Metal-binding sites of N-acetylneuraminic acid

Article abstract of DOI:10.1002/zaac.201200415

Sialic acid represents a group of thirty derivates of neuraminic acid with various substituents at the amino residue and the alcoholic hydroxy groups. We analysed the behaviour of the tetracoordinated metal ions palladium(II) and silicon(IV) against the most important derivative N-acetylneuraminic acid (NANA). The molecular structures were assigned by a combined ¹H, ¹³C and ²⁹Si NMR-spectroscopic approach. Despite the presence of many different functional groups, the coordination chemistry of NANA with Pd^II follows established rules. Coordination via the N-acetyl-group - sterically impossible with Pd^II - was realised with Si^IV. Copyright

Full text of DOI:10.1002/zaac.201200415

ARTICLE
DOI: 10.1002/zaac.201200415
Metal-binding Sites of N-Acetylneuraminic Acid
Sarah Illi,[a] Johanna Schulten,[a] and Peter Klüfers*[a]
Keywords: Palladium; Silicon; N-acetylneuraminic acid; NMR spectroscopy
1
Abstract. Sialic acid represents a group of thirty derivates of neur-
structures were assigned by a combined H, 13C and 29Si NMR-spec-
aminic acid with various substituents at the amino residue and the troscopic approach. Despite the presence of many different functional
alcoholic hydroxy groups. We analysed the behaviour of the tetracoor-
dinated metal ions palladium(II) and silicon(IV) against the most im-
groups, the coordination chemistry of NANA with PdII follows estab-
lished rules. Coordination via the N-acetyl-group – sterically imposs-
portant derivative N-acetylneuraminic acid (NANA). The molecular ible with PdII – was realised with SiIV.
hydroxyl groups. In the pH range 2–7, species of the type
Introduction
[M(NANAH–1)2] were postulated and, above pH 7, the species
[M(NANAH–1)2(OH)]–.[5] By potentiometric titrations the
binding sites cannot be identified for sure, because the depro-
tonated or protonated sites cannot be localised.
The thirty derivatives of neuraminic acid occur at the non-
reducing ends of glycoproteins, glycolipides and polysaccha-
rides.[1,2] These glycoconjugates can be found in microbes,
protozoa, in cells and tissues of higher animals. They are usu-
ally located on the outer cell membrane[3,4] and are involved
in various biological processes.[5] N-Acetylneuraminic acid
(NANA), the N-acetyl derivate of sialic acid, is the most abun-
dant sialic acid in humans and has been isolated from body
fluids. Women with endometrial cancer and patients with pros-
tatic or bladder cancer have significantly higher NANA levels
than healthy people.[6] The level of NANA concentration can
also be increased by other diseases, hence it is not suitable for
an initial diagnosis of cancer. Its quantitative measurement,
however, may help in monitoring the progress of surgical ther-
apy and diagnosis of metastasis.[7] NANA has gained special
interest among organic and medical chemists who are engaged
in synthesising a library of derivatives.
In this work, new results using further metal ions coordinat-
ing NANA, PdII and SiIV, are presented. By using 13C NMR
spectroscopy we were now able to identify the actual binding
sites at NANA. To probe the metal-binding sites, PdIIN2-type
fragments (N2 = bidentate nitrogen ligand) which we recently
introduced as tools for the analysis of carbohydrates and carbo-
hydrate derivatives in aqueous solution were employed in this
work.[11,12] For the investigation of the NANA binding sites,
Pd-tmen, an aqueous solution of [Pd(tmen)(OH)2] (tmen =
N,N,N’,N’-tetramethylethane-1,2-diamine), and Pd-chxn, an
aqueous solution of [Pd(chxn)(OH)2] (chxn = (1R,2R)-cyclo-
hexane-1,2-diamine) were used. And to identify the silicon-
binding sites, the diol-protecting reagent bis(tert-butyl) bis(tri-
fluoromethanesulfonato)silicon (DTBS triflate) was used.
DTBS compounds are used especially for the protection of the
3’,5’-hydroxy function of ribofuranosyl moieties in RNA syn-
thesis.[13–15] DTBS derivates of the aldopentoses were ana-
lysed in a recently published work by our group.[16]
An investigation of NANA complexes with alkali and alka-
line earth metals suggested that the glycerol side chain is in-
volved in the binding;[8] 13C and H NMR studies confirmed
1
the coordination of NANA towards calcium(II).[9,10] Further-
more, the coordination ability of NANA towards biologically
important metal ions such as copper(II), cobalt(II) and towards
toxic metal ions such as lead(II) and cadmium(II) was investi-
gated in aqueous solution by means of potentiometry, UV and
NMR spectroscopy. Both binary and ternary systems were
studied by Saladini et al., where the authors suggest that
NANA coordinates all metal ions through the carboxylic
group, pyranosidic ring oxygen and glycerol chain alcoholic
Results and Discussion
N-Acetylneuraminic acid is a reducing sugar which exists in
aqueous solution in various cyclic and acyclic forms (Figure
1). With the hydroxy function on the N-acetylated carbon atom
C5 missing, no furanoses are observed. In the major isomer,
the β-pyranose, the N-acetyl group, the adjacent hydroxy
group, the glycerol chain on C6, and the carboxy function are
all oriented equatorially in the observed 2C5 conforma-
tion.[17,18]
* Prof. Dr. P. Klüfers
Fax: +49-89-2180-77407
E-Mail: kluef@cup.uni-muenchen.de
[a] Department Chemie
In the equimolar reaction of NANA with Pd-tmen (0.45 m
in D2O) or Pd-chxn (0.30 m in D2O), the monometallated
complexes [Pd(tmen)(NeuNAc1,2H–2-κ2O1,2)] (1a) and
Ludwig-Maximilians-Universität München
Butenandtstr. 5–13(D)
81377 München, Germany
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/zaac.201200415 or from the author [Pd(chxn)(NeuNAc1,2H–2-κ2O1,2)] (1b) were obtained
Z. Anorg. Allg. Chem. 2013, 639, (1), 77–83
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
77

S. Illi, J. Schulten, P. Klüfers
ARTICLE
Figure 1. N-Acetylneuraminic acid isomers in aqueous solution. The
percentages of cyclic and acyclic forms were determined at pH 2.[18]
(Figure 2). On monometallation, the PdII fragment was che-
lated by the carboxyl residue and by the adjacent hydroxy
group after its deprotonation in a five-membered chelate ring.
The structures of both species were determined by standard
NMR techniques, including 2D spectra (Table 1, Figure 3).
By increasing the PdII concentration to a Pd:NANA
molar ratio of 2:1, the dimetallated complexes
[{Pd(tmen)}2(NeuNAc1,2,8,9H–4-1κ2O1,2:2κ2O8,9)] (2a) and
Figure 2. Species detected in PdII-containing solutions of N-acetyl-
neuraminic acid (see text).
[{Pd(chxn)}2(NeuNAc1,2,8,9H–4-1κ2O1,2:2κ2O8,9)] (2b) were group relative to the glycerol chain, the formation of a chelate
formed. 13C NMR spectroscopy revealed that a second PdII ring via the N-acetyl group is not possible due to vicinal
fragment coordinated at the terminal diol function of the glyc- acetyl-hydroxyl repulsion.[20]
erol chain. This result matches the rules: from experiments
By adding one equivalent of sodium hydroxide to the reac-
with sugar alcohols it is known that the stability of a PdII che- tion mixture of NANA with two equivalents of Pd-tmen and
late depends on the configuration of the diol moiety and de- Pd-chxn, the carboxylate residue was set free and the monome-
creases in the order threo Ͼ terminal Ͼ erythro.[19] With the tallated species [Pd(tmen)(NeuNAc8,9H–2-κ2O8,9)]– (3a) and
C7-C8 diol being configured erythro, the observed terminal [Pd(chxn)(NeuNAc8,9H–2-κ2O8,9)]– (3b) were formed. The
coordination confirms the rule (Table 1).
structures of these species were derived from 13C NMR spectra
At higher PdII-tmen:NANA molar ratios, the concentration (Table 1, Figure 3). The shifts observed for the structure deter-
of 2a increased. At a 5:1 ratio, only the dimetallated species minations lay in the typical range between 8 ppm and 14 ppm
was observed. Even with this high concentration of palladi- for carbon atoms bound to the coordinated carboxy and alkoxy
um(II), no coordination of a third palladium moiety via the oxygen atoms.
acetyl amino function was observed. Generally, a palladium(II)
For the investigation of silicon binding sites on NANA, we
atom is able to bind a deprotonated amido ligand at a relatively used bis(tert-butyl) bis(trifluoromethanesulfonato)silicon
low pH level. As recently published, the binding properties of (DTBS triflate). We performed several investigations, includ-
the palladium reagents with deprotonated acetyl amino func- ing aldopentoses as an example for reducing carbohydrates,[16]
tions depend on the steric requirements introduced by the and tartaric acid as a model compound for the competition
acetyl residue which is forced into coplanar arrangement with between carboxy and hydroxy groups. NANA combines these
the chelate ring. Depending on the position of the N-acetyl different functional groups and offers, in addition, the N-acetyl
78
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© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Metal-binding Sites of N-Acetylneuraminic Acid
Table 1. 13C NMR chemical shifts (δ /ppm) and shift differences (Δδ) of [Pd(tmen)(NeuNAc1,2H–2-κ2O1,2)] (1a), [Pd(chxn)(NeuNAc1,2H–2-
κ2O1,2)]
(1b),
[{Pd(tmen)}2(NeuNAc1,2,8,9H–4-1κ2O1,2:2κ2O8,9)]
(2a),
[{Pd(chxn)}2(NeuNAc1,2,8,9H–4-1κ2O1,2:2κ2O8,9)]
(2b),
[Pd(tmen)(NeuNAc8,9H–2-κ2O8,9)]– (3a) and [Pd(chxn)(NeuNAc8,9H–2-κ2O8,9)]– (3b) in D2O, referenced to an internal methanol signal
(49.5 ppm).
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
NeuNAc
1a
δ
δ
Δδ
δ
Δδ
δ
Δδ
δ
Δδ
δ
Δδ
δ
Δδ
172.7
185.3
12.6
185.8
13.1
177.9
5.2
185.7
13.0
186.6
13.9
177.8
5.1
93.2
102.1
8.9
101.1
7.9
97.2
4.0
102.5
9.3
102.1
8.9
97.2
4.0
36.7
42.0
5.3
43.0
6.3
39.7
3.0
42.5
5.8
42.2
5.5
39.3
2.6
64.6
67.3
2.7
66.9
2.3
67.2
2.6
67.9
3.3
67.7
3.1
67.8
3.2
49.9
52.9
3.0
53.6
3.7
53.8
3.9
53.2
3.3
53.1
3.2
53.1
3.2
68.0
69.5
1.5
70.3
2.3
70.2
2.2
69.6
1.6
70.7
2.7
70.5
2.5
66.1
69.0
2.9
70.1
4.0
71.5
5.4
69.5
3.4
70.1
4.0
70.6
4.5
68.2
70.9
2.7
79.0
10.8
79.4
11.2
71.3
3.1
80.2
12.0
79.8
11.6
61.0
63.4
2.4
70.7
9.7
71.4
10.4
63.9
2.9
70.8
9.8
171.4
174.8
3.4
174.8
3.4
175.3
3.9
175.3
3.9
175.2
3.8
175.2
3.8
19.9
22.3
2.4
22.4
2.5
22.9
3.0
22.7
2.8
22.9
3.0
22.9
3.0
2a
3a
1b
2b
3b
74.4
13.4
Figure 3. 13C NMR spectra of N-acetylneuraminic acid. a) free NANA (β-pyranose), b) NANA in Pd-tmen at a molar ratio of 1:1, c) NANA
in Pd-tmen at a molar ratio of 1:2 and d) NANA in Pd-tmen with NaOH at a molar ratio of 1:2:1. All measurements were performed at
room temperature in D2O. The numbering follows IUPAC rules, see Figure 1. [Pd(tmen)(NeuNAc1,2H–2-κ2O1,2)] (1a) marked with a ring,
[{Pd(tmen)}2(NeuNAc1,2,8,9H–4-1κ2O1,2:2κ2O8,9)] (2a) marked with a rhombus and [Pd(tmen)(NeuNAc8,9H–2-κ2O8,9)]– (3a) marked with a
star.
Z. Anorg. Allg. Chem. 2013, 77–83
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79

S. Illi, J. Schulten, P. Klüfers
ARTICLE
group. In contrast to the Pd experiments, it was not possible suitable method to assign the type of chelate formed. Silicon
to work in aqueous solution with oxophilic SiIV compounds. atoms, as part of five-membered chelate rings on the one hand,
The solvent of choice was dried N,N-dimethylformamide.
In contrast to our results with PdII, in the equimolar reaction in clearly-resolved shift regions for DTBS carbohydrates.
of NANA with DTBS triflat the first chelate ring with the The analysis of the mono-metallated product thus was sup-
and those of six-membered rings or higher on the other, occur
DTBS unit was formed at the glycerol chain. In Si(tBu)2(Neu- ported by a signal in the 29Si NMR spectrum at –8.5 ppm –
NAc7,9H–2-κ2O7,9) (4) NANA is coordinated via O7 and O9 typical for six-membered chelate rings. By increasing the sili-
in a six-membered chelate ring (Figure 4). SiIV as a small, con concentration the coordination of the acidic carboxy oxy-
highly charged Lewis acid obviously prefers binding the more gen was also realised. In the reaction mixture of NANA with
basic oxygen atoms. Compound 4 was identified using 1D and the double molar amount of DTBS triflate, the di-metallated
2D NMR spectroscopy, the 13C NMR shifts are in the typical
range for silicon-coordinated carbohydrates: +3.1 and +5.0
ppm for the coordinated carbon atoms C7 and C9 and –6.7
ppm for C8. Negative CIS (coordination-induced shift) values
were characteristic for the inner carbon atom in six-membered
chelate rings (Table 2). 29Si NMR spectroscopy was used as a
product Si2(tBu)4(NeuNAc1,2,7,9H–4-1κ2O1,2:2κ2O7,9
)
(5)
was identified. The CIS values for the carbon atoms C7, C8
and C9 corresponded those of the Si1 product. The additional
coordination via the acidic carboxy group with C1 and the
adjacent hydroxy group on C2 caused shifts of –3.4 ppm for
C1 and +3.6 ppm for C2 in the 13C NMR spectrum (Figure 5).
The surprising negative CIS value for the coordinated carboxy
group was validated by analysing simple carbonic acids such
as tartaric acid. For several carboxy groups coordinated by
DTBS, negative shifts were observed and partially documented
by single X-Ray analysis.[21]
In contrast to palladium, a tri-metallated product was formed
using three equivalents of DTBS triflate, namely Si3(tBu)6-
(NeuNAc1,2,4,5,7,9H–6-1κ2O1,2:2κO4,2κN5:3κ2O7,9
)
(6).
Compared to the analysed Si1 and Si2 species which displayed
a degree of impurity (educt and species other stoichiometries),
6 is uniquely distinct and extraordinarily pure. To form a third
chelate ring at the NANA backbone, the N-acetyl nitrogen
atom had to be involved (excluding an eight-membered chelate
ring formed by C4 and C8). Competing Si3 species that were
considered without the N-acetyl function had to be derived
from the open-chain enol or ketohydrate form. The latter needs
one equivalent of water for its formation and was thus ex-
cluded. Analysing the 13C NMR spectrum, the enol form could
be excluded as well, because, in the range for double-bonded
carbon atoms, no signal was detected. In addition to the CIS
values for C1, C2, C7, C8 and C9, the signals of the carbon
atoms C4 and C5 were shifted as expected for 6: C4, bonded
to the coordinating oxygen, exhibits a signal shift of +5.0 ppm,
C5, bonded to the coordinating nitrogen, a shift of –3.3 ppm.
The 29Si NMR spectrum shows, besides the two signals for the
former discussed chelate rings, an additional signal at –5.1
ppm. A signal of this magnitude correlates to a six-membered
chelate ring if a 1,3-diol is bonded. With a coordinating nitro-
gen atom, the rules of carbohydrate NMR spectroscopy seem
Figure 4. Species detected in DTBS-containing solutions of N-acetyl-
neuraminic acid. The compounds shown were observed increasing the
molar ratio from 1:1 (4) to 1:3 (6).
Table 2. 13C NMR chemical shifts (δ /ppm) and shift differences (Δδ) of Si(tBu)2(NeuNAc7,9H–2-κ2O7,9) (4), Si2(tBu)4(NeuNAc1,2,7,9H–4-
1κ2O1,2:2κ2O7,9) (5) and Si3(tBu)6(NeuNAc1,2,4,5,7,9H–6-1κ2O1,2:2κO4,2κN5:3κ2O7,9) (6) in DMF.
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
NeuNAc
4
δ
δ
Δδ
δ
Δδ
δ
172.0
170.8
–1.2
168.6
–3.4
167.8
–4.2
96.0
94.8
–1.2
99.6
3.6
99.0
3.0
40.9
39.2
–1.7
40.6
–0.3
41.1
0.2
67.0
67.0
0.0
66.5
–0.5
72.0
5.0
54.4
53.2
–1.2
52.3
–2.1
51.1
–3.3
71.8
69.1
–2.7
71.7
–0.1
71.6
–0.2
70.6
75.6
5.0
76.0
5.4
75.7
5.1
71.0
64.3
–6.7
64.7
–6.3
64.6
–6.4
65.2
68.3
3.1
68.8
3.6
68.6
3.4
173.1
173.5
0.4
172.1
–1.0
22.7
22.2
–0.5
22.7
0.0
5
6
170.5
–2.6
22.7
0.0
Δδ
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© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Metal-binding Sites of N-Acetylneuraminic Acid
Figure 5. 13C NMR spectra of N-acetylneuraminic acid. a) free NANA (β-pyranose), b) NANA with tBu2Si(OTf)2 at a molar ratio of 1:3. All
measurements were performed at room temperature in DMF. The numbering follows IUPAC rules, see Figure 1.
to be not simply transferable. To confirm the result we thus the α-hydroxy carbonic acid unit in a five-membered chelate
analysed model compounds which have carboxy, hydroxy, ring, Si2(tBu)4(NeuNAc1,2,7,9H–4-1κ2O1,2:2κ2O7,9) (5). In ad-
amino groups or N-acetyl amino groups, and found equivalent dition, the small, highly-charged Lewis acid SiIV binds via the
values.[22]
deprotonated N-acetyl function if three equivalents
of DTBS triflate are used. Si3(tBu)6(NeuNAc1,2,4,5,7,9H–6-
1κ2O1,2:2κO4,2κN5:3κ2O7,9) (6) is formed: a tri-metallated
compound, which was not observed even with five equivalents
of PdII. As an overall result, even the binding-site pattern of
as complicated a carbohydrate derivative as NANA may be
unravelled through a combination of NMR results and the ap-
plication of rules derived from simpler model compounds.
A final comment regarding isolated compounds: Many
attempts to isolate the discussed compounds as a solid have
failed despite the utilisation of several techniques successfully
used with palladium and silicon carbohydrate species in the
past. Based on the enormous complexity of NANA, it is neces-
sary to develop new methods for species with hydroxy, car-
boxy and amido groups.
Conclusions
N-Acetylneuraminic acid is a very important biomolecule
and the knowledge of its metal-binding properties should be
a matter of course. As a reducing carbohydrate, NANA
exists in various cyclic and acyclic forms. The β-pyranose
is the most stable one in the free carbohydrate. We analysed
NANA coordination towards PdII and SiIV. In recent investi-
gations the rules of the carbohydrate chemistry of these
atoms were unravelled. On this solid foundation, attempts at
analysing a polyfunctional biomolecule such as NANA
seems promising despite the fact that some important func-
tional groups are present in the same molecule: various
hydroxy functions, a carboxy and a N-acetyl group. With
PdII, two different binding sites were observed: the α-hy-
droxy carbonic acid unit and the glycerol side chain. Com-
plying with the rules, [Pd(tmen)(NeuNAc1,2H–2-κ2O1,2)] (1a)
formed with an equimolar amount of PdII reagent,
[{Pd(tmen)}2(NeuNAc1,2,8,9H–4-1κ2O1,2:2κ2O8,9)] (2a) with
an excess of the PdII reagent and [Pd(tmen)(NeuNAc8,9H–2-
κ2O8,9)]– (3a) after addition of NaOH. With Pd-chxn, the same
coordination pattern was obtained. All compounds were ana-
lysed using the various NMR-spectroscopic techniques. Even
with a high concentration of PdII, no coordination via the N-
acetyl group was detected, but this bonding pattern was
achieved with SiIV. The results using DTBS triflate also agreed
with the rules of carbohydrate chelation. The first DTBS unit
bonded in a six-membered chelate ring on the glycerol side
Experimental Section
Methods and Materials
All chemicals were purchased and used without further purification:
N-acteylneuraminic acid (ABCR), N,N,N’,N’-tetramethylethane-1,2-
diamine
(Sigma-Aldrich),
(1R,2R)-(–)-1,2-diaminocyclohexane
(ABCR), deuterium oxide (Eurisotop), palladium(II) chloride (Alfa
Aesar), silver(I) oxide (Merck), bis(tert-butyl) bis(trifluoromethanesul-
fonato) silicon (Aldrich: bis(tert-butyl)silyl bis(trifluoromethanesul-
fonate)), N,N-dimethylformamide D7 (Eurisotop). Reactions contain-
ing silicon compounds were carried out using standard Schlenk tech-
nique in a nitrogen atmosphere.
NMR Spectroscopy
NMR spectra were recorded at room temperature with Jeol ECX400/
chain, Si(tBu)2(NeuNAc7,9H–2-κ2O7,9) (4), the second one on ECP 400 (1H: 400 MHz, 13C{1H}: 100 MHz) spectrometers. In aque-
Z. Anorg. Allg. Chem. 2013, 77–83
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81

S. Illi, J. Schulten, P. Klüfers
ARTICLE
ous solution the signals of methanol, which was added to the sample,
Table 3. Applied ratios for the reaction of N-acetylneuraminic acid
were used as an internal secondary reference for the chemical shift. with Pd-tmen (0.45 m, 2.0 mL, 0.90 mmol).
The signals of the deuterated solvent were used as an internal second-
N
euNAc
sodium hydroxide
ary reference for 13C NMR spectra using DMF D7. 29Si shift values
were referenced externally to TMS. NMR signals were assigned by
1H–1H COSY45, 1H–13C HMQC and 1H–13C HMBC experiments.
Shift differences are given as δ(Ccomplex) – δ(Cfree sugar). To assign the
signal sets to individual species, first of all coupling constants J were
analysed applying the Karplus relationship to identify the correct an-
omer. Afterwards, CIS values were used to assign the correct chelation
site. The values for the free N-acetylneuraminic acid were taken from
our own measurements in D2O in neutral aqueous solution and DMF
D7.
1:1:1
1:2:1
1:2:1
1:3:1
1:5:1
280 mg (0.90 mmol)
140 mg (0.45 mmol)
140 mg (0.45 mmol)
93 mg (0.30 mmol)
56 mg (0.18 mmol)
–
–
18 mg (0.45 mmol)
–
–
1H NMR Data of N-Acetylneuraminic Acid Complexes:
[Pd(tmen)(NeuNAc1,2H–2-κ2O1,2)] (1a): 1H NMR: δ = 3.87–4.00 (m,
4H, H9a, H4, H6, H8), 3.76 (t, 1H, H5, J4,5 = 10.1 Hz, J5,6
10.1 Hz), 3.65 (dd, 1H, H9b, J8,9b = 6.3 Hz, J9a,9b = –11.7 Hz), 3.48
(d, 1H, H7, J6,7 = 8.8 Hz), 2.36 (dd, 1H, H3a, J2,3a = 4.5 Hz, J3a,3b
3
3
=
3
2
3
2
3
Syntheses
= –12.4 Hz), 1.98 (s, 3H, H11), 1.66 (t, 1H, H3b, J2,3b = 12.1 Hz,
2J3a,3b = –12.4 Hz) ppm.
Preparation of [Pd(tmen)(OD)2]: To the brown suspension of PdCl2
(2.50 g, 14.1 mmol) in water (85 mL), concentrated hydrochloric acid
(2.50 mL) was added and the mixture was stirred for 30 min, yielding a
brown solution. Under stirring, on the dropwise addition of N,N,N’,N’-
tetramethylethane-1,2-diamine (3.25 g, 27.97 mmol) in water (90 mL)
a yellow precipitate of [Pd(tmen)Cl2] was formed. After stirring for
30 min, the yellow complex was filtered through a G4 filter, washed
with cold water and dried in vacuo. The yield was 3.84 g (93%). Anal.
calcd. for C6H16Cl2N2Pd: C, 24.55; H, 5.49; N, 9.54; Cl, 24.16. Found:
C, 24.37; H, 5.36; N, 9.50; Cl, 24.12.
[{Pd(tmen)}2(NeuNAc1,2,8,9H–4-1κ2O1,2:2κ2O8,9)] (2a): 1H NMR:
δ = 4.15–4.20 (m, 1H, H4 and H7), 3.96–4.02 (m, 1H, H6), 3.74–3.80
2
(m, 1H,H5), 3.42–3.48 (m, 1H, H9a), 3.10 (d, 1H, H9a, J9a,9b
=
–9.9 Hz), 3.03–3.05 (m, 1H, H8), 1.98 (s, 3H, H11), 1.68 (t, 1H, H3b,
2
3J2,3b = 12.1 Hz, J3a,3b = –12.4 Hz) ppm.
[Pd(tmen)(NeuNAc8,9H–2-κ2O8,9)]– (3a): 1H NMR: δ = 4.10–4.14
(m, 2H, H4, H6), 4.04–4.08 (m, 1H, H7), 3.83–3.91 (m, 1H, H5),
3.36–3.46 (m, 1H, H9a), 2.94–3.08 (m, 2H, H9b, H8), 1.98 (s, 3H,
3
2
H11), 1.62 (t, 1H, H3b, J2,3b = 12.1 Hz, J3a,3b = –12.4 Hz) ppm.
A suspension of [Pd(tmen)Cl2] (1.32 g, 4.50 mmol) and silver(I) oxide
(1.15 g, 4.95 mmol) in D2O (15 mL) was stirred under nitrogen and
the exclusion of light at 40 °C. After 30 min AgCl was removed by
filtration through a G4 filter under nitrogen atmosphere, leaving a clear
yellow solution of [Pd(tmen)(OD)2] (“Pd-tmen”). The alkaline ca. 0.45
m solution was stored at –60 °C. 13C{1H} NMR (D2O): δ = 50.2 (4C,
CH3), 62.1 (2C, CH2) ppm.
N-Acetylneuraminic Acid Solutions in Pd-chxn: N-Acetylneur-
aminic acid was dissolved in Pd-chxn (ca. 0.30 m solution in D2O) at
4 °C. The yellow solution was stirred for about 2 h at 4 °C and sub-
sequently either placed immediately under NMR investigation or
stored at –60 °C. The used rates are listed in Table 3, the respective
13C{1H} NMR N-acetylneuraminic acid shifts are listed in Table 4.
Preparation of [Pd(chxn)(OD)2]: To a suspension of PdCl2 (5.00 g,
28.2 mmol) in water (50 mL) at 45 °C potassium chloride (4.20 g,
56.3 mmol) was added and the mixture was stirred for 10 min until a
brown solution of K2[PdCl4] was formed. (1R,2R)-cyclohexane-1,2-
diamine (3.22 g, 28.2 mmol) dissolved in water (95 mL) and HCl (5
mL, 37%) was slowly added. After the reaction mixture was stirred
for 1 h at 45 °C, the pH was raised by a solution of NaOH (2 m) to
7.0 and the complex precipitated from the solution. The reaction mix-
ture was stirred for 3 h at 45 °C (during the first hour, the pH value
was repeatedly adjusted). After the suspension was cooled to room
temperature, the yellow complex was filtered through a G4 filter,
washed with cold water (5 ϫ 20 mL) and dried in vacuo. The yield
was 7.7 g (93%). Anal. calcd. for C6H14Cl2N2Pd: C, 24.72; H, 4.84;
N, 9.61; Cl, 24.32. Found: C, 24.70; H, 4.73; N, 9.58; Cl, 24.35.
Table 4. Applied ratios for the reaction of N-acetylneuraminic acid
with Pd-tmen (0.30 m, 2.0 mL, 0.60 mmol).
NeuNAc
sodium hydroxide
1:1:1
1:2
186 mg (0.60 mmol)
93 mg (0.30 mmol)
–
–
1:2:1:1 93 mg (0.30 mmol)
12 mg (0.30 mmol)
1H NMR Data of N-Acetylneuraminic Acid Complexes:
[Pd(chxn)(NeuNAc1,2H–2-κ2O1,2)] (1b): 1H NMR: δ = 3.92 (ddd,
3
3
3
1H, H8, J7,8 = 9.1 Hz, J8,9a = 2.8 Hz, J8,9b = 6.6 Hz), 3.84 (d, 1H,
3
H9a, J8,9a = 2.8 Hz), 3.77–3.83 (m, 1H, H4), 3.77 (s, 1H, H6), 3.66–
3.72 (m, 1H, H5), 3.56 (d, 1H, H9b, J8,9b = 6.6 Hz), 3.39 (d, 1H, H7,
3J7,8 = 9.1 Hz), 2.21–2.26 (m, 1H, H3a), 1.85–1.90 (m, 3H, H11), 1.63
3
3
2
(t, 1H, H3b, J2,3b = 12.1 Hz, J3a,3b = –12.1 Hz) ppm.
A suspension of [Pd(chxn)Cl2] (3.28 g, 11.25 mmol) and silver(I) ox-
ide (2.80 g, 12.08 mmol) in D2O (25 mL) was stirred under nitrogen
and the exclusion of light at 40 °C. After 2 h AgCl was removed by
filtration through a G4 filter under nitrogen atmosphere, leaving a clear
yellow solution of [Pd(chxn)(OD)2] (“Pd-chxn”). The alkaline ca. 0.3
m solution was stored at –60 °C. 13C{1H} NMR (D2O): δ = 24.3 (2C,
γ-CH2), 33.7 (2C, β-CH2), 60.9 (2C, α-CH2) ppm.
Preparation of Si(tBu)2(NeuNAc7,9H–2-κ2O7,9) (4) in Solution: To
a cooled (0 °C) suspension of N-acetylneuraminic acid (320 mg,
1.0 mmol) in N,N-dimethylformamide D7 (4 mL) Si(tBu)2(OTf)2 (0.33
mL, 1.0 mmol) was added dropwise.
After stirring for 3 h, 1H, 13C, 29Si and 2D NMR spectra were re-
corded.
N-Acetylneuraminic Acid Solutions in Pd-tmen: N-Acetylneur-
aminic acid was dissolved in Pd-tmen (ca. 0.45 m solution in D2O) at
4 °C. The yellow solution was stirred for about 2 h at 4 °C and sub-
sequently either placed immediately under NMR investigation or
stored at –60 °C. The used rates are listed in Table 3.
29Si{1H}-NMR: δ = –8.5 ppm.
Preparation of Si2(tBu)4(NeuNAc1,2,7,9H–4-1κ2O1,2:2κ2O7,9) (5) in
Solution: To a cooled (0 °C) suspension of N-acetylneuraminic
82
www.zaac.wiley-vch.de
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 2013, 77–83

Metal-binding Sites of N-Acetylneuraminic Acid
acid (320 mg, 1.0 mmol) in N,N-dimethylformamide D7 (4 mL)
Si(tBu)2(OTf)2 (0.65 mL, 2.0 mmol) was added dropwise.
References
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After stirring for 3 h 1H, 13C, 29Si and 2D NMR spectra were recorded.
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1H NMR: δ = 4.21 (t, 1H, H5, J4,5 = J5,6 = 9.8 Hz), 4.09–4.14 (m,
1H, H6), 3.96–4.03 (m, 1H, H4), 3.90–3.86 (m, 1H, H9a), 3.80–3.87
(m, 1H, H7), 3.68–3.65 (m, 2H, H8 and H9b), 2.14–1.86 (m, 2H, H3a
and H3b) ppm.
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N-acetylneuraminic acid (160 mg, 0.50 mmol) in N,N-dimethylform-
amide D7 (4 mL) Si(tBu)2(OTf)2 (0.52 mL, 1.6 mmol) was added
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3
3
1H NMR: δ = 4.65 (dd, 1H, H4, J3,4 = 6.9 Hz, J4,5 = 9.3 Hz), 4.36
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3
3
3
(dd, 1H, H5, J4,5 = J5,6 = 9.9 Hz), 4.22 (d, 1H, H6, J6,7 = 10.4 Hz),
3.93 (dd, 1H, H9a, J8,9a = 3.5 Hz, 2J9a,9b = –9.2 Hz), 3.86 (d, 1H, H7,
3
3J7,8 = 8.8 Hz), 3.70–3.59 (m, 2H, H8 and H9b), 2.23–2.18 (m, 1H,
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29Si{1H}-NMR: δ = 18.4, –5.1, –8.4 ppm.
For all compounds, the 13C NMR signals of the tBu function were
found in the ranges 25.5–28.7 ppm (CH3) and 19.3–22.7 ppm (Cquart);
1
the shift range of H NMR signals was 1.32–0.87 ppm.
Supporting Information (see footnote on the first page of this article):
All coloured figures
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Acknowledgement
This work was funded by the Deutsche Forschungsgemeinschaft
(Grant Kl 624/11–1). Johnson-Matthey (Reading, UK) is gratefully ac-
knowledged for a generous loan of palladium chloride. The authors
thank Christiane Schwegler for her valuable support.
Received: September 18, 2012
Published Online: October 30, 2012
Z. Anorg. Allg. Chem. 2013, 77–83
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
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