The metal-binding sites of glycose phosphates

Article abstract of DOI:10.1039/b909431h

In aqueous solution, the reducing sugar phosphates d-arabinose 5-phosphate, d-ribose 5-phosphate, d-fructose 1,6-bisphosphate, d-fructose 6-phosphate, d-glucose 6-phosphate and d-mannose 6-phosphate provide metal-binding sites at their glycose core on rea

Full text of DOI:10.1039/b909431h

View Article Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/dalton | Dalton Transactions
The metal-binding sites of glycose phosphates†
Kathrin Gilg, Tobias Mayer, Natascha Ghaschghaie and Peter Kl u¨ fers*
Received 12th May 2009, Accepted 23rd June 2009
First published as an Advance Article on the web 31st July 2009
DOI: 10.1039/b909431h
In aqueous solution, the reducing sugar phosphates D-arabinose 5-phosphate, D-ribose 5-phosphate,
D-fructose 1,6-bisphosphate, D-fructose 6-phosphate, D-glucose 6-phosphate and D-mannose
II
III
6
-phosphate provide metal-binding sites at their glycose core on reaction with Pd (en) or M (tacn)
residues (M = Ga, Co; en = ethylenediamine, tacn = 1,4,7-triazacyclononane). The individual species
were detected by one- and two-dimensional NMR spectroscopy. The coordination patterns are related
to the metal-binding modes of the respective parent glycoses. In detail, ribo- and arabinofuranose
1
,3
phosphate favour kO coordination, whereas the ketofuranose core of fructose phosphate and fructose
2
,3
bisphosphate provides the kO chelator thus maintaining the configuration of the respective major
solution anomer. On palladium excess, D-fructose 6-phosphate is metallated twice in a unique
1
,3
2,4
kO : kO metallation pattern. Dimetallation is also found for the aldohexose phosphates. A mixed
II
III
glycose-core–phosphate chelation was detected for Pd (en) and M (tacn) residues with M = Al, Ga in
the pH range just above the physiological pH for the D-fructose 1,6-bisphosphate ligand. The results are
discussed in relation to D-fructose-1,6-bisphosphate-metabolism in class-II aldolases.
Introduction
Reducing glycose phosphates play a decisive role in all organ-
isms’ metabolism. Glucose polymers such as starch and their
degradation products are converted into D-glucose 6-phosphate in
the glycolytic pathway, the next step of which is the isomerisation
to D-fructose 6-phosphate. Similarly, D-mannose is phosphory-
lated to D-mannose 6-phosphate. Both aldohexose-metabolising
pathways join in the next step, the formation of D-fructose 1,6-
Biomolecules such as amino acids, nucleosides and nucleotides, as
well as their oligo- and polymeric forms are, from the viewpoint
of coordination chemistry, typical ambident ligands. Depending
on factors such as stoichiometry, pH value and the kind of central
metal, these bioligands may act as chelators of variable denticity
and ligator-atom type. However, though a marked flexibility with
respect to their metal-binding properties is observed, these ligands
all have in common the fact that they adapt their ligator-atom
pattern on the level of conformational changes: their configuration
is invariable.
2
bisphosphate. This latter tetraanion is well-suited to demonstrate
the principles of the ligand-library approach to an aldose’s
coordination chemistry. Restricting this introductory discussion
to the bidentate chelating mode, the bisphosphate’s parent glycose,
the ketohexose D-fructose, is expected to provide various cis-
Reducing sugars (aldoses and ketoses = glycoses) constitute
a unique bioligand class. In contrast to the well-established
bioligands mentioned above, glycoses provide ligands of both
adaptable configuration and conformation to a metal. Even a
simple molecule such as the parent glycose of one of the ligands
in this work, the aldopentose D-arabinose, is able to provide
various types of bidentate chelators to a metal, among them diol
functions supplied by a- and b-D-arabinofuranose and a- and
1
,3 and cis-1,2-chelating diol functions through both the two
fructofuranose (a- and b-D-Fruf ) and the two fructopyranose (a-
and b-D-Frup) anomers. Examples of actually observed bonding
modes include the dianionic b-D-Fruf 1,2H-2-kO chelator with
silicon and the tetraanionic b-D-Frup2,3,4,5H-4-kO : kO bis-
chelator with boron.
On 1,6-bisphosphorylation, the pure hydroxy-function-based
fructose ligand turns into a true ambident molecule by now bear-
ing distinctly different functions. Thus, at first glance, the ligand
properties of the parent glycose that are diverse anyway, appear to
be further complicated. However, there is a marked shrinking of
the fructose’s original ligand library. Since the 6-position is blocked
by a phosphate function, only two of the previous four isomers of
the fructose core are accessible, namely the two furanose anomers.
Hence, an aqueous D-fructose 1,6-bisphosphate solution is made
1,2
2,3
4,5
3,4
1
b-D-arabinopyranose. Bearing in mind the interconvertibility of
the various isomers, D-arabinose, though being a single substance,
provides a central metal with a dynamic ligand library. This unique
property is shared by glycose derivatives whose derivatisation had
not affected the glycose’s anomeric centre (C1 for an aldose, C2
for a ketose). Such reducing glycose derivatives include glycuronic
acids, deoxy glycoses, glycosamines (2-amino-2-deoxy-glycoses)
and the focus of this work, the non-anomerically phosphorylated
glycose phosphates.
up of these two furanose anomers only, the minor a-D-Fruf 1,6P
2
5
(
12%) and the major b-D-Fruf 1,6P isomer (88%).
2
To assess the metal-binding sites of a glycose phosphate, the
major form of D-fructose 1,6-bisphosphate, the b anomer, may
be chosen as an example (Scheme 1). Various regions appear to
be attractive for a metal fragment: the phosphate residues, mixed
glycose-core–phosphate chelates, and chelates that are provided by
the glycose core solely after deprotonation of a cis-diol function.
Department of Chemistry and Biochemistry of the Ludwig Maximilian
University Munich, Butenandtstr. 5-13, D-81377 Munich, Germany.
E-mail: kluef@cup.uni-muenchen.de; Fax: +49 89 2180 77407
†
Electronic supplementary information (ESI) available: Coupling con-
stants and derived conformations. See DOI: 10.1039/b909431h
7
934 | Dalton Trans., 2009, 7934–7945
This journal is © The Royal Society of Chemistry 2009

View Article Online
A specific bonding situation was adjusted by the addition of a
stoichiometric amount of base needed for the deprotonation of the
metal-binding hydroxy functions of the glycose core. An example
may be derived from Scheme 1. M is to be considered a fragment
with three vacant coordination sites. In an attempt to bind M to
the deprotonated 2,3-cis-diol function and one phosphate-oxygen
donor, two equivalents of base were added.
The investigated reducing glycose phosphates, all of which
occur in metabolic pathways, were chosen to address the various
issues of glycose-phosphate–metal interactions: (1) basic rules
of glycose-core–metal chelation are derived from aldopentose
Scheme
1 The potential bidentate metal-binding sites of
b-D-fructofuranose 1,6-bisphosphate (in its protonation state about the
physiological pH): (1) formation of a chelate with glycose-core hydroxy
functions and a metal M sufficiently acidic to force deprotonation;
5
-phosphates; (2) D-fructose 1,6-bisphosphate then shifts the focus
(
2) formation of eight- (left M¢) and seven-membered (right M¢) chelate
rings by attack of a metal M¢ to a mixed glycose-core–phosphate ligand set;
3) transformation of a phosphate residue to a bridging ligand by a metal
fragment M , as described in ref. 8 for M
= Cu
to mixed chelates and to the physiological pH range; (3) for
the related D-fructose 6-phosphate dimetallation is probed, and,
finally, (4) preliminary results on the aldohexose 6-phosphates
highlight their close relationship to the parent aldohexoses
(
n
n
2
.
(
Scheme 2).
It might be expected that the mixed sites would not play a
considerable role as a glycose phosphate’s metal-binding site since
the ring sizes of the tentative chelates are rather large. Hence the
left phosphate group in Scheme 1 would be, together with the
4
-hydroxy function, part of an unfavourable eight-membered
chelate ring. In the case of a 1-phosphorylated ketose, however,
it should be noted that a less unstable seven-membered ring is
6
possible (the right phosphate group in Scheme 1). Accordingly,
the association of magnesium and zinc ions to the 1-phosphate
3
1
group of D-fructose 1,6-bisphosphate has been deduced from
NMR spectra.
P
7
Among the factors that govern the actual binding site of an
individual central metal, the interplay of the metal’s Lewis acidity
and the solution pH appears decisive. Hence, the design of a special
phosphate-directed assembly of two moderately acidic copper(II)
centres without the supply of additional base enabled the Tanase
group to isolate a phosphate-bonded coordination compound
8
of the D-Fruf 1,6P
2
ligand. Neither metallation of a reducing
glycose-phosphate’s glycose core nor the mixed phosphate–glycose
ligation has yet been unequivocally demonstrated outside of an
enzyme’s active site. Even a stronger Lewis acid such as the
aluminium(III) centre has been found to be exclusively phosphate-
9
Scheme 2 Formulae and symbols used for the glycose phosphates
used in this work. Top: D-arabinofuranose 5-phosphate, D-ribofuranose
5-phosphate; middle: D-fructofuranose 6-phosphate, D-fructofuranose
binding in a study on D-glucose 6-phosphate. However, stable
metal complexes of the parent glycoses of this work have been
10–14
found with di- and trivalent Lewis acidic metal centres.
1
,6-bisphosphate; bottom: the pyranose forms of D-mannose 6-phosphate
Among the enzymes that act on sugar phosphates, class-II
aldolase shows the closest relationship to the complexes of this
work. Class-II aldolase cleaves D-fructose 1,6-bisphosphate to
dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate.
Two recent single-crystal analyses of substrate-bonding class-II
and D-glucose 6-phosphate, the latter two aldohexose 6-phosphates being
capable of forming furanose isomers, in principle. Wavy bonds indicate
a/b anomerism.
15,16
aldolases are available.
In both diffraction studies, the substrate
Experimental
is not captured in its furanose ground state but in an activated,
open-chain keto form. A mononuclear zinc centre, whose Lewis
acidity is high due to its fixation at the protein via neutral ligands
only (three histidine residues), is chelated by O3 and O4 of the
fructose core. No information is presently available about the
initial binding of ground-state D-fructofuranose 1,6-bisphosphate
to the metal centre.
Herein we describe the formation of metal complexes of various
reducing glycose phosphates. The metal centres have been chosen
to bear sufficient Lewis acidity to make the glycose core the
ligand in an aqueous environment somewhere on the pH scale.
Methods and materials
All chemicals were purchased and used without further purifi-
cation: disodium D-arabinose 5-phosphate, disodium D-ribose
5-phosphate dihydrate, dipotassium D-fructose 6-phosphate,
dipotassium D-glucose 6-phosphate hydrate, sodium D-mannose
6-phosphate, cobalt(II) chloride hexahydrate, gallium chloride
(Sigma-Aldrich), aluminium chloride hexahydrate (Fluka), deu-
terium oxide (Eurisotop), active charcoal, ethylenediamine
(Gr u¨ ssing), trisodium D-fructose 1,6-bisphosphate octahydrate
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7934–7945 | 7935

View Article Online
1
3
Table 1
C NMR shifts of metal complexes of the aldopentose 5-phosphates of D-arabinose and D-ribose. Dd is the difference of a shift in the presence
of the metal probe and the free glycose phosphate. Note the large Dd values in the case of five-membered palladacycles
C1
C2
C3
C4
C5
1
,3
[
[
[
[
[
[
[
[
Pd(en)(a-D-Araf 5P1,3H-2-kO )]2-
d
104.4
3.3
110.8
15.5
96.4
0.0
111.6
15.2
102.1
0.8
100.6
4.0
82.4
1.0
88.7
12.5
72.4
1.5
82.5
11.6
87.8
12.3
72.8
2.1
76.8
1.1
78.9
4.5
88.1
5.6
81.3
0.6
86.1
3.4
79.7
-3.0
84.7
2.7
85.2
1.2
85.3
2.3
84.7
0.5
64.6
1.1
65.5
0.9
64.4
0.4
64.6
0.6
66.2
1.4
65.1
0.8
64.3
0.1
64.1
1.0
a
a
a
a
Dd
d
Pd(en)(b-D-Araf 5P1,2H-2-kO )]2-
1,2
a
Dd
d
Pd(en)(a-D-Ribf 5P1,3H-2-kO )]2-
1,3
70.1
-0.3
71.7
1.3
a
Dd
d
Pd(en)(a-D-Ribf 5P1,2H-2-kO )]2-
1,2
Dd
d
Pd(en)(b-D-Ribf 5P2,3H-2-kO )]2-
2,3
83.4
12.5
73.7
3.0
72.9
2.3
a
a
a
a
a
a
a
a
Dd
d
Ga(tacn)(a-D-Ribf 5PH-3-kO )]2-
1
,2,3
a
a
a
Dd
d
Ga(Me
3
tacn)(a-D-Ribf 5PH-3-kO )]2-
1,2,3
100.3
3.7
103.1
3.5
71.9
1.3
84.7
13.9
Dd
d
Co(tacn)(a-D-Ribf 5PH-3-kO )]2-
1
,2,3
75.0
2.3
Dd
a
The metal-binding site.
(
Applichem), (Aldrich), palladium(II) chloride (Fluka), silver(I)
Batches of [Pd(en)Cl
silver(I) oxide (2.40 g, 10.41 mmol) in D
2
] (2.00 g, 8.42 mmol) were suspended with
O (16.8 mL) under a
oxide (VWR), 1,4,7-triazacyclononane trihydrochloride (TCI
Europe), and sodium hydroxide pellets (Biesterfeld-Gra e¨ n). Re-
actions were carried out using standard Schlenk techniques in a
nitrogen atmosphere.
2
nitrogen atmosphere and the exclusion of light. The mixture was
◦
heated to 40 C for 15 min and filtrated. The resulting clear, yellow,
◦
ca. 0.5 M solution of [Pd(en)(OD)
2
] (“Pd-en”) was stored at -60 C.
1
3
C NMR (D
2
O): d [ppm] = 45.4.
NMR spectroscopy
Glycose phosphate solutions in Pd-en. Under ice cooling, 0.2–
0
.7 mmol of glycose phosphate was dissolved in a threefold
NMR spectra were recorded at room temperature on Jeol ECP
amount of Pd-en (ca. 0.5 M solution in D O). The yellow solution
was stirred for about 2 h and immediately placed under NMR
investigation. The solutions were either investigated immediately
or stored at -60 C. For D-ribose 5-phosphate, solutions of 1 : 1
molar ratio were investigated as well. The respective C NMR
2
1
13
27
31
2
70 ( H: 270 MHz, C: 67.9 MHz, Al: 70.4 MHz, P: 109 MHz,
7
1
1
13
1
Ga: 82.4 MHz), Jeol ECX 400/ECP 400 ( H: 400 MHz, C{ H}:
3
1
1
1
00 MHz, P: 109 MHz) and Jeol ECP 500 ( H: 500 MHz,
◦
1
3
1
C{ H}: 125 MHz) spectrometers. The signals of the deuterated
1
3
1
3
1
solvent ( C) and the residual protons therein ( H) were used as
glycose signals are collected in Table 1.
an internal secondary reference for the chemical shift. When D
was used as a solvent, a small capillary tube containing [D ]DMSO
was added to the sample tube (5 mm) in order to obtain a reference
2
O
1
31
6
H and P NMR data of D-arabinose 5-phosphate complexes.
1
,3 2-
1
[Pd(en)(a-D-Araf 5P1,3H-2-kO )] : H NMR (D
3.09 (s, 1H, H3), 3.40 (s, 1H, H2), 3.57–3.66 (m, 2H, H5a, H5b),
4.23 (t, 1H, H4, J4,5 5.2 Hz), 4.43 (s, 1H, H1). P NMR (D
d [ppm] = 4.39.
2
O): d [ppm] =
1
3
signal in the C spectra. Shift differences are given as d(Ccomplex) -
d(Cfree sugar). The values for the free glycose phosphates were taken
3
31
2
O):
from our own measurements in D
2
O and were thus in neutral
1
,2 2-
1
aqueous solution.
[Pd(en)(b-D-Araf 5P1,2H-2-kO )] :
H
NMR
(D
2
O):
3
d [ppm] = 3.47 (t, 1H, H2, J2,3 3.6 Hz), 3.57–3.66 (m, 1H,
3
H4), 4.02 (m, 2H, H5a, H5b), 4.12 (t, 1H, H3, J3,4 4.1 Hz), 4.91
Syntheses
3
31
(
d, 1H, H1, J1,2 3.3 Hz). P NMR (D
2
O): d [ppm] = 4.75.
Preparation of [Pd(en)(OD) ]. To the brown suspension of
2
1
31
H and P NMR data of D-ribose 5-phosphate complexes.
palladium(II) chloride (10.16 g, 57.4 mmol) in water (50 mL),
concentrated hydrochloric acid (10 mL) was added. Two thirds of a
solution of ethylenediamine (13.5 mL, 202 mmol) in water (30 mL)
1
,3 2-
1
[
Pd(en)(a-D-Ribf 5P1,3H-2-kO )] : H NMR (D
2
O): d [ppm] =
3
3
.05 (d, 1H, H3, J3,4 5.2 Hz), 3.49–3.54 (m, 2H, H5), 3.86 (t, 1H,
3
3
H2, J2,3 5.2 Hz), 4.19 (t, 1H, H4, J4,5a 5.0 Hz), 4.32 (d, 1H, H1,
was added dropwise, yielding a pink precipitate. After warming to
3
31
◦
J
1,2 4.4 Hz). P NMR (D
2
O): d [ppm] = 4.67.
NMR (D
ppm] = 3.49–3.54 (m, 2H, H5), 3.56–3.57 (m, 1H, H3), 3.57 (d,
6
0 C and the addition of the remaining ethylenediamine solution,
1
,2 2-
1
[
Pd(en)(a-D-Ribf 5P1,2H-2-kO )] :
H
2
O):
d
the solid dissolved. The mixture was filtrated and adjusted to pH 2
[
with half-concentrated hydrochloric acid. The reaction tube was
3
3
◦
1H, H2), 4.17 (t, 1H, H4, J4,5a 5.2 Hz, J4,5b 5.0 Hz), 4.92 (d, 1H,
stored at 0 C for 2 h, and a yellow precipitate of [Pd(en)Cl
2
]
3
31
H1, J1,2 3.3 Hz). P NMR (D
2
O): d [ppm] = 4.67.
formed. The solid was filtrated and dried. The remaining solution
was again adjusted to pH 2. More product precipitated in the
course of 2 h. The combined yellow solids were dried in vacuum
2,3 2- 1
[
Pd(en)(b-D-Ribf 5P2,3H-2-kO )] : No assigned H data avail-
3
1
able due to multiple cross-peak overlap. P NMR (D
ppm] = 4.78.
2
O): d
[
(
9.95 g, 73% yield). Anal. calcd for C
2
H
8
N
2
Cl
2
Pd: C, 10.12; H,
1
31
3
1
.40; Cl, 29.86; N, 11.80. Found: C, 10.15; H, 3.41; Cl, 29.87; N,
1.67.
H and P NMR data of D-fructose 1,6-bisphosphate com-
2
,3 4-
1
plexes. [Pd(en)(b-D-Fruf 1,6P
2
2,3H-2-kO )] : H NMR (D O):
2
7
936 | Dalton Trans., 2009, 7934–7945
This journal is © The Royal Society of Chemistry 2009

View Article Online
2
3
d [ppm] = 3.44 (dd, 1H, H1a, J1a,1b 10.7 Hz, J1a,P 5.0 Hz), 3.59 (d,
hydroxide (0.048 g, 1.20 mmol) were dissolved in D
2
O (2 mL).
3
1
H, H3, J3,4 6.6 Hz), 3.67–3.73 (m, 1H, H5), 3.84 (dd, 1H, H1b,
1b,P 2.7 Hz), 3.88–4.04 (m, 2H, H6a, H6b), 4.4 (t, 1H, H4, J4,5
A colourless solution was obtained after vigorous stirring for
3
3
71
J
5 min at room temperature. Ga NMR (82.4 MHz, H
2
O): d =
3
1
-
31
1
13
1
7
.2 Hz). P NMR (D
2
O): d [ppm] = 4.43, 4.79.
222.2 ([Ga(OH)
(100.6 MHz, D
NMR (399.4 MHz, D
4
] ), 110.2. P{ H} NMR: d = 3.6. C{ H} NMR
2
,4 4-
1
1
[
Pd(en)(a-D-Fruf 1,6P
2
2,4H-2-kO )] : H NMR (D
2
O): d
2
O): ribose signals see Table 1, 42.6–41.7 (tacn). H
3
[
ppm] = 3.11 (s, 1H, H4), 3.60 (s, 1H, H3), 3.66–3.71 (m, 2H,
2
O): d = 5.28 (d, 1H, J1,2 = 3.7 Hz, H1),
3
1
3
3
3
H1, H6), 3.88–3.94 (m, 2H, H6 bzw, H1), 4.26 (m, 1H, H5).
P
4.25 (t, 1H, J4,5 = 5.3 Hz, H4), 4.20 (dd, 1H, J2,1 = 3.7 Hz, J2,3
=
3
NMR (D
2
O): d [ppm] = 4.64, 5.58.
5.1 Hz, H2), 3.88 (d, 1H, J3,2 = 5.1 Hz, H3), 3.76–3.70 (m, 2H,
H5/H5¢), tacn: 3.32–3.11 (m), 2.92–2.84 (m).
1
31
H and P NMR data of D-fructose 6-phosphate complexes.
Essentially the same result was obtained by the use of
the N,N¢,N¢¢-trimethyl derivative of 1,4,7-triazacyclononane ( C
NMR resonances in Table 1).
1
,3
2,4 2-
1
[
[
(
7
Pd
2
(en)
2
(a-D-Fruf 6PH-4-kO : kO )] : H NMR (D
2
O): d
1
3
2
ppm] = 2.66 (d, 1H, H1a, J1a,1b 11.6 Hz), 2.85 (s, 1H, H3), 2.88
3
3
d, 1H, H1b), 2.90 (s, 1H, H4), 4.35 (t, 1H, H5, J5,6a 6.0 Hz, J5,6
3
1
.7 Hz), 4.55–4.60 (m, 2H, H6a, H6b). P NMR (D
.03.
Pd(en)(b-D-Fruf 6P2,3H-2-kO )] : H NMR (D
.30 (d, 1H, H1a, J1a,1b 12.1 Hz), 3.49 (d, 1H, H3, J3,4 6.9 Hz),
.61 (d, 1H, H1b), 3.58–3.62 (m, 1H, H5), 3.87–3.93 (m, 1H, H6a),
.96–4.01 (m, 1H, H6b), 4.51 (t, 1H, H4, J4,5 7.7 Hz). P NMR
O): d [ppm] = 4.75.
Pd(en)(a-D-Fruf 6P2,4H-2-kO )] :
ppm] = 3.17 (s, 1H, H4), 3.52 (s, 1H, H3), 3.46–3.56 (m, 1H,
H1a), 3.64–3.66 (m, 1H, H1b), 3.66–3.72 (m, 1H, H6a), 3.97–4.01
2
O): d [ppm] =
2
,3,P
3-
[
Al(tacn)(b-D-Fruf1,6P 2,3H-2-kO )] . Aluminium chlo-
2
5
2
,3 2-
1
ride hexahydrate (0.048 g, 0.20 mmol), 1,4,7-triazacyclononane
trihydrochloride (0.048 g, 0.20 mmol), trisodium D-fructose 1,6-
bisphosphate (0.110 g, 0.20 mmol) and sodium hydroxide (0.048 g,
[
2
O): d [ppm] =
2
3
3
3
3
3
31
1.20 mmol) were dissolved in D
2
O (2 mL). A colourless solution
was obtained after vigorous stirring for 30 min at room temper-
(
D
[
2
2
7
-
2
,4 2-
1
ature. Al NMR (70.4 MHz, H
2
O): d = 80.4 ([Al(OH)
4
] ), 40.0
H
NMR (D
2
O):
d
3
1
1
(
broad). P{ H} NMR (109.4 MHz, H
P NMR (109.4 MHz, H
2
O): d = 3.8, 3.7, -2.9.
[
3
1
3
2
O): d = 3.7 (t, JP,H = 4.7 Hz), -2.9
3
3
13
1
3
3
31
(dd, JP,H = 4.7 Hz, JP,H = 24.7 Hz). C{ H} NMR (100.6 MHz,
(
m, 1H, H6b), 4.23 (t, 1H, H5, J5,6a 4.6 Hz, J5,6b 5.0 Hz).
P
D
6
2
O): d = 105.3 (C2), 80.6 (C3), 79.5 (C4), 78.3 (C5), 72.3 (C1),
NMR (D
2
O): d [ppm] = 4.34.
1
4.3 (C6), 44.6–43.5 (tacn). H NMR (399.4 MHz, D
2
O): d = 4.10
1
31
H and P NMR data of D-glucose 6-phosphate complexes.
3
(
d, 1H, J3,4 = 6.9 Hz, H3), 3.95–3.83 (m, 2H, H6), 3.77 (dd, 1H,
4,3 = 6.9 Hz, J4,5 = 9.1 Hz, H4), 3.65 (m, 3H, J4,5 = 9.1 Hz,
H1/H5).
1
,2
3,4 2-
1
[
[
Pd
2
(en)
2
(a-D-Glcp6PH-4-kO : kO )] : H NMR (D
2
O): d
3
3
3
J
3
ppm] = 2.66 (dd, 1H, H2, J2,3 9.0 Hz), 2.89–2.93 (m, 1H, H4),
3
.60–3.64 (m, 1H, H6a), 3.72–3.76 (m, 1H, H5), 3.83–3.87 (m, 1H,
3
31
H6b), 4.23–4.27 (m, 1H, H3), 4.90 (d, 1H, H1, J1,2 3.9 Hz).
NMR (D
O): d [ppm] = 5.39.
Pd (en) (b-D-Glcp6PH-4-kO : kO )] : H NMR (D O): d
P
2,3,P
3-
[
Ga(tacn)(b-D-Fruf1,6P 2,3H-2-kO )] . Gallium chloride
2
2
(0.035 g, 0.20 mmol), 1,4,7-triazacyclononane trihydrochloride
(0.048 g, 0.20 mmol), trisodium D-fructose 1,6-bisphosphate
(0.110 g, 0.20 mmol) and sodium hydroxide (0.048 g, 1.20 mmol)
1
,2
3,4 2-
1
[
2
2
2
3
[
3
ppm] = 2.90–2.95(m, 1H, H4), 2.94–2.99(m, 1H, H2, J2,3 9.1Hz),
3
.20 (t, 1H, H3, J3,4 9.1 Hz), 3.32–3.36 (m, 1H, H5), 3.54–3.58
were dissolved in D O (2 mL). A colourless solution was obtained
2
3
1
(
(
m, 1H, H6a), 3.83–3.87 (m, 1H, H6b), 4.25 (s, 1H, H1). P NMR
31
1
after vigorous stirring for 15 min at room temperature. P{ H}
D
2
O): d [ppm] = 5.24.
31
NMR (161.8 MHz, D
2
O): d = 4.8 (broad), 0.6 (broad). P NMR
1
31
13
1
H and P NMR data of D-mannose 6-phosphate complexes.
Pd(en)(b-D-Manp6P1,2H-2-kO )] : H NMR (D
.08–3.12 (m, 1H, H5), 3.45 (dd, 1H, H3, J3,4 3.6 Hz), 3.65–3.66
m, 1H, H4), 3.66–3.72 (m, 1H, H6a/b), 3.72 (d, 1H, H2, J2,3
.9 Hz), 3.82–3.93 (m, 1H, H6a/b), 4.19 (s, 1H, H1). P NMR
O): d [ppm] = 5.31.
Pd (en) (b-D-Manp6PH-4-kO : kO )] : H NMR (D
(161.8 MHz, D
(125.7 MHz, D
2
O): d = 4.8 (broad), 0.6 (broad). C{ H} NMR
2
1
,2 2-
1
[
3
(
9
2
O): d [ppm] =
O): d = 103.0 (C2), 77.5 (C3), 77.5 (C4), 77.5
3
1
(C5), 70.4 (C1), 62.4 (C6), 41.0–40.3 (tacn). H NMR (500.2 MHz,
3
3
D
2
O): d = 4.07 (d, 1H, JH3,H4 = 6.6 Hz, H3), 3.95–3.84 (m, 3H,
3
1
H6/H6¢/H1), 3.74–3.67 (m, 3H, H1¢/H4/H5).
(
D
[
2
1
,2
3,4 2-
1
2
2
2
O): d
III
1,2,3 2-
Cobalt(III) complexes. [Co (tacn)(a-D-Ribf 5PH-3-kO )] :
[
1
(
5
ppm] = 3.08–3.12 (m, 1H, H5), 3.19–3.23 (m, 1H, H2), 3.37 (t,
To a solution of cobalt(II) chloride hexahydrate (0.048 g,
3
3
H, H4, J4,5 9.6 Hz), 3.62–3.66 (m, 1H, H3, J3,4 9.6 Hz), 3.82–3.93
0
.20 mmol) in D
drochloride (0.048 g, 0.20 mmol), disodium D-ribose 5-phosphate
0.055 g, 0.20 mmol) and sodium hydroxide (0.048 g, 1.2 mmol)
2
O (2.0 mL), 1,4,7-triazacyclononane trihy-
3
1
m, 2H, H6a, H6b), 4.09 (s, 1H, H1). P NMR (D
2
O): d [ppm] =
.34.
[
(
2
,3 2-
1
Pd(en)(a-D-Manp6P2,3H-2-kO )] :
H
NMR (D O):
2
d
were added under continuous stirring at room temperature. After
3
3
[
ppm] = 2.90 (dd, 1H, H3, J3,4 8.8 Hz), 3.56 (d, 1H, H2, J2,3
the addition of 0.2 g active charcoal, the mixture was heated to
3
1
5
.9 Hz), 3.61–3.64 (m, 1H, H5), 3.86–3.92 (m, 2H, H6), 4.13 (t,
◦
5
0 C for 2 h under vigorous stirring. A clear pink-red solution
3
31
H, H4, J4,5 9.6 Hz), 4.75 (s, 1H, H1). P NMR (D
2
O): d [ppm] =
1
3
1
was obtained after filtration and centrifugation. C{ H} NMR
100.5 MHz, D O): ribose signals see Table 1, tacn: 50.9–48.5.
A 0.1 ppm split of the ribose-C2/C4 signal is observed by using
.24.
(
2
III
Glycose phosphate ligation to an M (tacn) residue
1
H
2
O instead of D
2
O. H NMR (399.8 MHz, D
2
O): d = 4.50 (d,
3
3
Aluminium and gallium complexes. [Ga(tacn)(a-D-Ribf 5PH-3
-
1H, JH1,H2 = 3.30 Hz, H1), 4.04 (t, 1H, JH4,H5 = 5.50 Hz, H4),
1
,2,3 2-
kO )] : gallium chloride (0.035 g, 0.20 mmol), 1,4,7-
triazacyclononane trihydrochloride (0.048 g, 0.20 mmol), dis-
odium D-ribose 5-phosphate (0.055 g, 0.20 mmol) and sodium
3.83–3.81 (m, 1H, H2), 3.69–3.60 (m, 2H, H5/H5¢), 3.25 (d, 1H,
3
J
H3,H2 = 4.12 Hz, H3), tacn: 3.35–3.30 (m), 3.19–2.95 (m), 2.83–
2.63 (m).
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7934–7945 | 7937

View Article Online
Results and discussion
Consistency checks were performed as the last step of species
assignment. Thus both chemical shifts and CIS values of similar
partial structure show close resemblance through the series of
species. An example is given in the Conclusions section.
The experimental strategy
The maximum number of metal–spectator-ligand fragments that
are bonded to a glycose phosphate was not initially clear.
Hence, various metal : glycose-phosphate molar ratios were used
to elucidate the stoichiometries of the various solution species. A
typical series of spectra and their interpretation is shown below
in the D-fructose 6-phosphate chapter. If a number is given for
the relative amount of an individual species, it was derived by the
D-Arabinose 5-phosphate: a furanose ligand with a remote
phosphate function
D-Arabinose 5-phosphate, in comparison to D-arabinose, is con-
figurationally restricted to the two furanose anomers since the
phosphate residue blocks the 5-position which is required for the
formation of the pyranoses. The aqueous equilibrium mixture thus
consists of only two significant species, the a- (57.3%) and the
b-furanose (40.4%; minor species: 2.2% aldehyde hydrate and 0.2%
1
integration of H NMR spectra.
To ascertain the assignments made, basically the same exper-
1
13
31
imental strategy was applied. 1D H, C, P, DEPT-135 and
D HMQC and COSY NMR spectra of the reaction mixtures
2
5
aldehyde).
were recorded. From the 2D spectra, peaks were assigned to
their carbon atoms and to the various species. Furthermore, the
Dissolution of D-arabinose 5-phosphate in excess Pd-en (molar
Pd : glycose ratio of 3 : 1) resulted in the formation of two
monometallated species (Table 1, Scheme 3). The main prod-
3
1
13
characteristic P– C coupling supported the assignments. This
coupling led to a signal splitting of carbon atoms adjacent to the
phosphate group and of the ones next to those carbons. Hence,
due to the presence of two terminal phosphate groups in D-fructose
uct (66%) was the six-ring chelate [Pd(en)(a-D-Araf 5P1,3H-2
-
1,3 2-
kO )] . In terms of coupling constants, the furanose confor-
2
mation was found close to E which resembles a contraction of
1
,6-bisphosphate, C1, C2, C5 and C6 showed split signals in NMR
the O
2
ligand set to match the binding sites of the central metal.
1
spectra for this compound. In most cases, H NMR signals as well
as H,H coupling constants were also accessible and were used
for configurational and conformational analysis according to a
The minor product (34%) was the five-ring chelate [Pd(en)(b-D-
1,2 2-
3
Araf 5P1,2H-2-kO )] with a furanose conformation between E
3
and T
2
. Since no other signals were observed, the phosphate
17
13
modified Karplus relation. In addition, C spectra were used
to calculate coordination-induced shifts (CISs) which support the
determination of the metal-binding site. In particular, the case of
five-membered palladacycles was clearly indicated by typical CIS
values of about 10 ppm downfield.
group thus remained uncoordinated despite the metal-excess
conditions. Obviously, the formation of an eight-ring chelate such
as the left one in Scheme 1 is unfavourable and the tentative
1,2
3,P
-
dinuclear species [Pd
2
(en) (b-D-Araf 5PH-3-kO : kO )] , which
2
is derived from the right-most species in Scheme 3, was not
detected. This statement holds also for solutions that exactly match
the stoichiometry of the tentative dinuclear complex.
3
1
P NMR spectroscopy was used to detect the incorporation
of a phosphate group into a chelate ring, a case that was
observed for D-fructose 1,6-bisphosphate at pH values close to
3
1
neutrality. In the absence of such rings, both the P chemical shifts
3
1
1
and the P– H coupling constants resembled the values of free
7
D-fructose 1,6-bisphosphate at the respective pH. If, instead, the
phosphate group contributes to a chelate ring, its characteristic
rotamer distribution about the phosphate–methylene bond freezes
to a conformer compatible with the chelate. To assess the
P–O–C–C torsion angle in these phospha cycles, Karplus curves
derived for the related case of a nucleotide’s conformation about
18,19
the P–O–C5–C4 axis may be used.
However, it should be noted
that a detailed conformational analysis is hampered by the fact
that the torsion about the neighbouring axis, the C2–C1 bond,
remains unknown (a ketose lacks a proton at its anomeric centre;
hence, a useful number such as a related H– H coupling constant
is not available).
Scheme
3
Products of the reaction of Pd-en with D-arabinose
1
1
5
-phosphate.
To obtain the spectroscopic results, side reactions had to be
avoided. Particularly annoying reactions were observed in two
cases: (1) the redox stability of palladium(II)-containing solutions
may be so low that Fehling-like reactions proceeded even under
cooling. Hence, only limited results were obtained for equimolar
solutions of the aldohexose 6-phosphates which revealed them-
selves as particularly prone to palladium precipitation. (2) If the
Al(tacn) fragment was reacted with a glycose phosphate and the
required amount of base was added to trigger tridentate metal-
binding by the glycose core, the known formation of a N-glycoside
Since only monometallated products were observed, a similar
result was obtained on dissolution of D-arabinose 5-phosphate in
an equimolar amount of Pd-en. As expected, the only difference
between the spectra stems from the occurrence of unmetallated
glycose phosphate in the metal-reduced batch. With its two-
species metallation behaviour, D-arabinose 5-phosphate exhibits
a distinctly simpler coordination chemistry towards palladium(II)
than the parent glycose, D-arabinose. At varying metal supply, the
latter forms no less than five metallation products, since pyranose
20
1,3
1,2
of tacn took place. This side reaction was not observed with the
other metal–amine–glycose combinations of this work.
ligands add to the kO - and kO -bonding furanoses that are
1,12
common to the glycose and its 5-phosphate.
7
938 | Dalton Trans., 2009, 7934–7945
This journal is © The Royal Society of Chemistry 2009

View Article Online
Scheme 4 Products of the reaction of Pd-en with D-ribose 5-phosphate.
D-Ribose 5-phosphate: bi- and tridentate glycose-core chelators
BIDENTATE PALLADIUM(II) CHELATION. D-Ribose 5-phosphate,
like D-arabinose 5-phosphate, is restricted to its two furanose
isomers. At aqueous equilibrium, 35.6% a- and 63.9% b-furanose
were detected by NMR spectroscopy (minor species: 0.5% hydrate
5
and 0.1% aldehyde). In contrast to D-arabinose 5-phosphate, the
2
-hydroxy function is cis to the 1- and 3-hydroxy groups in D-
ribose 5-phosphate. Hence, one more bidentate chelation mode,
2
,3
kO , was expected.
In fact, upon the reaction of Pd-en with D-ribose 5-phosphate
at a molar Pd : glycose ratio of 3 : 1, two major and one
minor species were observed (Scheme 4). With [Pd(en)(a-D-
1
,3 2-
2
Ribf 5P1,3H-2-kO )] (40%, conformation close to E) and
1
,2 2-
2
[
Pd(en)(a-D-Ribf 5P1,2H-2-kO )] (20%, conformation T
3
or
[uncertainty due to missing coupling constants because of
signal overlap]), one of the major and the minor species resemble
3
T
2
1
,3
1,2
the furanose-kO and furanose-kO species of the arabinose
analogue. The second major species was assigned to [Pd(en)(b-D-
2
,3 2-
3
Ribf 5P2,3H-2-kO )] (40%, T ). This species was not detected
2
with the parent glycose, D-ribose, on dissolution in the related
II
reagent Pd–chxn, an aqueous solution of [Pd {(R,R)-chxn}(OH)
2
]
1
2,3
(
chxn = cyclohexane-1,2-diamine). Obviously, the kO chelator
gains stability with the phosphorylated glycose. The C1 and C2
signals of this species were broadened at the 3 : 1 molar ratio of
Pd : glycose due to rapid mutarotation under these base-excess
conditions. At an equimolar ratio, however, sharp signals of the
major b anomer arose in addition to small signals of the minor
1
3
a anomer. C NMR shifts of the products are listed in Table 1.
1
3
13
Particularly well resolved C NMR spectra were obtained with
the related Pd–chxn solvent. Fig. 1 (top) shows the respective
spectrum. A characteristic property of the palladium probes
becomes apparent, namely the ability to develop all the bidentate
members of a glycose-based ligand library. In contrast, tridentate
probes such as those described in the next paragraph normally find
much fewer bonding partners in a glycose’s equilibrium mixture.
Fig. 1 Top: C NMR spectrum of the ribose part of ribose 5-phos-
phate in a Pd–chxn solution at a molar Pd : D-Ribf 5P ratio of
1
: 1. For each of the three species, the anomer and the Pd-bond-
ing site is indicated in a short notation such as a1,3 for the main
II
1,3 2-
[
Pd {(R,R)-chxn}(a-D-Ribf 5P1,3H-2-kO )] species. The signals of the
main species are connected to the respective signals of free a-D-Ribf 5P.
Note the relatively small downfield shift of this six-ring chelate compared
with the larger shifts of the two five-ring chelates. Note as well a residual
13
amount of free ribose phosphate in the Pd–chxn solution. Middle:
C
NMR spectrum of the equilibrium mixture of the minor a- and the
major b-D-ribose 5-phosphate anomer. Note the splitting of the C5 and,
particularly, the C4 signal due to the P– C coupling. Bottom: C NMR
spectrum of the ribose part of the same solution after reaction with
the Ga(tacn) precursor. Note the complete transformation of the b to
a anomer and the downfield shift on coordination.
Tridentate metal(III) chelation. The a-D-ribofuranose part of
D-ribose 5-phosphate is able to cover a fac-O position of a
3
31
13
13
coordination polyhedron. This is demonstrated (1) by the reaction
III
of the Ga (tacn) residue (tacn = 1,4,7-triazacyclononane) with
D-ribose 5-phosphate and the required amount of base, and (2) by
the reaction of cobalt(II) and tacn with D-ribose 5-phosphate and
the required amount of base on exposure to air. Solutions of a pH
close to 11.5 resulted. As attempted, the NMR spectra (Table 1)
is particularly useful in this case since it is the minor component
of an aqueous D-ribose 5-phosphate solution which provides the
III
III
showed the signals of a single species each, the [M (tacn)(a-D-
ligand. On reaction with the M (tacn) precursors, the minor
1
,2,3 2-
Ribf 5PH-3-kO )] ion (Scheme 5; M = Co, Ga). It should be
a-anomer was enriched at the expense of the major b-isomer. The
mutarotation equilibrium progressively readjusts in the course of
noted that to view a glycose solution as a dynamic ligand library
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7934–7945 | 7939

View Article Online
1
3
Table 2
C NMR shifts of the products from the reaction of various metal probes with D-fructose 1,6-bisphosphate and the hexose 6-phosphates of
D-fructose, D-glucose and D-mannose (molar Pd : glycose ratio of 3 : 1). Note the large Dd values in the case of five-membered pallada cycles
C1
66.4
0.0
65.5
0.2
64.0
0.7
64.2
1.0
62.8
-0.5
102.4
9.8
106.0
9.5
106.5
12.5
107.9
13.9
94.4
0.0
C2
C3
C4
C5
C6
2,3H-2-kO )]4-
2
,3
d
114.7
13.1
109.0
3.5
111.5
6.6
114.7
12.8
107.9
3.0
83.3
11.3
85.2
10.5
82.7
11.3
85.8
14.4
81.2
10.4
105.2
5.1
87.5
11.0
82.7
0.6
87.1
4.9
87.4
11.7
83.1
0.9
86.4
13.7
87.1
11.5
72.3
-0.4
84.1
11.4
79.7
9.7
79.7
4.9
78.7
1.5
80.3
3.9
79.6
4.7
79.0
2.6
78.9
9.4
80.3
10.8
67.2
1.0
75.4
9.2
80.9
0.7
88.6
6.2
89.0
8.1
80.6
0.3
88.2
7.3
74.0
2.5
75.9
0.0
74.9
-0.8
74.7
-1.0
71.8
-0.3
78.3
0
66.2
1.2
64.9
0.2
66.8
2.9
66.0
1.2
65.0
1.1
63.3
0.2
63.8
0.6
63.7
0.6
63.6
0.5
64.4
1.4
64.2
0
62.4
-1.8
[
[
[
[
[
[
[
[
[
[
[
[
Pd(en)(b-D-Fruf 1,6P
Pd(en)(a-D-Fruf 1,6P
Pd (en)
(a-D-Fruf 6PH-4-kO : kO )]2-
Pd(en)(b-D-Fruf 6P2,3H-2-kO )]2-
2
a
a
a
a
a
a
a
a
a
a
a
a
a
Dd
d
2,4H-2-kO )]4-
2
,4
2
a
Dd
d
1,3
2,4
2
2
a
a
a
a
Dd
d
2,3
Dd
d
Pd(en)(a-D-Fruf 6P2,4H-2-kO )]2-
2,4
a
a
a
Dd
d
1,2
3,4
Pd
Pd
2
2
(en)
2
2
(a-D-Glcp6PH-4-kO : kO )]2-
a
a
a
a
a
a
Dd
d
1,2
3,4
(en)
(b-D-Glcp6PH-4-kO : kO )]2-
Dd
d
Pd(en)(b-D-Manp6P1,2H-2-kO )]2-
1
,2
Dd
d
1,2
3,4
Pd
2
(en)
2
(b-D-Manp6PH-4-kO : kO )]2-
a
a
a
a
a
Dd
d
Pd(en)(a-D-Manp6P2,3H-2-kO )]2-
2
,3
70.0
3.5
79.5
4.4
77.5
2.4
Dd
d
Al(tacn)(b-D-Fruf 1,6P
Ga(tacn)(b-D-Fruf 1,6P
2
2,3H-3-kO )]3-
2
,3,P
72.3
6.2
70.4
4.3
80.6
5.6
77.5
2.5
Dd
d
2,3H-3-kO )]3-
2
,3,P
103.0
2.9
77.5
-0.9
2
Dd
a
The metal-binding site.
5
-phosphate but carries a phosphoryloxymethyl group instead
of the arabinose H1 atom. Aqueous solutions of D-fructose 1,6-
bisphosphate consist of 13.1% a- and 86.0% b-furanose (minor
5
species: 0.9% open-chain keto form). Hence, the arabinose
derivative’s a anomer, which is capable of providing the six-ring
chelator, is no longer the major species in the free glycose
phosphate’s solution equilibrium.
The anomeric composition of the free glycose phosphate
was nearly maintained after metallation by a threefold molar
amount of Pd-en (Scheme 6). The major species was [Pd(en)(b-D-
2
,3
4
4
Fruf 1,6P
2
2,3H-2-kO )] (81%, conformation between E and T
3
),
2
,4
the minor species was [Pd(en)(a-D-Fruf 1,6P
conformation close to E). The chemical shifts of the compounds
2
2,4H-2-kO )] (19%,
Scheme 5 The only product of the reaction of D-ribose 5-phosphate with
III
3
precursors of the M (tacn) fragment (M = Co, Ga).
are collected in Table 2.
the chelation reaction and, eventually, the D-ribose 5-phosphate is
present as the a-configured ligand exclusively (Fig. 1).
Bidentate glycose-core–phosphate chelation. In agreement with
the bonding of only one palladium centre to the D-arabinose
D-Fructose 1,6-bisphosphate: cooperative glycose-core–phosphate
chelation
5
-phosphate anomers, an equimolar solution of Pd-en and the
D-fructose 1,6-bisphosphate tetraanion resulted in the formation
2
,3
2,4
CHELATION BY THE GLYCOSE CORE. D-Fructose 1,6-
of the expected kO - and the kO -bonded species. It is recalled
bisphosphate has the same furanoidic backbone as D-arabinose
at this point that the lack of a mixed glycose-core–phosphate
Scheme 6 The major (right) and minor (left) products of the reaction of Pd-en with D-fructose 1,6-bisphosphate.
940 | Dalton Trans., 2009, 7934–7945
7
This journal is © The Royal Society of Chemistry 2009

View Article Online
Scheme 7 Tentative formulae of two species that are related to the species in Scheme 6 by the addition of a proton to O3/O4.
chelation for arabinose and ribose 5-phosphate was connected
with unsuitably large eight-membered chelate rings in these cases.
In the case of a 1-phosphorylated ketose, however, a mixed ligand
set would result in a less unsuitable seven-membered chelate ring.
To trigger the formation of this bonding mode, one equivalent
of acid was added for the reasons that have been discussed in
the Introduction (in fact, Pd-en was reacted with an equivalent
amount of trisodium D-fructose 1,6-bisphosphate). The pH value
of the resulting solution was close to 8. Hence, the observed
species distribution is regarded to be relevant for D-fructose 1,6-
bisphosphate complexation with Lewis acids similarly strong as
palladium(II) at the physiological pH. As a result, the spectro-
Tridentate glycose-core–phosphate chelation. In order to con-
firm the tentative mixed-ligand set of the last subchap-
III
ter, M (tacn)-based metal fragments were used. Since these
fragments are selective probes of a fac-tridentate pattern, only a
single reaction product, resulting from the exclusive coordination
of the b anomer of a D-fructose 1,6-bisphosphate hexaanion, was
expected in terms of chelate-ring-size considerations (Scheme 8).
2
,3
2,4
scopical analysis revealed three species: the kO - and the kO -
bonded species derived above, and a third species of intermediate
quantity. The signals of the latter species vanished not only on
the addition of more Pd-en but also of sodium hydroxide. It thus
appears reasonable to assume the participation of the 1-phosphate
group to the metal-binding site of the new species since phosphate
ligation does not require base addition. Arabinose 5-phosphate,
being de-1-phosphoryloxymethyl-fructose 1,6-bisphosphate, did
not show a third species under the same reaction conditions
which demonstrates that, in fact, the 1-phosphate residue provides
the metal-binding oxygen atom. These arguments led us to the
formulation given in Scheme 7 which shows two anomers that
exhibit seven-membered chelate rings in a mixed glycose-core–
phosphate ligation. No data are available to decide which formula
correctly represents the species.
Scheme 8 The only product of the reaction of equimolar amounts
3+
of {M(tacn)} and D-fructose 1,6-bisphosphate tetraanion, and two
equivalents of base (M = Al, Ga).
III
In fact, the reaction of the M (tacn) fragments with D-fructose
1,6-bisphosphate and the required amount of base consistently
resulted in the formation of a single species for M = Al and Ga
III
in solutions of pH 10. (The Al –tacn probe could be included
despite the N-glycosylation result with ribose 5-phosphate since
the pH value is too low for the side reaction in this part of the
investigation.) As attempted, the spectra showed the formation
of a single species. The fixation of the 1-phosphate residue into
3
1
P NMR spectra support these conclusions. The pH 8 solution
contained four types of D-fructose 1,6-bisphosphate ions: the two
fructose-core-metallated species, free bisphosphate and the mixed-
ligand species. Seven of the eight phosphate residues within these
species are not involved in metal-binding. In agreement with their
3
1
1
a chelate ring became most directly apparent from the P– H
coupling between the 1-phosphate residue and the methylene
3
1
31
similar chemical environment, their P resonances superimposed
in a range of 4 to 6 ppm. However, the resonance of the mixed-
ligand species close to 10 ppm was well-separated from the
protons bonded to C1 in proton-coupled P NMR spectra of the
Al-containing solutions. The coupling pattern of the 6-phosphate
7
was in agreement with a non-chelating residue (triplet, 4.7 Hz).
3
1
1
group of seven. More significant, the P– H coupling constants
markedly changed. In the case of free phosphate residues, the
specific rotamer distribution of D-fructose 1,6-bisphosphate in
The signal of the 1-phosphate, however, occurred as a doublet
of doublets (4.7 and 24.7 Hz) and shows, with these values, a
particularly large deviation from non-chelating phosphate.
3
1
1
an aqueous environment resulted in a mean P– H coupling
7
constant of about 6 Hz. The respective constants of the 10 ppm
D-Fructose 6-phosphate: a dinucleating ligand
signal, however, were 13 and 14 Hz. As has been noted above,
the actual conformation on metal-binding was not unravelled
since both the correct anomer and the rotation angle about the
C2–C1 axis, which also determines the conformation, remain
D-Fructose 6-phosphate, which consists mainly of 16.1% a- and
81.8% b-furanose at aqueous equilibrium (minor species: 2.2%
open-chain keto form), resembles D-arabinose 5-phosphate in its
1
1
5
unknown due to the lack of an appropriate H– H coupling in a
ketose.
C2/C6 part. The only difference is the hydroxymethyl function
with C1 which substitutes H1 of the aldopentose analogue.
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7934–7945 | 7941

View Article Online
Scheme 9 Products of the reaction of Pd-en with D-fructose 6-phosphate. The symbols refer to Fig. 2.
1
3
Fig. 2
C NMR spectra of D-fructose 6-phosphate in Pd-en at molar Pd : glycose ratios of 3 : 1 (top), 2 : 1 (middle) and 1 : 1 (bottom). The symbols refer
31
13
to Scheme 9. Note the splitted C5 and C6 signals as a result of P– C coupling.
3
Accordingly, the monometallation products shown in Scheme 9
were expected.
Since D-fructose 6-phosphate has four hydroxy functions at the
glycose core, dimetallation seemed possible. In fact, variation of
furanose ring was close to E. The major species in an equimolar
2
,3 2-
solution was the [Pd(en)(b-D-Fruf 6P2,3H-2-kO )] ion (90%),
the minor species the [Pd(en)(a-D-Fruf 6P2,4H-2-kO )] ion
(10%). The furanose conformation of the monometallated species
2
,34 2-
4
4
3
the molar ratio of palladium and glycose phosphate revealed a
dimetallated product. Fig. 2, which is illustrative of the general
strategy applied in this work to assure a given stoichiometry by the
variation of the solution composition, shows in its bottom part—
at an equimolar mixture of metal and glycose phosphate—the two
signal sets that were expected for the monometallated analogues of
the arabinose phosphate species. At a higher metal supply, a single
dimetallated species gained significance. Its formula is depicted
is between E and T
3
for the former, close to E for the latter.
Chemical shifts for all three compounds are given in Table 2. It
should be noted that the CIS values of the dimetallated species
reflect the unusual coordination pattern of two internested six-
membered chelate rings. This motif is unprecedented as is the CIS
pattern.
D-Glucose 6-phosphate and D-mannose 6-phosphate
1
,3
2,4 2-
in Scheme 9 (right). This [Pd
2
(en) (a-D-Fruf 6PH-4-kO : kO )]
2
ion was the major species at metal excess (61% at a 3 : 1 molar ratio
of palladium and glycose phosphate). The conformation of the
It may be stated as an interim result that a phosphate residue
reduces the number of potential ligands of a parent glycose if it
7
942 | Dalton Trans., 2009, 7934–7945
This journal is © The Royal Society of Chemistry 2009

View Article Online
substitutes a hydroxy function at the glycose core which is needed
for semiacetal formation. Hence, in all cases discussed so far,
only furanose ligands had to be considered since the terminal
hydroxy function of the respective glycose is essential for pyranose
formation but was blocked by the phosphate residue. This is not the
case for the aldohexose 6-phosphates of D-glucose and D-mannose
which have both their 5- and 4-hydroxy functions free for pyranose
and furanose formation. However, only first results on the ligand
behaviour can be reported herein for the aldohexose 6-phosphates
due to technical reasons. Among the glycose phosphates of this
study, aldohexose 6-phosphate solutions in Pd-en are, by far, the
most unstable with respect to palladium(II) reduction. We were
able, hence, to record NMR spectra of metal-excess solutions
large extent due to its enhancement by the 2-axial hydroxy
4
function of the C
1
-pyranose form. Thus the dominance of the
a-pyranose in a D-mannose solution (62.2% a-D-Manp, 32.9%
b-D-Manp, 0.6% a-D-Manf , 0.2% b-D-Manf ) was shared by
the 6-phosphate (71% a-D-Manp6P and 29% b-D-Manp6P from
1
21
H data). On dissolution in a threefold molar amount of
Pd-en, the b-pyranose was the only anomer that was capable of
1
,2
forming a kO chelate due to the 2-axial substituent. However,
2
,3
an unusual kO ligand was also detected. Due to the anomeric
effect, its 1-hydroxy function was in the a position (Scheme 10
and Table 2). The similarity with the parent glycose was recog-
nisable. However, the dimetallated form appeared suppressed
for the 6-phosphate. Thus, the three species in the bottom part
1
,2 2-
(
3 : 1 molar ratio of Pd and glycose phosphate), but no data are
of Scheme 10, [Pd(en)(b-D-Manp6P1,2H-2-kO )] , [Pd
2
(en)
2
(b-
-
1
,2
3,4 2-
available for equimolar solutions. Keeping in mind these restric-
tions, the interpretation of the available data was straightforward
due to the close similarity of the parent aldohexoses and their
D-Manp6PH-4-kO : kO )] , and [Pd(en)(a-D-Manp6P2,3H-2
2
,3 2-
kO )] , occurred in a relative amount of 37 : 31 : 31, compared
to 21 : 67 : 13 for D-mannose in Pd–chxn at the lower 2 : 1 molar
ratio of Pd : glycose (3 : 1 solutions of D-mannose in Pd–chxn
also exclusively showed the dimetallated species). It should be
noted that kO -bonded species has the same significance for
6
-phosphates.
D-glucose 6-phosphate solution thus resembles, with its
1
A
2
,3
equilibrium composition of 36% a- and 64% b-D-glucose
1
2,3
6
(
-phosphate (from H NMR spectra), the values of D-glucose
both D-mannose and its 6-phosphate. In contrast, the kO -
21
37.6% a- and 62% b-pyranose, 0.1% a- and 0.3% b-furanose).
bonded ribofuranose ligand described above was found for the
Metal excess conditions (3 : 1 Pd : glycose) in the Pd–chxn
reagent transformed D-glucose to two dimetallated products,
5-phosphate but not for the parent glycose.
1
,2
3,4
6
5% [Pd{(R,R)-chxn}(a-D-GlcpH-4-kO : kO )] and 35% of the
Conclusions
respective b isomer (Scheme 10 and Table 2). Glucofuranose
ligands were detectable as tentative minor species at a lower
metal supply. The result for D-glucose 6-phosphate, dissolved in a
threefold molar amount of Pd-en, was substantially the same. The
Glycose phosphates have been regarded as ligands for many
decades. However, it was solely their phosphate function that
appeared to provide the significant metal-binding site—even in
1
,2
3,4
9
two dimetallated species, [Pd
2
(en)
2
(a-D-Glcp6PH-4-kO : kO )]
the case of as strong a Lewis acid as the trivalent aluminium ion.
(
75%) and the b analogue (25%) are depicted in Scheme 10.
Recent structural work on D-fructose-1,6-bisphosphate bonding
As for D-glucose, the additional negative charge on the O1
atom after deprotonation and bonding to palladium(II) made the
anomeric effect become more dominant, and the a anomer became
enriched.
The anomeric effect dominated the metallation pattern of both
D-mannose 6-phosphate and its parent glycose to a particularly
to an acidic Zn(His) centre in the active site of class-II aldolase,
however, shifts the focus of interest to the metal-binding sites with
3
1
5,16
glycose-core participation.
We have demonstrated that for the
II
III
III
III
metal centres Pd , Al , Ga , and Co , each of them bearing
amine spectator ligands, in fact, two more bonding modes are
accessible in aqueous solution: at a higher pH, the coordination of
Scheme 10 Products of the reaction of Pd-en with D-glucose 6-phosphate (top) and D-mannose 6-phosphate (bottom). All ligands were found as stable
4
1
C chairs.
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7934–7945 | 7943

View Article Online
solely the glycose core to the metal, and, close to the physiological
pH, mixed glycose-core–phosphate chelation.
The assumption that the seven-membered chelate rings of
a mixed phosphate–glycose-core metal-binding site might be
formed was substantiated for 1-phosphorylated ketoses. The most
important biomolecule of this kind, D-fructose 1,6-bisphosphate,
belongs to this special group of reducing glycose derivatives. On
addition of the required equivalents of base, the glycose core of
D-fructose 1,6-bisphosphate can be addressed as the only ligand.
The same holds for a mixed glycose-core–phosphate chelation
which is provoked by adding the required, smaller, equiv-
alents of base. The latter solutions adjusted to pH values
of about 8 (Pd) or 10 (Al, Ga) when the mixed bonding
mode was attempted. A special and remarkable detail with
respect to recent aldolase-related work should be noted for
In order to optimise the glycose-core–metal interaction, we have
taken advantage of a recently established concept. Reducing sugars
are able to adapt their chelating functions to the requirements of
a metal by adjusting both their conformation and configuration.
Since the various isomers are interconvertible under the reaction
conditions applied, they provide a dynamic ligand library to
a metal. Among the sugar phosphates, important metabolic
intermediates belong to this class of reducing glycose derivatives.
Most of them bear a phosphate function at the glycose’s non-
reducing terminus. Examples from this work are the aldopentose
5
-phosphates and fructose 6-phosphate, where we find molecules
II
whose d position is phosphorylated and thus are restricted
to the glycose’s furanose form. In retrospect, the coordination
chemistry of this subclass of reducing glycose phosphates is
particularly straightforward since the phosphate residue is spa-
tially well-separated from the glycose core. Hence, in either
case we have found that the glycose core exhibits the typical
coordination chemistry of the parent furanose. No mixed glycose-
core–phosphate chelating has ever been observed. Taken together
with the published knowledge, these ligands are expected to bind,
for a given central metal, via their phosphate function at a low pH,
and via their glycose core at a higher pH.
A similar separation of the phosphate function and the glycose
core is given for the aldohexopyranose 6-phosphates, whose
coordination chemistry is particularly close to that of the parent
aldohexoses. It should be noted, however, that a closer vicinity
of the phosphate group and a glycose-core hydroxy function is
given for the respective furanoses that have a 5-hydroxy function
adjacent to the phosphate moiety and thus might be able to
establish seven-membered chelates of considerable stability. Due
to the low thermal stability of the respective solutions, we were
not able to further clarify this point by using a palladium-based
probe.
Pd chelation. The almost neutral solutions in which a mixed
glycose-core–phosphate chelator was detected, contained this
III
species not as the major component as the M (tacn) systems
do. The major species in pH 8 Pd-en is the same as that
of an alkaline solution, namely the only-glycose-core-bonding
2,4-furanose ligand.
A final methodological comment on the various metals might
be given. The trivalent metals have been shown to use the dynamic
ligand library provided by a reducing glycose or its derivatives
in the sense that usually one isomer is bonded and enriched.
Palladium(II), however, is different. As has been shown for the
II
glycoses themselves, the Pd centre forms chelates of similar
stability with practically all of the expected ligands, and thus
II
displays the entire library. Using probes such as the Pd (en)
fragment thus results in the accumulation of many spectral data
and the possibility to correlate them. Scheme 11 shows, as an
example, such a data collection which serves as an additional
check of the assignments made. Cross-checking CIS values is one
method of comparing NMR data. The same close relationships
are obtained with the chemical shifts directly. In Scheme 11, the
D-arabinofuranose core is the common partial structure that is
also present in the D-fructofuranose derivatives of this work. It is
Scheme 11 CIS values of furanose ligands on bonding to palladium(II) via six- and five-membered chelate rings: D-arabinose 5-phosphate (top),
D-fructose 6-phosphate (middle), and D-fructose 1,6-bisphosphate (bottom).
7
944 | Dalton Trans., 2009, 7934–7945
This journal is © The Royal Society of Chemistry 2009

View Article Online
obvious that the CIS values of carbon atoms in a related chemical
environment are similar.
7 G. B. van den Berg and A. Heerschap, Arch. Biochem. Biophys., 1982,
2
19, 268–276.
8
M. Kato, T. Tanase and M. Mikuriya, Inorg. Chem., 2006, 45, 2925–
941.
9 D. Champmartin, P. Rubini, A. Lakatos and T. Kiss, J. Inorg. Biochem.,
001, 84, 13–21.
0 J. Burger, C. Gack and P. Kl u¨ fers, Angew. Chem., Int. Ed. Engl., 1995,
4, 2647–2649.
1 P. Kl u¨ fers and T. Kunte, Angew. Chem., Int. Ed., 2001, 40, 4210–
212.
12 P. Kl u¨ fers and T. Kunte, Chem.–Eur. J., 2003, 9, 2013–2018.
2
Acknowledgements
2
1
1
The authors thank Martin Rieger and Mark Dethlefsen for
contributing to this work during their research courses. Sandra
Albrecht is acknowledged for technical assistance. The palladium
part of this work was supported by the Deutsche Forschungsge-
meinschaft (grant Kl624/12–1).
3
4
1
1
1
3 J. Burger and P. Kl u¨ fers, Z. Anorg. Allg. Chem., 1997, 623, 1547–1554.
4 P. Kl u¨ fers and T. Kunte, Z. Anorg. Allg. Chem., 2004, 630, 553–557.
5 S. D. Pegan, K. Rukseree, S. G. Franzblau and A. D. Mesecar, J. Mol.
Biol., 2009, 386, 1038–1053.
6 A. Galkin, Z. Li, L. Li, L. Kulakova, L. R. Pal, D. Dunaway-Mariano
and O. Herzberg, Biochemistry, 2009, 48, 3186–3196.
References
1
1
Y. Arendt, O. Labisch and P. Kl u¨ fers, Carbohydr. Res., 2009, 344, 1213–
224.
D. Voet and J. G. Voet, in Biochemistry, Wiley, New York, USA, 2004,
pp. 581–622.
1
17 C. A. G. Haasnoot, F. A. A. M. de Leeuw and C. Altona, Tetrahedron,
1980, 36, 2783–2792.
2
18 J. P. Marino, H. Schwalbe and C. Griesinger, Acc. Chem. Res., 1999,
3
4
P. Kl u¨ fers, F. Kopp and M. Vogt, Chem.–Eur. J., 2004, 10, 4538–4545.
S. P. Draffin, P. J. Duggan and G. D. Fallon, Acta Crystallogr., Sect. E,
32, 614–623.
19 V. Sychrovsk y´ , Z. Vok a´ cˇ ov a´ , J. Sˇ poner, N. Sˇ pa cˇ kov a´ and B. Schneider,
2
004, 60, o1520-o1522.
J. Pierce, A. S. Serianni and R. Barker, J. Am. Chem. Soc., 1985, 107,
448–2456.
G. Liang, D. Chen, M. Bastian and H. Sigel, J. Am. Chem. Soc., 1992,
14, 7780–7785.
J. Phys. Chem. B, 2006, 110, 22894–22902.
20 T. Tanase, H. Inukai, T. Onaka, M. Kato, S. Yano and S. J. Lippard,
Inorg. Chem., 2001, 40, 3943–3953.
21 Y. Zhu, J. Zajicek and A. S. Serianni, J. Org. Chem., 2001, 66, 6244–
6251.
5
6
2
1
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7934–7945 | 7945