Oxime-based synthesis of new chromogenic and fluorogenic oligosaccharides

Article abstract of DOI:10.1002/ejoc.200800855

A new simple approach for the synthesis of labelled oligosaccharides is described. Free synthetic carbohydrate recognition building blocks were conjugated with fluorogenic and chromogenic dyes under mild experimental conditions by using oxime ligation. The resulting oligosaccharide probes presenting well-defined anomer configuration might find broad interests for recognition studies with a large panel of carbohydrate-binding proteins. Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

Full text of DOI:10.1002/ejoc.200800855

SHORT COMMUNICATION
DOI: 10.1002/ejoc.200800855
Oxime-Based Synthesis of New Chromogenic and Fluorogenic
Oligosaccharides
Olivier Renaudet*[a] and Pascal Dumy*[a]
Keywords: Carbohydrate / Chemoselectivity / Fluororescent probes / Chromophores
A new simple approach for the synthesis of labelled oligosac-
charides is described. Free synthetic carbohydrate recogni-
tion building blocks were conjugated with fluorogenic and
chromogenic dyes under mild experimental conditions by
using oxime ligation. The resulting oligosaccharide probes
presenting well-defined anomer configuration might find
broad interests for recognition studies with a large panel of
carbohydrate-binding proteins.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2008)
phore or a chromophore with a well-defined anomer con-
figuration. We report herein a new oxime-based strategy
through the synthesis of dabsyl and dansyl-labelled α--
man, α--fuc, β--Lac as well as cancer-related Tn antigen
(Figure 1).
Introduction
The understanding of the molecular basis of carbo-
hydrate–protein interactions remains a major challenge in
glycomics research.[1] For this purpose, diverse analytical
tools have been developed either in solution or with immo-
bilized proteins or carbohydrates, not only to assess the
binding parameters, but also to discover new active carbo-
hydrate-based therapeutics or diagnostics.[2] For example,
inhibition experiments such as hemagglutination (HIA)[3]
or enzyme-like lectin assays (ELLA)[4] are commonly used
to reflect the relative affinity of a carbohydrate-based ligand
for a lectin by calculating an IC50 value. Furthermore, accu-
rate association constants can be obtained through experi-
ments such as isothermal titration calorimetry (ITC),[5] sur-
face plasmon resonance (SPR),[6] fluorescence polarization
(FP)[7] or frontal affinity chromatography (FAC).[8] By con-
trast with techniques that involve unlabelled protein or li-
gand, most of the recognition assays require the labelling
of one conterpart to allow for the rapid monitoring of the
binding event.
The development of an efficient labelling method for bio-
molecules, in particular for carbohydrate-based ligands, re-
mains therefore necessary. Indeed, only few reports involv-
ing amide coupling, thiol/maleimide ligation or reductive
amination have been reported so far.[9] In pursuing our re-
search related to the design and the preparation of bioactive
multitopic neoglycopeptides,[10] we focused recently our ef-
forts on exploring a simple and efficient synthetic alterna-
tive to prepare diverse oligosaccharides bearing a fluoro-
Figure 1. General structure of fluorogenic and chromogenic carbo-
hydrate probes.
Results and Discussion
Due to the importance of the spatial display of the sugar
ligand in the protein binding site, the reporter molecule has
to be incorporated through a linker in a well-defined an-
omer configuration to both prevent steric hindrance and
preserve the recognition efficiency. In order to by-pass the
problems related to the control of stereoselectivity encoun-
tered during glycosylation, we have selected oxime ligation
as a suitable and direct method to incorporate a dye with a
pre-defined anomer position. Indeed, oxime bond forma-
[a] Département de Chimie Moléculaire, UMR-CNRS 5250 &
ICMG FR 2607, Université Joseph Fourier,
38041 Grenoble Cedex 9, France
Fax: +33-4-76514946
E-mail: olivier.renaudet@ujf-grenoble.fr
pascal.dumy@ujf-grenoble.fr
Supporting information for this article is available on the
WWW under http://www.eurjoc.org or from the author.
Eur. J. Org. Chem. 2008, 5383–5386
© 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
5383

O. Renaudet, P. Dumy
SHORT COMMUNICATION
tion relies on the high reactivity between aminooxy and al-
A few oligosaccharides bearing an α- or β-aminooxy
dehyde (or ketone) functions as illustrated in a number of function were prepared next from the corresponding glyco-
examples in the last decade.[11] Particularly, aminooxy syl fluorides.[14] We focused on α--Man (a), α--Fuc (b), α-
carbohydrates were shown to provide useful building blocks -GalNac (c) and β--Lac (d) (Scheme 2) as carbohydrate-
for the preparation of glycoconjugates presenting well-de- based recognition motifs, because they are closely involved
fined anomer configuration by condensation with other free in numbers of physiological and/or pathological pro-
biomolecules containing aldehydes.[12] In this report, we cesses.[15]
have investigated for the first time the labelling of amino-
oxy-containing oligosaccharides with different reporter
molecules bearing the complementary aldehyde function.
We have first selected the dansyl moiety since its fluores-
cence properties (λex = 334 nm; λem = 540 nm) make its de-
rivatives useful as a detection agent as well as for binding
or competition assays by using FP.[7] Alternatively, we chose
dabsyl as a chromophore as it allows a facile and rapid
visual detection (λabs = 466 nm).
The dye functionalization with an aldehyde linker was
realized according to the route described in Scheme 1. 4-
Aminobutyraldehyde diethylacetal (1) was condensed with
dansyl chloride (2) in dimethylformamide (DMF) in the
presence of diisopropylethylamine (DIEA) as a base.[13] Af-
ter silica gel chromatography, compound 3 was obtained in
80% yield. A similar procedure was applied to prepare 4
from the commercially available dabsyl chloride.
Scheme 2. Synthesis of fluorogenic and chromogenic probes 5a–d
and 6a–b.
The further conjugation of these aminooxy building
blocks a–d with the probes 3–4 was realized with a one-pot
procedure. The cleavage of the acetal protecting group of
3–4 and the final oxime ligation with a–d were achieved in
aqueous acetic acid at room temperature. As illustrated by
the RP-HPLC monitoring of the condensation between
dabsyl derivative 4 and lactose derivative d [see peaks at
retention time (tR) = 4.5 min and 11.2 min, respectively, on
the RP-HPLC profiles given in Figure 2], the treatment of
Scheme 1. Synthesis of dansyl and dabsyl probes 3–4.
4 under these mild acidic conditions led to the formation
Figure 2. Reverse-phase HPLC of the crude one-pot deprotection/coupling reaction mixture for lactosyl-dabsyl probe 6d at four different
times (from 1 to 270 min). This reaction was performed between a twofold excess of aminooxy lactose d in an AcOH/H2O/CH3CN
mixture and acetal-protected dabsyl derivative 4 (HPLC elution conditions: linear gradient A/B, 95:5 to 0:100 in 15 min, flow: 1.3 mL/
min, λ = 214 nm; column: nucleosil 100 Å, 5 µm C18 particles, 250ϫ4.6 mm; solvent A: 0.09% TFA, solvent B: 0.09% TFA in 90%
acetonitrile).
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New Chromogenic and Fluorogenic Oligosaccharides
Figure 3. (A) UV spectrum of 6d at different concentrations in CH3CN/phosphate buffer (5 m, pH = 7.4) (1:1) at 23 °C: (a) 1.25 m,
(b) 0.62 m, (c) 0.31 m, (d) 0.15 m, (e) 0.08 m, (f) 0 m. (B) Fluorescence spectrum of 5d (1 m) in CH3CN/phosphate buffer (5 m
pH = 7.4) (1:1) at 23 °C: (a) emission spectrum (λem. = 540 nm), (b) excitation spectrum (λex. = 334 nm).
of the aldehyde function within 4 h (tR = 9.3 min on the showing a maximum absorbance wavelength at 460 nm
RP-HPLC profiles given in Figure 2). The resulting alde- (Figure 3 A). We next performed fluorescence studies with
hyde was found to react simultaneously with the free 5d, which gave excitation and emission spectra (Figure 3 B)
aminooxy carbohydrate d to afford 6d (tR = 7.5 min on the in agreement with the fluorescent properties of the dansyl
RP-HPLC profiles given in Figure 2). The completeness of probe (maximum wavelength at 334 nm and 540 nm, respec-
the one-pot deprotection/oxime coupling reactions was en- tively). More interestingly, we measured the fluorescence
sured after 270 min as confirmed by the clean crude reac- anisotropy values of 5a and 5d in HEPES aqueous buffer,
tion mixture observed in the last RP-HPLC profile (Fig- then after adding increasing amounts of the mannose-bind-
ure 2).
ing lectin ConcanavalinA (ConA) for 5a or the galactose-
After removal of excess unreacted carbohydrate by semi- binding lectin from Arachis hypogaea (PNA) for 5d (see
preparative RP-HPLC, the dabsyl-functionalized lactosyl Supporting Information).[17] As expected, whereas low fluo-
compound 6d was obtained in a yield (ca. 80%) similar to rescence anisotropy values were measured for both 5a and
those usually observed in oxime conjugation between ami- 5d without lectin, we observed a significant increase of an-
nooxy and free aldehyde building blocks. The fluorogenic isotropy in the presence of ConA or PNA, respectively, thus
and chromogenic oligosaccharides 5/6a–d were obtained confirming that our dansyl probes might provide a useful
similarly with good yields and purity. Each compound was tool to analyse carbohydrate–protein interactions.
fully characterized by RP-HPLC, mass spectrometry and
NMR spectroscopy (Table 1 and Supporting Information).
Furthermore, we observed that the labelled sugars were ob-
tained as a (Z)/(E) isomer mixture. Each isomer was as-
Conclusions
We have explored for the first time an oxime-based strat-
signed by further NMR experiments with the (E) isomer
egy as a simple method for the preparation of new labelled
carbohydrates with well-defined anomer configuration.
First, dansyl and dabsyl probes 3 and 4 bearing an acetal-
protected linker have been prepared. The final aldehyde re-
lease and the subsequent coupling reaction with free
aminooxy carbohydrates building blocks a–d [α--Man (a),
α--Fuc (b), β--Lac (d) and cancer-related antigen Tn (c)]
occurred following a one-pot procedure under mild acidic
conditions. The resulting fluorogenic and chromogenic
carbohydrates 5/6a–d were thus obtained in good yields and
purity without using a coupling reagent or a deprotection
step. We thus anticipate that our approach could be further
extended to other fluorescent probes largely employed for
biomolecules labelling, such as fluorescein and rhodamine
derivatives. These new labeled compounds might find prom-
ising applications for the measurement of binding interac-
tions with a large panel of proteins by using different tech-
niques. For example, fluorescence polarization assays or
HPLC screening procedure with cocktails of carbohydrates
being the major compound (60–70%).[16] However, several
attempts to separate both isomers by changing the RP-
HPLC elution gradient were unsuccessful.
Table 1. Analytical data for compounds 5/6a–d.
[a]
Probe
Yield
tR
MS[b]
(Z)/(E)[c]
5a
5b
5c
5d
6a
6b
6c
6d
83% (21 mg)
87% (21 mg)
65% (20 mg)
54% (10 mg)
89% (25 mg)
85% (33 mg)
84% (21 mg)
82% (33 mg)
5.6 min 498.11 (498.19)
5.9 min 482.11 (482.19)
5.6 min 539.12 (539.21)
5.2 min 660.14 (660.24)
7.9 min 552.17 (552.21)
8.1 min 536.16 (536.24)
7.9 min 593.15 (593.21)
7.5 min 714.18 (714.26)
0.4:0.6
0.35:0.65
0.35:0.65
0.35:0.65
0.4:0.6
0.4:0.6
0.35:0.65
0.3:0.7
[a] Reverse-phase HPLC retention time tR under conditions de-
scribed in Figure 2. [b] Mass spectrometry was performed by the
electrospray ionisation method in the positive mode. Calculated
masses are given in parentheses [M + H]+. [c] Determined by NMR
spectroscopy by using DPFGSE NOE experiments.[16]
Spectral data were finally collected for the lactosyl deriv- might give useful information on lectin glycopatterning.[18]
atives 5d and 6d. The UV properties of the red compound Further investigations in this direction are currently in pro-
6d was firstly recorded from low to high concentrations, gress in our laboratory.
Eur. J. Org. Chem. 2008, 5383–5386
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O. Renaudet, P. Dumy
SHORT COMMUNICATION
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Experimental Section
Typical Procedure. Synthesis of 5a: A solution of α--Man-ONH2
(a) (10 mg, 0.051 mmol) and protected dansyl derivative 3 (40 mg,
0.10 mmol) in AcOH/H2O/CH3CN (3:2:5) (6 mL) was stirred at
room temperature for 4 h. The crude mixture was purified by semi-
preparative RP-HPLC to obtain 5a in 83% yield (21 mg,
0.042 mmol) as a lyophilized powder. (E)/(Z) oxime ratio (deter-
mined by NMR spectroscopy):[16] 0.6:0.4. 1H NMR (300 MHz,
[D4]MeOH): δ = 8.59–8.53 (m, 2 H), 8.27 (br. d, J = 7.3 Hz, 1 H),
7.71 (br. t, J = 7.9 Hz, 2 H), 7.58 (br. d, J = 5.2 Hz, 1 H), 7.32 (t,
J = 5.7 Hz, 0.6 H), 6.69 (t, J = 5.5 Hz, 0.4 H), 5.32 (d, J = 1.6 Hz,
0.4 H), 5.24 (d, J = 1.6 Hz, 0.6 H), 3.89 (dd, J = 1.6, 3.3 Hz, 0.4
H), 3.85 (dd, J = 1.6, 3.3 Hz, 0.6 H), 3.79–3.59 (m, 4 H), 3.50–3.44
(m, 1 H), 3.12 (s, 3 H), 3.10 (s, 3 H), 2.94–2.88 (m, 2 H), 2.31–2.24
(m, 1 H), 2.13–2.07 (m, 1 H), 1.63–1.54 (m, 2 H) ppm. 13C NMR
(75 MHz, [D4]MeOH): δ = 154.7, 154.3, 137.8, 137.6, 130.9, 130.6,
129.9, 129.8, 129.6, 129.5, 129.0, 128.9, 125.7, 125.6, 123.4, 123.1,
117.9, 117.8, 103.4, 103.2, 75.4, 75.1, 72.8, 72.7, 70.8, 68.3, 62.7,
62.7, 46.5, 46.5, 43.5, 43.1, 27.4, 27.3, 24.1 ppm. Analytical RP-
HPLC (5–100% B in 15 min, λ = 250 nm): tR = 5.6 min. ES-MS
(positive mode): calcd. for C22H32N3O8S 498.19; found 498.11 [M
+ H]+.
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Supporting Information (see footnote on the first page of this arti-
cle): Detailed description of synthetic procedure, 1H and 13C NMR
spectra, HPLC, MS analysis of each new carbohydrate probe and
fluorescence anisotropy assay titration curves.
Acknowledgments
This work was supported by the Centre National pour la Recherche
Scientifique (CNRS), the Université Joseph Fourier (UJF) and
COST D-34. The authors thank Prof. J. Garcia for performing the
DPFGSE NOE experiments.
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Received: September 4, 2008
Published Online: September 30, 2008
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Eur. J. Org. Chem. 2008, 5383–5386