity and ellipsoids are drawn at 50% probability.
prone than3 to dissociate into two dinuclear complexes in solution.
Both exogenous carboxylates (the acetate and the pivalate) bridge
in an unsymmetrical syn,syn-m-1,3 manner with shorter bonds
˚
to Zn1 (2: Zn1–O5 1.9860(17), Zn2–O4 2.0741(17) A and 3:
˚
Zn1–O5 2.006(3), Zn2–O4 2.069(3) A). The phenolate is also
unsymmetrically bridging with shorter bonds to Zn1 (2: Zn1–
8186 | Dalton Trans., 2010, 39, 8183–8194
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Despite the unsymmetrical coordination of the bridging car-
boxylates, the two C–O bonds in each bridging carboxylate moiety
have similar bond distances (2: O1–C8 1.262(3), O2–C8 1.250(3),
(n2 and n3 in Fig. 6(a)) that can be assigned to antisymmetric
carboxylate stretches (for 3 these are unresolved, cf. experimental
section) and the corresponding symmetric stretches are assigned
to resonances at 1436 and 1414 cm-1 (n5 and n6). The peak at
1485 cm-1 (n4) and the cluster of peaks with intermediate intensity
˚
O4–C27 (1.259(3), O–5C27 (1.259(3) A and 3: O1–C8 1.265(5),
˚
O2–C8 1.247(5), O4–C27 (1.250(5), O5–C27 (1.265(5) A). This
observation is consistent with the delocalized nature of the C O
around 1570 cm-1 (not labelled) are assigned to aromatic C C
=
=
bond and in contrast with what is observed for the protonated
carboxylates in the structure of H4L, where the corresponding
distances are significantly different (O1–C8 1.232(7), O2–C8
stretches. The difference (Dn) between the symmetric (nsym) and
antisymmetric (nasym) carboxylate stretches is often used to infer
the type of coordination to the metal. A Dn > 200 is usually taken
to indicate monodentate coordination to the metal while Dn <
200 indicates bridging and/or chelating coordination if an ionic
carboxylate salt can be ruled out.32 For 2 in KBr, the differences are
either 173 and 186 cm-1 (n2 - n5 and n3 - n6) or 164 and 195 cm-1
(n2 - n6 and n3 - n5) for the carboxylate resonance depending on
the assignment of the symmetric and antisymmetric stretches for
each carboxylate. As discussed below, the former assignment is
more reasonable.
˚
1.310(7) A). Although pivalate is a slightly stronger base, and
hence a stronger donor, the difference in pKa is not large
(pKa(HPiv) = 5.03; pKa(HOAc) = 4.76) and the oxygen to zinc
distances (vide supra) for the two different carboxylate bridges
are fairly similar for 2 and 3. The bonds trans to these donors
are however slightly longer for 3 (Zn1–N2 2.221(3), Zn2–N4
˚
˚
2.147(3) A) than for 2 (Zn1–N2 2.1736(19), Zn2–N4 2.111(2) A).
The three metal–metal distances in the tetranuclear structure of
i
˚
˚
2 are Zn1–Zn2 3.4019(4) A, Zn1–Zn2 4.7630(4) A and Zn1–
i
˚
Zn1 4.9326(4) A which may be compared to the corresponding
i
˚
˚
distances in 3, i.e. Zn1–Zn2 3.3949(6) A, Zn1–Zn2 4.9327(6) A
i
˚
and Zn1–Zn1 5.1398(7) A. The distances found in the trinuclear
active sites discussed above (Fig. 1) are: alkaline phosphatase (3.9,
˚
˚
4.9 and 7.1 A), nuclease P1 (3.2, 4.7, 5.8 A) and phospholipase C
˚
(3.3, 4.7, 6.0 A). Furthermore, the distances between Zn1 and Zn2
in 2 and 3 are similar to those in the closely related tetranuclear Zn
24
complex [{Zn2(ICIMP)(O2CCHPh2)}2]2+ (Zn1–Zn2 3.459(1) A)
˚
where diphenylacetate acts as exogenous carboxylate. The trend
with unsymmetrical bridges carries over to this complex but the
imidazole groups, that are stronger s-donors, have shorter bonds
to the Zn ions than the corresponding pyridyl groups in 2 and 3.
Comparing the structures of the coordinated and non-
coordinated IPCPMP ligand, there is little change in the distances
and angles in the carbon backbone of the ligand upon coordina-
tion, with the exception of the C–C bond between the carbonyl
carbon (C8) and the a-carbon (C7), where the distance increases
significantly (H4L: 1.477(8) A, 2: 1.520(3) A and 3: 1.521(5) A).
This change indicates that significant strain is introduced upon
coordination of Zn. The only other notable change is that
mentioned above for the C–O bonds of the carboxylates, that
become more symmetric when bridging between two metals than
when protonated and hydrogen-bonded to other heteroatoms.
These differences are well illustrated by the IR spectra of the ligand
compared to that of the complex (vide infra).
Fig. 6 FTIR spectra of [{Zn2IPCPMP(OAc)}2]2+ 2 in KBr (a), acetoni-
trile (b) and a mixture of acetonitrile and water 1 : 1 (v/v) (c).
˚
˚
˚
Complex
upon dissolution to
2
is readily soluble in acetonitrile, and
concentration of 25 mM (w.r.t.
a
[{Zn2(IPCPMP)(OAc)}2][PF6]2) the FTIR spectrum changes
to that in Fig. 6(b). In acetonitrile solution, the resonances at n3
and v6 are absent and have been replaced by resonances at 1638
(n1) and 1395 cm-1 (n7). These new bands correspond well to a
monodentate carboxylate group with Dn = 243 cm-1, suggesting
that the tetranuclear complex dissociates into two dinuclear
entities (2*) in this solvent. The dinuclear zinc complex 2* is likely
to retain its structure (cf. Fig. 5(b)). This observation supports
the assignment mentioned above, (i.e. n2 - n5 represents one
carboxylate, and n3 - n6 the other).
For solubility reasons, i.e. compatibility between substrate and
Zn complex, and in order to be able to compare the present results
with related studies,33–40 the reactivity studies were performed in
a 1 : 1 acetonitrile–water mixture. Therefore this solvent combina-
tion was also studied by FTIR (Fig. 6(c)). The analysis becomes
much more difficult, however, as water can form hydrogen bonds
to the oxygen atom(s) of the carboxylate moieties, forming pseudo-
bridged systems.32 This would explain the disappearance of the n1
resonance and possibly the formation of the strong but broad and
poorly defined peaks around 1590 cm-1. Due to the rather broad
and undefined features of the spectrum of 2 in acetonitrile–water
no further conclusions can be drawn.
IR spectroscopy
Due to the characteristic signatures of carboxylate functional-
ities in infrared spectroscopy, FTIR was used to study H4L
(H4IPCPMP(PF6)2), 2 and 3 in the solid state as KBr pellets,
and 2 in various solutions. The antisymmetric and symmetric
stretches for the carboxylic acid group in H4L occur at 1710 and
1229 cm-1, respectively, whereas in 2 and 3 they are shifted to about
1600 and 1420 cm-1, as is usually observed upon coordination
of a carboxylate group. This change correlates well with the
observed change in carboxylate bond lengths upon coordination
(vide supra). The FTIR spectra of 2 and 3 in KBr are very
similar except that the peaks for 3 are broader and less well
defined. The spectrum of 2 correlates very well with the solid
state structure. There are two peaks at 1609 and 1600 cm-1
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Table 4 Calculated overall stability constants (logb) and derived stepwise
dissociation constants (pK) for the proton and Zn2+ complexes of L
(IPCPMP) at 25 ◦C with I = 0.2 M (KCl)
Equilibrium processes
logb
pKa
L2- + H+ = HL-
9.28 (1)
14.71(1)
17.12(2)
18.75(2)
11.2(1)
15.25(2)
8.38(6)a
1.67(3)
9.28
5.43
2.41
1.63
L2- + 2H+ = H2L
L2- + 3H+ = H3L+
L2- + 4H+ = H4L2+
Zn2+ + L2- = [ZnL]
2Zn2+ + L2- = [Zn2L]2+
2Zn2+ + L2- + H2O = [Zn2L(OH)]+ + H+
2Zn2+ + L2- + 2H2O = [Zn2L(OH)2] + 2 H+
[Zn2L]2+ + 2H2O = [Zn2L(OH)2]+ + 2H+
[Zn2L]2+ + H2O = [Zn2L(OH)]+ + H+
[Zn2L(OH)]+ + H2O = [Zn2L(OH)2] + H+
13.58
6.87b
6.71c
a Due to the cooperativity of the deprotonation of two water molecules,
the formation of the dihydroxo species is favoured in this system, while
the presence of the monohydroxo species is almost negligible. b The pK
value refers to the first dissociation process of a coordinated water in the
dinuclear complex. c The pK value refers to the second dissociation process
of a coordinated water in the dinuclear complex.
Coordination in aqueous solution
To further study the species distribution in aqueous solution, a
potentiometric titration with H4IPCPMP(PF6)2 (H4L) and ZnCl2
was performed. The determined stability constants are shown in
Table 4 along with the calculated acid dissociation constants (pKa)
for the stepwise deprotonation processes. The titration curves are
shown in the ESI (Fig. S2).† First, the acid dissociation constants
of the ligand were determined. As seen in the species distribution
diagram in Fig. 7(a), formation of H4L only occurs at very low
pH and all basic positions are not protonated within the pH range
of this study. The first proton of [H4IPCPMP]2+ is completely
released at about pH 3, most probably from the carboxylic acid
moiety. The next two deprotonation steps are likely to occur at
the two ammonium groups; the large difference between these two
pKa values may be due to different degrees of hydrogen bonding to
nearby functional groups. The fairly low pKa values may also be
related to hydrogen bonding to the phenolic proton which makes
further protonation of the amine to form the ammonium cation
less favourable, thus giving a large Ka for the deprotonation of the
ammonium moiety.41
Fitting of the data from potentiometric titrations of 1 : 1 and
2 : 1 mixtures of Zn2+ and 1.2 mM H4IPCPMP(PF6)2 (H4L)
resulted in the calculated stability constants listed in Table 4.
Species distributions are depicted in Fig. 7(b) and (c), respectively.
When one equivalent of Zn2+ is added, a mononuclear species
([ZnL]) is the most prevalent species over a very large pH range.
Deprotonation of metal bound water only occurs at very high pH
and then only with the simultaneous formation of a dinuclear
species ([Zn2L(OH)2]). This observation indicates that in the
mononuclear complex [ZnL], the zinc ion is coordinated in the
tetradentate pocket of the ligand that contains the carboxylate
donor, since the highly anionic environment is expected to make
deprotonation of coordinated water molecules less favourable.
It is not possible to determine the exact pKa value of this
process on the basis of the pH range used for the potentiometric
studies but these studies indicate that the value is higher than 9.
Regarding the preferred coordination site of the zinc ion, we have
found that the preparation of a mononuclear Fe(III) complex of
Fig. 7 (a) Species distribution curves for IPCPMP (L). (b) For 1 : 1
Zn2+ : IPCPMP and (c) 2 : 1 Zn2+ : IPCPMP. Cligand = 1.2 ¥ 10-3 M. In
(c) are also included the pH dependence of the initial rates for the cleavage
of BDNPP ( ◊ ◊ ◊ ᭹ ◊ ◊ ◊ ) and HPNP (---ꢀ---).
(H4IPCPMP(PF6)2 leads exclusively to coordination of the ferric
ion in the tetradentate pocket.42 A surprising feature is the high
stability of the mononuclear species in relation to the correspond-
ing dinuclear one when two equivalents of Zn2+ are used. In the
pH range 3.5–6.5, the ratio of dinuclear vs. mononuclear complex
is about 3 : 1 and a large fraction (~20%) of the Zn2+ remains
non-coordinated up to just above pH 6. This can be explained by
the very different coordinating abilities of the two side arms of
the IPCPMP ligand. The chelate effect of the tridentate pocket
is less than that for the tetradentate pocket, and it is likely that
the metal affinity of the tridentate pocket at low to intermediate
pH is significantly less than that of the tetradentate pocket. A
similar behaviour was found for N,N¢-bis(5-methyl-imidazole-4-
ylmethyl)-1,3-diaminopropan-2-ol (bimido) which only has two
coordinating groups on either side of the alcohol groups.43 In the
case of bimido, it was found that only a mononuclear complex
was stable between pH 4.5–7 in the presence of two equivalents
of zinc; this observation could be explained by a crystal structure
of this species, revealing a non-coordinated alcohol group and
coordination of the single zinc ion by all the other groups in
the ligand. A similar phenomenon is unlikely to occur in the
present case due to the rigidity of the phenol linker of IPCPMP
(L). It is also interesting to note that the stoichiometry upon
deprotonation of the metal-coordinated water molecules implies
the formation of two hydroxido groups ([ZnL(OH)2]) with an
overall stability constant logb = 1.67(3). This process can be
separated into two deprotonation steps having close to identical
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pKa values of 6.87 and 6.71, as calculated from the corresponding
stability constants in Table 4. Values as low as this for the protolysis
of coordinated water at Zn2+ is often taken as evidence for the
formation of bridging hydroxido groups between the two zinc
ions,44–46 suggesting that [Zn2L(OH)2] has the structure depicted
in Fig. 8(a). There are however mononuclear zinc complexes that
have been reported to have aqua ligands with pKa < 7 although
they include highly specialized bulky ligands with hydrophobic
metal compartments and the actual species is not always clearly
identified.21,46–48 As noted by Buchholz et al.49 a strict correlation
between pKa and terminally or bridging coordination mode of the
water molecule is difficult to find. Furthermore, it is difficult to
rationalize the close to identical pKa values for the formation of
these two bridges since the first deprotonation should influence
the second considerably.
[{Zn2(IPCPMP)(OAc)}2][PF6]2 (2) were made but no peaks could
be discerned due to the large buffer-derived peaks that obscure
all other peaks. However, when ESI spectra of 2 were recorded
in water–MeOH (1 : 1 v/v) solutions, where the pH was adjusted
by addition of dilute NaOH, a peak attributable to [Zn2L(OH)2]
([Zn2(IPCPMP)(O)(OH)], [Zn2(IPCPMP)(O)2]) could be detected
at 611 ( 0.6) amu at pH 7.0 and 7.5, along with a more intense peak
at 595 amu attributable to [Zn2L(OH)] ([Zn2(IPCPMP)(O)]), as
well as a peak envelope for [Zn2(IPCPMP)(OAc)] (Fig. S4, ESI†).
Reactivity studies
The reactivity of 2 towards hydrolysis of phosphate diesters
was investigated using 2,4-bis-dinitrophenol-phosphate (BDNPP,
Scheme 1) as a substrate. The initial rates were measured by
following the absorbance of the formed 2,4-nitrophenolate (pKa =
4.07, e = 12 100 M-1 cm-1) at 400 nm in buffered water–acetonitrile
1 : 1 (v/v) solution with 0.1 M NaClO4 concentration in order to
maintain ionic strength. The pH dependence of the reactivity is
displayed in Fig. 9 along with that previously determined for the
transesterification of the substrate 2-hydroxypropyl-4-nitrophenol
phosphate (HPNP).22 For BDNPP, the initial rates show a
sigmoidal dependence on the pH, reaching a modest maximum
rate 127 times faster than that of the non-catalyzed reaction (at
pH 7.5). The change to zero order with respect to hydroxide
concentration at pH > 7.5 clearly indicates that a pre-equilibrium
process involving deprotonation of a functional group is important
for reactivity. The pKa of this process has been estimated to 6.63(5)
by fitting a sigmoidal Boltzmann function to the data. The curve
also follows the formation of the hydroxido species [Zn2L(OH)2]
(Fig. 7(c)) as determined by the potentiometric studies, and the
calculated pKa values agree well with those determined from
the kinetic measurements. This indicates that the active species
is [Zn2L(OH)2], or possibly [Zn2L(OH)] since this species forms
to some extent at the same pH and may be favoured when a
bridging acetate or substrate is present. Another possibility is
that the active species, which binds and activates the substrate,
is [Zn2L] (or possibly [ZnL]) and that the substrate is attacked
by a non-coordinating hydroxide. Morrow and co-workers53 have
shown that a dinuclear Zn(II) complex of the ligand 1,3-bis(1,4,7-
triazacyclonon-1-yl)-2-hydroxypropane is more strongly inhibited
Fig. 8 Proposed structures of the species [Zn2L(OH)2] identified in
the potentiometric studies with two bridging (a) or two terminal but
hydrogen bonded (b) hydroxido groups. In (c) a third proposal displaying
hydrogen bonding between two terminal hydroxides and a coordinated
water molecule is shown.
The very similar values for the deprotonation constants (pKa
6.87 and 6.73) may suggest a cooperative effect where the
deprotonation of one water on one Zn favours the deprotonation
of the second water molecule (on the other Zn ion) through
hydrogen bonding (Fig. 8(b)). Low pKa values in the range of
7–8 have been found for diaqua species with a hydrogen bonded
Zn–OH ◊ ◊ ◊ H2O–Zn structure, but then only deprotonation of one
of the water molecules was detected.50,51 This O2H3 unit was
˚
favoured for metal–metal distances >4 A, larger than that in
2.52 If this type of hydrogen bonding motif is responsible for
the low pKa of the coordinated water molecules, involvement
of a third water molecule to stabilize the two hydroxides in an
O3H4 unit may explain the observed behaviour (Fig. 8(c)). When
two equivalents of Zn2+ were used, precipitation was observed
at pH > 8. Titrations using 1.5 equivalents do not have this
problem and the resultant curve gives no indication of further
deprotonation of metal-coordinated water even though there
should still be one site available for water coordination due to the
non-coordinating isopropyl group. Probably the highly negative
charge contributed by four anionic coordinating groups makes
further metal hydrolysis in [Zn2L(OH)2] unfavourable.
The existence of the mononuclear and dinuclear complexes
discussed above is indirectly supported by ESI-MS experiments
of a 2 : 1 solution of ZnCl2–H4IPCPMP(PF6)2 at pH 7.8, which
show peaks for the species [ZnL]+, [Zn2LCl]+ and [Zn2L(OH)Cl]K,
but no species corresponding to [Zn2L(OH)2] could be observed
(see ESI, Fig. S2†). In an effort to detect [Zn2L(OH)2] un-
der conditions that are similar to those used in the reactivity
studies (vide infra), ESI-measurements of buffered solutions of
Fig. 9 Graphs displaying the pH dependence of the hydrolysis of BDNPP
(᭹) and the transesterification of HPNP(ꢀ) catalyzed by 2.
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at pH lower than that for maximal rate. If a similar situation
were to occur in the present system, the maximum rate reached at
about pH 8 is then a result of the balance between a decrease in
reactivity due to decreasing concentration of [Zn2L] and increase
in reactivity due to increasing hydroxide concentration.
It is interesting to note that the transesterification of HPNP
(a RNA mimic, Scheme 1) does not follow the pH dependence
observed for BDNPP. Although the curve still is sigmoidal, the
rate increment starts at much higher pH and thus does not appear
to be associated with the formation of the deprotonated species
[Zn2L(OH)2] (Fig. 7(c)). The maximum rate that is reached at
about pH 9.5 could be a result of a pre-equilibrium deprotonation
of the hydroxyl moiety of the substrate. Metal coordination of this
hydroxyl moiety is necessary in order to induce a sufficiently low
pKa value to achieve complete protolysis at pH 9.5–10. A pKa
of 12.8 has been determined for a non-coordinated 2¢-hydroxyl
group of an analogue to HPNP that contains a ribose unit and
an ethyl group on the phosphate.54 The difference in pKa for non-
coordinated HPNP is expected to be small and a lowering of
3–4 units upon coordination could be feasible considering that
water coordinated to hydrated Zn(II) reduces its pKa to 9.6.55 For
both substrates, a decline in activity is observed at high pH (10–
10.5); this decline may be associated with decomposition of the
zinc complex, and such decomposition may also cause or add to
the observed pH dependence for HPNP transesterification.
The dependence of the initial rate on complex and substrate
concentration was studied in order to further elucidate the
reactivity. As shown in Fig. 10(a), there is a strict first order
dependence of the initial rate on the catalyst concentration for both
HPNP and BDNPP and second order rate constants of k2nd,HPNP
=
Fig. 10 The dependence on concentrations of complex (a) and substrate
(b) for the transesterification of HPNP (᭹) and the hydrolysis of BDNPP
(ꢀ) catalyzed by 2. In (a) the complex concentration is given for the
dinuclear entity 2* and [BDNPP] = 1.0 mM. In (b) [2*] = 5.0 ¥ 10-5 M.
0.032(3) M-1 s-1 and k2nd,BDNPP = 0.034(1) M-1 s-1 could be calculated
from the slope and the substrate concentration, assuming that the
latter is constant for initial rate conditions. Here, the concentration
is given for the assumed dinuclear complex (2*) that will form upon
dissociation of the tetranuclear dimer. Fig. 10(b) shows saturation
kinetics for the substrate dependence indicating binding of the
substrate in a pre-equilibrium step. This data could be fitted to
the Michaelis–Menten equation (eqn (1)) yielding values for Km
and Vmax that are reported in Table 5 and used to calculate other
parameters.
Zn complexes from symmetric ligands containing alkoxide or
phenolate groups as bridges (KM = 5.4–16 mM),43,44,56,57 while a
calix[4]arene based complex displays much stronger binding (KM
=
0.018 mM).58 The kcat for HPNP transesterification enhanced by
2 (1.2 ¥ 10-4 s-1) is however significantly lower compared to all of
these complexes (6.4 ¥ 10-4–0.01 s-1)43,44,56–58 and this is 560 times
(kcat/kuncat) that of the uncatalyzed reaction (kuncat = 2.13 ¥ 10-7 s-1
at pH 8.5)33 and an overall efficiency (kcat/KM) of 0.033 M-1 s-1.
The kcat for BDNPP hydrolysis was calculated to 6.4 ¥ 10-4 s-1
and due to the weak binding the efficiency of the complex to
promote BDNPP hydrolysis is quite low (kcat/Km = 0.040 M-1 s-1)
but there is still a 2 ¥ 104 fold rate acceleration (kcat/kuncat) compared
to the uncatalyzed reaction (kuncat = 3.2 ¥ 10-8 s-1 in water–
acetonitrile 1 : 1 (v/v);59 previous studies on BDNPP hydrolysis34–40
in this solvent mixture have used a kuncat value for solutions with
Vmax[S]
n0 =
(1)
Km +[S]
The KM values indicate that the complex binds HPNP more
strongly than BDNPP (3.6 and 16 mM respectively), possibly
due to the more electron rich nature of the phosphate moiety
in HPNP. However, involvement of the 2-hydroxyl group in the
coordination of the HPNP cannot be excluded. The binding
of HPNP is stronger than for several other similar dinuclear
Table 5 Second order rate constants and kinetic parameters from the fitting of the Michaelis–Menten equation to the dependence of substrate
concentration for the transesterification of HPNP and the hydrolysis of BDNPP at pH 8.5
a
k2nd
V
max/10-9 M s-1b
K
M/mMb
K
ass/M-1c
k
cat/10-4 s-1d
k
cat/KM/M-1 s-1
k
cat/kuncat [104]
BDNPP
HPNP
0.034(1)
0.032(3)
32(10)
5.9(7)
16(5)
3.6(6)
64
280
6.4
1.2
0.040
0.033
2.0
0.056
a Second order rate constant defined by v0 = k2nd[2*][Sub] where [Sub] is the substrate concentration. b The standard errors from the non-linear curve
d
fitting are given within parentheses. c Kass = 1/KM kcat = Vmax/[2*]
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only water (1.8 ¥ 10-7 s-1) and for 2 this would yield kcat/kuncat
3.8 ¥ 103).
=
by the nucleophile coordinated to Zn1. The ligand IPCPMP has
recently been shown to be able to form heterodinuclear complexes
with various metals65 and further investigations are being directed
toward studying the effect of metal substitution on the above
phosphoryl transfer reactions.
To our knowledge, only a few studies on hydrolysis of BDNPP
by zinc complexes have been reported and only pseudo-first order
rate constants with the complexes in excess were calculated so
no straightforward comparison can be made here.60–63 Neves
and coworkers34–40 have tested several heterodinuclear complexes
for activity towards BDNPP cleavage. The pH optima of these
complexes and 2 differ, but a comparison of kcat at respective
pH optimum makes it clear that 2 rivals the heterodinuclear
complexes with respect to kcat although it binds the substrate
much more weakly and hence suffers from lower overall efficiency
(kcat/KM 0.0398 M-1 s-1 (2) vs. 0.075–0.16 M-1 s-1).34–40 The
catalytic efficiency is also low when compared to the dinuclear
nickel complex of 2-[N-(2-(pyridyl-2-yl)ethyl)(1-methylimidazol-
2-yl)aminomethyl]-4-methyl-6-[N-(2-(imidazol-4-yl)ethyl)amino
methyl]phenol),64 which has been reported to have a kcat/KM value
of 68 M-1 s-1, and displays both rapid turnover (kcat = 0.386 s-1)
and tight substrate binding (KM = 5.67 mM).
Experimental
Materials
All solvents were of at least 99.5% purity and used as received
or dried either by distillation from CaH2 (methanol, 2-propanol)
or by keeping over molecular sieves in a sealed bottle over night
˚
˚
(acetone 3 A molecular sieves, dichloromethane 4 A molecular
sieves). Reagents were of at least 99% purity, obtained from
commercial sources and used as received. The starting mate-
rials N-(ethoxycarbonylmethyl)-N-((2-pyridylmethyl)amine (b),66
2-(hydroxymethyl)-6-carbaldehyde-4-methyl-phenol25 (c), the bar-
ium salt of 2-hydroxypropyl-p-phenylphosphate67 and the sodium
salt of bis(2,4-nitrophenyl)phosphate59,68 were synthesized accord-
ing to literature procedures.
Conclusions
Physical measurements
In the present work, the complete synthesis and structural char-
acterization of the unsymmetrical dinucleating ligand IPCPMP
has been presented and its ability to coordinate Zn(II) has
been studied. In the solid state, two tetranuclear complexes
have been structurally characterized but both IR spectroscopy
and mass spectra indicate that the tetranuclear complex
[{Zn2IPCPMP(OAc)}2][PF6]2 (2) dissociates into two dinuclear
entities when dissolved in acetonitrile or an acetonitrile–water
mixture. A zinc complex of the unsymmetrical ligand has been
shown to be a better promoter of the transesterification of HPNP
(an RNA mimic) than a similar but symmetric ligand (BCPMP),
probably due to lower coordination number on one of the Zn(II)
ions in the IPCPMP case.22 The pH dependence of the activity
suggests that the hydroxyl group of the HPNP is activated by
coordination and is deprotonated before its nucleophilic attack on
the phosphorus center (Scheme 3(a)). The mode of coordination
of the phosphate moiety has not been determined. A detailed
investigation of the reactivity of [{Zn2IPCPMP(OAc)}2][PF6]2 (2)
towards the hydrolysis of BDNPP (a DNA mimic) has been
undertaken and the pH dependence indicates that a metal-
bound water molecule needs to be deprotonated for activity.
Potentiometric titration studies indicate that a terminally bound
hydroxide is the active nucleophile, and weak binding of the
substrate in an pre-equilibrium step leads us to propose a
mechanism in agreement with Scheme 3(b), where the substrate
binds in a monodentate fashion to Zn2 before being attacked
UV-vis spectroscopy and kinetic measurements were performed
on a Varian 300 Bio UV/vis spectrophotometer equipped with
a 12-position thermostated cell changer. Infrared spectra were
collected on a Nicolet Avatar 360 FTIR spectrometer for solid
KBr discs and a Digilab Excalibur FTIR spectrometer equipped
with an Axiom Analytical DPR-210 dipper system and a MCT
detector for liquid samples. Fast atom bombardment (FAB+) and
high resolution mass spectra were measured on a JEOL SX-102
instrument. NMR spectroscopy was performed on a Varian Inova
500 spectrophotometer in CDCl3 solution unless noted otherwise
and referenced to the residual proton signal of the solvent.
Syntheses
N-isopropyl-N-(2-pyridylmethyl)amine (a). A total of 4.50 ml
(0.0420 mmol) pyridyl carboxaldehyde was dissolved in 200 ml
dichloromethane and 8.0 ml (93 mmol) isopropyl amine was
added. The mixture was stirred at room temperature for
10 min before adding 20.7
g (0.0977 mmol) of sodium
tris(acetoxy)borohydride and the solution was left stirring at
room temperature for 16 h after which the reaction was complete
according to TLC (heptane : ethyl acetate : triethylamine 2 : 1 : 1).
The reaction mixture was washed with 3 ¥ 150 ml saturated
Na2CO3(aq) and the water phases were extracted with 3 ¥ 50 ml
dichloromethane. The combined organic phases were dried over
Na2SO4(s), filtered and evaporated to yield 6.24 g (98.8%) of
a yellowish oil that was pure according to NMR. 1H NMR
(500 MHz) CDCl3: d 8.54 (d 1H), 7.61 (t 1H), 7.28 (d 1H), 7.13
(t 1H), 3.88 (s 2H), 2.85 (sept. 1H), 1.81 (s (br), 1H), 1.10 (d 6H).
2-(N -isopropyl-N -(2-pyridylmethyl)amino-methyl-6-hydroxy-
methyl-4-methylphenol (d). A total of 5.019 g (0.03002 mol) of
2-(hydroxymethyl)-6-carbaldehyde-4-methyl-phenol and 3.276 g
(0.0218 mmol) of (a) was dissolved in 80 ml of dichloromethane
under an inert atmosphere in a dry round-bottom flask with
Scheme 3 Proposals for the nucleophilic attack in the cleavage of (a)
HPNP and (b) BDNPP catalyzed by the proposed active species formed
from [{Zn2(IPCPMP)(OAc)}2][PF6]2
˚
4 g of 4 A molecular sieves to remove water formed in the
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reaction. After stirring at room temperature for 45 min, 14.0 g of
sodium tris(acetoxy)borohydride was added. After 3 h of stirring
at room temperature, the reaction was complete according to
TLC (chloroform : heptane 3 : 1 with NH3 extracted from 25%
aqueous ammonia) and was quenched by addition of 100 ml of
saturated NaHCO3(aq). The pH was adjusted to 8 with saturated
Na2CO3(aq). The organic phase was further washed two times
with saturated Na2CO3 before drying over Na2SO4(s), filtering
and removal of solvent under vacuum. The crude product was
purified by column chromatography on silica using CHCl3 with 2%
methanol as eluent yielding 4.81 g (73.3%). 1H NMR (500 MHz)
CDCl3: d 8.55 (ddd, 1H, J = 0.9 Hz, J = 1.8 Hz, J = 4.9 Hz) 7.66
(dt, 1H, J = 1.8 Hz, J = 7.7 Hz) 7.33 (d, 1H, J = 7.8 Hz) 7.18
(ddd, 1H, J = 1.0 Hz, J = 4.9 Hz, J = 7.5 Hz) 6.91 (d, 1H, J =
1.6 Hz) 6.75 (d, 1H, J = 1.5 Hz) 3.12 (td, 1H, J = 6.5 Hz, J =
13.1 Hz).
(s, 2H) 3.69 (s, 2H) 3.68 (sept, 1H, J = 6.6 Hz) 2.14 (s, 3H) 1.44 (d,
6H, J = 6.6 Hz). IR (KBr) cm-1 3680 (w, O–H), 3300 (s, br, O–H),
=
3118 (w, C–H etc.), 2987 (w, C–H etc.), 1710 (s, C O), 1616 (m,
=
=
=
C C arom.), 1598 (m, C C arom.), 1538 (m, C C arom.), 1491
=
=
=
(m, C C arom.), 1468 (m, C C arom.), 1442 (m, C C arom.),
1393 (m), 1229 (s, C–OH carbox. acid), 843 (vs, PF6), 558 (s, PF6).
Elem. Anal. C26H36F12N4O4P2 Calc.: C, 41.17; H, 4.78; F, 30.06;
N, 7.39; Found: C, 41.72; H, 5.264; F, 29.92; N, 8.008.
[{Zn2(IPCPMP)(OAc)}2][PF6]2 (2).
A total of 30.1 mg
(0.0388 mmol) of (H4L) and 6.3 mg (0.12 mmol, 3 eq.) of sodium
methoxide were dissolved in 0.9 ml methanol and then transferred
to a solution of 18.7 mg (0.0852 mmol) zinc acetate dihydrate
in 0.9 ml water. A white precipitate formed upon addition but it
slowly dissolved again. A microcrystalline material (28 mg, 92%)
was formed upon slow evaporation of solvent. Crystals suitable
for X-ray crystallography were grown from methanol solution by
slow evaporation.
2-(N-isopropyl-N-((2-pyridyl)methyl)aminomethyl)-6-(N-(ethoxy-
carbonylmethyl)-N-((2-pyridyl)methyl)amino methyl)-4-methyl-
phenol (e). To 1.11 g (3.68 mmol) of (d) dissolved in 10 ml
of dichloromethane, 1.00 ml (3.8 mmol) of SOCl2 dissolved in
5 ml of dichloromethane was added dropwise over 30 min. After
stirring at room temperature for 4 h, the solvent and remaining
SOCl2 was removed under vacuum. The remaining yellowish
solid was dissolved in 6 ml of ethanol. This solution was added
dropwise over 45 min to a solution of 0.787 g (2.28 mmol) of (b) in
18 ml ethanol and 2.5 ml of triethylamine. The reaction mixture
was then refluxed for 1.5 h after which the solvent was removed
under reduced pressure (rotary evaporator). The remaining solid
was dissolved in aqueous phosphate buffer (0.1 M, pH 7) and
extracted with 3 ¥ 100 ml of dichloromethane. The organic phases
were combined, dried over Na2SO4, filtered and evaporated to
yield a brown crude product that was purified by chromatography
on silica using a mixture of heptane, toluene and triethylamine
(4 : 2 : 1) as eluent. This yielded 0.857 g (48.8%) of a yellowish
product. 1H NMR (500 MHz) CDCl3: d 8.50 (d, 1H, J = 6.1 Hz),
8.49 (d, 1H, J = 5.9 Hz) 7.61 (t, 2H, J = 7.7 Hz) 7.53 (d, 1H, J =
7.3 Hz) 7.42 (d, 1H, J = 6.1 Hz) 7.13 (t, 1H, J = 4.3 Hz) 7.11 (t,
1H, J = 4.9 Hz) 6.97 (s, 1H) 6.85 (s, 1H) 4.15 (q, 2H, J = 7.1H
z) 3.95 (s, 2H) 3.83 (s, 2H) 3.76 (s, 2H) 3.73 (s, 2H) 3.38 (s, 2H)
3.03 (s, 1H) 2.21 (s, 3H) 1.24 (t, 3H, J = 7.2 Hz) 1.10 (d, 6H, J =
4.4 Hz).
[{Zn2(IPCPMP)(OAc)}2](PF6)2
Elem.
Anal.
Calc.
C56H66F12N8O10P2Zn4 C, 43.04; H, 4.26; N, 7.17; Found C, 42.8; H
4.4; N, 7.0. IR (KBr) cm-1: 2964 (w, C–H); 2921 (w, C–H); 1609 (s,
anti-sym. –CO2); 1598 (s, anti-sym. –CO2); 1563 (m, C C arom.);
=
=
1486 (m, C C arom.); 1435 (m, sym. –CO2); 1414 (m, sym. –CO2);
844 (s); 559 (m); UV/vis (acetonitrile, nm): 234 (sh); 260; 298;
FAB+ MS: m/z (64Zn) 829 ([Zn2(IPCPMP)(OAc)2] + NB+, NB+ =
3-nitrobenzyl cation, matrix); 633 ([Zn2(IPCPMP)(OAc)]+); 619
([Zn2(IPCPMP)(O2CH)]+); 591 ([Zn2(IPCPMP)(OH)]+); 1411
([{Zn2(IPCPMP)(O2CCH3)}2]2+.PF6 ).
-
[Zn2(IPCPMP)(O2CC(CH3)3]2[PF6]2 (3). A total of 30.0 mg
(0.0386 mmol) of (H4L) was dissolved in 1.0 ml ethanol. To this
solution 12.1 mg (0.0888 mmol) ZnCl2 and 9.7 mg (0.0782 mmol)
sodium pivalate, each dissolved in 0.3 ml ethanol, were added.
When 43.1 ml (0.3091 mmol) triethyl amine was added a white
precipitate formed. This precipitate was filtered off and the
remaining solution was left to slowly evaporate which yielded
crystalline material.
[{Zn2(IPCPMP)(O2CC(CH3)3}2](PF6)2·2H2O,2C2H5OH Elem.
Anal. Calc. C64H86F12N8O12P2Zn4 C, 44.93; H, 5.07; N, 6.55;
Found C, 44.5; H, 5.1; N, 6.4; IR (KBr) cm-1: 2970 (w);
2921 (w); 2871 (w); 1609 (s, br, anti-sym. –CO2); 1562 (m);
1482 (m); 1442 (w, sym. –CO2); 1418 (m, sym. –CO2); 844
(s); 553 (m); UV/vis (acetonitrile, nm): 233 (sh); 261; 298;
FAB+ MS: m/z (64Zn) 675 ([Zn2(IPCPMP)(O2CC(CH3)3]+); 1495
2-(N-isopropyl-N-((2-pyridyl)methyl)aminomethyl)-6-(N-(car-
boxylmethyl)-N-((2-pyridyl)methyl)aminomethyl)-4-methylphenol
H4IPCPMP(PF6)2·2H2O (H4L). To 0.925 g (1.941 mmol) of
(e) dissolved in 12 ml of a 1 : 1 (v/v) ethanol–water mixture
was added 5.83 ml of a 1.0 M NaOH(aq) solution (5.83 mmol)
and the resultant solution was left stirring for 1.5 h. Then 1 M
HCl (aq) was added dropwise until pH was 1–2 and thereafter
1.34 g (7.98 mmol) of NaPF6 was added under stirring. The
white precipitate formed was collected by filtration and washed
three times with small amounts of cold water and then dried under
vacuum in a dessicator overnight to yield 1.22 g (81.0%) of a white
powder. Crystals of X-ray quality were grown from a mixture of
methanol and water. 1H-NMR (500 MHz) CD3CN d 8.48 (d, 1H,
J = 5.8 Hz) 8.46 (d, 1H, J = 4.8 Hz) 8.25 (t, 1H, J = 7.9 Hz)
7.78 (t, 1H, J = 7.8 Hz) 7.73 (t, 1H, J = 6.8 Hz) 7.61 (d, 1H, J =
8.0 Hz) 7.34 (dd, 1H, J = 5.3 Hz, J = 7.1 Hz) 7.25 (d, 1H, J =
7.8 Hz) 6.93 (s, 2H) 4.35 (s, 2H, br) 4.18 (s, 2H, br) 4.09 (s, 2H) 3.83
([{Zn2(IPCPMP)(O2CC(CH3)3)}2]2+·PF6 ).
-
X-Ray structure determinations†
The crystals of 2 and H4L were immersed in cryo-oil, mounted
in a Nylon loop, and measured at a temperature of 100 K. The
X-ray diffraction data were collected on a Nonius KappaCCD
diffractometer using Mo-Ka radiation (l = 0.71073 A). The
˚
Denzo-Scalepack or EvalCCD program packages were used for
cell refinements and data reductions. The structures were solved
by direct methods using the SHELXS-97 programs69 with the
WinGX graphical user interface. A semi-empirical absorption
correction (SADABS or Xprep in SHELXTL) was applied to all
data. Structural refinements were carried out using SHELXL-
97. In 2, the H2O, OH and NH hydrogen atoms were located
from the difference Fourier map but constrained to ride on their
8192 | Dalton Trans., 2010, 39, 8183–8194
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parent atom, with Uiso = 1.5. Other hydrogens were positioned
of 200 mL min-1. The temperature of the drying gas (N2) was
300 ◦C. The pressure of the nebulizating gas (N2) was 10 psi. The
spectrometer was operated at Ultra Scan Mode and was calibrated
with ESI Tuning Mix.
geometrically and constrained to ride on their parent atoms,
˚
with C–H = 0.98–1.00 A and Uiso = 1.2–1.5 Ueq (parent atom).
The crystallographic details are summarized in Table 1. Bond
lengths and angles for the organic framework of 2, 3 and H4L
is displayed in Table 2 while selected hydrogen bonds for H4L can
be found in Table S1 (ESI).† Relevant distances and angles for
the metal coordination in 2 and 3 are shown in Table 3. To allow
easy comparison, the labels of the atoms for 2 and 3 have been
renumbered to fit those of H4L and hence are different from the
labels in the CIF files already published.22
Kinetic measurements
The increase in concentration of the products, p-nitrophenol
and 2,4-dinitrophenol, were monitored at 25 ◦C by UV/vis
spectroscopy at 400 nm in quartz suprasil cuvettes using a Cary 300
Bio spectrophotometer equipped with a 12 position thermostated
cell changer. Substrate and catalyst concentrations were 0.80 mM
and 0.25 mM respectively and the ionic strength and pH were
kept constant by using total concentrations of 0.1 M NaClO4
and 0.01 M buffer (MES pH5-6.5, MOPS 7.0–7.5, EPPS 8.0–
8.5, CHES 9.0–9.5, CAPS 10.0–11.0). The pH of the buffer was
adjusted in standard solutions using a calibrated pH meter before
addition to the cuvettes. Each cuvette was prepared by consecutive
addition of 980 ml acetonitrile, 30 ml of a 0.0167 M standard
solution of the complex in acetonitrile–H2O (2 : 1 v/v) and 970 ml
of a 0.0207 M buffer solution containing 0.207 M NaClO4. After
mixing, the zero absorption was measured. Then 20 ml of a 0.080 M
standard solution of the substrate in H2O was added and after
quick mixing the increase in absorption over time was measured
at 400 nm, initially every minute but after 4 h every 5 min and
after 8 h every 15 min. The initial rates were calculated by fitting
a straight line to the curve corresponding to abs < 5% of the
maximum absorption at full conversion (usually the first 30 min).
The dissociation constant of the phenol products (pKa,PNP = 7.15,
pKa,BNP = 4.07) were taken into account when calculating the total
concentration of phenol from the absorption of the phenolate at
400 nm (ePNP = 18 500 M-1 cm-1, eBNP = 12 100 M-1 cm-1). Complex
and substrate concentration dependence were studied at pH 8.5
for both the BDNPP and HPNP cleavage. The measurement and
preparation of the solutions in the cuvettes were made in the
same manner as above with total NaClO4 concentration 0.1 M
but with total buffer concentration increased to 0.050 M. For
the dependence on complex concentration different volumes of
stock solution of [{Zn2(IPCPMP)(OAc)}2][PF6]2 (2) (2.0 mM) was
added to yield total concentrations of 0.020, 0.050, 0.1, 0.25, 0.5
and 1.0 mM of the postulated dinuclear complex (2*) in the cuvette
while keeping the substrate concentration constant at 1.0 mM. The
second order rate constant k2nd was determined from the slope of
Potentiometric studies
The pH-potentiometric titrations were performed in the pH range
2.0–11.0 or until precipitation occurred on samples of 10.00 cm3
at an ionic strength of 0.2 M (KCl) and at 25.0 0.1 ◦C. During
the titrations purified, strictly oxygen-free argon was continuously
bubbled through the samples. Samples were always freshly pre-
pared and since H4L (H4IPCPMP(PF6)2) was only slightly soluble
in water, its maximum concentration, what could be used in
the samples during the titrations, was 0.0015 M. The metal ion
stock solutions were prepared from ZnO (Reanal) dissolved in a
known amount of HCl solution (0.1 M). The concentration of
the metal ion stock solution was determined gravimetrically via
precipitation of quinolin-8-olates. The HCl concentration of the
Zn(II) solution and the exact concentration of the carbonate-free
KOH titrant were determined by pH-potentiometry. The metal
to ligand ratios were, 1.5 : 1 and 2 : 1. The pH-metric titrations
were performed with a Radiometer pHM84 instrument equipped
with a Metrohm combined electrode (type 6.0234.100). Titrant
KOH was added from a Metrohm 715 Dosimat auto burette. The
electrode system was calibrated according to Irving et al.70 so that
the pH-meter readings could be directly converted into hydrogen
ion concentration. The water ionization constant (pKW) obtained
was 13.75 0.01. The pH-metric results were utilized to find the
stoichiometry of species and to calculate the stability constants.
The calculations were made with the aid of the computer program
PSEQUAD.71
Electrospray mass spectrometric studies
Electrospray ionization time-of-flight mass spectrometric (ESI-
TOF MS) analysis was carried out on a Bruker BIOTOF II ESI-
TOF instrument and regular ESI measurements were carried out
on a Brucker HCT Ultra instrument. The metal to ligand ratio was
2 : 1. For the Zn2+-IPCPMP system, 0.0007 M ligand concentration
at pH 7.80 was used. The solutions were introduced directly into
the ESI source by a syringe pump (Cole-Parmer Ins. Comp. type
74900) at a flow rate of 2 mL h-1. The temperature of drying gas
(N2) was 100 ◦C. The pressure of the nebulizating gas (N2) was
30 psi. Voltages applied at the capillary entrance, capillary exit
and the first and the second skimmers were -4500, 120, 40 and
30 V, respectively. The spectrum was accumulated and recorded
by a digitalizer at a sampling rate of 2 GHz. The spectrometer was
operated at unit mass resolution and was calibrated with sodium
trifluoroacetate. For the measurements in absence of buffer, the pH
values were adjusted with sodium hydroxide in a water–MeOH
mixture (1 : 1 v/v). The solutions were introduced directly into
the ESI source by a syringe pump (KD Scientific) at a flow rate
2
the straight line fitted to the data, weighting each point by 1/s .
The values are shown in Table 5. For the dependence on substrate
concentration a similar addition of stock solutions of NaBDNPP
(40.0 mM) and Ba(HPNP)2 (20 mM) yielded total concentrations
of 0.25, 0.5, 1.0, 2.0 4.0 and 6.0 mM in the cuvette while the
complex concentration was kept constant at 0.050 mM. Initial
rates were measured three times for each concentration as above
and the results were fitted to the Michaelis–Menten equation
2
weighting each point by 1/s where s is the standard deviation
of each respective point. The fitted parameters KM and Vmax and
their standard errors along with calculated values of Kass and kcat
are shown in Table 5.
Acknowledgements
This research has been supported by a grant from the Swedish
Research Council (VR), and has been carried out within the
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The Royal Society of Chemistry 2010
Dalton Trans., 2010, 39, 8183–8194 | 8193
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framework of the International Research Training Group Metal
Sites in Biomolecules–Structures, Regulation and Mechanisms
(www.biometals.eu). We wish to thank Dr Luca Zuppiroli for
assistance with the mass spectrometry measurements. E.Cs. and
E.F. thank OTKA-NKTH CK77586 for financial support.
38 M. Lanznaster, A. Neves, A. J. Bortoluzzi, V. V. E. Aires, B. Szpoganicz,
H. Terenzi, P. C. Severino, J. M. Fuller, S. C. Drew, L. R. Gahan, G. R.
Hanson, M. J. Riley and G. Schenk, JBIC, J. Biol. Inorg. Chem., 2005,
10, 319–332.
39 A. Neves, M. Lanznaster, A. J. Bortoluzzi, R. A. Peralta, A. Casellato,
E. E. Castellano, P. Herrald, M. J. Riley and G. Schenk, J. Am. Chem.
Soc., 2007, 129, 7486–7487.
40 G. Schenk, R. A. Peralta, S. C. Batista, A. J. Bortoluzzi, B. Szpoganicz,
A. K. Dick, P. Herrald, G. R. Hanson, R. K. Szilagyi, M. J. Riley, L. R.
Gahan and A. Neves, J. Biol. Inorg. Chem., 2008, 13, 139–155.
41 Isolated tertiary amines commonly have pKa values around 9–10; H. K.
Hall, J. Am. Chem. Soc., 1957, 79, 5441–5444.
42 M. Jarenmark, H. Carlsson, V. Trukhan, M. Haukka, S. E. Canton, M.
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