Synthesis, crystal structure and investigation of mononuclear copper(II) and zinc(II) complexes of a new carboxylate rich tripodal ligand and their interaction with carbohydrates in alkaline aqueous solution

Article abstract of DOI:10.1016/j.jinorgbio.2015.04.012

A new carboxylate rich asymmetric tripodal ligand, N-[2-carboxybenzomethyl]-N-[carboxymethyl]-β-alanine (H₃camb), and its di-copper(II), (NH₄)₂[1]₂, and di-zinc(II), ((CH₃)₄ N)₂[

Full text of DOI:10.1016/j.jinorgbio.2015.04.012

Synthesis, Crystal Structure and Investigation of Mononuclear Copper(II) and
Zinc(II) Complexes of a New Carboxylate Rich Tripodal Ligand and Their
Interaction with Carbohydrates in Alkaline Aqueous Solution
Christopher D. Stewart, Mayra Pedraza, Hadi Arman, Hua-Jun Fan, Ed-
uardo Luiz Schilling, Bruno Szpoganicz, Ghezai T. Musie
PII:
DOI:
Reference:
S0162-0134(15)00106-3
doi: 10.1016/j.jinorgbio.2015.04.012
JIB 9712
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
12 February 2015
17 April 2015
19 April 2015
Please cite this article as: Christopher D. Stewart, Mayra Pedraza, Hadi Arman,
Hua-Jun Fan, Eduardo Luiz Schilling, Bruno Szpoganicz, Ghezai T. Musie, Synthe-
sis, Crystal Structure and Investigation of Mononuclear Copper(II) and Zinc(II) Com-
plexes of a New Carboxylate Rich Tripodal Ligand and Their Interaction with Carbo-
hydrates in Alkaline Aqueous Solution, Journal of Inorganic Biochemistry (2015), doi:
1
0.1016/j.jinorgbio.2015.04.012
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ACCEPTED MANUSCRIPT
Synthesis, Crystal Structure and Investigation of Mononuclear Copper(II) and Zinc(II)
Complexes of a New Carboxylate Rich Tripodal Ligand and Their Interaction with
Carbohydrates in Alkaline Aqueous Solution
a
a
a
b
Christopher D. Stewart, Mayra Pedraza, Hadi Arman, Hua-Jun Fan, Eduardo Luiz
c
c
a
Schilling, Bruno Szpoganicz, Ghezai T. Musie *
a
Department of Chemistry, The University of Texas at San Antonio, San Antonio, TX, 78249,
United States
b
Department of Chemistry, Prairie View A&M University, Prairie View, TX 77446, United
States
c
Departamento de Química - Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil
Corresponding Author
*
Fax (210)458-7428 (G.T.M); E-mail: ghezai.musie@utsa.edu (G.T.M)
1

ACCEPTED MANUSCRIPT
ABSTRACT
A new carboxylate rich asymmetric tripodal ligand, N-[2-carboxybenzomethyl]-N-
[
(
carboxymethyl]-β-alanine (H camb), and its di-copper(II), (NH ) [1] , and di-zinc(II),
3 4 2 2
(CH ) N) [2] complexes have been synthesized as carbohydrate binding models in aqueous
3
4
2
2,
solutions. The ligand and complexes have been fully characterized using several techniques,
including single crystal X-ray diffraction. The interactions of (NH ) [1] and ((CH ) N) [2]
2
4
2
2
3 4
2
with D-glucose, D-mannose, D-xylose and xylitol in aqueous alkaline media were investigated
1
3
using UV-vis and C-NMR spectroscopic techniques, respectively. The molar conductance,
NMR and ESI-MS studies indicate that the complexes dissociate in solution to produce the
-
-
-
-
respective complex anions, 1 and 2 . Complexes 1 and 2 showed chelating ability towards the
naturally abundant and biologically relevant sugars, D-glucose, D-mannose, D-xylose, and
xylitol. The complex ions bind to one molar equivalent of the sugars, even in the presence of
stoichiometric excess of the substrates, in solution. Experimentally obtained spectroscopic data
and computational results suggest that the substrates bind to the metal center in a bidentate
fashion. Apparent binding constant values, pKapp, between the complexes and the substrates
were determined and a specific mode of substrate binding is proposed.
The pKapp and
relativistic density functional theory (DFT) calculated Gibbs free energy values indicate that D-
mannose displayed the strongest interaction with the complexes. Syntheses, characterizations,
detailed substrate binding studies using spectroscopic techniques, single crystal X-ray diffraction
and geometry optimizations of the complex-substrates with DFT calculations are also reported.
Keywords: Metal complex, Monosaccharides, sugar-metal complex interaction, Binding
constants, Mutarotation,
2

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Introduction
Sugar–metal ion interactions are not only ubiquitous in biological processes but are also
vital in cell functions [1-5]. Although some effort has been directed towards understanding
carbohydrate recognition, unlike various other metalloenzymes, the study of carbohydrates in
biological systems using synthetic model complexes is largely unexplored. One of the
contributing factors for the absence of such studies has been the lack of technological advances
in the realm of high magnetic field NMR and other quantitative spectroscopic methods to handle
the complicated equilibrium and polyfunctional nature of carbohydrates and other natural
products in solution. However, NMR spectroscopy and several other techniques have developed
significantly in recent times. As the consequence of the developments, the study of complicated
systems including carbohydrates has been attracting considerable interest. Recently, some
synthetic receptors have been used to investigate carbohydrate recognition in relation to the
important roles they play in biological processes [3, 4]. One of such recognition which is being
investigated with ever increasing interest includes simple metal ions in carbohydrate interactions
[
6-13]. This particular interaction has important implications in a variety of biological systems
such as support in membrane systems, cell–cell adhesion, intercellular recognition, signal
transduction, fertilization, and as targets of bacterial or viral infections of cells [1-5]. Although
understanding carbohydrate-metal ion coordination chemistry is of fundamental importance to
these systems, structural and functional investigations of metal complexes with carbohydrates
has been limited to complexes derived from amino sugars [14-16]. Aside from the biological
relevance, carbohydrate interaction with metal ions has also been a subject of intense research in
the field of enantioselective catalysis of organic reactions [17-24].
To understand the carbohydrate-metal ion interactions, some model complexes have been
prepared and their interactions reported in the literature [25-28]. In the past several years, the
study of carbohydrate-transition metal ion interactions in chemistry and biology has been
pursued by several research groups [2, 14-16, 24, 29-32]. For example, synthetic strategies for
2
+
3+
2+
3+
2+
2+
2+
2+
2+
VO , Cr , Mn , Fe , Co , Ni , Cu , Zn , and MoO carbohydrate complexes have been
2
developed [6, 7, 11-14, 28, 31-36]. Furthermore, the biologically relevant aspects of
3
+
3+
2+
2+
carbohydrate complexes of Fe , Cr , VO , and Zn , have also been reported [32-35, 37-43]. It
2
+
2+
2+
has also been elucidated that carboxylato-bridged dinuclear complexes of Mg , Mn , Co ,
2
+
2+
Ni , and Zn , are involved in non-redox active enzymatic processes [33, 44-51].
3

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In an attempt to contribute to the understanding of carbohydrate-metal complex chemistry,
recently, we reported new polynuclear iron(III) and copper(II) complexes and their interaction
with several monosaccharides in aqueous media [34]. Thus, the main focus of the current
manuscript is on the interaction of newly synthesized mononuclear copper(II) and zinc(II)
complexes of a carboxylate rich asymmetric tripodal ligand, N-[2-carboxybenzomethyl]-N-
[
carboxymethyl]-β-alanine (H camb) with the monosaccharides in alkaline aqueous media.
3
Herein, we report the syntheses and characterizations of H camb, (NH ) [1] and ((CH ) N) [2]
2
3
4 2
2
3 4
2
complexes, solution behavior and the level and mode of their interaction with D-glucose, D-
mannose, D-xylose and xylitol in alkaline aqueous media. Also reported is relativistic density
functional theory (DFT) modeling study on the complexes and their interaction with the
substrates.
2
. Experimental
2
.1. General Remarks
All starting materials were purchased from commercial sources and were used without
further purification. Elemental analyses were determined by Atlantic Microlab, 6180 Atlantic
Blvd. Suite M, Norcross, GA. FTIR spectra were recorded on solid samples using a Bruker
Vector 22 FTIR-ATR spectrometer. Molar conductivities were measured in aqueous solution of
-3
the complexes (10 M) using a Corning Model 441 conductivity meter.
2
.2. UV-Vis Spectroscopy
All experiments were performed on an Agilent 8453 diode array UV-visible (UV-Vis)
spectrophotometer with 1 cm quartz cell at room temperature over a range of 200-900 nm. An
Eppendorf Research micropipette was used to measure volumes. All experiments were carried
out in degassed nanopure water, in which pH of the solutions were adjusted using NaOH
solution. Each concentration was made and measured three times and the data points were
averaged. Standard deviation was applied to these averages. The detailed experimental procedure
used for the sugar titrations was adopted from our earlier published work [34].
4

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2
.3. NMR Spectroscopy
1
13
H and C NMR spectra of H camb and (CH ) N) [2] were obtained in D O solution with a
3
3 4
2
2
1
3
Varian Inova 500 NMR spectrophotometer. All C NMR experiments for the determination of
binding interactions were performed on the same NMR spectrophotometer at room temperature.
The experiments were done in D O solution, for which pH was adjusted to pH 12.5 with NaOH
2
prior to the use for each set of NMR titration. The pH of the resulting solution was measured
immediately after mixing.
2
.4. Mass Spectrometry
Electrospray ionization time-of-flight mass-spectrometry (ESI-TOF-MS) spectrometry data
was collected using a Bruker Daltonics micrOTOF instrument at the RCMI program. Data was
collected for a m/z range of 100 – 2000 in negative ion mode. Samples were delivered as either
dilute (1 – 2 mg/mL) aqueous or methanol solutions with a relatively moderate flow rate of 0.90
mL/h. In all the measurements, the setting of the nebulization gas, N , was 45.0 psi, the capillary
2
potential was 4 kV, the drying gas was 5.0 L/min, the skimmer was set to -33 V, and the
hexapole RF was set to 300 Vpp. Data was analyzed and the spectra plots were generated using
the Mass software package [52, 53]. Simulations of the stable isotope patterns were made using
Molecular Weight Calculator (Matthew Monroe, PNNL, Richland WA, U.S.A.) software.
2
.5. Potentiometric Titration
The potentiometric studies were carried out in doubly distilled and boiled water solution with
a Metrohm 848 Titrino plus automatic titrator combined with an Ag/AgCl electrode calibrated to
+
read –log[H ] directly, designated as pH. The electrode was calibrated using the data obtained
-1
from a potentiometric titration of a known volume of a standard 0.0100 mol L HCl solution
with standard 0.100 M KOH. The experiments were carried out in a 50 mL sealed thermostated
cell at 25ºC and bubbled with argon to ensure an inert atmosphere and the ionic strength adjusted
to 0.100 M by addition of KCl. In a typical experiment the mass equivalent to 0.05 mmol of the
H camb ligand was weighted in an analytical balance and dissolved in 30 mL of water and 20
3
mL of 0.0100 M HCl solution to ensure the complete protonation and an initial pH value near 2.
The titration was carried out by the addition of 0.05 ml aliquots of a standard CO -free 0.100 M
2
5

ACCEPTED MANUSCRIPT
KOH solution pH 12 was reached. Computation of results were carried out with BEST7 program
and species distribution calculations with SPECIES program [54].
2
.6. Computational Studies
The theoretical calculations were carried out using the Gaussian 09 implementation of
B3LYP DFT [55-57]. The geometry optimization were carried out using a 6-31G(d) basis set on
C, N, O and H atoms where LANL’s double zeta basis set (LANL2DZ) with effective core
potentials (ECP) were used for metals [58-60]. All structures were fully optimized with
polarizable continuum model (PCM) and frequency analyses were performed to ensure a
minimum state was achieved [61, 62]. To test the robustness and reliability of the B3LYP
functional used in the simulations, all geometries were also optimized under parameterized DFT
functional M06-2X, which have shown promise for noncovalent interactions [63]. The
thermodynamic functions, including enthalpies, entropies and free energies, were calculated at
2
98.15 K and 1 atm within the harmonic potential approximation at optimized structures. In
order to examine basis set effect, Dunning’s correlation consistent triple zeta basis set cc-pVTZ
for all atoms were implemented with B3LYP functions to perform the single point energy (SPE)
calculation upon the aforementioned optimized geometries [64].
2
2
.7. Syntheses
.7.1. Synthesis of N-[2-carboxybenzomethyl]-β-alanine, H cbal
2
The ligand has been prepared according to our previously published procedure [65]. The
product was collected by filtration, washed with water, methanol and dried at 80C. The product
1
was confirmed by elemental analysis and H NMR spectroscopy. Yield: 8.5 g (76%). Anal.
Calcd. for C H NO : C, 59.19%; H, 5.87%; N, 6.27%. Found: C, 59.08%; H, 5.93%; N,
11
13
4
1
6
.20%. H NMR for the sodium salt of the compound (500 MHz, D O, 25°C, δ): 7.66 (d, 1H, J
2
=
7.0 Hz), 7.48 (m, 2H), 7.45 (t, 1H, J = 7.0 Hz), 4.11 (s, 2H), 3.05 (t, 2H, J = 7.5 Hz), 2.52 (t,
2
H, J = 7.5 Hz).
2
.7.2. Synthesis of N-[2-carboxybenzomethyl]-N-[carboxymethyl]-β-alanine, H camb
3
An aqueous solution containing H cbal (3.50 g, 13.5 mmol), NaOH (1.62 g, 40.5 mmol), and
2
N,N-Diisopropylethylamine (2.82 mL, 16.2 mmol) in 10 mL deionized water was prepared. In a
6

ACCEPTED MANUSCRIPT
separate container an aqueous solution of bromoacetic acid (2.25 g, 16.2 mmol) in 10 mL
deionized water was similarly prepared. The above two solutions were combined (pH = 11 after
mixing) in a 100 mL round bottom flask and refluxed for 4 hours with stirring. The pH of the
solution had decreased to 9 over the 4 hour reflux. The resulting solution was acidified with 6M
HCl to pH = 3 while being kept at 90°C, after which a white precipitate formed over a period of
1
8
0 minutes. The product was collected by filtration, washed with water, methanol and dried at
1
13
0C. The product was characterized as H camb by elemental analysis, ESI-MS, H and C
3
NMR spectroscopy. Yield: 2.81 g (74%). Anal. Calcd. for C H NO : C, 55.51%; H, 5.38%;
13
15
6
N, 4.98%. Found: C, 55.39%; H, 5.43%; N, 5.03%. ESI-MS m/z (%): 280.0867 (80)
–
–
–
1
[
C H NO ] ; 302.0624 (95) [C H NO Na] ; 324.0535 (100) [C H NO Na ] . H NMR for
13 14 6 13 13 6 13 12 6 2
the sodium salt of the compound (500 MHz, D O, 25°C, δ): 7.51 (d, 1H, J = 8.1 Hz), 7.34 (m,
2
3
H), 3.80 (s, 2H, J = 7.6 Hz), 3.06 (s, 2H), 2.75 (t, 2H, J = 7.5 Hz), 2.36 (t, 2H, J = 7.5 Hz).
.7.3. Synthesis of (NH ) [Cu (camb) ], (NH ) [1]
2
4
2
2
2
4 2
2
An aqueous solution (1.0 mL) containing [Cu(NH ) ]SO · H O (0.1880 g, 0.77 mmol) was
3
4
4
2
added drop wise, at ambient temperature, to a stirring 1.0 mL aqueous suspension of the ligand
H camb (0.2150 g, 0.76 mmol). After complete addition the resulting dark blue solution was
3
allowed to stir for 24 hours at ambient temperature, after which the volume of solvent was
removed by heating to 70°C and the resulting blue powder was redissolved in 2.0 mL of water.
The solution was filtered to remove any undissolved solids and the product was precipitated
using acetone to afford the blue complex characterized as (NH ) [1] after filtering and drying
4
2
2
over calcium sulfate. Na [1] complex was synthesized in similar manner and X-ray quality
2
2
single crystals were grown by acetone diffusion into the concentrated solution in 30 days. Yield:
.2640 g (48%). Anal. Calc. for C H N O Cu : C, 43.39; H, 4.48; N, 7.79. Found: C, 43.21;
0
2
6
32
4
12
2
-
H, 4.47; N, 7.88%. ESI-MS m/z (%): 341.0016 (100) [C H CuNO ] ; 683.0242 (29)
1
3
12
6
-
-
[
(
C H Cu N O ] ; 705.0004 (7) [C H Cu N NaO ] . IR (Solid on ATR): ν 3234 (w) 3038
26 25 2 2 12 26 24 2 2 12
w) 2900, 2866 (vw) 2793 (br), 1631, 1598, 1571 (s), 1464, 1441 (m), 1368, 1361, 1334 (s),
239, 1156, 1112, 1083, 1011 (m), 975 (w), 942, 921 (m), 866 (s), 807 (m), 767, 717, 673, 626
1
-1
-1
-1
(
s) 596, 572 (m) (cm ). UV-Vis (H O) λ /nm (ε/L mol cm ): 725 (161); Molar conductance,
2 max
-1
2
–1
Λ : (H O) = 128  cm mol .
M
2
7

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2
.7.4. Synthesis of (CH ) N) [Zn (camb) ], ((CH ) N) [2]
2
3
4
2
2
2
3 4
2
A methanol solution (5.0 mL) containing Zn(NO ) ∙ 6H O (0.1058 g, 0.36 mmol) was added
3
2
2
drop wise, at ambient temperature, to a stirring 5.0 mL methanol solution of the ligand H camb
3
(
0.1000 g, 0.36 mmol) and (CH ) NOH (0.2255 g, 1.25 mmol). After complete addition the
3 4
resulting colorless solution was allowed to stir at ambient temperature for 2 hours. X-ray quality
single crystals of [(CH ) N)] [2] were grown in 3 days by slow acetone vapor diffusion into the
3
4
2
2
methanoic solution of the complex. Yield: 0.2633 g (88%). Anal. Calc. for C H N O Zn : C,
3
4
60
6
15
2
4
4.21; H, 6.55; N, 9.10. Found: C, 44.83; H, 6.43; N, 9.09%. ESI-MS m/z (%): 342.0021 (100)
-
-
[
C H NO Zn] ; 760.1113 (2) [C H N O Zn ] . IR (Solid on ATR): ν 3031 (w) 2955 (w)
13 12 6 30 36 3 12 2
1
644 (m), 1615, 1583 (s) 1489, 1468, 1447, 1421, 1396 (m) 1346, 1330 (s), 1258, 1232, 1148,
-1
-
1
118, 989 (m), 954, 855, 759, 721, 666 (s) (cm ); Molar conductance, Λ : (H O) = 118 
M
2
1
2
–1
cm mol .
2
.8. X-ray Crystallography and Data Analysis
The data was collected at 98(2) K using a Rigaku AFC12/Saturn 724 CCD fitted with Mo Kα
radiation ( = 0.71073 Å). Data collection and unit cell refinement were performed using
CRYSTAL CLEAR software [66]. The total number of data was measured in the range 3.09° <
θ < 27.5° using ω scans. Data processing and absorption correction, giving minimum and
maximum transmission factors, was accomplished with CRYSTAL CLEAR and ABSCOR,
respectively [66, 67]. The structure was refined with Olex2 [68] using SHELX-2014 by direct
2
methods and refined (on F ) using full-matrix, least-squares techniques [69]. All non-hydrogen
atoms, for all structures, were refined with anisotropic displacement parameters. All carbon
bound hydrogen atom positions were determined by geometry and refined by a riding model. In
the case of (Na) [1] complex, electron density peaks were used to identify oxygen bound
2
2
hydrogen atom for the O9 and the hydrogen atom on O7 was placed on a calculated position, and
the displacement parameters were set to 1.5 times the displacement parameters of the bonded
atoms.
3
. Results and discussion
3
.1. Synthesis of the Ligand and Metal Complexes.
8

ACCEPTED MANUSCRIPT
The asymmetric tri-carboxylate amine tripodal ligand, H camb, has been synthesized in two
3
reaction steps, Scheme 1. The synthesis of the precursor ligand, H cbal, was accomplished by
2
the condensation of stoichiometric amounts of 2-carboxybenzaldehyde and -alanine in presence
of NaOH in methanol under refluxing conditions for 4 hr, followed by subsequent reduction
using NaBH [65]. Alkylation of the secondary amine group of H cbal with stoichiometric
4
2
quantities of bromoacetic acid in the presence of NaOH/N,N-Diisopropylethylamine followed by
acidification of the solution with HCl yielded a pure H camb as white powder.
3
<
Scheme 1>
The H camb ligand has been fully characterized using various analytical techniques such as
3
1
13
elemental analysis, ESI-MS, FTIR, H and C NMR spectroscopies. Additionally, the pKa
values for the ligand have been determined using potentiometric titration methods. The titration
curve for H camb ligand has two observable buffer regions. One region involves the
3
consumption of three equivalents of the base and is attributed to the protonation/deprotonation
equilibria of the three carboxylic groups is at 2.5 to 4.5 pH range. The second buffer region
which involves the consumption of one equivalent of the base due to the
protonation/deprotonation of the amino group is observed after pH 5. The calculated equilibrium
constant values for each of the protonation/deprotonation steps involved are shown in Scheme 2.
2
-
The calculated species distribution as a function of pH indicates that HL as the main species in
3
-
the pH values above 4.3 and less than 10.2, the completely deprotonated species, L ,
predominates at pH values 10.2 while the di-, tri- and tetra-protonated species have a maximum
formations at pH values of 3.8, 2.8 and 2.0, respectively.
<
Scheme 2>
The ligand, H camb, was specifically designed for the investigation for two reasons.
3
Firstly, its carboxylate rich coordination environment mimics the coordination environment of an
active site of the metalloprotein that involves carbohydrates, xylose/glucose isomerase (XGI),
Fig. 1.
<
Fig 1>
9

ACCEPTED MANUSCRIPT
Secondly, H camb as a mono-nucleating ligand gives a structural complementarity to our
3
reported di-nucleating ligand, H ccdp; and thus, it presents an opportunity to study and contrast
5
the effect of metal nuclearity on the complex-carbohydrate interaction without significantly
altering the molecular structure of the ligands [33, 34]. For comparison reasons, the molecular
structures of H ccdp and H camb ligands are depicted in Fig. 2.
5
3
<
Fig 2>
The direct synthetic routes to (NH ) [1] and ((CH ) N) [2] are described in Scheme 3. The
4
2
2
3 4
2
2
synthesis of (NH ) [1] in aqueous solution is accomplished by reacting [Cu(NH ) ]SO ·H O and
4
2
2
3 4
4
2
H camb in 1:1 mole ratio in ambient conditions. Similarly, a reaction of equimolar amounts of
3
Cu(NO ) salt and H camb in presence of four equivalents of NaOH in water produced Na [1] .
3
2
3
2
2
Both the (NH ) [1] and Na [1] complexes are stable under standard conditions, soluble in
4
2
2
2
2
water and insoluble in most common organic solvents. While single crystals of the sodium salt
of the complex suitable for X-ray analysis were grown by slow acetone diffusion into an aqueous
solution of the reaction mixture, similar crystallization effort on the ammonium salt has not
produced crystals suitable for diffraction. Correspondingly, the synthesis of ((CH ) N) [2] was
3
4
2
2
carried by the reaction of Zn(NO ) ∙6H O and H camb in a 1:1 molar ratio in the presence of an
3
2
2
3
3
.5 equivalents of (CH ) NOH as a base in methanol under ambient conditions. Slow acetone
3 4
diffusion into the reaction mixture produced X-ray quality single crystals characterized as
(CH ) N) [2] . The product is stable under ambient conditions, exceedingly soluble in water,
(
3
4
2
2
and moderately soluble in most common organic solvents. Characterization of (Na) [1] ,
2
2
(
NH ) [1] and ((CH ) N) [2] has been carried out using a variety of techniques including
4 2 2 3 4 2 2
1
13
elemental analysis, FTIR, UV-Vis, H NMR, C NMR, ESI-MS, and single crystal X-ray
diffraction.
<
Scheme 3>
3
.2. Spectroscopic Characterizations.
The UV-vis spectra of (NH ) [1] (2.50 mM) were recorded in H O at pH 7.0 and 12.5. The
4
2
2
2
spectrum in water at pH = 7 has only one d–d transition band with the λmax centered at 725 nm,
1
0

ACCEPTED MANUSCRIPT
while in pH = 12.5 the λmax is blue-shifted to 704 nm. Solution characterizations of
1
3
(
(CH ) N) [2] dissolved in D O was performed using C-NMR spectroscopy. The spectrum
3 4 2 2 2
displayed fourteen well resolved NMR signals corresponding to the 14 different sets of carbons
+
in the complex, including the carbon from the (CH ) N counter ion which appears at 55 ppm.
3
4
The chemical shifts for the aromatic, methylene and carboxylates carbons positioned around
1
38-129, 60-33, and 182-178 ppm, respectively.
In order to gain further insight into the behavior of the complexes in aqueous solution, the
ESI-MS spectra of complexes (NH ) [1] and ((CH ) N) [2] were recorded and analyzed. The
4
2
2
3 4
2
2
negative ion mode ESI-MS spectra of the complexes in aqueous media are shown in Fig. 3.
Besides to the value of the m/z ratio, the distinct copper and zinc nuclei isotope distribution
patterns have been used in the identification and determination of the number of metal ions
present in the molecular ion peaks. The ESI-MS spectrum of (NH ) [1] dissolved in nanopure
4
2
2
water with the pH adjusted to 12.5 with NaOH, is shown in Fig. 3(a). The spectrum contains
-
-
-
signals corresponding to [Cu(camb)] , [Cu(C H NO )] and [Cu(camb)(CH COOH)] at m/z =
12
12
4
3
3
4
41 (100%), 297 (26%) and 401 (15%), respectively. The data indicates that the ion at m/z =
-
01 losses a mass that corresponds to CH CO H to produce [Cu(camb)] at m/z = 341,
3
2
-
subsequently followed by a loss of CO from the ligand to from the [Cu(C H NO )] at m/z =
2
12 12
4
2
97. The experimentally obtained and the simulated isotope distribution patterns for the
-
molecular ion [Cu(camb)] at m/z = 341 are shown in Fig. 3(c).
<
Fig. 1>
The comparison of the distribution patterns for the experimentally obtained and the simulated
data shows an excellent agreement to one another. Similarly, the ESI-MS spectrum for
(
(CH ) N) [2] under the same experimental conditions was obtained and is presented in Fig.
3 4 2 2
-
1
(b). The spectrum contains signals corresponding to molecular ions [Zn(camb)] at m/z = 342
-
-
(
100%), [Zn(C H NO )] at m/z = 342 (16%), and [Zn (camb) + ((CH ) N)] at m/z = 760
12 12 4 2 2 3 4
-
(
2%). The ESI-MS data indicates that the ion at m/z = 760 dissociates to give [Zn(camb)] at m/z
-
=
342, which subsequently losses a CO from the ligand to produce [Zn(C H NO )] at m/z =
2
12 12
4
-
2
98. The experimentally obtained and simulated isotope distribution patterns for [Zn(camb)] at
m/z = 342 are shown in Fig. 3(d). Once again, the two distribution patterns are in agreement to
one another. The data obtained in this ESI-MS study not only helps to establish the m/z ratio
1
1

ACCEPTED MANUSCRIPT
corresponding species in solution, but also exposes the stability of the ions at pH 12.5.
−
3
Furthermore, molar conductance of 10 M aqueous solutions of complexes (NH ) [1] and
4
2
2
(
(CH ) N) [2] at different temperatures were measured. The values obtained for the complexes
3 4 2 2
-1
2
–1
in water (25°C) were 118 and 128  cm mol , respectively. The molar conductance values in
-1
2
-1
aqueous solutions fall in the range 118–131  cm mol , suggesting that these are 1:1
electrolytes [70]. The solution behavior and dissociation of the complexes into their respective
mono-anionic complexes are depicted on Scheme 4.
<
Scheme 4>
The FTIR spectra of solid samples of complexes (Na) [1] , (NH ) [1] and ((CH ) N) [2]
2
2
2
4 2
2
3 4
2
were recorded and analyzed, Supporting Information Fig. S1. When comparing the IR spectrum
of (Na) [1] and (NH ) [1] , the spectra for Na [1] notably lacks the asymmetric ν (N-H)
2
2
4 2
2
2
2
as
-1
vibration at 3234 cm whereas the spectra for (NH ) [1] contains a broad O-H stretching
4
2
2
-1
vibration at 3370 cm . This difference is attributed to the absence of ammonium cations and the
presence of water molecules in the solids sample Na [1] . Deacon and Phillips had previously
2
2
examined the FTIR spectra of many metal-carboxylate complexes with known X-ray crystal
structures and were able to draw useful conclusions for the correlations between carboxylate
stretching frequencies and their geometries [71]. Similarly, FTIR analysis of zinc(II)
carboxylates has been extensively studied as a means to correlate their spectra with their known
crystal structures [72]. The IR spectra of (NH ) [1] (Supporting Information, Fig. S1(b)) shows
4
2
2
-
-1
three strong asymmetric ν (COO ) vibrations at 1631, 1598, and 1571 cm , and three strong
as
-
-1
-
symmetric ν (COO ) vibrations at 1368, 1361, and 1334 cm . The difference, ∆ (∆ = ν (COO )
s
as
-
-1
−
ν (COO )), between the carboxylate stretches of 203, 264, and 270 cm is attributed to the
s
difference in binding modes between the carboxylates. The ∆ value of 203 is characteristic of a
-1
monodentate monatomic bridging carboxylate, while the ∆ values of 264 and 270 cm are
characteristic of a monodentate carboxylate binding mode [71, 72]. In the IR spectra obtained
-
for ((CH ) N) [2] (Supporting Information, Fig. S1(c)), three strong asymmetric ν (COO )
3
4
2
2
as
-1
-
vibrations at 1617, 1585, and 1569 cm , and three strong symmetric ν (COO ) vibrations at
s
-1
1
423, 1348, and 1332 cm were observed. The significant difference in the magnitude of the
-1
separation between the carboxylate stretches, ∆, of 146, 269, and 253 cm is attributed to the two
-1
different binding modes of the carboxylates in the complex. The lower ∆ value of 146 cm is
1
2

ACCEPTED MANUSCRIPT
-1
characteristic of syn–anti bridging coordination while the larger ∆ values of 269 and 253 cm are
characteristic of monodentate carboxylate binding [71, 72].
3
.3. X-ray Molecular Structure Characterization
Detailed crystal structure analysis of complexes is described herein. The crystal structural
data and selected metric data for the complexes are given in Tables 1 and 2, respectively. The
thermal ellipsoid representations of the single crystal X-ray molecular structures for Na [1] and
2
2
(
(CH ) N) [2] are depicted in Fig. 4 and 5, respectively.
3 4 2 2
<
Table 1>
3
.4. Crystal Structure of Na [1]
2 2
-
The complex crystallized in the monoclinic P 2 /n space group with two mononuclear [1]
1
1
1
anions bridged by the O(5) and O(5A) of the β-alanine arms of the ligands in a μ :η η (O,O)
2
+
fashion forming a centrosymmetric dimer. Two Na ions are present as counter ions to balance
-
the charge and link adjacent [1] units together forming a continuous coordination chain,
[
Na Cu (camb) (H O) ]n [73]. The continuous coordination chain of the complex is shown in
2 2 2 2 5
the Supporting Information Fig. S2(A). The thermal ellipsoid diagram for the dicopper complex
2
-
anion, [1] , with atomic numbering scheme is shown in Fig. 4 the B3YLP generated
2
electrostatic potential map is shown in the Supporting Information Fig. S2(B). Each copper(II)
ion is in a penta-coordinated environment with a [NO ] donor set. The Addison parameter,
4
defined as τ = (β – α)/60, is a structural index parameter used to systematize the deviation
between square pyramidal and trigonal bi-pyramidal geometries in penta-coordinate species [74].
<
Fig. 4>
When applied to the complex, the calculated Addison parameter of 0.13 indicates a distorted
square pyramidal geometry around the copper center. The O(3) atom of the acetate arm of the
ligand opens at 10.5° out of the square plane of the base defined by O(1), O(5A), N(1), O(3) and
the Cu(1). This distortion most likely arises from the steric hindrance imposed by the shorter
acetate arm of the ligand. The long Cu(1) –O(5) bond length appears longer than normal at
2
.222 Å; while the remaining bond lengths and angles appear typical for coordination complexes
of this nature [75]. The dinuclear copper(II) core of the complex anion shown in two views in
1
3

ACCEPTED MANUSCRIPT
Fig. 4(a) and 4(b) exists as a planar four membered ring with alternating copper and oxygen
atoms forming a corners of a rhomboid geometry. The Cu(1)---Cu(1A) and O(5)---O(5A)
distances across the core are 3.188 and 2.719 Å, respectively. The rest of the metric data is
present in Table 2.
3
.5. Crystal Structure of ((CH ) N) [2]
3 4 2 2
The single crystal X-ray structure analysis reveals that the complex consists two zinc(II) ions,
3
-
two camb ligands and two tetramethylammonium as counter cations. Where the methyl groups
2
-
of the ammonium cations are disordered. A structural view of the anion of complex [2]2 is
depicted in Fig. 5 and the B3YLP generated electrostatic potential map is shown in the
Supporting Information Fig. S3. Using the Addison parameter, the coordination geometry
around each of the zinc(II) centers is best described as a slightly distorted trigonal bipyramidal
geometry [75]. While the trigonal plane of the bipyramidal geometry is defined by O(1), O(3)
3
-
and O(5) atoms of one camb ligand, the axial positions are taken by N(1) of the same ligand
3
-
and O(6) of the second camb of the complex.
<
Fig. 5>
Whereas the benzoate and the acetate groups are bound as a terminal ligands, the propionate arm
1
1
of the ligand bridges between the two zinc(II) centers in a rare syn,anti-μ :η η fashion.
2
Structural search performed on the core of complex as shown in Fig. 6 (c) in the Cambridge
Structural Database (CSD) returned with no crystal structure match. Although no coordination
mode of this nature in di-zinc(II) complexes has been reported in the literature, Lalioti and
1
1
1 1
coworkers have observed four syn,syn-μ :η η and two syn,anti-μ :η η coordination modes of a
2
2
-
bridging CH CO ions in the crystal structure of the hexa-zinc(II) complex obtained by the
3
2
.
reaction of Zn(CH CO ) 2H O and d-2-pyridyl ketone (C NH ) CO [76]. The two zinc centers
3
2 2
2
5
4 2
and the bridging propionate arms in crystal structure of (CH ) N) [2] form a unique octagonal
3
4
2
2
cavity core as shown in Fig. 6(c) and 6(d). The Zn(II)---Zn(II) and O---O distances across the
cavity are 4.353 and 4.372 Å, respectively. The Zn–O (bridging carboxylate) bond length of
2
.0514(17) Å is slightly longer than the 1.999(2) Å observed for Zn–O (terminal carboxylate).
<
Fig. 6>
1
4

ACCEPTED MANUSCRIPT
However, the Zn–O (benzoate) bond length at 1.9773(17) Å is comparable to the Zn–O (terminal
carboxylate) distance and shorter than the Zn–N bond length, 2.185(2). The distortion from the
ideal trigonal bipyramidal around the zinc centers is particularly reflected in the bond angles of
o
o
N(1) –Zn–O(6), O(3)–Zn(1)–N(1) and O(1)–Zn(1)–O(5) at 171.57(7) , 81.04(7) , and
o
1
01.00(7) , respectively. As presented in Table 2, most of the observed metric values for the
complex are well within the range of previously reported similar di-zinc(II) complexes in the
literature [44, 77]. However, the observed Zn(II)---Zn(II) distance in the complex is slightly
longer than bond distances reported for similar di-nuclear zinc(II) complex in the literature [33,
4
5, 78-81].
<
Table 2>
3
.6. Carbohydrate/Metal Complex Binding Studies
Room temperature molecular interactions between D-glucose, D-mannose, D-xylose, and
xylitol with (NH ) [1] and ((CH ) N) [2] at pH 12.5 were studied using UV-vis and NMR
4
2
2
3 4
2
2
spectrometry, respectively. The percentage distribution of the main equilibrium structures of the
substrates under investigation are illustrated in Supporting Information, Scheme S1. In an effort
1
to obtain further insights into the stability of the complexes in solution, UV-vis and H NMR
techniques were applied to study (NH ) [1] and [(CH ) N)] [2] at pH 12.5, respectively. The
4
2
2
3 4
2
2
UV-vis absorbance values at wavelengths 680, 704 and 720 nm for (NH ) [1] vs time for the
4
2
2
respective bands were monitored. The plots of the data are shown in the Supporting Information
Fig. S4. The data shows no decay of absorption bands for extended period of time under the
experimental conditions. Similarly, the stability of ((CH ) N) [2] in the experimental conditions
3
4
2
2
1
were studied by monitoring the H NMR signal intensity of the aromatic protons at 7.31 ppm of
the complex. The plot of the signal intensity values vs time showing a straight line with a zero
slope is presented in the Supporting Information, Fig. S5. Therefore, the data establishes the
stabilities of (NH ) [1] and ((CH ) N] [2] in solution under the operative conditions. Several
4
2
2
3 4
2
2
copper and zinc complexes found in literature with similar structural features to (NH ) [1] and
4
2
2
(
(CH ) N) [2] have also been shown to be stable over a wide pH range in aqueous solutions [12,
3 4 2 2
3
-
1
3]. In an effort to assess the stability of the complexes towards displacement of the camb
ligand from the complexes by any of the substrates under the experimental conditions, several
control studies were carried out in aqueous solutions and their UV-vis spectroscopy, NMR and
1
5

ACCEPTED MANUSCRIPT
electrospray ionization time-of-flight mass spectrometry were studied. In a typical experiment,
1
3
UV-Vis in case of the copper(II) and C NMR in case of the zinc(II), a spectrum of a known
concentration of a complex in the presence of stoichiometric excess of a substrate in one hand
and a spectrum of a free metal ion and a substrate under the same experimental conditions in the
other hand were recorded and analyzed. The results of this exercise clearly indicate that the
3
-
camb ligand remains firmly bound to the metal centers in the presence of the substrates under
the experimental conditions.
Systematic addition of substoichiometric amounts of the substrates into aqueous solutions of
(
NH ) [1] at pH 12.5 resulted in a significant reduction in the absorbance value at λ = 704
4 2 2 max
nm. The reductions in the absorption value continued with each aliquot added until a 1:1 molar
-
ratio of [1] to substrate was reached, at which point further additions of substrate did not bring
change to the absorbance value. The decrease in absorbance was accompanied by a blue shift in
the spectra with all of the substrates investigated. A typical change in the λmax and absorbance
-
values with the addition of substoichiometric amounts of the substrates into [1] solution is
shown in Fig. 7. This representative particular set of data was obtained for the titration of a 2.5
o
mM (NH ) [1] solution with aliquots of xylitol at 25 C and pH of 12.5. Similar data for the rest
4
2
2
of the systems is provided in the Supplementary Information, Fig. S6. Treatment of the data
using the Rose–Drago method allowed determination of the binding stoichiometry between
substrates and metal complex [82, 83].
<
Fig. 7>
Representative binding isotherms, shown in Fig. 8, are plots of the change in absorbance at
7
04 nm (ΔA704nm) versus the substrate equivalents added, SE = [S]/[M ], where [S] is the moles
1
of the substrate and [M ] is the moles of (NH ) [1] . The graphs shows a large change in
1
4 2
2
absorbance with each aliquot of substrate added up to a 1.0 equivalent of substrate, followed by a
plateau where additional aliquots of substrate do not significantly affect the absorbance values
even up to 5.0 equivalents of substrate to the (NH ) [1] solution. The binding isotherms provide
4
2
2
further spectroscopic evidence of a 1:1 binding interaction between the substrate and the
complex.
<
Fig. 8>
1
6

ACCEPTED MANUSCRIPT
The method established by Rose & Drago for determining the number of spectroscopic
states, and hence the number of species present in solution, has been used with great success in
previously reported studies [11, 13, 33, 34, 82]. Full mathematical justification of the Rose-
Drago method as it relates to the present study is provided in the Supplementary Information
section. Connors relates the method to a chemical equilibrium (eq. 1) in which the apparent
-1
-1
binding constant is given by the equation pKapp = log(Kapp ), where Kapp is defined in eq. 2 [82].
[
M] + [S] ↔ [MS]
eq. 1
eq. 2
ꢅꢆꢇꢈ
ꢂꢃ
ꢄ
ꢀ
ꢁꢁ
ꢉꢅꢆꢈꢊꢅꢇꢈꢋ
When applied to a system in which the only absorbing species present in solution are the
metal complex [M] and the substrate-bound metal complex [MS], a two-state system can be
observed by plotting the change in absorbance (ΔA) at a specific wavelength (n = 1) of two
different concentrations (j and k) versus the ΔA at a different wavelength (n = 2) of
concentrations j and k; {(A – A ) versus (A – A ), where j ≠ k}. If only one absorbing
1
j
1k
2j
2k
species is present in solution the graph will contain two overlapping lines with identical slopes.
In the case of a two-state system, the graph will contain two different lines with unique slopes
both of which intersect at the origin. The method also provides insight into systems in which
there are more than two spectroscopic states. When a single substrate binds in different modes to
a metal complex in solution then multiple spectroscopic states could exist. When this situation
arises it can be clearly seen in the Rose–Drago plot by the deviation from linearity of the data to
a non-linear quadratic slope [83].
When the UV-Vis data obtained from the titration of (NH ) [1] with xylitol in aqueous
4
2
2
solution at pH = 12.5 was treated with the Rose–Drago method of analysis, a linear plot was
observed, shown in Fig. 9. The presence of two linear functions with different slopes which pass
-
through the origin indicates a 1:1 binding interaction between [1] and xylitol as expected for
two-state systems. When the data for the titration of (NH ) [1] with D-glucose, D-mannose, and
4
2
2
D-xylose were subjected to the same treatment, similar results were obtained, Supporting
Information Fig. S7. For each graph it can be seen that there are two plots with different linear
slopes which both pass through the origin, again suggesting the presence of only one substrate-
bound species in solution for D-glucose, D-mannose, and D-xylose. Evidence of multiple
1
7

ACCEPTED MANUSCRIPT
equilibria in solution is not present and a 1:1 [substrate]/[complex] ratio persists even when up to
a five-fold excess of substrate is added.
<
Fig. 9>
The apparent binding constant, pK , values between (NH ) [1] and the substrates were
app
4 2
2
calculated and are presented in Table 3. Also present in Table 3, for comparison purposes, are
pKapp values for other copper(II) complexes found in the literature. Based on the value of the
binding constants, the most tightly bound substrate to (NH ) [1] is D-mannose with a pK
app
4
2
2
value of 2.71 ± 0.15. Subsequent to the D-mannose are the pKapp values for D-xylose, D-
glucose, and xylitol. When the binding constant values for (NH ) [1] with D-glucose and D-
4
2
2
mannose are compared with other mono-nuclear copper(II) complexes values reported in the
literature (Table 3), they are consistently smaller. The binding constant values reported in
literature for mono-nuclear copper(II) complexes with D-mannose are 3.44 ± 0.04 for
2
+
2+
2+
[
[
Cu(bpdpo)] , 3.07 ± 0.03 for [Cu(paeo)] , 3.68 ± 0.12 for [Cu(DIEN)] , 3.38 ± 0.19 for
2
+
2+
Cu(HEN)] , and 3.05 ± 0.41 for [Cu(styDIEN)] [12, 13].
Table 3>
Similarly, the binding constant values reported in literature for mono-nuclear copper(II)
<
2
+
2+
complexes with D-glucose are 3.66 ± 0.02[12] for [Cu(bpdpo)] , 3.15 ± 0.02 for [Cu(paeo)] ,
2
+
2+
2+
3
.73 ± 0.12 for [Cu(DIEN)] , 3.61 ± 0.17 for [Cu(HEN)] , and 3.37 ± 0.31 for [Cu(styDIEN)]
[
12, 13]. While the complex reported herein is mono-anionic in solution, all the previously
reported studies on mono-nuclear copper(II) complexes involved are di-cationic copper(II)
species. Hence, the main cause for the observed lower binding affinity of the complex in this
study could well be the charge difference between the complex ions. Direct comparison of the
pKapp values for D-xylose and xylitol with other mono-nuclear copper(II) complexes reported in
literature was not possible because of the lack of reported data. However, the binding constant
values for (NH ) [1] with D-xylose and xylitol are in the range of several di-nuclear first row
4
2
2
transition metal complexes reported in recent literature. The list of these data is presented in the
Supporting Information, Table S1 and Fig. S8.
1
8

ACCEPTED MANUSCRIPT
1
3
3
.7. C NMR Spectroscopy and Substrate Binding with ((CH ) N) [2]
3 4 2 2
To get insight into specific binding to the metal centers using NMR spectroscopy, we have
synthesized the new zinc(II) complex of the H camb ligand, and investigated its interaction with
3
1
3
the substrates. A representative C NMR spectra for ((CH ) N) [2] , D-glucose, and the
3
4
2
2
reaction mixture of ((CH ) N) [2] and D-glucose are shown in Fig. 10. The best spectrum
3
4
2
2
resolution for the reaction mixture was obtained when using 1:2 molar ratio of ((CH ) N) [2] to
3
4
2
2
1
3
D-glucose. The assigned chemical shifts in Fig. 10(a) are typical for C NMR spectra of α- and
β-D-glucopyranose in aqueous alkaline medium [33]. The most informative feature in the
spectra can be easily identified when comparing the spectra of the free D-glucose and
(
(CH ) N) [2] D-glucose reaction mixture, Fig. 10(a) and 10(c) respectively.
3 4 2 2 /
<
Fig. 10>
Firstly, the intrinsic equilibrium between α and β forms at the anomeric carbon is disrupted,
which results in α/β population inversion upon interaction with the metal complex. A proposed
metal complex induced mutarotation of D-glucose responsible for the α/β population inversion is
shown in Scheme 5.
<
Scheme 5>
Similar mutarotaion of D-glucose induced by its interaction with MCl (M = Cu and Cr) in
2
methylimidazolium based ionic liquids have been reported in the literature [84]. Secondly, the
binding sites of D-glucose to the complex are indicated by a characteristic “coordination-induced
shift” (CIS), about 2 ppm up-field chemical shift, by the carbons bearing the coordinating
oxygen atoms to the zinc(II) center. The complete resonance shifts for all the substrates are
1
present in Table 4. While the most change in the CIS is observed for the anomeric carbon (C )
2
and C , the rest show insignificant change during the substrate metal complex interaction. The
NMR study clearly indicates that ((CH ) N) [2] competitively binds to the substrates in solution
3
4
2
2
at pH 12.5. Recent literature has examples of some metal ions interacting with D-glucose and D-
1
3
xylose [14, 16, 31, 85]. Klüfer and co-workers reported a X-ray single crystal structure and C
NMR spectral characterization of a D-glucose bound dipalladium(II) complex [8]. Their study
1
4
revealed that C –O to C –O are deprotonated and the α-D-glucopyranose tetraanion is bridging
2
+
in a bis(chelate) fashion between two different Pd(en) units.
1
9

ACCEPTED MANUSCRIPT
<
Table 4>
3
.8. Density Functional Theory Calculations
To get insight into the solution coordination chemistry and energetics involved, we
performed comparative theoretical studies using DFT on our system. Calculations have been
-
-
performed on complex ions, 1 and 2 , with the substrates in different coordination modes at the
B3LYP and M06-02 functional with 6-31G*/LANL2DZ, cc-pVTZ, and 6-311G** basis set.
When (NH ) [1] and [(CH ) N)] [2] dissociate to their respective monomeric complex ions in
4
2
2
3 4
2
2
solution, as shown in Scheme 5, the metal centers take a pseudo-trigonal bipyramidal geometry.
As depicted in Fig. 11, the metal centers of the complexes are coordinated to the oxygen atoms
3
-
of the three carboxylate arms of camb to form the trigonal bases. The optimized geometry
-
parameters for complex 2 , for example, gave the N–Zn–O bond angles at 85.32º, 100.5º, 101.4º,
and O–Zn–O bond angles at 114.2º, 121.0º and 122.0º. Similar optimization was carried out for
-
complex 1 and the metric data is presented in the Supporting Information Table S2. The metric
data is consistent with pseudo-trigonal planar geometry around the metal centers. As the result
of the geometry around the metal centers, in the presence of the coordinating sugars, two
possible products are expected in solution. The first possibility is where the substrate acts as a
monodentate ligand and binds through the anomeric oxygen to take the axial position to form the
trigonal bipyramidal geometry around the metal center. The second scenario is where a substrate
1
2
behaves as a bidentate ligand coordinating via C –O and C –O, as observed in our NMR studies,
to produce an octahedral geometry where the oxygen atoms of the substrate take one equatorial
and one axial position around the metal center. Due to high flexibility and reorganization ability
3
-
of the tripodal camb ligand around the metal centers, the formation of penta- and hexa-
coordination geometries upon binding to the substrates is expected. Recently, Martin and
coworkers reported the flexibility of cobalt complexes with a related tripodal aminotrisphenolate
ligand scaffold which easily accommodated the two geometries when reacted with mono- and
bidentate exogenous ligands [86].
<
Fig. 11>
-
-
The penta- and hexa-coordinated 1 and 2 conformation with substrate were optimized, Fig.
1
1, and their thermodynamic parameters and the binding energy with the substrate were
2
0

ACCEPTED MANUSCRIPT
determined. Whereas the Gibbs free energy for the trigonal bipyramidal [1--D-mannose] is only
-3.05 kcal/mol, the octahedral complex is -76.42 kcal/mol. The observed difference in energy is
3
-
partly due to the different reorganization and adaptability of the camb ligand around the metal
center when binding the substrate. For example, while the arrangement and thus metric values of
3
-
-
the camb ligand in the penta-coordination product reassembles that of 1 , major rearrangement
were required to accommodate the bidentate substrate. The Metal–O bond lengths increase by
0
.07-0.31 Å where metal–N bond length increase by up to 0.29 Å for the trigonal bipyramidal
and the octahedral complexes, Supporting Information Table S2. The most significant changes
for the octahedral geometry comes from the bond angles, for example, N–Zn–O bond angle
change from ~100 º to 154 º, and 170 º, and 174 º respectively. The results of the DFT study
1
3
1
corroborate the experimentally observed C-NMR chemical shifts which suggested the C and
2
C substrate coordination mode to the metal centers of the complexes, vide supra. The
calculated binding energy values in the form of solvated Gibbs free energy for the hexa-
coordinated anomeric substrate bound complexes are presented Table 5. A representative
-
-
optimized structure between 2 and -D-glucopyranose and between 2 and β-D-glucopyranose is
shown in Fig. 10. While the optimized structure involving the β-D-glucopyranose shows a H-
6
3-
bonding interaction between the C –OH and the carboxylate arm of the camb ligand at a 1.928
Å distance, such interaction was absent in the case of α-D-glucopyranose, Fig. 12. Instead, a
4
3
long range interaction at 2.325 Å between C –OH and C –OH was observed. Due to steric
congestions around the metal centers, coordination of two different sugar molecules in a
monodentate fashion at the same time is highly unlikely. The structural optimization and energy
minimizations for the rest possible interactions are presented in Table 5. The study indicates
-
-
that, generally, the -anomers of the substrate bind to the metal centers of 1 and 2 at lower
energy than their -anomer counterparts.
<
Fig. 12>
-
For example, the binding energy of β-mannose with 2 is -31.49 kcal/mol while the α-mannose
bound at -22.84 kcal/mol with the same complex. With the exception of xylose, the trend
observed in the calculated binding energy values are in accordance to the trend observed in the
-
experimentally determined Kapp values between 2 and the substrates. Upon close examination of
the optimized geometries involving xylose, the M–O(substrate) bond distances were found to be
2
1

ACCEPTED MANUSCRIPT
slightly longer than the bond lengths for the other substrates. Furthermore, the C–C bond lengths
of the coordinated xylose reassemble to those of free xylose substrate. The effect of having less
6
σ-induction due to the missing C group on the ring of the pentose is factor to the observed
calculated free energy value.
<
Table 5>
4
. Conclusions
The rapidly increasing number of the publications dealing with coordination chemistry of
the carbohydrates in the last few decades is associated with the extensive advancement of
technological methods such as high field multinuclear NMR, X-ray, CD, FTIR, Raman
spectroscopy and molecular modeling calculation programs. Such technological advances has
made possible to assign the coordinating hydroxides and conformational changes due to metal
coordination of carbohydrates. To take advantage of such prospects, we synthesized and
investigated new copper(II) and zinc(II) complexes, (NH ) [1] and ((CH ) N) [2] , for potential
4
2
2
3 4
2
2
interaction with biologically important carbohydrates in aqueous alkaline solution. Both
NH ) [1] and ((CH ) N) [2] ) are very soluble in water and stable in a wide range of pHs.
(
4
2
2
3 4
2
2
Solution studies using NMR and ESI-MS techniques indicate that the complexes dissolve and
-
-
-
-
dissociate to produce complex anions 1 and 2 . In aqueous alkaline media, complexes 1 and 2
showed chelating ability towards the abundant and biologically relevant sugars, D-glucose, D-
mannose, D-xylose, and xylitol. Our investigation into the interaction of the substrates with the
complexes revealed that only 1:1 substrate/complex molar ratio are formed and sustained in
solution, even in the presence of stoichiometric excess of substrate. The determined binding
constant, pK , values ranging from 2.33 to 2.71 are 1 - 2 orders of magnitude lower than the
app
values reported for the multinuclear copper(II), cobalt(II) and iron(III) complexes in the
literature. Based on the pKapp values, the most tightly bound substrate to the complexes is D-
mannose. This observation was also supported by the Gibbs free energy results obtained from
the Density Functional Theory calculations on the system. Although direct structural evidence,
from single crystal X-ray structure, for these complexes is presently unavailable, the NMR study
on the zinc(II) complex suggested that binding of the carbohydrates with these types of
1
complexes most likely occurs through the hydroxyl groups attached to the anomeric carbon, C ,
2
and C . The results of this investigation will positively contribute to the field of carbohydrate
2
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recognition in aqueous media. Additionally, the present study provides important structural and
functional information relevant to various sugar-metabolizing metalloenzymes and catalysis.
5
. Abbreviations
bpdpo – N,N’-1,3-bis[(pyridin-2-ylmethyl)amino]propan-2-ol
CIS – Coordination Induced Shift
DFT – Density Functional Theory
DIEN – Diethylenetriamine
ECP – Effective Core Potentials
ESI-MS – Electron-Spray Ionization Mass Spectroscopy
H cbal – N-[2-carboxybenzomethyl]-β-alanine
2
H camb – N-[2-carboxybenzomethyl]-N-[carboxymethyl]-β-alanine
3
H ccdp – N,N’-Bis[2-carboxybenzomethyl]-N,N’-Bis[carboxymethyl]-1,3-diaminopropan-2-ol
5
HEN – N-(2-hydroxyethyl)ethylenediamine
hpnbpda – N,N’-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N’-diacetic acid
paeo – N-2-[(pyridin-2-ylmethyl)amino]ethanol
PCM – Polarizable Continuum Model
SPE – Single Point Energy
styDIEN – 4-(N-vinylbenzyl)-diethylenetriamine
tcdc – N,N,N’,N’-tetrakis(sodium carboxylatemethyl)-2,6-diaminocresolate
Acknowledgments
Financial support from the Welch Foundation in the form of Grant AX-1540 and the Department
of Chemistry at UTSA are greatly appreciated. The authors also would like to thank the
Chemistry Department, University of Texas at San Antonio, for funds to upgrade the X-ray
instrument and computers. Special thanks to Dr. David Black for helping with the ESI-MS
measurements and valuable discussions on the data. Mass spectroscopic analysis was supported
by a grant from the National Institute on Minority Health and Health Disparities
(G12MD007591).
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