Sialic acid C-glycosides with aromatic residues: Investigating enzyme binding and inhibition of Trypanosoma cruzi trans-sialidase

Article abstract of DOI:10.1039/c0ob01176b

Several α-configured C-sialosides were synthesised by cross metathesis and further synthetic derivatisation to obtain ligands for Trypanosoma cruzi trans-sialidase (TcTS), a key enzyme in Chagas disease. Affinities of these compounds to immobilised TcTS w

Full text of DOI:10.1039/c0ob01176b

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PAPER
Sialic acid C-glycosides with aromatic residues: Investigating enzyme binding
and inhibition of Trypanosoma cruzi trans-sialidase†
Sebastian Meinke, Andreas Schroven and Joachim Thiem*
Received 15th December 2010, Accepted 23rd March 2011
DOI: 10.1039/c0ob01176b
Several a-configured C-sialosides were synthesised by cross metathesis and further synthetic
derivatisation to obtain ligands for Trypanosoma cruzi trans-sialidase (TcTS), a key enzyme in Chagas
disease. Affinities of these compounds to immobilised TcTS were measured by surface plasmon
resonance (SPR). The KD values thus obtained are in the lower millimolar range and will be discussed.
The results show the importance of addressing Tyr119 and Trp312 side chains of TcTS in target oriented
ligand synthesis, since these amino acids constitute the acceptor binding region in the active site of
TcTS. The best ligand showed a significant decrease of TcTS activity in a preliminary NMR based
inhibition assay.
several efforts have been made previously in this area,6–8 no strong
Introduction
inhibitor for TcTS is known to date.
The important roles that oligosaccharide structures play in cell
For investigation of possible ligands for TcTS, analogues of
biology lead to increasing interest in research concerning enzymes
involved in synthesis or degradation of such structures. Elucidating
a-sialosides are of particular interest. In this paper a rational syn-
thetic approach is presented, based on qualitative considerations
the enzyme mechanisms as well as the biological context in
deduced from the crystal structure of TcTS in complex with sialyl
lactose published by Amaya et al.9
which glycosyltransferases and glycosidases display their activity
may induce more effective as well as novel approaches to defeat
Ligand design for TcTS should particularly address the fact that
infections. Of particular interest are enzymes belonging to the
TcTS shows additional binding of acceptor molecules effected by
two aromatic amino acid side chains (Tyr119 and Trp312, see Fig. 1),9
responsible for the transfer of Neu5Ac and the negligible amount
of hydrolysis occurring in TcTS catalyzed enzymatic reactions.3
sialidase family (EC 3.2.1.18), because neuraminic acid (Neu5Ac)
plays an important role in Nature as a major constituent of
a variety of glycoconjugates occurring in animals and many
pathogens.1 Terminal sialylation of glycans is associated with many
processes such as cell recognition and cell differentiation. Either it
allows recognition by a suitable receptor protein, or the presence
of Neu5Ac is able to mask recognition sites.
In the South American trypanosomiasis (Chagas disease)2
trans-sialidase of Trypanosoma cruzi (TcTS) causes the transfer
of Neu5Ac from a human host cell to the cell epitope of the
pathogen.3 This unusual transfer mechanism enables the pathogen
to protect its own cell surface against recognition by the human
immune system. Because T. cruzi trypomastigotes lacking trans-
sialidase are less efficient in cell invasion,4 this enzyme seems to
play a crucial role in T. cruzi infection. Therefore, development
of ligands as potential inhibitors for TcTS can be considered as
a most promising goal in defeating T. cruzi infection.5 Although
Fig. 1 Tyr119 and Trp312 in the acceptor binding site (TcTS in complex
University of Hamburg, Faculty of Science, Department of Chemistry, Insti-
tute of Organic Chemistry, Martin-Luther-King Platz 6, 20146, Hamburg,
(Germany). E-mail: thiem@chemie.uni-hamburg.de; Fax: (+49) 40-42838-
4325
† Electronic supplementary information (ESI) available: General methods,
synthesis of new compounds, 1H and 13C NMR spectra. See DOI:
10.1039/c0ob01176b
with sialyl lactose9).
Another intended modification consists of adding an aromatic
residue to O-9 of the glycerol side chain. The vicinity of Trp120
(Fig. 2) suggests a positive effect of this derivatisation.
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Fig. 4 Intended derivatisations.
equilibration of the intermediate cis-product that is supposed
to react faster than the thermodynamically more stable trans-
product.13
Fig. 2 Trp120 contact with the glycerol chain.9
With the nitro-compound complete conversion was ob-
served, however a 1 : 5 mixture of cis/trans compounds resulted
(Scheme 1). While isomerisation of the trans-product after the
sodium methanolate deacetylation step (7c) might be explained
by a base catalyzed mechanism,14 the cis/trans mixture in the
metathesis product 5c is probably caused by photoisomerisation.
In case of the methoxy-substituted styrene, the conversion was
incomplete, however, only the trans configured product 5b could
be obtained. A proposed lower reactivity of the intermediate p-
methoxybenzylidene ruthenium complex in comparison to the un-
substituted benzylidene complex is reported by Grubbs et al.,13,15
so it is possible that decomposition of the catalyst occurred before
the reaction was completed.
Finally, the presence of several arginine residues in the active
site would allow for an ionic interaction, especially for Arg53 close
to O-4 of sialic acid (Fig. 3). Therefore we decided to introduce a
carboxylate in this position.
Deprotection of 5a–c in two steps led straight forwardly to the
C-sialoside ligands 9a–c. The lower yields in case of the nitro-
compounds might be due to degradation processes, indicated by a
deepening in the original yellow colour of these compounds over
the time.
The recently reported hydrogenated metathesis products of a-
an b-configured C-sialosides 10 and 11 (Scheme 2)11 were also
deprotected to give the ligands 12 and 13.
Fig. 3 Arginine side chains in proximity to O-4.
The deacetylated nitro-substituted metathesis product 7c could
easily be hydrogenated to give the saturated amino-substituted
C-sialoside 14 (Scheme 3).
Deprotection to C-sialoside ligand 15 was conducted with
triethylamine in water to avoid loss of product during the
ion exchange neutralisation step necessary in sodium hydroxide
saponification.
The synthesised compounds should then be studied in SPR
affinity measurements with immobilised recombinant trans-
sialidase. Resulting dissociation constants (KD) should provide a
more detailed picture of structural requirements for TcTS binding,
hence enabling the synthesis of competitive inhibitors against
Chagas disease.
Acetylation of 14 gave the N-acetyl derivative 16. In this case
the deprotection steps succeeded in average yields partly due to
slight base lability of the aromatic N-acetyl group and partly due
to the pronounced hydrophilicity of the compounds 16, 17 and 18
during chromatography.
Results and Discussion
Synthesis of the ligands
Aromatic residues for the acceptor binding site. Allyl C-
sialosides10 are useful precursors for the planned derivatisations
(Fig. 4). Recently we reported on an approach for adding
substituents to allyl-C-sialosides by olefin cross metathesis, leading
to C-sialosides with both aromatic and carbohydrate residues.11 In
this work we employed also p-methoxy- and p-nitro-substituted
styrenes, synthesised by Wittig reaction of the corresponding
aldehydes,12 to further explore this method and fine-tune the
electronic properties of the intended ligands. Both the second
generation Grubbs catalyst (1) and the second generation Grubbs–
Hoveyda (2) catalyst, were employed in our studies.
Reductive benzylation of the glycerol side-chain. The 9-
hydroxyl group of sialic acid is supposed to bind to the indole
nitrogen of Trp120 as supported by data of the crystal structure of
TcTS (see Fig. 2).9 Therefore, an additional aromatic ring in this
position was supposed to be advantageous for TcTS binding of
these ligands.
Application of a method for 9-O-benzylation of sialic acid
derivatives recently reported by Ernst et al.16 to compound 6 gave
8,9-O-benzylidene acetal 19 (1 : 1 mixture of diastereomers) as well
as 7,9-O-benzylidene acetal 20 as a single species displaying an all
equatorial six-membered chair as resolved by two dimensional
nmr spectroscopy (Scheme 4). Interestingly, compound 20 did
Only trans-configured cross metathesis product could be ob-
tained with unsubstituted styrene 4a.11 This can be explained by
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Scheme 1 a) Ruthenium catalyst 1 or 2, CH2Cl2, reflux; b) NaOMe, MeOH, rt; c) 0.1 M NaOH, H2O, rt.
Scheme 2 a) 0.1 M NaOH, H2O, rt.
not react in a subsequent reductive cleavage, however, the 8,9-
O-benzylidene acetal 19 gave the 9-O-benzylated C-sialoside 21 in
51% yield.
While saponification of 21 afforded C-sialoside 22, acetylation
to protected compound 23 enabled an additional metathesis step
(Scheme 5), leading to an interesting molecule with two aromatic
Scheme 3 a) H2, Pd/C, MeOH, rt; b) Et3N, H2O, rt; c) Ac2O, pyridine,
rings in terminal positions. The yield in the cross metathesis
reaction with styrene was satisfactory in comparison to non-
benzylated compounds.11
rt; d) NaOMe, MeOH, rt; e) 0.1 M NaOH, H2O, rt.
Utilising the method of Zbiral17 for compound 7a the glycerol
side chain was protected by an isopropylidene group to yield 27.
The subsequent oxidation by pyridinium chlorochromate lead to
the 4-ketone 28 in satisfactory yield, which by Wittig reaction
gave the unsaturated ethyl ester 29 in 45% yield (Scheme 6). In the
subsequent hydrogenation step only the product with equatorial
orientation of the C4 substituent 30 was formed, and the final
two step deprotection procedure gave C-sialoside 32 bearing two
carboxyl groups.
Introducing the carboxylate in the 4-position. The presence of
several arginine residues in the active site leads to the idea to
place an additional carboxylate residue as a useful substituent
in the Neu5Ac ring system. Aside from the important binding
of the Neu5Ac carboxylate to arginine, there are two further
arginine residues located near the 4-hydroxyl group. One of these
arginines (Arg53) is supposed to interact with the 4-hydroxyl group
via hydrogen bonding (see Fig. 3).9
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Scheme 5 a) catalyst 1, CH2Cl2, reflux; b) NaOMe, MeOH, rt; c) 0.1 M
NaOH, H2O, rt.
Table 1 Influence of aromatic residue and configuration on binding
Compound
KD (mM)
8
n. d.a
9a
12
13
3.6 0.6
0.16 0.02
n. d.a
Scheme 4 a) BADMA, pTSA, MeCN, 0 ◦C; b) BH3–Me3N, AlCl3, THF,
H2O, rt; c) 0.1 M NaOH, H2O, rt; d) Ac2O, pyridine.
Surface plasmon resonance affinity measurements
Recombinant trans-sialidase18 was immobilised on a commer-
cially available CM5 sensor chip (BIACORE) using the amino
coupling method as described by the manufacturer. The affinity
measurements were performed on a BIACORE T100 instrument
with Tris-HCl (pH = 7.5, 100 mM) as running buffer using 7–10
different dilutions of synthetic ligands up to 5 mM. The KD-values
were calculated using OriginPro 7.5 and the BIACORE evaluation
software.
The KD values obtained of these ligands vary from 0.16 to
14 mM and thus are in the range of common monovalent
carbohydrate protein interactions.19,20
The results in Table 1 clearly indicate the impact of an aromatic
group attached to the ligand considered to mimic the sugar
residue of the natural substrate bound between two aromatic
amino acid side chains (Tyr119 and Trp312, see Fig. 1). A missing
aromatic residue as in compound 8 as well as compounds with
a b-configured residue as in 13 clearly show no binding, which
indicate the specific interactions in the active site of TcTS.
a The KD values obtained were in the molar range.
Hydrogenation of the double bond increases affinity for TcTS
(compound 12 in comparison to compound 9a), probably due to
an increased flexibility of the phenylpropyl residue. Compound
12 shows the best KD value (0.16 mM) in this work, and its SPR
response curves and the resulting curve fit is shown as an example
in Fig. 5.
A further improvement of binding by para-substituents attached
to the aromatic ring was hitherto not observed (Table 2).
The roughly doubled KD of the nitro-compound 9c measured
as a mixture of cis/trans isomers could be interpreted as a
significantly reduced binding of the cis-isomer. However, since
the proportion of the cis-isomer is low (ca. 10%), the binding of
the trans-isomer is likely to be the main cause for the overall KD
value of 9c.
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Scheme 6 a) 2,2-Dimethoxy propane, H+ (ion exchange), acetone, rt; b) PCC, mol sieve 4 A, CH2Cl2, rt; c) Ph3P CHCO2Et, THF, rt; d) H2, Pd/C,
˚
MeOH, rt; e) H+ (ion exchange), H2O, 60 ◦C; f) 0.1 M NaOH, H2O, rt.
Table 2 Aromatic C-sialosides with substituents
Compound
KD (mM)
9c
9b
15
18
11.5 1.9a
5.0 3.0
0.35 0.12b
3.9 1.4b
a cis/trans-mixture. b Errors calculated on basis of average percental
aberration of KD values.
aromatic group attached to the glycerol side chain to be located in
the acceptor binding site as shown by X-ray crystal structure.
The possibility of an additional binding to Trp120 as mentioned
above is eliminated by the fact that two aromatic residues in the
same molecule do not improve binding as demonstrated with
compound 26. Based on the results of Withers et al.21 it can
be assumed that steric hindrance between two aromatic rings in
the acceptor binding site is responsible for decrease in affinity of
compound 26.
Fig. 5 Response curves and curve fit for ligand 12.
The hydrophilic amino group in compound 15 with a KD value
similar to compound 12 seems to eliminate the possibility of mere
hydrophobic interaction in the binding of aromatic C-sialosides
to TcTS.
The trend for better binding associated with greater flexibility
is obvious with the KD values of 15 and 18.
Ligand binding is also enabled by an aromatic group connected
to the glycerol side chain as in compound 23 (Table 3). Very likely
this binding event also occurs between Tyr119 and Trp312 (see Fig. 1)
as suggested by recent results of Withers et al.,21 who observed an
Interestingly, the active site tolerates the additional carboxylate
in compound 32, although no stronger binding results from
this synthetic variation. Perhaps stronger binding due negatively
charged carboxylate is counteracted by steric demands.
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Table 3 Other C-sialoside derivatives
Compound
KD (mM)
22
0.37 0.07
26
2.0 0.3
32
33
34
3.5 0.4
Fig. 6 Transfer rates measured by NMR. Compound 12 leads to a
decrease in enzyme activity at a concentration of 1 mM.
14
5
Conclusions
Cross metathesis turned out to be a useful tool in synthesising a se-
ries of differently substituted aromatic C-sialosides. This method,
once established, allows to use various substituted styrenes, thus
providing the means to fine-tune electronic properties of ligands.
This approach should prove generally useful in the field of ligand
synthesis regarding studies of carbohydrate protein interactions.
The KD values obtained by SPR and discussed above provide
experimental evidence to qualitative considerations deduced by
inspection of the crystal structure of TcTS in complex with sialyl
lactose.9 The most convincing result is the considerable influence
on ligand binding by addition of an aromatic ring to the a-C-
sialoside, which clearly indicates the importance of mimicking
the acceptor molecule in TcTS ligand design. Additionally it was
shown that two aromatic rings as in compound 26 are sterically
too demanding for the acceptor binding site between Tyr119 and
Trp312. Compound 12 with the best affinity (KD = 0.16 mM)
represents a novel class of C-glycoside ligands for TcTS. Such C-
glycosides do not only provide hydrolytic stability but also allow
for versatile derivatisation in the development of stronger binding
ligands.
0.65 0.85
The chain elongated C-analogue of a(2–6)-sialyl galactose 3311
does also not show any increase of affinity, which is probably
caused by mimicking the “wrong” a(2–6)-linkage which is not
recognised by this enzyme. A corresponding decrease in binding
by an a(2–6)-sialyl galactose analogue containing a phosphonate
group in comparison to its a(2–3)-isomer was observed by
Streicher et al.8
As reference the glycal of Neu5Ac (Neu5Ac2en, compound 34)
was tested under similar conditions. Neu5Ac2en is known as an
inhibitor of viral and bacterial sialidases,22 but shows only weak
inhibition towards TcTS.23,24 Since TcTS displays a mechanism
similar to that of sialidases,9,25 Neu5Ac2en can be considered as
transition state analogue for TcTS and should naturally show
affinity to this enzyme. Interestingly, the KD value of 34 is in
the submillimolar range, while a poor inhibitory constant of
12.3 mM is reported in the literature.5 Since it is generally difficult
to compare KD and Ki values, this suggests that further studies are
needed regarding these compounds.
A preliminary NMR based inhibition assay demonstrated that
inhibition of TcTS by this type of compounds is possible. Selective
derivatisation of 12 should permit the synthesis of more effective
inhibitors for TcTS.
Experimental
NMR based inhibition assay
General methods
Ligand 12 showed the highest affinity towards TcTS in the
SPR experiments (KD = 0.16 mM) and was therefore subjected
to a preliminary inhibition assay based on the TcTS catalyzed
sialylation of methyl allolactoside employing pNP-sialoside as
donor substrate in D2O. Transfer rates were determined by proton
NMR.18 As shown in Fig. 6 we observed a considerable decrease
in trans-sialidase activity at a ligand concentration of 1 mM.
Commercially available starting materials were used without
further purification. Solvents were dried according to standard
methods. TLC was performed on precoated aluminium plates
(Silica Gel 60 F254, Merck 5554) employing UV-absorption and
charring with 10% H2SO4 in ethanol for visualisation. For column
chromatography Silica Gel 60, 230–400 mesh, 40–63 mm (Merck)
was used.
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1H NMR and 13C NMR spectra were recorded on Bruker
AMX-400 (400.25 MHz for 1H, 100.65 MHz for 13C), Bruker AV-
room temperature until TLC showed complete conversion of
the starting material. Column chromatography on silica gel
(dichloromethane/methanol) yielded the product.
1
400 (400.25 MHz for H, 100.65 MHz for 13C) and on Bruker
DRX-500 (500.13 MHz for 1H, 125.77 MHz for 13C) at
300 K. Chemical shifts were calibrated to solvent residual peaks.26
The signals were assigned by HH-COSY, HSQC, HMBC and if
necessary NOESY experiments. J values are given in Hz.
Optical rotations were measured using a Perkin Elmer 241 (546
nm) or a Kru¨ss Optronic P8000 (589 nm) at 20 ◦C; [a]D values are
given in 10-1 deg cm2 g-1.
Mass spectra were recorded on a Bruker Biflex III in positive
reflector mode (MALDI-TOF), on a VG Analytical VG/70-250 F
(FAB) and on a Thermo Finnigan MAT 95 XL mass spectrometer
(ESI).
Allyl C-sialosides 3, 6, 810 and substituted styrenes 4b and 4c12
were synthesised as described and the data were consistent with
those published. The syntheses of C-sialosides 5a, 10, 11 and 33
were published before in a short communication.11
The synthetic routes leading to compounds 9b, 22 and 32
are shown in the following section as examples for the three
derivatisation methods employed in this work. The complete
experimental data for all new compounds can be found in the
ESI.†
(E)-Methyl-5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-
3,5-dideoxy-2-C-(3-(4-methoxyphenyl)-prop-2-enyl)-D-erythro-L-
manno-nononate (5b). a) Compound 3 (240 mg, 466 mmol) was
reacted with 4b (540 mg, 4.02 mmol) according to GP1 using
catalyst 2 (25 mg, 40 mmol) to give 5b in a mixture with starting
material 3 (136 mg, 47%; calculated from NMR) as a colourless
oil;
b) 7b (13 mg, 29 mmol) was acetylated according to GP2 to give
5b (18 mg, 100%) as a colourless oil;
[a]2D5 -13.8 (c 1 in CHCl3); dH (500 MHz, CDCl3) 7.31 (2 H, d,
J 8.7, arom. H), 6.84 (2 H, d, J 8.7, arom. H), 6.31 (1 H, d, J
15.8, -CH CH–Ph), 6.00 (1 H, ddd, J 15.8, 8.0 and 6.8, -CH2–
CH ), 5.40 (1 H, ddd, J8,7 6.9, J8,9b 6.0, J8,9a 2.6, H-8), 5.34 (1
H, dd, J7,8 6.9, J7,6 1.1, H-7), 5.17 (1 H, d, JNH,5 9.6, NH), 4.84 (1
H, ddd, J4,3ax 11.7, J4,5 10.0, J4,3eq 4.6, H-4), 4.42 (1 H, dd, J9a,9b
12.4, J9a,8 2.6, H-9a), 4.15 (1 H, dd, J9b,9a 12.4, J9b,8 6.0, H-9b),
4.05–4.00 (2 H, m, H-5, H-6), 3.80 (3 H, s, arom. OCH3), 3.70
(3 H, s, OCH3), 2.65–2.50 (2 H, m, -CH2–CH ), 2.51 (1 H, dd,
J3eq,3ax 12.9, J3eq,4 4.6, H-3eq), 2.12, 2.11, 2.02, 2.01, (each 3 H,
4x s, 4x AcCH3), 1.87 (3 H, s, NHAcCH3), 1.83 (1 H, dd, J3ax,3eq
12.9, J3ax,4 11.7, H-3ax); dC (125 MHz, CDCl3) 171.77 (CO2CH3),
171.23, 170.86, 170.43, 170.35, 170.24 (5x AcC O), 159.26 (arom.
C), 133.63 (-CH CH–Ph), 129.96 (arom. C), 127.69 (arom. C),
120.48 (-CH2–CH ), 114.04 (arom. C), 80.97 (C-2), 73.66 (C-6),
70.37 (C-4), 69.87 (C-8), 68.08 (C-7), 62.55 (C-9), 55.43 (arom.
OCH3), 52.52 (OCH3), 49.81 (C-5), 43.74 (-CH2-CH ), 37.52
(C-3), 23.35 (NHAcCH3), 21.26, 21.04 (2 ¥ AcCH3), 20.94 (2 ¥
AcCH3); HRMS (FAB): m/z calcd. for [M+H]+: 622.249966;
found: 622.250854.
Syntheses
General procedure for cross metathesis with styrenes (GP1).
The allyl C-sialoside was dissolved under nitrogen in anhydrous
dichloromethane to yield a 0.002 M solution and the styrene (8–10-
fold excess) was added. After addition of the ruthenium catalyst,
the reaction was stirred under reflux for 24 h. The removal of
the solvent in vacuo and column chromatography on silica gel
(toluene/acetone or pure ethyl acetate) yielded the product.
General procedure for acetylation (GP2). The compound was
dissolved in pyridine and an excess of acetic anhydride was added.
The solution was stirred at room temperature until TLC indicated
complete consumption of starting material. The solvent was
removed in vacuo and the residue was co-evaporated with toluene
before column chromatography (silica gel, toluene/acetone or
pure ethyl acetate).
(E)-Methyl-5-acetamido-2,6-anhydro-3,5-dideoxy-2-C-(3-(4-
methoxyphenyl)-prop-2-enyl)-D-erythro-L-manno-nononate (7b).
Compound 5b (136 mg, 0.230 mmol) was deacetylated according
to GP3 to give 7b (97 mg, 100%) as a colourless oil; [a]2D5 +4.2
(c 1 in MeOH); dH (400 MHz, [D4]MeOH) 7.28 (2 H, d, J 8.8,
arom. H), 6.84 (2 H, d, J 8.8, arom. H), 6.38 (1 H, d, J 15.8,
-CH CH–Ph), 6.05 (1 H, ddd, J 15.8, 7.5 and 7.5, -CH2–CH ),
3.89 (1 H, ddd, J8,7 8.9, J8,9b 5.7, J8,9a 2.8, H-8), 3.85 (1 H, dd,
General procedure for deacetylation (GP3). The compound
was dissolved in dry methanol and sodium methoxide (1 molar) in
dry methanol was added drop wise until a pH of 9–10. The reaction
was stirred at room temperature until TLC showed complete
deacetylation. After neutralisation with acidic ion exchanger and
filtration the solvent was removed in vacuo. Column chromatogra-
phy on silica gel (dichloromethane/methanol) yielded the product.
J
9a,9b 11.3, J9a,8 2.8, H-9a), 3.77 (3 H, s, arom. OCH3), 3.75 (3 H, s,
OCH3), 3.73 (1 H, dd, J5,6 10.2, J5,4 10.1, H-5), 3.68-3.60 (2 H, m,
H-4, H-9b), 3.52 (1 H, dd, J6,5 10.2, J6,7 1.6, H-6), 3.51 (1 H, dd,
J
7,8 8.9, J7,6 1.6, H-7), 2.65–2.59 (3 H, m, -CH2–CH and H-3eq),
2.00 (3 H, s, AcCH3), 1.65 (1 H, dd, J3ax,3eq 13.1, J3ax,4 11.5, H-3ax);
dC (100 MHz, [D4]MeOH) 175.24 (2 ¥ C O), 160.72 (arom.
C), 134.74 (-CH CH–Ph), 131.34 (arom. C), 128.47 (arom. C),
121.66 (-CH2–CH ), 114.96 (arom. C), 82.19 (C-2), 75.95 (C-6),
72.91 (C-8), 70.26 (C-7), 69.14 (C-4), 64.67 (C-9), 55.71 (arom.
OCH3), 54.19 (C-5), 53.04 (OCH3), 44.90 (-CH2–CH ), 41.46
(C-3), 22.64 (AcCH3); HRMS (ESI): m/z calcd. for [M+Na]+:
476.1891; found: 476.1897.
General procedure for ester saponification (GP4). The ester was
dissolved in aqueous sodium hydroxide (0.1 M) and stirred at
room temperature until TLC showed complete conversion. After
neutralisation with acidic ion exchanger and filtration the material
was freeze dried.
General procedure for catalytic hydrogenation (GP5). The
unsaturated compound was dissolved in methanol under nitrogen.
After adding a catalytic amount of palladium on charcoal (5%,
wet) the reaction vessel was carefully evaporated and flushed
with hydrogen. The reaction was stirred under hydrogen at
(E)-5-Acetamido-2,6-anhydro-3,5-dideoxy-2-C-(3-(4-methoxy-
phenyl)-prop-2-enyl)-D-erythro-L-manno-nononic acid (9b). Com-
pound 7b (12 mg, 26 mmol) was saponified according to GP4 to
give 9b (12 mg, 100%) as a yellow amorphous solid; [a]2D5 +7.0 (c
0.5 in MeOH); dH (400 MHz, D2O) 7.41 (2 H, d, J 8.8, arom. H),
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6.97 (2 H, d, J 8.8, arom. H), 6.49 (1 H, d, J 15.9, -CH CH–Ph),
6.12 (1 H, ddd, J 15.9 and 2 ¥ 7.5, -CH2–CH ), 3.89 (1 H, ddd,
J8,7 8.6, J8,9b 6.2, J8,9a 2.7, H-8), 3.90–3.84 (1 H, m, H-9a), 3.83 (3
H, s, arom. OCH3), 3.83–3.78 (1 H, m, H-5), 3.74 (1 H, ddd, J4,3ax
11.7, J4,5 10.0, J4,3eq 4.6, H-4), 3.66 (1 H, dd, J6,5 10.2, J6,7 1.5, H-6),
3.66–3.62 (1 H, m, H-9b), 3.59 (1 H, dd, J7,8 8.6, J7,6 1.5, H-7), 2.66
(1 H, dd, J3eq,3ax 13.0, J3eq,4 4.6, H-3eq), 2.65–2.64 (2 H, m, -CH2–
CH ), 2.05 (3 H, s, AcCH3), 1.69 (1 H, dd, J3ax,3eq 13.0, J3ax,4 11.7,
H-3ax); dC (100 MHz, D2O) 176.75 (C O), 132.95 (-CH CH–
Ph), 130.37 (arom. C), 127.58 (arom. C), 121.67 (-CH2–CH ),
114.32 (arom. C), 81.01 (C-2), 73.76 (C-6), 71.90 (C-8), 68.38,
68.35 (C-4, C-7), 62.80 (C-9), 55.43 (arom. OCH3), 52.24 (C-5),
43.05 (-CH2–CH ), 39.71 (C-3), 22.06 (AcCH3); MS (MALDI-
TOF): m/z = 440.5 ([M+H]+), 462.4 ([M+Na]+), 478.4 ([M+K]+);
MS (FAB): m/z = 462.3 ([M+Na]+), 478.3 ([M+K]+).
(1 H, dd, J7,8 9.7, J7,6 1.1, H-7), 2.54–2.42 (3 H, m, CH2, H-3eq),
1.99 (3 H, s, AcCH3), 1.54 (1 H, dd, J3ax,3eq 12.8, J3ax,4 11.9, H-3ax);
dC (125 MHz, [D4]MeOH) 133.41 (-CH CH2), 129.50, 128.84,
127.50 (arom. C), 118.89 (-CH CH2), 102.24 (PhCH), 81.77
(C-2), 80.78 (C-7), 73.16 (C-6), 72.62 (C-9), 69.76 (C-4), 61.36
(C-8), 52.75 (C-5), 52.55 (OCH3), 45.58 (CH2), 41.11 (C-3), 22.93
(AcCH3); HRMS (FAB): m/z calcd. for [M+H]+: 436.197142;
found: 436.197968.
Methyl-5-acetamido-2,6-anhydro-9-O-benzyl-3,5-dideoxy-2-C-
(prop-2-enyl)-D-erythro-L-manno-nononate (21). Compound 19
(160 mg, 367 mmol), trimethyl amine borane adduct (0.11 g,
1.5 mmol) and aluminium chloride (294 mg, 2.20 mmol) were
dissolved in 8.4 mL dry tetrahydrofuran. After stirring for 5 min
water (9.9 mL, 0.55 mmol) was added and the reaction was
stirred at room temperature for 4 h. After TLC showed complete
consumption of starting material the reaction was stopped by
adding water (4.2 mL) and 0.1 molar HCl (4.2 mL). The solution
was diluted with dichloromethane (42 mL) and washed twice with
5% sodium bicarbonate solution (2 ¥ 20 mL) and with water
(20 mL). The organic phase was dried over sodium sulphate,
filtrated and concentrated in vacuo. The residue was purified by
column chromatography (dichloromethane/methanol) to give 21
Methyl-5-acetamido-2,6-anhydro-8,9-O-benzyliden-3,5-dide-
oxy-2-C-(prop-2-enyl)-D-erythro-L-manno-nononate (19) and
methyl-5-acetamido-2,6-anhydro-7,9-O-benzyliden-3,5-dideoxy-2-
C-(prop-2-enyl)-D-erythro-L-manno-nononate (20). Compound
6 (200 mg, 0.576 mmol) and benzaldehyde dimethyl acetal
(173 mL, 1.15 mmol) were dissolved in dry acetonitrile. The
solution was cooled to 0 ◦C and a catalytic amount (8 mg,
0.04 mmol) of p-toluenesulfonic acid monohydrate was added.
The reaction was stirred at room temperature until TLC showed
complete consumption of the starting material. After adding a
few drops of triethyl amine the solvent was removed in vacuo
and the products were separated by column chromatography
(dichloromethane/methanol); 19 (143 mg, 57%, 1 : 1 mixture
◦
(82 mg, 51%) as a white solid; mp 101 C; [a]2D5 -104 (c 0.2 in
CH2Cl2); dH (500 MHz, [D4]MeOH) 7.38–7.31 (5 H, m, arom. H),
5.84–5.74 (1 H, m, CH CH2), 5.11–5.06 (2 H, m, CH CH2),
4.59–4.57 (2 H, m, PhCH2), 3.97 (1 H, ddd, J8,7 8.8, J8,9b 5.7, J8,9a
2.3, H-8), 3.78 (1 H, dd, J9a,9b 10.3, J9a,8 2.3, H-9a), 3.75 (3 H, s,
OCH3), 3.70 (1 H, dd, J5,4 10.2, J5,6 10.2, H-5), 3.65–3.58 (1 H,
m, H-4), 3.62 (1 H, dd, J9b,9a 10.3, J9b,8 5.7, H-9b), 3.55 (1 H, dd,
J7,8 8.8, J7,6 1.4, H-7), 3.51 (1 H, dd, J6,5 10.2, J6,7 1.4, H-6), 2.57
(1 H, dd, J3eq,3ax 13.2, J3eq,4 4.6, H-3eq), 2.54–2.44 (2 H, m, CH2),
1.98 (3 H, s, AcCH3), 1.45 (1 H, dd, J3ax,3eq 13.2, J3ax,4 11.4, H-3ax);
dC (100 MHz, [D4]MeOH) 175.22 (C-1), 139.85 (arom C), 132.94
(-CH CH2), 129.33 (arom. C), 128.84 (arom. C), 128.59 (arom.
C), 119.37 (-CH CH2), 81.75 (C-2), 75.78 (C-6), 74.41 (PhCH2),
72.90 (C-9), 71.82 (C-8), 70.16 (C-7), 69.04 (C-4), 54.17 (C-5),
52.95 (OCH3), 45.71 (CH2), 41.45 (C-3), 22.63 (AcCH3); HRMS
(FAB): m/z calcd. for [M+H]+: 438.212792; found: 438.211823.
◦
of diastereomers): mp 112 C; [a]2D5 +83 (c 0.1 in CH2Cl2); dH
(500 MHz, [D4]MeOH) 7.50–7.46 (2 H, m, arom. H), 7.38–7.35
(3 H, m, arom. H), 5.87 (0.5 H, s, PhCHI), 5.85–5.74 (2 H, m,
I
CH CH2 , CH CH2II), 5.76 (1 H, s, PhCHII), 5.12–5.06 (4 H,
I
m, CH CH2 , CH CH2II), 4.40–4.34 (2 H, m, H-8), 4.27 (1 H,
dd, J9a,9b 8.5, J9a,8 6.7, H-9aI), 4.24 (1 H, dd, J9a,9b 8.3, J9a,8 5.2,
H-9aII), 4.11 (1 H, dd, J9b,9a 8.5, J9b,8 7.6, H-9bI), 4.09 (1 H, dd,
J9b,9a 8.3, J9b,8 7.3, H-9bII), 3.82 (1 H, dd, J7,8 5.2, J7,6 1.1, H-7I),
3.75 (1 H, dd, J5,4 10.2, J5,6 10.2, H-5I), 3.74 (1 H, dd, J5,4 10.2,
J5,6 10.2, H-5II), 3.69–3.59 (3 H, m, H-7II, H-4I, H-4II), 3.66 (3
H, s, OCH3), 3.65 (3 H, s, OCH3), 3.50 (1 H, dd, J6,5 10.2, J6,7
1.1, H-6II), 3.47 (1 H, dd, J6,5 10.2, J6,7 1.1, H-6I), 2.56–2.44 (6
5-Acetamido-2,6-anhydro-9-O-benzyl-3,5-dideoxy-2-C-(prop-
2-enyl)-D-erythro-L-manno-nononic acid (22). Compound 21
(62 mg, 0.14 mmol) was saponified according to GP4 to give 22
(60 mg, 100%) as a white solid; mp 147 ◦C; [a]D25 -22 (c 0.1 in H2O);
I
H, m, CH2 , CH2II, H-3eqI, H-3eqII), 2.01 (3 H, s, AcCH3), 1.98 (3
H, s, AcCH3), 1.54 (1 H, dd, J3ax,3eq 12.8, J3ax,4 11.7, H-3ax), 1.53 (1
H, dd, J3ax,3eq 12.8, J3ax,4 11.7, H-3ax); dC (125 MHz, [D4]MeOH)
139.59 (arom. C), 133.21, 133.16 (-CH CH2), 130.31, 130.14,
129.23, 127.97, 127.68 (arom. C), 119.26, 119.17 (-CH CH2),
105.08 (PhCHII), 104.84 (PhCHI), 81.99, 81.88 (C-2), 78.11,
77.92 (C-8), 76.72, 76.31 (C-6), 70.93, 70.89 (C-7), 69.07, 68.92
(C-4), 68.59, 68.48 (C-9), 54.11 (C-5), 52.46, 52.43 (OCH3), 45.64,
45.61 (CH2), 41.27 (C-3), 22.72 (AcCH3); HRMS (FAB): m/z
calcd. for [M+H◦]+: 436.197142; found: 436.197968; 20 (88 mg,
35%): mp 83–84 C; [a]2D5 -21.4 (c 0.5 in MeOH); dH (500 MHz,
[D4]MeOH) 7.59–7.56 (2 H, m, arom. H), 7.37–7.30 (3 H, m,
arom. H), 5.88–5.75 (1 H, m, CH CH2), 5.43 (1 H, s, PhCH),
5.07–5.03 (2 H, m, CH CH2), 4.27 (1 H, dd, J9a,9b 10.6, J9a,8 5.4,
H-9a), 4.04 (1 H, ddd, J8,9b 10.0, J8,7 9.7, J8,9a 5.4, H-8), 4.02 (1
H, dd, J5,6 10.5, J5,4 10.3, H-5), 3.87 (1 H, dd, J6,5 10.5, J6,7 1.1,
H-6), 3.74 (3 H, s, OCH3), 3.60 (1 H, ddd, J4,3ax 11.9, J4,5 10.3,
J4,3eq 4.7, H-4), 3.56 (1 H, dd, J9b,9a 10.6, J9b,8 10.0, H-9b), 3.55
d
H (500 MHz, [D4]MeOH) 7.37–7.23 (5 H, m, arom. H), 5.94–5.85
(1 H, m, CH CH2), 5.08–4.99 (2 H, m, CH CH2), 4.59 (1 H, d,
J 12.3, PhCH2a), 4.54 (1 H, d, J 12.3, PhCH2b), 3.99 (1 H, ddd,
J8,7 8.8, J8,9b 5.7, J8,9a 2.2, H-8), 3.76 (1 H, dd, J9a,9b 10.4, J9a,8 2.2,
H-9a), 3.70 (1 H, ddd, J4,3ax 11.4, J4,5 9.8, J4,3eq 4.7, H-4), 3.61 (1
H, dd, J5,6 0.4, J5,4 9.8, H-5), 3.60 (1 H, dd, J9b,9a 10.4, J9b,8 5.7,
H-9b), 3.54 (1 H, dd, J6,5 10.4, J6,7 1.9, H-6), 3.52 (1 H, dd, J7,8 8.8,
J
7,6 1.9, H-7), 2.63 (1 H, dd, J3eq,3ax 12.6, J3eq,4 4.7, H-3eq), 2.48-2.38
(2 H, m, CH2), 1.99 (3 H, s, AcCH3), 1.45 (1 H, dd, J3ax,3eq 12.6,
J3ax,4 11.4, H-3ax); dC (125 MHz, [D4]MeOH) 175.47 (AcC O),
134.62 (CH CH2), 129.29 (arom. C), 128.80 (arom. C), 128.51
(arom. C), 117.87 (CH CH2), 75.31 (C-6), 74.30 (PhCH2), 72.75
(C-9), 71.98 (C-8), 70.34 (C-7), 69.78 (C-4), 54.47 (C-5), 45.95
(CH2), 42.16 (C-3), 22.54 (AcCH3); HRMS (FAB): m/z calcd. for
[M+H]+: 424.197142; found: 424.196808.
4494 | Org. Biomol. Chem., 2011, 9, 4487–4497
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(E)-Methyl-5-acetamido-2,6-anhydro-3,5-dideoxy-8,9-O-iso-
propyliden-2-C-(3-phenylprop-2-enyl)-D-erythro-L-manno-nono-
nate (27). Compound 7a (80 mg, 0.19 mmol) was dissolved in
dry acetone (27 ml) under argon atmosphere. After addition of
2,2-dimethoxypropane (240 ml, 1.95 mmol) and ion exchanger
(DOWEX 50 ¥ 8, H+, 80 mg) the solution was stirred overnight
at room temperature. When the consumption of starting material
was complete (TLC) the yellow solution was filtered, the solvent
removed in vacuo and the residue purified by column chromatogra-
phy (dichloromethane/methanol, containing 0.5% triethyl amine)
to give 27 (70 mg, 80%); mp 178 ◦C; [a]D25 +47 (c 0.15 in CH2Cl2);
dH (400 MHz, CDCl3) 7.34–7.26 (4 H, m, arom. H), 7.24–7.19
(1 H, m, arom. H), 6.41 (1 H, d, J 15.9, CH CH–Ph), 6.08
(1 H, ddd, J 15.9 and 2 ¥ 7.5, -CH CH–Ph), 5.62 (1 H, d,
JNH,5 7.7, NH), 4.32 (1 H, ddd, J8,7 6.4, J8,9a 6.3, J8,9b 6.3, H-
8), 4.14 (1 H, dd, J9a,9b 8.6, J9a,8 6.3, H-9a), 4.07 (1 H, dd, J9b,9a
8.6, J9b,8 6.3, H-9b), 3.80 (1 H, ddd, J5,6 10.1, J5,4 10.0, J5,NH 7.7,
H-5), 3.75-3.66 (1 H, m, H-4), 3.71 (3 H, s, OCH3), 3.58 (1 H,
dd, J7,8 6.4, J7,6 1.0, H-7), 3.37 (1 H, dd, J6,5 10.1, J6,7 1.0, H-6),
2.73–2.60 (3 H, m, CH2, H-3eq), 2.05 (3 H, s, AcCH3), 1.62 (1 H,
dd, J3ax,3eq 13.0, J3ax,4 11.0, H-3ax), 1.40 (3 H, s, C(CH3)2), 1.37 (3
H, s, C(CH3)2); dC (100 MHz, CDCl3) 173.10, 172.85 (AcC O),
137.10 (arom. C), 134.38 (CH CH–Ph), 128.71, 127.69, 126.41
(arom. C), 122.79 (-CH CH–Ph), 108.71 (C(CH3)2), 81.11 (C-
2), 75.74 (C-8), 75.55 (C-6), 70.16 (C-7), 79.11 (C-4), 66.89 (C-9),
53.86 (C-5), 52.29 (OCH3), 43.64 (CH2), 40.50 (C-3), 26.98, 25.70
(C(CH3)2), 23.40 (AcCH3); HRMS (FAB): m/z calcd. for [M+H]+:
464.228442; found: 464.228058.
at room temperature. When TLC showed complete conversion the
solution was filtered, concentrated in vacuo and the residue was
purified by column chromatography (dichloromethane/methanol)
to give 29 (19 mg, 45%) as a white solid; mp 122 ◦C; [a]D25 -38 (c 0.15
in MeOH); dH (500 MHz, [D4]MeOH) 7.37–7.35 (2 H, m, arom.
H), 7.30–7.26 (2 H, m, arom. H), 7.22–7.18 (1 H, m, arom. H),
6.47 (1 H, d, J 15.9, CH CH–Ph), 6.23 (1 H, ddd, J 15.9 and 2 ¥
7.5, -CH CH–Ph), 5.76 (1 H, dd, J 1.3 and 1.2, HC–CO2Et),
4.72 (1 H, dd, J5,6 10.4, J 1.2, H-5), 4.46 (1 H, d, J3a,3b 13.9, H-3a),
4.26 (1 H, ddd, J8,9b 7.0, J8,9a 6.5, J8,7 5.2, H-8), 4.19–4.13 (2 H, m,
OCH2CH3), 4.09 (1 H, dd, J9a,9b 8.4, J9a,8 6.5, H-9a), 4.04 (1 H, dd,
J
9b,9a 8.4, J9b,8 7.0, H-9b), 3.82 (1 H, dd, J6,5 10.4, J6,7 1.2, H-6), 3.76
(1 H, dd, J7,8 5.2, J7,6 1.2, H-7), 3.62 (3 H, s, OCH3), 2.71 (2 H, d,
J 7.5, -CH2CH ), 2.30 (1 H, dd, J3b,3a 13.9, J 1.3, H-3b), 2.03 (3
H, s, AcCH3), 1.39 (3 H, s, C(CH3)2), 1.33 (3 H, s, C(CH3)2), 1.26
(3 H, t, J 7.1, OCH2CH3); dC (125 MHz, [D4]MeOH) 154.62 (C-4),
135.19 (CH CH–Ph), 129.55, 128.44, 127.27 (arom. C), 124.43
(-CH CH–Ph), 114.90 ( HC–CO2Et), 84.66 (C-2), 78.25 (C-
6), 78.23 (C-8), 70.74 (C-7), 67.17 (C-9), 61.21 (OCH2CH3), 52.35
(OCH3), 51.81 (C-5), 44.45 (-CH2CH ), 36.92 (C-3), 26.92, 25.78
(C(CH3)2), 22.73 (AcCH3), 14.56 (OCH2CH3); HRMS (FAB):
m/z calcd. for [M+H]+: 532.254657; found: 532.253052.
Methyl-5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-ethoxy-car-
bonylmethyl-8,9-O-isopropyliden-2-C-(3-phenylpropyl)-D-erythro-
L-manno-nononate (30). Compound 29 (60 mg, 0.11 mmol), was
hydrogenated according to GP5 to give 30 (60 mg, 100%) as a
white solid; mp 115 ◦C; [a]D25 -73 (c 0.1 in MeOH); dH (500 MHz,
[D4]MeOH) 7.27–7.22 (2 H, m, arom. H), 7.18–7.13 (3 H, m, arom.
H), 4.22 (1 H, ddd, J8,7 7.0, J8,9b 6.9, J8,9a 6.5, H-8), 4.15–4.08 (2
H, m, OCH2CH3), 4.04 (1 H, dd, J9a,9b 8.4, J9a,8 6.5, H-9a), 3.97 (1
H, dd, J9b,9a 8.4, J9b,8 6.9, H-9b), 3.73–3.67 (1 H, m, H-5), 3.71 (3
H, s, OCH3), 3.61 (1 H, dd, J7,8 7.0, J7,6 1.5, H-7), 3.54 (1 H, dd,
J6,5 9.9, J6,7 1.5, H-6), 2.65–2.51 (2 H, m, CH2c), 2.49 (1 H, dd,
J 15.4 and 4.3, CH2CO2Et), 2.33 (1 H, dd, J3eq,3ax 13.4, J3eq,4 3.6,
H-3eq), 2.11 (1 H, dd, J 15.4 and 8.7, CH2CO2Et), 2.03–1.96 (1 H,
m, H-4), 1.94 (3 H, s, AcCH3), 1.73–1.68 (3 H, m, CH2a, CH2b),
1.54–1.46 (1 H, m, CH2b), 1.41–1.33 (1 H, m, H-3ax), 1.37 (3 H, s,
C(CH3)2), 1.33 (3 H, s, C(CH3)2), 1.25 (3 H, t, J 7.1, OCH2CH3);
dC (125 MHz, [D4]MeOH) 129.41 (arom. C), 129.35 (arom. C),
126.87 (arom. C), 109.54 (C(CH3)2), 81.61 (C-2), 78.05 (C-8),
77.04 (C-6), 70.55 (C-7), 67.09 (C-9), 61.64 (OCH2CH3), 52.40 (C-
5), 50.52 (OCH3), 40.67 (CH2a), 38.88 (C-3), 38.77 (CH2CO2Et),
36.69 (CH2c), 36.28 (C-4), 26.96 (C(CH3)2), 26.62 (CH2b), 25.81
(C(CH3)2), 22.55 (AcCH3), 14.54 (OCH2CH3); HRMS (FAB):
m/z calcd. for [M+H]+: 536.285957; found: 536.286560.
(E)-Methyl-5-acetamido-2,6-anhydro-3,5-dideoxy-8,9-O-iso-
propyliden-2-C-(3-phenylprop-2-enyl)-D-glycero-D-galacto-non-4-
ulosonate (28). Compound 27 (70 mg, 0.15 mmol) was dissolved
in dry dichloromethane (4 mL). After addition of powdered mol
˚
sieve (4 A, 280 mg) and pyridinium chlorochromate (130 mg,
603 mmol) the solution was stirred at room temperature until TLC
showed complete conversion. The solution was diluted with diethyl
ether, filtrated and concentrated in vacuo. The residue was purified
by column chromatography (dichloromethane/methanol) to give
28 (45 mg, 65%) as a colourless oil; [a]2D5 +21 (c 0.2 in CH2Cl2); dH
(500 MHz, CDCl3) 7.38–7.29 (5 H, m, arom. H), 6.47 (1 H, d, J
15.7, CH CH–Ph), 6.15 (1 H, d, J 15.7, J 7.5, -CH CH–Ph),
4.67 (1 H, dd, J5,6 10.7, J5,NH 6.8, H-5), 4.09 (1 H, ddd, J8,7 9.0,
J8,9b 5.2, J8,9a 3.7, H-8), 3.96 (1 H, dd, J9a,9b 11.2, J9a,8 3.7, H-9a),
3.78 (1 H, dd, J9b,9a 11.2, J9b,8 5.2, H-9b), 3.76 (3 H, s, OCH3),
3.63 (1 H, dd, J6,5 10.7, J6,7 1.6, H-6), 3.53 (1 H, dd, J7,8 9.0,
J7,6 1.6, H-7), 3.22 (1 H, d, J3a,3b 14.0, H-3a), 2.84–2.76 (3 H, m,
H-3b, CH2), 2.09 (3 H, s, AcCH3), 1.37 (3 H, s, C(CH3)2), 1.36
(3 H, s, C(CH3)2); dC (125 MHz, CDCl3) 135.63 (CH CH–Ph),
128.68, 127.97, 126.39 (arom. C), 121.21 (CH CH–Ph), 111.89
(C(CH3)2), 83.47 (C-2), 78.54 (C-6), 70.62 (C-8), 70.10 (C-7), 64.56
(C-9), 56.95 (C-5), 53.36 (OCH3), 46.63 (C-3), 44.04 (CH2), 27.74,
26.42 (C(CH3)2), 23.15 (AcCH3); HRMS (FAB): m/z calcd. for
[M+H]+: 462.212792; found: 462.211456.
Methyl-5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-ethoxy-car-
bonylmethyl-2-C-(3-phenylpropyl)-D-erythro-L-manno-nononate
(31). Compound 30 (58 mg, 0.11 mmol) was suspended in water
and a catalytic amount of ion exchanger (DOWEX 50 ¥ 8, H+)
was added. The suspension was stirred overnight at 60 ◦C and
became clear during the course of the reaction. After TLC showed
complete consumption of the starting material the solution was
filtered◦and freeze dried to give 31 (40 mg, 75%) as a white solid;
mp 62 C; [a]2D5 -42 (c 0.1 in MeOH); dH (500 MHz, [D4]MeOH)
7.26–7.22 (2 H, m, arom. H), 7.16–7.13 (3 H, m, arom. H), 4.18–
4.08 (2 H, m, OCH2CH3), 3.86–3.80 (2 H, m, H-8, H-9a), 3.79
(3 H, s, OCH3), 3.69 (1 H, dd, J5,4 10.5, J5,6 10.2, H-5), 3.65–
3.60 (1 H, m, H-9b), 3.58 (1 H, dd, J6,5 10.2, J6,7 1.5, H-6),
Methyl-5-acetamido-2,6-anhydro-3,4,5-trideoxy-4-ethoxy-car-
bonylmethyliden-8,9-O-iso-propyliden-2-C-((E)-3-phenyl-prop-2-
enyl)-D-glycero-D-galacto-nononate (29). Compound 28 (36 mg,
78 mmol) was dissolved in dry tetrahydrofuran and ethoxycar-
bonylmethylene triphenylphosphorane (54 mg, 0.16 mmol) was
added under argon atmosphere. The solution was stirred for 24 h
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3.49 (1 H, dd, J7,8 8.6, J7,6 1.5, H-7), 2.62–2.51 (3 H, m, CH2c,
CH2CO2H), 2.43 (1 H, dd, J3eq,3ax 13.3, J3eq,4 3.7, H-3eq), 2.19–
2.12 (1 H, m, CH2CO2H), 2.04–1.98 (1 H, m, H-4), 1.96 (3 H, s,
AcCH3), 1.75–1.67 (3 H, m, CH2a, CH2b), 1.50–1.41 (1 H, m,
CH2b), 1.45 (1 H, dd, J3ax,3eq 13.3, J3ax,4 12.7, H-3ax), 1.26 (3 H, t, J
7.1, OCH2CH3); dC (100 MHz, [D4]MeOH) 179.27 (C-1), 175.86
(CH2CO2Et), 129.42 (arom. C), 129.36 (arom. C), 126.88 (arom.
C), 81.54 (C-2), 76.59 (C-6), 73.05 (C-8), 70.26 (C-7), 64.57 (C-
9), 61.67 (OCH2CH3), 52.97 (OCH3), 50.51 (C-5), 43.66 (CH2a),
39.19 (C-3), 38.51 (CH2CO2Et), 36.63 (CH2c), 36.41 (C-4), 26.49
(CH2b), 22.51 (AcCH3), 14.55 (OCH2CH3); MS (MALDI-TOF):
m/z 496.5 ([M+H]+), 518.5 ([M+Na]+), 534.4 ([M+K]+).
NMR based inhibition assay
TRIS-HCl buffer (100 mM, pH 7.5) was lyophilised and redis-
solved in the same volume of D2O twice. The same was done to a
solution of TcTS (0.9 mg mL-1) in TRIS-HCl buffer.
Methyl a-allolactoside (35 mmol) and pNP-sialoside (25 mmol)27
were dissolved in 1 mL of TRIS-HCl (D2O) and 80 mL of the
TcTS solution (D2O) were added. The mixture was incubated at
◦
23 C. Samples (100 mL) were taken after the indicated reaction
time (Fig. 6) and added to previously prepared NMR tubes
containing a 1 : 1 mixture of D2O/[D4]MeOH. The ratio of free
p-nitrophenol to pNP-sialoside was determined by the ratio of
integrated aromatic proton signals in the proton NMR spectra.
Since hydrolysis can be neglected in the presence of a suitable
acceptor molecule,3 conversion rates can be measured easily with
this method.
5-Acetamido-2,6-anhydro-3,4,5-trideoxy-4-carboxymethyl-2-
C-(3-phenylpropyl)-D-erythro-L-manno-nononic acid (32). Com-
pound 31 (40 mg, 81 mmol) was saponified according to GP4 to
give 32 (18 mg, 49%) as a white solid; mp 183–185 ◦C; [a]D25 -32.2
(c 0.45 in MeOH); dH (400 MHz, [D4]MeOH) 7.23–7.07 (5 H, m,
arom. H), 3.86 (1 H, ddd, J8,7 8.9, J8,9b 5.6, J8,9a 2.6, H-8), 3.80 (1 H,
dd, J9a,9b 11.3, J9a,8 2.6, H-9a), 3.62–3.56 (2 H, m, H-6, H-9b), 3.46
(1 H, dd, J7,8 8.9, J7,6 2.1, H-7), 3.44 (1 H, dd, J5,4 10.4, J5,6 10.4, H-
5), 2.58–2.52 (2 H, m, CH2c), 2.52 (1 H, dd, J3eq,3ax 13.2, J3eq,4 3.5,
H-3eq), 2.34 (1 H, dd, J 14.5 and 5.4 Hz, CH2COOH), 2.16–2.09
(1 H, m, H-4), 2.04 (1 H, dd, J 14.5 and 6.6, CH2COOH), 1.96
(3 H, s, AcCH3), 1.88–1.59 (4 H, m, CH2a, CH2b), 1.26 (1 H, dd,
J3ax,3eq 13.2, J3ax,4 12.3, H-3ax). dC (100 MHz, [D4]MeOH) 179.86
(C-1), 175.26 (CH2COOH), 129.47 (arom. C), 129.16 (arom. C),
126.51 (arom. C), 82.78 (C-2), 76.89 (C-6), 72.95 (C-8), 70.79 (C-7),
64.67 (C-9), 52.98 (C-5), 42.52 (CH2a), 41.92 (CH2COOH), 41.12
(C-3), 37.40 (CH2c), 37.03 (C-4), 27.24 (CH2b), 22.49 (AcCH3);
HRMS (FAB): m/z calcd. for [M+H]+: 454.207707; found:
454.209564.
Acknowledgements
Support of this work by the Deutsche Forschungsgemeinschaft
(SFB 470, A5) and the Max-Buchner-Forschungsstiftung is grate-
fully acknowledged.
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