Synthesis and solvodynamic diameter measurements of closely related mannodendrimers for the study of multivalent carbohydrate-protein interactions

Article abstract of DOI:10.3762/bjoc.10.157

This paper describes the synthesis of three closely related families of mannopyranoside-containing dendrimers for the purpose of studying subtle structural parameters involved in the measurements of multivalent carbohydrate-protein binding interactions. T

Full text of DOI:10.3762/bjoc.10.157

Synthesis and solvodynamic diameter
measurements of closely related
mannodendrimers for the study of multivalent
carbohydrate–protein interactions
Yoann M. Chabre, Alex Papadopoulos, Alexandre A. Arnold and René Roy*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2014, 10, 1524–1535.
Pharmaqam, Department of Chemistry, Université du Québec à
Montréal, P. O. Box 8888, Succ. Centre-ville, Montréal, Québec,
Canada H3C 3P8
doi:10.3762/bjoc.10.157
Received: 04 March 2014
Accepted: 11 June 2014
Published: 04 July 2014
Email:
René Roy* - roy.rene@uqam.ca
This article is part of the Thematic Series "Multivalent glycosystems for
nanoscience".
* Corresponding author
Keywords:
Guest Editor: B. Turnbull
carbohydrates; click chemistry; dendrimers; glycodendrimers; lectins;
multivalent glycosystems
© 2014 Chabre et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
This paper describes the synthesis of three closely related families of mannopyranoside-containing dendrimers for the purpose of
studying subtle structural parameters involved in the measurements of multivalent carbohydrate–protein binding interactions.
Toward this goal, two trimers 5 and 9, three 9-mers 12, 17, 21, and one 27-mer 23, varying by the number of atoms separating the
anomeric and the core carbons, were synthesized using azide–alkyne cycloaddition (CuAAc). Compound 23 was prepared by an
efficient convergent strategy. The sugar precursors consisted of either a 2-azidoethyl (3) or a prop-2-ynyl α-D-mannopyranoside (7)
derivative. The solvodynamic diameters of 9-mer 12, 17, and 21 were determined by pulsed-field-gradient-stimulated echo (PFG-
STE) NMR experiments and were found to be 3.0, 2.5, and 3.4 nm, respectively.
Introduction
Multivalent carbohydrate–protein interactions are at the fore- resides in the modifications of both the aglycon [17-19] and
front of a wide range of biological events which have triggered substituent residues [20-22] of the targeted sugar moieties
a plethora of versatile synthetic methods for the design of potent through extensive studies of quantitative structure–activity rela-
inhibitors and glycomimetics [1-4]. Among the diverse strate- tionships (QSARs). In most of the studies related to aglycon
gies leading to efficient ligands, glycopolymers [1,5-7], glyco- modifications, it was concluded that aromatic glycosides
dendrimers [7-14], and sugar rods [15,16] have retained most possessed improved binding properties due to the ubiquitous
attention. An additional approach that has gained keen interest presence of aromatic amino acids in the cognate binding sites
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[23-25]. This is also supported by the recent findings that the predicting which structural parameters would be optimal for the
sugar backbones themselves also possess a hydrophobic side binding interactions.
that orients the sugars in appropriate aromatic amino acid rich
pockets [26-28].
In order to gain more insight into this direction, we designed
herein three families of closely related mannopyranoside clus-
Unfortunately, due to the inherent complexity of studying ters (glycodendrimers) aimed at evaluating their relative
multivalent binding interactions, researchers have used experi- binding abilities against the hometetrameric leguminous lectin
mental conditions that often biased the intrinsic phenomena ConA from Canavalia ensiformis by inhibition of haemaggluti-
under investigations [29]. For instance, when evaluating ther- nation and by turbidimetry. The latter would allow us to
modynamic parameters by isothermal calorimetry (ITC), scien- measure relative kinetic factors involved in the cross-linking
tists used either truncated versions of for instance, tetrameric lattice formation using soluble partners.
lectins such as ConA, or diluted conditions to avoid precipita-
tion of the complexes [30,31]. Alternatively, the application of Results and Discussion
surface plasmon resonance (SPR) also creates artificial situa- In order to critically evaluate the subtle structural parameters
tions not sufficiently related to the natural cellular events, thus imparted by glycodendrimers in deciphering their relative ther-
requiring complex mathematical algorithms [32]. Most solid- modynamic and kinetic abilities towards multivalent lectins, we
phase immunoassays (ELLA, ELISA) also fall under the same designed three families of closely related mannopyranoside
criticism by providing unusually high (or too close) sugar densi- dendrimers. Scheme 1 describes the preparation of trimers 5 and
ties. Also important and in spite of the two decades of glyco- 9 built around benzene-1,3,5-tricarboxamide (BTA or trimes-
dendrimer chemistry [7], there is still no general rule to allow amide core) having respectively nine and ten atoms between the
Scheme 1: Synthesis of mannosylated trimers 5 and 9 using trimesic acid core transformed into propargylated (2) and azidopropylated (6) scaffolds
and then coupled by “click chemistry” with either 2-azidoethyl (3) or propargyl (7) mannopyranosides.
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anomeric and the benzene carbon, hence differing by a distance The synthesis of 5 was accomplished starting from commercial
of only ~1.5 Å. Schemes 2–4 illustrate the syntheses of 9-mers trimesic acid chloride 1 which was readily transformed into
12 and 21 using the same trimesic acid core, together with a known tripropargyl amide derivative 2 [33] using propargyl-
phloroglucinol template to initiate the synthesis of homologue amine according to Scheme 1. Amide 2 was conjugated to
17, but incorporating 2-amino-2-hydroxymethylpropane-1,3- peracetylated 2-azidoethyl α-D-mannopyranoside 3 [34] under
diol as a branching unit (TRIS) at the G(1) level. Thus, com- classical copper-catalyzed dipolar cycloaddition (CuAAc) to
pounds 12, 17, and 21 differ by having nine atoms between the afford 4 in 56% yield. Structure 4 was readily characterized by
anomeric carbon and the focal quaternary carbon of TRIS fol- the absence of acetylenic protons at δ 3.16 ppm, the appearance
lowed by two, four, and nine atoms to reach the benzene of identical triazole protons (3H) at δ 7.74 ppm relative to the
carbon, respectively (~4, 6, and 12 Å). Finally, the synthesis of anomeric signal (3H) at δ 4.81 ppm and corresponding HRMS
a 27-mer mannosylated dendrimer 23 is shown in Scheme 5.
data. Zemplén deprotection (NaOMe, MeOH) afforded 5 in
Scheme 2: Divergent CuAAc “click reaction” between propargylated core 10 and azide 3 to afford 9-mer 12.
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94% yield. Synthesis of the related homolog 9, prepared in 74% The syntheses of 9-mers 12, 17 and 21 are illustrated in
overall yield from known 6 [17] by an analogous click chem- Schemes 2–5 and follow a conceptually identical strategy to the
istry, is also described in Scheme 1. To this end, trichloride 1 one described above for trimers 5 and 9. Toward this goal,
was treated as above with 3-azido-1-propanamine to provide 6 propargylated 9-mer scaffold 10 [17] was treated under the
in 87% yield. Azide–alkyne cycloaddition of 6 with prop-2-ynyl same CuAAc conditions with azide 3 to provide peracetylated
α-D-mannopyranoside 7 [35] gave 8 (79%) which was de-O- 11 in 83% yield which upon Zemplén de-O-acetylation gave 12
acetylated under Zemplén conditions (NaOMe, MeOH, 95%) to in essentially quantitative yield (Scheme 2). Complete spectral
give 9.
characterization (1H, 13C NMR and HRMS) concluded for the
Scheme 3: Divergent CuAAc synthesis of “extended” 9-mer 17 using phloroglucinol (13) as core, bromoacylated TRIS as linker and mannopyranosyl-
azide 3.
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aforementioned structure having twelve atoms in the linking clear advantages of providing an easier purification process
arm (see Supporting Information File 1).
from partially substituted end-products together with a better
assessment of complete surface group modifications. Hence,
Analogously, the extended 9-mer glycodendrimer 17, known 14 [36] was first cycloadded to mannosylazide 3 under
possessing fourteen atoms between the anomeric carbon and the the above CuAAc conditions. The “click reaction” proceeded
benzene carbon, was prepared according to Scheme 3. Thus, exceptionally efficiently and provided bromoacylated dendron
phloroglucinol (13) was carefully O-alkylated with the previ- precursor 18 in 94% yield. Substitution of the bromide by azide
ously synthesized bromoacetylated TRIS derivative 14 [36] also proceeded uneventfully (NaN3, DMF, rt, 16 h) to afford
using K2CO3 in DMF to provide 15 in 43% yield. Again, the intermediate glycodendron 19 in 93% yield. Finally, coupling of
structural integrity of 15 was fully assessed by the simplicity of the propargylated core 2 with azidodendron 19 under the typical
its 1H NMR symmetrical patterns that showed the character- CuAAc conditions gave peracetylated intermediate 20 which
istic singlets for the three amide protons at δ 6.85 ppm, relative was readily deprotected to give 9-mer 21 in 84% overall yield.
to the three benzene protons (δ 6.17 ppm) and the six O-acyl All spectral characteristics concurred to the expected structural
protons at δ 4.36 ppm (core) compared with the peripheral integrity of 21 (see Supporting Information File 1).
acetylenic methylenes (18H), inner methylene of TRIS (18H),
and the terminal alkyne protons (9H) at δ 4.16, 3.87, and Finally, a 27-mer mannosylated G(1)-dendrimer 23 was
2.48 ppm, respectively.
similarly prepared using an accelerated convergent strategy
(Scheme 5). This time, the nonapropargylated scaffold 10 was
Toward the last and further extended 9-mer 21, a convergent “clicked” under CuAAc with trimeric azidodendron 19 to give
strategy was rather adopted (Scheme 4). This strategy has the 22 in an acceptable yield of 63% after silica gel column chro-
Scheme 4: Convergent synthesis of further “extended” 9-mer 21 using mannosylated bromoacyl dendron 18 transformed into azide 19 followed by
CuAAc coupling to tripropargylated core 2.
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Scheme 5: Convergent assembly of 27-mer 23 using key propargylated scaffold precursors 10 and mannosylated azidodendron 19. Insert: Zoom
section of HRMS (+TOF) spectrum for deprotected G(1)-mannodendrimer 23 illustrating observed and theoretical isotopic distributions for [M + 7H]7+
adduct.
matography, corresponding to an excellent 95% yield per indi- meric mannosides and ConA, we determined their relative
vidual dendron’s incorporation. The complete disappearance of diffusivity measurements by NMR spectroscopy. In fact, diffu-
propargylic signals in the 1H NMR spectrum supported sion NMR spectroscopy has recently become a method of
complete conversion. Note that working with peracetylated choice to access information about sizes and shapes of macro-
sugar precursors allows less tedious purification practices as molecular species by measuring their diffusion coefficients in a
opposed to working with unprotected sugars which often neces- given solvent [17,37]. The size of nonavalent compounds 12,
sitate purification by cumbersome dialysis followed by HPLC 17, and 21, and more particularly their solvodynamic radii, was
treatment. Here again, the complete structural integrity of the thus estimated with the help of pulsed-field-gradient stimulated
final product can be readily confirmed from its characteristic echo (PFG-STE) NMR experiments using bipolar pulse pairs-
spectral identification. Ultimately, dendrimer 23 was depro- longitudinal-eddy-current delay (BPP-LED) in D2O at 25 °C.
tected under the usual Zemplén conditions in 82% yield. Once Stimulated echoes were used since they avoid signal attenua-
again, all the relative integrations for each proton presented on tion due to transverse relaxation while bipolar gradient pulses
the surface were in perfect agreement with those of the middle reduce gradient artefacts [38]. The diffusion rates (D) were
and internal regions. Interestingly, high resolution mass spec- calculated from the decay of the signal intensity of the common
trometry (+TOF technique) resulted in the formation of multi- H-5 proton (δ = 2.98 ppm) located on each epitope with
charged adducts that matched the expected theoretical patterns, increasing field gradient strength (Figure 1a). In all cases,
especially the one corresponding to [M + 7H]7+, as illustrated in monoexponential behavior was observed (Figure 1b), which
Scheme 5 (insert).
was manifested as a linear decay of the logarithm of the signal
intensity as a function of the gradient strength. This behavior is
consistent with a spherical and unimolecular character of the
glycodendrimers, confirming the absence of aggregation
phenomena in aqueous solution under the working concentra-
NMR diffusion studies
To accurately estimate the various structural factors involved in
the intricate binding interactions between our synthetic multi-
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Figure 1: a) Decay of 1H signal for the nonavalent mannosylated compound 12 in D2O during the PFGSTE experiment. The gradient strength is
increased linearly between 1.8 and 54.2 G·cm−1; b) characteristic echo decays of the H-5 resonances (δ = 2.98 ppm) as a function of squared
gradient strength located in 12 (full circles) and 21 (full triangles) with δ = 4 ms and Δ = 50 ms (Δ = 40 ms for 17 (circles)). Notably, such linear
behavior was also obtained for the decay of the signal intensities of other protons located either in internal regions of the conjugates on aromatic or
branching sections, or in the peripheral saccharidic belt (results not shown).
tions. The corresponding solvodynamic diameters (ds = 2 × rs) of the dendrons that emanate from 1,3,5-O-alkylations on the
can be calculated using the Stokes–Einstein equation and the aromatic core in 17, compared to the one generated in BTAs-
viscosity of pure D2O (Table 1).
centered structures 12 and 21, could explain this observation.
Also, these discrepancies might result from the general
As expected, nonavalent conjugates 12, 17, and 21 presented amphiphilic behavior of this kind of macromolecules [39]. In
solvodynamic diameters in the range of roughly 3 nm when fact, these glycoclusters shared common structural factors with
considering the decay of distinctive and common H-5 signals. hydrophilic peripheral moieties and an aromatic central core but
These values remained consistent with similar congeners the introduction of distinct functionalized linkers may change
described earlier and harboring different epitopes [17]. The the overall hydrophobic/hydrophilic balances of the structures.
variation of the complexity of anchoring functionalities in the As such, they could engage supplementary intramolecular
middle region with the incorporation of amide functions and hydrogen bonding or hydrophobic interactions that could
triazole groups is responsible for a diameter enhancement for 21 mediate their three-dimensional arrangement in aqueous media.
when compared with 12, as expected. On the other hand, rather Moreover, it is also reported that the relative spatial distribu-
counter-intuitive tendencies were observed since the apparently tion of the branches around the C=O-centered BTAs strongly
slightly extended structure 17 was measured as the smallest depends on the nature of the substituents [40]. This hypothesis
molecule of the family in water. A specific spatial arrangement can partly explain the discrepancy observed for the calculated
Table 1: Determination of diffusion data and solvodynamic diameters of nonavalent conjugates 12, 17, and 21 by diffusion NMR experiments.
Entry
Compound
D [× 10−10 m2s−1]a,b,c
Solvodynamic diameter [ds, nm]d
1
2
3
12
17
21
1.33
1.62
1.17
3.0 (2.9)
2.5 (2.3)
3.4 (2.9)
aSee general procedures and Supporting Information File 1 for extraction of the diffusion rate and calibration of the gradient strength. D was deter-
mined from the decay of the H-5 resonance (δ = 2.98 ppm). bViscosity of D2O at 25 °C: ηD2O = 1.097 × 10−3 Pa s. cThe error associated with the
measurement was estimated from repeated calculations of the diffusion coefficients to be below 10%. dResults in parentheses correspond to the
average value calculated from the decays of 4 or 5 different proton signals.
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diameter of 21 (Table 1, entry 3). In fact, diffusion data for 21 254 nm light, a mixture of iodine/silica gel and/or mixture of
ranged from 1.61 × 10−10 m2s−1 for central CHar to ceric ammonium molybdate solution (100 mL H2SO4, 900 mL
1.17 × 10−10 m2s−1 for H-5, indicating a heterogeneity in diffu- H2O, 25 g (NH4)6Mo7O24H2O, 10 g Ce(SO4)2) and subsequent
sivity depending on the proton location within the same mole- development by gentle warming with a heat-gun. Purifications
cule. As a consequence, the calculated ds value based on the were performed by flash column chromatography using
utilization of an average value of diffusion data (D) extracted silica gel from Silicycle (60 Å, 40–63 µm) with the indicated
from signal decays of distinct protons located at different levels eluent.
in the molecule differ from that obtained with the decay of
peripheral H-5 signal only. This heterogeneity was less pro- NMR, IR, and MS spectroscopy
nounced for 17 and absent for 12 that presented consistent 1H NMR and 13C NMR spectra were recorded at 300 or
values of D ranging from 1.51 to 1.33 × 10−10 m2s−1 for 600 MHz and 75 or 150 MHz, respectively, on a Bruker spec-
protons in the core or the periphery. Interestingly, calculation of trometer (300 MHz) and Varian spectrometer (600 MHz). All
the extended conformation (MM2, Chem3D) of the linkers in NMR spectra were measured at 25 °C in indicated deuterated
12, 17, and 21 showed lengths of 14.8, 17.1, and 21.8 Å, res- solvents. Proton and carbon chemical shifts (δ) are reported in
pectively.
ppm and coupling constants (J) are reported in Hertz (Hz). The
resonance multiplicity in the 1H NMR spectra are described as
“s” (singlet), “d” (doublet), “t” (triplet), and “m” (multiplet) and
Conclusion
The syntheses of three related families of mannosylated glyco- broad resonances are indicated by “br”. Residual protic solvent
clusters and glycodendrimers were efficiently accomplished of CDCl3 (1H, δ 7.27 ppm; 13C, δ 77.0 ppm (central resonance
around a benzene core and using the CuAAc methods now of the triplet)), D2O (1H, δ 4.79 ppm and 30.89 ppm for CH3 of
routinely used in this field [9,41,42]. The targeted compounds acetone for 13C spectra of de-O-acetylated compounds), MeOD
were based on trimesic acid scaffold which is known to prop- (1H, δ 3.31 ppm and 13C, δ 49.0 ppm). 2D Homonuclear corre-
erly expose the surface sugar groups to tetrameric lectins such lation 1H-1H COSY together with 2D heteronuclear correlation
as ConA [43] and the LecA lectin from Pseudomonas aerugi- 1H-13C HSQC experiments were used to confirm NMR peak
nosa [17]. With these closely related families of mannosylated assignments.
dendrimers in hand, together with their known relative size in
solution, we are now well positioned to evaluate their binding Fourier transform infrared (FTIR) spectra were obtained with
behavior against their cognate proteins and this work will be Thermo-scientific, Nicolet model 6700 equipped with ATR.
published in due course [44].
The absorptions are given in wavenumbers (cm−1). The inten-
sity of the bands is described as s (strong), m (medium) or w
The study of subtle structural variations and the nature of (weak). Melting points were measured on a Electrothermal
anchoring functions, as observed in diffusivity experiments, MEL-TEMP apparatus and are uncorrected.
could represent a first step towards rational interpretation to
explain the differential kinetic behavior within a closely related Accurate mass measurements (HRMS) were performed on a
family of glycoclusters.
LC–MSD–ToF instrument from Agilent Technologies in posi-
tive electrospray mode. Low-resolution mass spectra were
performed on the same apparatus or on a LCQ Advantage ion
trap instrument from Thermo Fisher Scientific in positive elec-
Experimental
General remarks
All reactions in organic medium were performed in standard trospray mode (Mass Spectrometry Laboratory (Université de
oven-dried glassware under an inert atmosphere of nitrogen Montréal), or Plateforme analytique pour molécules organiques
using freshly distilled solvents. CH2Cl2 was distilled from CaH2 (Université du Québec à Montréal), Québec, Canada). Either
and DMF from ninhydrin, and kept over molecular sieves. protonated molecular ions [M + nH]n+ or adducts [M + nX]n+
Solvents and reagents were deoxygenated when necessary by (X = Na, K, NH4) were used for empirical formula confirma-
purging with nitrogen. Water used for lyophilization of final tion.
dendrimers was nanopure grade, purified through Barnstead
NANOPure II Filter with Barnstead MegOhm-CM Sybron NMR diffusion measurements were performed at 25 °C on a
meter. All reagents were used as supplied without prior purifi- Varian Inova Unity 600 spectrometer (Varian, Walnut Creek,
cation unless otherwise stated, and obtained from Sigma- CA, USA) operating at a frequency of 599.95 MHz for 1H using
Aldrich Chemical Co. Ltd. Reactions were monitored by analyt- a 5 mm broadband z-gradient temperature-regulated probe. The
ical thin-layer chromatography using silica gel 60 F254 temperature was calibrated with 1,2-ethanediol according to a
precoated plates (E. Merck) and compounds were visualized by standard procedure [38]. The diffusion experiment employed a
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bipolar pulse-field gradients stimulated echo sequence as period. After removal of the solvents with a Pasteur pipette, the
proposed by Wu et al [45]. The gradient pulse duration δ was residue was dissolved in H2O (3 mL), and the pH was adjusted
4 ms and the diffusion times (Δ) were 40 to 50 ms to ensure that to 7 by the addition of ion-exchange resin (Amberlite IR 120
the echo intensities were attenuated by at least 80%. A complete H+). After filtration, the solvent was removed under vacuum
attenuation curve was obtained by measuring 30 gradient with a rotary evaporator and lyophilized to yield the fully
strengths, which were linearly incremented between 1.8 and deprotected glycocluster.
54.2 Gcm−1. Hard 90° 1H pulses of 15 μs were used and 36 k
data points were recorded with 16 scans acquired for each Synthesis of peracetylated trivalent derivative 8: To a solu-
gradient’s strength. A recycle delay of 3.0 s was used. The tion of triazido core 6 (50.0 mg, 109 μmol, 1.00 equiv) and
gradient strength was calibrated by back calculation of the coil mannoside 7 (158 mg, 409 μmol, 3.75 equiv) in a THF/H2O
constant from diffusion experiments on H2O traces in D2O mixture (1:1, 6 mL) were added sodium ascorbate (19.4 mg,
(D = 1.90 × 10−9 m2 s−1) [46].
98.1 μmol, 0.90 equiv) and CuSO4·5H2O (24.5 mg, 98.1 μmol,
0.90 equiv). The reaction mixture was stirred at 50 °C for 3 h
Diffusion rates were extracted from the slope of the straight then at room temperature for an additional 16 h period. Ethyl
lines obtained by plotting ln(I) against the gradient-pulse power acetate (10 mL) was added and the resulting solution was
squared according to the following equation: ln(I) = poured in a separatory funnel containing 35 mL of EtOAc and
−Dγ2G2δ2(Δ − δ/3 − τ/2) + ln(I0) where I is the relative inten- 30 mL of a saturated aqueous solution of NH4Cl. Organics were
sity of a chosen resonance (I = I0exp−[Dγ2G2δ2(Δ − δ/3 − washed with (2 × 25 mL) of saturated NH4Claq, water
τ/2)]), G = gradient strength (T/m), γ = proton gyromagnetic (2 × 20 mL) and brine (1 × 10 mL). The organic phase was then
ratio, D = diffusion rate (m2 s−1), δ = gradient duration, Δ = dried over MgSO4 and concentrated under reduced pressure.
diffusion delay, and τ = pulse length for bipolar pulses. All Column chromatography on silica (DCM/MeOH 98:2 to 94:6)
diffusion spectra were processed in Mat NMR [47].
afforded the desired compound 8 (138 mg, 86.0 μmol, 79%) as
a viscous oil. Rf 0.34 (95:5 DCM/MeOH); 1H NMR (600 MHz,
CDCl3) δ (ppm) 8.27 (s, 3H, CHar), 7.79 (s, 3H, CHtriazole),
7.72 (t, J = 5.3 Hz, 3H, NH), 5.29–5.19 (m, 9H, H2, H3, H4),
4.92 (sapp, 3H, H1), 4.77–4.62 (2 × d, J = 12.4 Hz, 6H, OCH2),
Glycodendrimer synthesis
Procedure A: multiple CuAAc couplings on
polypropargylated cores
To a solution of polypropargylated core (1.00 equiv) and 4.54 (t, J = 6.4 Hz, 6H, NtriazoleCH2), 4.28 (dd, J = 12.4 Hz, J =
complementary azido synthon (1.25 equiv/propargyl) in a THF/ 5.4 Hz, 3H, H6b), 4.11–4.03 (m, 6H, H5 + H6a), 3.55 (m, 6H,
H2O mixture (1:1) were added sodium ascorbate (0.30 equiv/ NHCH2), 2.28 (m, 6H, CH2CH2CH2), 2.12, 2.10, 2.02, 1.96
propargyl) and CuSO4·5H2O (0.30 equiv/propargyl). The reac- (4s, 36H, COCH3); 13C{1H} NMR (150 MHz, CDCl3) δ (ppm)
tion mixture was stirred at 50 °C for 3 h then at room tempera- 170.8, 170.1, 170.0, 169.7 (COCH3), 166.1 (CONH), 143.5
ture for an additional 16 h period. Ethyl acetate (10 mL) was (Ctriazole), 134.9 (Carom), 128.5 (CHarom), 123.9 (CHtriazole),
added and the resulting solution was poured in a separatory 96.7 (C1), 69.3 (C2), 69.0 (C3), 68.7 (C5), 65.9 (C6), 62.3 (C4),
funnel containing 25 mL of EtOAc and 30 mL of a saturated 60.7 (OCH2), 48.3 (CH2Ntriazole), 37.5 (NHCH2), 29.9
aqueous solution of NH4Cl. Organics were washed with (CH2CH2CH2), 20.9, 20.8, 20.7, 20.7 (COCH3); MS (+TOF-
(2 × 25 mL) of saturated NH4Claq, water (2 × 20 mL) and brine MS, m/z): [M + H]+ calculated for C69H90N12O33, 1615.6;
(1 × 10 mL). The organic phase was then dried over MgSO4 found, 1615.6.
and concentrated under reduced pressure. Column chromatog-
raphy on silica (DCM/MeOH 100:0 to 90:10) afforded the Synthesis of nonapropargylated core 15: To a solution of
desired glycocluster.
phloroglucinol (13, 10.0 mg, 79.3 μmol, 1.00 equiv) in anhy-
drous DMF (3 mL) was added under nitrogen anhydrous
K2CO3 (previously heated at 250 °C under vaccum, 39.5 mg,
285 μmol, 3.60 equiv). After 10 min of vigorous stirring,
Procedure B: Zemplén de-O-acetylation procedure
for insoluble hydroxylated derivatives
The acetylated compound was dissolved in anhydrous MeOH tripropargylated synthon 14 (93.0 mg, 285 μmol, 3.60 equiv)
and a solution of sodium methoxide (1 M in MeOH, 5 µL every was added into the solution under inert atmosphere and the
20 min until precipitation) was added. An additional 100 µL reaction mixture was allowed to stir at 65 °C for 39 h. In the
was then injected and the heterogeneous reaction mixture was end, the dark-brown heterogeneous reaction was poured in
stirred at room temperature for 24 h. The solvent was then 30 mL of EtOAc and organics were washed with a saturated
removed with a Pasteur pipette and a mixture of anhydrous aqueous solution of NH4Cl (2 × 30 mL) then water (2 × 20 mL)
MeOH/DCM (4:1, 5 mL) was added to the residual white foam. and brine (10 mL). The organic phase was then dried over
A vigorous agitation is maintained for an additional 15 min MgSO4 and concentrated under reduced pressure. Column chro-
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Beilstein J. Org. Chem. 2014, 10, 1524–1535.
matography on silica (EtOAc/hexane 40:60 to 50:50) afforded 1.00 equiv) in dry DMF (1.5 mL) was added under a nitrogen
the desired compound 15 (32.0 mg, 33.8 μmol, 43%) as a color- atmosphere sodium azide (7.3 mg, 112 μmol, 1.50 equiv). After
less oil. Rf 0.27 (1:1 EtOAc/hexane); 1H NMR (300 MHz, stirring overnight at room temperature, the solvent was removed
CDCl3) δ (ppm) 6.85 (s, 3H, NH), 6.17 (s, 3H, CHar), 4.36 (s, under vaccum. Ethyl acetate (20 mL) was added and the
6H, OCH2CONH), 4.16 (m, 18H, OCH2C≡CH), 3.87 (br s, resulting solution was poured in a separatory funnel containing
18H, HNCqCH2O), 2.48 (m, 9H, OCH2C≡CH); 13C{1H} NMR 20 mL of EtOAc and 30 mL of a saturated aqueous solution of
(75 MHz, CDCl3) δ (ppm) 167.3 (CONH), 159.0 (CarOCH2), NH4Cl. Organics were washed with 2 × 30 mL of saturated
95.8 (CHar), 79.4 (OCH2C≡CH), 74.9 (OCH2C≡CH), 68.3 NH4Claq, water (2 × 30 mL) and brine (20 mL). The organic
(HNCqCH2O), 67.5 (OCH2CONH), 59.2 (Cq), 58.6 phase was then dried over MgSO4 and concentrated under
(OCH2C≡CH); HRMS (+TOF-HRMS, m/z): [M + H]+ calcu- reduced pressure to furnish the desired compound 19 (110 mg,
lated for C51H57N3O15, 952.3862; found, 952.3843 (Δ = −2.10 69.9 μmol, 93%) as a white solid. Rf 0.47 (94:6 DCM/MeOH);
ppm); [M + Na]+: calculated for 974.3682; found, 974.3662 (Δ mp 62–65 °C (not corrected); 1H NMR (300 MHz, CDCl3) δ
= −2.05 ppm).
(ppm) 7.68 (br s, 3H, CHtriazole), 6.69 (br s, 1H, NH), 5.27–5.18
(m, 9H, H2, H3, H4), 4.80 (d, J = 1.3 Hz, 1H, H1), 4.61–4.58 (br
Synthesis of bromoacylated dendron 18: To a solution of s, 12H, OCH2Ctriazole + NtriazoleCH2), 4.23–4.00 (m, 11H,
tripropargylated synthon 14 (140.0 mg, 393.0 μmol, 1.00 equiv) OCH2CH2 + H6a + N3CH2CONH), 3.90–3.81 (m, 9H, H6b +
and mannoside 3 (616 mg, 1.48 mmol, 3.75 equiv) in a THF/ NHCqCH2O), 3.60 (m, 3H, H5), 2.12, 2.08, 2.03, 1.98 (4s, 36H,
H2O mixture (1:1, 6 mL) were added sodium ascorbate COCH3); 13C{1H} NMR (75 MHz, CDCl3) δ (ppm) 170.4,
(70.0 mg, 354 μmol, 0.90 equiv) and CuSO4·5H2O (88.4 mg, 169.9, 169.8, 169.5, (COCH3), 166.7 (CONH), 144.9 (Ctriazole),
354 μmol, 0.90 equiv). The reaction mixture was stirred at 123.7 (CHtriazole), 97.4 (C1), 69.0 (C2), 68.8 (C3), 68.8 (C5),
50 °C for 3 h then at room temperature for an additional 16 h 68.4 (NHCqCH2O), 66.1 (C6), 65.6 (C4), 64.5 (OCH2Ctriazole),
period. Ethyl acetate (20 mL) was added and the resulting solu- 62.1 (OCH2CH2), 59.9 (Cq), 52.5 (CH2N3), 49.5 (CH2Ntriazole),
tion was poured in a separatory funnel containing 40 mL of 20.7, 20.7, 20.6, 20.6 (COCH3); IR (cm−1): 2934, 2361, 2338,
EtOAc and 30 mL of a saturated aqueous solution of NH4Cl. 2107 (N3), 1751, 1734, 1540, 1373, 1218, 1045, 761; HRMS
Organics were washed with 2 × 35 mL of saturated NH4Claq, (+TOF-HRMS, m/z): [M + H]+ calculated for C63H87N13O34,
water (2 × 30 mL) and brine (20 mL). The organic phase was 1570.5551; found, 1570.5543 (Δ = −0.51 ppm); [M + Na]+
then dried over MgSO4 and concentrated under reduced pres- calculated for 1592.5371; found, 1592.5366 (Δ = −0.31 ppm).
sure. Column chromatography on silica (DCM/MeOH 99:1 to
96:4) afforded the desired compound 18 (594 mg, 369.4 μmol, Synthesis of peracetylated 27-mer derivative 22: To a solu-
94%) as a white solid. Rf 0.47 (94:6 DCM/MeOH); mp tion of nonapropargylated core 10 (4.6 mg, 5.38 μmol,
68–72 °C (not corrected); 1H NMR (300 MHz, CDCl3) δ (ppm) 1.00 equiv) and trimannosylated dendron 19 (95.0 mg,
7.68 (br s, 3H, CHtriazole), 6.89 (br s, 1H, NH), 5.24–5.18 (m, 60.5 μmol, 11.25 equiv) in a THF/H2O mixture (1:1, 3 mL)
9H, H2, H3, H4), 4.80 (d, J = 1.3 Hz, 1H, H1), 4.61–4.58 (br s, were added sodium ascorbate (2.9 mg, 15 μmol, 2.70 equiv) and
12H, OCH2Ctriazole + NtriazoleCH2), 4.17–4.00 (m, 11H, CuSO4·5H2O (3.6 mg, 15 μmol, 0.90 equiv). The reaction mix-
OCH2CH2 + H6a + BrCH2CONH), 3.94–3.78 (m, 9H, H6b + ture was stirred at 50 °C for 3 h then at room temperature for an
NHCqCH2O), 3.60 (m, 3H, H5), 2.12, 2.08, 2.03, 1.98 (4s, 36H, additional 16 h period. Ethyl acetate (10 mL) was added and the
COCH3); 13C{1H} NMR (75 MHz, CDCl3) δ (ppm) 170.5, resulting solution was poured in a separatory funnel containing
169.9, 169.9, 169.5, (COCH3), 165.6 (CONH), 145.0 (Ctriazole), 25 mL of EtOAc and 30 mL of a saturated aqueous solution of
123.7 (CHtriazole), 97.4 (C1), 69.1 (C2), 68.9 (C3), 68.8 (C5), NH4Cl. Organics were washed with 2 × 25 mL of saturated
68.4 (NHCqCH2O), 66.2 (C6), 65.6 (C4), 64.6 (OCH2Ctriazole), NH4Claq, water (2 × 20 mL) and brine (10 mL). The organic
62.1 (OCH2CH2), 60.2 (Cq), 49.6 (CH2Ntriazole), 29.7 (CH2Br), phase was then dried over MgSO4 and concentrated under
20.8, 20.7, 20.6, 20.6 (COCH3); IR (cm−1): 2956, 2937, 2361, reduced pressure. Column chromatography on silica (DCM/
2337, 1751, 1734, 1540, 1370, 1218, 1045, 759; HRMS (+TOF- MeOH 98:2 to 90:10) afforded the desired compound 22
HRMS, m/z): [M + 2H]2+ calculated for C63H87BrN10O34, (50.0 mg, 3.33 μmol, 63%) as a yellowish oil. Rf 0.72 (90:10
804.2358; found, 804.2356 (Δ = −0.18 ppm); [M + H] + calcu- DCM/MeOH); 1H NMR (600 MHz, CDCl3) δ (ppm) 8.27 (m,
lated for 1607.4642, found: 1607.4620 (Δ = −1.36 ppm); [M + 3H, CHar), 7.79 (s, 9H, CHint-triazole), 7.75 (s, 27H,
Na]+ calculated for 1629.4462; found, 1629.4448 (Δ = −0.84 CHext-triazole), 7.34–7.31 (m, 12H, NH), 5.23–5.18 (m, 81H, H2,
ppm).
H3, H4), 5.05 (br s, 18H, NtriazoleCH2CONH), 4.81 (sapp, 27H,
H1), 4.62–4.53 (m, 126H, OCH2Ctriazole + NtriazoleCH2),
Synthesis of azidoacylated dendron 19: To a stirring solution 4.20–3.64 (m, 207H, OCH2 + H6 + NHCqCH2O + H5), 2.11,
of brominated trivalent dendron 18 (121.0 mg, 75.2 μmol, 2.08, 2.01, 1.96 (4s, 324H, COCH3); 13C{1H} NMR (150 MHz,
1533

Beilstein J. Org. Chem. 2014, 10, 1524–1535.
CDCl3) δ (ppm) 170.6, 170.5, 170.0, 169.9, 169.9, 169.7, 169.6 Acknowledgements
(COCH3), 168.4 (CONH), 165.4 (CONH), 144.9 + 144.8 This work was supported by a discovery grant from the
(Cext-triazole), 144.5 (Cint-triazole), 135.6 (Carom), 128.6 (CHarom), National Science and Engineering Research Council of Canada
124.9 (CHint-triazole), 124.0 (CHext-triazole), 97.5 (C1), 69.1 (C2), (NSERC) and by a Canadian Research Chair in Therapeutic
69.0 (C3), 68.7 (C5), 68.4 (NHCqCH2O), 66.2 (C6), 65.6 (C4), Chemistry. YMC is grateful to the FQRNT (Québec) for a
64.5 (OCH2Ctriazole), 62.1 (OCH2), 60.4 (Cq), 52.4 (Ntria- scholarship. We are thankful to Dr. A. Furtos (Université de
zoleCH2CONH), 49.5 (CH2Ntriazole), 20.8, 20.8, 20.7, 20.7 Montréal) for HRMS determination.
(COCH3); MS (+TOF-MS, m/z): [M + H]+ calculated for
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Supporting Information
Supporting Information File 1
Experimental procedures, characterization data, NMR, IR
and mass spectra and NMR diffusion experiments.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-10-157-S1.pdf]
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